Cell-permeable antibody compositions and methods of use

Abstract

Compositions and methods for delivering nucleic acid cargo into cells are provided. The compositions typically include (a) a 4H2 monoclonal antibody or an antigen-binding cell-permeable fragment thereof; a monovalent, bivalent, or multivalent single-chain variable fragment (scFv); or a diabody; or a humanized form or variant thereof; and (b) a nucleic acid cargo comprising, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex. Also provided are compositions and methods for increasing the activation of immune receptors, such as cGAS and TLR7, in cells of a subject. The methods typically include administering an effective amount of a 4H2 antibody to a subject. The subject may be healthy or may have a disease or disorder, such as cancer or an infectious disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 68 / 379,121, filed October 11, 2022, and U.S. Provisional Patent Application No. 68 / 379,123, filed October 11, 2022, the entire contents of each of which are specifically incorporated herein.

[0002] Reference to sequence listing The Sequence Listing, submitted as a text file named "YU8475PCT.xml" and 27,711 bytes in size, created on October 11, 2023, is hereby incorporated by reference herein pursuant to 37 CFR § 1.52(e)(5).

[0003] FIELD OF THE INVENTION The present invention relates generally to the field of intracellular delivery of nucleic acids for applications including, but not limited to, in vitro, ex vivo, and in vivo gene therapy and gene editing, and / or to enhancing immune responses, particularly through modulation of immune receptors, and its applications including, but not limited to, the treatment of cancer and infectious diseases and improved vaccination. [Background technology]

[0004] Background of the Invention gene therapy Gene therapy includes a variety of applications ranging from gene replacement and knockdown to vaccination for inherited or acquired diseases such as cancer. Viral vectors and synthetic liposomes have now emerged as the vehicles of choice for many applications, but both have limitations and risks, including complex construction, limited packaging capacity, and unfavorable immunological characteristics that limit the application of gene therapy and inhibit the potential for preventative gene therapy (Seow and Wood, Mol Ther. 17(5): 767-777 (2009)).

[0005] The in vivo uptake and distribution of nucleotide in cells and tissues has been observed (Huang, et al., FEBS Lett., 558(1-3):69-73 (2004)).In addition, for example, Nyce et al. have shown that when antisense oligodeoxynucleotide (ODN) is inhaled, it binds to endogenous surfactant (lipid produced by lung cells), and is taken up by lung cells without the need for additional carrier lipids (Nyce, et al., Nature, 385:721-725 (1997)), and small molecule nucleic acid is taken up by T24 bladder cancer tissue culture cells (Ma, et al., Antisense Nucleic Acid Drug Dev., 8:415-426 (1998)), and there is still a need for improved nucleic acid transfection technology, especially for in vivo application. AAV9, the viral vector still typically used in humans, was discovered in 2003 (Robbins, "Gene therapy pioneer says the field is behind - and that delivery technology is embarrassing," Stat, November, 2019).

[0006] Accordingly, it is an object of the present invention to provide compositions and methods of use thereof for improved delivery of nucleic acids into cells.

[0007] Modulating the immune response GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses through the production of the second messenger cGAMP, which in turn activates the adaptor STING (Chen, et al., Nat Immunol (2016)17(10):1142-9.10.1038 / ni.3558). The cGAS-STING pathway mediates protective immune defenses against infections by a wide variety of DNA-containing pathogens (e.g., microbial DNA) as well as detecting tumor-derived DNA and generating intrinsic antitumor immunity. The STING pathway and its role in immune modulation and cancer development have been reviewed, for example, in Corrales, et al., Cell Res (2017) 27(1):96-108.10.1038 / cr.2016; Corrales, et al., J Clin Invest (2016) 126(7):2404-11.10.1172 / JCI86892; Rivera Vargas, et al., Eur J Cancer (2017) 75:86-97.10.1016 / j.ejca.2016.1; Qiao, et al., Curr Opin Immunol (2017) 45:16-20.10.1016 / j.coi.2016.12.005; He, et al., Cancer Lett (2017) 402:203-12.10.1016 / j.canlet.2017.05.026.

[0008] For example, in the tumor microenvironment, T cells, endothelial cells, and fibroblasts produce type I IFN when stimulated with STING agonists ex vivo (Corrales, et al., Cell Rep (2015) 11(7):1018-30.10.1016 / j.celrep.2015.04.031). In contrast, the majority of studies have shown that tumor cells can inhibit STING pathway activation, potentially leading to immune evasion during cancer development (He, et al., Cancer Lett (2017) 402:203-12.10.1016 / j.canlet.2017.05.026; Xia, et al., Cancer Res (2016) 76(22):6747-59.10.1158 / 0008-5472.CAN-16-1404). For example, evidence indicates that activation of the STING pathway leads to the induction of spontaneous antitumor T cell responses accompanied by the expression of type I IFN genes (Chen, et al., Nat Immunol (2016) 17(10):1142-9.10.1038 / ni.3558; Barber, et al., Nat Rev Immunol (2015) 15(12):760-70.10.1038 / nri3921; Woo, et al., Immunity (2014) 41(5):830-42.10.1016 / j.immuni.2014.10.017). Furthermore, the host STING pathway is required for efficient DC-mediated cross-priming of tumor Ag-specific CD8+ T cells (Woo, et al., Immunity (2014) 41(5):830-42.10.1016 / j.immuni.2014.10.017; Deng, et al., Immunity (2014) 41(5):843-52.10.1016 / j.immuni.2014.10.019). Based on these results, direct pharmacological stimulation of the STING pathway is being explored as a cancer treatment.

[0009] The development of STING agonists has been proposed for several different therapeutic purposes, including not only cancer but also use as vaccine adjuvants and against chronic viral or bacterial infections.

[0010] With the expanding range of clinical applications, there is an increasing desire for improved compositions and methods for modulating the cGAS-STING pathway and other immune response receptor signaling pathways.

[0011] It is therefore also an object of the present disclosure to provide improved compositions and methods of use for increasing the activity of immune receptors such as pattern recognition receptors (PPRs), including cGAS and toll-like receptors (e.g., TLR7). [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Seow and Wood, Mol Ther. 17(5): 767-777 (2009) [Non-patent document 2] Huang, et al., FEBS Lett., 558(1-3):69-73 (2004) [Non-patent document 3] Nyce, et al., Nature, 385:721-725(1997) [Non-patent document 4] Ma, et al., Antisense Nucleic Acid Drug Dev., 8:415-426(1998) [Non-Patent Document 5] Robbins, "Gene therapy pioneer says the field is behind - and that delivery technology is embarrassing," Stat, November, 2019 [Non-patent document 6] Chen, et al., Nat Immunol (2016)17(10):1142-9.10.1038 / ni.3558

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[0013] Summary of the Invention Compositions and methods of use for delivering nucleic acid cargo into cells are provided. The compositions typically include (a) a 4H2 monoclonal antibody or a cell-permeable fragment thereof; a monovalent, bivalent, or multivalent single-chain variable fragment (scFv); or a diabody; or a humanized form or variant thereof; and (b) a nucleic acid cargo comprising, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex. Exemplary 4H2 antibodies and fragments and fusion proteins thereof include those having: (i) a combination of the CDR of SEQ ID NO: 1 (optionally the CDR of SEQ ID NO: 2-4) and the CDR of SEQ ID NO: 5 (optionally the CDR of SEQ ID NO: 6-8); (ii) a combination of a first, second, and third heavy chain CDR selected from SEQ ID NO: 1 (optionally the CDR of SEQ ID NO: 2-4) and a first, second, and third light chain CDR selected from SEQ ID NO: 5 (optionally the CDR of SEQ ID NO: 5-8); (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain having an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5 and a light chain having an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1; and (v) a humanized form of (iv).

[0014] In some embodiments, the antibody or fragment or fusion protein may be bispecific, eg, contain binding sequences that target a cell type, tissue, or organ of interest.

[0015] The nucleic acid cargo may be composed of DNA, RNA, modified nucleic acid, including but not limited to PNA, or a combination thereof. 4H2 binds to guanosine. Thus, the cargo typically comprises one or more guanine nucleobases, preferably one or more guanosine nucleosides. The nucleic acid cargo is typically a functional cargo, such as a functional nucleic acid (e.g., inhibitory RNA), mRNA, or a vector, such as an expression vector. The nucleic acid cargo, including a vector, may comprise a nucleic acid sequence encoding a polypeptide of interest, operably linked to an expression control sequence. The vector may be, for example, a plasmid. Typically, the cargo is not, for example, randomly sheared or fragmented genomic DNA.

[0016] In some embodiments, the cargo comprises or consists of a nucleic acid encoding a Cas endonuclease, a gRNA, or a combination thereof. In some embodiments, the cargo comprises or consists of a nucleic acid encoding a chimeric antigen receptor polypeptide. In some embodiments, the cargo is a functional nucleic acid such as an antisense molecule, an siRNA, a microRNA (miRNA), an aptamer, a ribozyme, an RNAi, or an external guide sequence, or a nucleic acid construct encoding the same.

[0017] The cargo may comprise or consist of a plurality of single nucleic acid molecules, or a plurality of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid molecules. In some embodiments, the cargo nucleic acid molecules comprise or consist of nucleic acid molecules between about 1 nucleobase and about 25,000 nucleobases in length. The cargo may be a single-stranded nucleic acid, a double-stranded nucleic acid, or a combination thereof.

[0018] Also provided is a pharmaceutical composition comprising the complex and a pharmaceutically acceptable excipient. In some embodiments, the complex is encapsulated in a polymer nanoparticle. The targeting moiety, cell-penetrating peptide, or a combination thereof may be directly or indirectly associated, linked, conjugated, or otherwise bound to the nanoparticle.

[0019] Also provided is a method for delivering nucleic acid cargo into cells by contacting cells with an effective amount of the complex alone or the complex encapsulated in nanoparticles. The contacting can be performed in vitro, ex vivo, or in vivo. In some embodiments, the ex vivo treated cells are administered to a subject in need thereof in an effective amount, for example, in an effective amount for treating one or more symptoms of a disease or disorder.

[0020] In some embodiments, the contacting is carried out in vivo after administration to a subject in need thereof.The subject may have a disease or disorder, such as a genetic disorder or cancer.The composition may be administered to the subject by, for example, injection or infusion, in an effective amount to reduce one or more symptoms of the disease or disorder in the subject.

[0021] Applications of the compositions and methods are also provided, including but not limited to gene therapy and CAR T cell manufacturing / generation / therapy.

[0022] Also provided are compositions and methods for increasing the activation of cGAS and / or other immune receptors (e.g., pattern recognition receptors such as TLR7) in cells of a subject in need thereof. The methods typically include administering an effective amount of a 4H2 antibody to the subject. Exemplary 4H2 antibody forms include, but are not limited to, intact monoclonal antibodies and cell-permeable fragments thereof, such as monovalent, bivalent, or multivalent single-chain variable fragments (scFv), diabodies, and the like. The antibody may be a humanized form, chimeric form, or variant thereof.

[0023] Exemplary 4H2 antibodies and fragments and fusion proteins thereof include, for example, (i) a combination of the CDR of SEQ ID NO: 1 (optionally the CDR of SEQ ID NO: 2-4) and the CDR of SEQ ID NO: 5 (optionally the CDR of SEQ ID NO: 6-8); (ii) a combination of the first, second, and third heavy chain CDRs selected from SEQ ID NO: 1 (optionally the CDR of SEQ ID NO: 2-4) and the first, second, and third light chain CDRs selected from SEQ ID NO: 5 (optionally the CDR of SEQ ID NO: 5-8); (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain having an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 5 and a light chain having an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1; and (v) a humanized form of (iv).

[0024] In some embodiments, the subject has cancer or an infectious disease. In some embodiments, the subject does not have cancer. Thus, methods of treating a subject for cancer and an infectious disease are also provided. In some embodiments, the subject is a healthy subject.

[0025] In some embodiments, the compositions and / or methods comprise administering to the subject an additional agent, hi some embodiments, the additional agent is a nucleic acid cargo, an immunostimulatory nucleic acid, one or more vaccine components, an immune checkpoint modulator that induces, augments, or enhances an immune response, and combinations thereof.

[0026] In certain embodiments, a method of treating cancer or an infectious disease comprises administering to a subject in need thereof an effective amount of a combination of a 4H2 antibody and an immune checkpoint modulator that induces, augments, or enhances an immune response.

[0027] Immune checkpoint modulators typically induce immune responses against cancer or infectious diseases. Immune checkpoint modulators can, for example, reduce immune inhibitory pathways, such as the PD-1 pathway. Thus, the modulator can be a PD-1 antagonist, a PD-1 ligand antagonist, or a CTLA4 antagonist. In some embodiments, the immune checkpoint modulator enhances immune activation pathways. Immune checkpoint modulators can, for example, be small molecules, antibodies, CAR-T cells, or oncolytic viruses.

[0028] In another specific embodiment, a method for treating cancer or an infectious disease comprises administering to a subject in need thereof an effective amount of a combination of a 4H2 monoclonal antibody and an immunostimulatory nucleic acid. In some embodiments, the immunostimulatory nucleic acid is a STING agonist.

[0029] In another specific embodiment, the method for vaccinating a subject comprises administering to the subject a 4H2 antibody and one or more vaccine components. The one or more vaccine components can include, for example, an antigen, a nucleic acid encoding an antigen, an adjuvant, a nucleic acid encoding an adjuvant, or a combination thereof. The antigen can be derived from, for example, a bacterium or a virus.

[0030] In some embodiments, administering a combination of 4H2 and an additional agent to a subject results in an increased immune response and / or an additive or greater than additive reduction in one or more symptoms of cancer or an infectious disease compared to the increased immune response and / or reduction in one or more symptoms of cancer or an infectious disease achieved by administering either agent alone in the absence of the other.

[0031] In some embodiments, the 4H2 antibody is administered to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before the administration of the additional agent, or any combination thereof. In other embodiments, the additional agent is administered to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before the administration of the 4H2 antibody, or any combination thereof.

[0032] Any of the methods may further include a therapeutic agent or intervention, such as a chemotherapeutic agent, an anti-infective agent, surgery, radiation therapy, or a combination thereof.

[0033] The nucleic acid cargo or nucleotide, nucleoside, or nucleobase may enhance the cell penetration of the 4H2 antibody and / or the activation of cGAS and / or another pattern recognition receptor, such as TLR7, by the 4H2 antibody. Thus, any of the compositions and methods of the present disclosure may further comprise a nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo. In some embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo is an additional drug. In some embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo is not an additional drug (i.e., is administered in combination with an additional drug). In a preferred embodiment, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo is complexed with the 4H2 antibody. In a preferred embodiment, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo contains guanine or guanosine and is complexed with the 4H2 antibody. The nucleic acid cargo may be composed of, for example, DNA, RNA, PNA, phosphorodiamidate morpholino oligomers (PMOs), or other modified nucleic acids, nucleic acid analogs, or modified nucleotide, nucleoside, or nucleobase analogs, or combinations thereof. [Brief explanation of the drawings]

[0034] [Figure 1A-B]Figures 1A-1C show that 4H2 is a DP-sensitive, cell-permeable anti-GUO autoantibody. Figure 1A shows Western blot analysis of lysates from Cal12T cells treated with 0-1 mg / mL 4H2 for 24 hours, probed with a primary actin antibody as a loading control, an anti-mouse secondary antibody to detect the actin primary antibody, and 4H2 (both mouse). The 4H2 HC and LC migrated at their expected molecular weights, indicating that this antibody was not significantly degraded after 24 hours of intracellular permeabilization. Figure 1B shows Western blot analysis of lysates from Cal12T cells treated with control medium, IgG control, or 4H2 for 24 hours. The IgG control had no effect on total ERK1 / 2 or pERK1 / 2, whereas 4H2 reduced pERK1 / 2 but not total ERK1 / 2. FIG. 1C is a dot plot showing ImageJ quantification of 4H2 fluorescence (intracellular penetration) in Cal12T cells treated with or without DP. [Figure 1C] Same as above. [Figure 2A-B] Figures 2A-2D show that 4H2 penetrates glioma cells in a GUO-responsive manner and crosses a transwell model of the BBB. Figures 2A-2C are plots showing the effect of supplementing cell culture medium with ADE or GUO on the efficiency of intracellular penetration into GSCs, as assessed by ImageJ quantification of DX1 or 4H2 fluorescent signals. ADE enhanced the penetration of DX1 (Figure 2A), but not 4H2 (Figure 2B). GUO significantly enhanced the intracellular penetration of 4H2 (Figure 2C). Figure 2D is a graph showing the results of a transwell model of the BBB using hCMEC / D3 BECs and NHA, used to assess the translocation of 4H2 across the barrier from the apical to the basolateral chamber. 4H2 crossed the barrier, and transport was inhibited by the nucleoside transport inhibitor, DP. [Figure 2C-D] Same as above. [Figure 3]Figures 3A-3B show that 4H2 localizes to orthotopic brain tumors and prolongs survival in a GBM model. Figure 3A shows Kaplan-Meier survival plots for mice bearing GSC-derived orthotopic GBM tumors treated with IgG control (N = 4) or 4H2 (N = 5). 4H2 increased median survival by 66% compared with mice treated with IgG control (**P < 0.01, log-rank test), with 40% of the 4H2-treated group surviving to study completion compared with 0% of the IgG control group. Figure 3B shows Kaplan-Meier survival plots for mice bearing GL261-derived orthotopic GBM tumors treated with IgG control (N = 6), 4H2 (N = 6), anti-PD1 (N = 6), anti-PD1 + IgG control (N = 7), or anti-PD1 + 4H2 (N = 7). 4H2 increased median survival by 32% compared with the IgG control (*P=0.03, log-rank test), and when 4H2 was combined with anti-PD1, it increased median survival by 50% compared with the anti-PD1 + IgG control (*P=0.02, log-rank test). Survival to study completion was 33% and 29% with 4H2 alone or 4H2 + anti-PD1, respectively, compared with 0% in all other groups. [Figure 4A]Figure 4A is a bar graph showing quantification of TUNEL staining by ImageJ, demonstrating a relative fold increase in TUNEL signal in 4H2-treated mice compared with the IgG control of 4.5 ± 0.6 (**P < 0.01). Figure 4B is a bar graph showing relative CD8 cell counts per high-power field (HPF) based on anti-CD8 immunostaining of sections from GBM brain tumors in mice after treatment with the IgG control or 4H2. 4H2 increased CD8 content in tumors by approximately 53%, with a relative count of 1.53 ± 0.15 in 4H2-treated mice compared with 1.00 ± 0.04 in IgG control-treated mice (*P < 0.03). These data demonstrate 4H2-mediated stimulation of T cell infiltration into GBM tumors. Figures 4C and 4D show that 4H2 does not improve survival in an immunodeficient orthotopic GBM model. Kaplan-Meier survival plots are shown for athymic nude mice bearing PPQ orthotopic GBM brain tumors treated with either IgG control (N = 4 and 6, respectively) or 4H2 (N = 4 and 6, respectively) in weekly (Figure 4C) or twice-weekly (Figure 4D) cycles. In this immunodeficient model, 4H2 did not significantly affect median survival compared to the IgG control, thus demonstrating the importance of a functional immune system for the effects of 4H2 on survival. [Figure 4B-D] Same as above. [Figure 5A-B]Figures 5A-5D are images of Western blots demonstrating 4H2 binding to cGAS. Antibody content and bound proteins were isolated using protein G beads from GSCs treated with either the IgG control or 4H2. Western blots of the input and protein G pulldowns were probed for the G proteins Ras and cGAS. No binding to Ras was observed with either the IgG control or 4H2 (Figure 5A). However, 4H2 showed clear association with cGAS, exceeding the background signal detected with the IgG control (Figure 5B). Purified cGAS (nucleic acid) was incubated with the IgG control or 4H2, and then the antibody and bound proteins were pulled down with protein G. 4H2 showed enhanced binding to cGAS compared to background IgG control binding. The presence of nucleic acid reduced 4H2 binding to cGAS, whereas nonspecific association of the IgG control with cGAS was unaffected (Figure 5C). Equivalent IgG control and 4H2 content in the pulldown samples was confirmed by anti-IgG Western blot (Figure 5D). Figures 5E and 5G are images of the Western blots, and Figure 5F is a bar graph showing that 4H2 interacts with cGAS in a nucleic acid-dependent manner. Purified recombinant cGAS was incubated with IgG control or 4H2 + / - nuclease (benzonase). The antibody and bound proteins were then isolated with protein G beads, and cGAS pulldown was visualized by Western blot and quantified using ImageJ. In the absence of nuclease, the interaction between 4H2 and cGAS was demonstrated by a ∼6-fold increase in cGAS pulldown compared to the IgG control (***P < 0.001), whereas the addition of nuclease eliminated this interaction. [Figure 5C-F] Same as above. [Figure 5G] Same as above. [Figure 6A-B]Figures 6A-6D show that 4H2 enhances cGAS activity. Figure 6A is a line graph showing that 4H2 causes a dose-dependent increase in cGAS activity. cGAS activity was assayed by measuring the relative production of cGAMP from ATP and GTP in the presence of IgG control or 4H2. Figure 6B is an image of a blot showing that 4H2 induces nuclear translocation of NF-kB in GSCs. The cytoplasmic and nuclear contents of GSCs treated with IgG control or 4H2 were separated and analyzed by Western blot probing NF-kB and lamin B1 as a loading control. GSCs transfected with control or cGAS siRNA were treated with IgG control or 4H2. Figure 6C is an image of a cGAS Western blot confirming successful knockdown. Figure 6D is a line graph showing the results of a colony formation assay demonstrating the cGAS-dependent toxicity of 4H2 to GSCs. Figure 6E is a bar graph showing that 4H2 induces nuclear translocation of NF-κB. Cytoplasmic and nuclear contents of PPQ cells treated with IgG control or 4H2 were analyzed by Western blot probing NF-κB and lamin B1 as a loading control. Relative nuclear content of NF-κB was quantified using ImageJ. 4H2 increased relative nuclear NF-κB by 2.2 ± 0.2-fold (*P < 0.05). Figure 6F is a bar graph showing the surviving fraction determined by colony formation assay in Cal12T lung cancer cells transfected with control or cGAS siRNA and treated with IgG control or 4H2 (D) (*P < 0.05), demonstrating the cGAS-dependent toxicity of 4H2. [Figure 6C-E] Same as above. [Figure 6F] Same as above. [Figure 7A]Figures 7A-7B show that 4H2 binds to DNA and RNA. Figure 7A is an image showing the binding of 4H2 to circular and linearized pcDNA3 plasmid DNA, as assessed by 1% agarose EMSA. 4H2 produced a shift consistent with binding to both forms of DNA, but not the IgG control. Figure 7B is an image showing the binding of 4H2 to total RNA and mRNA, as assessed by 1% agarose EMSA. 4H2 produced a shift consistent with binding to both forms of RNA, but not the IgG control. [Figure 7B] Same as above. [Figure 8] Figures 8A-8B are bar graphs showing that 4H2 delivers DNA and mRNA to glioma cells. pGL4.13 (luc2 / SV40) complexed with DX1 or 4H2 was added to U87 glioma cells, and luciferase activity was assayed 24 hours later (Figure 8A). Luc mRNA complexed with DX1 or 4H2 or encapsulated in MC3-LNP lipid nanoparticles was added to U87 glioma cells, and luciferase activity was assayed 24 hours later (Figure 8B). [Figure 9] Figures 9A-9B are images showing that 4H2 mediates localized gene therapy in the CNS. 4H2 / Cre mRNA was injected into the brain of Ai9 Cre reporter mice, and Cre recombinase activity was assessed by RFP fluorescence 24 hours later. RFP signals were visualized within the localized area of ​​the injection track (Figure 9A). Ai9 Cre reporter mice treated with intraocular injection of 4H2 / Cre mRNA were assessed for RFP signals 24 hours later. RFP signals visualized in the retina demonstrated 4H2-mediated retinal gene therapy (Figure 9B). [Figure 10A]Figures 10A-10B are images demonstrating 4H2 mRNA delivery in vivo. Nude mice bearing H358 flank tumors received a single intratumoral injection of DX1 or a mixture of 4H2 and Luc mRNA (w / w = 3). Luc expression was assessed by IVIS at 6, 24, and 72 hours. 4H2 / Luc mRNA successfully mediated Luc expression, whereas minimal signal was detected in tumors injected with DX1 / Luc mRNA (Figure 10A). C57 / BL6 mice were intramuscularly injected with 4H2 / Luc mRNA (left quadriceps w / w = 3, right quadriceps w / w = 1). Luc expression was assessed by IVIS at 6 and 24 hours. 4H2 / Luc mRNA successfully mediated Luc expression (Figure 10B). [Figure 10B] Same as above. [Figure 11A] Figures 11A-11B are a series of representative IVIS images (Figure 11A) and corresponding bar graphs (Figure 11B) of luminescence for untreated mice and mice treated with 4H2 alone, 4H2 + NF2 DNA, or 4H2 + NF2 mRNA in a luciferase-expressing HEI193 xenograft model. [Figure 11B] Same as above. [Figure 12A-B] Figure 12A is a schematic diagram showing the design of the 4H2-CD5 bispecific antibody. Figures 12B and 12C are a series of representative FACS plots (Figure 12B) and corresponding bar graphs (Figure 12C) showing the expression of DeRed tumor cells isolated from an Ai9 mouse model bearing MC38 tumors. [Figure 12C] Same as above. [Figure 13]Figures 13A and 13B are images of Western blots and corresponding graphs (determined by ImageJ) showing results for cell lysates from glioma stem-like cells (GSCs) treated with IgG control or 4H2 and probed for TLR7. Figure 13C is an image of a Western blot. Antibodies and bound proteins from lysates of GSCs treated with IgG control or 4H2 were pulled down with protein G beads and then analyzed by Western blot for TLR7. Blots are representative of two independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention I. Definition As used herein, the term "single-chain Fv" or "scFv" refers to a single-chain variable fragment comprising a light chain variable region (VL) and a heavy chain variable region (VH) in a single polypeptide chain, wherein the light chain variable region (VL) and the heavy chain variable region (VH) are joined by a linker that enables the scFv to form the desired structure for antigen binding (i.e., the VH and VL of the single polypeptide chain associate with each other to form the Fv). The VL and VH regions may be derived from a parent antibody or may be synthesized chemically or recombinantly.

[0036] As used herein, the term "variable region" is intended to distinguish such domains of immunoglobulins from domains shared extensively by antibodies (e.g., antibody Fc domains). Variable regions contain "hypervariable regions" whose residues are responsible for antigen-binding. The hypervariable regions comprise amino acid residues from the "complementarity determining regions" or "CDRs" (i.e., typically about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and about residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and about residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

[0037] As used herein, the terms "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0038] As used herein, the term "antibody" refers to a natural or synthetic antibody that binds to a target antigen. The term encompasses polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term also encompasses binding proteins, fragments, and polymers of those immunoglobulin molecules, as well as human or humanized versions of immunoglobulin molecules, that bind to a target antigen.

[0039] As used herein, the term "cell-permeable antibody" refers to an immunoglobulin protein, fragment, variant, or fusion protein based thereon that is transported into the cytoplasm of a living mammalian cell. As used herein, the term "cell-permeable anti-guanosine antibody" refers to an antibody or antigen-binding fragment or molecule thereof that is transported into the cytoplasm of a living mammalian cell and binds to guanosine. In some embodiments, the antibody is transported into the cytoplasm of the cell without the aid of a carrier or conjugate. In other embodiments, the antibody is conjugated to a cell-permeable moiety, such as a cell-permeable peptide.

[0040] In addition to intact immunoglobulin molecules, fragments of immunoglobulin molecules, binding proteins of immunoglobulin molecules, polymers of immunoglobulin molecules, chimeric antibodies containing sequences from more than one species, class, or subclass of immunoglobulin, such as human or humanized antibodies, and recombinant proteins containing at least the idiotype of an immunoglobulin that specifically binds DNA, are also encompassed by the term "antibody." Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic activity is tested according to known clinical testing methods.

[0041] As used herein, the term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and the reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A polypeptide variant may be naturally occurring, e.g., an allelic variant, or it may be a variant not known to occur naturally.

[0042] Modifications and changes can be made to the structure of the disclosed polypeptides, and still obtain molecules with similar characteristics to the polypeptides (e.g., conservative amino acid substitutions).For example, certain amino acids in the sequence can be substituted with other amino acids without appreciable loss of activity.Because the biological functional activity of a polypeptide is determined by its interaction ability and properties, certain amino acid sequence substitutions can be made in the polypeptide sequence, and still obtain polypeptides with similar properties.

[0043] When making such changes, the hydropathic index of amino acids can be taken into consideration.The importance of the amino acid hydropathic index in conferring interactive biological function to a polypeptide is generally understood in the art.It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indexes or scores, and still result in polypeptides with similar biological activity.Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0044] The relative hydropathicity of amino acids is believed to determine the secondary structure of the resulting polypeptide, which in turn determines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. When making such changes, substitution of amino acids with a hydropathic index within ±2 is preferred, substitution of amino acids with a hydropathic index within ±1 is particularly preferred, and substitution of amino acids with a hydropathic index within ±0.5 is even more particularly preferred.

[0045] Substitutions of similar amino acids can also be made on the basis of hydrophilicity, particularly when the resulting biologically functional equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, polypeptide. In making such changes, substitution of amino acids having hydrophilicity values ​​within ±2 is preferred, with substitution of amino acids having hydrophilicity values ​​within ±1 being particularly preferred, and substitution of amino acids having hydrophilicity values ​​within ±0.5 being even more particularly preferred.

[0046] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include the following (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg:Lys), (Asn:Gln, His), (Asp:Glu, Cys, Ser), (Gln:Asn), (Glu:Asp), (Gly:Ala), (His:Asn, Gln), (Ile:Leu, Val), (Leu:Ile, Val), (Lys:Arg), (Met:Leu, Tyr), (Ser:Thr), (Thr:Ser), (Tip:Tyr), (Tyr:Trp, Phe), and (Val:Ile, Leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of the above polypeptides. In particular, embodiments of the polypeptides may encompass variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0047] As used herein, the term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the reference nucleic acid sequence and the candidate sequence and introducing gaps, if necessary, to achieve maximum percent sequence identity.Alignment for determining percent sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequence being compared, can be determined by known methods.

[0048] As used herein, the term "specifically binds" refers to an antibody that binds to its cognate antigen (e.g., guanosine) but does not significantly bind to other antigens. For an antibody to specifically bind to a target under such conditions, the antibody must be selected for its specificity for the target. Various immunoassay formats can be used to select antibodies with specific immunoreactivity to a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies with specific immunoreactivity to a protein. For descriptions of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York. An antibody has an affinity constant (Ka) with its second molecule of about 10 5 mol -1 (e.g., 10 6 mol -1 , 10 7 mol -1 , 10 8 mol -1 , 10 9 mol -1 , 10 10 mol -1 , 10 11 mol -1 , and 10 12 mol -1 It is preferred that the antibody "specifically binds" to an antigen that is greater than 1000 kJ / s (or greater).

[0049] As used herein, the term "monoclonal antibody" or "MAb" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., an antibody in which the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules.

[0050] As used herein, the term "subject" refers to any individual targeted for administration. The subject may be a vertebrate, e.g., a mammal. Thus, the subject may be a human. The term does not denote a particular age or sex.

[0051] As used herein, the term "effective amount" means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration may merely require a reduction or alteration, not necessarily elimination. The exact dosage will vary depending on various factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, and the route of administration and pharmacokinetics of the administered agent.

[0052] As used herein, the term "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject without causing any undesirable biological effects or interacting adversely with any of the other components of the pharmaceutical composition containing it.

[0053] As used herein, the term "carrier" or "excipient" refers to an organic or inorganic, natural or synthetic inactive ingredient in a formulation that is combined with one or more active ingredients. The carrier or excipient will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0054] As used herein, the term "treating" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term encompasses active treatment, which is treatment specifically directed at ameliorating a disease, pathological condition, or disorder, and also encompasses causal treatment, which is treatment directed at eliminating the cause of the associated disease, pathological condition, or disorder. In addition, this term encompasses palliative treatment, which is treatment designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; preventative treatment, which is treatment directed at minimizing or partially or completely inhibiting the occurrence of the associated disease, pathological condition, or disorder; and supportive treatment, which is treatment used to supplement another specific therapy directed at ameliorating the associated disease, pathological condition, or disorder.

[0055] As used herein, "targeting moiety" refers to a substance that can direct particles or molecules to receptor sites of selected cells or tissue types, can act as a binding molecule, or can function to couple or bind other molecules.As used herein, "directing" refers to preferentially binding a molecule to selected cells or tissue types.As discussed below, targeting moieties can be used to direct intracellular materials, molecules, or drugs.

[0056] As used herein, the terms "inhibit" or "reduce" mean to decrease an activity, response, condition, disease, or other biological parameter. These can include, but are not limited to, the complete elimination of the activity, response, condition, or disease. These can include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount therebetween compared to native or control levels.

[0057] As used herein, a "fusion protein" refers to a polypeptide formed by joining two or more polypeptides by a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide. Fusion proteins can be formed by chemical coupling of the constituent polypeptides or can be expressed as a single polypeptide from a nucleic acid sequence encoding a single, continuous fusion protein. A single-chain fusion protein is a fusion protein having a single, continuous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join two genes in frame into a single nucleic acid sequence and then express the nucleic acid in an appropriate host cell under conditions that produce the fusion protein.

[0058] The recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually set forth herein.

[0059] Use of the term "about" is intended to describe values ​​that are approximately + / - 10% above or below the stated value. In other embodiments, values ​​may range approximately + / - 5% above or below the stated value; in other embodiments, values ​​may range approximately + / - 2% above or below the stated value; and in other embodiments, values ​​may range approximately + / - 1% above or below the stated value. The foregoing ranges are intended to be clear from the context, and no further limitation is intended to be implied.

[0060] All methods described herein can be performed in any suitable order unless otherwise specified or otherwise clearly contradicted by context. Any and all examples presented herein, or the use of exemplary language (e.g., "such as"), are intended solely to better illuminate the embodiments and do not purport to limit the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0061] Disclosed herein are materials, compositions, and components that can be used for, in conjunction with, or used to prepare the disclosed methods and compositions, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each of the various individual and collective combinations and permutations of these compounds is specifically contemplated and described herein, even though specific reference to each may not be explicitly disclosed. For example, when a ligand is disclosed and discussed, and several modifications that can be made to several molecules, including the ligand, are discussed, all possible combinations and permutations of the ligand and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, if classes of molecules A, B, and C and classes of molecules D, E, and F are disclosed, and an example molecular combination AD is disclosed, each is individually and collectively contemplated, even if each is not individually described. Thus, in this example, from the disclosure of A, B, and C; D, E, and F; and combination example AD, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF should be considered specifically contemplated and disclosed. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, from the disclosure of A, B, and C; D, E, and F; and combination example AD, the subgroups AE, BF, and CE should be considered specifically contemplated and disclosed. Furthermore, each of the materials, compositions, components, etc. contemplated and disclosed above can also be specifically and independently included in or excluded from any group, subgroup, list, set, etc. of such materials.

[0062] These concepts apply to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any particular embodiment or combination of methods of embodiments of the present disclosure, and that each such combination is specifically contemplated and should be considered disclosed.

[0063] All methods described herein can be performed in any suitable order unless otherwise specified or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") presented herein is intended solely to better illuminate the embodiments and does not pose a limitation on the scope of the embodiments unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0064] II. Composition It has been discovered that 4H2 antibodies aid in the delivery of nucleic acids across the plasma membrane into the cytoplasm. Thus, compositions and methods are provided for using 4H2 to enhance the delivery of nucleic acid constructs. Typically, an effective amount of 4H2 antibody is contacted with the nucleic acid desired to be delivered into a cell. Typically, contact is carried out for a period of time sufficient for 4H2 and the nucleic acid cargo to form a non-covalent complex. The complex and the cell are contacted for a period of time sufficient for the nucleic acid cargo to be delivered into the cell. The cargo may accumulate in greater quantity, in higher quality (e.g., more intact, functional, etc.), or at a faster rate, or a combination thereof, compared to when the cell is contacted with the nucleic acid cargo in the absence of the antibody. Because the antibody serves as a delivery vehicle, this delivery system is typically non-viral.

[0065] Multiple cell-permeable anti-DNA autoantibodies have been isolated from mouse models of SLE. While the majority of these antibodies penetrate the nuclei of live cells, the anti-GUO autoantibody 4H2 is distinguished by its cytoplasmic localization. The epitope on GUO to which 4H2 binds has been mapped to a G protein binding site, consistent with reports of anti-GUO autoantibody binding in human SLE patient sera (Colburn, et al., Journal of Rheumatology 30(5): 993-97 (2003)). Furthermore, 4H2 penetrates cultured cells and reduces cAMP levels in those cells, consistent with interference with G protein signaling (Colburn & Green, Clin Chim Acta 370: 9-16 (2006)). The results shown below indicate that 4H2 penetration into the cytoplasm is coupled to nucleoside transport, and that 4H2 binds to and mediates the delivery of nucleic acids, and also binds to and enhances the activity of cGAS, causing cGAS-dependent toxicity to tumor cells. The results also indicate that 4H2 causes activation of TLR7, as evidenced by the induction of TLR7 by 4H2 (truncated forms of TLR7 are active). Furthermore, pull-down assays indicate that 4H2 binds to the truncated forms of TLR7. Thus, compositions and methods for modulating cGAS and other pattern recognition receptors, such as TLR7, are also provided.

[0066] A.4H2 antibody Although generally referred to herein as "4H2," "4H2 antibody," or "4H2 antibodies," it will be understood that unless otherwise specified (e.g., in the Experimental Examples), fragments and binding proteins, including whole immunoglobulins as well as antigen-binding fragments, variants, and fusion proteins disclosed herein, such as scFvs, di-scFvs, tri-scFvs, and other single-chain variable fragments, chimeric and humanized forms, and other cell-permeable nucleic acid transport molecules, are encompassed by the phrases "4H2," "4H2 antibody," and "4H2 antibodies" and are also expressly provided for use in the compositions and methods disclosed herein. Antibodies are also referred to herein as cell-permeable and binding proteins.

[0067] In a preferred embodiment, the 4H2 antibody is transported into the cytoplasm of the cell without the aid of a carrier or conjugate.

[0068] Antibodies that can be used in the compositions and methods include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domain differs in sequence among antibodies and is used to determine the binding and specificity of each particular antibody for its specific antigen. However, variability is usually not evenly distributed throughout the variable domains of antibodies. The variability is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FR regions that primarily adopt a beta-sheet configuration, connecting the three CDRs, which form loops that connect, and in some cases, form part of the beta-sheet structure. The CDRs of each chain are held in close proximity by the FR regions, and the CDRs of the other chain contribute to forming the antigen-binding site of antibodies. Thus, antibodies typically contain at least the CDRs necessary to maintain binding to guanosine.

[0069] The 4H2 hybridoma was previously generated from the MRLmpj / lpr lupus mouse model. 4H2 does not localize to lysosomes or endosomes, where cargo molecules are often destroyed with other delivery vehicles, such as the TAT peptide. 4H2 is a cell-permeable lupus anti-guanosine antibody that can reduce ERK and Akt phosphorylation in cells and is toxic to cancer cells with various mutations of the small GTPase K-Ras, but does not have significant toxicity to cells with wild-type K-Ras. See International Application Publication Nos. WO 2015 / 134607 and WO 2017 / 218824, each of which is specifically incorporated by reference in its entirety.

[0070] The 4H2 antibody is typically a monoclonal 4H2, or a variant, derivative, fragment, fusion, or humanized form thereof that binds to the same or different epitope(s) as 4H2.

[0071] 1. Antibody sequence a.4H2 light chain variable region The amino acid sequence of the kappa light chain variable region (VL) of mAb 4H2 is [ka] is.

[0072] [ka] The complementarity determining regions (CDRs) containing the

[0073] b.4H2 heavy chain variable region The amino acid sequence of the heavy chain variable region (VH) of mAb 4H2 is: [ka] is.

[0074] [ka] The complementarity determining regions (CDRs) containing the

[0075] 2. Antibody morphology Exemplary antibodies that can be used include whole immunoglobulins of any class (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The variable domain differs in sequence among antibodies and is used to determine the binding and specificity of each particular antibody for its specific antigen. However, variability is usually not evenly distributed throughout the variable domains of antibodies. The variability is typically concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FR regions that primarily adopt a beta-sheet configuration, connecting the three CDRs, which form loops that connect, and in some cases, form part of the beta-sheet structure. The CDRs of each chain are held in close proximity by the FR regions, and the CDRs of the other chain contribute to forming the antigen-binding site of antibodies. Thus, the antibody may contain the CDR components necessary to penetrate into cells and bind to guanosine.

[0076] The antibody may be a humanized or chimeric antibody, or a fragment, variant, or fusion protein thereof. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues from a non-human source introduced into it. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. Antibody humanization techniques generally involve using recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.

[0077] A 4H2 antibody may consist of an antibody fragment or fusion protein that contains one or more CDRs that are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical in amino acid sequence to the CDR(s) of 4H2 or a variant or humanized form thereof (e.g., the CDR(s) of any of SEQ ID NOS: 1 and 5, e.g., SEQ ID NOS: 2, 3, 4 and 6, 7, 8, respectively). Determining the percent identity of two amino acid sequences can be determined by BLAST protein comparison. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the CDRs of the above preferred variable domains (e.g., SEQ ID NOs: 1 and 5) without any alterations or with up to 0, 1, 2, 3, 4, or 5 alterations per CDR (i.e., selected independently for each CDR) or across all CDRs in total.

[0078] The 4H2 antibody may consist of an antibody fragment or fusion protein comprising a variable heavy and / or variable light chain amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the variable heavy and / or light chain amino acid sequence of 4H2 or a humanized form thereof (e.g., SEQ ID NOs: 5 and 1).

[0079] Preferably, the antibody comprises a combination of heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3.

[0080] Thus, in some embodiments, the cell-permeable antibody contains the CDRs of SEQ ID NOs: 5 and 1, or the entire heavy and light chain variable regions; or humanized forms thereof.

[0081] Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and therefore may induce undesired immune responses when administered to humans. Therefore, humanized 4H2 antibodies, antibody fragments, and fusions are provided. Humanized antigen-binding molecules can reduce the likelihood that antibodies, antibody fragments, or scFvs will induce undesired immune responses when administered to humans.

[0082] Humanized forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include those in which residues from a complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are neither found in the recipient antibody nor in the imported CDR or framework sequences. Generally, humanized antibodies contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions correspond to those of a human immunoglobulin consensus sequence. The humanized antibody optimally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0083] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues, often referred to as "import" residues, are typically obtained from an "import" variable domain. Antibody humanization techniques generally involve manipulating the DNA sequence encoding one or more polypeptide chains of an antibody molecule using recombinant DNA technology. Humanization can essentially be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, humanized forms of non-human antibodies (or fragments thereof) are chimeric antibodies or fragments in which substantially less of the human variable domain than the intact human variable domain has been substituted by the corresponding sequence from the non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0084] The selection of human variable domains, both light and heavy, used to generate a humanized antibody is crucial to reduce antigenicity. According to the "best fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework (FR) for the humanized antibody. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies.

[0085] It is further important that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are preferably prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen(s). In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0086] Also encompassed are biologically active antibody fragments, which may contain insertions, deletions, substitutions, or other selected modifications of particular regions or particular amino acid residues, whether or not linked to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified antibody or antibody fragment.

[0087] Techniques can also be adapted to produce single-chain antibodies specific to the antigenic proteins of the present disclosure. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can be created by fusing the heavy-chain variable domain and the light-chain variable domain using a short peptide linker, thereby reconstituting an antigen-binding site on a single molecule. Single-chain antibody variable fragments (scFvs) have been developed in which the C-terminus of one variable domain is joined to the N-terminus of the other variable domain with a peptide or linker of, for example, 15-25 amino acids, without significantly disrupting antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind in their proper conformational orientation.

[0088] 4H2 antibodies can be modified to improve their therapeutic potential. For example, in some embodiments, the cell-permeable 4H2 antibody is conjugated with another antibody specific for a second therapeutic target, for example, in the cytoplasm and / or nucleus of the target cell. For example, the cell-permeable 4H2 antibody can be a fusion protein containing 4H2 Fv and a single-chain variable fragment of a monoclonal antibody that specifically binds to the second target. In other embodiments, the cell-permeable 4H2 antibody is a bispecific antibody that has a first heavy chain and a first light chain derived from 4H2 and a second heavy chain and a second light chain derived from a monoclonal antibody that specifically binds to the second target.

[0089] In some embodiments, the second target is specific to a target cell type, tissue, organ, etc. Thus, the second heavy chain and the second light chain can serve as a targeting moiety that targets and delivers the complex to a target cell type, tissue, organ, etc. In some embodiments, the second heavy chain and the second light chain are specific to hematopoietic stem cells, CD34 + In some embodiments, the second heavy chain and the second light chain target thymus, spleen, or cancer cells, for example, by targeting a receptor or ligand expressed in the desired cell type.

[0090] In some embodiments, particularly those for targeting T cells in vivo, immune cell or T cell markers, such as CD3, CD5, CD7, or CD8, can be targeted, e.g., for in vivo production of CAR T cells. For example, both anti-CD8 antibodies and anti-CD3 Fab fragments have been used to target T cells in vivo (Pfeiffer, et al., EMBO Mol Med., 10(11)(2018). pii: e9158.doi: 10.15252 / emmm.201809158., Smith, et al., Nat Nanotechnol., 12(8):813-820(2017). doi: 10.1038 / nnano.2017.57). Thus, in some embodiments, the 4H2 antibody or antigen-binding fragment or fusion protein is a bispecific antibody portion capable of specifically binding to CD3, CD5, CD7, CD8, or another immune cell (e.g., T cell) marker, or a marker of a particular tissue such as the thymus, spleen, or liver.

[0091] Exemplary fragments and fusions include, but are not limited to, single-chain antibodies, single-chain variable fragments (scFv), di-scFv, tri-scFv, diabodies, triabodies, tetrabodies, disulfide-linked Fv (sdFv), Fab', F(ab')2, Fv, and single-domain antibody fragments (sdAb).

[0092] For example, bivalent single-chain variable fragments (di-scFvs) can be engineered by linking two scFvs. This can be done by creating a single peptide chain with two VH and two VL regions, resulting in tandem scFvs. scFvs can also be designed with a linker peptide that is too short (approximately 5 amino acids) for the two variable regions to fold together, allowing the scFvs to form dimers. This type is known as a diabody. Diabodies have been shown to have dissociation constants up to 40-fold lower than those of corresponding scFvs, meaning they have much higher affinity for their targets. Even shorter linkers (one or two amino acids) result in the formation of trimers (triabodies or tribodies). Tetrabodies have also been created, which exhibit even higher affinity for their targets than diabodies. In some embodiments, a 4H2 antibody may contain two or more linked single-chain variable fragments of 4H2 (e.g., 4H2 di-scFv, 4H2 tri-scFv) or conservative variants thereof. In some embodiments, a 4H2 antibody is a diabody or triabody (e.g., 4H2 diabody, 4H2 triabody).

[0093] In some embodiments, antibodies are conjugated or fused to a cell-penetrating moiety, such as a cell-penetrating peptide, to facilitate cell entry. Examples of cell-penetrating peptides include, but are not limited to, polyarginine (e.g., R9), antennapedia sequence, TAT, HIV-Tat, penetratin, Antp-3A (Antp mutant), buforin II, transportan, MAP (model amphipathic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol), and BGTC (bis-guanidinium-Tren-cholesterol). In other embodiments, antibodies are modified using TransMabs™ technology (InNexus Biotech., Inc., Vancouver, BC).

[0094] The function of an antibody or a fragment thereof can be enhanced by coupling it with a therapeutic agent. Such coupling of an antibody or fragment with a therapeutic agent can be achieved by creating an immunoconjugate comprising the antibody or antibody fragment and the therapeutic agent, or by creating a fusion protein comprising the antibody or antibody fragment and the therapeutic agent, or by linking the antibody or fragment with a nucleic acid such as DNA or RNA (e.g., siRNA) so as to comprise the antibody or antibody fragment and the therapeutic agent.

[0095] A recombinant fusion protein is a protein created by genetic engineering of a fusion gene. This genetic engineering typically involves removing the stop codon from the cDNA sequence encoding the first protein, and then adding the cDNA sequence of the second protein in frame by ligation or overlap extension PCR. The DNA sequence is then expressed in cells as a single protein. The protein can be engineered to contain the complete sequence of both original proteins, or only a portion of one of them. When the two entities are proteins, a linker (or "spacer") peptide is often also added, which increases the likelihood that the proteins will fold independently and behave predictably.

[0096] In some embodiments, the cell-permeable antibody is modified to alter its half-life. In some embodiments, it is desirable to increase the half-life of the antibody so that it remains in the circulation or at the treatment site for a longer period of time. For example, it may be desirable for the antibody's titer to be maintained in the circulation or at the treatment site for an extended period of time. In other embodiments, the half-life of the 4H2 antibody is shortened to reduce potential side effects. Antibody fragments, such as 4H2Fv, may have a shorter half-life than full-sized antibodies. Other methods of altering half-life are known and can be used in the described methods. For example, the antibody can be engineered with an Fc variant that has an extended half-life, for example, using Xtend™ antibody half-life extension technology (Xencor, Monrovia, CA).

[0097] a. linker The term "linker" as used herein includes, but is not limited to, peptide linkers. Peptide linkers can be of any size, provided they do not interfere with the binding of the variable region to its epitope. In some embodiments, the linker comprises one or more glycine and / or serine amino acid residues. Monovalent single-chain antibody variable fragments (scFvs) typically connect the C-terminus of one variable domain to the N-terminus of the other variable domain via a peptide or linker of 15 to 25 amino acids. The linker is selected to allow the heavy and light chains to bind in their proper conformational orientation. Linkers in diabodies, triabodies, etc. typically contain shorter linkers than those of the monovalent scFvs described above. Di-scFvs, tri-scFvs, and other multivalent scFvs typically contain three or more linkers. The linkers can be the same or different in length and / or amino acid composition. Thus, the number of linkers, the composition of the linker(s), and the length of the linker(s) can be determined based on the desired valency of the scFv, as known in the art. The linker(s) can enable or drive the formation of di-scFvs, tri-scFvs, and other multivalent scFvs.

[0098] For example, the linker can contain 4 to 8 amino acids. In certain embodiments, the linker contains the amino acid sequence GQSSRSS (SEQ ID NO: 10). In other embodiments, the linker contains 15 to 20 amino acids, e.g., 18 amino acids. In certain embodiments, the linker contains the amino acid sequence GQSSRSSSGGGSSGGGGS (SEQ ID NO: 11). Other flexible linkers include, but are not limited to, the amino acid sequences Gly-Ser, Gly-Ser-Gly-Ser (SEQ ID NO: 12), Ala-Ser, Gly-Gly-Gly-Ser (SEQ ID NO: 13), (Gly-Ser) (SEQ ID NO: 14), (Gly-Ser) (SEQ ID NO: 15), and (Gly-Gly-Gly-Gly-Ser) (SEQ ID NO: 16).

[0099] Other exemplary linkers include, for example: RADAAPGGGGSGGGGSGGGGS (SEQ ID NO: 17) and ASTKGPSVFPLAPLESSGS (SEQ ID NO: 18).

[0100] b. Exemplary 4H2 scFv Sequences Those skilled in the art will appreciate that exemplary fusion proteins, or domains thereof, can be utilized to construct the fusion proteins discussed in more detail above. For example, in some embodiments, the scFv comprises an scFv comprising a V variable region (SEQ ID NO: 1, or a functional variant or fragment thereof) linked to a V variable domain (e.g., SEQ ID NO: 5, or a functional variant or fragment thereof). In some embodiments, the di-scFv comprises a first scFv comprising a V variable region (SEQ ID NO: 1, or a functional variant or fragment thereof) linked to a V variable domain (e.g., SEQ ID NO: 5, or a functional variant or fragment thereof), linked to a second scFv comprising a V variable region (e.g., SEQ ID NO: 1, or a functional variant or fragment thereof) linked to a V variable domain (e.g., SEQ ID NO: 5, or a functional variant or fragment thereof). In some embodiments, the tri-scFv comprises a di-scFv linked to a third scFv domain comprising a V variable region (e.g., SEQ ID NO: 1, or a functional variant or fragment thereof) linked to a V variable domain (e.g., SEQ ID NO: 5, or a functional variant or fragment thereof).

[0101] The Vk variable region and VH variable domain can be linked, for example, by a linker (e.g., (GGGGS)3 (SEQ ID NO: 19) alone or in combination with a (6 amino acid) light chain CH1 (e.g., RADAAP (SEQ ID NO: 20)). Other suitable linkers are described above and known in the art. The scFv can be linked by a linker (e.g., the first 13 amino acids of human IgG CH1 (e.g., ASTKGPSVFPLAP (SEQ ID NO: 21)) alone or in combination with a swivel sequence (e.g., LESSGS (SEQ ID NO: 22)). Other suitable linkers are described above and known in the art.

[0102] In some embodiments, the fusion protein comprises an additional domain. For example, in some embodiments, the fusion protein comprises a sequence that enhances solubility. In some embodiments, the fusion protein comprises one or more domains that enhance the purification, isolation, capture, identification, separation, etc. of the fusion protein. Exemplary domains include, for example, Myc tags and / or His tags. Other replaceable and additional domains are discussed in more detail above.

[0103] Exemplary scFv molecules are also provided. [ka] Single underline: 4H2 VL sequence Double underline: linker sequence Dashed underline: 4H2 VH sequence Wavy underline: His6 tag

[0104] Examples of scFvs include those in which the N-terminal sequence of 4H2 VH of SEQ ID NO: 9 is linked to the C-terminus of the 4H2 VL of SEQ ID NO: 9, or those in which the N-terminal sequence of 4H2 VL of SEQ ID NO: 9 is linked to the C-terminus of the 4H2 VH of SEQ ID NO: 9. The linker of SEQ ID NO: 9 can be replaced with alternative linkers, including but not limited to, those disclosed herein. Typically, linkers are about 10 to about 25 amino acids and typically contain glycine. The His6 tag of SEQ ID NO: 9 can be replaced with another tag, moved to the N-terminus of the scFv, or deleted entirely. In some embodiments, the 4H2 VL, 4H2 VH, or a combination thereof is a variant or humanized form of the 4H2 VL and / or 4H2 VH of SEQ ID NO: 9. In some embodiments, the 4H2 VL and / or 4H2 VH domains are truncated at both the N-terminus and C-terminus compared to the 4H2 VL and / or 4H2 VH of SEQ ID NO: 9. The scFv may comprise the three CDRs of 4H2 VL and / or 4H2 VH of SEQ ID NO: 9 or a humanized form thereof. In some embodiments, the antibody, or fragment or fusion thereof, has at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent sequence identity to SEQ ID NO: 9. In some embodiments, the antibody, or fragment or fusion thereof, has a VL domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent sequence identity to the 4H2 VL domain of SEQ ID NO: 9. In some embodiments, the antibody, or fragment or fusion thereof, has a VH domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent sequence identity to the 4H2 VH domain of SEQ ID NO: 9.

[0105] SEQ ID NO: 9 and its humanized forms and variants can be used in any of the compositions and methods disclosed herein. In some embodiments, SEQ ID NO: 9 or its humanized forms or variants are used in therapeutic methods, such as the methods disclosed herein, without conjugation with nanocarriers or therapeutic agents. Thus, in some embodiments, SEQ ID NO: 9 or its humanized forms or variants is the only therapeutic agent. In some embodiments, SEQ ID NO: 9 or its humanized forms or variants is not a therapeutic agent (e.g., is merely a targeting moiety), or is one of two or more therapeutic agents.

[0106] c. Exemplary 4H2 Bispecific Antibodies An exemplary bispecific antibody is utilized in Example 13 below. This antibody has a format according to Figure 12A and the following heavy and light chain variable region sequences: [ka] [ka]

[0107] It will be understood that this antibody is merely exemplary, and that other formats, alternative sequences, particularly framework sequences, and even other second arm binding domains targeting antigens other than CD5 are expressly provided. For example, in some embodiments, the CDRs of SEQ ID NOs: 1, 5, 23, and 24 or humanized forms thereof (e.g., having one, two, or three mutations, e.g., conservative substitutions, per CDR or across CDRs) are also provided as chimeric or humanized bispecific antibodies having human heavy and light chain variable region frameworks and, optionally, constant domains.

[0108] The predicted CDRs of 4H2 are underlined above and are expressly provided. The predicted CDRs of anti-CD5 are underlined above and are expressly provided as follows: [ka]

[0109] For example, a 4H2-CD5 bispecific antibody may be composed of an antibody fragment or fusion protein containing one or more CDRs that are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical in amino acid sequence to the CDR(s) of 4H2 or a variant or humanized form thereof (e.g., the CDR(s) of any of SEQ ID NOS: 1 and 5, e.g., SEQ ID NOS: 2, 3, 4, and 6, 7, and 8, respectively), in combination with an anti-CD5 antibody fragment or fusion protein or a variant or humanized form thereof (e.g., the CDR(s) of any of SEQ ID NOS: 23 and 24, e.g., SEQ ID NOS: 25, 26, 27, and 28, 29, and 30, respectively). Determining the percent identity of two amino acid sequences can be determined by BLAST protein comparison. In some embodiments, the antibody comprises the CDRs of one, two, three, four, five, or all six of the above preferred variable domains (e.g., SEQ ID NOs: 1 and 5 and / or 23 and 24) without any alterations or with up to 0, 1, 2, 3, 4, or 5 alterations per CDR (i.e., selected independently for each CDR) or across all CDRs in total.

[0110] A 4H2-CD5 bispecific antibody may be composed of an antibody fragment or fusion protein comprising a variable heavy and / or variable light chain amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the variable heavy and / or light chain of 4H2 or a humanized form thereof (e.g., SEQ ID NOs: 5 and 1), and the variable heavy and / or light chain of anti-CD5 or a humanized form thereof (e.g., SEQ ID NOs: 24 and 23).

[0111] Preferably, the bispecific antibody comprises a combination of the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3, respectively, of 4H2 in combination with a heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 combination of anti-CD5.

[0112] Thus, in some embodiments, the cell-permeable bispecific antibody contains the CDRs, or the entire heavy and light chain variable regions, of SEQ ID NOs: 5 and 1 and 24 and 23 or humanized forms thereof.

[0113] B. Additional medications The results described below indicate that extracellular nucleic acids facilitate intracellular penetration by 4H2. Furthermore, cGAS activation by cytosolic DNA results in the endogenous production of cyclic GMP-AMP, a unique second messenger that binds to stimulator of interferon genes (STING) and leads to the activation of TANK-binding kinase 1 (TBK1) and IRF3, thereby resulting in the transcription of genes encoding type I interferons (Pesiridis and Fitzgerald, Nature Reviews Genetics volume 20, pages 657-674 (2019)). The experimental results below also indicate that 4H2 activates cGAS and other pattern recognition receptors (PPRs), such as TLR7. Such activation may be due to direct binding and activation by 4H2, or may be due to indirect binding through simultaneous interactions between immune receptors, 4H2, and cytoplasmic nucleic acids and / or GTP.

[0114] Therefore, the compositions of the present disclosure can be used to facilitate the delivery of nucleic acid cargo.In addition or alternatively, 4H2 antibody can be used with or without the aid of nucleic acid cargo to modulate immune response.For example, in some embodiments, compositions and methods include nucleic acid and / or GTP (also referred to as nucleic acid cargo) to facilitate the intracellular penetration of 4H2 and / or the activation of another PRR, such as cGAS and / or TLR7.

[0115] Furthermore, STING agonists have been proposed for several different therapeutic purposes, including for the treatment of cancer, infectious diseases, and as vaccine adjuvants. See, for example, Pesiridis and Fitzgerald, Nature Reviews Genetics volume 20, pages 657-674 (2019), the entire contents of which are expressly incorporated herein by reference. Thus, in some embodiments, the disclosed compositions and methods further include an additional agent for these applications. Non-limiting examples of additional agents include, but are not limited to, additional STING agonists, vaccine compositions, and immune checkpoint inhibitors, each of which is discussed in more detail below. In some embodiments, the additional agent is a nucleic acid (e.g., a nucleic acid encoding a vaccine component such as an immunostimulatory oligonucleotide or a peptide antigen). Such additional agent nucleic acid may be a nucleic acid cargo and, in addition to or instead of, may be separately administered to a subject.

[0116] Therefore, any additional agent may be present in the same or different mixture as the 4H2 antibody, and may be administered at the same or different time point as the 4H2 antibody. In some embodiments, such as when the additional agent is a nucleic acid cargo, the additional agent and the 4H2 antibody are contacted to form a complex, and then administered to a subject. The interaction between the antibody and the nucleic acid cargo is non-covalent. In such embodiments, the complex can be administered to a subject. Although referred to as cargo, the cargo nucleic acid disclosed herein may be administered separately, and therefore is not necessarily the cargo of the 4H2 antibody under these conditions.

[0117] 1. Cargo Nucleic acid cargo is also provided.As discussed in more detail below, the 4H2 antibody of the present disclosure can be used to deliver nucleic acid cargo to cells for any purpose.In certain embodiments, cargo can also be used to increase the cell penetration of 4H2 antibody and / or increase the activation of other PRRs, such as cGAS and / or TLR7.As used in the nucleic acid delivery method presented herein, typically, 4H2 is contacted with cells as a complex with nucleic acid cargo.The interaction between antibody or binding protein and nucleic acid cargo is non-covalent.

[0118] The nucleic acid cargo can be single-stranded or double-stranded, or a single base, nucleoside, or nucleobase, or multiple thereof. In some embodiments, the cargo is GTP, GDP, GMP, cGAMP, or cGMP. The nucleic acid cargo can be or include DNA, RNA, a nucleic acid analog, or a combination thereof. As discussed in more detail below, the nucleic acid analog may be modified at the base moiety, sugar moiety, or phosphate backbone. Such modifications may, for example, improve the stability, hybridization, or solubility of the nucleic acid. 4H2 binds to guanosine. Thus, the cargo typically includes one or more guanine nucleobases, preferably one or more guanosine nucleosides.

[0119] The nucleic acid cargo may be functional, in the sense that it is or encodes a biologically active agent when delivered into a cell, or it may be non-functional and simply facilitate delivery of 4H2 to the cytoplasm and / or activation of its cGAS and / or another PRR, such as TLR7. Exemplary cargoes are discussed in more detail below, and include, for example, mRNA or DNA encoding a polypeptide of interest, including, for example, expression constructs and vectors, inhibitory nucleic acids such as siRNA, or nucleic acids encoding inhibitory nucleic acids, including, for example, expression constructs and vectors, or non-coding RNA or DNA.

[0120] The compositions of the present disclosure may comprise a plurality of a single nucleic acid cargo molecule. In some embodiments, the composition comprises a plurality of a variety of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different nucleic acid molecules.

[0121] In some embodiments, the cargo molecule is 0.001 kilobase, 0.01 kilobase, 1 kilobase, tens of kilobases, hundreds of kilobases, thousands of kilobases, tens of thousands of kilobases, and / or hundreds of thousands of kilobases in length.

[0122] In some embodiments, for example, the cargo is between 0.001 kb and 100 kb, or between 0.001 kb and 50 kb, or between 0.001 kb and 25 kb, or between 0.001 kb and 12.5 kb, or between 0.001 kb and 10 kb, or between 0.001 kb and 8 kb, or between 0.001 kb and 5 kb, or between 0.001 kb and 2.5 kb, or between 0.001 kb and 1 kb, or between 0.01 kb and 100 kb, or between 0.01 kb and 50 kb, or between 0.01 kb and 25 kb, or between 0.01 kb and 12.5 kb, or between 0.01 kb and 10 kb, or between 0.01 kb and 8 kb, or between 0.01 kb and 5 kb, inclusive. The length may be between 0.01 kb and 2.5 kb, or between 0.01 kb and 1 kb, or between 0.1 kb and 100 kb, or between 0.1 kb and 50 kb, or between 0.1 kb and 25 kb, or between 0.1 kb and 12.5 kb, or between 0.1 kb and 10 kb, or between 0.1 kb and 8 kb, or between 0.1 kb and 5 kb, or between 0.1 kb and 2.5 kb, or between 0.1 kb and 1 kb, or between 1 kb and 100 kb, or between 1 kb and 50 kb, or between 1 kb and 25 kb, or between 1 kb and 12.5 kb, or between 1 kb and 10 kb, or between 1 kb and 8 kb, or between 1 kb and 5 kb, or between 1 kb and 2.5 kb.

[0123] In some embodiments, for example, the cargo may be between 0.2kb and 10kb, or between 0.2kb and 5kb, or between 0.2kb and 2.5kb, or between 0.2kb and 1kb, or between 0.2kb and 0.5kb, or between 0.2kb and 0.25kb, or between 0.5kb and 10kb, or between 0.5kb and 5kb, or between 1kb and 5kb, or between 1kb and 3kb, or between 2kb and 10kb, or between 3kb and 5kb.

[0124] It will be understood that for specific applications, the nucleic acid cargo may be one or more distinct lengths, for example, within one of the aforementioned ranges (inclusive), with each specific value expressly disclosed. For example, the size may be as small as a single base or a nucleic acid base. In an exemplary application, the cargo is a cyclic dinucleotide such as cGAMP, a STING agonist. In other embodiments, the cargo is a short oligomer. For example, an 8-mer oligomer can be used for antisense or splice switching. Slightly longer oligomers (e.g., 18-20 mers) can be used for gene editing.

[0125] a. Cargo type Nucleic acid cargo can be nucleic acid, and can be an isolated nucleic acid composition.As used herein, " isolated nucleic acid " refers to nucleic acid that is separated from other nucleic acid molecules present in mammalian genome, including the nucleic acid that is usually adjacent to one or both sides of the nucleic acid in mammalian genome.When the term " isolated " is used herein in reference to nucleic acid, it also encompasses any combination of non-naturally occurring nucleic acid sequences, because such non-naturally occurring sequences are not found in nature and do not have immediate neighboring sequences in naturally occurring genome.

[0126] An isolated nucleic acid can be, for example, a DNA molecule, provided that one of the nucleic acid sequences normally found immediately adjacent to the DNA molecule in a naturally occurring genome has been removed or is absent. Thus, isolated nucleic acid includes, but is not limited to, DNA molecules that exist as separate molecules independent of other sequences (e.g., chemically synthesized nucleic acids, or cDNAs, or genomic DNA fragments generated by PCR or restriction endonuclease treatment), as well as recombinant DNA molecules incorporated into vectors, autonomously replicating plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, or herpes viruses), or into the genomic DNA of prokaryotes or eukaryotes. Furthermore, isolated nucleic acids can include engineered nucleic acids, such as recombinant DNA molecules that are part of hybrid or fusion nucleic acids. For example, a nucleic acid present among hundreds to millions of other nucleic acids in a cDNA or genomic library, or in a gel slice containing a genomic DNA restriction enzyme digest, is not considered an isolated nucleic acid.

[0127] Nucleic acid sequences encoding polypeptides include genomic sequences. Exon-deleted mRNA / cDNA sequences are also disclosed. Other nucleic acid sequences encoding polypeptides, such as polypeptides comprising the above-identified amino acid sequences and fragments and variants thereof, are also disclosed. The nucleic acid encoding the polypeptide can be optimized for expression in a selected expression host. To account for differences in codon usage between the organism from which the nucleic acid sequence is derived and the expression host, codons can be replaced with alternative codons encoding the same amino acid. In this way, nucleic acids can be synthesized using codons preferred by the expression host.

[0128] The nucleic acid can be in sense or antisense orientation, or can be complementary to a reference sequence that encodes, for example, a polypeptide.

[0129] i. Vector The cargo may be a vector, for example, a vector encoding a polypeptide(s) and / or functional nucleic acid(s). Nucleic acids, such as those described above, can be inserted into a vector for expression in a cell. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment can be inserted to bring about replication of the inserted segment. The vector may be an expression vector. An "expression vector" is a vector containing one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0130] The nucleic acid in the vector may be operably linked to one or more expression control sequences. For example, the control sequences can be incorporated into a gene construct, thereby effectively controlling the expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that is typically composed of a region of a DNA molecule within 100 nucleotides upstream of the transcription start site (generally near the initiation site of RNA polymerase II). To place a coding sequence under the control of a promoter, it is necessary to position the translation start site of the translational reading frame of the polypeptide 1 to approximately 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function at various distances from the transcription site. Enhancers may be located downstream of the transcription start site. A coding sequence is "operably linked to" and "under the control" of an expression control sequence if RNA polymerase in the cell is capable of transcribing the coding sequence into mRNA that can then be translated into the protein encoded by the coding sequence.

[0131] Suitable expression vectors include, but are not limited to, plasmids, cosmids, and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0132] In some embodiments, the cargo is delivered intracellularly and remains extrachromosomal. In some embodiments, the cargo is introduced into a host cell and integrated into the host cell's genome. As discussed in more detail below, the composition can be used in gene therapy methods. Gene therapy methods can include introducing into a cell a polynucleotide that alters the genotype of the cell. The introduction of the polynucleotide can correct, replace, or otherwise alter an endogenous gene by genetic recombination. The method can include introducing an entire replacement copy of a defective gene, a heterologous gene, or a small nucleic acid molecule such as an oligonucleotide. For example, the corrective gene can be introduced into a non-specific location within the host's genome.

[0133] In some embodiments, the cargo is a vector. Methods for constructing expression vectors containing gene sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Expression vectors generally contain regulatory sequences and elements necessary for the translation and / or transcription of the inserted coding sequence, which may be, for example, a polynucleotide of interest. To help control the expression of the desired gene product, the coding sequence can be operably linked to a promoter and / or enhancer. Promoters used in biotechnology are of different types depending on the type of gene expression control intended. Promoters can generally be divided into constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0134] For example, in some embodiments, the polynucleotide of interest is operably linked to a promoter or other regulatory elements known in the art. Thus, the cargo can be a vector, such as an expression vector. Engineering a polynucleotide for expression in prokaryotic or eukaryotic systems can be performed by techniques commonly known to those skilled in the art of recombinant expression. Expression vectors typically contain one of the compositions of the present disclosure under the control of one or more promoters. To place a coding sequence "under the control" of a promoter, the 5' end of the translation initiation site of the reading frame is generally positioned approximately 1-50 nucleotides "downstream" of the selected promoter (i.e., 3' to the selected promoter). The "upstream" promoter stimulates transcription of the inserted DNA, promoting expression of the encoded recombinant protein or functional nucleic acid. This is the meaning of "recombinant expression" in the context used herein.

[0135] Many standard techniques are available for constructing expression vectors containing appropriate nucleic acids and transcriptional / translational control sequences to effect protein or peptide or functional nucleic acid expression in a variety of host expression systems.

[0136] Expression vectors for use in mammalian cells usually contain an origin of replication (if necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. The origin of replication can be provided by constructing the vector to contain an exogenous origin, such as from SV40 or other viral (e.g., polyoma, adeno, VSV, BPV) sources, or can be provided by the replication mechanism of the host cell chromosome. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0137] Promoters can be derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). Furthermore, it may be possible, and even desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell system.

[0138] Several viral-based expression systems are available; for example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40 (SV40). Both the early and late promoters of SV40 virus are useful because they are easily obtained from the virus as a fragment that also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication is included.

[0139] When adenovirus is used as an expression vector, the coding sequence can be ligated to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the protein in infected hosts.

[0140] Specific initiation signals may also be required for efficient translation of the compositions of the present disclosure. These signals include the ATG initiation codon and adjacent sequences. It may be necessary to provide additional exogenous translational control signals, including the ATG initiation codon. One of skill in the art can readily determine this requirement and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be in-frame (or in phase) with the reading frame of the desired coding sequence. These exogenous translational control signals and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by including appropriate transcription enhancer elements or transcription terminators.

[0141] For eukaryotic expression, it is typically also desirable to incorporate an appropriate polyadenylation site into the transcription unit if one was not contained within the original cloned segment. Typically, the poly A addition site is located approximately 30-2000 nucleotides "downstream" of the protein's termination site, at a position prior to the termination of transcription.

[0142] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines can be engineered that stably express protein-encoding constructs. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with vectors controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introducing the foreign DNA, engineered cells can be grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form nests, which can then be cloned and expanded into cell lines.

[0143] ii. mRNA The cargo may be mRNA.

[0144] Chemical structures that can promote stability and / or translation efficiency can also be used. For example, the RNA can have a 5'UTR and a 3'UTR. The length of the 3'UTR can be, for example, more than 100 nucleotides. In some embodiments, the 3'UTR sequence is between 100 and 5000 nucleotides. In some embodiments, the 5'UTR is between 0 and 3000 nucleotides in length. The length of the 5'UTR sequence and the 3'UTR sequence added to the coding region can be changed by various methods, including, but not limited to, designing PCR primers that anneal with different regions of the UTR. Using this method, those skilled in the art can modify the length of the 5'UTR and the 3'UTR as needed to achieve optimal translation efficiency after delivery of the transcribed RNA.

[0145] 5'UTR and 3'UTR can be the naturally occurring endogenous 5'UTR and 3'UTR of gene of interest.Alternatively, UTR sequence that is not endogenous to gene of interest can be added by incorporating the UTR sequence into forward primer and reverse primer, or by any other modification of template.The use of UTR sequence that is not endogenous to gene of interest can be useful for modifying RNA stability and / or translation efficiency.For example, it has been found that AU-rich element in 3'UTR sequence can reduce mRNA stability.Therefore, based on the characteristics of UTR well known in the art, 3'UTR can be selected or designed to increase the stability of RNA transcription.

[0146] In some embodiments, the 5'UTR contains the Kozak sequence of the endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. Although the Kozak sequence can increase the efficiency of translation of some RNA transcripts, it is believed that it is not necessary for all RNAs to enable efficient translation. The necessity of the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to prevent exonuclease degradation of mRNA.

[0147] In some embodiments, the mRNA has a cap at the 5' end, a 3' poly(A) tail, or a combination thereof, which determines ribosome binding, initiation of translation, and stability of the mRNA within the cell.

[0148] The 5' cap provides stability to the RNA molecule. 7 G(5')ppp(5')G,m 7The 5' cap may be a G(5')ppp(5')A, G(5')ppp(5')G, or G(5')ppp(5')A cap analog, all of which are commercially available. The 5' cap may be an anti-reverse cap analog (ARCA) (Stepinski, et al., RNA, 7:1468-95(2001)) or any other suitable analog. The 5' cap can be incorporated using techniques known in the art (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95(2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966(2005)).

[0149] The RNA may also contain an internal ribosome entry site (IRES) sequence, which may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates translation initiation.

[0150] In general, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[0151] Poly(A) segments can be generated during PCR by using a reverse primer containing a poly(T) tail, e.g., a 100-T tail (the size can be, e.g., 50-5000 Ts), or by any other method after PCR, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs, reducing their degradation. Additionally or alternatively, the poly(A) tail of an RNA can be extended after in vitro transcription using a poly(A) polymerase, such as E. coli poly(A) polymerase (E-PAP).

[0152] Furthermore, mRNA stability can be increased by attaching different chemical groups to the 3' end. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into poly(A) tails using poly(A) polymerase. ATP analogs can further increase RNA stability. Suitable ATP analogs include, but are not limited to, cordycepin and 8-azaadenosine.

[0153] B. Cargo sequence i. A polypeptide of interest The cargo may encode one or more proteins. The cargo may be a polynucleotide, which may be monocistronic or polycistronic. In some embodiments, the polynucleotide is multigenic. The polynucleotide may be, for example, an expression construct such as mRNA or a vector.

[0154] The cargo may encode one or more polypeptides of interest. The polypeptide may be any polypeptide. For example, the polypeptide encoded by the polynucleotide may be a polypeptide that provides a therapeutic or preventive effect to an organism or can be used to diagnose a disease or disorder in an organism. For example, a polypeptide that can function as a ligand or receptor for cells of the immune system or that can function to stimulate or inhibit the immune system of an organism can be encoded by a polynucleotide(s) that expresses the polypeptide to treat cancer, autoimmune disorders, parasitic infections, viral infections, bacterial infections, fungal infections, or other infections.

[0155] In some embodiments, the polynucleotide supplements or replaces a defective polynucleotide in the organism.

[0156] In certain embodiments, the polynucleotide encodes dystrophin, utrophin, or a combination thereof. Such compositions can be administered in an effective amount to treat a subject for a dystrophy, particularly a muscular dystrophy, such as Duchenne muscular dystrophy.

[0157] In another specific embodiment, the polynucleotide encodes an antigen, e.g., an antigen that can be utilized in vaccine formulations and related methods. In a specific embodiment, the polynucleotide encodes a viral antigen(s), e.g., a SARS-CoV-2 antigen(s). Accordingly, provided are compositions and methods of use thereof for protection from and treatment of SARS-CoV-2 virus and viral infections and diseases associated therewith, including COVID-19.

[0158] In some embodiments, the polynucleotide comprises a selectable marker, e.g., a selectable marker effective in eukaryotic cells, e.g., a drug resistance selectable marker. The selectable marker gene can encode a factor necessary for the survival or growth of transformed host cells grown in a selective culture medium. Typical selectable genes encode proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, kanamycin, gentamicin, Zeocin, or tetracycline, complement auxotrophic deficiencies, or supply vital nutrients not available from the culture medium.

[0159] In working Example 12 below, tumor growth is reduced by nucleic acids encoding wild-type merlin (i.e., NF2), the protein that is mutated in neurofibromatosis type 2. Thus, in some embodiments, the nucleic acid encodes a wild-type or other compensatory variant of an oncogenic protein such as merlin.

[0160] In some embodiments, the polynucleotide comprises a reporter gene. A reporter gene is typically a gene that is not present or expressed in host cells. A reporter gene typically encodes a protein that causes some phenotypic changes or enzymatic properties. Examples of such genes are provided in Weising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the glucuronidase (GUS) gene and the GFP gene.

[0161] ii.Functional nucleic acid The cargo can be or encode a functional nucleic acid. A functional nucleic acid is a nucleic acid molecule that has a specific function, such as binding to a target molecule or catalyzing a specific reaction. As discussed in more detail below, functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNAs, miRNAs, aptamers, ribozymes, RNAi, and external guide sequences, and cyclic dinucleotides. Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional nucleic acid molecules can have novel activities that are independent of any other molecules.

[0162] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptide, or carbohydrate chain.Therefore, functional nucleic acid can interact with the mRNA or genomic DNA of target polypeptide, or can interact with polypeptide itself.In many cases, functional nucleic acid is designed to interact with other nucleic acids based on the sequence homology between target molecule and functional nucleic acid molecule.In other cases, the specific recognition between functional nucleic acid molecule and target molecule is not based on the sequence homology between functional nucleic acid molecule and target molecule, but is based on the formation of tertiary structure, which allows specific recognition to occur.

[0163] Therefore, the composition can comprise one or more functional nucleic acids designed to reduce the expression of a gene or its gene product.For example, functional nucleic acids or polypeptides can be designed to target mRNA and reduce or inhibit its expression or translation, or to reduce or inhibit protein expression, reduce protein activity, or increase protein degradation.In some embodiments, the composition comprises a vector suitable for in vivo expression of functional nucleic acids.

[0164] (1) Antisense The functional nucleic acid may be an antisense molecule or may encode it. Antisense molecules are designed to interact with target nucleic acid molecules through either standard or non-standard base pairing. The interaction between the antisense molecule and the target molecule is designed to promote the destruction of the target molecule, for example, by ribonuclease (RNAse) H-mediated RNA-DNA hybrid degradation. Alternatively, the antisense molecule is designed to interfere with the processing function that normally occurs on the target molecule, such as transcription or replication. Antisense molecule design can be based on the sequence of the target molecule. There are many methods for optimizing antisense efficiency by finding the most accessible region of the target molecule. Exemplary methods include in vitro selection experiments and DNA modification tests using DMS and DEPC. Antisense molecules are designed to bind to the target molecule within 10 -6 Less than or 10 -6 , 10 -8 Less than or 10 -8 , 10 -10 Less than or 10 -10 , or 10 -12 Less than or 10 -12 Dissociation constant (K d ) is preferably bonded.

[0165] (2) RNA interference In some embodiments, functional nucleic acid induces gene silencing by RNA interference.Genetic expression can also be silenced by RNA interference (RNAi) with high specificity and effectiveness.This silencing was originally observed by adding double-stranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). Once inside the cell, dsRNA is cleaved by the ribonuclease (RNase) III-like enzyme Dicer into 21-23 nucleotide long double-stranded small interfering RNAs (siRNAs) containing two-nucleotide overhangs at the 3' end (Elbashir, et al. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In an ATP-dependent step, siRNAs are incorporated into a multisubunit protein complex, commonly known as the RNAi-induced silencing complex (RISC), which guides the siRNA to the target RNA sequence (Nykanen, et al. (2001) Cell, 107:309-21). At some point, the siRNA duplex unwinds, and the antisense strand remains bound to RISC, presumably directing the degradation of the complementary mRNA sequence by a combination of endonucleases and exonucleases (Martinez, et al. (2002) Cell, 110:563-74). However, the effects or uses of iRNA or siRNA are not limited to any type of mechanism.

[0166] Small interfering RNA (siRNA) is double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or even inhibiting gene expression.In one example, siRNA induces homologous RNA molecule, for example, mRNA, to be specifically degraded in the region where the sequence between siRNA and target RNA is identical.For example, WO02 / 44321 discloses that when base pairing with 3' protruding end, the siRNA can sequence-specifically degrade target mRNA, and this patent document is incorporated herein by reference for the method of making these siRNA.

[0167] In mammalian cells, synthetic short double-stranded RNA can be used to mimic the siRNA produced by Dicer enzyme to achieve sequence-specific gene silencing (Elbashir, et al. (2001) Nature, 411:494 498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can be chemically synthesized or in vitro synthesized, or can be the result of short double-stranded hairpin-like RNA (shRNA) being processed into siRNA inside the cell. Synthetic siRNA is generally designed using algorithms and conventional DNA / RNA synthesizers. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using a kit such as Ambion's SILENCER® siRNA Construction Kit.

[0168] The production of siRNA from vector is more commonly achieved by transcription of short hairpin RNAse (shRNA).Kits for constructing vectors with shRNA are available, such as Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™ inducible RNAi plasmids and lentiviral vectors.

[0169] In some embodiments, the functional nucleic acid is an siRNA, shRNA, miRNA. In some embodiments, the composition comprises a vector that expresses the functional nucleic acid.

[0170] (3) Aptamers The functional nucleic acid may be or encode an aptamer. An aptamer is a molecule that interacts, preferably specifically, with a target molecule. Typically, an aptamer is a small nucleic acid molecule ranging from 15 to 50 bases in length that folds into a defined secondary and tertiary structure, such as a stem-loop or a G-quadruplex. Aptamers can bind to small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. Aptamers bind to target molecules in a 10-fold manner. -12 K less than M d Aptamers can bind very tightly to target molecules at 10 -6 Less than 10 -8 Less than 10 -10 Less than or equal to 10 -12 Less than K d Aptamers can bind to target molecules with very high specificity. For example, aptamers have been isolated that have a binding affinity difference of more than 10,000 times between a target molecule and another molecule that differs only at a single position on the molecule. The K d is the K for background binding molecules dPreferably, the background molecule is at least 10, 100, 1000, 10,000, or 100,000 times lower. When comparing molecules such as polypeptides, preferably the background molecule is a different polypeptide.

[0171] (4) Ribozyme The functional nucleic acid may be or encode a ribozyme. Ribozymes are nucleic acid molecules capable of catalyzing either intramolecular or intermolecular chemical reactions. Preferably, ribozymes catalyze intermolecular reactions. There are several different types of ribozymes that catalyze nuclease or nucleic acid polymerase-type reactions based on ribozymes found in natural systems, such as hammerhead ribozymes. Some ribozymes are not found in natural systems but have been engineered to catalyze novel specific reactions. Preferred ribozymes cleave RNA or DNA substrates, and more preferably those that cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate, followed by cleavage. This recognition is often primarily based on canonical or non-canonical base pairing interactions. Because target substrate recognition is based on the target substrate sequence, this property makes ribozymes particularly good candidates for target-specific cleavage of nucleic acids.

[0172] (5) External guide arrangement The functional nucleic acid can be or encode an external guide sequence. An external guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule, forming a complex that is recognized by RNase P, which then cleaves the target molecule. An EGS can be designed to specifically target a selected RNA molecule. RNase P assists in the processing of transfer RNA (tRNA) within cells. Bacterial RNase P can be recruited to cleave virtually any RNA sequence by using an EGS to mimic the natural tRNA substrate in the target RNA:EGS complex. Similarly, eukaryotic EGS / RNase P-directed RNA cleavage can be used to cleave desired targets within eukaryotic cells. Representative examples of how to create and use EGS molecules to facilitate cleavage of a variety of different target molecules are known in the art.

[0173] Methods for making and using vectors for expressing functional nucleic acids in vivo, such as antisense oligonucleotides, siRNAs, shRNAs, miRNAs, EGSs, ribozymes, and aptamers, are known in the art.

[0174] (6) Cyclic dinucleotide In some embodiments, the 4H2 antibody is co-administered in combination with an immunostimulatory oligonucleotide. The immunostimulatory oligonucleotide may be a cargo and therefore may be administered as a complex with the antibody or separately. In some embodiments, the immunostimulatory oligonucleotide is a cyclic dinucleotide.

[0175] The functional nucleic acid may be or encode a cyclic dinucleotide, which directly binds to the STING adaptor protein, resulting in the production of IFN-β (Zhang, et al., Mol Cell., 51(2):226-35 (2013). doi: 10.1016 / j.molcel.2013.05.022.). Several standard and non-standard dinucleotides are known in the art, including, but not limited to, GMP-AMP (cGAS), 2'3'-cGAMP, 2'3'-cGAMP, 3'3'-cGAMP, c-di-GMP, 2'2'-cGAMP, 2'3'-cGAM(PS)2(Rp / Sp), 3'3'-cGAMP Fluorinated, c-di-GMP Fluorinated, or 2'3'-c-di-GMP, c-di-AMP, c-di-GMP, cAIMP(CL592), cAIMP Difluor(CL614), cAIM(PS)2 Difluor(Rp / Sp)(CL656), c-di-AMP Fluorinated, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'3'-c-di-AM(PS)2(Rp,Rp), c-di-GMP Fluorinated, 2'3'-c-di-GMP, c-di-IMP, and DMXAA.

[0176] (7) Immunostimulatory oligonucleotides In some embodiments, the immunostimulatory oligonucleotide is or encodes an oligonucleotide ligand, examples of which include, but are not limited to, pattern recognition receptor (PRR) ligands.

[0177] Examples of PRRs include the Toll-like family of signaling molecules, which play a role in the initiation of the innate immune response and also influence the later, more antigen-specific adaptive immune response. Thus, oligonucleotides can serve as ligands for Toll-like family signaling molecules, such as Toll-like receptor 9 (TLR9).

[0178] For example, in humans, unmethylated CpG sites can be detected by TLR9 on plasmacytoid dendritic cells and B cells (Zaida, et al., Infection and Immunity, 76(5):2123-2129, (2008)). Thus, the sequence of the oligonucleotide can contain one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. Although "p" refers to the DNA phosphodiester backbone, in some embodiments, oligonucleotides containing CG can have a modified backbone, for example, a phosphorothioate (PS) backbone.

[0179] In some embodiments, the oligonucleotide may contain more than one CG dinucleotide, either consecutively or separated by an intervening nucleotide(s). The CpG motif(s) may be present within the oligonucleotide sequence. Many nucleotide sequences stimulate TLR9, and the number and location of CG dinucleotides may vary, and the base sequence adjacent to the CG dimer may be precise.

[0180] Typically, CG ODNs are classified based on their sequence, secondary structure, and effect on human peripheral blood mononuclear cells (PBMCs). There are five classes: class A (type D), class B (type K), class C, class P, and class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61(3): 195-204 (2009), incorporated herein by reference). CG ODNs can stimulate the production of type I interferon (e.g., IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also potent activators of natural killer (NK) cells through indirect cytokine signaling. Several classes are potent stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20): 10833-7 (1997); Dalpke, AH, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun. 164(3):1617-2 (2000), each of which is incorporated herein by reference).

[0181] Other PRR Toll-like receptors include TLR3 and TLR7, which can recognize double-stranded RNA, single-stranded RNA, and small double-stranded RNA, respectively, as well as the retinoic acid-inducible gene I (RIG-I)-like receptor, or RIG-I, and melanoma differentiation-associated gene 5 (MDA5), which is best known as a cytosolic RNA-sensing receptor.

[0182] RIG-I (retinoic acid-inducible protein 1, also known as Ddx58) and MDA-5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard) belong to the RIG-I-like receptor (RLR) family and are cytoplasmic RNA helicases that are crucial for the host antiviral response.

[0183] RIG-I and MDA-5 sense double-stranded RNA (dsRNA), a replication intermediate of RNA viruses, and signal through the mitochondrial antiviral signaling protein MAVS (also known as IPS-1, VISA, or Cardif), thereby leading to the production of type I interferons (IFN-α and IFN-β).

[0184] RIG-I detects viral RNAs that exhibit uncapped 5'-diphosphate / triphosphate ends and short, blunt-ended double-stranded segments, two essential features that facilitate their differentiation from self-RNA. The characteristics of the MDA-5 physiological ligand have not yet been fully characterized. However, it has been observed that RIG-I and MDA-5 exhibit different dependencies on dsRNA length: RIG-I preferentially binds short dsRNA, whereas MDA-5 preferentially binds long dsRNA. Consistent with this, RIG-I and MDA-5 bind the synthetic dsRNA analog poly(I:C) with different length preferences.

[0185] Under some circumstances, RIG-I can also indirectly sense dsDNA. Viral dsDNA can be transcribed by RNA polymerase III into dsRNA with a 5'-triphosphate moiety. Poly(dA:dT), a synthetic analog of B-form DNA, therefore constitutes another RIG-I ligand.

[0186] Exemplary RIG-I ligands include, but are not limited to, 5'ppp-dsRNA, which is a specific agonist of RIG-I; 3p-hpRNA, which is a specific agonist of RIG-I; poly(I:C) / LyoVec complexes, which are recognized by RIG-I and / or MDA-5 depending on the size of poly(I:C); and poly(dA:dT) / LyoVec complexes, which are indirectly recognized by RIG-I.

[0187] In some embodiments, the oligonucleotide contains a functional ligand of TLR3, TLR7, TLR8, TLR9, or a RIG-I-like receptor, or a combination thereof.

[0188] Examples of immunostimulatory oligonucleotides, and methods for making them, are known in the art and commercially available. See, e.g., Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporated herein by reference.

[0189] c. Cargo composition The nucleic acid cargo of the present disclosure may typically be or include a DNA or RNA nucleotide that includes a heterocyclic base (nucleobase), a sugar moiety linked to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl functional group of the sugar moiety. Major naturally occurring nucleotides include uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases, and ribose or deoxyribose sugars linked by phosphodiester bonds.

[0190] In some embodiments, cargo comprises or is composed of chemically modified nucleotide analogues to improve stability, half-life, or specificity or affinity for target receptors compared to DNA or RNA counterparts.Chemical modifications include chemical modifications of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof.As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide that has one or more chemical modifications of heterocyclic bases, sugar moieties, or phosphate moieties.In some embodiments, the charge of modified nucleotides is reduced compared to DNA or RNA of the same nucleobase sequence.For example, oligonucleotides can have low negative charge, can be uncharged, or can have positive charge.

[0191] Typically, nucleoside analogs support bases capable of hydrogen bonding with standard polynucleotide bases by Watson-Crick base pairing, where the analog backbone presents the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases of a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). In some embodiments, the analog has a substantially uncharged phosphorus-containing backbone.

[0192] i. Heterocyclic bases The main naturally occurring nucleotides include uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases. Cargos can contain chemical modifications to their nucleobase components. Chemical modification of heterocyclic bases or heterocyclic base analogs can be effective in increasing binding affinity or stability when binding to target sequences. Chemically modified heterocyclic bases include, but are not limited to, inosine, 5-(1-propynyl)uracil (pU), 5-(1-propynyl)cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5- and 2-amino-5-(2'-deoxy-beta-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.

[0193] ii. Sugar modification The cargo may include nucleotides with modified sugar moieties or sugar moiety analogs. Modifications of the sugar moiety include, but are not limited to, 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME), and 2'-O-(N-(methyl)acetamido) (2'-OMA). The 2'-O-aminoethyl sugar moiety is particularly preferred because it is protonated at neutral pH, thus suppressing charge repulsion between the TFO and the target duplex. This modification stabilizes the conformation of the C3' end of the ribose or dexyribose and also forms a bridge with the i-1 phosphate within the purine strand of the duplex.

[0194] In some embodiments, the nucleic acid is a morpholino oligonucleotide.Morpholino oligonucleotides are typically composed of two morpholino monomers containing a purine or pyrimidine base pairing moiety that is effective for base-specific hydrogen bonding with bases in a polynucleotide, linked by a phosphorus-containing linkage of 1 to 3 atoms in length connecting the morpholino nitrogen of one monomer to the 5' exocyclic carbon of the adjacent monomer.The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, or thymine.The synthesis, structure, and binding characteristics of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

[0195] An important property of morpholino-based subunits is their ability to link in oligomeric form, typically via stable, uncharged backbone linkages; the polymers formed have high T , even for short oligomers of 10-14 bases, to complementary bases in target nucleic acids, including target RNAs. mThese include the ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, or inosine) so that the oligomer can hybridize with a target nucleic acid; the ability of the oligomer to be actively transported into mammalian cells; and the ability of the oligomer:RNA heteroduplex to resist ribonuclease (RNAse) degradation.

[0196] In some embodiments, oligonucleotides use morpholino-based subunits with base-pairing moieties joined by uncharged linkages, as described above.

[0197] The morpholino oligonucleotide can be, for example, a phosphorodiamidate morpholino oligomer.

[0198] iii. Internucleotide linkages Oligonucleotides are connected by internucleotide bonds, which refer to the chemical linkage between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides can increase the binding affinity or stability of the oligonucleotide or reduce its susceptibility to nuclease digestion. Cationic modifications, including but not limited to, diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP), can be particularly useful because they reduce electrostatic repulsion between the oligonucleotide and the target. Modifications to the phosphate backbone can also include replacing one of the non-bridging oxygens in the phosphodiester linkage with a sulfur atom. This substitution creates a phosphorothioate internucleoside linkage instead of a phosphodiester linkage. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo.

[0199] Examples of modified nucleotides with reduced charge include modified internucleotide linkages, such as phosphate analogs with achiral and uncharged intersubunit linkages (e.g., Sterchak, EP et al., Organic. Chem., 52:4202, (1987)), and uncharged morpholino-based polymers with the above-mentioned achiral intersubunit linkages (see, e.g., U.S. Patent No. 5,034,506).Some internucleotide linkage analogs include morpholidates, acetals, and polyamide-linked heterocycles.

[0200] In another embodiment, the cargo is composed of a locked nucleic acid. Locked nucleic acid (LNA) is a modified RNA nucleotide (see, for example, Braasch, et al., Chem. Biol., 8(1):1-7 (2001)). LNA forms a more stable hybrid with DNA than a DNA / DNA hybrid, a property similar to that of a peptide nucleic acid (PNA) / DNA hybrid. Therefore, LNA can be used in exactly the same way as a PNA molecule. In some embodiments, the binding efficiency of LNA can be increased by adding a positive charge. LNA can be produced using a commercially available nucleic acid synthesizer and standard phosphoramidite chemistry.

[0201] In some embodiments, the cargo is composed of peptide nucleic acid. Peptide nucleic acid (PNA) is a synthetic DNA mimic in which the entire phosphate backbone of an oligonucleotide is replaced with repeating N-(2-aminoethyl)-glycine units, and phosphodiester bonds are typically replaced with peptide bonds. Various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNA maintains the same spacing of heterocyclic bases as conventional DNA oligonucleotides, but is an achiral, neutrally charged molecule. Peptide nucleic acid is composed of peptide nucleic acid monomers.

[0202] Other backbone modifications include peptide and amino acid variations and modifications.Therefore, the backbone structure of oligonucleotides such as PNA can be peptide linkages or non-peptide peptide linkages.Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linker), and amino acids such as lysine, which are particularly useful when a positive charge is desired for PNA.Methods for chemical assembly of PNA are well known.See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.

[0203] The cargo optionally contains modifications at one or more terminal residues or at either or both ends to increase the stability and / or affinity of the oligonucleotide to its target.Commonly used positively charged moieties include the amino acids lysine and arginine, but other positively charged moieties can also be useful.The cargo can also be further modified to add a terminal cap using a propylamine group to prevent degradation.Procedures for adding 3' or 5' caps to oligonucleotides are well known in the art.

[0204] In some embodiments, the nucleic acid may be single-stranded or double-stranded.

[0205] iv. Fine-tuning of bonds The sequence of the cargo can be modified to take these properties into account and to fine-tune the strength of binding between the cargo and the 4H2-binding protein.

[0206] The 4H2 antibody binds to guanosine, and therefore, increasing the number of guanines and / or selecting the location of guanines within a polynucleotide sequence can be used to increase antibody binding, create antibody binding sites, increase the number of antibodies that bind to a single polynucleotide, and / or target antibodies to bind to specific locations along the polynucleotide. Additionally or alternatively, reducing the number of guanines within a polynucleotide and / or selecting locations within the polynucleotide sequence where guanines are absent can be used to increase antibody binding, reduce or eliminate antibody binding sites, reduce the number of antibodies that bind to a single polynucleotide, and / or target antibodies to bind to alternative locations along the polynucleotide.

[0207] For example, any of the cargoes of the present disclosure may comprise or consist of guanine (G) (e.g., mono-G, di-G, or poly-G) alone or in combination with two, three, four, or more of adenine (A), thymine (T), cytosine (C), uracil (U), or inosine (I). In some embodiments, a synthetic non-coding sequence is added to the cargo, e.g., to increase or decrease binding to a 4H2-binding protein. Such a sequence may, but need not, be at the 5' or 3' end of the nucleic acid cargo. The cargo may be single-stranded or double-stranded DNA or RNA.

[0208] Additionally or alternatively, these binding properties can be explained by rational design of the cargo nucleic acid sequence using codon optimization to preferentially increase binding (e.g., preference for guanine) or decrease binding (e.g., preference for adenine (A), thymine (T), cytosine (C), uracil (U), or inosine (I)).

[0209] 2. Vaccine formulation Vaccines seek to induce a strong immune response. The 4H2 antibodies described herein can be administered as a component of a vaccine to enhance the immune response associated with the vaccine. In some embodiments, the vaccines disclosed herein comprise a 4H2 antibody, an antigen(s), and, if necessary, an adjuvant(s) of other additional agents.

[0210] a. Antigen Antigens can be peptides, proteins, polysaccharides, sugars, lipids, nucleic acids, or combinations thereof. Antigens can be derived from transformed cells, such as cancer or leukemia cells, and can be whole cells or immunogenic components thereof. Suitable antigens are known in the art and are available from commercial, governmental, and scientific sources.

[0211] The antigen may be a purified or partially purified polypeptide derived from a tumor, or a recombinant polypeptide produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. The antigen may be DNA or RNA (e.g., mRNA) encoding all or part of an antigenic protein. The DNA may be in the form of vector DNA, such as a viral vector, or plasmid DNA.

[0212] Antigens may be provided as single antigens or in combination. Antigens may be provided as complex mixtures of polypeptides or nucleic acids.

[0213] The antigen may be, for example, a tumor antigen or may be derived from an infectious agent or disease against which vaccination is desired, such as polio, tetanus, flu, hepatitis B, hepatitis A, hepatitis C, rubella, Hib, measles, whooping cough (pertussis), pneumococcal disease, HIV, SAR-CoV-2, or any of the other infectious diseases and diseases discussed in more detail below.

[0214] i. Viral antigensViral antigens can be isolated from any virus, including, but not limited to, viruses from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire strain, Reston strain, Ivory strain), etc. Coast strain, or Sudan strain), Flaviviridae, (e.g., hepatitis C virus, dengue virus 1, dengue virus 2, dengue virus 3, and dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., human herpesviruses 1, 3, 4, 5, and 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A, B, and C), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., human immunodeficiency virus (HIV) 1 and HIV2, Lentiviruses, etc.), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or a portion of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.

[0215] Viral antigens may be derived from specific strains or combinations of strains, such as SAR-CoV-2, papillomavirus, herpesvirus, i.e., herpes simplex 1 and 2; hepatitis virus, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV), tick-borne encephalitis virus; parainfluenza virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, and lymphocytic choriomeningitis virus.

[0216] ii. Bacterial antigens Bacterial antigens include, but are not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobi um, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Helicobacter, Haemophilus, Haemophilus influenzae type b (Hib), Hyphomicrobium, Legionella, Leptospira, Listeria, Meningococcus They may be of any bacterial origin, including A, B and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Rhodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.

[0217] iii. Parasite antigens For example, parasitic antigens can be derived from parasites, including, but not limited to, antigens from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni. These include all or part of sporozoan antigens, Plasmodium antigens, such as circumsporozoite proteins, sporozoite surface proteins, liver-stage antigens, apical membrane-associated proteins, or merozoite surface proteins.

[0218] iv. Tumor antigens Antigens include tumor antigens, including tumor-associated antigens or tumor-specific antigens, for example, but not limited to, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, Mage-A1, 2, 3, 4, 6, 10, 12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA(MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3(CA 27.29 BCAAs), CA 195, CA 242, CA-50, CAM43, CD68 KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein, cyclophilin C-related protein), TAAL6, TAG72, TLP, and TPS. Tumor antigens such as BCG can also be used as adjuvants to stimulate immune responses.

[0219] B. adjuvant If desired, the vaccines described herein may include an adjuvant. The adjuvant may be one or more of the following, but is not limited to: oil emulsions (e.g., Freund's adjuvant); saponin preparations; virosomes and virus-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; inorganic / mineral-containing compositions (e.g., inorganic salts, such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes; muramyl peptides; imidazoquinolone compounds; and surfactants (e.g., lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0220] Adjuvants may also include immune modulators, such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon gamma), macrophage colony-stimulating factors, and tumor necrosis factors. In addition to PD-1 antagonists, other costimulatory molecules, including other polypeptides of the B7 family, can be administered. Such proteinaceous adjuvants may be provided as full-length polypeptides or active fragments thereof, or in the form of RNA or DNA, such as plasmid DNA.

[0221] 3. Immune checkpoint modulators The 4H2 antibody can be administered in combination with an immune checkpoint modulator.

[0222] Immune checkpoints can be stimulatory or inhibitory, and tumors can use these checkpoints to protect themselves from attack by the immune system. Currently approved checkpoint therapies block inhibitory checkpoint receptors, but research into therapies that activate stimulatory checkpoints is also ongoing. Thus, immune checkpoint modulators can block inhibitory checkpoints or activate stimulatory checkpoints. Typically, immune checkpoint modulators induce or otherwise activate or augment an immune response against target cells, such as cancer cells or infected cells. Thus, in some embodiments, the immune checkpoint modulator can be a chimeric antigen receptor (CAR) directed to a cell, such as a CAR-T cell. In another embodiment, the immune checkpoint modulator can be an oncolytic virus.

[0223] In a preferred embodiment, the immune checkpoint modulator blocks an inhibitory checkpoint. Blocking the negative feedback of signal transduction on immune cells thus leads to an enhanced immune response against tumors. Thus, in some embodiments, the immune checkpoint modulator is administered to a subject in an effective amount to block an inhibitory checkpoint. Exemplary compounds are, for example, those that block or otherwise inhibit PD-1, PD-L1, or CTLA4.

[0224] a.PD-1 antagonist In some embodiments, the active agent is a PD-1 antagonist. T cell activation typically depends on antigen-specific signals following contact between the T cell receptor (TCR) and antigenic peptides presented via the major histocompatibility complex (MHC), while the extent of this response is controlled by positive and negative antigen-independent signals emanating from various costimulatory molecules. The latter are generally members of the CD28 / B7 family. Conversely, programmed death-1 (PD-1) is a member of the CD28 family of receptors that delivers negative immune responses when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the strength and / or duration of T cell responses. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, and include compounds or agents that bind to and block a ligand of PD-1, thereby interfering with or inhibiting binding of the ligand to the PD-1 receptor, or that directly bind to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[0225] In some embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without inducing inhibitory signal transduction and also binds to a ligand of the PD-1 receptor, reducing or inhibiting the induction of signal transduction through the PD-1 receptor by the ligand. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and induce inhibitory signal transduction, fewer cells can be compromised by negative signals delivered by PD-1 signal transduction, and a more robust immune response can be achieved.

[0226] PD-1 signaling is thought to be driven by binding of PD-1 ligands (e.g., B7-H1 or B7-DC) in close proximity to peptide antigens presented by the major histocompatibility complex (MHC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Thus, proteins, antibodies, or small molecules that prevent simultaneous ligation of PD-1 to the TCR on the T cell membrane are also useful PD-1 antagonists.

[0227] In preferred embodiments, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signal transduction by binding to a ligand of PD-1 or to PD-1 itself, particularly if such binding is not followed by simultaneous ligation to PD-1 and the TCR, and thus inhibitory signal transduction through the PD-1 receptor is not elicited. Other PD-1 antagonists include antibodies that bind to PD-1 or its ligands, such as PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), and other antibodies.

[0228] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications: PCT / IL03 / 00425(Hardy et al., WO / 2003 / 099196) PCT / JP2006 / 309606(Korman et al., WO / 2006 / 121168) PCT / US2008 / 008925(Li et al., WO / 2009 / 014708) PCT / JP03 / 08420(Honjo et al., WO / 2004 / 004771) PCT / JP04 / 00549(Honjo et al., WO / 2004 / 072286) PCT / IB2003 / 006304(Collins et al., WO / 2004 / 056875) PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174) PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874) PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533) Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0229] A specific example of an anti-PD-1 antibody is the human anti-PD-1 antibody MDX-1106 (Kosak, US20070166281 (published July 19, 2007) paragraph 42), preferably administered at a dose of 3 mg / kg.

[0230] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following publications: PCT / US06 / 022423 (WO / 2006 / 133396, published December 14, 2006) PCT / US07 / 088851 (WO / 2008 / 083174, published July 10, 2008) US2006 / 0110383 (released May 25, 2006)

[0231] A specific example of an anti-B7-H1 antibody is the human anti-B7-H1 antibody MDX-1105 (WO / 2007 / 005874, published January 11, 2007).

[0232] Regarding anti-B7-DC antibodies, see 7,411,051, 7,052,694, 7,390,888, and U.S. Patent Application Publication No. 2006 / 0099203.

[0233] The antibody may be a bispecific antibody comprising an antibody that binds to the PD-1 receptor cross-linked with an antibody that binds to a ligand of PD-1, such as B7-H1. In some embodiments, the PD-1 binding moiety reduces or inhibits signal transduction through the PD-1 receptor.

[0234] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, as well as active fragments of any of the foregoing, and fusion proteins incorporating any of these. In a preferred embodiment, the fusion protein comprises a soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, without incorporating all or part of the transmembrane portion of human B7-DC.

[0235] The PD-1 antagonist may be a fragment of mammalian B7-H1, preferably of mouse or primate, preferably human, origin, where the fragment binds to and blocks PD-1 but does not effect inhibitory signal transduction through PD-1. The fragment may be part of a fusion protein, e.g., an Ig fusion protein.

[0236] Other useful polypeptide PD-1 antagonists include those that bind to PD-1 receptor ligands. These include PD-1 receptor proteins or soluble fragments thereof that can bind to PD-1 ligands, such as B7-H1 or B7-DC, and prevent the binding of these ligands to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind to the protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such fragments also include soluble ECD portions of PD-1 proteins containing mutations, such as the A99L mutation, that enhance binding to natural ligands (Molnar et al., PNAS, 105:10483-10488(2008)). Also useful are B7-1 or soluble fragments thereof that are capable of binding to the B7-H1 ligand and preventing the binding of that ligand to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction.

[0237] Both DNA and RNA antisense nucleic acids and siRNA molecules against PD-1 and B7-H1 may be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 in T cells and the production of T cell ligands, such as B7-H1, PD-L1, and / or PD-L2. For example, siRNA (e.g., about 21 nucleotides long, specific to the gene encoding PD-1 or the gene encoding the PD-1 ligand, and oligonucleotides can be easily purchased commercially) complexed with a carrier such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8): 2231-2244 (2009)) can be easily taken up by cells expressing PD-1 and its ligand, reducing the expression of these receptors and ligands, thereby reducing inhibitory signal transduction in T cells and activating them.

[0238] Exemplary PD-1 inhibitors include, but are not limited to, the following: Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda) was developed by Merck and was first approved by the Food and Drug Administration in 2014 for the treatment of melanoma. Nivolumab (Opdivo) was developed by Bristol-Myers Squibb and was first approved by the FDA in 2014 for the treatment of melanoma. Pidilizumab, CureTech AMP-224, GlaxoSmithKline and MediImmune AMP-514, GlaxoSmithKline and MediImmune PDR001, Novartis Cemiplimab, Regeneron and Sanofi Exemplary PD-L1 inhibitors include, but are not limited to, the following: Atezolizumab (Tecentriq) is a fully humanized IgG1 (immunoglobulin 1) antibody developed by Roche Genentech. Atezolizumab was approved by the FDA in 2016 for urothelial carcinoma and non-small cell lung cancer. Avelumab (Bavencio) is a fully human IgG1 antibody developed by Merck Serono and Pfizer. Avelumab was approved by the FDA for the treatment of metastatic Merkel cell carcinoma. Avelumab was unsuccessful in a Phase III clinical trial for gastric cancer. Durvalumab (Imfinzi) is a fully human IgG1 antibody developed by AstraZeneca. Durvalumab is approved by the FDA for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation. ·BMS-936559, Bristol-Myers Squibb CK-301, Checkpoint Therapeutics, see e.g., Iwai, et al., Journal of Biomedical Science, (2017) 24:26, DOI 10.1186 / s12929-017-0329-9.

[0239] b. CTLA4 antagonist Other molecules useful for mediating the effects of T cells on immune responses are also contemplated as active agents. For example, in some embodiments, the molecule is an agent that binds to an immune response-mediating molecule other than PD-1. In a preferred embodiment, the molecule is a CTLA4 antagonist, such as an antagonistic anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies are described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0240] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and can range from 0.1 to 100 mg / kg, with narrower ranges of 1 to 50 mg / kg being preferred, and 10 to 20 mg / kg being more preferred. Suitable doses for human subjects are between 5 mg / kg and 15 mg / kg, with 10 mg / kg of antibody (e.g., a human anti-PD-1 antibody such as MDX-1106) being most preferred.

[0241] Specific examples of CTLA antagonists include ipilimumab, also known as MDX-010 or MDX-101, a human anti-CTLA4 antibody preferably administered at a dose of about 10 mg / kg, and tremelimumab, a human anti-CTLA4 antibody preferably administered at a dose of about 15 mg / kg. See also Sammartino, et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0242] In other embodiments, the antagonist is a small molecule. A series of small molecule organic compounds have been shown to bind to B7-1 ligand and prevent its binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368(2002)). Such small molecule organic compounds can be administered alone or together with anti-CTLA4 antibody to reduce the inhibitory signal transduction of T cells.

[0243] C. Cells directed by chimeric antigen receptors The modulator may be a cell directed by a chimeric antigen receptor. The term "chimeric antigen receptor" or alternatively "CAR" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that confer specificity to an immune effector cell for cancer cells and involve intracellular signal generation. In some embodiments, a CAR comprises at least an antigen-binding domain, such as an extracellular binding domain, a transmembrane domain, and an intracytoplasmic signaling domain (also referred to as an "intracellular signaling domain") including a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In one embodiment, the stimulatory molecule is a zeta chain ("zeta stimulatory domain") associated with the T cell receptor complex. In one embodiment, the intracytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule (e.g., 4-1BB (i.e., CD137), CD27, and / or CD28). In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In various embodiments, CAR is a fusion protein that fuses single-chain variable fragment (scFv) with CD3-zeta transmembrane domain.However, other intracellular signaling domains such as CD28, 41-BB and Ox40 can also be used in various combinations to generate desired intracellular signals.In some embodiments, the CAR disclosed herein comprises an extracellular binding domain.

[0244] The term "antigen-binding domain" is used in the context of this disclosure to refer to the portion of a CAR that specifically recognizes and binds to an antigen of interest. The "antigen-binding domain" can be derived from a binding protein disclosed herein, such as an antibody or a fragment thereof. In some embodiments, the "binding domain" is a single-chain variable fragment (scFv). In certain embodiments, the "binding domain" comprises a complementarity-determining region of a binding protein disclosed herein. In this embodiment, the cells targeted by the CAR can represent a 4H2 cell-permeable antibody (assuming it penetrates cancer cells) that induces cGAS / STING signaling, or a combination thereof, in combination with an immune checkpoint modulator that induces, augments, or enhances an immune response. For example, the binding domain can represent a cell-permeable antibody, and the engineered T cells can represent an immune cell modulator. In another example, cells targeted by a CAR disclosed herein are administered with a cell-permeable 4H2 antibody disclosed herein.

[0245] The terms "zeta" or "CD3-zeta" are used herein to define the protein provided under GenBan Accession No. BAG36664.1, or equivalent residues from non-human species, and "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" is defined as amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, sufficient to functionally transmit the initial signal required for T cell activation.

[0246] The term "immune effector cell" is used herein to refer to a cell that participates in an immune response (e.g., promoting an immune effector response). Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid phagocytes. In some embodiments, the immune effector cell(s) are allogeneic. In some embodiments, the immune effector cell(s) are autologous. In some embodiments, the immune checkpoint modulator is a CAR-directed T cell (CAR-T cell). Exemplary CAR-T cells include axicabtagene ciloleucel (KTE-C19, Axi-cel), tisagenlecleucel, and lisocabtagene maraleucel (liso-cel; JCAR017).

[0247] Activation and expansion of immune effector cells, such as T cells, can generally be carried out using previously described methods, for example, those described in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. As a general example, a population of immune effector cells, e.g., regulatory T cell depleted cells, can be expanded by contacting them with a surface that is bound to an agent that stimulates signals associated with the CD3 complex and a ligand that stimulates costimulatory molecules on the surface of T cells.

[0248] d. oncolytic viruses The modulator may be an oncolytic virus. The term "oncolytic virus" is used in the context of the present disclosure to refer to a virus that can infect and reduce the growth of cancer cells. For example, an oncolytic virus can inhibit cell proliferation. In some embodiments, an oncolytic virus can kill cancer cells. In some embodiments, an oncolytic virus preferentially infects and inhibits the growth of cancer cells compared to corresponding normal cells. In another embodiment, an oncolytic virus preferentially replicates in and inhibits the growth of cancer cells compared to corresponding normal cells.

[0249] In some embodiments, oncolytic viruses can naturally infect cancer cells and reduce their growth. Examples of such viruses include Newcastle disease virus, vesicular stomatitis virus, myxoma virus, reovirus, Sindbis virus, measles virus, and coxsackie virus. Oncolytic viruses naturally infect cancer cells and can generally target cancer cells by exploiting the cellular abnormalities present in these cells to reduce their growth. For example, oncolytic viruses can exploit defects in surface-bound receptors, activated oncogenes, such as Ras, Akt, p53, and / or interferon (IFN) pathways.

[0250] In another embodiment, the oncolytic virus encompassed in the present disclosure is engineered to infect and reduce the growth of cancer cells.Exemplary viruses suitable for such engineering include oncolytic DNA viruses, such as adenovirus, herpes simplex virus (HSV) and vaccinia virus; and oncolytic RNA viruses, such as lentivirus, reovirus, coxsackievirus, Seneca Valley virus, poliovirus, measles virus, Newcastle disease virus, vesicular stomatitis virus (VSV) and parvovirus, such as rodent protoparvovirus H-1PV.In some embodiments, oncolytic virus comprises the backbone of the virus mentioned above.

[0251] In some embodiments, the tumor specificity of an oncolytic virus can be engineered by mutating or deleting a gene or genes that are required for viral survival in normal cells but not in cancer cells. For example, an oncolytic virus can be engineered by mutating or deleting a gene encoding thymidine kinase, an enzyme required for nucleic acid metabolism. In this example, the virus becomes dependent on cellular thymidine kinase expression, which is high in proliferating cancer cells but repressed in normal cells. In another example, an oncolytic virus is engineered to contain a capsid protein that binds to a tumor-specific cell surface molecule. In some embodiments, the capsid protein is a fiber, penton, or hexon protein. In another example, an oncolytic virus is engineered to contain a tumor-specific cell surface molecule in order to transduce and target cancer cells. Exemplary tumor-specific cell surface molecules can include integrins, EGF receptor family members, proteoglycans, disialogangliosides, B7-H3, CA-125, EpCAM, ICAM-1, DAF, A21, integrin-α2β1, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, CEA, tumor-associated glycoprotein, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD155, MUC1, tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB, CC chemokine receptor, PSMA, RON receptor, and cytotoxic T lymphocyte antigen 4.

[0252] The oncolytic virus can be replication competent. In some embodiments, the oncolytic virus selectively replicates in cancer cells relative to corresponding normal cells.

[0253] For example, conditional replication can be achieved by inserting a tumor-specific promoter that drives the expression of a critical gene(s). Such promoters can be identified based on the difference in gene expression between tumors and corresponding surrounding tissues. Exemplary native promoters include AFP, CCKAR, CEA, erbB2, Cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, survivin, TRP1, STAT3, hTERT, and Tyr. Exemplary composite promoters include AFP / hAFP, SV40 / AFP, CEA / CEA, PSA / PSA, SV40 / Tyr, and Tyr / Tyr.

[0254] Various viruses can be engineered as outlined in the examples mentioned above.Oncolytic viruses can be, for example, modified HSV, lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV) or its derivatives, such as self-complementary AAV (scAAV), or non-integrating AV.Oncolytic viruses can be modified HSV.Oncolytic viruses can be modified lentivirus.Other exemplary viruses include vaccinia virus, vesicular stomatitis virus (VSV), measles virus, and Maraba virus.

[0255] In other examples, the oncolytic virus can be of various AV or AAV serotypes. In some embodiments, the oncolytic virus is serotype 1. In another example, the oncolytic virus is serotype 2. In another example, the oncolytic virus is serotype 3, 4, 7, 8, 9, 10, 11, 12, or 13. In another example, the oncolytic virus is serotype 5. In another example, the oncolytic virus is serotype 6.

[0256] Exemplary oncolytic viruses include T-Vec (HSV-1; Amgen), JX-594 (Vaccina; Sillajen), JX-594 (AdV; Cold Genesys), and Reolysin (Reovirus; Oncolytics Biotech). Other examples of oncolytic viruses are disclosed in WO2003 / 080083, WO2005 / 086922, WO2007 / 088229, WO2008 / 110579, WO2010 / 108931, WO2010 / 128182, WO2013 / 112942, WO2013 / 116778, WO2014 / 204814, WO2015 / 077624 and WO2015 / 166082, WO2015 / 089280.

[0257] e. Other immune checkpoint modulators Other immune checkpoint targets include, but are not limited to, ICOS, OX40, GITR, 4-1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, PARP, and others, and are targets for cancer treatment alone and in combination with anti-PD-1 compounds, anti-PD-L1 compounds, and anti-CTLA compounds. See, e.g., Iwai, et al., Journal of Biomedical Science. 24(1): 26. doi:10.1186 / s12929-017-0329-9; Donini, et al., J Thorac Dis. 2018 May;10(Suppl 13):S1581-S1601. doi: 10.21037 / jtd.2018.02.79. Thus, in some embodiments, the 4H2 antibody is administered in combination with a compound targeting ICOS, OX40, GITR, 4-1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, or PARP, or a combination thereof, alone or in combination with a compound targeting PD-1, PD-L1, and / or CTLA. In another embodiment, the immune checkpoint modulator is an antibody disclosed in WO2016 / 013870.

[0258] C. Pharmaceutical Compositions The compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0259] The compositions are preferably used for therapeutic use in combination with a suitable pharmaceutical carrier. Such compositions comprise an effective amount of the composition and a pharmaceutically acceptable carrier or excipient.

[0260] The composition may be formulated for local, topical, or systemic administration in a suitable pharmaceutical carrier. Typical carriers and preparation methods are disclosed in Remington's Pharmaceutical Sciences, 15th Edition by E. W. Martin (Mark Publishing Company, 1975). The antibody or complex formed thereby can also be encapsulated in suitable biocompatible particles formed from biodegradable or non-biodegradable polymers or proteins or liposomes for cell targeting. Such systems are well known to those skilled in the art. In some embodiments, the antibody or complex formed thereby is encapsulated in nanoparticles.

[0261] Injectable formulations can be provided in unit dosage form, for example, in ampoules or multi-dose containers, optionally with added preservatives. The compositions can take the form of sterile aqueous or non-aqueous solutions, suspensions, and emulsions, and in certain embodiments, can be isotonic with the subject's blood. Examples of non-aqueous solvents include fixed oils, including polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil, and mineral oil, injectable organic esters such as ethyl oleate, or synthetic monoglycerides or diglycerides. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers (e.g., those based on Ringer's dextrose). The material can be a solution, emulsion, or suspension (e.g., incorporated into particles, liposomes, or cells). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Trehalose can typically be added to the pharmaceutical composition in an amount of 1-5%. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5.

[0262] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, and surfactants. Carrier formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those skilled in the art can easily determine various parameters for preparing and formulating the compositions without resorting to undue experimentation.

[0263] The composition, alone or in combination with other suitable components, can also be made into an aerosol formulation (i.e., "nebulized") to be administered by inhalation. The aerosol formulation can be placed into a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and air. For administration by inhalation, the compound is delivered from a pressurized pack or nebulizer using a suitable propellant, in a form that provides an aerosol spray.

[0264] In some embodiments, the formulation comprises pharmaceutically acceptable carriers and formulation components, such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, preservatives, solubilizers, or stabilizers.

[0265] Nucleic acid can be conjugated with lipophilic groups such as cholesterol and lauric acid and lithocholic acid derivatives with C32 functionality to improve intracellular uptake.For example, cholesterol has been demonstrated to enhance the uptake and stability of siRNA in serum in vitro (Lorenz, et al., Bioorg. Med. Chem. Lett., 14(19):4975-4977 (2004)) and in vivo (Soutschek, et al., Nature, 432 (7014):173-178 (2004)).In addition, it has been shown that steroid-conjugated oligonucleotides can bind to different lipoproteins in bloodstream, such as LDL, thereby protecting their integrity and facilitating biodistribution (Rump, et al., Biochem. Pharmacol., 59(11):1407-1416 (2000)). Other groups that can be combined or conjugated with the above-mentioned nucleic acids to enhance cellular uptake include acridine derivatives; cross-linking agents, such as psoralen derivatives, azidophenacyl, proflavine, and azidoproflavine; artificial endonucleases; metal complexes, such as EDTA-Fe(II) and porphyrin-Fe(II); alkylating moieties; nucleases, such as alkaline phosphatase; terminal transferase; abzymes; cholesteryl moieties; lipophilic carriers; peptide conjugates; long-chain alcohols; phosphate esters; radioactive markers; non-radioactive markers; carbohydrates; and polylysine or other polyamines. Levy et al.'s U.S. Patent No. 6,919,208 also describes a method for enhanced delivery. These pharmaceutical preparations can be prepared in a manner known per se, for example, by conventional mixing, dissolving, granulating, triturating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0266] Further carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing antibodies or antibody-formed complexes, where the matrices are in the form of shaped particles, such as films, liposomes, or microparticles. Implantation includes inserting implantable drug delivery systems, such as microspheres, hydrogels, polymer reservoirs, cholesterol matrices, polymer systems, such as matrix erosion and / or diffusion systems, and non-polymer systems. Inhalation includes administering the composition using an aerosol in an inhaler, either alone or in combination with an absorbable carrier. For systemic administration, it may be preferable to encapsulate the composition in liposomes.

[0267] The compositions can be delivered using invasive devices such as vascular or urinary catheters, and using interventional devices such as stents having drug delivery capabilities and configured as expandable devices or stent grafts in a manner that allows for tissue-specific uptake of the drug and / or nucleotide delivery system.

[0268] The formulation can be delivered by diffusion or by degradation of the polymer matrix using biodegradable implants.In certain embodiments, the administration of the formulation can be designed to provide sequential exposure to the composition over a certain period of time, for example, over hours, days, weeks, months or years.This can be achieved, for example, by repeated administration of the formulation or by a sustained-release or controlled-release delivery system, in which the composition is delivered over a long period of time without repeated administration.

[0269] Other suitable delivery systems include sustained-release, delayed-release, sustained-release, or controlled-release delivery systems. Such systems can avoid repeated administration and often provide increased convenience for patients and physicians. Many types of release delivery systems are available and known to those skilled in the art. These release delivery systems include, for example, polymer-based systems, such as polylactic acid and / or polyglycolic acid, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and / or combinations thereof. Microcapsules of the aforementioned polymers containing nucleic acids are described, for example, in U.S. Pat. No. 5,075,109. Other examples include non-polymeric systems based on lipids, including sterols such as cholesterol, cholesterol esters, and fatty acids, or neutral lipids such as monoglycerides, diglycerides, and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. The formulation may be, for example, a microsphere, a hydrogel, a polymer reservoir, a cholesterol matrix, or a polymer system. In some embodiments, the system may allow for sustained or controlled release of the composition, for example, by controlling the diffusion or erosion / degradation rate of the formulation containing the antibody or a complex formed from the antibody.

[0270] The composition can be formulated for pulmonary or mucosal administration.Administration can include delivering the composition to the mucous membranes of the lungs, nose, oral cavity (sublingual, buccal), vagina or rectum.The term aerosol as used herein refers to any preparation of fine mist, which can be a solution or suspension, regardless of whether it is produced using a propellant.Aerosol can be produced using standard techniques such as ultrasonication or high pressure processing.

[0271] For administration via the upper respiratory tract, the preparation can be formulated as a solution or suspension in, for example, buffered or unbuffered water or isotonic saline, for intranasal administration as droplets or spray.Such solution or suspension is preferably isotonic with respect to nasal secretion, and has approximately the same pH, for example, from about pH 4.0 to about pH 7.4, or from pH 6.0 to pH 7.0.The buffer solution should be physiologically compatible, and merely an example thereof includes phosphate buffer.

[0272] Particle delivery vehicles can be used to deliver compositions to target cells. Nanoparticles generally refer to particles that fall within the range of 500 nm to less than 0.5 nm, preferably between 50 nm and 500 nm in diameter, and more preferably between 50 nm and 300 nm in diameter. The cellular internalization of polymer particles is highly dependent on their size, and the cellular internalization of nanoparticle polymer particles is much more efficient than that of microparticle polymer particles. For example, Desai et al. demonstrated that the uptake of 100 nm diameter nanoparticles by cultured Caco-2 cells is approximately 2.5 times higher than that of 1 μM diameter microparticles (Desai et al., Pharm. Res., 14:1568-73(1997)). Nanoparticles also have a higher ability to diffuse deeper into tissues in vivo.

[0273] In some embodiments, the delivery vehicle is a dendrimer.

[0274] Examples of preferred biodegradable polymers include synthetic polymers that degrade by hydrolysis, such as poly(hydroxy acids), e.g., polymers and copolymers of lactic acid and glycolic acid, other degradable polyesters, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), and poly(amine-co-ester) polymers, such as those described in Zhou, et al., Nature Materials, 11:82-90 (2012) and WO2013 / 082529, U.S. Patent Application Publication No. 2014 / 0342003, and WO2016 / 081621.

[0275] In some embodiments, particularly those targeting T cells in vivo, immune cell or T cell markers, such as CD3, CD7, or CD8, or markers of target tissues such as the liver, can be targeted, e.g., for in vivo production of CAR T cells. For example, anti-CD8 antibodies and anti-CD3 Fab fragments have both been used to target T cells in vivo (Pfeiffer, et al., EMBO Mol Med., 10(11) (2018). pii: e9158.doi: 10.15252 / emmm.201809158., Smith, et al., Nat Nanotechnol., 12(8):813-820 (2017). doi: 10.1038 / nnano.2017.57). Thus, in some embodiments, the particle or other delivery vehicle comprises a targeting moiety specific for CD3, CD7, CD8, or another immune cell (e.g., T cell) marker, or a marker for a particular tissue, such as the thymus, spleen, or liver. The binding moiety can be, for example, an antibody or antigen-binding fragment thereof.

[0276] The targeting moiety can be directly or indirectly associated, linked, conjugated, or otherwise bound to the nanoparticle or other delivery vehicle.The targeting molecule can be a protein, peptide, nucleic acid molecule, sugar, or polysaccharide that binds to a receptor or other molecule on the surface of the targeted cell.The specificity and avidity of binding to the graft can be modulated by selecting the targeting molecule.

[0277] Examples of moieties include those for delivery of molecules to specific cells, e.g., hematopoietic stem cells, CD34 + The targeting moiety includes a targeting moiety that delivers the antibody to cells, T cells, or any other preferred cell type, as well as to receptors and ligands expressed in the preferred cell type. Preferably, the moiety targets hematopoietic stem cells. Examples of molecules that target the extracellular matrix ("ECM") include glycosaminoglycans ("GAGs") and collagen. In one embodiment, the outer surface of the polymer particle can be modified to enhance the ability of the polymer particle to interact with selected cells or tissues. The above method of inserting an adapter element conjugated with a targeting molecule into the particle is preferred. However, in another embodiment, the outer surface of a polymer microparticle or nanoparticle having a carboxy terminus can be linked to a targeting molecule having a free amine terminus.

[0278] Other useful ligands that can be attached to polymeric microparticles and nanoparticles include pathogen-associated molecular patterns (PAMPs). PAMPs target Toll-like receptors (TLRs) on the surface of cells or tissues, or transmit signals inside cells or tissues, thereby potentially increasing uptake. PAMPs that can be conjugated to or encapsulated with particle surfaces include unmethylated CpG DNA (bacteria), double-stranded RNA (viruses), lipopolysaccharide (bacteria), peptidoglycan (bacteria), lipoarabinomannan (bacteria), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacteria), flagellin (bacteria), poly(inosinic-cytidylic) acid (bacteria), lipoteichoic acid (bacteria), or imidazoquinoline (synthetic).

[0279] In another embodiment, the outer surface of particles can be treated with mannose amine, thereby mannosylating the outer surface of particles.This treatment allows particles to be bound to the mannose receptor on the surface of antigen-presenting cells in target cells or tissues.Alternatively, the surface conjugation with the immunoglobulin molecules that contain Fc part (targeting Fc receptor), heat shock protein part (HSP receptor), phosphatidylserine (scavenger receptor) and lipopolysaccharide (LPS) are additional receptor targets in cells or tissues.

[0280] Lectins can be covalently attached to the microparticles and nanoparticles to target them specifically to mucin and mucosal cell layers.

[0281] The selection of the targeting moiety depends on the method of administration of the nanoparticle composition and the cells or tissues to be targeted. Targeting molecules can generally increase the binding affinity of particles to cells or tissues, or target nanoparticles to specific tissues within an organ or specific cell types within a tissue. In some embodiments, the targeting moiety targets the thymus, spleen, or cancer cells.

[0282] Covalently binding any of the natural components of mucin, either in pure or partially purified form, to particles reduces the surface tension at the interface between the beads and the gastrointestinal tract, increasing the solubility of the beads in the mucin layer. Attaching polyamino acids containing additional pendant carboxylic acid side chains, such as polyaspartic acid and polyglutamic acid, increases bioadhesion. Using polyamino acids with molecular weights in the range of 15,000 to 50,000 kDa, chains of 120 to 425 amino acid residues are attached to the particle surface. The polyamino chains increase bioadhesion by entangling the mucin chains and by increasing the carboxyl charge.

[0283] III.How to use A. Nucleic Acid Delivery A method for enhancing the delivery of a nucleic acid construct using a 4H2 antibody is provided. Typically, an effective amount of the 4H2 antibody is first contacted with a nucleic acid cargo desired to be delivered into a cell. For example, the nucleic acid cargo and the antibody can be mixed in solution for a sufficient time for the nucleic acid cargo and the antibody to form a complex. The mixture is then contacted with the cells. In other embodiments, the cargo and the antibody are added to a solution containing or otherwise housing cells, and the complex is allowed to form in the presence of the cells. The complex can be contacted with the cells in vitro, ex vivo, or in vivo. Thus, in some embodiments, a solution of the complex is added to cells in culture or injected into an animal to be treated. Treatment can be, for example, by administering a mixture of the antibody and the nucleic acid cargo to a subject in need thereof via simple IV administration.

[0284] The compositions and methods may include one, two, three, four, five, six, seven, eight, nine, ten, or more different nucleic acid constructs formed from RNA, DNA, PNA, or other modified nucleic acids, or combinations thereof.

[0285] An effective or therapeutically effective amount of a composition can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, or to otherwise produce a desired pharmacological and / or physiological effect, e.g., to reduce, inhibit, or reverse one or more of the pathophysiological mechanisms of the underlying disease or disorder.

[0286] An effective amount can also be an amount effective to increase the rate, quantity, and / or quality of delivery of the nucleic acid cargo compared to administration of the cargo in the absence of the antibody. The formulation of the composition is made to suit the administration format. Pharmaceutically acceptable carriers are determined in part by the specific composition to be administered and the specific method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions containing the conjugate. The exact dosage will vary depending on various factors, such as subject-dependent variables (e.g., age, immune system health, clinical symptoms, etc.).

[0287] The composition can be administered to or otherwise contacted with target cells once daily, twice daily, or three times daily; once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, seven times weekly, or once monthly, twice monthly, three times monthly, four times monthly, five times monthly, six times monthly, seven times monthly, or eight times monthly. For example, in some embodiments, the composition is administered every two or three days, or about two to about four times a week on average. Thus, in some embodiments, the composition is administered as part of a dosing regimen comprising two or more separate treatments.

[0288] Dosage regimens include a maintenance regimen, in which the dosage remains the same between two or more administrations, an ascending regimen, in which the dosage is increased between two or more administrations, a tapering regimen, in which the dosage is decreased between two or more administrations, or a combination thereof.

[0289] In some embodiments, the initial dose can be low. Dose escalation can continue until a sufficient biochemical or clinical response is achieved. The clinical response depends on the disease or disorder being treated and / or the desired outcome. In some embodiments, the dosage can be increased until a therapeutic effect is confirmed, preferably at a high dose that not only confirms a therapeutic effect but also does not induce undesirable toxicity or the toxicity is tolerable. The dosage can then be maintained or gradually reduced to a maintenance dose. This method can be used to standardize, optimize, or customize the dose level, dosing frequency, or duration of treatment.

[0290] Typically, particularly for in vivo administration, the antibody and nucleic acid are mixed for a period of time, e.g., at room temperature, prior to administration. In some embodiments, the complexation time ranges, for example, from 1 minute to 30 minutes, or from 10 minutes to 20 minutes, inclusive, with a preferred complexation time of about 15 minutes. The antibody dose can range from 0.0001 mg to 1 mg, inclusive, with a preferred dose being about 0.1 mg. The nucleic acid dose can range from 0.001 μg to 100 μg, inclusive, with a preferred dose being 10 μg. The experiments below utilized 4H2 / mRNA ratios of 1:1 w / w and 3:1 w / w, although other ratios are contemplated. In some embodiments, antibody:nucleic acid ratios are utilized, e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5 w / w.

[0291] In some embodiments, the RNA and / or DNA cargo is mixed with carrier DNA. The carrier DNA can be, for example, plasmid DNA or low molecular weight DNA, e.g., derived from salmon sperm. In some embodiments, the carrier DNA is non-coding DNA. The carrier DNA can be single-stranded or double-stranded, or a combination thereof. In some embodiments, the carrier DNA is composed of a nucleic acid of 1-10, 1-100, 1-1,000, or 1-10,000 nucleotides in length, or any subrange or integer, or combination thereof. Typically, the carrier DNA is not conjugated or otherwise covalently linked to an antibody. Typically, the carrier DNA is co-incubated with the cargo nucleic acid and antibody and co-delivered as a complex therewith.

[0292] 1. In vitro and ex vivo methods For in vitro and ex vivo methods, cells are typically contacted with the composition during culturing.For ex vivo methods, cells can be isolated from a subject and contacted with the composition ex vivo to produce cells containing cargo nucleic acid(s).In a preferred embodiment, cells are isolated from the subject to be treated or from an isogenic host.Target cells can be contacted with the composition after being removed from the subject.Antibody and cargo can be contacted with cells together or separately, or as a pre-formed complex.

[0293] 2. In vivo methods In some embodiments, delivery of nucleic acid cargo to cells in vivo is used for gene editing and / or treatment of a disease or disorder in a subject. Typically, a composition comprising an antibody-nucleic acid cargo complex can be administered directly to a subject for in vivo therapy.

[0294] Generally, the method of administering compounds, including antibody, oligonucleotide and related molecules, is well known in the art.In particular, the administration route that has been used for nucleic acid therapeutics provides the preferred administration route and formulation for the donor oligonucleotide described above, along with the currently used formulation.Preferably, composition is injected or infused into animals.

[0295] The compositions can be administered by several routes, including but not limited to intravenous, intraperitoneal, intraamniotic, intramuscular, subcutaneous, or topical (sublingual, rectal, intranasal, pulmonary, rectal mucosa, and vaginal), and oral (sublingual, buccal).

[0296] In some embodiments, the composition is formulated for pulmonary delivery, such as intranasal administration, or for oral inhalation. Administration of the formulation can be achieved by any acceptable method that allows the conjugates to reach their target. Depending on the condition being treated, administration can be localized (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic. Compositions and methods for in vivo delivery are also discussed in WO2017 / 143042.

[0297] The method can also include administering an effective amount of an antibody-nucleic acid complex composition to an embryo or fetus or its pregnant mother in vivo.In some methods, the composition is delivered intrauterinely by injecting and / or injecting the composition into a vein or artery of the embryo or fetus, for example, the vitelline vein or umbilical vein, or into the amniotic cavity.See, for example, Ricciardi, et al., Nat Commun. 2018 Jun 26;9(1):2481. doi: 10.1038 / s41467-018-04894-2 and WO2018 / 187493.

[0298] 3.Application 4H2 antibody can be used to deliver nucleic acid cargos, such as mRNA, functional nucleic acids, DNA expression constructs, vectors, etc., encoding polypeptides or functional nucleic acids of interest into cells to express or inhibit polypeptides in cells. The compositions and methods can be used across a variety of different applications. Non-limiting examples include CRISPR and gRNA expression vectors + / - editing DNA, delivery of large DNA (plasmids and expression vectors), gene replacement and gene therapy, such as delivery of DNA and / or RNA, siRNA, mRNA, etc., for generating CAR-T cells in vivo or ex vivo and simplifying CAR-T cell production in vivo or ex vivo. Exemplary applications of gene therapy / gene editing and immunomodulation, particularly chimeric antigen receptor T cell generation, are discussed below.

[0299] a. Gene therapy and gene editing In some embodiments, the composition is used for gene editing. For example, this method can be particularly useful for treating genetic defects, disorders and diseases caused by single gene mutation, for example, for correcting genetic defects, disorders and diseases caused by point mutation.When target gene contains mutation that causes genetic disorder, this method can be used for mutagenic repair, can return the DNA sequence of target gene to its original normal one.Target sequence can be present in the coding DNA sequence of gene or present in intron.Target sequence can also be present in the DNA sequence that regulates the expression of target gene, including promoter or enhancer sequence.

[0300] In the method herein, the cell contacted with the complex can be administered to a subject.The subject can have diseases or disorders such as hemophilia, muscular dystrophy, hemoglobinopathy, cystic fibrosis, xeroderma pigmentosum, or lysosomal storage disease, or inherited or acquired diseases of the retina, eye, brain, spine, or coronary artery or other vascular diseases.In such an embodiment, gene modification, gene replacement, gene addition, or a combination thereof can be performed in an effective amount to reduce one or more symptoms of disease or disorder in the subject.

[0301] In some embodiments, the composition of the present disclosure is used for retinal gene therapy.Inherited retinal diseases (IRDs) are typically caused by a single gene mutation, and include, but are not limited to, type 2 Leber congenital amaurosis (LCA), choroideremia (CHM), Stargardt's disease, retinitis pigmentosa (e.g., mutations in RHO, USH2A, and RPGR), and X-linked retinoschisis (XLRS).Various administration routes can be used, including intravitreal, subretinal, and suprachoroidal, and can result in different biodistributions.See also Gupta, et al., "Gene Therapy for Inherited Retinal Disease," Review of Ophthalmology, May 10, 2022.

[0302] In some embodiments, the compositions and methods of the present disclosure are used to induce or enhance the repair of damaged endothelial cells, for example, during revascularization.Therefore, the compositions and methods of the present disclosure can be used as an adjunct to cardiovascular surgery and other interventions.For example, revascularization is a procedure that can restore blood flow in blocked arteries or veins.The compositions and methods of the present disclosure can be used in conjunction with such interventions, for example, to reduce the expression or biological activity of pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α, to increase the growth and proliferation of endothelial cells, and / or to reduce neointimal hyperplasia (for example, smooth muscle cell growth, proliferation, migration, etc.).

[0303] In some embodiments, the compositions and methods of the present disclosure involve localized delivery to a site at or adjacent to the site in need of treatment, including, but not limited to, the brain, ear, and skin, where such delivery can be used to treat diseases associated therewith.

[0304] In some embodiments, the cargo comprises a nucleic acid encoding a nuclease, a donor oligonucleotide, or a nucleic acid encoding a donor oligonucleotide, or a combination thereof.

[0305] 1. Gene-editing nucleases The nucleic acid cargo includes one or more elements that encode for inducing single-strand or double-strand breaks in the genome of the target cell, optionally but preferably in combination with other elements such as donor oligonucleotides, and / or, particularly in the case of CRISPR / Cas, other elements of the system, such as gRNA. The composition can be used, for example, to reduce or otherwise alter the expression of a target gene.

[0306] (1) Strand break-inducing element CRISPR / Cas In some embodiments, the nucleic acid cargo comprises one or more elements of a CRISPR / Cas-mediated genome editing composition, a nucleic acid encoding one or more elements of a CRISPR / Cas-mediated genome editing composition, or a combination thereof. As used herein, a CRISPR / Cas-mediated genome editing composition refers to the elements of a CRISPR system necessary to perform CRISPR / Cas-mediated genome editing in a mammalian subject. As discussed in more detail below, a CRISPR / Cas-mediated genome editing composition typically comprises one or more nucleic acids encoding a crRNA, a tracrRNA (or a chimera thereof, also referred to as a guide RNA or single guide RNA), and a Cas enzyme, such as Cas9. A CRISPR / Cas-mediated genome editing composition may optionally comprise a donor polynucleotide capable of recombination integration into a target site (e.g., a single-strand or double-strand break site induced by Cas9) or a site adjacent thereto in the genome of a target cell.

[0307] The CRISPR / Cas system has been adapted for use in eukaryotic organisms as gene editing (silencing, enhancing, or changing specific genes) (see, for example, Cong, Science, 15:339 (6121) :819-823 (2013) and Jinek, et al., Science, 337 (6096) :816-21 (2012)). By transfecting cells with the necessary elements, including cas genes and specially designed CRISPRs, the genome of an organism can be cut and modified at any desired location. Methods for preparing compositions for genome editing using the CRISPR / Cas system are described in detail in WO2013 / 176772 and WO2014 / 018423, the entire contents of which are expressly incorporated herein by reference.

[0308] The delivery methods disclosed herein are suitable for use with many variations of CRISPR / Cas systems.

[0309] Generally, the term "CRISPR system" collectively refers to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA-processed partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences (e.g., direct repeat-spacer-direct repeat) operably linked to a guide sequence may also be referred to as a pre-crRNA (pre-CRISPR RNA) before processing by nucleases, or as a crRNA after processing.

[0310] As discussed in more detail below, in some embodiments, the tracrRNA and crRNA are linked to form a chimeric crRNA-tracrRNA hybrid, in which the mature crRNA is fused to a partial tracrRNA via a synthetic stem-loop to mimic the natural crRNA:tracrRNA duplex, as described in Cong, Science, 15:339 (6121):819-823 (2013) and Jinek, et al., Science, 337 (6096):816-21 (2012)). A single fusion crRNA-tracrRNA construct is also referred to herein as a guide RNA or gRNA (or single guide RNA (sgRNA)). Within the sgRNA, the crRNA portion can be identified as the "target sequence," and the tracrRNA is often referred to as the "scaffold."

[0311] In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0312] Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of target sequences (also referred to as protospacers in the context of endogenous CRISPR systems). In the context of CRISPR complex formation, "target sequence" refers to a sequence that guide sequences are designed to be complementary to, and hybridization of the target sequence and guide sequence promotes the formation of CRISPR complexes. The target sequence can be any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0313] In the target nucleic acid, each protospacer is accompanied by a protospacer adjacent motif (PAM), and PAM recognition is specific to each CRISPR system. In the Streptococcus pyogenes CRISPR / Cas system, the PAM is the nucleotide sequence NGG. In the Streptococcus thermophiles CRISPR / Cas system, the PAM is the nucleotide sequence NNAGAAW. The tracrRNA duplex directs Cas to a DNA target consisting of the protospacer and the required PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.

[0314] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs of the target sequence). All or a portion of a tracr sequence can also form part of a CRISPR complex, such as, for example, by hybridization with all or a portion of a tracr mate sequence operably linked to the guide sequence.

[0315] Many resources are available to assist practitioners in determining appropriate target sites once a desired DNA target sequence has been identified. For example, numerous public resources, including a bioinformatics-generated list of approximately 190,000 potential sgRNAs targeting over 40% of human exons, are available to assist practitioners in selecting a target site and designing an associated sgRNA to generate a nick or double-stranded break at that site. See also crispr.u-psud.fr / , a tool designed to help scientists discover CRISPR targeting sites in a wide range of species and generate appropriate crRNA sequences.

[0316] In some embodiments, one or more vectors that drive the expression of one or more elements of the CRISPR system are introduced into target cells, so that the expression of the elements of the CRISPR system directs the formation of CRISPR complexes at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector with one or more additional vectors that provide any components of the CRISPR system that are not included in the first vector. The CRISPR system elements combined in a single vector can be arranged in any suitable orientation, for example, one element can be located 5' (its "upstream") or 3' (its "downstream") relative to the second element. The coding sequence of one element can be located on the same strand or the opposite strand of the coding sequence of the second element, and can be oriented in the same direction or in the opposite direction. In some embodiments, a single promoter drives expression of transcripts encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and tracr sequence embedded within one or more intron sequences (e.g., each present in a different intron, two or more present in at least one intron, or all present in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

[0317] In some embodiments, the vector comprises one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about one or more, about two or more, about three or more, about four or more, about five or more, about six or more, about seven or more, about eight or more, about nine or more, about ten or more, or more) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence and, optionally, downstream of a regulatory element operably linked to the tracr mate sequence, such that, in eukaryotic cells, after the guide sequence is inserted into the insertion site, expression of the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, vector comprises two or more insertion sites, and each insertion site is located between two tracr mate sequences, so that each site can insert a guide sequence.In this configuration, two or more guide sequences can comprise two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof.When using multiple different guide sequences, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in cells. For example, a single vector can contain about or more than about 1, about or more than about 2, about or more than about 3, about or more than about 4, about or more than about 5, about or more than about 6, about or more than about 7, about or more than about 8, about or more than about 9, about or more than about 10, about or more than about 15, or about or more than about 20 guide sequences.In some embodiments, vectors containing about or greater than about 1, about or greater than about 2, about or greater than about 3, about or greater than about 4, about or greater than about 5, about or greater than about 6, about or greater than about 7, about or greater than about 8, about or greater than about 8, about or greater than about 9, about or greater than about 10 such guide sequences can be provided and delivered to cells as needed.

[0318] In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence that encodes a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. In some embodiments, modified CRISPR enzyme has DNA cleavage activity, such as Cas9.In some embodiments, CRISPR enzyme directs the cleavage of one or both strands at the location of target sequence, such as within target sequence and / or within the complement of target sequence.In some embodiments, CRISPR enzyme directs the cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of target sequence.

[0319] In some embodiments, the vector encodes a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme, so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of S.pyogenes Cas9 converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that turn Cas9 into a nickase include, but are not limited to, H840A, N854A, and N863A. As a further example, two or more catalytic domains (RuvC I, RuvC II, and RuvC III) of Cas9 can be mutated to create a mutant Cas9 that substantially lacks all DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to create a Cas9 enzyme that substantially lacks all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity if the DNA cleavage activity of the mutated enzyme is less than about 25%, less than 10%, even less than 5%, even less than 1%, even less than 0.1%, even less than 0.01%, or even less than its unmutated form.

[0320] In some embodiments, the enzyme coding sequence encoding CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells.Eukaryotic cells can be the cells of specific organisms, for example, mammals, including but not limited to, human, mouse, rat, rabbit, dog or non-human primates, or can be derived from them.Generally, codon optimization refers to the process of modifying nucleic acid sequence to enhance expression in target host cell by replacing at least one codon (for example, about 1 or more than about 1, about 2 or more than about 2, about 3 or more than about 3, about 4 or more than about 4, about 5 or more than about 5, about 10 or more than about 10, about 15 or more than about 15, about 20 or more than about 20, about 25 or more than about 25, about 50 or more than about 50 codons) of native sequence with the codon that is more frequently or most frequently used in the gene of the host cell, while maintaining native amino acid sequence. Different species exhibit particular biases for certain codons for particular amino acids. Codon bias (differences in codon usage between organisms) is often thought to correlate with the efficiency of messenger RNA (mRNA) translation, which in turn depends, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules.

[0321] The predominance of tRNA selected in cells generally reflects the codon that is most frequently used in peptide synthesis.Therefore, gene can be adjusted based on codon optimization for optimal gene expression in a given organism.For example, codon usage tables are readily available in "Codon Usage Database", and these tables can be adapted in several ways.See Nakamura, Y., et al., Nucl. Acids Res., 28:292 (2000).Computer algorithms are also available for codon optimization of specific sequences for the expression in specific host cells, such as Gene Forge (Aptagen; Jacobus, PA).In some embodiments, one or more codons (for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding CRISPR enzyme correspond to the codon that is most frequently used for specific amino acid.

[0322] In some embodiments, the vector encodes a CRISPR enzyme that comprises one or more nuclear localization sequences (NLS).When there are more than one NLS, each NLS can be selected independently of other NLSs, and thus a single NLS can be present in more than one copy, and / or can be present in one or more copies in combination with one or more other NLSs.In some embodiments, an NLS is considered to be near the N-terminus or C-terminus if the nearest amino acid of the NLS is present within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain from the N-terminus or C-terminus.

[0323] Generally, one or more NLSs are strong enough to drive the accumulation of detectable amounts of CRISPR enzyme in the nucleus of eukaryotic cells.In general, the strength of nuclear localization activity can be derived from the number of NLSs in CRISPR enzyme, the specific NLS(s) used, or a combination of these factors.

[0324] The detection of nuclear accumulation can be carried out by any suitable technique.For example, to visualize the location within the cell, for example, detectable marker can be fused to CRISPR enzyme, and combined with a means for detecting the location of the nucleus (for example, a nuclear-specific stain such as DAPI).Cell nucleus can also be isolated from cell, and then its contents can be analyzed by any suitable process for detecting protein, for example, immunohistochemistry, Western blotting or enzyme activity assay.Nuclear accumulation can also be determined indirectly by, for example, assaying the effect of CRISPR complex formation (for example, assaying for DNA breakage or mutation in target sequence, or assaying for the change in gene expression activity caused by CRISPR complex formation and / or CRISPR enzyme activity), compared with the control that is not exposed to CRISPR enzyme or complex, or is exposed to CRISPR enzyme that lacks one or more NLS.

[0325] In some embodiments, one or more of the elements of the CRISPR system are under the control of an inducible promoter, which may include an inducible Cas, such as Cas9.

[0326] Cong, Science, 15:339 (6121):819-823 (2013) reports that heterologous expression of Cas9, tracrRNA, and pre-crRNA (or Cas9 and sgRNA) can achieve targeted cleavage of mammalian chromosomes. Thus, the CRISPR system, and thus the cargo nucleic acid, utilized in the methods disclosed herein is a vector system that may include one or more vectors encoding elements of a CRISPR system, which may include a first regulatory element operably linked to a CRISPR / Cas system chimeric RNA (chiRNA) polynucleotide sequence, where the polynucleotide sequence includes (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, which may optionally include at least one or more nuclear localization sequences. Elements (a), (b), and (c) can be arranged in a 5' to 3' orientation, where Cas9 and CRISPR RNA are located on the same or different vectors of the system, and when transcribed, the tracr mate sequence hybridizes with the tracr sequence, and the guide sequence directs the sequence-specific binding of the CRISPR complex to the target sequence; and the CRISPR complex can include (1) a guide sequence that hybridizes with the target sequence, and (2) a tracr mate sequence that hybridizes with the CRISPR enzyme complexed with the tracr sequence, where the enzyme-coding sequence encoding the CRISPR enzyme further encodes a heterologous functional domain. In some embodiments, one or more of the vectors also encode an appropriate Cas enzyme, such as Cas9. Different genetic elements can be under the control of the same promoter or different promoters.

[0327] While specificity may vary among different engineered CRISPR systems, the overall methodology is similar. Practitioners interested in using CRISPR technology to target a DNA sequence (identified using one of the many available online tools) can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (approximately 20 nucleotides), a form of tracrRNA sequence (scaffold), and an appropriate promoter and elements necessary for proper processing in eukaryotic cells. Such vectors are commercially available (see, for example, Addgene). Many systems rely on custom-made complementary oligos that anneal to form double-stranded DNA and are then cloned into the sgRNA expression plasmid. Coexpression of the sgRNA and an appropriate Cas enzyme from the same or separate plasmids in transfected cells results in a single- or double-stranded break (depending on the activity of the Cas enzyme) at the desired target site.

[0328] (2) Zinc finger nuclease In some embodiments, the element that induces single-strand or double-strand breaks in the genome of the target cell is a nucleic acid construct or construct that encodes a zinc finger nuclease (ZFN). Thus, the nucleic acid cargo can encode a ZFN.

[0329] ZFNs are typically fusion proteins containing a DNA binding domain derived from a zinc finger protein and a cleavage domain linked thereto. The most common cleavage domain is the type IIS enzyme Fok1. Fok1 catalyzes double-stranded cleavage of DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al. Proc., Natl. Acad. Sci. USA 89 (1992):4275-4279; Li et al. Proc. Natl. Acad. Sci. USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad. Sci. USA 91:883-887 (1994a); Kim et al. J. Biol. Chem. 269:31, 978-31, 982 (1994b). One or more of these enzymes (or enzymatically functional fragments thereof) can be used as a source of the cleavage domain.

[0330] DNA-binding domains can, in principle, be designed to target any genomic location of interest and may be a tandem array of Cys2His2 zinc fingers, each typically recognizing 3–4 nucleotides within the target DNA sequence. Cys2His2 domains have the general structure: Phe (sometimes Tyr)-Cys-(2–4 amino acids)-Cys-(3 amino acids)-Phe (sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His. By linking multiple fingers (the number varies; published studies have used 3–6 fingers per monomer), ZFN pairs can be designed to bind genomic sequences 18–36 nucleotides in length.

[0331] Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. Rational design, for example, involves using a database containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to that particular triplet or quadruplet sequence. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Patent Application Publication Nos. 2002 / 0165356; 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Nos. WO98 / 53059 and WO2003 / 016496.

[0332] (3) Transcription activator-like effector nuclease In some embodiments, the element that induces single-strand or double-strand breaks in the genome of the target cell is a nucleic acid construct or construct that encodes a transcription activator-like effector nuclease (TALEN).Therefore, the nucleic acid cargo can be one that encodes a TALEN.

[0333] TALENs share a similar overall architecture to ZFNs, with the primary difference being that their DNA-binding domain is derived from TAL effector proteins, transcription factors derived from plant pathogenic bacteria. The DNA-binding domain of TALENs is a tandem array of amino acid repeats, each approximately 34 residues long. The repeats are highly similar to one another; typically, repeats differ primarily at two positions (amino acids 12 and 13, termed repeat variable diresidues, or RVDs). Each RVD defines preferential binding to one of four possible nucleotides; that is, each TALEN repeat binds a single base pair, although the NN RVD has been shown to bind adenine in addition to guanine. While DNA binding by TAL effectors is mechanistically less well understood than that of zinc finger proteins, their ostensibly simpler code may prove highly beneficial for engineered nuclease design. TALENs also cleave as dimers, have relatively long target sequences (the shortest bond reported to date is 13 nucleotides per monomer), and appear to have less stringent requirements for the length of the spacer between binding sites than ZFNs. Monomeric and dimeric TALENs can contain more than 10, more than 14, more than 20, or more than 24 repeats.

[0334] A method for engineering TALs to bind to specific nucleic acids is described in Cermak, et al., Nucl. Acids Res. 1-11 (2011). US Patent Application Publication No. 2011 / 0145940 discloses TAL effectors and methods for using them to modify DNA. Miller et al. Nature Biotechnol 29: 143 (2011) reports the creation of a site-specific nuclease architecture for TALENs by linking a truncated TAL variant with the catalytic domain of Fok1 nuclease. The resulting TALENs were shown to induce gene modification in immortalized human cells. The general design principles for TALE-binding domains can be found, for example, in WO2011 / 072246.

[0335] ii. Donor polynucleotide The nuclease activity of the genome editing system described herein cuts the target DNA, resulting in single-strand or double-strand breaks in the target DNA. Double-strand breaks can be repaired by cells in at least two ways: non-homologous end joining and homology-directed repair. In non-homologous end joining (NHEJ), double-strand breaks are repaired by direct ligation of the cut ends. Therefore, new nucleic acid material is not inserted at the site, but some nucleic acid material may be lost, resulting in a deletion. In homology-directed repair (HDR), a donor polynucleotide that is homologous to the cut target DNA sequence is used as a template to repair the cut target DNA sequence, thereby transferring genetic information from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material can be inserted / copied at the site.

[0336] Thus, in some embodiments, the nucleic acid cargo is or comprises a donor polynucleotide. Modification of the target DNA resulting from NHEJ and / or homologous recombination repair can be used to induce gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

[0337] Thus, DNA cleavage by a genome editing composition can be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and repairing the sequence in the absence of a donor polynucleotide exogenously provided to a cell. Alternatively, when the genome editing composition includes a donor polynucleotide sequence that includes at least a segment having homology to the target DNA sequence, the method can be used to add, i.e., insert or replace, nucleic acid material into the target DNA sequence (e.g., to "knock in" a nucleic acid encoding a protein, siRNA, miRNA, etc.), to add a tag (e.g., 6xHis, fluorescent protein (e.g., green fluorescent protein; yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g., a promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation), etc. Thus, the compositions can be used to modify DNA in a site-specific or "targeted" manner, for example, for gene knockout, gene knock-in, gene editing, gene tagging, etc., used in gene therapy, for example.

[0338] In applications where it is desired to insert a polynucleotide sequence into a target DNA sequence, the polynucleotide containing the donor sequence to be inserted is also provided to cells. " Donor sequence " or " donor polynucleotide " or " donor oligonucleotide " refers to the nucleic acid sequence that is inserted into the cleavage site. The donor polynucleotide typically contains sufficient homology to the genomic sequence at the cleavage site to support the homologous recombination repair between the donor polynucleotide and the genomic sequence to which it has homology, and for example, contains 70%, 80%, 85%, 90%, 95%, or 100% homology to the nucleotide sequence located on both sides of the cleavage site, for example, within about 50 bases or less, for example, within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or the nucleotide sequence located immediately on both sides of the cleavage site. Donor sequence is typically not identical to the genome sequence that it replaces, and donor sequence can contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to genome sequence, as long as there is sufficient homology to support homology-directed repair.In some embodiments, donor sequence comprises the non-homologous sequence flanked by two regions of homology, and therefore, the homology-directed repair between target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence in target region.

[0339] b. Immune modulation i.CAR T cells The compositions and methods of the present disclosure are particularly useful in the context of preparing lymphocytes expressing immune receptors, particularly chimeric immune receptors (CIRs), such as chimeric antigen receptors (CARs). Artificial immune receptors (also known and referred to herein as chimeric T cell receptors, chimeric immune receptors, chimeric antigen receptors (CARs), and chimeric immune receptors (CIRs)) are engineered receptors that graft selected specificities onto cells. Cells modified according to the methods discussed can be used in various immunotherapies to treat cancer, infectious diseases, inflammation, and autoimmune diseases, as discussed in more detail below.

[0340] In a particularly preferred embodiment, mRNA or DNA encoding the chimeric antigen receptor cargo is delivered to immune cells such as lymphocytes.

[0341] The cargo can be delivered to immune cells in vivo, ex vivo, or in vitro. In a preferred embodiment, the cargo is mRNA, which may allow for one or more of reduced cost, ease of manufacturing, and reduced side effects (e.g., cytokine storm, neurotoxicity, graft-versus-host disease, etc.). In certain embodiments, immune cells (e.g., T cells) are collected from a subject in need of CAR T cell therapy, and the compositions and methods disclosed herein are used to deliver mRNA encoding one or more CAR T cell constructs to the collected cells, which are then returned to the subject. In some embodiments, the process from initial collection of the cells to their return to the subject takes a week or less, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In certain embodiments, the process from initial collection of the cells to their return to the subject occurs in 1 or 2 days, or in less than 1 day, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours.

[0342] Strategies for designing and developing chimeric antigen receptors are described in Dotti, et al., Immunol Rev. 2014 January;257(1): . doi:10.1111 / imr.12131 (35 pages), which are expressly incorporated herein by reference in their entireties, as well as Dotti, Molecular Therapy, 22(5):899-890 (2014); Karlsson, et al., Cancer Gene Therapy, 20:386-93 (2013); Charo, et al., Cancer Res., 65(5):2001-8 (2005); Jensen, et al., Immunol Rev., 257(1): 127-144 (2014); Eaton, et al., Gene Therapy, 9:527-35 (2002); Barrett, et al., Annu Rev Med., 65: 333-347 (2014), Cartellieri, et al., Journal of Biomedicine and Biotechnology, Volume 2010, Article ID 956304, 13 pages doi:10.1155 / 2010 / 956304; and U.S. Patent Application Publication Nos. 2015 / 0017120, 2015 / 0283178, 2015 / 0290244, 2014 / 0050709, and 2013 / 0071414.

[0343] CARs combine the antigen-binding properties of monoclonal antibodies with the lytic potential and self-renewal of T cells, offering several advantages over conventional T cells (Ramos and Dotti, Expert Opin Biol Ther., 11:855-873 (2011); Curran, et al., J Gene Med., 14:405-415 (2012); Maher, ISRN Oncol. 2012:278093 (2012)). CAR-T cells recognize and kill cancer cells independently of major histocompatibility complex (MHC). Therefore, target cell recognition is unaffected by some of the mechanisms by which tumors escape MHC-restricted T cell recognition, such as downregulation of human leukocyte antigen (HLA) class I molecules and defects in antigen processing.

[0344] Chimeric immunoreceptors were first developed in the 1980s and initially comprised the variable (antigen-binding) region of a monoclonal antibody and the constant regions of the T cell receptor (TCR) α and β chains (Kuwana, et al., Biochem Biophys Res Commun., 149:960-968(1987)). In 1993, this design was modified to include an ectodomain derived from a single-chain variable fragment (scFv) derived from the antigen-binding regions of both the heavy and light chains of a monoclonal antibody, a transmembrane domain, and an endodomain with a signaling domain derived from CD3-ζ. Later CARs generally followed a similar structural design, with a costimulatory signaling endodomain. Thus, the CAR constructs utilized in the methods herein may comprise an antigen-binding domain or ectodomain, a hinge domain, a transmembrane domain, an endodomain, and combinations thereof.

[0345] In some embodiments, the ectodomain is an scFv. The affinity of the scFv predicts CAR function (Hudecek, et al., Clin Cancer Res., 19(12):3153-64 (2013); Chmielewski, et al., J Immunol., 173:7647-7653 (2004)). Antigen binding and subsequent activation can also be modified by adding a flexible linker sequence to the CAR, thereby allowing the expression of two separate scFvs that can recognize two different antigens (Grada, et al., Mol Ther Nucleic Acids, 2:e105 (2013)) (referred to as tandem CAR (TanCAR)). Tandem CARs may be more effective at killing cancers that express low levels of each antigen individually, and may also reduce the risk of tumor immune escape due to variants that lose a single antigen.Other ectodomains include those from IL13Rα2 (Kahlon, et al., Cancer Res., 64:9160-9166(2004); Brown, et al., Clin Cancer Res., 18(8):2199-209(2012); Kong, et al., Clin Cancer Res., 18:5949-5960(2012)), NKG2D-ligands and CD70 receptors, peptide ligands (e.g., T1E peptide ligands), and so-called "universal ectodomains" (e.g., avidin ectodomains designed to recognize targets contacted with biotinylated monoclonal antibodies or FITC-specific scFvs designed to recognize targets contacted with FITC-labeled monoclonal antibodies (Zhang, et al., Blood, 106:1544-1551(2005); Barber, et al., Exp Hematol., 36:1318-1328(2008), Shaffer, et al., Blood, 117:4304-4314(2011), Davies, et al., Mol Med., 18:565-576(2012), Urbanska, et al., Cancer Res., 72:1844-1852(2012), Tamada, et al., Clin Cancer Res.,18:6436-6445(2012)).

[0346] In some embodiments, CAR comprises hinge region.Ectodomain is important for CAR specificity, but the sequence connecting ectodomain and transmembrane domain (hinge region) can also affect CAR-T cell function by making the length and flexibility of CAR different.Hinge can include, for example, the CH2CH3 hinge or its fragment derived from immunoglobulin such as IgG1. For example, Hudecek et al. (Hudecek, et al., Clin Cancer Res., 19(12):3153-64 (2013)) compared the effects of a CH2-CH3 hinge [229 amino acids (AA)], a CH3 hinge (119 AA), and a short hinge (12 AA) on the effector function of T cells expressing a third-generation ROR1-specific CAR and found that T cells expressing the "short hinge" CAR had superior anti-tumor activity, while other researchers found that the CH2-CH3 hinge impaired epitope recognition of a first-generation CD30-specific CAR (Hombach, et al., Gene Ther., 7:1067-1075(2000)).

[0347] Between the hinge (or ectodomain if no hinge domain is present) and the signaling endodomain, there is typically a transmembrane domain, most typically derived from CD3-zeta, CD4, CD8, or CD28 molecules. Like the hinge, the transmembrane domain can also influence the effector function of CAR-T cells.

[0348] Upon antigen recognition, activation and costimulatory signals are transmitted from the CAR endodomain to the T cell. T cell activation relies on phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic CD3-ζ domain of the TCR complex (Irving, et al., Cell, 64:891-901(1991)). While most CAR endodomains contain an activation domain derived from CD3-ζ, other CAR endodomains may contain ITAM-containing domains, such as the Fc receptor for IgE-γ domain (Haynes, et al., J Immunol., 166:182-187(2001)).

[0349] The target specificity of cells expressing a CAR is determined by the antigen recognized by the antibody / ectodomain. The compositions and methods of the present disclosure can be used to create constructs targeting any antigen, and cells expressing the constructs. In the context of immunotherapy, particularly cancer immunotherapy, numerous antigens and suitable ectodomains for targeting these antigens are well known. Unlike native TCRs, most scFv-based CARs recognize target antigens expressed on the cell surface, rather than internal antigens processed and presented by the cellular MHC. However, CARs can recognize structures other than protein epitopes, including carbohydrates and glycolipids (Dotti, et al., Immunol Rev. 2014 January;257(1):. doi:10.1111 / imr.12131 (35 pages)), thus offering the advantage over classical TCRs of expanding the pool of potential target antigens. Preferred targets include antigens expressed only in cancer cells or their surrounding stroma (Cheever, et al., Clin Cancer Res., 15:5323-5337 (2009)), such as the glioma cell-specific splice variant of EGFR (EGFRvIII) (Sampson, et al., Semin Immunol., 20(5):267-75 (2008)). However, human antigens meet this requirement, and the majority of target antigens are expressed at low levels on normal cells (e.g., GD2, CAIX, HER2) and / or in a lineage-restricted manner (e.g., CD19, CD20).

[0350] Preferred targets, and CARs that target them, are known in the art (see, e.g., Dotti, et al., Immunol Rev. 2014 January;257(1): . doi:10.1111 / imr.12131 (35 pages). For example, CAR targets for hematological malignancies include, but are not limited to, CD19 (e.g., B cells) (Savoldo, et al., J Clin Invest., 121:1822-1826(2011); Cooper, et al., Blood, 105:1622-1631(2005); Jensen, et al., Biol Blood Marrow Transplant (2010); Kochenderfer, et al., Blood, 119:2709-2720(2012); Brentjens, et al., Molecular Therapy, 17:S157 (2009), Brentjens, et al., Nat Med., 9:279-286(2003), Brentjens, et al., Blood, 118:4817-4828(2011), Porter, et al., N Engl J Med., 365:725-733(2011), Kalos, et al., Sci Transl Med., CD20 (e.g., B cells) (Jensen, et al., Biol Blood Marrow Transplant (2010), Till, et al., Blood, 112:2261-2271(2008), Wang, et al., Hum Gene Ther., 18:712-725(2007), Wang, et al., Mol Ther., 9:577-586(2004), Jensen, et al., Biol Blood Marrow Transplant, 4:75-83(1998)); CD22 (e.g. B cells) (Haso, et al., Blood, 121:1165-1174(2013)); CD30 (e.g., B cells) (Di Stasi, et al., Blood, 113:6392-6402(2009); Savoldo, et al., Blood, 110:2620-2630(2007); Hombach, et al., Cancer Res., 58:1116-1119(1998)); CD33 (e.g., myeloid) (Finney, et al., J Immunol., 161:2791-2797(1998)); CD70 (e.g., B cells / T cells) (Shaffer, et al., Blood, 117:4304-4314(2011)); CD123 (e.g., myeloid) (Tettamanti, et al., Br J Haematol., 161:389-401(2013)); kappa (e.g., B cells) (Vera, et al., Blood, 108:3890-3897(2006)); Lewis Y (e.g., myeloid) (Peinert, et al., Gene Ther., 17:678-686(2010); Ritchie, et al., Mol Ther. (2013)); NKG2D ligands (e.g., myeloid) (Barber, et al., Exp Hematol., 36:1318-1328(2008); Lehner, et al., PLoS One., 7:e31210 (2012); Song, et al., Hum Gene Ther., 24:295-305(2013); Spear, et al., J Immunol. 188:6389-6398 (2012); ROR1 (e.g., B cells) (Hudecek, et al., Clin Cancer Res. (2013)).

[0351] CAR targets for solid tumors include, but are not limited to, B7H3 (e.g., sarcoma, glioma) (Cheung, et al., Hybrid Hybridomics, 22:209-218(2003)); CAIX (e.g., kidney) (Lamers, et al., J Clin Oncol., 24:e20-e22. (2006)); Weijtens, et al., Int J Cancer, 77:181-187(1998)); CD44 v6 / v7 (e.g., cervix) (Hekele, et al., Int J Cancer, 68:232-238(1996)); Dall, et al., Cancer Immunol Immunother, 54:51-60(2005); CD171 (e.g., neuroblastoma) (Park, et al., Mol Ther., 15:825-833(2007)); CEA (e.g., colon) (Nolan, et al., Clin Cancer Res., 5:3928-3941(1999)); EGFRvIII (e.g., glioma) (Bullain, et al., J Neurooncol. (2009); Morgan, et al., Hum Gene Ther., 23:1043-1053(2012)); EGP2 (e.g., carcinoma) (Meier, et al., Magn Reson Med., 65:756-763(2011); Ren-Heidenreich, et al., Cancer Immunol Immunother., 51:417-423(2002)); EGP40 (e.g., colon) (Daly, et al., Cancer Gene Ther., 7:284-291(2000);EphA2(e.g., glioma, lung)(Chow, et al., Mol Ther., 21:629-637(2013));ErbB2(HER2)(e.g., breast, lung, prostate, glioma)(Zhao, et al., J Immunol., 183:5563-5574(2009), Morgan, et al., Mol Ther., 18:843-851(2010), Pinthus, et al., 114:1774-1781(2004), Teng, et al., Hum Gene Ther., 15:699-708(2004), Stancovski, et al., J Immunol., 151:6577-6582(1993), Ahmed, et al., Mol Ther., 17:1779-1787(2009), Ahmed, et al., Clin Cancer Res., 16:474-485(2010), Moritz, et al., Proc Natl Acad Sci USA, 91:4318-4322(1994)); ErbB receptor family (e.g., breast, lung, prostate, glioma) (Davies, et al., Mol Med., 18:565-576(2012)); ErbB3 / 4 (e.g., breast, ovary) (Muniappan, et al., Cancer Gene Ther., 7:128-134(2000); Altenschmidt, et al., Clin Cancer Res., 2:1001-1008(1996)); HLA-A1 / MAGE1 (e.g., melanoma) (Willemsen, et al., Gene Ther., 8:1601-1608(2001); Willemsen, et al., J Immunol., 174:7853-7858(2005)); HLA-A2 / NY-ESO-1 (e.g., sarcoma, melanoma) (Schuberth, et al., Gene Ther., 20:386-395(2013)); FR-α (e.g., ovarian) (Hwu, et al., J Exp Med., 178:361-366(1993); Kershaw, et al., Nat Biotechnol., 20:1221-1227(2002); Kershaw, et al., Clin Cancer Res., 12:6106-6115(2006); Hwu, et al., Cancer Res., 55:3369-3373(1995)); FAP (e.g., cancer-associated fibroblasts) (Kakarla, et al., Mol Ther. (2013)); FAR (e.g., rhabdomyosarcoma) (Gattenlohner, et al., Cancer Res., 66:24-28(2006)); GD2 (e.g., neuroblastoma, sarcoma, melanoma) (Pule, et al., Nat Med., 14:1264-1270(2008); Louis, et al., Blood, 118:6050-6056(2011); Rossig, et al., Int J Cancer., 94:228-236(2001)); GD3 (e.g., melanoma, lung cancer) (Yun, et al., Neoplasia., 2:449-459(2000)); HMW-MAA (e.g., melanoma) (Burns, et al., Cancer Res., 70:3027-3033(2010)); IL11Rα (e.g., osteosarcoma) (Huang, et al., Cancer Res., 72:271-281(2012)); IL13Rα2 (e.g., glioma) (Kahlon, et al., Cancer Res., 64:9160-9166(2004); Brown, et al., Clin Cancer Res. (2012); Kong, et al., Clin Cancer Res., 18:5949-5960(2012); Yaghoubi, et al., Nat Clin Pract Oncol., 6:53-58(2009)); Lewis Y (e.g., breast / ovarian / pancreatic) (Peinert, et al., Gene Ther., 17:678-686(2010); Westwood, et al., Proc Natl Acad Sci USA, 102:19051-19056(2005); Mezzanzanica, et al., Cancer Gene Ther., 5:401-407(1998)); mesothelin (e.g., mesothelioma, breast, pancreas) (Lanitis, et al., Mol Ther., 20:633-643(2012); Moon, et al., Clin Cancer Res., 17:4719-4730(2011)); Mue1 (e.g., ovarian, breast, prostate) (Wilkie, et al., J Immunol., 180:4901-4909(2008)); NCAM (e.g., neuroblastoma, colorectal) (Gilham, et al., J Immunother., 25:139-151(2002)); NKG2D ligands (e.g., ovarian, sarcoma) (Barber, et al., Exp Hematol., 36:1318-1328(2008); Lehner, et al., PLoS One, 7:e31210 (2012); Song, et al., Gene Ther., 24:295-305(2013); Spear, et al., J Immunol., 188:6389-6398(2012)); PSCA (e.g., prostate, pancreatic) (Morgenroth, et al., Prostate, 67:1121-1131(2007); Katari, et al., HPB, 13:643-650(2011)); PSMA (e.g., prostate) (Maher, et al., Nat Biotechnol., 20:70-75(2002); Gong, et al., Neoplasia., 1:123-127(1999)); TAG72 (e.g., colon) (Hombach, et al., Gastroenterology, 113:1163-1170(1997); McGuinness, et al., Hum Gene Ther., 10:165-173(1999)); VEGFR-2 (e.g., tumor vasculature) (J Clin Invest., 120:3953-3968(2010); Niederman, et al., Proc Natl Acad Sci USA, 99:7009-7014(2002)).

[0352] ii. Metabolic stability In some embodiments, the metabolic stability of cells (e.g., CAR cells) is improved by endowing the cells with the ability to produce growth factors that are limiting in vivo. In some embodiments, a nucleic acid cargo encoding an anti-apoptotic factor, such as BCL-XL, is transiently delivered to the cells. B-cell lymphoma-extra-large (Bcl-XL, or BCL2-like 1 isoform 1) is a transmembrane protein within mitochondria. Bcl-XL is a member of the Bcl-2 family of proteins and acts as a pro-survival protein in the intrinsic apoptotic pathway, preventing the release of mitochondrial contents, such as cytochrome c, thereby leading to caspase activation. Both amino acid and nucleic acid sequences encoding BCL-XL are known in the art, including, for example, UniProtKB-Q07817 (B2CL1_HUMAN), Isoform Bcl-X(L) (identifier: Q07817-1) (amino acid sequence); ENA|U72398|U72398.1 Human Bcl-x beta (bcl-x) gene, complete cds, (genomic nucleic acid sequence); ENA|Z23115|Z23115.1 H. sapiens bcl-XL mRNA (mRNA / cDNA nucleic acid sequence).

[0353] In some embodiments, the nucleic acid cargo encodes a proliferation-inducing factor such as IL-2. Amino acid and nucleic acid sequences encoding IL-2 are known in the art, including, for example, UniProtKB-P60568 (IL2_HUMAN) (amino acid sequence); ENA|X00695|X00695.1 Human interleukin-2 (IL-2) gene and 5'-flanking region (IL-2) (gene nucleic acid sequence); and ENA|V00564|V00564.1 Human mRNA encoding interleukin-2 (IL-2) (mRNA / cDNA nucleic acid sequence).

[0354] However, the production of secreted IL-2 can have the undesirable side effect of stimulating the proliferation of lymphoma and Treg cells and impairing the formation of memory T cells (Zhang, et al., Nature Medicine, 11:1238-1243(2005)). Furthermore, the use of IL-2 in patients undergoing treatment with tumor-infiltrating lymphocytes (TILs) leads to increased toxicity (Heemskerk, et al., Human Gene Therapy, 19:496-510(2008)). To avoid this potential, in addition to or instead of IL-2, the nucleic acid cargo can encode a chimeric γc cytokine receptor (CγCR), such as one consisting of interleukin-7 (IL-7) and its associated IL-7Rα / CD127, which confers a cell-specific STAT5 cytokine signal independent of exogenous cytokines (Hunter, et al., Molecular Immunology, 56:1-11(2013)). This design is modular in that the IL-2Rβ / CD122 cytoplasmic chain can be swapped for the IL-7Rα / CD127 cytoplasmic chain to enhance Shc activity. The construct mimics wild-type IL-2 signaling in human CD8+ T cells (Hunter, et al., Molecular Immunology, 56:1-11 (2013)) and should therefore function similarly to IL-2 mRNA without the unwanted side effects.

[0355] Additionally or alternatively, other anti-apoptotic molecules and cytokines can be used to preserve cell viability in a native state. Exemplary factors include, but are not limited to: Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic factor (e.g., UniProtKB-Q07820 (MCL1_HUMAN) (amino acid sequence); ENA|AF147742|AF147742.1 Homo sapiens myeloid cell differentiation protein (MCL1) gene, promoter and complete cds (genomic nucleic acid sequence); ENA|AF118124|AF118124.1 Homo sapiens myeloid cell leukemia sequence 1 (MCL1) mRNA, complete cds. (mRNA / cDNA nucleic acid sequence)); IL-7, which is important for T cell survival and development (e.g., UniProtKB-P13232 (IL7_HUMAN) (amino acid sequence); ENA|EF064721|EF064721.1 Homo sapiens interleukin 7 (IL7) gene, complete cds. (genomic nucleic acid sequence); ENA|J04156|J04156.1 Human interleukin 7 (IL-7) mRNA, complete cds. (mRNA / cDNA nucleic acid sequence), and Examples of IL-15 include IL-15, which promotes the survival of T cells and NK cells (e.g., UniProtKB-P40933 (IL15_HUMAN) (amino acid sequence); ENA|X91233|X91233.1 H. sapiens IL15 gene (genomic nucleic acid sequence); ENA|U14407|U14407.1 Human interleukin 15 (IL15) mRNA, complete cds. (mRNA / cDNA nucleic acid sequence)) (Opferman, et al., Nature, 426: 671-676 (2003); Meazza, et al., Journal of Biomedicine & Biotechnology, 861920, doi:10.1155 / 2011 / 861920 (2011); Michaud, et al., Journal of Immunotherapy, 33:382-390 (2010)). These cytokine mRNAs can be used independently or in combination with BCL-XL, IL-2, and / or CγCR mRNA. Thus, in some embodiments, mRNA encoding MCL-1, mRNA encoding IL-7, mRNA encoding IL-15, or a combination thereof is delivered to a cell.

[0356] iii. Inhibitory CAR (iCAR) In some embodiments, T cell therapy is delivered via CAR cells, which have demonstrated long-term efficacy and curative potential for the treatment of some cancers, but their use is limited by damage to non-cancerous tissues, mimicking graft-versus-host disease after donor lymphocyte infusion. Any of the disclosed compositions and methods can be used in combination with nonspecific immunosuppression (e.g., high-dose corticosteroid therapy, which exerts cytostatic or cytotoxic effects on T cells to suppress immune responses), irreversible T cell elimination (e.g., so-called suicide engineering strategies), or a combination thereof. However, in some preferred embodiments, off-target effects are reduced by introducing a construct encoding an inhibitory chimeric antigen receptor (iCAR) into CAR cells. T cells with specificity for both tumors and off-target tissues can be restricted to tumors by using antigen-specific iCARs introduced into T cells, sparing off-target tissues (Fedorov, et al., Science Translational Medicine, 5:215ral72 (2013)). iCARs can contain a surface antigen recognition domain combined with a potent acute inhibitory signaling domain, so that T cell responsiveness is limited despite simultaneous engagement of an activating receptor (e.g., CAR). In a preferred embodiment, iCARs contain the signaling domain of an immunoinhibitory receptor (e.g., CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGF-beta receptor dominant-negative analog, etc.) fused to a single-chain variable fragment (scFv) specific for the inhibitory antigen via a transmembrane region that specifically inhibits T cell function upon antigen recognition. When CAR cells encounter cells (e.g., cancer cells) that do not express the inhibitory antigen, iCAR-transduced T cells can initiate a CAR-induced response against the CAR's target antigen.DNA iCARs using PSMA-specific scFvs carrying the inhibitory signaling domains of either CTLA-4 or PD-1 are discussed in (Fedorov, et al., Science Translational Medicine, 5:215ra172 (2013)).

[0357] Design considerations include the findings that PD-1 is a more potent inhibitor than CTLA-4, that CTLA-4 exhibits cytoplasmic localization unless the Y165G mutant is used, and that iCAR expression levels are important.

[0358] iCARs can be designed against cell-type specific surface molecules, hi some embodiments, iCARs are designed to prevent the reactivity of T cells, NK cells, or other immune cells against certain tissues or cell types.

[0359] iv. Reduction of endogenous inhibitory signaling In some embodiments, cells are contacted with nucleic acid cargos that reprogram cells to prevent the expression of one or more antigens.For example, in some embodiments, the nucleic acid cargo is or encodes an interfering RNA that prevents the expression of mRNA that encodes antigens such as CTLA-4 or PD-1.Using this method, universal donor cells can be prepared.The RNA used to change the expression of allogeneic antigens can be used alone or in combination with RNA that causes dedifferentiation of target cells.

[0360] While the above sections present compositions and methods utilizing inhibitory signaling domains, e.g., from CTLA-4 or PD-1, in engineered iCARs to limit on-target / off-tumor cytotoxicity, overall CAR cell on-tumor effector efficacy can also or alternatively be increased by reducing the expression of endogenous inhibitory signaling in CAR cells such that the CAR cells are resistant to the inhibitory signals of the hostile tumor microenvironment.

[0361] CTLA-4 and PD-1 inhibit T cells at different stages of activation and function. CTLA-4 regulates T cell responses to self-antigens, as knockout mice spontaneously develop organ damage due to highly activated tissue-infiltrating T cells without exposure to specific antigens (Tivol, et al., Immunity, 3:541-547(1995); Waterhouse, et al., Science, 270:985-988(1995)). Interestingly, conditional knockout of CTLA-4 in Treg cells reproduces global knockout, thus indicating that CTLA-4 functions normally in Tregs (Wing, et al., Science, 322:271-275(2008)). In contrast, PD-L1 knockout mice are prone to autoimmunity but do not spontaneously develop widespread inflammatory cell infiltration of normal organs, thus indicating that the primary physiological function of PD-L1 is to mediate inducible negative feedback regulation of ongoing tissue inflammation (Dong, et al., Immunity, 20:327-336(2004)). Indeed, according to the "adaptive resistance" hypothesis, the majority of tumors upregulate PD-L1 in response to IFNγ, a key cytokine released by effector T cells, including CART cells (Greenwald, et al., Annu Rev Immunol, 23:515-548(2005); Carreno, et al., Annu Rev Immunol, 20:29-53(2002); Chen, et al., The Journal of Clinical Investigation, 125:3384-3391(2015); Keir, et al., Annu Rev Immunol, 26:677-704(2008); Pentcheva-Hoang, et al., Immunological Reviews, 229:67-87(2009)).PD-L1 then delivers inhibitory signals to T cells, thereby reducing their proliferation and cytokine and perforin production (Butte, et al., Immunity, 27:111-122(2007); Chen, et al., Immunology, 4:336-347(2004); Park, et al., Blood, 116:1291-1298(2010); Wherry, et al., Nat Immunol, 12:492-499(2011); Zou, et al., Immunology, 8:467-477(2008)). Furthermore, reverse signaling from T cells through B7-H1 on cancer cells induces anti-apoptotic effects that counteract Fas-L signaling (Azuma, et al., Blood, 111:3635-3643(2008)). Azuma, et al., Blood, 111:3635-3643(2008).

[0362] Given the upregulation of B7-H1 by cancer cells and the association of B7-H1 expression with cancer progression and poor clinical outcomes (Flies, et al., Journal of Immunotherapy, 30:251-260(2007); Nishimura, et al., Immunity, 11:141-151(1999); Wang, et al., Curr Top Microbiol Immunol, 344:245-267(2011)), antibodies antagonizing the PD-1 pathway and CTLA-4 pathway have shown dramatic efficacy in solid tumors, particularly melanoma, and combining these two antibodies has shown even greater activity. The anti-CTLA-4 antibody ipilimumab improves overall survival in metastatic melanoma and is associated with increased T cell infiltration into the tumor and an increase in the intratumoral CD8+:Treg ratio, primarily due to inhibition of Treg cells (Hamid, et al., J Transl Med, 9:204 (2011); Ribas, et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 15:6267-6276 (2009); Twyman-Saint, et al., Nature, 520:373-377 (2015)).The anti-PD-1 antibody nivolumab has demonstrated an overall response rate of 30-40% in metastatic melanoma (Robert, et al., The New England Journal of Medicine, 372:320-330(2015); Topalian, et al., J Clin Oncol, 32:1020-1030(2014)), and similar findings have been observed in early phase clinical trials for other solid tumors, including metastatic kidney cancer, non-small cell lung cancer, and recurrent Hodgkin lymphoma (Ansell, et al., The New England Journal of Medicine, 372:311-319(2015); Brahmer, et al., J Clin Oncol, 28:3167-3175(2010); Topalian, et al., The New England Journal of Medicine, 366:2443-2454(2012)). Because resistance to anti-CTLA-4 antibodies in mouse melanoma models is due to upregulation of PD-L181, combining ipilimumab and nivolumab demonstrates additional efficacy in both mouse models and human patients (Larkin, et al., The New England Journal of Medicine, 373:23-34 (2015); Spranger, et al., J Immunother Cancer, 2, 3, doi:10.1186 / 2051-1426-2-3 (2014); Yu, et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 16:6019-6028 (2010)). Given the importance of checkpoint inhibition pathways, PD-1 / CTLA-4 blockade releases the brakes, while chimeric antigen receptors press the accelerator. Importantly, transient delivery can be used to release the brakes only transiently, so that these cells do not result in subsequent autoimmune disease.

[0363] (1).CRISPRi To avoid permanent genome modification and inactivation of inhibitory signals such as PD-1 and CTLA-4, the dCAS9 CRISPRi system (Larson, et al., Nat Protoc, 8:2180-2196 (2013)) can be used. Nucleic acids encoding enzymatically inactive dCAS9-KRAB-repression domains, fusion proteins, and sgRNAs against inhibitory signaling proteins (e.g., CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGF-beta receptor dominant-negative analogs, etc.) can be co-delivered into CAR cells. One or more sgRNAs can be used. The sgRNAs can be designed to target the proximal promoter and coding regions (non-template strands). An alternative approach utilizes the single-component Cpf1 CRISPR system, which allows smaller RNAs to be introduced and expressed by electroporation (Zetsche, et al., Cell, doi:10.1016 / j.cell.2015.09.038 (2015)). Any of the aforementioned RNA components may also be encoded in a DNA expression construct, such as a vector, e.g., a plasmid. Thus, either RNA, DNA, or a combination thereof may serve as the nucleic acid cargo.

[0364] Broad inhibition of CTLA-4 with ipilimumab results in autoimmune sequelae, but these side effects are thought to be reduced by limiting loss to CAR cells and the transient nature of mRNA delivery; inhibitory function is restored over time.

[0365] (2) Inhibitory RNA The nucleic acid cargo that can be delivered to cells can be or code for functional nucleic acids or polypeptides that are designed to target and reduce or inhibit the expression or translation of inhibitory signaling molecule mRNA, or to reduce or inhibit expression, reduce activity, or increase the degradation of inhibitory signaling molecule protein.Suitable techniques include, but are not limited to, antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex-forming molecules, RNAi, etc.In some embodiments, mRNA codes for antagonist polypeptides that reduce inhibitory signaling.

[0366] In some embodiments, cargo that is or encodes functional RNA suitable for reducing or silencing expression of CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGF beta receptor dominant negative analogs, and the like, alone or in combination, can be delivered to cells.

[0367] In some embodiments, the cargo is RNA or DNA encoding a polypeptide that reduces the bioavailability of, or functions as an antagonist or other negative regulator or inhibitor of, CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGF-beta receptor dominant negative analog, or another protein in an immune inhibitory pathway. The protein can be paracrine, endocrine, or autocrine. The protein can regulate cells intracellularly. The protein can be secreted and regulate the expressing cell and / or other (e.g., nearby) cells. The protein can be a transmembrane protein that regulates the expressing cell and / or other cells. The protein can be a fusion protein, such as an Ig fusion protein.

[0368] v. Proapoptotic factors Also provided are compositions and methods for activating and reactivating apoptotic pathways. In some embodiments, the nucleic acid is or encodes a factor or agent that activates, reactivates, or otherwise enhances or increases an intrinsic apoptotic pathway. Preferably, the factor activates, reactivates, or otherwise enhances an intrinsic apoptotic pathway in cancer (e.g., tumor) cells, and more preferably, is specific to or targeted to cancer cells.

[0369] In some embodiments, after delivery of anti-apoptotic or growth-promoting factors, such as those described above or otherwise known in the art, cells are more resistant to or less sensitive to induced apoptosis than untreated cells.The pro-apoptotic factor can, for example, induce or increase apoptosis in untreated cells compared to treated T cells, and is preferably selective for cancer cells.This regimen provides a two-pronged attack on cancer cells, one cellular and one molecular.

[0370] The intrinsic apoptotic pathway can be activated, reactivated, or otherwise enhanced by targeting BCL-2 family members. BCL-2 family members are classified into three subgroups based on their function and Bcl-2 homology (BH) domain: multidomain anti-apoptotic proteins (e.g., BCL-2 or BCL-XL), multidomain pro-apoptotic proteins (e.g., BAX and BAK), and BH3-only pro-apoptotic proteins (e.g., BIM). Members of the BH3-only subgroup, such as BIM, function as cell death sentinels located throughout the cell, ready to transmit various physiological and pathological signals of cellular injury to the central apoptotic machinery located in the mitochondria (Danial, et al., Cell, 116:205-219(2004)).

[0371] In some embodiments, the pro-apoptotic factor is a pro-apoptotic BH3 mimetic.A variety of pro-apoptotic BH3 mimetics can simulate the native pro-apoptotic activity of BIM, and can provide the ability to manipulate multiple points in the apoptotic pathway.For example, BIM SAHB (stabilized alpha helix of BCL-2 domain), ABT-737, and ABT-199 are pro-apoptotic BH3 mimetics designed through the structural study of the interaction between the pro-apoptotic BH3-only helix domain and the hydrophobic groove formed by the assembly of the BH1, BH2 and BH3 domains of anti-apoptotic proteins (Oltersdorf, et al., Nature, 435:677-681(2005)).

[0372] 4.Target cells In some embodiments, the conjugates of the present disclosure are targeted to one or more specific cell types or tissues. The target cells may be in vitro, ex vivo, or within a subject (i.e., in vivo). The applications discussed herein can be performed in vitro, ex vivo, or in vivo. For ex vivo applications, cells can be harvested or isolated and treated in culture. The ex vivo treated cells can be administered to a subject in need thereof in a therapeutically effective amount. For in vivo applications, the cargo can be passively delivered to the target cells, e.g., via circulation, local delivery, etc., of the composition, or can be actively targeted, e.g., using additional cell-, tissue-, or organ-specific targeting moieties. Thus, in some embodiments, the cargo is delivered to the target cells to the exclusion of other cells. In some embodiments, the cargo is delivered to the target cells and non-target cells.

[0373] The target cell can be selected by the practitioner based on the desired treatment and therapy and the intended effect of the nucleic acid cargo. For example, if the nucleic acid cargo is intended to induce cell death, the target cell can be a cancer cell. If the nucleic acid cargo is intended to induce genomic alteration, the target cell can be a stem cell. If the nucleic acid cargo encodes a chimeric antigen receptor, the target cell can be an immune cell.

[0374] 4H2 penetrates into cells in a dipyridamole-sensitive manner, which is enhanced by the addition of GUO, thus indicating nucleoside transporter-dependent transport facilitated by local nucleic acids.

[0375] In some embodiments, target cell expresses nucleoside transporter on their plasma membrane.The expression of nucleoside transporter is relatively ubiquitous, but its abundance varies between tissue types and cell types.For example, ENT2 expression has been confirmed in brain, heart, placenta, thymus, pancreas, prostate and kidney (Griffiths, et al., Biochem J, 1997.328(Pt 3): p.739-43; Crawford, et al., J Biol Chem, 1998.273(9): p.5288-93). ENT2 has one of the highest mRNA expression levels in skeletal muscle compared to other transporters (Baldwin, et al., Pflugers Arch, 2004.447(5): p. 735-43; Govindarajan, et al., Am J Physiol Regul Integr Comp Physiol, 2007.293(5): p. R1809-22). Thus, in some embodiments, the target cell is brain, heart, placenta, thymus, pancreas, prostate, kidney, or skeletal muscle.

[0376] Additional, non-limiting, exemplary target cells are discussed below.

[0377] i. Progenitor and stem cells The cells may be hematopoietic progenitor or stem cells. In some embodiments, particularly those relating to gene editing and gene therapy, the target cells may express CD34 + Hematopoietic stem cells. Hematopoietic stem cells (HSCs), such as CD34+ cells, are multipotent stem cells that give rise to all blood cell types, including red blood cells.

[0378] Those skilled in the art are able to isolate and enrich stem cells. + Methods for isolating and enriching cells and other cells are known in the art and are disclosed, for example, in U.S. Patent Nos. 4,965,204; 4,714,680; 5,061,620; 5,643,741; 5,677,136; 5,716,827; 5,750,397 and 5,759,793. As used herein, in the context of a composition enriched in hematopoietic progenitor and stem cells, "enriched" refers to a higher proportion of desirable elements (e.g., hematopoietic progenitor and stem cells) than that found in a natural cell source. The cell composition can be enriched by at least one order of magnitude, preferably two or three orders of magnitude, more preferably ten, one hundred, two hundred or one thousand orders of magnitude relative to the natural cell source.

[0379] For humans, CD34 +Cells can be harvested from umbilical cord blood, bone marrow, or from blood after cytokine mobilization, which is achieved by subcutaneously or intravenously injecting a donor with hematopoietic growth factors, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF), or stem cell factor (SCF), in sufficient amounts to induce hematopoietic stem cell migration from the bone marrow cavity into the peripheral circulation. Bone marrow cells can be initially obtained from any suitable bone marrow source, such as the tibia, femur, vertebrae, and other bone cavities. To isolate bone marrow, bones can be flushed with an appropriate solution, typically a balanced salt solution conveniently supplemented with fetal bovine serum or other naturally occurring factors, along with a low concentration of about 5-25 mM in an acceptable buffer. Convenient buffers include Hepes, phosphate buffer, lactate buffer, and the like.

[0380] Cells can be selected by positive and negative selection techniques. Cells can be selected using commercially available antibodies that bind to hematopoietic progenitor or stem cell surface antigens, such as CD34, using methods known to those skilled in the art. For example, the antibodies can be conjugated to magnetic beads and immunogenic procedures can be used to recover the desired cell type. Another technique involves the use of fluorescence-activated cell sorting (FACS). The CD34 antigen is found on progenitor cells within the hematopoietic system of non-leukemic individuals and is expressed in a population of cells (i.e., expressing the CD34 antigen) that are recognized by the monoclonal antibody My-10, which can be used to isolate stem cells for bone marrow transplantation. My-10 has been deposited at the American Type Culture Collection (Rockville, Md.) as HB-8483 and is commercially available as anti-HPCA1. Furthermore, negative selection of differentiated and "specialized" cells from human bone marrow can be used to select for virtually any desired cell marker. For example, progenitor or stem cells, most preferably CD34 + The cells are CD3 - , CD7 - , CD8 - , CD10 - , CD14 - , CD15- , CD19 - , CD20 - , CD33 - , class II HLA + and Thy-1 + It can be characterized as either:

[0381] Once progenitor cells or stem cells are isolated, they can be grown in any suitable medium.For example, progenitor cells or stem cells can be grown in the conditioned medium from stromal cells, such as those obtained from bone marrow or liver that secrete factors, or in a medium that contains cell surface factors that support the proliferation of stem cells.Hematopoietic cells can be removed from stromal cells using suitable monoclonal antibodies to remove unwanted cells.

[0382] The isolated cells are contacted with the complex of antibody and nucleic acid cargo ex vivo. The cells into which the cargo is delivered can be referred to as modified cells. The solution of the complex can simply be added to the cells in culture. It may be desirable to synchronize the cells in S phase. For example, methods for synchronizing cultured cells by double thymidine blockade are known in the art (Zielke, et al., Methods Cell Biol., 8:107-121(1974)).

[0383] The modified cells can be maintained or expanded in culture prior to administration to a subject. Culture conditions are generally known in the art depending on the cell type. +The conditions for maintaining hematopoietic cells have been particularly well studied, and several suitable methods are available. A common approach to expanding multipotent hematopoietic cells ex vivo is to culture purified progenitor or stem cells in the presence of early-acting cytokines such as interleukin-3. It has also been shown that including a combination of thrombopoietin (TPO), stem cell factor (SCF), and flt3 ligand (Flt-3L; i.e., the ligand for the flt3 gene product) in the nutrient medium for maintaining hematopoietic progenitor cells ex vivo was useful for expanding primitive (i.e., relatively undifferentiated) human hematopoietic progenitor cells in vitro, and that these cells were able to engraft in SCID-hu mice (Luens et al., 1998, Blood 91:1206-1215). In other known methods, cells can be maintained ex vivo in a nutrient medium containing mouse prolactin-like protein E (mPLP-E) or mouse prolactin-like protein F (mPIP-F; collectively mPLP-E / IF) (e.g., for minutes, hours, or for 3, 6, 9, 13, or more days) (U.S. Pat. No. 6,261,841). It will be appreciated that other suitable cell culture and expansion methods can be used as well. Cells can also be grown in serum-free medium, as described in U.S. Pat. No. 5,945,337.

[0384] In another embodiment, the modified hematopoietic stem cells are ex vivo transfected with CD4+ cells using specific combinations of interleukins and growth factors using methods well known in the art prior to administration to a subject. + The cells can be expanded ex vivo to a large number of cells, preferably at least 5-fold, more preferably at least 10-fold, and even more preferably at least 20-fold, compared to the original population of isolated hematopoietic stem cells.

[0385] In another embodiment, the cells can be dedifferentiated somatic cells. Somatic cells can be reprogrammed to become pluripotent stem-like cells that can be induced to become hematopoietic progenitor cells. The hematopoietic progenitor cells can then be transfected with CD34 + Can be treated with the composition described for cell.The representative somatic cells that can be reprogrammed include but are not limited to fibroblasts, adipocytes and muscle cells.In mice, hematopoietic progenitor cells have been successfully produced from induced stem-like cells (Hanna, J. et al. Science, 318:1920-1923(2007)).

[0386] To produce hematopoietic progenitor cells from induced stem-like cells, somatic cells are collected from a host. In a preferred embodiment, the somatic cells are autologous fibroblasts. The cells are cultured and transduced with vectors encoding Oct4, Sox2, Klf4, and c-Myc transcription factors. The transduced cells are cultured and screened for embryonic stem cell (ES) morphology and ES cell markers, including but not limited to AP, SSEA1, and Nanog. The transduced ES cells are cultured and induced to produce induced stem-like cells. The cells are then screened for CD41 and c-kit markers (early hematopoietic progenitor cell markers) and markers for myeloid and erythroid differentiation.

[0387] The modified hematopoietic stem cells or cells, including, for example, induced hematopoietic progenitor cells, are then introduced into a subject. Delivery of the cells can be effected using a variety of methods, including, most preferably, intravenous administration by infusion and direct depot injection into periosteal, bone marrow, and / or subcutaneous sites.

[0388] Subjects receiving the modified cells can be pretreated to condition the bone marrow to enhance cell engraftment. The recipient can be treated with radiation or chemotherapy prior to administration of the cells to enhance engraftment. Once the cells are administered, a period of time is generally required for the cells to engraft. It typically takes several weeks to several months for significant engraftment of hematopoietic stem or progenitor cells to be achieved.

[0389] A high percentage of modified hematopoietic stem cells may not need to engraft to achieve a significant prophylactic or therapeutic effect. Engrafted cells may expand over time after engraftment, resulting in an increasing percentage of modified cells. In some cases, only a small number or percentage of modified hematopoietic stem cells may need to engraft to provide a prophylactic or therapeutic effect.

[0390] In preferred embodiments, the cells administered to a subject are autologous cells, eg, cells derived from the subject, or syngeneic cells.

[0391] ii. Embryo In some embodiments, the compositions and methods can be used to deliver cargo to embryonic cells in vitro. The methods typically involve contacting an embryo in vitro with an effective amount of antibody-cargo DNA to improve cargo transduction into the embryo. The embryo can be a single-cell zygote, but treatments are also provided for male and female gametes before and during fertilization, as well as embryos with two, four, eight, or sixteen cells, and embryos including not only zygotes but also morulae and blastocysts. In some embodiments, the embryo is contacted with the composition during in vitro fertilization or on days 0-6 of post-fertilization culture.

[0392] The contacting may be by adding the composition to the liquid medium the embryo is in. For example, the composition may be pipetted directly into the embryo culture medium, whereupon the composition is taken up by the embryo.

[0393] iii.Immune cells In some embodiments, target cell is one or more types of immune cells.For example, different types of cells can be used for immunomodulation and CAR-based therapy or can be targeted in other ways.Preferred target / engineered T cells can vary according to tumor and the purpose of adoptive therapy. Typically, effector T cells are preferred because they secrete high levels of effector cytokines and are highly potent killers of tumor targets in vitro (Barrett, et al., Annu Rev Med., 65: 333-347 (2014)). CD3-CD56+ NK cells and CD3+CD8+ T cells are two complementary lymphocyte populations with robust CAR-mediated cytotoxicity. When used together with CD8+ T cells and CD4+ helper T cells, the presence of suppressor T-reg cells increases, attenuating the cytotoxicity of CD8+ T cells. Reprogrammed CD8+ T cells are preactivated and therefore act directly against tumor cells without the need for activation in lymph nodes, so support by CD4+ T cells is not essential.

[0394] Furthermore, naive T cells (Rosenberg, et al. Adv. Cancer Res., 25:323-388(1977)), central memory T cells (T CM There is evidence that the infusion of Th17 cells (Berger, et al. J. Clin. Invest., 118:294-305(2008)), Th17 cells (Paulos, et al., Sci. Transl. Med., 2:55-78(2010)), and memory T stem cells (Gattinoni, et al., Nat. Med., 17:1290-1297(2012)) may all have certain advantages in certain applications, for example, due to their high replicative capacity. Tumor infiltrating lymphocytes (TILs) also have certain advantages due to their antigen specificity and can be used in the delivery strategies disclosed herein.

[0395] Although the CARs and other delivery strategies disclosed herein are sometimes referred to as CAR cells, CAR immune cells, and CART cells (or CAR T cells), it will be understood that they can also be practiced with other cell types, particularly various types of immune cells, including those discussed herein (e.g., lymphocytes, natural killer cells, dendritic cells, B cells, antigen-presenting cells, macrophages, etc.) and those described elsewhere (see, e.g., Barrett, et al., Annu Rev Med., 65: 333-347 (2014)).

[0396] iv. Cancer cells and tumors In some embodiments, the target cells are cancer cells. In such embodiments, treatment methods are provided that may be useful for cancer, including tumor therapy.

[0397] The cargo that can be delivered to cancer cells includes but is not limited to constructs for expressing one or more apoptosis-promoting factors, immunogenic factors or tumor suppressors; gene editing compositions that target cancer genes, inhibitory nucleic acids; and other strategies discussed herein and elsewhere.In some embodiments, cargo is mRNA that encodes apoptosis-promoting factors or immunogenic factors that increase the immune response against cells.In other embodiments, cargo is siRNA that reduces the expression of oncogenes or other cancer-causing transcripts.

[0398] In mature animals, a balance between cell renewal and cell death is normally maintained in most organs and tissues. Various types of mature cells in the body have a finite lifespan, and as these cells die, new cells are generated through the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated to maintain a constant number of any particular cell type. Nevertheless, cells occasionally arise that no longer respond to normal growth control mechanisms. These cells give rise to clones of cells that can expand to a significant size and generate tumors or neoplasms. Tumors that are unable to grow indefinitely and do not extensively invade healthy surrounding tissue are benign. Tumors that continue to grow and become increasingly invasive are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells extrude from the tumor, invade blood or lymphatic vessels, and travel to other tissues, where they continue to proliferate. In this way, a primary tumor at one site can give rise to secondary tumors at another site.

[0399] The compositions and methods described herein may be useful for treating a subject with a benign or malignant tumor by slowing or inhibiting tumor growth in the subject, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0400] Treatable malignant tumors are herein classified according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors arising from endodermal or ectodermal tissues, such as the skin or the epithelial lining of internal organs and glands. The compositions of the present disclosure are particularly useful in treating carcinomas. Sarcomas occur less frequently and originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can manifest in multiple organs or tissues of the body, establishing cancer.

[0401] The types of cancer that can be treated using the provided compositions and methods include, but are not limited to, cancers, such as vascular cancers such as multiple myeloma, adenocarcinomas and sarcomas of the bone, bladder, brain, breast, cervix, colorectum, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus. In some embodiments, the compositions of the present disclosure are used to treat multiple cancer types simultaneously. The compositions can also be used to treat metastases or tumors in multiple locations.

[0402] B. Methods of Modulating the Immune Response Methods for increasing immune response are provided. For example, immune response against cancer and infectious diseases can be increased. Therefore, methods for treating cancer and infectious diseases in subjects, as well as methods for vaccinating both healthy and diseased subjects, are also provided. Immune response is also involved in wound healing, and therefore, methods for promoting wound healing are also provided. Immunomodulation is also involved in some autoimmune diseases, such as multiple sclerosis, and therefore, methods for promoting immunomodulation in multiple sclerosis are also provided. Thus, in some embodiments, the subject has a wound or multiple sclerosis.

[0403] The method typically involves administering an effective amount of a 4H2 antibody to a subject in need thereof to increase the activation of cGAS and / or another PRR, such as TLR7. In some embodiments, the composition and method increase the activation of cGAS and / or another PRR, such as TLR7, for example, by direct binding and activation by 4H2 or indirect binding through simultaneous interaction of cGAS and / or other PPRs, 4H2, and nucleic acids and / or GTP in the cytoplasm. Activated cGAS catalyzes the formation of cGAMP from the precursor molecules ATP and GTP, and the cGAMP produced by cGAS promotes the nuclear translocation of NF-kB. Thus, in some embodiments, the composition of the present disclosure increases cGAMP production and / or promotes the nuclear translocation of NF-kB. Typically, the method enhances an immune response by inducing or enhancing signaling through the cGAS / STING pathway.

[0404] In some embodiments, the compositions and methods include stimulating T cell proliferation, disrupting tumor vasculature, contributing to tumor cell death and apoptosis, enhancing the release of tumor-associated antigens, improving antigen-specific IgG responses through mechanisms dependent on T helper 1 (TH1), TH2, and / or TH17 cell responses, reducing viral or bacterial load, reducing susceptibility to viruses or bacteria, or any combination thereof. See also Motwani and Fitzgerald, Nature Reviews Genetics volume 20, pages 657-674 (2019), which is expressly incorporated by reference in its entirety and describes additional outcomes of enhanced cGAS / STING signaling. In some embodiments, the compositions and methods include increasing the recruitment of tumor-infiltrating lymphocytes (TILs) to tumors.

[0405] The results described below also indicate that 4H2 interacts with TLR7, and this interaction appears to be nucleic acid-dependent. The TLR family plays an important role in pathogen recognition and innate immune activation. TLRs recognize pathogen-associated molecular patterns (PAMPs) expressed on infectious agents and mediate the production of cytokines necessary for effective immune development. TLR7 is an intracellular pattern recognition receptor in endosomes that recognizes single-stranded RNA, a common feature of viral genomes internalized by macrophages and dendritic cells. For example, TLR7 recognizes the single-stranded RNA of viruses such as HIV and HCV. TLR7 can recognize GU-rich single-stranded RNA.

[0406] This adds another dimension to the use of 4H2 as an immune stimulator, as the results indicate that 4H2 activates at least cGAS and TLR7, and possibly other immune response-inducing receptors as well. Other immune receptors that may be activated by 4H2 include other PPRs, such as RIG-I-like receptors and other toll-like receptors, including, but not limited to, TLR3, TLR8, TLR9, etc. Other receptors include, but are not limited to, receptors whose ligands are referred to as cargo and / or adjuvants.

[0407] In certain embodiments, the method comprises administering to a subject in need thereof an effective amount of a 4H2 antibody in combination with one or more additional agents, such as a nucleic acid cargo, an immunostimulatory nucleic acid, a vaccine component(s), an immune checkpoint modulator, or a combination thereof. In some embodiments, the 4H2 antibody and the additional agent can be used in combination to enhance signaling by another immune receptor, such as cGAS / STING and / or a PPR (e.g., TLR7), to a greater extent than either agent used alone. For example, in some embodiments, e.g., in treating cancer, the enhanced activity is greater anti-tumor activity.

[0408] The 4H2 antibody, and / or immune checkpoint modulator can be administered locally or systemically to a subject, or can be coated onto or incorporated into a device.

[0409] The monotherapy and combination therapy and treatment regimens of the present disclosure typically include methods for treating a disease or a symptom thereof, or for achieving a desired physiological change, comprising administering an effective amount of a 4H2 antibody to an animal, e.g., a mammal, particularly a human, to treat a disease or infection, such as cancer, or a symptom thereof, or to effect a physiological change.

[0410] When administered in combination with an additional agent, the 4H2 antibody and the additional agent can be administered together, e.g., as part of the same composition, or separately, independently at the same time, or at different times (i.e., the administration of the 4H2 antibody and the administration of the immune checkpoint modulator are separated from each other by a finite amount of time). Thus, the terms "combination" or "combined" are used to refer to either the concomitant, simultaneous, or sequential administration of two agents. Combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines to the same subject; one agent given orally and the other by infusion or injection, etc.), or sequentially (e.g., one agent given first, followed by the second agent).

[0411] In some embodiments, the results achieved by the combination are partially or fully additive to the results achieved by each component alone. In some embodiments, the results achieved by the combination are more than additive to the results achieved by each component alone. In some embodiments, the effective amount of one or both agents used in combination is less than the effective amount of each agent when administered separately. In some embodiments, the amount of one or both agents used in combination therapy is sub-therapeutic when used alone.

[0412] The effect of combination therapy or its individual drug may depend on the disease or condition being treated or its progression.For example, in some embodiments, the combination can expand the range of subjects (for example, cancer or infectious disease type) that can be treated compared with each drug alone.Therefore, in some embodiments, the effect of combination on cancer or infectious disease can be compared with the effect of each drug alone on cancer or infectious disease.

[0413] Monotherapy and combination therapy treatment regimens may include one or more administrations of the 4H2 antibody. Combination therapy treatment regimens may include one or more administrations of the additional agent.

[0414] In some embodiments, the 4H2 antibody and the additional agent are administered sequentially, for example, as two or more different pharmaceutical compositions.In certain embodiments, the 4H2 antibody is administered before the first administration of the additional agent.In other embodiments, the additional agent is administered before the first administration of the 4H2 antibody.For example, the 4H2 antibody and the additional agent can be administered to the subject on the same day.Alternatively, the 4H2 antibody and the additional agent can be administered to the subject on different days.

[0415] Administration of the 4H2 antibody can occur at least 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, or 30 hours, at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 24 days, or 30 days before administration of the additional agent, or at least 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, or 30 hours after administration, or at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 24 days, or 30 days after administration of the additional agent. Alternatively, administration of the immune checkpoint modulator can occur at least 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, or 30 hours, at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 24 days, or 30 days before administration of the 4H2 antibody, or at least 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, or 30 hours after administration of the 4H2 antibody, or at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 20 days, 24 days, or 30 days after administration. In certain embodiments, the additive or greater than additive effect of the 4H2 antibody and the additional agent is evident 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, or more than 1 week after administration.

[0416] The dosing regimen or cycle of drug can be completely or partially overlapping, or can be sequential.For example, in some embodiments, all of the administration of 4H2 antibody is carried out before or after the administration of additional drug.Alternatively, the administration of one or more doses of 4H2 antibody and the administration of additional drug can be staggered in time, so that one or more doses of 4H2 antibody are administered, then one or more doses of additional drug are administered, then one or more doses of 4H2 antibody are administered; or one or more doses of additional drug are administered, then one or more doses of 4H2 antibody are administered, then one or more doses of additional drug are administered, etc., to form a uniform or non-uniform treatment course, all of which depends on the schedule selected or desired by the researcher or clinician who administers treatment.

[0417] Each effective amount of the agent can be administered as a single unit dosage (e.g., as a dosage unit) or as sub-therapeutic doses administered over a finite time interval. Such unit doses can be administered daily for a finite period of time, for example, up to 3 days, or up to 5 days, or up to 7 days, or up to 10 days, or up to 15 days, or up to 20 days, or up to 25 days are all specifically contemplated.

[0418] 1. Cancer treatment The treatments disclosed herein can be used to treat, reduce, and / or prevent cancer in a subject. Thus, the compositions can be administered in an effective amount to treat, reduce, and / or prevent cancer in a subject. An effective amount or therapeutically effective amount for treating cancer or its tumor is typically a dosage sufficient to reduce or prevent at least one symptom of cancer or otherwise produce a desired pharmacological and / or physiological effect. The symptom can be physical, such as tumor burden, or biological, such as reducing cancer cell proliferation or increasing cancer cell death. In some embodiments, the amount is effective to kill tumor cells or reduce or inhibit tumor cell proliferation or metastasis. In some embodiments, the amount is effective to reduce tumor burden. In some embodiments, the amount is effective to reduce or prevent at least one coexisting condition of cancer.

[0419] In mature animals, a balance between cell renewal and cell death is normally maintained in most organs and tissues. Various types of mature cells in the body have a finite lifespan, and as these cells die, new cells are generated through the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated to maintain a constant number of any particular cell type. Nevertheless, cells occasionally arise that no longer respond to normal growth control mechanisms. These cells give rise to clones of cells that can expand to a significant size and generate tumors or neoplasms. Tumors that are unable to grow indefinitely and do not extensively invade healthy surrounding tissue are benign. Tumors that continue to grow and become increasingly invasive are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells extrude from the tumor, invade blood or lymphatic vessels, and travel to other tissues, where they continue to proliferate. In this way, a primary tumor at one site can give rise to secondary tumors at another site.

[0420] The compositions and methods described herein are useful for treating a subject with a benign or malignant tumor by slowing or inhibiting tumor growth in the subject, reducing tumor growth or size, inhibiting or reducing tumor metastasis, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0421] Treatable malignant tumors can be classified according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the skin or the epithelial lining of internal organs and glands. The compositions of the present disclosure are particularly useful in treating carcinomas. Sarcomas occur less frequently and originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemias grow as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors can manifest in multiple organs or tissues of the body, establishing cancer.

[0422] The antigen-binding molecules of the present disclosure can be used to treat cells undergoing unregulated growth, invasion, or metastasis.

[0423] Cancer cells are characterized by mutations in one or more Ras genes or mutations in genes encoding other components of the Ras / MAPK signaling pathway, and are particularly good targets for the compositions of the present disclosure.

[0424] Cancerous cells can occur as a result of the somatic gain-of-function mutation of Ras gene, which leads to the activation of mutation of small GTPase Ras enzyme.The oncogenic mutation of H-Ras gene, N-Ras gene or K-Ras gene is most frequently associated with malignant diseases in humans.In certain embodiments, cells express the mutant form of small GTPase Ras family, such as K-Ras.In certain embodiments, cells do not express wild-type Ras gene.

[0425] Oncogenic mutations have also been identified in other upstream or downstream components of the Ras intracellular signaling pathway, including cytosolic kinases and membrane RTKs (Ras / MAPK pathway).

[0426] Oncogenic mutations in K-Ras gene can result in the constitutive activation of the resulting Ras protein.Exemplary mutations include mutations in codons 12, 13, and / or 61, which cause any change in the amino acid at position 12, 13, or 61 of K-ras protein.This includes, but is not limited to, K-ras amino acid 12 (glycine to aspartic acid, cysteine, serine, threonine, arginine, or valine) and amino acid 13 and 61 (glutamine to lysine, arginine, leucine, or aspartic acid).Another way to describe these exemplary K-Ras mutations in this context is G12A, G12C, G12D, G12S, G12I, G12R, G12V, G13C, G13D, G13S, Q61L, Q61R. Again, any change in the amino acid content at positions 12, 13, and 61 are considered exemplary mutations.

[0427] A representative, but non-limiting list of cancers that can be treated using the present compositions includes cancers of the blood and lymphatic system (including leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, solitary plasmacytoma, and multiple myeloma), cancers of the genitourinary system (including prostate cancer, bladder cancer, kidney cancer, urethral cancer, penile cancer, and testicular cancer), cancers of the nervous system (including meningioma, glioma, glioblastoma, astrocytoma, oligodendrocyte glioma, oligoastrocytoma, and ependymoma), cancers of the head and neck (including squamous cell carcinoma of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharynx, larynx, and paranasal sinuses), and cancers of the pancreas, including thyroid cancer, thyroid cancer, and thyroid cancer. cancers), lung cancer (including small cell lung cancer and non-small cell lung cancer), gynecological cancer (including cervical cancer, uterine cancer, vaginal cancer, vulvar cancer, ovarian cancer, and fallopian tube cancer), gastrointestinal cancer (including gastric cancer, small intestine cancer, colorectal cancer, liver cancer, hepatobiliary cancer, and pancreatic cancer), skin cancer (including melanoma, squamous cell carcinoma, and basal cell carcinoma), breast cancer (including ductal and lobular carcinoma and triple-negative breast cancer), and childhood cancer (including neuroblastoma, Ewing's sarcoma, Wilms' tumor, and medulloblastoma). Thus, in some embodiments, the present disclosure relates to methods of treating breast cancer, ovarian cancer, colon cancer, prostate cancer, lung cancer, brain cancer, skin cancer, liver cancer, gastric cancer, pancreatic cancer, or blood-based cancer. In some embodiments, the present disclosure relates to the treatment of glioblastoma.

[0428] Any of the methods of the present disclosure can be further used in combination with radiation therapy, chemotherapy (e.g., antineoplastic agents), or a combination thereof to treat any cancer, including carcinoma, glioma, sarcoma, or lymphoma. Examples of antineoplastic agents that can be combined with the antigen-binding molecules of the present disclosure include, but are not limited to, alkylating agents (e.g., temozolomide, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (e.g., fluorouracil, gemcitabine, methotrexate, cytosine arabinoside, fludarabine, and floxuridine), some antimitotic agents, and vinca alkaloids, such as cyclosporine, cyclosporine, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide. , vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin, and actinomycins such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), and topoisomerase inhibitors (including camptothecins, e.g., irinotecan and topotecan, and derivatives of epipodophyllotoxins, e.g., amsacrine, etoposide, etoposide phosphate, and teniposide).

[0429] Strategies combining STING immunotherapy with other immunomodulatory agents are being explored. The antitumor efficacy of cGAMP administered by it injection into B16.F10 tumors was enhanced when combined with anti-programmed death-1 (PD-1) and anti-cytotoxic T-lymphocyte-associated 4 (CTLA-4) antibodies (Demaria, et al., Proc Natl Acad Sci USA (2015) 112(50):15408-13.10.1073 / pnas.1512832112). In other studies, CDNs and anti-PD-1 therapy stimulated significantly stronger antitumor effects than monotherapy in mouse models of squamous cell carcinoma and melanoma (Gadkaree, et al., Head Neck (2017) 39(6):1086-94.10.1002 / hed.24704; Wang, et al., Proc Natl Acad Sci USA (2017) 114(7):1637-42.10.1073 / pnas.1621363114). Luo et al. showed promising results in the TC-1 tumor model by combining a STING-activating nanovaccine with an anti-PD-1 antibody, resulting in long-lasting antitumor memory (Luo, et al., Nat Nanotechnol (2017) 12(7):648-54.10.1038 / nnano.2017.52). Thus, a particularly preferred method for treating cancer involves administering to a subject a combination of a 4H2 antibody and a checkpoint modulator.

[0430] 2. Infection and Transformed Cells with Viruses In some embodiments, the compositions can be used to treat or prevent infection of cells by, for example, bacteria or viruses, such as oncoviruses. Thus, the compositions can be administered to treat localized or systemic infections.

[0431] They also have a slightly less expensive selection and they are able to do a lot of A ctinomyces、Anabaena、Bacillus、Bactero ides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type b(Hib)、Histoplasma、Hyphomicrobium、Legionella、Leishmania、Leptospira、Listeria、Meningococcus A. B. C. Methanobacterium Micrococcus isseria、Nitrobacter、Oscillatoria、Prochloron、Proteus、Pseudomonas、Phodospirillum、Ricke ttsia、Salmonella、Shigella、Spirillum、Spirochaeta、Staphylococcus、Streptococcus、Strepto myces、Sulfolobus、Thermoplasma、Thiobacillus、およびTreponema、Vibrio、Yersinia、Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum、Plasmodium vivax、Trypanosoma brucei、MosquitoesThese include infections caused by microorganisms including T. histolytica, T. gondii, T. trichomonas vaginalis, and T. mansoni.

[0432] Exemplary viruses that may be affected by the compositions of the present disclosure include human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-lymphotropic virus (HTLV), Kaposi's sarcoma-associated herpesvirus (HHV-8), Merkel cell polyomavirus, Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and human cytomegalovirus (CMV). Related infectious diseases include, but are not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), the common cold (e.g., human rhinovirus), coronavirus infection (e.g., SARS-CoV-2), Zika virus infection, dengue virus infection, and vesicular stomatitis virus (VSV) infection.

[0433] For example, the composition can be administered topically to treat viral skin diseases, such as herpes zoster or shingles, or genital warts. The composition can also be administered to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.

[0434] Other viral diseases that may be affected by administration of the compositions include Colorado tick fever (Coltivirus, caused by an RNA virus), West Nile fever (encephalitis, caused by a flavivirus present primarily in the Middle East and Africa), yellow fever, rabies (caused by several different strains of neurotropic viruses in the Rhabdoviridae family), viral hepatitis, gastroenteritis (viruses) - acute viral gastroenteritis caused by Norwalk virus, Norwalk-like virus, rotavirus, calicivirus, and astrovirus, poliomyelitis, influenza (flu), measles (rubella), Paramyxoviridae, mumps, respiratory syndromes including viral pneumonias and acute respiratory syndromes, including croup, caused by a variety of viruses collectively referred to as acute respiratory viruses, and respiratory disease caused by respiratory syncytial virus (RSV, the most dangerous cause of respiratory infection in young children).

[0435] In some embodiments, the compositions of the present disclosure are used to treat or prevent a viral infection or the spread or worsening of a viral infection. For example, in some embodiments, the compositions are used to treat or prevent a viral infection or the spread or worsening of a viral infection in a subject who has been exposed to or is at risk of being exposed to a virus such as those discussed herein.

[0436] 3. Vaccination The comp...

Claims

1. (a) an intact 4H2 monoclonal antibody, or a cell-permeable fragment thereof, optionally selected from a monovalent, bivalent, or multivalent single-chain variable fragment (scFv), or diabody; or a humanized, chimeric, or variant thereof; (b) a nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof; A composition comprising or consisting of:

2. (a) is (i) the combination of the CDRs of SEQ ID NO: 5 with the CDRs of SEQ ID NO: 1; (ii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:5 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:1; or (v) A humanized form of (iv) The composition of claim 1 comprising:

3. 3. The composition of claim 1 or 2, wherein (a) has the same or a different epitope specificity as monoclonal antibody 4H2.

4. The composition of any one of claims 1 to 3, wherein (a) is a recombinant antibody having the paratope of monoclonal antibody 4H2.

5. (a) (i) the combination of the CDRs of SEQ ID NO: 5 with the CDRs of SEQ ID NO: 1; (ii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:5 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:1; or (v) A humanized form of (iv) a binding protein comprising: (b) a nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof; A composition comprising:

6. 6. The composition of any one of claims 1 to 5, wherein (a) is bispecific.

7. The composition of claim 6 , wherein (a) targets a cell type of interest.

8. 8. The composition of claim 1, wherein (a) and (b) are non-covalently linked or associated.

9. 9. The composition of claim 1, wherein (a) and (b) form a complex.

10. 10. The composition of any one of claims 1 to 9, wherein (b) comprises DNA, RNA, PNA or other modified nucleic acid, or nucleic acid analog, or a combination thereof.

11. 11. The composition of claim 1, wherein (b) comprises mRNA.

12. 12. The composition of any one of claims 1 to 11, wherein (b) comprises a vector.

13. The composition of claim 12 , wherein the vector comprises a nucleic acid sequence encoding a polypeptide of interest operably linked to an expression control sequence.

14. The composition of claim 13 , wherein the vector is a plasmid.

15. 15. The composition of any one of claims 1 to 14, wherein (b) comprises a nucleic acid encoding a Cas endonuclease, a gRNA, or a combination thereof.

16. 16. The composition of any one of claims 1 to 15, wherein (b) comprises a nucleic acid encoding a chimeric antigen receptor polypeptide.

17. 17. The composition of claim 1, wherein (b) comprises a functional nucleic acid.

18. 18. The composition of any one of claims 1 to 17, wherein (b) comprises a nucleic acid encoding a functional nucleic acid.

19. 19. The composition of claim 17 or 18, wherein the functional nucleic acid is an antisense molecule, siRNA, miRNA, aptamer, ribozyme, RNAi, or external guide sequence.

20. 20. The composition of any one of claims 1 to 19, wherein (b) comprises a plurality of single nucleic acid molecules.

21. 20. The composition of any one of claims 1 to 19, wherein (b) comprises a plurality of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid molecules.

22. 22. The composition of any one of claims 1 to 21, wherein (b) comprises or consists of a nucleic acid molecule between about 1 nucleobase and 25,000 nucleobases in length.

23. 23. The composition of any one of claims 1 to 22, wherein (b) comprises or consists of a single-stranded nucleic acid, a double-stranded nucleic acid, or a combination thereof.

24. 24. The composition of any one of claims 1 to 23, further comprising carrier DNA.

25. 25. The composition of claim 24, wherein the carrier DNA is non-coding DNA.

26. 26. The composition of claim 24 or 25, wherein (b) is composed of RNA.

27. 27. A pharmaceutical composition comprising the composition of any one of claims 1 to 26 and a pharmaceutically acceptable excipient.

28. 28. The composition of claim 27, further comprising polymeric nanoparticles in which the complex of (a) and (b) is encapsulated.

29. 30. The composition of claim 28, wherein a targeting moiety, a cell-penetrating peptide, or a combination thereof is directly or indirectly associated, linked, conjugated, or otherwise attached to the nanoparticle.

30. 30. A method of delivering a nucleic acid cargo to a cell, comprising contacting said cell with an effective amount of a composition of any one of claims 1 to 29.

31. 31. The method of claim 30, wherein the contacting step is performed ex vivo.

32. 32. The method of claim 31, wherein the cell is a hematopoietic stem cell or a T cell.

33. 33. The method of any one of claims 30 to 32, further comprising administering the cells to a subject in need thereof.

34. 34. The method of claim 33, wherein the cells are administered to the subject in an amount effective to treat one or more symptoms of a disease or disorder.

35. 31. The method of claim 30, wherein the contacting step is performed in vivo after administration to a subject in need thereof.

36. 36. The method of any one of claims 33 to 35, wherein the subject has a disease or disorder.

37. 37. The method of claim 36, wherein the disease or disorder is a genetic disorder, cancer, or an infection or infectious disease.

38. 38. The method of claim 36 or 37, wherein (b) is delivered intracellularly to the subject in an amount effective to reduce one or more symptoms of the disease or disorder in the subject.

39. 30. A method of making the composition of any one of claims 1 to 29, comprising the step of incubating and / or mixing (a) and (b) at a suitable temperature for a length of time effective to form a complex of (a) and (b) prior to contacting with cells.

40. 30. A method of making the composition of any one of claims 1 to 29, comprising incubating and / or mixing (a) and (b) for about 1 minute to about 30 minutes, about 10 minutes to about 20 minutes, or about 15 minutes, optionally at room temperature or 37 degrees Celsius.

41. 10. A composition or method according to any one of the preceding claims, wherein the ratio of (a):(b) is between 1:3 and 5:1, optionally the ratio is 1:1 or 3:

1.

42. 1. A method of increasing immune receptor activation in cells of a subject in need thereof, comprising the step of: (a) administering an effective amount of an intact 4H2 monoclonal antibody, or optionally a cell-permeable fragment thereof selected from a monovalent, bivalent, or multivalent single-chain variable fragment (scFv), or a diabody; or a humanized, chimeric, or variant thereof; optionally, wherein the immune receptor is cGAS or another pattern recognition receptor (PRR), optionally a toll-like receptor, optionally TLR7.

43. 43. The method of claim 42, wherein the subject has cancer or an infectious disease.

44. 44. The method of claim 42 or 43, wherein the subject does not have cancer.

45. 45. The method of any one of claims 42 to 44, wherein the subject has a wound in need of healing.

46. 45. The method of any one of claims 42 to 44, wherein the subject has an immune dysregulation, and optionally the immune dysregulation is multiple sclerosis.

47. 47. The method of any one of claims 42 to 46, further comprising the step of: (b) administering an additional agent to the subject.

48. 48. The method of claim 47, wherein (b) is selected from a nucleic acid cargo that induces, augments, or enhances an immune response, an immunostimulatory nucleic acid, one or more vaccine components, an immune checkpoint modulator, and combinations thereof.

49. 1. A method for treating cancer or an infectious disease, comprising administering to a subject in need thereof: (a) an intact 4H2 monoclonal antibody, or a cell-permeable fragment thereof, optionally selected from a monovalent, bivalent, or multivalent single-chain variable fragment (scFv), or diabody; or a humanized, chimeric, or variant thereof; (b) an immune checkpoint modulator that induces, augments, or enhances an immune response; and The method comprises administering an effective amount of a combination of:

50. 50. The method of any one of claims 48-49, wherein said immune checkpoint modulator induces an immune response against said cancer or infectious disease.

51. 51. The method of any one of claims 48-50, wherein the immune checkpoint modulator reduces an immune inhibitory pathway.

52. 52. The method of claim 51, wherein the immune inhibitory pathway is the PD-1 pathway.

53. 53. The method of any one of claims 48-52, wherein the immune checkpoint modulator is selected from the group consisting of a PD-1 antagonist, a PD-1 ligand antagonist, and a CTLA4 antagonist.

54. 51. The method of any one of claims 48-50, wherein the immune checkpoint modulator increases an immune activation pathway.

55. 55. The method of any one of claims 48 to 54, wherein the immune checkpoint modulator is an antibody.

56. 55. The method of any one of claims 48 to 54, wherein the immune checkpoint modulator is a CAR-T cell.

57. 55. The method of any one of claims 48-54, wherein the immune checkpoint modulator is an oncolytic virus.

58. 1. A method for treating cancer or an infectious disease, comprising administering to a subject in need thereof: (a) an intact 4H2 monoclonal antibody, or a cell-permeable fragment thereof, optionally selected from a monovalent, bivalent, or multivalent single-chain variable fragment (scFv), or diabody; or a humanized, chimeric, or variant thereof; (b) immunostimulatory nucleic acids; The method comprises administering an effective amount of a combination of:

59. 59. The method of claim 48 or 58, wherein the immunostimulatory nucleic acid is a STING agonist.

60. 1. A method of vaccinating a subject, comprising administering to said subject: (a) an intact 4H2 monoclonal antibody, or a cell-permeable fragment thereof, optionally selected from a monovalent, bivalent, or multivalent single-chain variable fragment (scFv), or diabody; or a humanized, chimeric, or variant thereof; (b) one or more vaccine components; The method of claim 1, further comprising administering

61. 61. The method of claim 48 or 60, wherein the one or more vaccine components comprise an antigen, a nucleic acid encoding an antigen, an adjuvant, a nucleic acid encoding an adjuvant, or a combination thereof.

62. 62. The method of claim 61, wherein the antigen is derived from a bacterium or a virus.

63. 63. The method of any one of claims 48-62, wherein administering a combination of (a) and (b) results in a greater than additive reduction in one or more symptoms of cancer or infectious disease compared to the reduction in one or more symptoms of cancer or infectious disease achieved by administering (a) or (b) in the absence of the other.

64. 64. The method of any one of claims 48 to 63, wherein (a) is administered to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before (b) is administered to the subject, or any combination thereof.

65. 64. The method of any one of claims 48 to 63, wherein (b) is administered to the subject 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, or 24 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before (a) is administered to the subject, or any combination thereof.

66. 66. The method of any one of claims 42-65, further comprising administering to the subject one or more additional active agents selected from the group consisting of chemotherapeutic agents, anti-infective agents, and combinations thereof.

67. 67. The method of any one of claims 42 to 66, further comprising surgery or radiation therapy.

68. 68. The method of any one of claims 42 to 67, comprising a nucleic acid cargo.

69. 69. The method of claim 68, wherein (a) and the nucleic acid cargo form a complex.

70. 70. The method of claim 68 or 69, wherein (b) is the nucleic acid cargo, and optionally the nucleic acid cargo is composed of DNA, RNA, PNA, PMO, or other modified nucleic acid, or nucleic acid analog, or a combination thereof.

71. 70. The method of claim 68 or 69, wherein (b) is not a nucleic acid cargo.

72. (a) is (i) the combination of the CDRs of SEQ ID NO: 5 with the CDRs of SEQ ID NO: 1; (ii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:5 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:1; or (v) A humanized form of (iv) 72. The method of any one of claims 42 to 71, comprising:

73. 73. The method of any one of claims 42 to 72, wherein (a) comprises the same or a different epitope specificity as monoclonal antibody 4H2.

74. 74. The method of any one of claims 42 to 73, wherein (a) is a recombinant antibody having the paratope of monoclonal antibody 4H2.

75. (a) is (i) the combination of the CDRs of SEQ ID NO: 5 with the CDRs of SEQ ID NO: 1; (ii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:5 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:1; or (v) A humanized form of (iv) 75. The method of any one of claims 42 to 74, comprising:

76. 76. The method of any one of claims 42 to 75, wherein (a) is bispecific.

77. 77. The method of claim 76, wherein (a) targets a cell type of interest.

78. 78. A pharmaceutical composition comprising (a) and (b) according to any one of claims 48 to 77 and a pharmaceutically acceptable excipient.

79. 79. The pharmaceutical composition of claim 78, comprising a nucleic acid cargo.

80. 80. The pharmaceutical composition of claim 79, wherein (b) is the nucleic acid cargo.

81. 80. The pharmaceutical composition of claim 79, wherein (b) is not the nucleic acid cargo.

82. 82. The pharmaceutical composition of any one of claims 79 to 81, wherein (a) and the nucleic acid cargo form a complex.

83. 83. The pharmaceutical composition of claim 82, further comprising polymeric nanoparticles encapsulating (a), (b), the nucleic acid cargo, or a combination thereof.

84. 84. The pharmaceutical composition of any one of claims 78 to 83, wherein a targeting moiety, a cell-penetrating peptide, or a combination thereof is directly or indirectly associated, linked, fused, conjugated, or otherwise bound to (a), (b), the nucleic acid cargo, the nanoparticle, or a combination thereof.

85. (a) (i) the combination of the CDRs of SEQ ID NO: 5 with the CDRs of SEQ ID NO: 1; (ii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 2, 3, and 4, respectively; (iii) a humanized form of (ai) or (aii); (iv) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:5 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:1; or (v) A humanized form of (iv) a bispecific binding protein comprising: a binding domain that binds to an immune cell marker; A composition comprising:

86. 86. The composition of claim 85, wherein the immune cell marker is CD5.

87. the CD5-binding domain is (vi) a combination of the CDRs of SEQ ID NO: 24 and the CDRs of SEQ ID NO: 23; (vii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively; (viii) a humanized form of (iv) or (iii); (ix) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 24 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 23; or (x) Humanized forms of (ix) 87. The composition of claim 86, comprising:

88. (b) a nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof.

89. 90. A method of increasing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of claims 85 to 88.

90. 90. The method of claim 89, wherein the subject has cancer or an infectious disease.

91. (i) the combination of the CDRs of SEQ ID NO: 24 with the CDRs of SEQ ID NO: 23; (ii) a first, second, and third heavy chain CDR comprising the amino acid sequences of SEQ ID NOs: 25, 26, and 27, respectively, in combination with a first, second, and third light chain CDR comprising the amino acid sequences of SEQ ID NOs: 28, 29, and 30, respectively; (iii) a humanized form of (i) or (ii); (iv) a combination of a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 24 and a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 23; or (v) A humanized form of (iv) A binding protein, optionally an antibody, comprising:

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