Compositions and methods using cell penetrating antibodies

Abstract

Compositions for delivering nucleic acid cargo into cells and methods of using the same are provided. Compositions generally include (a) a 4H2 monoclonal antibody or an antigen-binding, cell-penetrating fragment thereof; a monovalent, divalent or multivalent single chain variable fragment (scFv); or a bispecific antibody fragment; 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 activation of immune receptors, such as cGAS and TLR7, in cells of a subject. The methods generally include administering to the subject an effective amount of a 4H2 antibody. The subject may be healthy and may also have a disease or condition, such as cancer or infection.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 68 / 379,121, filed October 11, 2022, and U.S. Provisional Application No. 68 / 379,123, filed October 11, 2022, the contents of each of which are expressly incorporated herein by reference.

[0003] Reference sequence list

[0004] The sequence list was submitted as a text file named “YU8475PCT.xml”, created on October 11, 2023, and is 27,711 bytes in size. It is hereby incorporated by reference in 37C.FR§1.52(e)(5). Technical Field

[0005] This invention relates generally to the field of intracellular delivery of nucleic acids, with applications including but not limited to in vitro, ex vivo, and in vivo gene therapy and gene editing and / or enhancing immune responses, particularly through the regulation of immune receptors, with applications including but not limited to treating cancer and infections and improving vaccination. Background Technology

[0006] Gene therapy

[0007] Gene therapy encompasses a range of applications, from gene replacement and knockout for genetic or acquired diseases such as cancer to vaccination. Viral vectors and synthetic liposomes have become the preferred vectors for many applications today, but both have limitations and risks, including manufacturing complexity, limited packaging capabilities, and unfavorable immunological properties, which limit the application of gene therapy and its potential for preventative gene therapy (Seow and Wood, Mol Ther. 17(5):767-777 (2009)).

[0008] In vivo uptake and distribution of nucleotides in cells and tissues has been observed (Huang et al., FEBS Lett., 558(1-3): 69-73 (2004)). Moreover, for example, studies by Nyce et al. have shown that antisense oligodeoxynucleotides (ODNs) are taken up by lung cells after inhalation, bind to endogenous surfactant (lipids produced by lung cells), and are taken up by lung cells without the need for additional carrier lipids (Nyce, et al., Nature, 385:721-725 (1997)), small nucleic acids are taken up by T24 bladder carcinoma tissue culture cells (Ma et al., Antisense Nucleic Acid Drug Dev., 8:415-426 (1998)), and there remains a need for improved nucleic acid transfection technology, particularly for in vivo applications. AAV9, discovered in 2003, remains a commonly used viral vector (Robbins, “Gene therapy pioneer says field is behind the curve — delivery technology is embarrassing,” Stat, Nov. 2019).

[0009] Accordingly, it is an object of the present application to provide compositions that improve the delivery of nucleic acids to cells and methods of using the same.

[0010] Modulating immune responses

[0011] GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses by producing the second messenger cGAMP. In turn, cGAMP activates the adaptor protein STING (Chen et al., Nat Immunol (2016) 17(10): 1142-9. 10.1038 / ni.3558). The cGAS-STING pathway not only mediates protective immune defenses against infection by a variety of DNA-containing pathogens (e.g., microbial DNA), but also detects tumor-derived DNA and generates intrinsic anti-tumor immunity. The STING pathway and its role in immune regulation and cancer development have been reviewed in, for example, 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.

[0012] For example, in the tumor microenvironment, T cells, endothelial cells, and fibroblasts produce type I IFNs upon stimulation by STING agonists in vitro and in vivo (Corrales et al., Cell Rep (2015) 11(7):1018-30.10.1016 / j.celrep.2015.04.031). In contrast, most studies suggest that tumor cells can suppress activation of the STING pathway, possibly leading to immune evasion during carcinogenesis (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, there is evidence that activation of the STING pathway is associated with inducing spontaneous anti-tumor T cell responses that involve 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). In addition, the host STING pathway is required for dendritic cell (DC)-mediated effective 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, people have begun to explore the use of drugs to directly stimulate the STING pathway to treat cancer.

[0013] In addition to cancer, it has also been suggested to develop STING agonists for a variety of different therapeutic purposes, including as vaccine adjuvants and for chronic viral or bacterial infections.

[0014] As the range of clinical applications continues to expand, modified compositions and methods for modulating the cGAS-STING pathway and other immune response receptor signaling pathways are also increasingly popular.

[0015] Accordingly, it is another object of the present disclosure to provide improved compositions and methods of use thereof to enhance the activity of immune receptors such as cGAS and pattern recognition receptors (PPRs), including toll-like receptors (such as TLR7). SUMMARY

[0016] The present disclosure provides compositions and methods of use thereof to deliver nucleic acid cargo into cells. The compositions generally include (a) a 4H2 monoclonal antibody or cell-penetrating fragment thereof; a monovalent, bivalent, or multivalent single-chain variable fragment (scFv); or a bispecific antibody fragment; or a humanized version thereof or a variant thereof; and (b) a nucleic acid cargo including, 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 generally non-covalently linked to form a complex. Exemplary 4H2 antibodies and fragments and fusion proteins thereof include those having (i) the CDRs of SEQ ID NO: 1 (optionally SEQ ID NOs: 2-4) in combination with the CDRs of SEQ ID NO: 5 (optionally SEQ ID NOs: 6-8); (ii) a combination of the first, second, and third heavy chain CDRs selected from SEQ ID NO: 1 (optionally SEQ ID NOs: 2-4) and the first, second, and third light chain CDRs selected from SEQ ID NO: 5 (optionally SEQ ID NOs: 5-8); (iii) a humanized version of (i) or (ii); (iv) a combination of a heavy chain having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5 and a light chain having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1; (v) a humanized version of (iv).

[0017] In certain embodiments, the antibody or fragment or fusion protein can be bispecific, e.g., can include a binding sequence that targets a cell type, tissue, or organ.

[0018] The nucleic acid cargo can consist of DNA, RNA, modified nucleic acids including but not limited to PNA, or a combination thereof. 4H2 binds to guanosine. Accordingly, the cargo generally includes one or more guanine nucleobases, preferably one or more guanine nucleosides. The nucleic acid cargo is generally a functional cargo, such as a functional nucleic acid (e.g., an inhibitory RNA), an mRNA, or a vector, e.g., an expression vector. The nucleic acid cargo, including the vector, can include a nucleic acid sequence encoding a polypeptide of interest operably linked to an expression control sequence. The vector can be, e.g., a plasmid or the like. The cargo is generally not randomly sheared or fragmented genomic DNA.

[0019] In certain 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.

[0020] The cargo can comprise or consist of a plurality of individual nucleic acid molecules or a plurality of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid molecules. In certain embodiments, the nucleic acid molecules of the cargo comprise or consist of nucleic acid molecules having a length between about 1 and about 25,000 nucleobases. The cargo can be a single-stranded nucleic acid, a double-stranded nucleic acid, or a combination thereof.

[0021] Also provided are pharmaceutical compositions comprising the complex and a pharmaceutically acceptable excipient. In certain embodiments, the complex is encapsulated in a polymeric nanoparticle. The targeting moiety, the cell-penetrating peptide, or a combination thereof can be associated with, linked to, conjugated to, or otherwise directly or indirectly attached to the nanoparticle.

[0022] Further, methods of delivering a nucleic acid cargo into a cell by contacting the cell with an effective amount of the complex, either alone or encapsulated in a nanoparticle, are also provided. The contacting can occur in vitro, ex vivo, or in vivo. In certain embodiments, an effective amount of the ex vivo treated cell is administered to a subject in need thereof, e.g., to treat one or more symptoms of a disease or disorder with the effective amount.

[0023] In some embodiments, the contacting occurs in vivo after administration to a subject in need thereof. The subject can have a disease or disorder, such as a genetic disease or cancer. An effective amount of the composition can be administered to the subject, e.g., by injection or infusion, to alleviate one or more symptoms of the disease or disorder in the subject.

[0024] Also provided are uses of the compositions and methods, including but not limited to gene therapy and CAR T cell manufacturing / formation / treatment.

[0025] Also provided are compositions and methods of enhancing cGAS and / or other immune receptor (such as a pattern recognition receptor, such as TLR7) activation in a cell of a subject in need thereof. These methods generally comprise administering to the subject an effective amount of a 4H2 antibody. Exemplary 4H2 antibody formats include, but are not limited to, intact monoclonal antibodies and cell-penetrating fragments thereof, such as monovalent, bivalent, or multivalent single-chain variable fragments (scFv), diabody fragments, and the like. The antibody can be a humanized version thereof, a chimeric version thereof, or a variant thereof.

[0026] Exemplary 4H2 antibodies, and fragments and fusion proteins thereof, include, for example, those having (i) the CDRs of SEQ ID NO: 1 (alternatively SEQ ID NOs: 2-4) in combination with the CDRs of SEQ ID NO: 5 (alternatively SEQ ID NOs: 6-8); (ii) a combination of the first, second, and third heavy chain CDRs selected from SEQ ID NO: 1 (alternatively SEQ ID NOs: 2-4) and the first, second, and third light chain CDRs selected from SEQ ID NO: 5 (alternatively SEQ ID NOs: 5-8); (iii) a humanized version of (i) or (ii); (iv) a combination of a heavy chain having an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5 and a light chain having an amino acid sequence that is at least 85% identical to SEQ ID NO: 1; (v) a humanized version of (iv).

[0027] In certain embodiments, the subject has a cancer or an infection. In certain embodiments, the subject does not have a cancer. Accordingly, methods of treating a cancer and an infection in a subject are also provided. In certain embodiments, the subject is a healthy subject.

[0028] In certain embodiments, the compositions and / or methods comprise administering to the subject an additional agent. In 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, increases, or enhances an immune response, and combinations thereof.

[0029] In one particular embodiment, the method of treating a cancer or an infection comprises administering to a subject in need thereof an effective amount of a 4H2 antibody in combination with an immune checkpoint modulator that induces, increases, or enhances an immune response.

[0030] The immune checkpoint modulator is generally capable of inducing an immune response against a cancer or an infection. For example, the immune checkpoint modulator can reduce an immune inhibitory pathway, such as the PD-1 pathway. Accordingly, the modulator can be a PD-1 antagonist, a PD-1 ligand antagonist, or a CTLA4 antagonist. In certain embodiments, the immune checkpoint modulator increases an immune activation pathway. The immune checkpoint modulator can be a small molecule, an antibody, a CAR-T cell, or an oncolytic virus.

[0031] In another particular embodiment, the method of treating a cancer or an infection comprises administering to a subject in need thereof an effective amount of a 4H2 monoclonal antibody in combination with an immunostimulatory nucleic acid. In certain embodiments, the immunostimulatory nucleic acid is a STING agonist.

[0032] In another specific embodiment, the method of vaccinating a subject comprises administering to the subject a 4H2 antibody and one or more vaccine components. For example, the one or more vaccine components can comprise 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.

[0033] In certain embodiments, administration of a combination of 4H2 and an additional drug to a subject results in an increase in an immune response and / or a reduction in one or more symptoms of a cancer or an infection compared to administration of either alone without the other.

[0034] In certain embodiments, a 4H2 antibody is administered to a subject 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof, prior to administration of the additional drug. In other embodiments, the additional drug is administered to a subject 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours, 1, 2, 3, 4, 5, 6, or 7 days, 1, 2, 3, or 4 weeks, or any combination thereof, prior to administration of the 4H2 antibody.

[0035] Any of the methods can further use a therapeutic agent or intervention, such as a chemotherapy drug, an anti-infective drug, surgery, radiation, or a combination thereof.

[0036] A nucleic acid cargo or nucleotide, nucleoside, or nucleobase can increase cellular penetration and / or activation of cGAS and / or another pattern recognition receptor, such as TLR7, by a 4H2 antibody. Accordingly, any of the compositions and methods disclosed can further comprise a nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo. In certain embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo is the additional drug. In certain embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo is not the additional drug (i.e., is further combined with the additional drug). In preferred embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo forms a complex with the 4H2 antibody. In preferred embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo contains guanine or guanosine and forms a complex with the 4H2 antibody. The nucleic acid cargo can consist of DNA, RNA, PNA, phosphorodiamidate morpholino oligomers (PMO), or other modified nucleic acids, nucleic acid analogs, or modified nucleotide, nucleoside, or nucleobase analogs, or a combination thereof. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figures 1A-1C 4H2 is shown to be a cell penetrating anti-GUO autoantibody sensitive to DP. Figure 1Ais an image of lysates of Cal12T cells treated with 0-1 mg / mL 4H2 for 24 hours, probed with an actin primary antibody, detected with an anti-mouse secondary antibody, and analyzed by western blot. 4H2 HC and LC run at their expected molecular weights (MW), indicating that the antibody was not significantly degraded after 24 hours of penetration into the cells. Figure 1B is an image showing total ERK1 / 2 and pERK1 / 2 western blot analysis of Cal12T cell lysates treated with control media, IgG control, or 4H2 for 24 hours. IgG control had no effect on total ERK1 / 2 and pERK1 / 2, while 4H2 decreased pERK1 / 2 but not total ERK1 / 2. Figure 1C is a dot plot showing quantification of 4H2 fluorescence in Cal12T cells with and without DP treatment (cell penetration) by ImageJ.

[0038] Figures 2A-2D shows that 4H2 penetrates glioma cells in a GUO-responsive manner and crosses a Transwell model of the BBB (blood brain barrier). Figures 2A-2C is a graph showing the effect of adding ADE or GUO to cell culture media on the efficiency of cell penetration by GSCs, as assessed by quantification of DX1 or 4H2 fluorescence signal by ImageJ. ADE enhanced the penetration of DX1 ( Figure 2A ), but had no effect on 4H2 ( Figure 2B ). GUO significantly enhanced the cell penetration of 4H2 ( Figure 2C ). Figure 2D is a graph showing the results of a BBB Transwell model using hCMEC / D3 brain microvascular endothelial cells (BECs) and normal human astrocytes (NHAs) to assess the transport of 4H2 across the barrier from the apical chamber to the basolateral chamber. The results show that 4H2 can penetrate the barrier, and that the nucleoside transport inhibitor DP inhibits the transport of 4H2.

[0039] Figures 3A-3B shows that 4H2 localizes to orthotopic brain tumors and extends survival time in a GBM model. Figure 3A is a Kaplan-Meier survival plot of GSC-derived orthotopic GBM tumor mice treated with IgG control (N=4) or 4H2 (N=5). 4H2 increased median survival of mice by 66% compared to mice treated with IgG control (**P<0.01, log-rank test), and the survival rate of mice treated with 4H2 was 40% at the end of the study, while the survival rate of mice treated with IgG control was 0% at the end of the study. Figure 3BKaplan-Meier survival plot of GL261-derived orthotopic GBM tumor mice treated with IgG control (6), 4H2 (6), anti-PD1 (6), anti-PD1 + IgG control (7), or anti-PD1 + 4H2 (7). 4H2 increased median survival by 32% compared to IgG control (*P=0.03, log-rank test); 4H2 in combination with anti-PD1 increased median survival by 50% compared to anti-PD1 + IgG control (*P=0.02, log-rank test). 4H2 alone or 4H2 + anti-PD1 resulted in 33% and 29% survival at the end of the study, respectively, while all other groups had 0% survival.

[0040] Figure 4A Histogram quantifying TUNEL staining with ImageJ showing a 4.5 ± 0.6-fold relative increase in TUNEL signal in mice treated with 4H2 compared to IgG control (**P<0.01). Figure 4B Histogram showing relative CD8 cell counts per high power field (HPF) based on anti-CD8 immunostaining of GBM brain tumor sections after mice were treated with IgG control or 4H2. 4H2 increased CD8 content in the tumor by about 53%, with a relative count of 1.53 ± 0.15 for 4H2-treated mice compared to 1.00 ± 0.04 for IgG control-treated mice (*P<0.03). These data demonstrate 4H2-mediated stimulation of T cell infiltration of GBM tumors. Figure 4C and 4D show that 4H2 did not improve survival in an immunodeficient orthotopic GBM model. Figure 4C and Figure 4D show that 4H2 did not improve survival in an immunodeficient orthotopic GBM model. Kaplan-Meier survival plots showing athymic nude mice with PPQ orthotopic GBM brain tumors treated with IgG control (N=4) or 4H2 (N=4) once per week ( Figure 4C ) or twice per week ( Figure 4D ). 4H2 did not significantly affect median survival compared to IgG control in this immunodeficient model, demonstrating the importance of a functional immune system for 4H2 to affect survival.

[0041] Figures 5A-5DThis is a Western blot image showing the binding of 4H2 to cGAS. Antibody content and binding proteins were isolated from IgG controls or 4H2-treated glioma stem cells (GSCs) using protein G beads. Western blotting of input and protein G pulldown was performed to detect G proteins Ras and cGAS. No binding of IgG controls or 4H2 to Ras was observed. Figure 5A However, 4H2 showed significant binding to cGAS, with the binding signal exceeding the background signal detected in the IgG control. Figure 5B Purified cGAS±nucleic acid was cultured with IgG controls or 4H2, followed by protein G pull-down of antibodies and binding proteins. The presence of nucleic acid reduced the binding of 4H2 to cGAS, but did not affect the non-specific binding of IgG controls to cGAS. Figure 5C Western blot analysis of anti-IgG confirmed that the levels of IgG control and 4H2 in the pull-down sample were equal. Figure 5D ). Figure 5E and 5G It is a Western blot image. Figure 5F The bar chart shows the interaction of 4H2 with cGAS in a nucleic acid-dependent manner. Purified recombinant cGAS was incubated with control IgG or a 4H2+ / - nuclease (benzonase). Antibody and binding proteins were then separated on Protein G beads, cGAS pulldown was visualized by Western blotting, and quantified using ImageJ. In the absence of nuclease, the interaction between 4H2 and cGAS resulted in approximately a 6-fold increase in cGAS pulldown compared to the IgG control group (***P<0.001), while the addition of nuclease eliminated this interaction.

[0042] Figures 6A-6D The results show that 4H2 can enhance the activity of cGAS. Figure 6A This is a line graph showing the dose-dependent increase in cGAS activity caused by 4H2. cGAS activity was detected by measuring the relative production of cGAMP from ATP and GTP in the presence of IgG control or 4H2. Figure 6B These are immunoblot images showing that 4H2 induces nuclear translocation of NF-κB in GSCs. Cytoplasmic and nuclear contents of GSCs treated with IgG control or 4H2 were separated and analyzed by Western blot with NF-κB and Lamin B1 as loading controls. GSCs transfected with control group or cGAS siRNA were treated with IgG control group or 4H2. Figure 6C These are western blot images confirming successful cGAS knockout. Figure 6Dis a line graph showing the results of colony formation assays demonstrating the cGAS-dependent toxicity of 4H2 on GSCs. Figure 6E is a histogram showing 4H2 induced nuclear translocation of NF-κΒ. Cytoplasmic and nuclear contents of PPQ cells treated with IgG control or 4H2 were analyzed by western blot for NF-κΒ, with Lamin B1 as a loading control. Nuclear relative content of NF-κΒ was quantified using ImageJ. 4H2 increased the relative content of nuclear NF-κΒ by 2.2 ± 0.2 fold (*P < 0.05). Figure 6F is a histogram showing the survival rate determined by colony formation assays in Cal12T lung cancer cells transfected with control or cGAS siRNA and treated with IgG control or 4H2, demonstrating the cGAS-dependent toxicity of 4H2. (*P < 0.05).

[0043] Figures 7A-7B shows that 4H2 binds to DNA and RNA. Figure 7A is an image showing the binding of 4H2 to circular and linearized pcDNA3 plasmid DNA assessed by 1% agarose EMSA. 4H2 but not IgG control caused a shift consistent with binding to both forms of DNA. Figure 7B is an image showing the binding of 4H2 to total DNA and mRNA assessed by 1% agarose EMSA. 4H2 but not IgG control caused a shift consistent with binding to both forms of RNA.

[0044] Figures 8A-8B is a histogram 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 measured 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 measured 24 hours later ( Figure 8B ).

[0045] Figures 9A-9B shows that 4H2 mediates local gene therapy in the central nervous system (CNS). 4H2 / Cre mRNA was injected into the brain of an Ai9 Cre reporter mouse and Cre recombinase activity was assessed by RFP fluorescence 24 hours later. RFP signal was observed in the local area of the injection track ( Figure 9A ). Ai9 Cre reporter mice were treated with intraocular injection of 4H2 / Cre mRNA and RFP signal was assessed 24 hours later. RFP signal observed in the retina demonstrates 4H2-mediated retinal gene therapy ( Figure 9B ).

[0046] Figures 10A-10B is an image showing 4H2 delivery of mRNA in vivo. Nude mice bearing H358 flank tumors received a single intratumoral injection of DX1 or 4H2 mixed with Luc mRNA (w / w = 3). Expression of Luc was assessed by IVIS after 6, 24 and 72 hours. 4H2 / Luc mRNA successfully mediated expression of Luc, while little signal was detected in tumors injected with DX1 / Luc mRNA. Figure 10A ) C57 / BL6 mice were injected intramuscularly with 4H2 / Luc mRNA (left quadriceps w / w = 3, right quadriceps w / w = 1). Expression of Luc was assessed with IVIS after 6 and 24 hours. 4H2 / Luc mRNA successfully mediated expression of Luc Figure 10B

[0047] Figures 11A-11B is a series of representative IVIS images Figure 11A ) and corresponding bar graphs Figure 11B showing luminescence in a luciferase-expressing HEI 193 xenograph model in untreated mice and mice treated with 4H2 alone, 4H2 + NF2 DNA, or 4H2 + NF2 mRNA.

[0048] Figure 12A is a schematic of the design of the 4H2-CD5 bispecific antibody. Figure 12B and 12C is a series of representative FACS plots Figure 12B ) and corresponding bar graphs Figure 12C showing expression in DeRed tumor cells isolated from an Ai9 mouse model bearing MC38 tumors.

[0049] Figure 13A and 13B are western blot images and corresponding graphs (determined by ImageJ) of cell lysates of glioma stem-like cells (GSCs) treated with IgG control or 4H2 and assayed for TLR7. Figure 13C is an image of a western blot. Protein G beads pulled down antibody and bound protein from GSC cleavage fluid treated with IgG control or 4H2 and then assayed for TLR7 immunoblot. Blots represent two independent experiments. DETAILED DESCRIPTION

[0050] I. DEFINITIONS

[0051] ​The term "single-chain Fv" or "scFv" as used herein 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, connected by a linker, which enables scFv to form the desired structure for antigen binding (i.e., the VH and VL in a single polypeptide chain associate with each other to form a Fv). The VL and VH regions can be derived from a parent antibody or can be synthesized de novo by chemical synthesis or recombinant synthesis.

[0052] The term "variable region" as used herein is designed to distinguish such domains of immunoglobulins from domains that are widely shared by antibodies (such as the Fc domain of an antibody). The variable region includes "hypervariable regions," whose residues are responsible for antigen binding. The hypervariable regions include amino acid residues derived from "complementarity determining regions" or "CDRs" (i.e., generally about residues 24-34 (LI), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and about residues 27-35 (HI), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues derived from "hypervariable loops" (i.e., residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and residues 26-32 (HI), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917).

[0053] The term "framework region" or "FR" residues, as used herein, refer to the variable region residues other than the hypervariable region residues as defined herein.

[0054] The term "antibody" as used herein refers to a natural or synthetic antibody that binds to a target antigen. The term includes polyclonal antibodies and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes binding proteins, fragments, polymers of these immunoglobulin molecules, and human or humanized immunoglobulin molecules that bind to a target antigen.

[0055] The term "cell-penetrating antibody" as used herein refers to an immunoglobulin, a fragment thereof, a variant thereof, or a fusion protein based thereon that can be transported into the cytoplasm of a living mammalian cell. The term "cell-penetrating anti-guanosine antibody" as used herein 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 certain embodiments, the antibody is transported into the cytoplasm of a cell without the aid of a carrier or conjugate. In other embodiments, the antibody is conjugated to a cell-penetrating moiety, such as a cell-penetrating peptide.

[0056] In addition to intact immunoglobulin molecules, the term "antibody" includes fragments of immunoglobulin molecules, binding proteins, and polymers, chimeric antibodies containing sequences derived from more than one species, class, or subclass of immunoglobulin, e.g., human antibodies or humanized antibodies, and recombinant proteins containing at least the specificity of an immunoglobulin that specifically binds to DNA. The intended activity of an antibody can be detected using the in vitro assay methods described herein or similar methods, and its therapeutic activity in vivo can then be detected according to known clinical assay methods.

[0057] The term "variant" as used herein refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications and retains essential properties. A typical variant of a polypeptide differs in amino acid sequence by one or more amino acids. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall. Variants of a polypeptide and the reference polypeptide can differ in amino acid sequence by the use of conservatively substituted amino acids. A variant of a polypeptide can be a naturally occurring variant, such as an allelic variant, or a variant that is not known to naturally occur.

[0058] Modifications and alterations of a polypeptide structure of the present disclosure can be made and still obtain a molecule with similar characteristics to the polypeptide (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Since the primary sequence of a polypeptide determines its interactive capability and properties, certain amino acid sequences can be replaced in a polypeptide sequence and still obtain a polypeptide with similar characteristics.

[0059] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a peptide is generally understood by those skilled in the art. It is accepted that certain amino acids can be substituted for other amino acids in certain contexts while preserving the biological activity of the peptide. Each amino acid has been assigned a hydropathic index: 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

[0060] It is generally believed that the relative hydropathicity of the amino acids determines 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 that an amino acid can be substituted for another amino acid with a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, substitution of amino acids with hydropathic indices of ±2 is preferred, ±1 is particularly preferred, and ±0.5 is even more particularly preferred.

[0061] Based on the hydropathicity of the amino acids, particularly where the resulting biologically functional equivalent polypeptide or peptide is used in an immunological context, substitution of like amino acids can be made. The following are the hydropathic values assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+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 with a similar hydropathic value and still obtain a biologically equivalent, particularly an immunologically equivalent, polypeptide. In such changes, substitution of amino acids with hydropathic values of ±2 is preferred, ±1 is particularly preferred, and ±0.5 is even more particularly preferred.

[0062] As described above, amino acid substitutions are typically based on the relative similarity of the side chains of amino acids, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the above-described characteristics into account, including (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu: Leu, Val), (Leu: He, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: He, Leu). Accordingly, embodiments of the present disclosure contemplate functional or biologically equivalent forms of the polypeptides described above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide of interest.

[0063] The term "percent (%) sequence identity" as used herein refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in the reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved using various methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Suitable parameters for measuring the alignment, including any algorithms used for alignment, and the measurement of percent sequence identity, can be determined by known methods.

[0064] The term "specifically binds" as used herein means that an antibody binds to its cognate antigen (e.g., guanosine) and does not bind significantly to other antigens. In this context, for an antibody to specifically bind to a target, the antibody must be selected for its specificity for the target. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to 5 mol -1 (eg., 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 or greater).

[0065] The term "monoclonal antibody" or "MAb" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies

[0066] The term "subject" as used herein refers to any individual to whom treatment is administered. The subject can be a vertebrate, e.g., a mammal. Thus, the subject can be a human. The term does not indicate a particular age or sex.

[0067] The term "effective amount" as used herein refers to the amount of a composition used that is sufficient to effectuate an improvement in one or more signs or symptoms of a disease or condition. Such improvement is only required to be a reduction or change, not necessarily elimination. The precise amount will vary depending on factors such as subject-related variables (e.g., age, immune system health, etc.), the disease or condition being treated, and the route of administration and pharmacokinetics of the agent administered.

[0068] The term "pharmaceutically acceptable" as used herein refers to a material that is biologically or otherwise, physiologically acceptable, i.e., the material can be administered to a subject, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0069] The term "carrier" or "excipient" as used herein refers to an organic or inorganic ingredient, natural or synthetic, in the formulation to which one or more active ingredients are combined. The choice of carrier or excipient depends primarily on the particular active ingredient with which the carrier or excipient is combined, as well as the particular form of administration of the pharmaceutical composition. As will be understood by those of skill in the art, the selection of a carrier or excipient is influenced by a number of factors, including, but not limited to, the particular active ingredient with which the carrier or excipient is combined, the particular form of administration of the pharmaceutical composition, and the biological compatibility of the carrier or excipient with the active ingredient.

[0070] The term "treatment" as used herein 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 includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term also includes palliative treatment, that is, treatment designed for the relief of symptoms rather than cure of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment directed to supplementing the effect of another specific therapy in the improvement of the associated disease, pathological condition, or disorder.

[0071] The term "targeting moiety" as used herein is a component that can direct a microparticle or molecule to a receptor site of a selected cell or tissue type, either as an attachment molecule, or for conjugating or attaching another molecule. "Directing" as used herein means preferential attachment of a molecule to a selected cell or tissue type. This can be used to direct cellular material, molecules, or drugs, as described below.

[0072] The term "inhibit" or "reduce" as used herein means to decrease an activity, reaction, condition, disease, or other biological parameter. This can include, but is not limited to, complete ablation of the activity, reaction, condition, or disease. This can also include, for example, a 10% decrease in the activity, reaction, condition, or disease, as compared to a natural level or a control level. Thus, the amount of reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of reduction in between, as compared to a natural level or a control level.

[0073] The term "fusion protein" as used herein refers to a polypeptide formed by linking two or more polypeptides together through a peptide bond formed between the amino terminus of one polypeptide and the carboxy terminus of another polypeptide. Fusion proteins can be formed by chemical coupling of the constituent polypeptides or can be expressed as a single polypeptide by a nucleic acid sequence encoding a single contiguous fusion protein. Single chain fusion proteins are fusion proteins having a single contiguous polypeptide backbone. Fusion proteins can be prepared using routine techniques in molecular biology, linking two genes in frame into a single nucleic acid sequence, which is then expressed in an appropriate host cell under conditions such that the fusion protein is produced.

[0074] Unless otherwise indicated herein, recitation of a numerical range using the term "about" is meant to describe values that are within + / - 10% of the value; in other embodiments, the range of values can be within + / - 5% of the value; in other embodiments, the range of values can be within + / - 2% of the value; in other embodiments, the range of values can be within + / - 1% of the value. The foregoing ranges are intended to be illustrative only and are not intended to further limit the scope of the application.

[0075] The term "about" is used to describe values that are within + / - 10% of the value; in other embodiments, the range of values can be within + / - 5% of the value; in other embodiments, the range of values can be within + / - 2% of the value; in other embodiments, the range of values can be within + / - 1% of the value. The foregoing ranges are intended to be illustrative only and are not intended to further limit the scope of the application.

[0076] Unless otherwise indicated or contradicted by context, all methods described herein can be performed in any suitable order. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments of the application and does not pose a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0077] The disclosed materials, compositions, and components can be used in, can be used in conjunction with, can be used in the preparation of, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds can not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a list of components is disclosed and a number of these components are discussed, each individual component and each combination and permutation of the components are specifically contemplated, even if not explicitly listed. As such, any combination of the disclosed materials and / or components, or any combination or permutation of individual steps of the disclosed methods, is specifically contemplated and should be considered disclosed. Thus, if there are a variety of components that can be added to a composition, and a number of these components are discussed, each individual component component, and each combination and permutation of the components, are specifically contemplated. Likewise, if there is a disclosure of a variety of steps in a process and a number of these steps are discussed, each individual step component, and each combination and permutation of the steps, are specifically contemplated. Likewise, if there is a disclosure of a variety of steps in a process and a number of these steps are discussed, each individual step component, and each combination and permutation of the steps, are specifically contemplated. In addition, any individual component or step of the disclosed methods and compositions can be used in any combination or permutation with any other individual component or step of the disclosed methods and compositions, even if such a combination or permutation is not explicitly disclosed.

[0078] These concepts apply to all aspects of the present application, including but not limited to steps in the methods of making and using the disclosed compositions. Thus, if additional steps can be performed, it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and each such combination is specifically contemplated and should be considered disclosed.

[0079] Unless otherwise stated, all methods described herein can be performed in any suitable order. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein, is intended to illuminate the application and is not intended to limit the scope of the application unless otherwise claimed. No language is intended to indicate that any unclaimed element is essential to the practice of the application.

[0080] II. Compositions

[0081] The present study found that the 4H2 antibody helps deliver nucleic acids across the plasma membrane into the cytoplasm. Accordingly, compositions and methods are provided for using 4H2 to enhance delivery of nucleic acid constructs. Typically, an effective amount of 4H2 antibody is contacted with a nucleic acid that needs to be delivered into a cell. Typically, the contact is for a time sufficient for the 4H2 and the nucleic acid cargo to form a non-covalent complex. The complex is contacted with a cell for a time sufficient for the nucleic acid cargo to be delivered into the cell. The nucleic acid cargo can accumulate in greater quantity, higher quality (e.g., more complete, more functional, etc.), or faster, or a combination thereof, compared to the nucleic acid cargo contacted with the cell in the absence of the antibody. The delivery system is typically non-viral due to the antibody as the delivery means.

[0082] A variety of cell-penetrating anti-DNA autoantibodies were isolated from a mouse model of systemic lupus erythematosus (SLE). While most of these antibodies penetrate the nucleus of living cells, anti-GUO autoantibody 4H2 is unique in its cytoplasmic localization. 4H2 binds to the same epitope on GUO as the binding site for G protein, which is consistent with reports of anti-GUO autoantibodies binding in human systemic lupus erythematosus (SLE) patient sera (Colburn et al., Journal of Rheumatology 30(5), 993-97 (2003)). In addition, 4H2 penetrates and reduces cAMP concentrations in cultured cells, which is consistent with interference with G protein signaling (Colburn & Green, Clin Chim Acta 370:9-16 (2006)). The results below show that cytoplasmic penetration by 4H2 is associated with nucleoside transport, 4H2 binds to and mediates transport of nucleic acids, binds to cGAS and enhances its activity, and produces cGAS-dependent toxicity to tumor cells. The results also show that 4H2 can cause activation of TLR7 (the cleaved form of TLR7 is active), as shown by 4H2 inducing TLR7. In addition, pulldown assays show that 4H2 binds to the cleaved form of TLR7. Accordingly, compositions and methods are also provided for modulating cGAS and other pattern recognition receptors, such as TLR7.

[0083] A. 4H2 antibody

[0084] While this document is generally referred to as “4H2,” “4H2 antibody,” or “4H2 antibodies,” it is understood that, unless otherwise stated (e.g., experimental examples), the phrases “4H2,” “4H2 antibody,” or “4H2 antibodies” disclosed herein include not only the whole immunoglobulin but also fragments and binding proteins, including antigen-binding fragments, variants, and fusion proteins such as scFv, di-scFv, tri-scFv, and other single-chain variable fragments, chimeric and humanized forms, and other cell-penetrating, nucleic acid-transporting molecules, and explicitly provided for the compositions and methods disclosed herein. Antibodies are also referred to herein as cell-penetrating proteins and binding proteins.

[0085] In a preferred embodiment, the 4H2 antibody can be transported into the cytoplasm of the cell without the aid of a carrier or conjugate.

[0086] Antibodies that can be used in compositions and methods include any class of whole immunoglobulins (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least an antigen-binding variable region of the antibody. The variable regions of different antibodies differ sequentially, which can be used to ensure the binding and specificity of each specific antibody to its specific antigen. However, the variability of antibody variable regions is generally not uniformly distributed. It is typically concentrated in three segments in the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frames (FRs). The variable regions of the natural heavy and light chains each include four FR regions, mostly in a β-sheet conformation, linked by three CDRs to form a loop connecting the β-sheet conformation, and in some cases, also forming part of the β-sheet conformation. The CDRs on each chain are tightly bound together through the FR regions and together with the CDRs on the other chain, constitute the antigen-binding site of the antibody. Therefore, antibodies typically contain at least the CDRs required to maintain guanosine binding.

[0087] 4H2 hybridomas have previously been generated from the MRLmpj / lpr lupus mouse model. 4H2 does not localize to lysosomes or endosomes, whereas with other delivery systems (such as TAT ​​peptides), the cargo molecule is often destroyed in lysosomes or endosomes. 4H2 is a cell-penetrating lupus antiguanosine antibody that reduces ERK and Akt phosphorylation in cells. It is toxic to cancer cells carrying a range of small GTPase K-Ras mutations, but shows no significant toxicity to cells carrying WT K-Ras. See published international applications WO 2015 / 134607 and WO 2017 / 218824, the entire contents of which are incorporated herein by reference.

[0088] 4H2 antibodies are typically monoclonal 4H2 or its variants, derivatives, fragments, fusions, or humanized forms that can bind to the same or different epitopes as 4H2.

[0089] 1. Antibody sequence

[0090] a.4H2 light chain variable region

[0091] The amino acid sequence of the kappa light chain variable region (VL) of mAb 4H2 is as follows:

[0092] DIVLTQSPATLSVTPGDRVSLSC RASQSISNYLH WYQQKSHESPRLLIK YASQSIS GIPSRFSGSGSGTDFTLSIISVETEDFGMYFC QQSNSWPLT FGAGTKLELK(SEQ ID NO:1).

[0093] The complementary decision regions (CDRs) are shown underlined, including RASQSISNYLH (SEQ ID NO:2); CDR L2:YASQSIS (SEQ ID NO:3); and CDR L3:QQSNSWPLT (SEQ ID NO:4).

[0094] b. 4H2 heavy chain variable region

[0095] The amino acid sequence of the heavy chain variable region (VH) of mAb 4H2 is as follows:

[0096] EVQLQQSGPELVKPGASVKMSCKASGYTFT DYYMN WVKQSHGKSLEWIG RVNPSNGGISYNQKFKG KATLTVDKSLSTAYMQLNSLTSEDSAVYYCAR GPYTMYY WGQGTSVTVSS(SEQ ID NO:5).

[0097] The complementarity determination regions (CDRs) are indicated by underscores and include CDR H1:DYYMN (SEQ ID NO:6); CDR H2:RVNPSNGGISYNQKFKG (SEQ ID NO:7); and CDR H3:GPYTMYY (SEQ ID NO:8).

[0098] 2. Forms of antibodies

[0099] Exemplary antibodies that can be used include any class of whole immunoglobulins (i.e., complete antibodies), fragments thereof, and synthetic proteins containing at least an antibody-antigen-binding variable region. The variable regions of different antibodies differ sequentially, which can be used for the binding and specificity of each particular antibody to its specific antigen. However, the variability of antibody variable regions is generally not uniformly distributed. It is typically concentrated in three segments in the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frames (FRs). The variable regions of both the natural heavy and light chains each contain four FR regions, predominantly in a β-sheet conformation, linked by three CDRs to form a loop connecting the β-sheet conformation, and in some cases, also forming part of the β-sheet conformation. The CDRs on each chain are tightly bound together by the FR regions and together with the CDRs on the other chain, constitute the antigen-binding site of the antibody. Therefore, antibodies can include CDR components required for cell penetration and guanosine binding.

[0100] Antibodies can be humanized antibodies or chimeric antibodies, or fragments thereof, their variants, or fusion proteins thereof. Methods for humanizing nonhuman antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “introduced” residues and are usually derived from an “introduced” variable region. Antibody humanization techniques typically involve using recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.

[0101] The 4H2 antibody may consist of an antibody fragment or a fusion protein comprising one or more CDRs (e.g., CDRs of any one of SEQ ID NO: 1 and 5, e.g., SEQ ID NO: 2-4 and 6-8, respectively) that are at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of the CDR of 4H2 or its variants or humanized forms thereof. The percentage of identity between the two amino acid sequences can be determined by BLAST protein alignment. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the aforementioned preferred variable regions of CDR (e.g., SEQ ID NO: 1 and 5) without any variation, or with a maximum of 0, 1, 2, 3, 4, or 5 variations per CDR (i.e., each CDR is selected independently), or the total variation of all CDRs.

[0102] The 4H2 antibody may consist of an antibody fragment or a fusion protein comprising 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% of the amino acid sequence of the variable heavy chain and / or light chain of 4H2 or its humanized form (such as SEQ ID NO: 5 and 1).

[0103] Preferably, the antibody comprises heavy chains CDR1, CDR2, and CDR3 that bind to light chains CDR1, CDR2, and CDR3.

[0104] Therefore, in some embodiments, the cell-penetrating antibody comprises the CDR of SEQ ID NO:5 and 1 or the entire heavy and light chain variable regions; or a humanized form thereof.

[0105] Many non-human antibodies (such as those derived from mice, rats, or rabbits) are naturally antigenic to humans and therefore can elicit adverse immune responses when administered to humans. Therefore, humanized 4H2 antibodies, antibody fragments, and fusions are provided. Humanized antigen-binding molecules can reduce the likelihood of adverse immune responses when antibodies, antibody fragments, or scFvs are administered to humans.

[0106] Humanized forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains, or fragments thereof, containing minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (receptor antibodies) where residues of the receptor antibody's complementarity-determining region (CDR) are replaced by residues of the CDR from a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capacity. In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also contain residues not present in the receptor antibody or in the introduced CDR or framework sequence. Typically, humanized antibodies will substantially include at least one (usually two) variable regions, where all or substantially all of the CDR regions correspond to the CDR regions of the non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of the human immunoglobulin consensus sequence. Humanized antibodies preferably include at least a portion of the immunoglobulin constant region (Fc), typically the constant region (Fc) of human immunoglobulins.

[0107] Methods for humanizing nonhuman antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “imported” residues and are usually derived from an “imported” variable region. Antibody humanization techniques generally involve using recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization is essentially achieved by replacing the CDR or CDR sequence of a rodent with the corresponding sequence of a human antibody. Thus, the humanized form of a nonhuman antibody (or fragment thereof) is a chimeric antibody or fragment in which essentially less than a complete human variable region has been replaced by the corresponding sequence of a nonhuman species. In practice, humanized antibodies are often human antibodies in which some of the CDR residues and possibly some of the FR residues are replaced by residues at similar sites in rodent antibodies.

[0108] To reduce antigenicity, it is crucial to select the light and heavy human variable regions for manufacturing humanized antibodies. Following a "best fit" approach, the variable region sequences of rodent antibodies are screened against an entire library of known human variable region sequences. The human sequence closest to the rodent sequence is accepted as the human frame (FR) for the humanized antibody. Alternatively, a frame derived from a consensus sequence of all human antibodies derived from a specific subset of the light or heavy chain can be used. The same frame can be used for multiple different humanized antibodies.

[0109] More importantly, antibodies must maintain high affinity for antigens and other beneficial biological properties while being humanized. To achieve this goal, humanized antibodies are preferably prepared using methods that analyze parental sequences and various conceptual humanized products by employing three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are available and are familiar to those skilled in the art. Existing computer programs can illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. By examining these displays, the potential roles of residues in the function of the candidate immunoglobulin sequence can be analyzed, i.e., the residues affecting the antigen-binding ability of the candidate immunoglobulin can be analyzed. In this way, FR residues can be selected and combined from consensus and import sequences to obtain the desired antibody properties, such as increased affinity for target antigens. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

[0110] In addition, it includes biologically active antibody fragments. These fragments, whether or not attached to other sequences, include insertions, deletions, substitutions, or other selective modifications to specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or impaired compared to an unmodified antibody or antibody fragment.

[0111] The techniques for producing specific single-chain antibodies against the antigen proteins disclosed herein can also be modified. Methods for producing single-chain antibodies are well known to those skilled in the art. Single-chain antibodies can reconstruct antigen-binding sites on a single molecule by fusing variable regions of the heavy and light chains together using short peptide linkers. In developed single-chain antibody variable fragments (scFvs), the C-terminus of one variable region is linked to the N-terminus of another variable region via a 15- to 25-amino acid peptide or linker without significantly disrupting antigen binding or binding specificity. The linker is selected to allow the heavy and light chains to bind together in the correct conformational orientation.

[0112] 4H2 antibodies can be modified to enhance their therapeutic potential. For example, in some embodiments, a cell-penetrating 4H2 antibody is conjugated to another antibody in the cytoplasm and / or nucleus of target cells that is specific to a second, such as a therapeutic target. For example, a cell-penetrating 4H2 antibody can be a fusion protein containing 4H2 Fv and a single-chain variable fragment of a monoclonal antibody that specifically binds to a second target. In other embodiments, a cell-penetrating 4H2 antibody is a bispecific antibody fragment having 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 a second target.

[0113] In some implementations, the second target is specific to target cell types, tissues, organs, etc. Therefore, the second heavy chain and the second light chain can serve as targeting components to target the complex to a specific cell type, tissue, or organ. In some implementations, the second heavy chain and the second light chain target hematopoietic stem cells and CD34. + Cells, T cells, cancer cells, infected cells, or any other preferred cell type, for example, by targeting receptors or ligands expressed on the preferred cell type. In some embodiments, the second heavy chain and the second light chain target the thymus, spleen, or cancer cells.

[0114] In some implementations, particularly those for targeting T cells in vivo, such as those for producing CAR T cells in vivo, immune cells or T cell markers, such as CD3, CD5, CD7, or CD8, may be targeted. For example, anti-CD8 antibodies and anti-CD3 Fab fragments have been used for targeting 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). Therefore, in some embodiments, the 4H2 antibody or antigen-binding fragment or fusion protein is a bispecific antibody portion that can specifically bind to CD3, CD5, CD7, CD8 or another immune cell (such as T cell) marker or a marker of a specific tissue such as the thymus, spleen or liver.

[0115] Exemplary fragments and fusions include, but are not limited to, single-chain antibodies, single-chain variable fragments (scFv), bivalent single-chain variable fragments (di-scFv), trivalent single-chain variable fragments (tri-scFv), bispecific antibody fragments (diabody), trispecific antibody fragments (triabody), tetravalent antibody fragments (teratbody), disulfide-linked variable fragments (sdFv), Fab', F(ab')2, variable fragments (Fv), and single-domain antibody fragments (sdAb).

[0116] For example, divalent single-chain variable fragments (di-scFvs) can be designed by linking two scFvs together. This can be achieved by producing a single peptide chain with two VH regions and two VL regions, resulting in a tandem scFv. scFvs can also be designed as linking peptides, but these linking peptides are too short for the two variable regions to fold together (approximately 5 amino acids), forcing the scFv to dimerize. This type is called a bispecific antibody fragment (diabody). Studies have shown that bispecific antibody fragments (diabody) have a dissociation constant 40 times lower than the corresponding scFv, meaning they have a much higher affinity for the target. Shorter linker arms (one or two amino acids) can lead to the formation of trimers (i.e., triabodies or tribodies). Furthermore, tetravalent antibody fragments (teratbody) are also produced. They have even higher affinity for the target compared to bispecific antibody fragments (diabody). In some embodiments, the 4H2 antibody may comprise two or more linked 4H2 single-chain variable fragments (such as 4H2 di-scFv, 4H2 tri-scFv) or conserved variants thereof. In some embodiments, the 4H2 antibody is a bispecific antibody fragment (diabody) or a trispecific antibody fragment (triabody) (such as 4H2 bispecific antibody fragment (4H2diabody), 4H2 trispecific antibody fragment (triabody)).

[0117] In some embodiments, the antibody is conjugated or fused to a cell-penetrating moiety (such as a cell-penetrating peptide) to facilitate antibody entry into cells. Examples of cell-penetrating peptides include, but are not limited to, polyarginine (such as R9), antennapediase sequences, TAT, HIV-Tat, penetrantin, Antp-3A (Antp mutant), Buforin II, Transportan, MAP (model amphiphilic peptide), K-FGF, Ku70, Prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (biguanidine-spermine-cholesterol), and BGTC (biguanidine-triethylenetetramine-cholesterol). In other embodiments, the antibody is delivered via TransMabs. TM Technically modified (InNexus Biotech., Inc., Vancouver, BC)

[0118] Antibody function can be enhanced by conjugating antibodies or fragments thereof to therapeutic agents. Conjugation of antibodies or antibody fragments to therapeutic agents can be achieved by creating immunoconjugates or fusion proteins, or by linking antibodies or antibody fragments to nucleic acids such as DNA or RNA (e.g., siRNA) that include both the antibody or antibody fragment and the therapeutic agent.

[0119] Recombinant fusion proteins are proteins created through genetic engineering using fusion genes. This typically involves removing a stop codon from the cDNA sequence encoding the first protein, and then adding the cDNA sequence of the second protein to the frame via ligation or overlap extension PCR. The DNA sequence is then expressed in the cell as a single protein. This protein can be engineered to include the complete sequences of both original proteins, or only a portion of one of them. If both entities are proteins, a linker arm (or “spacer”) is often added to make the protein more likely to fold independently and function as intended.

[0120] In some embodiments, cell-penetrating antibodies are modified to alter their half-life. In some embodiments, it is desirable to prolong the antibody's half-life, allowing it to remain in circulation or at the treatment site for a longer period. For example, it may be necessary to maintain antibody titers in circulation or at the treatment site for an extended time. In other embodiments, the half-life of 4H2 antibodies is shortened to reduce potential side effects. The half-life of antibody fragments such as 4H2Fv may be shorter than that of full-size antibodies. Other methods for altering half-life are known and can be used in these methods. Antibodies can be designed using Fc variants with extended half-lives, for example, using Xtend. TM Antibody half-life extension technology (Xencor, Monrovia, CA).

[0121] a. Connecting arm

[0122] The term "linker arm" as used herein includes, but is not limited to, peptide linkers. Peptide linkers can be of any size as long as they do not interfere with the binding of the variable region to the epitope. In some embodiments, the linker arm comprises one or more glycine and / or serine amino acid residues. In monovalent single-chain antibody variable fragments (scFvs), the C-terminus of one variable region is typically linked to the N-terminus of another variable region via a 15- to 25-amino acid peptide or linker arm. The linker arm is selected to allow the heavy and light chains to bind together in the correct conformational orientation. As mentioned above, the linkers of bispecific antibody fragments (diabody), trispecific antibody fragments (triabody), etc., are typically shorter than those of monovalent scFvs. Bivalent, trivalent, and other multivalent scFvs typically include three or more linkers. The length and / or amino acid composition of the linkers can be the same or different. Therefore, the number of linkers, their composition, and their length can be determined based on the desired titer of the scFv known in the art. The linker arm allows or drives the formation of bivalent, trivalent, and other multivalent scFvs.

[0123] For example, the linker arm may comprise 4-8 amino acids. In a specific embodiment, the linker arm comprises the amino acid sequence GQSSRSS (SEQ ID NO:10). In another embodiment, the linker arm comprises 15-20 amino acids, such as 18 amino acids. In a specific embodiment, the linker arm comprises the amino acid sequence GQSSRSSSGGGSSGGGS (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), (Gly4-Ser)2 (SEQ ID NO:14), (Gly4-Ser)4 (SEQ ID NO:15), and (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO:16).

[0124] Other exemplary connecting arms include, for example, RADAAPGGGGSGGGGSGGGGS (SEQ ID NO:17) and ASTKGPSVFPLAPLESSGS (SEQ ID NO:18).

[0125] b. Exemplary 4H2 scFv sequence

[0126] Those skilled in the art will understand that exemplary fusion proteins or their domains can be used to construct the fusion proteins discussed in more detail above. For example, in some embodiments, the scFv includes an scFv associated with a VH variable region (such as SEQ ID NO:5 or a functional variant thereof or a fragment thereof), the scFv including a Vk variable region (SEQ ID NO:1 or a functional variant thereof or a fragment thereof). In some embodiments, the di-scFv includes a first scFv including a Vk variable region (SEQ ID NO:1 or a functional variant thereof or a fragment thereof), associated with a VH variable region (e.g., SEQ ID NO:5 or a functional variant thereof or a fragment thereof), associated with a second scFv including a Vk variable region (such as SEQ ID NO:1 or a functional variant thereof or a fragment thereof), and associated with a VH variable region (such as SEQ ID NO:5 or a functional variant thereof or a fragment thereof). In some implementations, the trivalent monochain variable fragment (tri-scFv) includes a bivalent monochain variable fragment (di-scFv) connected to a third scFv structural domain, the third scFv structural domain including a Vk variable region (such as SEQ ID NO:1, or a functional variant thereof or a fragment thereof) connected to a VH variable region, and connected to a VH variable region (such as SEQ ID NO:5, or a functional variant thereof or a fragment thereof).

[0127] For example, the Vk variable region can be linked to the VH variable region alone, such as with a linker arm (e.g., (GGGGS)3 (SEQ ID NO:19)) or in combination with a linker arm and (6aa) of the light chain CH1 (e.g., RADAAP (SEQ ID NO:20)). The scFv can be linked alone with a linker arm (e.g., the 13 amino acids starting at human IgG CH1 (e.g., ASTKGPSVFPLAP (SEQ ID NO:21)) or in combination with a rotatable sequence (e.g., LESSGS (SEQ ID NO:22)). Other suitable linker arms have been discussed above and are known in the art.

[0128] In some embodiments, the fusion protein includes additional domains. For example, in some embodiments, the fusion protein includes sequences that improve solubility. In some embodiments, the fusion protein includes one or more domains that improve the purification, separation, capture, identification, isolation, etc., of the fusion protein. Exemplary domains include, for example, Myc tags and / or His tags. Other alternative and additional domains have been discussed in detail above.

[0129] An exemplary scFv molecule is also provided.

[0130] DIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSG TDFTLSIISVETEDFGMYFCQQSNSWPLTFGAGTKLELKADAAPGGGGSGGGGSGGGGSGE VQLQQSGPELVKPGAS VKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGRVNPSNGGISYNQKFKGKATLTVDKSLSTAYMQLNSLTSEDSAV YYCARGPY TM YYWGQGTSVTVSSHHHHHH (SEQ ID NO: 9)

[0131] Single underscore: 4H2 VL sequence

[0132] Double underscores: Connecting arm sequence

[0133] Dashed underline: 4H2 VH sequence

[0134] Wavy underline: His6 tag

[0135] For example, the scFv may include the C-terminus of the 4H2VL sequence of SEQ ID NO:9 linked to the N-terminal sequence of 4H2 VH of SEQ ID NO:9, or the C-terminus of the 4H2 VH sequence of SEQ ID NO:9 linked to the N-terminal sequence of 4H2 VL of SEQ ID NO:9. The linker arm of SEQ ID NO:9 may be replaced with alternative linkers, including but not limited to those disclosed herein. Typically, the linker arm is about 10 to about 25 amino acids, typically including glycine. The His6 tag of SEQ ID NO:9 may be replaced with another tag, moved to the N-terminus of the scFv, or completely deleted. In some embodiments, 4H2 VL, 4H2 VH, or combinations thereof are variants of 4H2 VL and / or 4H2 VH of SEQ ID NO:9, or humanized forms thereof. In some embodiments, the N-terminus and C-terminus of the 4H2 VL and / or 4H2 VH domains are truncated compared to 4H2 VL and / or 4H2 VH of SEQ ID NO:9. The scFv may include three CDRs of SEQ ID NO:9's 4H2 VL and / or 4H2 VH, or their humanized forms. In some embodiments, the antibody, fragment, or fusion thereof has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:9. In some embodiments, the VL domain of the antibody or fragment or fusion thereof has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the 4H2 VL domain of SEQ ID NO:9. In some embodiments, the VH domain of the antibody, fragment, or fusion thereof has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the 4H2 VH domain of SEQ ID NO:9.

[0136] SEQ ID NO:9, its humanized forms, and variants thereof can be used in any of the compositions and methods disclosed herein. In some embodiments, SEQ ID NO:9, its humanized forms, or variants thereof can be used in therapeutic methods, such as those disclosed herein, without conjugation to a nanocarrier or therapeutic agent. Thus, in some embodiments, SEQ ID NO:9, its humanized forms, or variants thereof are simply therapeutic agents. In some embodiments, SEQ ID NO:9, its humanized forms, or variants thereof are not therapeutic agents (e.g., only a targeting portion), or may be one of two or more therapeutic agents.

[0137] c. Exemplary 4H2 bispecific antibody

[0138] An exemplary bispecific antibody was used in Example 13 below. This antibody has... Figure 12A The format shown, along with the heavy and light chain variable region sequences:

[0139] 4H2 sequence

[0140] VL:

[0141] DIVLTQSPATLSVTPGDRVSLSC RASQSISNYLH WYQQKSHESPRLLIK YASQSIS GIPSRFSGSGSGTDFTLSIISVETEDFGMYFC QQSNSWPLT FGAGTKLELK(SEQ ID NO:1)

[0142] VH:

[0143] EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIG RVNPSNGGISYNQKFKG KATLTVDKSLSTAYMQLNSLTSEDSAVYYCAR GPYTMYY WGQGTSVTVSS(SEQ ID NO:5)

[0144] CD5 sequence

[0145] VL:

[0146] NIVMTQSPSSLSASVGDRVTITC QASQDVGTAVA WYOQKPDQSPKLLI YWTSTRHT GVPDRFTGSGSGTDFTLTISSLOPEDIATYFC HQYNSYNT FGSGTKLEIK(SEQ ID NO:23)

[0147] VH:

[0148] QVTLKESGPVLVKPTETLTLTC TFSGFSLSTSGMGVG WIRQAPGKGLEWVA HIWWDDDVY YNPSLKSRLTITKDASKDQVSLKLSSVTAADTAVYYCVR RRATGTGFDY WGQGTLVTVSS(SEQ ID NO:24)

[0149] This antibody is merely exemplary, and it is understood that other forms, alternative sequences, particularly framework sequences, and even other second-arm binding domains targeting antigens other than CD5 are also explicitly provided. For example, in some embodiments, chimeric or humanized bispecific antibodies are provided having CDRs of SEQ ID NO: 1, 5, 23, and 24 or their humanized forms (e.g., 1, 2, or 3 mutations, such as conserved substitutions, per CDR or in total), having a human heavy chain and light chain variable region framework and optionally constant domains.

[0150] The predicted CDR for 4H2 is underlined above. The predicted CDR for anti-CD5 is underlined above and explicitly provided:

[0151] CDR L1: QASQDVGTAVA (SEQ ID NO: 25); CDR L2: YWTSTRHT (SEQ ID NO: 26); HQYNSYNT CDR L3: (SEQ ID NO: 27).

[0152] CDR H1: TFSGFSLSTSGMGVG (SEQ ID NO: 28); CDR H2: HIWWDDDVY (SEQ ID NO: 29); CDR H3: RRATGTGFDY (SEQ ID NO: 30).

[0153] For example, a 4H2-CD5 bispecific antibody fragment may consist of an antibody fragment or a fusion protein comprising one or more CDRs having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the amino acid sequence of a CDR of 4H2 or its variants or humanized forms thereof (e.g., any one of SEQ ID NO:1 and 5, such as SEQ ID NO:2-4 and 6-8, respectively) and an anti-CD5 antibody fragment or its fusion protein or its variants or humanized forms thereof (e.g., any one of SEQ ID NO:23 and 24, such as SEQ ID NO:25-27 and 28-30, respectively). The percentage of identity between the two amino acid sequences can be determined by BLAST protein alignment. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the above-mentioned preferred variable regions CDR (e.g., SEQ ID NO: 1 and 5 and / or 23 and 24) without any variation, or each CDR (i.e., each CDR is selected independently) or all CDRs cumulatively have a maximum of 0, 1, 2, 3, 4, or 5 variations.

[0154] The 4H2-CD5 bispecific antibody may consist of an antibody fragment or a fusion protein comprising an amino acid sequence of a variable heavy chain and / or a variable light chain, the amino acid sequence of which has 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% identity with the amino acid sequence of the variable heavy chain and / or variable light chain of 4H2 or its humanized form (such as SEQ ID NO: 5 and 1) and the amino acid sequence of the variable heavy chain and / or light chain of antiCD5 or its humanized form (such as SEQ ID NO: 24 and 23).

[0155] Preferably, the bispecific antibody comprises a combination of heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2, CDR3 of each of 4H2, and a combination with the corresponding CDR region of the anti-CD5 antibody.

[0156] Therefore, in some embodiments, the cell-penetrating bispecific antibody comprises the CDR of SEQ ID NO:5 and 1 and 24 and 23 or the entire heavy and light chain variable regions; or a humanized form thereof.

[0157] B. Additional medications

[0158] The following results indicate that extracellular nucleic acids can facilitate cell penetration via 4H2. Furthermore, cytoplasmic DNA activation of cGAS leads to the endogenous production of cyclic GMP-AMP, a unique second messenger that binds to interferon gene stimulating factor (STING), resulting in the activation of TANK-binding kinase 1 (TBK1) and IRF3, thereby leading to transcription of genes encoding type I interferon (Pesiridis and Fitzgerald, Nature Reviews Genetics, Vol. 20, pp. 657-674 (2019)). The following experimental results show that 4H2 can activate cGAS and other pattern recognition receptors (PPRs), such as TLR7. This activation may be through direct binding and activation of 4H2, or indirectly through simultaneous interactions between immune receptors, 4H2, and cytoplasmic nucleic acids and / or GTP.

[0159] Therefore, the disclosed compositions can be used to facilitate the delivery of nucleic acid cargo. Alternatively or additionally, 4H2 antibodies can also be used to modulate immune responses with or without the aid of nucleic acid cargo. For example, in some embodiments, the compositions and methods include nucleic acids and / or GTP (also known as nucleic acid cargo) to facilitate 4H2 cell penetration and / or activation of cGAS and / or another PRR, such as TLR7.

[0160] Furthermore, STING agonists have been proposed for a variety of different therapeutic purposes, including the treatment of cancer, infections, and as vaccine adjuvants. See, for example, Pesiridis and Fitzgerald, Nature Reviews Genetics, Vol. 20, pp. 657–674 (2019), the entire contents of which are incorporated herein by reference. Therefore, in some embodiments, the disclosed compositions and methods include adjunctive pharmaceutical products that facilitate these applications. Non-limiting examples of adjunctive pharmaceutical products include, but are not limited to, adjunctive STING agonists, vaccine compositions, and immune checkpoint inhibitors, each of which will be discussed in more detail below. In some embodiments, the adjunctive pharmaceutical product is a nucleic acid (such as an immunostimulatory oligonucleotide, a nucleic acid encoding a vaccine component such as a peptide antigen, etc.). Adjunctive pharmaceutical products of this type of nucleic acid can be nucleic acid cargoes or adjuncts or substitutes given separately to the subject.

[0161] Therefore, any adjunct drug can be in the same or different mixtures as the 4H2 antibody and can be administered at the same or different times. In some embodiments, such as when the adjunct drug is a nucleic acid cargo, the adjunct drug and the 4H2 antibody are contacted and form a complex before administration to the subject. The interaction between the antibody and the nucleic acid cargo is non-covalent. In this embodiment, the complex can be administered to the subject. Although referred to as cargo, as disclosed herein, the cargo nucleic acid can also be administered alone, and therefore is not necessarily a cargo of the 4H2 antibody under these conditions.

[0162] 1. Goods

[0163] Nucleic acid cargo is also provided. As discussed in more detail below, the disclosed 4H2 antibody can be used to deliver the nucleic acid cargo to cells for any purpose. In specific embodiments, the cargo can also be used to increase cell penetration of the 4H2 antibody and / or increase the activation of cGAS and / or another PRR (such as TLR7). In the nucleic acid delivery methods provided herein, 4H2 is typically contacted with cells conjugated with the nucleic acid cargo. The interaction between the antibody or binding protein and the nucleic acid cargo is non-covalent.

[0164] Nucleic acid cargo can be single-stranded or double-stranded, and can be a single nucleotide, nucleotide, or nucleobase, or multiple nucleotides, nucleotides, or nucleobases. In some embodiments, the cargo is GTP, GDP, GMP, cGAMP, or cGMP. Nucleic acid cargo can be DNA, RNA, nucleic acid analogs, or combinations thereof, or include DNA, RNA, nucleic acid analogs, or combinations thereof. As described below, nucleic acid analogs can be modified on the base moiety, glycosyl moiety, or phosphate backbone. For example, such modifications can improve the stability, hybridization, or solubility of the nucleic acid. 4H2 can bind to guanosine. Therefore, the cargo typically includes one or more guanine nucleobases, preferably one or more guanine nucleosides.

[0165] Nucleic acid cargoes can be functional, i.e., pharmaceutical agents or agents that are biologically active once delivered into the cell, or non-functional, simply facilitating the delivery of 4H2 to the cytoplasm and / or activating cGAS and / or another PRR (such as TLR7). Exemplary cargoes will be discussed in more detail below, but include mRNA or DNA encoding a target polypeptide, such as including expression constructs and vectors, repressive nucleic acids (such as siRNA), or nucleic acids encoding repressive nucleic acids, such as including expression constructs and vectors, or non-coding RNA or DNA.

[0166] The disclosed compositions may include multiple individual nucleic acid cargo molecules. In some embodiments, the compositions include multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different nucleic acid molecules.

[0167] In some embodiments, the length of the cargo molecule is 0.001, 0.01, 1, 10's, 100's, 1,000's, 10,000's and / or 100,000's kilobases.

[0168] In some embodiments, for example, the weight of the goods may be between 0.001kb and 100kb, or between 0.001kb and 50kb, or between 0.001kb and 25kb, or between 0.001kb and 12.5kb, or between 0.001kb and 10kb, or between 0.001kb and 8kb, or between 0.001kb and 5kb, or between 0.001kb and 2.5kb, or between 0.001kb and 1kb, or between 0.01kb and 100kb, or between 0.01kb and 50kb. Between 0.01kb and 25kb, or between 0.01kb and 12.5kb, or between 0.01kb and 10kb, or between 0.01kb and 8kb, or between 0.01kb and 5kb, or between 0.01kb and 2.5kb, or between 0.01kb and 1kb, or between 0.1kb and 100kb, or between 0.1kb The values ​​between 0.1kb and 50kb, or between 0.1kb and 25kb, or between 0.1kb and 12.5kb, or between 0.1kb and 10kb, or between 0.1kb and 8kb, or between 0.1kb and 5kb, or between 0.1kb and 2.5kb, or between 0.1kb and 1kb, or between 1kb and 100kb, or between 1kb and 50kb, or between 1kb and 25kb, or between 1kb and 12.5kb, or between 1kb and 10kb, or between 1kb and 8kb, or between 1kb and 5kb, or between 1kb and 2.5kb, all include endpoint values.

[0169] In some implementations, for example, the weight of the goods 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.

[0170] It is understood that, in specific applications, the length of the nucleic acid cargo can be one or more discrete lengths, for example, falling within one of the ranges mentioned above (including the endpoints), the specific values ​​of each range being explicitly disclosed. For example, the size can be as small as a single nucleotide or nucleobase. In exemplary applications, the cargo is a cyclic dinucleotide, such as cGAMP, which is a STING agonist. In other embodiments, the cargo is a short oligomer. For example, oligomers as short as 8 meters can be used for antisense or splicing conversion. Slightly longer oligomers (such as polymers of 18 to 20 meters) can be used for gene editing.

[0171] a. Form of goods

[0172] Nucleic acid cargo is nucleic acid, which can be isolated nucleic acid compositions. As used herein, "isolated nucleic acid" refers to nucleic acid isolated from other nucleic acid molecules present in the mammalian genome, including those typically located on one or both sides of a nucleic acid in the mammalian genome. The term "isolated" in relation to nucleic acids also includes combinations with any non-naturally occurring nucleic acid sequence, because such non-naturally occurring sequences are not found in nature and have no directly adjacent sequences in naturally occurring genomes.

[0173] Isolated nucleic acids can be, for example, DNA molecules, provided that one of the nucleic acid sequences flanking the DNA molecule in the natural genome has been removed or deleted. Therefore, isolated nucleic acids include, but are not limited to, DNA molecules existing as separate molecules independent of other sequences (e.g., chemically synthesized nucleic acids or cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment), and recombinant DNA incorporated into vectors, autonomously replicating plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, or herpesviruses), or prokaryotic or eukaryotic genomic DNA. Furthermore, isolated nucleic acids can also include engineered nucleic acids, such as recombinant DNA molecules as part of hybrid or fusion nucleic acids. Nucleic acids present in hundreds to millions of other nucleic acids in gel sections containing restriction digests of genomic DNA, such as cDNA libraries or genomic libraries, cannot be considered isolated nucleic acids.

[0174] The nucleic acid sequences encoding the polypeptides include genomic sequences. mRNA / cDNA sequences with deleted exons are also disclosed. Other nucleic acid sequences encoding the polypeptides are also disclosed, such as polypeptides comprising the above-mentioned amino acid sequences, fragments thereof, and variants thereof. The nucleic acids encoding the polypeptides can be optimized for expression in selected expression hosts. Codons can be replaced with substitution codons encoding the same amino acids to resolve differences in codon usage between the source organism of the nucleic acid sequence and the expression host. In this way, nucleic acids can be synthesized using codons preferred by the expression host.

[0175] Nucleic acids can be sense or antisense oriented, and can also be complementary to the reference sequence encoding a polypeptide.

[0176] i. Carrier

[0177] The cargo can be a vector, such as a vector encoding polypeptides and / or functional nucleic acids. Nucleic acids, as described above, can be inserted into vectors for expression in cells. As used herein, a "vector" is a replicon, such as a plasmid, bacteriophage, virus, or granule, into which another DNA fragment can be inserted to achieve replication of the inserted fragment. A vector can also be an expression vector. An "expression vector" is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence.

[0178] The nucleic acids in the vector can be operatively linked to one or more expression control sequences. For example, control sequences can be integrated into a gene construct, thereby enabling the expression control sequences to effectively control the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence composed of a region of DNA molecule, typically located within 100 nucleotides upstream of the transcription start site (usually near the start site of RNA polymerase II). For a coding sequence to be controlled by a promoter, the translation start site of the polypeptide translation reading frame must be placed 1 to 50 nucleotides downstream of the promoter. Enhancers are expression-specific in terms of time, location, and level. Unlike promoters, enhancers can function at different locations from the transcription site.

[0179] Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe a coding sequence into mRNA, that coding sequence is "operably linked" and controlled by an expression-controlled sequence in the cell, which then translates the mRNA into the protein encoded by that coding sequence.

[0180] Suitable expression vectors include, but are not limited to, plasmids, granules, and viral vectors, such as those derived from bacteriophages, baculoviruses, tobacco mosaic virus, herpesviruses, cytomegaloviruses, retroviruses, vaccine viruses, adenoviruses, and adenovirus-associated vectors. Many vectors and expression systems are available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0181] In some embodiments, the cargo is delivered into the cell and held extrachromosomally. In some embodiments, the cargo is introduced into a host cell and integrated into the host cell's genome. As discussed in detail below, the composition can be used in gene therapy methods. Gene therapy methods may include introducing polynucleotides that alter the cell's genotype into the cell. The introduction of polynucleotides may correct, replace, or otherwise alter endogenous genes through gene recombination. Methods may 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, a corrective gene may be introduced into a nonspecific location in the host genome.

[0182] 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 DNA recombination techniques, synthetic techniques, and in vivo gene recombination. Expression vectors typically include regulatory sequences and necessary elements for the translation and / or transcription of the inserted coding sequence, for example, a target polynucleotide. The coding sequence can be operatively linked to a promoter and / or enhancer to help control the expression of the desired gene product. Promoters used in biotechnology vary depending on the intended type of gene expression control. They are generally classified as constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0183] For example, in some embodiments, the target polynucleotide is operatively linked to a promoter or other regulatory element known in the art. Thus, the cargo can be a vector, such as an expression vector. Engineered expression of the polynucleotide in prokaryotic or eukaryotic systems can be performed using techniques generally known to those skilled in the art of recombinant expression. Expression vectors typically comprise one of disclosed compositions controlled by one or more promoters. To make the coding sequence “controlled” by a promoter, the 5’ end of the translation start site of the reading frame is typically positioned “downstream” (i.e., the 3’ end) of the selected promoter by approximately 1 to 50 nucleotides. The “upstream” promoter stimulates transcription of the inserted DNA, promoting the expression of the recombinant protein or functional nucleic acid. This is the meaning of “recombinant expression” as used herein.

[0184] There are many standard techniques available for constructing expression vectors containing appropriate nucleic acids and transcription / translation control sequences to enable the expression of proteins, peptides, or functional nucleic acids in a variety of host expression systems.

[0185] Expression vectors used in mammalian cells typically include an origin of replication (if necessary), a promoter preceding the gene to be expressed, and any necessary ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. The origin of replication can be provided by constructing the vector, which can be exogenous, possibly derived from SV40 or other viruses (such as polyomaviruses, adenoviruses, VSVs, and BPV), or provided by the host cell's chromosomal replication mechanism. If the vector integrates into the host cell's chromosome, the latter is usually sufficient.

[0186] Promoters can be derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the adenovirus late promoter; the vaccine virus 7.5K promoter). Furthermore, it is possible, and may be ideal, to utilize promoters or control sequences that are typically associated with the desired gene sequence, provided that these control sequences are compatible with the host cell system.

[0187] Various virus-based expression systems can be utilized; for example, commonly used promoters are derived from polyomavirus, adenovirus 2, cytomegalovirus, and simian vacuolating virus 40 (SV40). Both early and late promoters of SV40 are useful because both can be readily obtained from the virus as fragments, including the SV40 origin of replication. Smaller or larger SV40 fragments can also be used, but must include approximately 250 bp of sequence extending from the HindIII site to the BglI site located at the viral origin of replication.

[0188] When using adenovirus as an expression vector, the coding sequence can be ligated to an adenoviral transcription / translation control complex, such as a late promoter and a triplet leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (such as the E1 or E3 region) enables the recombinant virus to survive and express the protein in an infected host. Efficient translation of the disclosed composition may also require specific initiation signals. These signals include the ATG start codon and neighboring sequences. Additionally, exogenous translation control signals, including the ATG start codon, may also be required. Those skilled in the art can readily identify this requirement and provide the necessary signals. It is well known that the start codon must be in frame (or in phase) with the desired coding sequence to ensure translation of the entire inserted fragment. These exogenous translation control signals and start codons come from a variety of sources, both natural and synthetic. Including appropriate transcriptional enhancer elements or transcription terminators can improve expression efficiency.

[0189] In eukaryotic expression, if the original clone fragment does not include a suitable polyadenylation site, then a suitable polyadenylation site usually needs to be added to the transcription unit. Typically, the polyadenylation site is located approximately 30 to 2000 nucleotides "downstream" of the protein termination site, i.e., before transcription termination.

[0190] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that can stably express constructs encoding proteins can be engineered. Unlike expression vectors containing viral replication origins, host cells can be transformed using vectors controlled by appropriate expression control elements (such as promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selective markers. After introducing exogenous DNA, engineered cells can be grown in enrichment media for 1-2 days and then transferred to selective media. Selective markers in the recombinant plasmid confer selective resistance to the cells, enabling them to stably integrate the plasmid into their chromosomes and grow into colonies, which can then be cloned and expanded into cell lines.

[0191] ii.mRNA

[0192] Goods can be mRNA

[0193] Chemical structures that improve stability and / or translation efficiency can also be used. For example, RNA can have 5' and 3' UTRs. For example, the length of the 3' UTR can exceed 100 nucleotides. In some embodiments, the 3' UTR sequence is between 100 and 5000 nucleotides. In some embodiments, the length of the 5' UTR is between 0 and 3000 nucleotides. The lengths of the 5' and 3' UTR sequences to be added to the coding region can be changed by various methods, including but not limited to designing PCR primers that anneal to regions different from the UTRs. Using this method, those skilled in the art can modify the lengths of the 5' and 3' UTRs to achieve optimal translation efficiency after transcribed RNA delivery.

[0194] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the target gene. Alternatively, non-target gene endogenous UTR sequences can be added by incorporating UTR sequences into the forward and reverse primers or by making any other modifications to the template. Using non-target gene endogenous UTR sequences can be used to alter RNA stability and / or translation efficiency. For example, it is well known that AU elements enriched in 3' UTR sequences reduce mRNA stability. Therefore, 3' UTRs can be selected or designed based on UTR characteristics well known in the art to improve the stability of transcribed RNA.

[0195] In some embodiments, the 5'UTR includes the Kozak sequence of the endogenous gene. Alternatively, when adding a non-target gene endogenous 5'UTR via PCR as described above, the consensus Kozak sequence can be redesigned by adding the 5'UTR sequence. Kozak sequences can improve the translation efficiency of some RNA transcripts, but it appears that not all RNAs require a Kozak sequence for efficient translation. The requirement for a Kozak sequence for many mRNAs is well known in the art. In other embodiments, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to inhibit the degradation of mRNA by exonucleases.

[0196] In some implementations, the 5' end of the mRNA has a cap, the 3' end has a poly(A) tail, or a combination thereof, which determines ribosome binding, translation initiation, and mRNA stability in the cell.

[0197] The 5' cap provides stability to RNA molecules. For example, the 5' cap can be an m 7 G(5')ppp(5')G,m 7 G(5')ppp(5')A, G(5')ppp(5')G, or G(5')ppp(5')A cap analogues are available commercially. The 5' cap may also be an anti-reverse cap analogue (ARCA) (Stepinski et al., RNA, 7:1468-95 (2001)) or any other suitable analogue. The 5' cap may 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. Res. Res. Commun., 330:958-966 (2005)).

[0198] RNA can also include an internal ribosome entry site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and promotes the initiation of translation.

[0199] Generally, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one implementation, the adenosine number in the poly(A) tail is between 100 and 5000.

[0200] Reverse primers containing a poly(T) tail, such as 100T tails (sizes can range from 50 to 5000T), can be used during PCR, or any other method, including but not limited to DNA ligation or in vitro recombination, can be used after PCR to generate poly(A) fragments. The poly(A) tail also provides stability to RNA and reduces its degradation. The poly(A) tail of RNA can be further or selectively extended post-transcriptionally using poly(A) polymerases (such as E. coli poly(A) polymerase (E-PAP)).

[0201] Furthermore, attaching different chemical groups to the 3' end can increase mRNA stability. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further enhance RNA stability. Suitable ATP analogs include, but are not limited to, cordiocipin and 8-azaadenosine.

[0202] b. Cargo sequence

[0203] i. Related peptides

[0204] The cargo may encode one or more proteins. The cargo may be a monocistronic or polycistronic polynucleotide. In some embodiments, the polynucleotide is multigenic. For example, the polynucleotide may be, for instance, mRNA or an expression construct, such as a vector.

[0205] The cargo may encode one or more target peptides. The peptide can be any peptide. For example, a peptide encoded by a polynucleotide may be a peptide that has therapeutic or preventative effects on an organism, or a peptide that can be used to diagnose diseases or conditions in an organism. For example, to treat cancer, autoimmune diseases, parasites, viruses, bacteria, fungi, or other infections, the polynucleotide to be expressed may encode a peptide that can act as a ligand or receptor for immune system cells, or that can stimulate or inhibit the organism's immune system.

[0206] In some implementations, polynucleotides can supplement or replace defective polynucleotides present in the organism.

[0207] In a specific embodiment, the polynucleotide encodes dystrophin, umtrophin, or a combination thereof. This composition can be administered in effective amounts to treat muscular dystrophy, particularly in subjects with muscular dystrophy, such as those with Duchenne muscular dystrophy.

[0208] In another specific embodiment, the polynucleotide encodes an antigen, such as an antigen that can be used in vaccine formulations and related methods. In one specific embodiment, the polynucleotide encodes a viral antigen, such as the SARS-CoV-2 antigen. Therefore, compositions for the prevention and treatment of SARS-CoV-2 virus and related viral infections and diseases (including COVID-19) and methods of using thereof are provided.

[0209] In some implementations, the polynucleotide includes a selective marker, such as a selective marker effective in eukaryotic cells, like a drug resistance selector. This selective marker gene can encode factors essential for the survival or growth of transformed host cells grown in selective media. Typical selector genes encode proteins that confer resistance to antibiotics or other toxins (such as ampicillin, neomycin, methotrexate, kanamycin, gentamicin, bleomycin, or tetracycline), compensate for auxotrophic deficiencies, or provide key nutrient components missing in the culture medium.

[0210] In the following working example 12, the nucleic acid encoding wild-type Merlin (a protein mutated in neurofibromatosis type 2) (i.e., NF2) reduces tumor growth. Therefore, in some embodiments, the nucleic acid encodes a wild-type or other compensatory variant of an oncogenic protein (such as Merlin).

[0211] In some embodiments, the polynucleotide includes a reporter gene. A reporter gene is typically a gene that is absent or not expressed in the host cell. Reporter genes typically encode proteins that provide a certain phenotypic change or enzymatic property. (Weising et al., Ann. Rev. Genetics, 22, 421 (1988)). Preferred reporter genes include glucuronidase (GUS) genes and GFP genes.

[0212] ii. Functional nucleic acids

[0213] Goods can be functional nucleic acids or nucleic acids encoding functional nucleic acids. Functional nucleic acids are nucleic acid molecules with specific functions, such as binding to target molecules or catalyzing specific reactions. As detailed below, functional nucleic acid molecules can be classified into the following non-restrictive categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, RNAi, external guide sequences, and cyclic dinucleotides. Functional nucleic acid molecules can act as effectors, inhibitors, regulators, and stimulators of the specific activities of target molecules, or they can possess novel activities independent of any other molecule.

[0214] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Therefore, functional nucleic acids can interact with the mRNA or genomic DNA of a target polypeptide, or with the polypeptide itself. Typically, functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other cases, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology, but rather on the formation of a tertiary structure that allows for specific recognition.

[0215] Therefore, the composition may include one or more functional nucleic acids designed to reduce the expression of a gene or its gene product. For example, the functional nucleic acid or peptide may be designed to target and reduce or inhibit the expression or translation of mRNA; or to reduce or inhibit the expression of a protein, decrease protein activity, or increase protein degradation. In some embodiments, the composition includes a vector suitable for in vivo expression of the functional nucleic acid.

[0216] (1) Antonyms

[0217] Functional nucleic acids can be antisense molecules or encode antisense molecules. Antisense molecules are designed to interact with target nucleic acid molecules through canonical or non-canonical base pairing. The interaction between antisense molecules and target molecules is designed to promote the destruction of target molecules through RNA-DNA hybridization degradation mediated by, for example, RNase H. Alternatively, antisense molecules are designed to interrupt processing functions that normally occur on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are many methods to optimize antisense efficiency by identifying the most accessible regions of the target molecule. Typical methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. The dissociation constant (Kd) of the antisense molecule binding to the target molecule is preferably less than or equal to 10. -6 10 -8 10 -10 Or 10 -12 .

[0218] (2) RNA interference

[0219] In some implementations, functional nucleic acids induce gene silencing via RNA interference. Gene expression can also be effectively silenced in a highly specific manner via RNA interference (RNAi). This silencing was initially observed through the addition of double-stranded RNA (dsRNA) (Fire et al. (1998), Nature, 391:806-11; Napoli et al. (1990), Plant Cell 2:279-89; Hannon et al. (2002), Nature, 418:244-51). Once dsRNA enters the cell, it is cleaved by the RNase III-like enzyme Dicer into a double-stranded small interfering RNA (siRNA) of 21-23 nucleotides in length, with a 2-nucleotide overhang at its 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, siRNA is integrated into a multi-subunit protein complex (often called the RNAi-induced silencing complex (RISC)), which guides the siRNA into the target RNA sequence (Nykanen et al. (2001), Cell, 107:309-21). The siRNA double strand will unwind at some point, while the antisense strand appears to remain bound to RISC and guide the degradation of the complementary mRNA sequence via a combination of endonucleases and exonucleases (Martinez et al. (2002), Cell, 110: 563-74). However, the function or use of iRNA or siRNA is not limited to any particular mechanism.

[0220] Short interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific posttranscriptional gene silencing, thereby reducing or even suppressing gene expression. In one instance, siRNA triggers specific degradation of homologous RNA molecules (such as mRNA) within a region of sequence identity between the siRNA and the target RNA. For example, WO 02 / 44321 discloses that siRNA, when paired with a 3' overhang, can cause sequence-specific degradation of target mRNA, and the method for preparing these siRNAs is incorporated herein by reference.

[0221] In mammalian cells, sequence-specific gene silencing can be achieved using artificially synthesized short double-stranded RNAs that mimic siRNA produced by the dicer enzyme (Elbashir et al. (2001), Nature, 411:494498)(Ui-Tei et al. (2000), FEBS Lett 479:79-82). siRNA can be synthesized chemically or in vitro, or it can be the result of processing short double-stranded hairpin-like RNA (shRNA) into siRNA within cells. siRNA synthesis is typically 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, Netherlands). siRNA can also be synthesized using Ambion's... siRNA construction kits and other reagent kits are synthesized in vitro.

[0222] A more common method for producing siRNA from vectors is through transcription of short hairpin RNAse (shRNA). Kits for producing shRNA vectors are available, such as Imgenex's Genesuppressor. TM Building kits and Invitrogen's BLOCK-IT TM Inducible RNAi plasmids and lentiviral vectors.

[0223] In some embodiments, the functional nucleic acid is siRNA, shRNA, or miRNA. In some embodiments, the composition includes a vector expressing the functional nucleic acid.

[0224] (3) Aptamers

[0225] Functional nucleic acids can be aptamers or coding aptamers. An aptamer is a molecule that interacts with a target molecule, preferably in a specific manner. Typically, aptamers are small nucleic acids of 15-50 bases in length that can fold into defined secondary and tertiary structures, such as stem-loops or G-tetramers. Aptamers can bind to small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. Aptamers can bind to molecules with a Kd of less than 10. -12 The target molecules of M bind very tightly. Preferred aptamers have a Kd value less than 10. -6 10 -8 10 -10Or 10 -12 The aptamer binds to the target molecule with extremely high specificity. For example, some isolated aptamers have binding affinities with the target molecule that differ from those of another molecule by more than 10,000 times, even though they differ only at one position. Preferably, the dissociation constant (Kd) of the aptamer with the target molecule is at least 10, 100, 1000, 10,000, or 100,000 times lower than that with the background molecules. When comparing molecules such as peptides, a different peptide is preferred as the background molecule.

[0226] (4) Ribozyme

[0227] Functional nucleic acids can be ribozymes or ribozyme-encoding ribonucleases. Ribozymes are nucleic acid molecules capable of catalyzing intramolecular or intermolecular chemical reactions. Preferably, ribozymes catalyze intermolecular reactions. There are many different types of ribozymes that catalyze reactions of the nuclease or nucleic acid polymerase type, which are based on ribozymes found in natural systems, such as hammerhead ribozymes. Some ribozymes are not present in natural systems but have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, more preferably RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate and subsequently cleaving it. This recognition is usually based primarily on canonical or non-canonical base pair interactions. Because target substrate recognition is based on the target substrate sequence, this characteristic makes ribozymes the best candidates for targeted and specific cleavage of nucleic acids.

[0228] (5) External guidance sequence

[0229] Functional nucleic acids can be external guide sequences or molecules encoding external guide sequences. An external guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex. RNase P recognizes this complex and then cleaves the target molecule. EGS can be engineered specifically for selected RNA molecules. RNase P helps process transportable ribonucleic acid (tRNA) within the cell. Bacterial RNase P can be recruited using EGS to cleave almost any RNA sequence, as EGS makes the target RNA:EGS complex mimic the natural tRNA substrate. Similarly, eukaryotic EGS / RNase P-guided RNA cleavage can be used to cleave desired targets within eukaryotic cells. Representative examples of how EGS molecules are manufactured and used to facilitate the cleavage of various target molecules are well known in the art.

[0230] Methods for preparing and using vectors for expressing functional nucleic acids (such as antisense oligonucleotides, siRNA, shRNA, miRNA, EGSs, ribozymes, and aptamers) in vivo are known in the art.

[0231] (6) Cyclic dinucleotides

[0232] In some embodiments, the 4H2 antibody is administered in combination with an immunostimulatory oligonucleotide. The immunostimulatory oligonucleotide may be commercially available and can therefore be administered in combination with the antibody or alone. In some embodiments, the immunostimulatory oligonucleotide is a cyclic dinucleotide.

[0233] Functional nucleic acids can be cyclic dinucleotides or encode cyclic dinucleotides. Circular dinucleotides bind directly to the STING adapter protein, thereby generating IFN-β (Zhang et al., Mol Cell., 51(2):226-35(2013). doi:10.1016 / j.molcel.2013.05.022.). Several typical and atypical 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), fluorinated 3'3'-cGAMP, fluorinated c-di-GMP, or 2'3'-c-di-GMP, c-di-AMP, c-di-GMP, c AIMP (CL592), cAIMP difluoride (CL614), cAIM(PS)2 difluoride (Rp / Sp) (CL656), fluorinated c-di-AMP, 2'3'-c-di-AMP, 2'3'-c-di-AM(PS)2(Rp,Rp), 2'3'-c-di-AM(PS)2(Rp,Rp), fluorinated c-di-GMP, 2'3'-c-di-GMP, c-di-IMP and DMXAA.

[0234] (7) Immunostimulatory oligonucleotides

[0235] In some implementations, the immunostimulatory oligonucleotide is an oligonucleotide ligand or encoding an oligonucleotide ligand. Examples include, but are not limited to, pattern recognition receptor (PRR) ligands.

[0236] Examples of pattern recognition receptors include Toll-like family signaling molecules that play a role in the initiation of innate immune responses, while also influencing subsequent and more antigen-specific adaptive immune responses. Therefore, oligonucleotides can serve as ligands for Toll-like family signaling molecules such as Toll-like receptor 9 (TLR9). For example, TLR9 on human plasmacytoid dendritic cells and B cells can detect unmethylated CpG sites (Zaida et al., Infection and Immunity, 76(5): 2123-2129, (2008)). Thus, oligonucleotide sequences can include one or more unmethylated cytosine-guanine (CG or CpG, interchangeable) dinucleotide motifs. "p" refers to the phosphodiester backbone of DNA; however, in some embodiments, oligonucleotides including CG may have a modified backbone, such as a phosphate thioester (PS) backbone.

[0237] In some implementations, the oligonucleotide may include more than one CG dinucleotide, which may be arranged sequentially or separated by intermediate nucleotides. The CpG motif may be located within the oligonucleotide sequence. Many nucleotide sequences can stimulate TLR9, but the number and position of the CG dinucleotides, as well as the precise base sequences flanking the CG dinucleotides, vary. Many nucleotide sequences can stimulate TLR9 through variations in the number and position of the CG dinucleotides, and variations in the precise base sequences flanking the CG dinucleotides.

[0238] CG ODNs are typically classified based on their sequence, secondary structure, and effects on human peripheral blood mononuclear cells (PBMCs). These five classes are A (type D), B (type K), C, P, and 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 interferons (such as IFNα) and induce dendritic cell (DC) maturation. Some types of ODNs are also potent activators of natural killer (NK) cells via indirect cytokine signaling. Some types of ODN are also strong stimulants for the maturation of human B cells and monocytes (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 included in this article by reference).

[0239] Other PRR Toll-like receptors include TLR3 and TLR7, which can recognize double-stranded RNA, single-stranded RNA, and short double-stranded RNA, respectively, as well as retinoic acid-induced gene I (RIG-I)-like receptors, namely RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are known RNA-sensing receptors in the cytoplasm.

[0240] RIG-I (retinoic acid-induced protein 1, also known as Ddx58) and MDA-5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard) are cytoplasmic RNA helicases belonging to the RIG-I-like receptor (RLR) family and are crucial for the host's antiviral response.

[0241] RIG-I and MDA-5 sense the replication intermediate double-stranded RNA (dsRNA) of RNA viruses and send signals through the mitochondrial antiviral signaling protein MAVS (also known as IPS-1, VISA, or Cardif) to produce type I interferons (IFN-α and IFN-β).

[0242] The viral RNA detected by RIG-I has an uncapped 5'-bisphosphate or triphosphate end and a short, blunt-ended double strand, two fundamental characteristics that help distinguish it from its own RNA. The characteristics of the MDA-5 physiological ligand are not fully understood. However, RIG-I and MDA-5 exhibit different dependencies on the length of double-stranded RNA (dsRNA): RIG-I selectively binds to short dsRNA, while MDA-5 selectively binds to long dsRNA. Consistent with this, RIG-I and MDA-5 show different length preferences when binding to the synthetic dsRNA analog Poly(I:C).

[0243] In some cases, RIG-I can also indirectly sense dsDNA. Viral dsDNA can be transcribed by RNA polymerase III into dsRNA with a 5'-triphosphate group. Therefore, the synthetic analog of type B DNA, Poly(dA:dT), constitutes another RIG-I ligand.

[0244] Exemplary RIG-I ligands include, but are not limited to, 5'ppp-dsRNA, RIG-I specific agonists; 3p-hpRNA, RIG-I specific agonists; Poly(I:C) / LyoVec complexes recognized by RIG-I and / or MDA-5 according to the poly(I:C) size; and Poly(dA:dT) / LyoVec complexes indirectly recognized by RIG-I.

[0245] In some implementations, the oligonucleotide contains a functional ligand of TLR3, TLR7, TLR8, TLR9, or a RIG-I-like receptor or a combination thereof.

[0246] Examples of immunostimulatory oligonucleotides and methods of their manufacture are known in the art and are commercially available, for example, see Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporat, which is incorporated herein by reference.

[0247] C. Composition of goods

[0248] The disclosed nucleic acid cargo can be DNA or RNA nucleotides or include DNA or RNA nucleotides. They typically consist of a heterocyclic base (nucleic acid base), a glycosyl group attached to the heterocyclic base, and a phosphate group that functionalizes the hydroxyl group of the glycosyl group through esterification. Major natural nucleotides include uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases, and ribose or deoxyribose linked by phosphodiester bonds.

[0249] In some embodiments, the cargo comprises or consists of chemically modified nucleotide analogs that, relative to their DNA or RNA counterparts, may have improved stability, half-life, or specificity or affinity for a target receptor. Chemical modifications include modifications to nucleobases, sugar molecules, nucleotide linkages, or combinations thereof. As used herein, “modified nucleotide” or “chemically modified nucleotide” refers to a nucleotide in which one or more components of a heterocyclic base, sugar group, or phosphate group have been chemically modified. In some embodiments, the modified nucleotide has a reduced charge compared to DNA or RNA with the same nucleobase sequence. For example, oligonucleotides may carry a low negative charge, no charge, or a positive charge.

[0250] Typically, nucleoside analogs support bases capable of forming hydrogen bonds with standard polynucleotide bases via Watson-Crick base pairing, where the analog backbone presents bases in a manner that allows the oligonucleotide analog molecule to form hydrogen bonds with bases in standard polynucleotides (such as single-stranded RNA or single-stranded DNA) in a sequence-specific manner. In some embodiments, the analog has a substantially uncharged phosphorus-containing backbone.

[0251] i. Heterocyclic bases

[0252] Naturally occurring major nucleotides include heterocyclic bases such as uracil, thymine, cytosine, adenine, and guanine. Goods may include chemical modifications to their nucleobase components. Chemical modifications of heterocyclic bases or heterocyclic base analogs can effectively improve affinity or stability for 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-oxoadenine, pseudocytosine, pseudoisocytosine, 5- and 2-amino-5-(2'-deoxy-β-D-furanoribosyl)pyridine (2-aminopyridine), and various pyrrolopyrimidine and pyrazolopyrimidine derivatives.

[0253] ii. Carbohydrate modification

[0254] The cargo molecule may also include nucleotides with modified glycosyl groups or glycosyl analogs. Glycosyl modifications include, but are not limited to, 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidinoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME), and 2'-O-(N-(methyl)acetamido) (2'-OMA). 2'-O-aminoethyl sugar substitutes are particularly preferred because they protonate at neutral pH, thereby suppressing charge repulsion between TFO and the target duplex. This modification stabilizes the C3'-inner conformation of ribose or deoxyribose and can also form a bridging structure with the i-1 phosphate in the double-stranded purine chain.

[0255] In some embodiments, the nucleic acid is a morpholine oligonucleotide. A morpholine oligonucleotide typically consists of two or more morpholine monomers containing purine or pyrimidine base-pairing moieties, which can bind to bases in a polynucleotide via base-specific hydrogen bonds. The purine or pyrimidine base-pairing moieties are typically adenine, cytosine, guanine, uracil, or thymine. The synthesis, structure, and binding properties of morpholine oligomers are detailed in U.S. Patents 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

[0256] Key properties of morpholino-based subunits typically include: the ability to link in oligomer form via a stable, uncharged backbone; and the ability to support nucleotide bases (such as adenine, cytosine, guanine, thymidine, uracil, or inosine), thereby enabling the formed polymer to bind with high T... M Hybridization with target nucleic acids (including target RNA) with complementary bases, and even with oligomers as short as 10-14 bases; oligomers can be actively transported into mammalian cells; and oligomers: RNA heteroduplexes have the ability to resist RNAse degradation.

[0257] In some embodiments, as described above, the oligonucleotide employs a morpholino subunit containing a base-pairing moiety and is linked by an uncharged linker arm. For example, the morpholino oligonucleotide can be a phosphodiamidomorpholino oligomer.

[0258] iii. Nucleotide linkage

[0259] Oligonucleotides are linked by nucleotide bonds, which are chemical linkages between two nucleoside units. Modifying the phosphate backbone of DNA or RNA oligonucleotides can increase their binding affinity or stability, or decrease their sensitivity to nuclease digestion. Cationic modifications, including but not limited to diethylethylenediamine (DEED) or dimethylaminopropylamine (DMAP), can be particularly useful due to their ability to reduce electrostatic repulsion between the oligonucleotide and its target. Modification of the phosphate backbone also includes replacing a non-bridging oxygen atom in a phosphodiester bond with a sulfur atom. This substitution creates a phosphate-thionucleotide inter-bond that replaces the phosphodiester bond. Oligonucleotides containing phosphate-thionucleotide inter-bonds have been shown to be more stable in vivo.

[0260] Examples of charge-reduced modified nucleotides include modified nucleotide linkages, such as phosphate analogs with achiral and uncharged subunit linkages (e.g., Stercak, EP, et al., Organic. Chem., 52:4202, (1987)), and uncharged morpholino polymers with achiral subunit linkages (as described above, see, for example, U.S. Patent 5,034,506). Some nucleoside linkage analogs include morpholino, acetal, and polyamide linkages with heterocycles.

[0261] In another embodiment, the cargo consists of locked nucleic acids. Locked nucleic acids (LNAs) are modified RNA nucleotides (e.g., see Braasch et al., Chem. Biol., 8(1):1-7(2001)). The hybrids formed by LNAs with DNA are more stable than DNA / DNA hybrids, a property similar to peptide nucleic acid (PNA) / DNA hybrids. Therefore, LNAs can be used like PNA molecules. In some embodiments, the binding efficiency of LNAs can be improved by adding a positive charge. Commercial nucleic acid synthesizers and standard phosphoramide chemistry methods can be used to manufacture LNAs.

[0262] In some implementations, the cargo consists of peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetically produced DNA mimics in which the phosphate backbone of an oligonucleotide is entirely replaced by repeating N-(2-aminoethyl)glycine units, and the phosphodiester bonds are typically replaced by peptide bonds. Various heterocyclic bases are linked to the backbone via methylene carbonyl bonds. Peptide nucleic acids (PNAs) maintain a similar heterocyclic base spacing to conventional DNA oligonucleotides, but are achiral and neutrally charged molecules. Peptide nucleic acids are composed of peptide nucleic acid monomers.

[0263] Other skeletal modifications include changes and modifications to peptides and amino acids. Therefore, the skeletal components of oligonucleotides such as PNA can be peptide chains or non-peptide peptide chains. Examples include acetyl caps, amino spacers (such as 8-amino-3,6-dioxanoic acid) (referred to herein as the O-linker arm), amino acids (such as lysine) (especially useful if a positive charge is desired for the PNA), etc. Chemical assembly methods for PNA are well known. See, for example, U.S. Patents 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571, and 5,786,571.

[0264] Optionally, the cargo includes one or more terminal residues, or is modified at one or both ends to increase stability and / or the oligonucleotide's affinity for its target. Commonly used positively charged groups include amino acids such as lysine and arginine, but other positively charged groups may also be useful. The cargo may be further modified to use a propylamine group for end capping to prevent degradation. 3' or 5' capping procedures for oligonucleotides are well known in the art.

[0265] In some implementations, nucleic acids can be single-stranded or double-stranded.

[0266] iv. Fine-tuning

[0267] These characteristics can be used to modify the cargo sequence and fine-tune the binding strength between the cargo and the 4H2 binding protein.

[0268] 4H2 antibodies bind to guanine, thus increasing the amount of guanine and / or selecting the position of guanine in the polynucleotide sequence to increase antibody binding affinity, create antibody binding sites, increase the number of antibodies binding to a single polynucleotide, and / or target antibodies to specific positions on the polynucleotide. Alternatively, reducing the amount of guanine in the polynucleotide and / or selecting guanine deletions in the polynucleotide sequence can also be used to increase antibody binding, reduce or remove antibody binding sites, reduce the number of antibodies binding to a single polynucleotide, and / or target antibodies to alternative positions on the polynucleotide.

[0269] For example, any disclosed cargo may comprise or consist of guanine (G) (e.g., single G, double G, or multiple G) alone or in combination with 2, 3, 4, or more adenine (A), thymine (T), cytosine (C), uracil (U), or inosine (I). In some embodiments, a synthetic non-coding sequence is added to the cargo to increase or decrease binding to 4H2-binding proteins. This sequence may, but is not necessarily, be located at the 5' or 3' end of the nucleic acid cargo. The cargo may be single-stranded or double-stranded DNA or RNA.

[0270] Alternatively, these binding properties can be used to increase binding preference (e.g., preference for guanine) or decrease binding preference (e.g., preference for adenine (A), thymine (T), cytosine (C), uracil (U), or inosine (I)) during the rational design of the nucleic acid sequence of the cargo.

[0271] 2. Vaccine preparations

[0272] Vaccines require a strong immune response. The 4H2 antibody described herein can be administered as a component of a vaccine to enhance the associated immune response. In some embodiments, the vaccine disclosed herein includes an adjuvant comprising the 4H2 antibody, an antigen, and optionally other additional pharmaceutical agents.

[0273] a. Antigen

[0274] Antigens can be peptides, proteins, polysaccharides, carbohydrates, lipids, nucleic acids, or combinations thereof. Antigens can originate from transformed cells such as cancer cells or leukemia cells, or they can be whole cells or their immunogenic components. Suitable antigens are known in the art and are available from commercial, governmental, and scientific sources.

[0275] Antigens can be purified or partially purified peptides derived from tumors, or recombinant peptides produced by expressing DNA encoding peptide antigens in a heterologous expression system. Antigens can be DNA or RNA (e.g., mRNA) encoding all or part of the antigen protein. The DNA can be in the form of vector DNA, such as viral vectors or plasmid DNA.

[0276] Antigens can be provided alone or in combination. Furthermore, antigens can also be provided as a complex mixture of peptides or nucleic acids.

[0277] For example, antigens can be tumor antigens, or they can be derived from infectious disease pathogens or diseases that require vaccination, such as polio, tetanus, influenza, hepatitis B, hepatitis A, hepatitis C, rubella, Hib, measles, pertussis, pneumococcal disease, HIV, SAR-CoV-2, or any other infection and disease discussed in detail below.

[0278] i. Viral antigens

[0279] Viral antigens can be isolated from any virus, including but not limited to viruses from any of the following families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, and Co. The family Coronaviridae (e.g., Coronaviridae, such as Severe Acute Respiratory Syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus strains such as Zaire, Reston, Ivory Coast, or Sudan types)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus type 1, Dengue virus type 2, Dengue virus type 3, and Dengue virus type 4), Hepadnaviridae, and Herpesviridae (e.g., Human herpesviruses 1, 3, 4, 5, and 6).And cytomegaloviruses), Hypoviridae, Hypoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (such as influenza A, B, and C viruses), Papovaviridae, Paramyxoviridae (such as measles virus, mumps virus, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (such as poliovirus, rhinovirus, hepatitis virus, and foot-and-mouth disease virus), Poxviridae (such as vaccinia virus and smallpox virus), Reoviridae (such as rotavirus), Retroviridae (such as lentiviruses, such as human immunodeficiency virus (HIV) 1 and HIV). 2) 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 part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2, and dengue D1NS3.

[0280] Viral antigens can originate from specific viral strains or combinations of viral strains, such as SAR-CoV-2, papillomavirus, herpesvirus (i.e., herpes simplex virus types 1 and 2); hepatitis viruses, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis 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 lymphochoroidal meningitis virus.

[0281] ii. Bacterial antigens

[0282] Bacterial antigens can originate from any bacteria, including but not limited to Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella pertussis, Borrelia, Campylobacter, Caurobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia coli, Francisella, Halobacterium, Heliobacter, Haemophilus influenzae, and Hemophilus influenzae type b. B, HIB), Hyphomicrobium, Legionella pneumophila, Leptospira, Listeria monocytogenes, Meningococcus A, B and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia (tsia), Salmonella, Shigella, Spirillum, Spirhaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio cholerae, Yersinia.

[0283] iii. Parasite antigens

[0284] Parasitic antigens can be acquired from parasites, such as, but not limited to, those 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 antigens include sporozoan antigens, Plasmodium antigens, such as cyclosporin, sporozoite surface proteins, hepatic antigens, apical membrane-associated proteins, or all or part of merozoite surface proteins.

[0285] iv. Tumor antigens

[0286] Antigens can be tumor antigens, including tumor-associated antigens or tumor-specific antigens, such as, but not limited to, α-actin-4, Bcr-Abl fusion protein, Casp-8, β-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, triose phosphate 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 (Pmel17), 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, b-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27).Tumor antigens, such as BCG, can also be used as adjuvants to enhance immunity. These include: 29 (BCAA), CA 195, CA 242, CA-50, CAM43, CD68 / KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclic protein C-associated protein), TAAL6, TAG72, TLP, and TPS. Tumor antigens, such as BCG, can also be used as adjuvants to enhance immunity.

[0287] b. Adjuvants

[0288] Optionally, the vaccines described herein may include adjuvants. Adjuvants may be, but are not limited to, one or more of the following: oil emulsions (such as Freund's adjuvant), saponin preparations, virions and virus-like particles, bacterial and microbial derivatives, immunostimulatory oligonucleotides, ADP-ribosylated toxins and their detoxified derivatives, alum, BCG, mineral-containing components (such as mineral salts, such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.), bioadhesives and / or mucus adhesives, microparticles, liposomes, polyoxyethylene ethers and polyoxyethylene ester preparations, polyphosphazenes, muramyl peptides, imidazoquinone compounds, and surfactants (such as lysophosphatidylcholine, poloxamer polyols, polyanionic peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0289] Adjuvants may also include immunomodulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12), interferons (e.g., interferon-γ), macrophage colony-stimulating factors, and tumor necrosis factor. In addition to PD-1 antagonists, other co-stimulatory molecules, including other peptides of the B7 family, may be administered. These protein adjuvants can be provided as full-length peptides or their active fragments, or as RNA or DNA (e.g., plasmid DNA).

[0290] 3. Immune checkpoint modulators

[0291] 4H2 antibodies can be used in combination with immune checkpoint modulators.

[0292] Immune checkpoints can be either stimulatory or inhibitory, and tumors can utilize these checkpoints to protect themselves from attacks by the immune system. Currently approved checkpoint therapies block inhibitory checkpoint receptors, but research is underway on therapies that activate stimulatory checkpoints. Therefore, immune checkpoint modulators can be modulators that block inhibitory checkpoints or modulators that activate stimulatory checkpoints. Typically, immune checkpoint modulators are modulators that can induce or otherwise activate or increase an immune response against target cells, such as cancer cells or infected cells. Thus, in some embodiments, immune checkpoint modulators can be chimeric antigen receptor (CAR)-directed cells, such as CAR-T cells. In another embodiment, immune checkpoint modulators can be oncolytic viruses.

[0293] In a preferred embodiment, the immune checkpoint modulator blocks inhibitory checkpoints. Therefore, blocking negative feedback signaling to immune cells can enhance the immune response against tumors. Thus, in some embodiments, an effective amount of the immune checkpoint modulator is administered to the subject to block inhibitory checkpoints. Exemplary compounds are those that block or otherwise inhibit, for example, PD-1, PD-L1, or CTLA4.

[0294] a. PD-1 antagonists

[0295] In some implementations, the active agent is a PD-1 antagonist. T cell activation typically depends on antigen-specific signaling following contact between the T cell receptor (TCR) and an antigenic peptide presented via the major histocompatibility complex (MHC), and the extent of this response is controlled by antigen-independent positive and negative signals from various co-stimulatory molecules. These are typically members of the CD28 / B7 family. Conversely, programmed death-1 (PD-1), a member of the CD28 receptor family, elicits a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response, thereby reducing T cell proliferation and / or the intensity and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Patents 8,114,845, 8,609,089 and 8,709,416, including compounds or agents that bind to and block PD-1 ligands to interfere with or inhibit the binding of ligands to PD-1 receptors, or compounds or agents that directly bind to and block PD-1 receptors without inducing inhibitory signal transduction through PD-1 receptors.

[0296] In some implementations, PD-1 receptor antagonists bind directly to the PD-1 receptor without triggering inhibitory signal transduction, while also binding to ligands of the PD-1 receptor to reduce or inhibit ligand-triggered signal transduction via the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are attenuated by the negative signal transmitted by PD-1 signaling, thereby enabling a more robust immune response.

[0297] It is currently believed that PD-1 signaling is driven by binding to PD-1 ligands (such as B7-H1 or B7-DC) and being closely adjacent to peptide antigens presented by the major histocompatibility complex (MHC) (e.g., see Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)). Therefore, proteins, antibodies, or small molecules that can prevent the co-binding of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0298] In a preferred embodiment, 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 PD-1 itself, particularly where the co-binding of PD-1 with the TCR does not follow such a binding, thereby preventing the triggering of inhibitory signal transduction through the PD-1 receptor. Other PD-1 antagonists include antibodies that bind to PD-1 or PD-1 ligands such as PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), as well as other antibodies.

[0299] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following literature:

[0300] PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196)

[0301] PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168)

[0302] PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708)

[0303] PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771)

[0304] PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286)

[0305] PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875)

[0306] PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174)

[0307] PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874)

[0308] PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533)

[0309] Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0310] A specific example of an anti-PD-1 antibody is MDX-1106 (see Kosak, US20070166281 (published July 19, 2007), paragraph 42), which is a human anti-PD-1 antibody, preferably administered at a dose of 3 mg / kg.

[0311] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following literature:

[0312] PCT / US06 / 022423 (WO / 2006 / 133396, published on December 14, 2006)

[0313] PCT / US07 / 088851 (WO / 2008 / 083174, published on July 10, 2008)

[0314] US2006 / 0110383 (Published on May 25, 2006)

[0315] A specific example of an anti-B7-H1 antibody is MDX-1105 (WO / 2007 / 005874, published on January 11, 2007), which is a human anti-B7-H1 antibody.

[0316] For information on anti-B7-DC antibodies, please refer to 7,411,051, 7,052,694, 7,390,888 and U.S. Publication Application 2006 / 0099203.

[0317] The antibody can be a bispecific antibody, including an antibody that binds to the PD-1 receptor, which is bridged to a PD-1 ligand (such as B7-H1). In some embodiments, the PD-1 binding portion may reduce or inhibit signal transduction through the PD-1 receptor.

[0318] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC peptides, including homologs and variants of these peptides, active fragments of any of the aforementioned peptides, and fusion proteins incorporating any of these peptides. 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) and does not contain all or part of the transmembrane region of human B7-DC.

[0319] PD-1 antagonists can also be fragments of mammalian B7-H1, preferably derived from mice or primates, and more preferably humans, wherein the fragment binds to and blocks PD-1, but does not cause inhibitory signal transduction via PD-1. The fragment can also be part of a fusion protein, such as an Ig fusion protein.

[0320] Other useful peptide PD-1 antagonists include peptides that bind to ligands of the PD-1 receptor. These peptides include the PD-1 receptor protein or soluble fragments thereof, which bind to PD-1 ligands (such as B7-H1 or B7-DC) and prevent binding to the endogenous PD-1 receptor, thereby inhibiting inhibitory signal transduction. B7-H1 can also bind to protein B7.1 (Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). These fragments also include soluble ECD portions of the PD-1 protein, which include mutations, such as the A99L mutation, that increase binding to the native ligand (Molnar et al., PNAS, 105:10483-10488 (2008)). B7-1 or soluble fragments thereof that can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby inhibiting inhibitory signal transduction, are also useful.

[0321] PD-1 and B7-H1 antisense nucleic acids (DNA and RNA) and siRNA molecules can also be PD-1 antagonists. These antisense molecules can prevent the expression of PD-1 on 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 in length, specific to genes encoding PD-1 or PD-1 ligands, and readily commercially available) complexed with a vector such as polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest. 119(8):2231-2244(2009)) is readily taken up by cells expressing PD-1 and PD-1 ligands, reducing the expression of these receptors and ligands, thereby reducing inhibitory signal transduction in T cells and activating T cells.

[0322] Exemplary PD-1 inhibitors include, but are not limited to,

[0323] Pembrolizumab (formerly known as MK-3475 or lambolizumab, Keytruda) was developed by Merck and first approved by the U.S. Food and Drug Administration in 2014 for the treatment of melanoma.

[0324] Nivolumab (Opdivo), developed by Bristol-Myers Squibb, was first approved by the U.S. Food and Drug Administration in 2014 for the treatment of melanoma.

[0325] -pidilizumab, developed by CureTech

[0326] AMP-224, developed by GlaxoSmithKline and MedImmune

[0327] AMP-514, developed by GlaxoSmithKline and MedImmune

[0328] - Novartis' PDR001

[0329] - Cemiliplimab, developed by Regeneron and Sanofi

[0330] Exemplary PD-L1 inhibitors include, but are not limited to,

[0331] Atezolizumab (Tecentriq) is a fully humanized IgG1 (immunoglobulin 1 antibody) developed by Roche Genentech. In 2016, the FDA approved atezolizumab for the treatment of urothelial carcinoma and non-small cell lung cancer.

[0332] Avelumab (Bavencio) is a fully human IgG1 antibody developed by Merck Serono and Pfizer. Avelumab has been approved by the FDA for the treatment of metastatic Merkel cell carcinoma. However, its Phase III clinical trial for gastric cancer failed.

[0333] Durvalumab (Imfinzi) is a fully human IgG1 antibody developed by AstraZeneca. Durvalumab has been approved by the FDA for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer following chemotherapy.

[0334] BMS-936559 developed by Bristol-Myers Squibb

[0335] CK-301 developed by Checkpoint Therapeutics

[0336] See, for example, Iwai et al., Journal of Biomedical Science, (2017) 24:26, DOI 10.1186 / s12929-017-0329-9.

[0337] b. CTLA4 antagonists

[0338] Other molecules that contribute to mediating T-cell effects in the immune response may also be considered as active agents. For example, in some embodiments, the molecule is a formulation that binds to a non-PD-1 immune response mediating molecule. In a preferred embodiment, the molecule is a CTLA4 antagonist, such as an anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies are described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0339] The dosages of 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, preferably a narrower range of 1 to 50 mg / kg, and more preferably a range of 10 to 20 mg / kg. For human subjects, a suitable dosage is 5 to 15 mg / kg, and most preferably 10 mg / kg of antibody (e.g., human anti-PD-1 antibody, such as MDX-1106).

[0340] Specific examples of CTLA antagonists include ipilimumab, also known as MDX-010 or MDX-101, a human anti-CTLA4 antibody, with a preferred dose of approximately 10 mg / kg, and tremelimumab, a human anti-CTLA4 antibody, with a preferred dose of approximately 15 mg / kg. See also Sammartino et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0341] In other implementations, the antagonist is a small molecule. A range of small organic compounds have been shown to bind to the B7-1 ligand, preventing binding to CTLA4 (see Erbe et al., J. Biol. Chem., 277:7363-7368 (2002)). These small organic molecules can be administered alone or in combination with anti-CTLA4 antibodies to reduce inhibitory signal transduction in T cells.

[0342] c. Chimeric antigen receptor-guided cells

[0343] The modulator can be a chimeric antigen receptor-directed cell. As used herein, the term "chimeric antigen receptor" or "CAR" refers to a group of polypeptides, typically two in the simplest embodiment, that, when present in immune effector cells, provide the cells with specificity for cancer cells and generate intracellular signaling. In some embodiments, the CAR includes at least an antigen-binding domain, such as an extracellular binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to as an "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulating molecule and / or a co-stimulating molecule as defined below. In one embodiment, the stimulating molecule is a zeta chain (ζ chain) ("zeta stimulating domain") associated with the T cell receptor complex. In one embodiment, the cytoplasmic signaling domain also includes one or more functional signaling domains derived from at least one co-stimulating molecule (e.g., 4-1BB (i.e., CD137), CD27, and / or CD28). In some embodiments, the CAR includes a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes a functional signaling domain derived from a stimulating molecule. In various embodiments, the CAR is a fusion protein of a single-stranded variable fragment (scFv) fused to the CD3-zeta transmembrane domain. However, other intracellular signaling domains, such as CD28, 41-BB, and Ox40, can also be used in various combinations to provide the desired intracellular signaling. In some embodiments, the CAR disclosed herein includes an extracellular binding domain.

[0344] As used herein, the term "antigen-binding domain" refers to the portion of a CAR that specifically recognizes and binds to a target antigen. The "antigen-binding domain" may be derived from the binding proteins disclosed herein, such as antibodies or fragments thereof. In some embodiments, the "binding domain" is a single-chain variable fragment (scFv). In some embodiments, the "binding domain" includes the complementarity-determining region of a binding protein disclosed herein. In this embodiment, the CAR-guided cell may represent a combination of a 4H2 cell-penetrating antibody (assuming it penetrates cancer cells) or a combination thereof that induces cGAS / STING signaling, along with an immune checkpoint modulator that induces, increases, or enhances an immune response. For example, the binding domain may represent a cell-penetrating antibody, and the modified T cell may represent an immune cell modulator. In another example, the CAR-guided cell disclosed herein is administered together with the cell-penetrating 4H2 antibody disclosed herein.

[0345] As used herein, the terms “zeta” or “CD3-zeta” are used to define proteins provided in GenBan Acc. BAG36664.1, or equivalent residues derived from non-human species. “Zeta-stimulatory domain” or alternatively “CD3-zeta-stimulatory domain” is defined as an amino acid residue of the zeta chain cytoplasmic domain or a functional derivative thereof that is sufficient to functionally deliver the initial signal required for T cell activation.

[0346] As used herein, the term "immune effector cell" refers to a cell that participates in an immune response (such as promoting an immune effector reaction). Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes. In some embodiments, the immune effector cells are heterologous. In some embodiments, the immune effector cells are autologous. In some embodiments, the immune checkpoint regulator is a CAR-guided T cell (CAR-T cell). Exemplary CAR-T cells include Axicabtagene ciloleucel (KTE-C19, Axi-cel), Tisagenlecleucel, and Lisocabtagene Maraleucel (liso-cel; JCAR017).

[0347] Immune effector cells, such as T cells, can typically be activated and expanded using methods previously described, such as those described in U.S. Patents 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. For example, immune effector cell populations (e.g., exhausted T regulatory cells) can be expanded by contact with a surface coated with a formulation that stimulates CD3 complex-related signaling and a ligand that stimulates co-stimulatory molecules on the T cell surface.

[0348] d. Oncolytic virus

[0349] The regulator can be an oncolytic virus. In this disclosure, the term "oncolytic virus" refers to a virus capable of infecting cancer cells and reducing their growth. For example, an oncolytic virus can inhibit cell proliferation. In some embodiments, the oncolytic virus can kill cancer cells. In some embodiments, the oncolytic virus preferentially infects cancer cells and inhibits their growth compared to corresponding normal cells. In another embodiment, the oncolytic virus preferentially replicates in cancer cells and inhibits their growth compared to corresponding normal cells.

[0350] In some implementations, 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 capable of naturally infecting and reducing the growth of cancer cells typically target cancer cells by exploiting cellular aberrations occurring within them. For example, oncolytic viruses may utilize defects in surface attachment receptors, activated oncogenes (such as Ras, Akt, p53), and / or interferon (IFN) pathways.

[0351] In another embodiment, the oncolytic viruses included in this disclosure are engineered to infect cancer cells and reduce their growth. 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 parvoviruses such as rodent proparvovirus H-1PV. In some embodiments, the oncolytic virus includes the backbone of the aforementioned viruses.

[0352] In some embodiments, oncolytic viruses can be engineered to be tumor-specific by mutating or deleting genes that are necessary for viral survival in normal cells but not in cancer cells. For example, oncolytic viruses can be engineered by mutating or deleting genes encoding thymidine kinase (an enzyme required for nucleic acid metabolism). In this example, the virus depends on the expression of cellular thymidine kinase, which is highly expressed in proliferating cancer cells but suppressed in normal cells. In another example, oncolytic viruses are engineered to include capsid proteins that bind to tumor-specific cell surface molecules. In some embodiments, the phage protein is a fibrin, pentamer, or hexamer. In yet another example, oncolytic viruses are engineered to include tumor-specific cell surface molecules for transduction targeting cancer cells. Exemplary tumor-specific cell surface molecules may include integrins, members of the epidermal growth factor receptor family, proteoglycans, disialotyl-gangliosides, 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 glycoproteins, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD155, MUC1, tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor α, transmembrane glycoprotein NMB, CC chemokine receptor, PSMA, RON-receptor, and cytotoxic T-lymphocyte antigen 4.

[0353] Oncolytic viruses can replicate. In some implementations, oncolytic viruses can selectively replicate in cancer cells compared to corresponding normal cells.

[0354] Conditional replication can be achieved, for example, by inserting a tumor-specific promoter to drive the expression of key genes. This promoter can be determined based on differences in gene expression between the tumor and the corresponding surrounding tissues. Exemplary natural promoters include AFP, CCKAR, CEA, erbB2, Cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, Survivin, TRP1, STAT3, hTERT, and Tyr. Exemplary compound promoters include AFP / hAFP, SV40 / AFP, CEA / CEA, PSA / PSA, SV40 / Tyr, and Tyr / Tyr.

[0355] Various viruses can be engineered using the methods described in the examples above. For instance, oncolytic viruses can be modified HSV, lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV), or recombinant forms such as recombinant adeno-associated virus (rAAV) or its derivatives, such as self-complementary adeno-associated virus (scAAV) or non-integrating adenovirus. Oncolytic viruses can be modified HSV oncolytic viruses or modified lentiviruses. Other example viruses include vaccine viruses, vesicular stomatitis virus (VSV), measles virus, and maraba virus.

[0356] In other instances, the oncolytic virus can be one of various AV or AAV serotypes. In some embodiments, the oncolytic virus is serotype 1. In another instance, the oncolytic virus is serotype 2. In other instances, the oncolytic virus is serotype 3, 4, 7, 8, 9, 10, 11, 12, or 13. In another instance, the oncolytic virus is serotype 5. In yet another instance, the oncolytic virus is serotype 6.

[0357] 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 WO 2003 / 080083, WO 2005 / 086922, WO 2007 / 088229, WO 2008 / 110579, WO2010 / 108931, WO 2010 / 128182, WO 2013 / 112942, WO 2013 / 116778, WO 2014 / 204814, WO2015 / 077624, WO 2015 / 166082, and WO 2015 / 089280.

[0358] e. Other immune checkpoint modulators

[0359] 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, etc., and are targeted alone for cancer treatment or in combination with anti-PD-1, anti-PD-L1, and anti-CTLA compounds for cancer treatment. For example, see 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. Therefore, in some embodiments, the 4H2 antibody is administered in combination with or in combination with compounds targeting ICOS, OX40, GITR, 4-1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B7-H3, KIR, or PARP, or in combination thereof, either alone or in combination with compounds targeting PD-1, PD-L1, and / or CTLA. In another embodiment, the immune checkpoint modulator is an antibody disclosed in WO 2016 / 013870.

[0360] C. Pharmaceutical Composition

[0361] This composition can be used in combination with a pharmaceutically acceptable carrier for therapeutic purposes.

[0362] The composition is preferably combined with a suitable pharmaceutical carrier for therapeutic purposes. Such compositions comprise an effective amount of the composition and a pharmaceutically acceptable carrier or excipient.

[0363] The composition can be a formulation administered topically, locally, or systemically in a suitable drug carrier. Typical carriers and preparation methods are disclosed in the 15th edition of Remington's Pharmaceutical Sciences, by E.W. Martin (Mark Publishing Company, 1975). Antibodies or complexes formed therefrom can also be encapsulated in suitable biocompatible particles formed from biodegradable or non-biodegradable polymers, proteins, or liposomes for cell targeting. Such systems are well known to those skilled in the art. In some embodiments, antibodies or complexes formed therefrom are encapsulated in nanoparticles.

[0364] Injectable formulations may be available in single-dose form, such as ampoules or multi-dose containers, and may optionally contain preservatives. Compositions may be in the form of sterile aqueous or non-aqueous solutions, suspensions, and emulsions, and in some embodiments may be isotonic with the subject's blood. Examples of non-aqueous solvents include polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or non-volatile oils including synthetic monoglycerides or diglycerides. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including physiological saline and buffer media. Parenteral carriers include sodium chloride solution, 1,3-butanediol, Ringer's glucose, glucose and sodium chloride, emulsified Ringer's solution, or non-volatile oils. Intravenous carriers include liquids and nutritional supplements, as well as electrolyte supplements (such as Ringer's glucose-based supplements). Materials may be solutions, emulsions, or suspensions (e.g., incorporated into particles, liposomes, or cells). Typically, a pharmaceutically acceptable salt is used in the formulation to make it isotonic. Trehalose may be added to the pharmaceutical composition, usually in an amount of 1-5%. The pH of the solution is preferably around 5 to 8, more preferably around 7 to 7.5.

[0365] 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 readily determine the various parameters for preparing and formulating compositions without requiring extensive experimentation.

[0366] The compositions can also be formulated into aerosol formulations (i.e., they can be “nebulized”) alone or in combination with other suitable ingredients for inhalation. Aerosol formulations can be contained in acceptable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, and air. For inhalation administration, the compounds can be applied as an aerosol spray using a pressurized pack or nebulizer with a suitable propellant.

[0367] In some embodiments, the compound includes a pharmaceutically acceptable carrier having a formulation component, such as a salt, carrier, buffer, emulsifier, diluent, excipient, chelating agent, preservative, solubilizer, or stabilizer.

[0368] Nucleic acids can be conjugated with lipophilic groups such as cholesterol, lauric acid, and lithocholic acid derivatives with a C32 functional group to enhance cellular uptake. For example, cholesterol has been shown to improve the uptake and serum stability of siRNA 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, studies have shown that steroid-conjugated oligonucleotides bind to different lipoproteins in the blood (such as low-density lipoprotein (LDL)) to protect their integrity and promote biodistribution (Rump et al., Biochem. Pharmacol, 59(11):1407-1416(2000)). Other groups that can be linked to or conjugated to the aforementioned nucleic acids to increase cellular uptake include: acridine derivatives; cross-linking agents such as psoralen derivatives, azidobenzoyl, propofol, and azidopropofol; artificial endonucleases; metal complexes such as EDTA-Fe(II) and porphyrin-Fe(II); alkylating groups; nucleases such as alkaline phosphatase; terminal transferases; antibody enzymes; cholesterol groups; lipophilic carriers; peptide conjugates; long-chain alcohols; phosphate esters; radiolabeled substances; non-radiolabeled substances; carbohydrates; and polylysine or other polyamines. U.S. Patent 6,919,208 to Levy et al. also describes methods for enhancing delivery. These pharmaceutical formulations can be manufactured using methods known per se, such as conventional mixing, dissolving, granulating, grinding, emulsifying, encapsulating, embedding, or lyophilizing processes.

[0369] Other carriers include sustained-release formulations, such as solid hydrophobic polymer semi-permeable matrices containing antibodies or complexes thereof, in the form of shaped particles, such as films, liposomes, or microparticles. Implantation includes insertion into implantable 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 administration of the composition with an aerosol in an inhaler, or administration alone or attached to an absorbable carrier. For systemic administration, the composition is preferably encapsulated in liposomes.

[0370] Invasive devices (such as vascular or urinary catheters) and interventional devices (such as stents with drug delivery capabilities and configured as dilatation devices or stent grafts) can be used to deliver the composition in a manner that enables tissue-specific absorption of the drug and / or nucleotide delivery systems.

[0371] Biodegradable implants can be used to deliver formulations via diffusion or degradation through a polymer matrix. In some embodiments, the administration of the formulation can be designed to provide continuous exposure to the composition over a period of time (e.g., hours, days, weeks, months, or years). This can be achieved, for example, by repeated administration of the formulation or by a sustained or controlled-release delivery system in which the composition can be administered long-term without repeated administration.

[0372] Other suitable delivery systems include sustained-release, extended-release, continuous-release, or controlled-release delivery systems. In many cases, these systems can avoid repeated administration, providing greater convenience for subjects and physicians. Many types of release delivery systems are available and are well known to those skilled in the art. For example, they include polymer-based systems such as polylactic acid and / or polyglycolic acid, polyanhydride, polycaprolactone, copolyoxalate, polyesteramide, polyorthoester, polyhydroxybutyrate, and / or combinations thereof. Microcapsules of the above polymers containing nucleic acids are described, for example, in U.S. Patent 5,075,109. Other examples include lipid-based non-polymer systems, including sterols such as cholesterol, cholesterol esters, fatty acids, or neutral fats such as monoglycerides, diglycerides, and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silica gel systems; peptide-based systems; wax-coated systems; compressed tablets using conventional adhesives and excipients; or partially fused implants. Formulations can be, for example, microspheres, hydrogels, polymer reservoirs, cholesterol matrices, or polymer systems. In some embodiments, the system can achieve sustained or controlled release of the composition by controlling the diffusion or erosion / degradation rate of the formulation containing the antibody or the complex formed therefrom.

[0373] The composition can be formulated for pulmonary or mucosal application. Application may include delivery of the composition to the mucosa of the lungs, nasal cavity, oral cavity (sublingual, oral), vagina, or rectum. As used herein, the term aerosol refers to any formulation of fine particulate matter, which may be a solution or suspension, whether or not it is generated using a propellant. Aerosols can be produced using standard techniques such as ultrasonic or high-pressure processing.

[0374] When administered via the upper respiratory tract, the formulation may be prepared as a solution, such as water or isotonic saline, a buffer or non-buffered solution, or a suspension, for intranasal administration as drops or sprays. Preferably, these solutions or suspensions are isotonic relative to nasal secretions and have approximately the same pH value, for example, from about pH 4.0 to about pH 7.4, or from about pH 6.0 to about pH 7.0. The buffer should be physiologically compatible; simple examples include phosphate buffer.

[0375] The composition can be delivered to target cells using particulate delivery carriers. Nanoparticles generally refer to particles with diameters ranging from 500 nm to less than 0.5 nm, preferably from 50 to 500 nm, and more preferably from 50 to 300 nm. The internalization of polymer particles within cells is highly dependent on their size; nanopolymer particles are internalized much more efficiently than micron-sized polymer particles. For example, studies by Desai et al. showed that 100 nm diameter nanoparticles were absorbed by cultured Caco-2 cells approximately 2.5 times more than 1 μM diameter particles (Desai et al., Pharm. Res., 14:1568-73 (1997)). Nanoparticles also have a greater ability to diffuse deep into tissues in vivo.

[0376] In some embodiments, the delivery carrier is a dendritic polymer.

[0377] Preferred examples of biodegradable polymers include synthetic polymers that can be degraded by hydrolysis, such as poly(hydroxy acids), polymers and copolymers of lactic acid and glycolic acid, other biodegradable polyesters, polyanhydrides, poly(orthoesters), polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), poly(lactide-co-caprolactone), and poly(amine-co-esters), such as those described in Zhou et al., Nature Materials, 11:82-90 (2012) and WO 2013 / 082529, U.S. Application Publication 2014 / 0342003 and WO 2016 / 081621.

[0378] In some embodiments, particularly those for in vivo targeting of T cells, such as for the in vivo production of CAR T cells, the target may be an immune cell or T cell marker, such as CD3, CD7, or CD8, or a marker of a target tissue, such as the liver. For example, anti-CD8 antibodies and anti-CD3 Fab fragments have been used for in vivo targeting of T cells (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). Therefore, in some embodiments, the particles or other delivery carriers include a targeting portion specific to CD3, CD7, CD8, or another immune cell (such as T cell) marker, or a marker of a specific tissue such as the thymus, spleen, or liver. For example, the binding site can be an antibody or its antigen-binding fragment.

[0379] The targeting moiety can be associated, linked, conjugated, or otherwise directly or indirectly attached to nanoparticles or other delivery carriers. The targeting moiety can be a protein, peptide, nucleic acid molecule, sugar, or polysaccharide that binds to receptors or other molecules on the surface of target cells. The specificity and affinity of the graft can be modulated by selecting the targeting moiety.

[0380] Examples of such components include, for instance, targeted components that can deliver molecules to specific cells, such as hematopoietic stem cells and CD34. + Antibodies against cells, T cells, or any other preferred cell type, and receptors and ligands expressed on the preferred cell type. Preferably, the portion targets hematopoietic stem cells. Examples of molecules targeting the extracellular matrix (“ECM”) include glycosaminoglycans (“GAG”) and collagen. In one embodiment, the outer surface of the polymer particle may be modified to enhance the particle’s ability to interact with selected cells or tissues. Preferably, the adapter element conjugated to the targeting portion is inserted into the particle using the method described above. However, in another embodiment, the outer surface of polymer microparticles or nanoparticles having carboxyl-terminated ends may be linked to the targeting portion having free amine-terminated ends.

[0381] Other useful ligands attached to polymer microparticles and nanoparticles include pathogen-associated molecular patterns (PAMPs). PAMPs target Toll-like receptors (TLRs) on the surface of cells or tissues, or signal into the interior of cells or tissues, potentially increasing uptake. PAMPs conjugated to or co-encapsulated on the particle surface can include: unmethylated CpG DNA (bacteria), double-stranded RNA (viruses), lipopolysaccharide (bacteria), peptidoglycan (bacteria), lipoarabinomannan (bacteria), yeast polysaccharide (yeast), proto-lipoproteins such as MALP-2 (bacteria), flagellin (bacteria), poly(inosine-cytidine) (bacteria), lipoteichoic acid (bacteria), or imidazoquinone (synthetic).

[0382] In another embodiment, the outer surface of the particles can be treated with mannosamine to mannose-encapsulate the outer surface of the particles. This treatment allows the particles to bind to target cells or tissues on the surface of antigen-presenting cells via mannose receptors. Additionally, surface conjugation with immunoglobulin molecules containing an Fc moiety (targeting Fc receptor), heat shock protein molecules (HSP receptor), phosphatidylserine (scavenger receptor), and lipopolysaccharide (LPS) represents additional receptor targets on the cells or tissues.

[0383] Lectins can be covalently attached to microparticles and nanoparticles, giving them targeting specificity for mucins and mucosal cell layers.

[0384] The choice of targeting portion depends on the method of application of the nanoparticle composition and the cells or tissues to be targeted. The targeting portion typically increases the binding affinity of the particles to cells or tissues, or targets the nanoparticles to specific tissues within an organ or specific cell types within a tissue. In some embodiments, the targeting portion targets the thymus, spleen, or cancer cells.

[0385] Any natural component of mucin, covalently attached to microparticles in purified or partially purified form, can reduce the surface tension at the bead-gut interface and increase the solubility of beads in the mucin layer. Attachment of polyamino acids containing attached dangling carboxylic acid side groups (such as polyaspartic acid and polyglutamic acid) can increase 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 generated that attach to the particle surface. Polyamino chains can enhance bioadhesion through chain entanglement and increased carboxyl charge within the mucin chain.

[0386] III. Instructions for Use

[0387] A. Delivery of nucleic acids

[0388] A method for enhancing nucleic acid construct delivery using 4H2 antibodies is provided. Typically, an effective amount of 4H2 antibody is first contacted with the nucleic acid cargo to be delivered into cells. For example, the nucleic acid cargo and antibody may be mixed in solution for a sufficient time to allow the nucleic acid cargo and antibody to form a complex. The mixture is then contacted with cells. In other embodiments, the cargo and antibody are added to a solution containing cells or otherwise soaking cells to form a complex in the presence of cells. The complex may contact cells in vitro, ex vivo, or in vivo. Thus, in some embodiments, the complex solution is added to cultured cells or injected into an animal to be treated. Treatment may be, for example, administered via simple intravenous injection, to a subject in need.

[0389] 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.

[0390] An effective or therapeutically effective amount of the composition may be a dose sufficient to treat, inhibit or alleviate one or more symptoms of a disease or condition, or a dose that otherwise provides the desired pharmacological and / or physiological effects, such as reducing, inhibiting or reversing one or more pathophysiological mechanisms of a disease or condition.

[0391] The effective amount can also be the amount that, relative to the administration of nucleic acid cargo in the absence of antibodies, can effectively improve the rate, quantity, and / or quality of nucleic acid cargo delivery. The formulation of the composition should be adapted to the method of administration.

[0392] The pharmaceutically acceptable carrier portion depends on the specific composition being administered and the specific method of administration. Therefore, there are a variety of suitable formulations for pharmaceutical compositions containing complexes. The precise dosage varies depending on various factors, such as subject-dependent variables (e.g., age, immune system health, clinical symptoms, etc.).

[0393] It can be applied once, twice, or three times daily; once, twice, three times, four times, five times, six times, or seven times weekly; or once, twice, three times, four times, five times, six times, seven times, or eight times monthly, or otherwise applied to contact with target cells. For example, in some embodiments, the composition is applied every two or three days, or on average about two to four times per week. Thus, in some embodiments, the composition is applied as part of a dosage regimen comprising two or more individual treatments.

[0394] Dosing regimens include maintenance regimens (the dose remains constant between two or more administrations), escalation regimens (the dose is increased between two or more administrations), de-escalation regimens (the dose is reduced between two or more administrations), or combinations thereof.

[0395] In some embodiments, the initial dose may be low. The dose may be increased until a satisfactory biochemical or clinical response is achieved. The clinical response will depend on the disease or condition being treated and / or the desired outcome. In some embodiments, the dose may be increased until a therapeutic effect is established, preferably without causing undesirable toxicity or bringing it within an acceptable range. The dose may then be maintained or steadily reduced to a maintenance dose. These methods can be used to standardize, optimize, or customize the dose level, frequency, or duration of treatment.

[0396] Generally, antibodies and nucleic acids are mixed for a period of time before administration, especially in vivo, for example at room temperature. In some embodiments, the complexation time ranges from, for example, 1 minute to 30 minutes (inclusive), or 10 minutes to 20 minutes (inclusive), with a preferred complexation time of about 15 minutes. The antibody dose ranges from 0.0001 mg to 1 mg (inclusive), with a preferred dose of about 0.1 mg. The nucleic acid dose ranges from 0.001 μg to 100 μg (inclusive), with a preferred dose of 10 μg. In the experiments below, the 4H2 / mRNA ratio is 1:1 w / w and 3:1 w / w, but other ratios are also possible. In some embodiments, the antibody to nucleic acid ratio can be 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.

[0397] In some embodiments, the RNA and / or DNA cargo is mixed with the vector DNA. The vector DNA can be, for example, plasmid DNA or low molecular weight DNA, such as DNA derived from salmon sperm. In some embodiments, the vector DNA is non-coding DNA. The vector DNA can be single-stranded or double-stranded, or a combination thereof. In some embodiments, the vector DNA consists of nucleic acids of lengths of 1-10, 1-100, 1-1,000, or 1-10,000 nucleotides or any subrange or integer thereof or a combination thereof. The vector DNA is generally not conjugated to or otherwise covalently linked to an antibody. The vector DNA is typically co-incubated with the cargo nucleic acid and antibody and co-delivered as a complex.

[0398] 1. In vitro and ex vivo methods

[0399] For in vitro and ex vivo methods, cells are typically contacted with the composition during culture. For ex vivo methods, cells can be isolated from a subject and contacted with the composition ex vivo to produce cells containing cargo nucleic acids. In a preferred embodiment, the cells are isolated from a subject to be treated or a syngeneic host. Target cells can be removed from the subject before contact with the composition. Antibodies and cargoes can be contacted with the cells together or separately, or as a pre-formed complex.

[0400] 2. In vivo methods

[0401] In some embodiments, nucleic acid cargo is delivered in vivo to cells for gene editing and / or treatment of a subject's disease or condition. Compositions typically including antibody-nucleic acid cargo complexes can be directly administered to the subject for in vivo treatment.

[0402] Generally, methods of administering compounds (including antibodies, oligonucleotides, and related molecules) are well known in the art. In particular, the administration routes already used in nucleic acid therapy and the formulations currently in use provide preferred administration routes and formulations for the aforementioned donor oligonucleotides. It is preferred to inject or infuse the composition into an animal.

[0403] The composition can be administered via a variety of routes, including but not limited to intravenous injection, intraperitoneal injection, intraamniotic injection, intramuscular injection, subcutaneous injection, or local (sublingual, rectal, intranasal, pulmonary, rectal mucosa, and vaginal) and oral (sublingual, oral).

[0404] In some embodiments, the composition is formulated for pulmonary administration, such as intranasal administration or oral inhalation. Administration of the formulation can be achieved by any acceptable method to deliver the complex to its target. Depending on the disease being treated, administration can be local (i.e., application to a specific area, physiological system, tissue, organ, or cell type) or systemic. In vivo delivery compositions and methods are also discussed in WO 2017 / 143042.

[0405] These methods may also include administering an effective amount of the antibody-nucleic acid complex composition to the embryo or fetus or its pregnant mother in vivo. In some methods, the composition is delivered intrauterinely by injection and / or infusion into a vein or artery, such as the yolk sac or umbilical vein, or by injection into the amniotic sac of the embryo or fetus. See, for example, Ricciardi et al., Nat Commun. 2018 26 June; 9(1):2481. doi:10.1038 / s41467-018-04894-2, and WO 2018 / 187493.

[0406] 3. Application

[0407] Nucleic acid cargoes encoding target peptides or functional nucleic acids, such as mRNA, functional nucleic acids, DNA expression constructs, vectors, etc., can be delivered into cells using 4H2 antibodies to express or inhibit peptides in cells. These compositions and methods can be used for a wide variety of applications. Non-limiting examples include: CRISPR and gRNA expression vector + / - DNA editing, delivery of large DNA (plasmids and expression vectors), gene replacement and gene therapy, DNA and / or RNA delivery, for example, for in vivo or in vitro CAR-T cell generation and simplification of in vivo or in vitro CAR-T cell production, siRNA delivery, mRNA delivery, etc. Exemplary applications related to gene therapy / gene editing and immune modulation, particularly chimeric antigen receptor T cell production, will be discussed below.

[0408] a. Gene therapy and gene editing

[0409] In some embodiments, the composition can be used for gene editing. For example, these methods are particularly suitable for treating genetic defects, symptoms, and diseases caused by mutations in a single gene, such as correcting genetic defects, symptoms, and diseases caused by point mutations. If the target gene contains mutations that cause genetic diseases, these methods can be used for mutation repair, restoring the DNA sequence of the target gene to normal. The target sequence can be in the coding DNA sequence of the gene or introns. The target sequence can also be within the DNA sequence that regulates the expression of the target gene, including promoter or enhancer sequences.

[0410] In the methods described herein, cells that have been in contact with the complex may be administered to a subject. The subject may have hemophilia, muscular dystrophy, globulinosis, cystic fibrosis, xeroderma pigmentosum, or lysosomal storage disease, or a hereditary or acquired disease of the retina, eye, brain, or spine, or coronary artery or other vascular disease. In these embodiments, gene modification, gene replacement, gene addition, or a combination thereof can effectively reduce one or more symptoms of the subject's disease or condition.

[0411] In some embodiments, the disclosed compositions can be used for retinal gene therapy. Inherited retinal diseases (IRDs) are typically caused by single-gene mutations, including but not limited to Leber congenital amblyopia type 2 (LCA), choroidal hematuria (CHM), Stargardt disease, retinitis pigmentosa (such as mutations in RHO, USH2A, and RPGR), and X-linked retinoschisis (XLRS). Different routes of administration are available, including intravitreal, subretinal, and suprachoroidal, providing different biodistributions. See also Gupta et al., “Gene Therapy for Inherited Retinal Disease,” Review of Ophthalmology, May 10, 2022.

[0412] In some embodiments, the disclosed compositions and methods can be used to induce or enhance the repair of damaged endothelial cells, for example, during revascularization. Therefore, the disclosed compositions and methods can be used as adjuncts to cardiovascular surgery and other interventions. For example, revascularization is a treatment that can restore blood flow to obstructed arteries or veins. The disclosed compositions and methods can be used in conjunction with such interventions to reduce the expression or bioactivity of pro-inflammatory cytokines (such as IL-6, IL-8, and TNF-α), increase endothelial cell growth and proliferation, and / or reduce neointimal hyperplasia (such as the growth, proliferation, and migration of smooth muscle cells).

[0413] In some embodiments, the disclosed compositions and methods include local delivery to a site of treatment or an adjacent site. Such local sites include, but are not limited to, the brain, ear, and skin, and such delivery can be used to treat associated diseases.

[0414] In some implementations, the cargo includes nucleic acids encoding nucleases, donor oligonucleotides, or nucleic acids encoding donor oligonucleotides, or combinations thereof.

[0415] 1. Gene editing nuclease

[0416] Nucleic acid cargoes include nucleic acids encoding one or more elements that induce single- or double-strand breaks in the target cell genome, optionally, but preferably, cargoes 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. These compositions can be used to reduce or otherwise modify the expression of target genes.

[0417] (1). Chain breakage induction element

[0418] CRISPR / Cas

[0419] In some embodiments, the nucleic acid cargo includes one or more elements of a CRISPR / Cas-mediated genome editing composition, nucleic acids encoding one or more elements of a CRISPR / Cas-mediated genome editing composition, or combinations thereof. As used herein, a CRISPR / Cas-mediated genome editing composition refers to a CRISPR system element required for CRISPR / Cas-mediated genome editing in a mammalian subject. As discussed in detail below, a CRISPR / Cas-mediated genome editing composition typically includes one or more nucleic acids encoding crRNA, tracrRNA (or a chimera thereof, also known as guide RNA or single guide RNA), and a Cas enzyme (such as Cas9). A CRISPR / Cas-mediated genome editing composition may optionally include a donor polynucleotide that can be recombined into the genome of a target cell at or near a target site (e.g., a Cas9-induced single-strand or double-strand break site).

[0420] The CRISPR / Cas system has been adapted for gene editing (silencing, enhancing, or altering specific genes) in eukaryotes (e.g., see Cong, Science, 15:339(6121):819-823(2013) and Jinek et al., Science, 337(6096):816-21(2012)). By transfecting cells with desired elements, including Cas genes and specially designed CRISPR, an organism's genome can be cut and modified at any desired location. WO 2013 / 176772 and WO 2014 / 018423 describe in detail methods for preparing compositions for genome editing using the CRISPR / Cas system, the entire contents of which are hereby incorporated by reference.

[0421] The delivery method disclosed in this paper is applicable to various variants of the CRISPR / Cas system.

[0422] Generally speaking, the "CRISPR system" refers to the collective term for transcripts and other elements involved in expressing or directing the activity of CRISPR-related ("Cas") genes. This includes sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr-mate sequences (in endogenous CRISPR systems, these include "direct repeats" and partially direct repeats processed by tracrRNA), guide sequences (also called "spacer bases" in endogenous CRISPR systems), or other sequences and transcripts derived from CRISPR loci. One or more tracr-paired sequences operatively linked to the guide sequence (such as direct repeat-spacer base-direct repeat) may also be referred to as pre-crRNA (pre-CRISPRRNA) before processing or as crRNA after nuclease processing.

[0423] As detailed below, in some implementations, tracrRNA and crRNA are linked to form a chimeric crRNA-tracrRNA hybrid, wherein the mature crRNA is fused to a portion of the tracrRNA via a synthetic stem-loop to mimic the natural crRNA:tracrRNA double strand, as described in Cong, Science, 15:339(6121):819-823 (2013) and Jinek et al., Science, 337(6096):816-21 (2012). The single fused crRNA-tracrRNA construct is also referred to herein as guide RNA or gRNA (or single guide RNA (sgRNA)). In sgRNA, the crRNA portion may be defined as the “target sequence,” while the tracrRNA is often referred to as the “scaffold.”

[0424] 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 specific organism, such as Streptococcus pyogenes, which includes an endogenous CRISPR system.

[0425] Typically, a CRISPR system is characterized by having elements that promote the formation of a CRISPR complex (also known as a protospacer in endogenous CRISPR systems) at a target sequence site. In the case of CRISPR complex formation, the "target sequence" refers to a sequence to which the guide sequence is designed to be complementary; hybridization between the target sequence and the guide sequence promotes CRISPR complex formation. The target sequence can be any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the cell nucleus or cytoplasm.

[0426] In the target nucleic acid, each protospacer is associated with a protospacer adjacent motif (PAM), and the recognition of this motif is CRISPR system specific. In the *Streptococcus pyogenes* CRISPR / Cas system, the PAM is the nucleotide sequence NGG. In the *Streptococcus thermophilus* CRISPR / Cas system, the PAM is the nucleotide sequence NNAGAAW. The tracrRNA duplex guides Cas to the DNA target site, composed of the protospacer and the necessary PAM, through a heteroduplex formed between the crRNA spacer region and the protospacer DNA.

[0427] Typically, in an endogenous CRISPR system, the formation of the CRISPR complex (including a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins) results in the 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 from the target sequence). All or part of the tracr sequence can also form part of the CRISPR complex, for example, through full or partial hybridization with a tracr pairing sequence operatively linked to the guide sequence.

[0428] Once the desired DNA target sequence is identified, numerous resources are available to help researchers determine suitable target sites. For example, many public resources, including a bioinformatics-generated list of approximately 190,000 potential sgRNAs that can target over 40% of human exons, can help researchers select target sites and design relevant sgRNAs to affect gaps or double-strand breaks at the site. See also crripr.u-psud.fr / , a tool designed to help scientists find CRISPR targets and generate appropriate crRNA sequences across multiple species.

[0429] In some implementations, a vector driving the expression of one or more elements of a CRISPR system is introduced into target cells, thereby directing the expression of CRISPR system elements at one or more target sites to form the CRISPR complex. For example, the Cas enzyme, the guide sequence linked to the tracr pairing sequence, and the tracr sequence can be operatively linked to different regulatory elements on different vectors, respectively. Alternatively, two or more elements expressed by the same or different regulatory elements can be combined in a single vector, with one or more additional vectors providing any elements of the CRISPR system 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 located at the 5' (“upstream”) or 3' (“downstream”) of the second element. The coding sequence of one element can be located on the same or opposite strand of the coding sequence of the second element, and in the same or opposite orientation. In some embodiments, a single promoter drives the expression of a tracr sequence encoding a CRISPR enzyme and one or more guide sequences, a tracr pairing sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded with one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr pairing sequence, and tracr sequence are operably linked to and expressed by the same promoter.

[0430] In some embodiments, the vector includes one or more insertion sites, such as restriction endonuclease recognition sequences (also known as “cloning sites”). In some embodiments, one or more insertion sites (e.g., approximately or more than approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of the vector. In some embodiments, the vector includes an insertion site upstream of a tracr chaperone sequence and optionally downstream of a regulatory element operatively linked to the tracr chaperone sequence, such that, upon insertion of a guide sequence into the insertion site, the guide sequence, upon expression, can guide the CRISPR complex to sequence-specific binding to a target sequence in eukaryotic cells. In some embodiments, the vector includes two or more insertion sites, each located between two tracer sequences, to insert a guide sequence at each site. In this arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations thereof. When using multiple different guide sequences, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within cells. For example, a single vector may include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 guide sequences. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 such vectors containing guide sequences may be provided and may be selectively delivered to cells.

[0431] In some embodiments, the vector includes a regulatory element operatively linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as the Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas1O, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, their homologs or modifications thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleaving activity, such as Cas9. In some embodiments, the CRISPR enzyme directs the cleavage of one or both strands at a location on the target sequence, such as within the target sequence and / or within its complementary sequence. In some embodiments, the CRISPR enzyme directs the cleavage of one or both strands within approximately 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 the target sequence.

[0432] In some embodiments, the vector-encoded CRISPR enzyme is mutated relative to the corresponding wild-type enzyme, thereby rendering the mutant CRISPR enzyme unable to cleave one or both strands of a target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine (D10A) in the RuvC I catalytic domain of Streptococcus pyogenes Cas9 transforms Cas9 from a two-strand nuclease into a nicking enzyme (cleaving a single strand). Other examples of mutations that make Cas9 a nicking enzyme include, but are not limited to, H840A, N854A, and N863A. As another example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvCIII) can be mutated to produce a mutant Cas9 that substantially lacks all DNA cleaving activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially lacks all DNA cleaving activity. In some implementations, CRISPR enzymes are considered to be substantially lacking all DNA cleavage activity when the DNA cleavage activity of the mutant enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or lower relative to its non-mutant form.

[0433] In some implementations, the enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells can be cells of a specific organism or cells derived from a specific organism, such as mammals, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance its expression in a host cell by replacing at least one codon in the natural sequence with a codon that is more frequently or most frequently used in the host cell's gene (e.g., approximately or more than approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons), while maintaining the natural amino acid sequence. Different species exhibit specific preferences for certain codons of specific amino acids. Codon preference (differences in codon use between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which is thought to depend on factors such as the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules.

[0434] The dominance of selected tRNAs in a cell typically reflects the most frequently used codons in peptide synthesis. Therefore, based on codon optimization, genes can be tailored for optimal gene expression in a specific organism. Codon usage tables are available on websites such as the Codon Usage Database, and these tables can be modified in various ways. See Nakamura, Y., et al. Nucl. Acids Res., 28:292 (2000). Computer algorithms, such as GeneForge (Aptagen; Jacobus, PA), can also be used to codon-optimize specific sequences for expression in specific host cells. In some implementations, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding the CRISPR enzyme correspond to the most frequently used codons for a specific amino acid.

[0435] In some embodiments, the vector-encoded CRISPR enzyme includes one or more nuclear localization sequences (NLS). When more than one NLS is present, each NLS can be selected independently of the others, such that a single NLS can exist in more than one copy, and / or combine with one or more other NLS present in one or more copies. In some embodiments, an NLS is considered to be near the N or C terminus when the nearest amino acid of the NLS is within the range of about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain from the N or C terminus.

[0436] Typically, the strength of one or more NLSs is sufficient to drive the CRISPR enzyme to accumulate in a detectable amount in the nucleus of a eukaryotic cell. The strength of nuclear localization activity can generally be derived from the number of NLSs in the CRISPR enzyme, the specific NLS used, or a combination of these factors.

[0437] Accumulation within the cell nucleus can be detected using any suitable technique. For example, a detectable marker can be fused to a CRISPR enzyme, allowing visualization of the intracellular location, such as when combined with methods for detecting nuclear location (e.g., nucleus-specific staining agents like DAPI). The nucleus can also be isolated from the cell and its contents analyzed using any suitable method for protein detection, such as immunohistochemistry, Western blot, or enzyme activity assays. Accumulation in the nucleus can also be indirectly determined compared to a control group not exposed to the CRISPR enzyme or complex, or to a control group exposed to a CRISPR enzyme lacking one or more NLS, for example, by detecting the effect of CRISPR complex formation (e.g., detecting DNA cleavage or mutation at the target sequence, or detecting changes in gene expression activity affected by CRISPR complex formation and / or CRISPR enzyme activity).

[0438] In some implementations, one or more elements of the CRISPR system are controlled by an inducible promoter, including an inducible Cas, such as Cas9.

[0439] Cong, Science, 15:339(6121):819-823 (2013) reported that heterologous expression of Cas9, tracrRNA, and pre-crRNA (or Cas9 and sgRNA) can achieve targeted cleavage of mammalian chromosomes. Therefore, the CRISPR system used in the method disclosed herein, and the cargo nucleic acid, is a vector system that may include one or more vectors encoding CRISPR system elements, which may include a first regulatory element operatively linked to a CRISPR / Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence includes (a) a guide sequence capable of hybridizing to a target sequence in eukaryotic cells, (b) a tracr pairing sequence, and (c) a tracr sequence; and a second regulatory element operatively linked to an enzyme-coding sequence encoding a CRISPR enzyme, which may selectively include at least one or more nuclear localization sequences. Elements (a), (b), and (c) may be arranged in a 5' to 3' orientation, wherein Cas9 and CRISPR RNA reside on the same or different vectors of the system, wherein during transcription, the tracr pairing sequence hybridizes with the tracr sequence, the guide sequence guides the CRISPR complex to sequence-specific binding with the target sequence, and wherein the CRISPR complex may comprise a CRISPR enzyme complexed with (1) the guide sequence hybridizing with the target sequence and (2) the tracr pairing sequence hybridizing with the tracr sequence, wherein the enzyme-coding sequence encoding the CRISPR enzyme further encodes a heterologous functional domain. In some embodiments, one or more vectors also encode a suitable Cas enzyme, such as Cas9. Different genetic elements may be controlled by the same or different promoters.

[0440] While the specific details of different engineered CRISPR systems may vary, the overall approach is similar. Researchers intending to target DNA sequences using CRISPR technology (identified using one of several online tools) insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid includes the target sequence (approximately 20 nucleotides), a form of tracrRNA sequence (scaffold), and a suitable promoter and the necessary elements for proper processing in eukaryotic cells. This vector is commercially available (see, for example, Addgene). Many systems rely on custom-made complementary oligonucleotides, which are annealed to form double-stranded DNA and then cloned into the sgRNA expression plasmid. The sgRNA is co-expressed in transfected cells with an appropriate Cas enzyme derived from the same or different plasmids, resulting in single-stranded or double-strand breaks at the desired target site (depending on the activity of the Cas enzyme).

[0441] (2) Zinc finger nucleases

[0442] In some implementations, the element that induces single-strand or double-strand breaks in the target cell genome is one or more nucleic acid constructs encoding zinc finger nucleases (ZFNs). Therefore, the nucleic acid cargo can encode a ZFN.

[0443] ZFNs are typically fusion proteins, including a DNA-binding domain derived from a zinc finger protein linked to a cleavage domain. The most common cleavage domain is the IIS-type enzyme Fok1. Fok1 catalyzes double-stranded DNA cleavage, with one strand containing 9 nucleotides from its recognition site and the other strand containing 13 nucleotides. For example, see U.S. Patents 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 fragments thereof) can be used as a source of cleavage domains. The DNA-binding domain can, in principle, be designed to target any genomic location. It can be a tandem array of Cys2His2 zinc fingers, each typically recognizing three to four nucleotides in the target DNA sequence. The Cys2His2 domain has a general structure: Phe (sometimes Tyr)-Cys-(2 to 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 zinc fingers together (the number varies: in published studies, three to six zinc fingers were used per monomer), ZFN pairs can be designed to bind to genomic sequences 18–36 nucleotides long.

[0444] Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. For example, rational design includes using a database comprising trimer (or tetramer) nucleotide sequences and individual zinc finger amino acid sequences, wherein each trimer or tetramer nucleotide sequence is associated with one or more amino acid sequences of a zinc finger that binds to a specific trimer or tetramer sequence. See, for example, U.S. Patents 6,140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Publication Nos. 2002 / 0165356; 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Publications WO 98 / 53059 and WO 2003 / 016496.

[0445] (3) Transcription activator-like effector proteins nucleases

[0446] In some implementations, the element that induces single-strand or double-strand breaks in the target cell genome is one or more nucleic acid constructs encoding a transcription activator-like effector protein nuclease (TALEN). Therefore, the nucleic acid cargo can encode TALEN.

[0447] The overall structure of TALENs is similar to that of ZFNs, with the main difference being that the DNA-binding domain originates from TAL effector proteins, which are transcription factors in plant pathogens. The DNA-binding domain of TALENs is a tandem array of amino acid repeat sequences, each approximately 34 residues long. These repeat sequences are very similar to each other; typically, they differ primarily at two positions (amino acids 12 and 13, called repeat variable diresidues, or RVDs). Each RVD specifies the preferential binding of one of four possible nucleotides, meaning each TALEN repeat sequence binds a single base pair, but NN RVDs are known to bind adenine in addition to guanine. Compared to zinc finger proteins, the mechanism of DNA binding in TAL effectors is less understood, but their seemingly simpler code may be very beneficial for the design of engineered nucleases. TALENs can also be cleaved in dimer form, with relatively long target sequences (the shortest target sequence reported to date binds 13 nucleotides per monomer), and the requirements for the spacing between binding sites appear to be less stringent than those for ZFNs. Monomeric and dimer TALENs can include more than 10, 14, 20, or 24 repeats.

[0448] Cermak et al., Nucl. Acids Res. 1-11 (2011). U.S. Publication 2011 / 0145940 discloses TAL effectors and methods for modifying DNA with them. Miller et al., Nature Biotechnol 29:143 (2011), reported the preparation of TALENs for site-specific nuclease structures by linking truncated variants of TAL to the catalytic domain of Fokl nuclease. The results showed that TALENs could induce gene modification in immortalized human cells. General design principles for TALLE-binding domains can be found, for example, in WO 2011 / 072246.

[0449] ii. Donor polynucleotides

[0450] The nuclease activity of the genome editing system described in this article can cleave target DNA, producing single-strand or double-strand breaks. Cells can repair double-strand breaks in at least two ways: non-homologous end joining (NHEJ) and homologous directed repair (HDR). In NHEJ, double-strand breaks are repaired by directly joining the broken ends together. Therefore, although some nucleic acid components may be lost, resulting in deletion, no new nucleic acid components are inserted into the site. In HDR, a donor polynucleotide homologous to the cleaved target DNA sequence is used as a template for repairing the cleaved target DNA sequence, causing genetic information to be transferred from the donor polynucleotide to the target DNA. Therefore, new nucleic acid components can be inserted / replicated into the site.

[0451] Therefore, in some implementations, the nucleic acid cargo is a donor polynucleotide or includes a donor polynucleotide. NHEJ and / or homology-directed repair modifications to target DNA can be used to induce gene correction, gene replacement, gene markers, transgene insertion, nucleotide deletion, gene damage, gene mutation, etc.

[0452] Therefore, the DNA cleavage of genome editing compositions can be used to remove nucleic acid components from the target DNA sequence by cutting the target DNA sequence and allowing the cell to repair the sequence in the absence of exogenously provided donor polynucleotides. Alternatively, if the genome editing composition includes a donor polynucleotide sequence that at least includes a fragment homologous to the target DNA sequence, these methods can be used to add nucleic acid components to the target DNA sequence, i.e., insert or replace nucleic acid components (e.g., “knock-in” nucleic acids encoding proteins, siRNAs, miRNAs, etc.), add tags (e.g., 6xHis, fluorescent proteins (such as green fluorescent protein, yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), add regulatory sequences to genes (such as promoters, polyadenylation signals, internal ribosome entry sequences (IRES), 2A peptides, start codons, stop codons, splicing signals, localization signals, etc.), modify nucleic acid sequences (e.g., introduce mutations), etc. Therefore, the compositions can be used to modify DNA in a site-specific (i.e., “targeted”) manner, such as gene knockout, gene knock-in, gene editing, gene tagging, etc., as used in gene therapy.

[0453] In applications requiring the insertion of a polynucleotide sequence into a target DNA sequence, it is also necessary to provide the cell with a polynucleotide including the donor sequence to be inserted. The term "donor sequence," "donor polynucleotide," or "donor oligonucleotide" refers to the nucleic acid sequence inserted at the cleavage site. The donor polynucleotide typically possesses sufficient homology to the genomic sequence at the cleavage site, for example, 70%, 80%, 85%, 90%, 95%, or 100% homology to nucleotide sequences flanking the cleavage site, such as within approximately 50 bases or less of the cleavage site, for example, within approximately 30 bases, approximately 15 bases, approximately 10 bases, approximately 5 bases, or immediately adjacent to the cleavage site, to support homology-directed repair between the cleavage site and a homologous genomic sequence. The donor sequence is typically not identical to the genomic sequence it replaces. Instead, provided sufficient homology exists to support homology-directed repair, the donor sequence may include at least one or more single-base variations, insertions, deletions, inversions, or rearrangements relative to the genomic sequence. In some implementations, the donor sequence includes a non-homologous sequence with two homologous regions on its flanks, such that homologous directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence into the target region.

[0454] b. Immune regulation

[0455] i.CAR T cells

[0456] The disclosed compositions and methods are particularly suitable for preparing lymphocytes expressing immune receptors, especially chimeric immune receptors (CIRs), such as chimeric antigen receptors (CARs). Artificial immune receptors (also referred to herein as chimeric T-cell receptors, chimeric immune receptors, chimeric antigen receptors (CARs), and chimeric immune receptors (CIRs)) are engineered receptors that can be selectively transplanted onto cells. As discussed in detail below, cells modified according to the methods discussed can be used in various immunotherapies for the treatment of cancer, infections, inflammation, and autoimmune diseases.

[0457] In a particularly preferred embodiment, mRNA or DNA encoding chimeric antigen receptor cargo is delivered to immune cells, such as lymphocytes.

[0458] The cargo can be delivered to immune cells in vivo, ex vivo, or in vitro. In a preferred embodiment, the cargo is mRNA, which reduces cost, facilitates manufacturing, and minimizes one or more side effects such as cytokine storm, neurotoxicity, graft-versus-host disease, etc. In a specific embodiment, immune cells (such as T cells) are collected from a subject requiring CAR T-cell therapy, mRNA encoding one or more CAR T-cell constructs is delivered into the collected cells using the compositions and methods disclosed herein, and the cells are then returned to the subject. In some embodiments, the entire process from initial cell collection to return of the cells to the subject takes one week or less, such as 1, 2, 3, 4, 5, 6, or 7 days. In a specific embodiment, the process from initial cell collection to return of the cells to the subject is completed within 1 or 2 days, or within 1 day, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours.

[0459] The design and development strategies of chimeric antigen receptors were reviewed by Dotti et al. in Immunol Rev. 2014 January; 257(1): doi:10.1111 / imr.12131 (35 pages), the full contents of which are incorporated herein by reference. Other relevant references include 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., AnnuRev. Med., 65:333-347 (2014), Cartellieri, et al., Journal of Biomedicine and Biotechnology, 2010, Article ID 956304, 13 pages, doi:10.1155 / 2010 / 956304; and U.S. Publications 2015 / 0017120, 2015 / 0283178, 2015 / 0290244, 2014 / 0050709 and 2013 / 0071414.

[0460] CARs combine the antigen-binding properties of monoclonal antibodies with the lytic and self-renewal capabilities of T cells, offering several advantages over traditional 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 do not rely on the major histocompatibility complex (MHC) to recognize and kill cancer cells. Therefore, target cell recognition is not affected by some mechanisms that allow tumors to evade MHC-restricted T cell recognition, such as downregulation of human leukocyte antigen (HLA) class I molecules and antigen processing defects.

[0461] Chimeric immune receptors were initially developed in the 1980s, initially comprising the variable region (antigen-binding region) of a monoclonal antibody and the constant regions of the α and β chains of the T-cell receptor (TCR) (Kuwana et al., Biochem Biophys Res Commun., 149:960-968 (1987)). In 1993, this design was modified by adding extracellular domains of single-chain variable fragments (scFv) derived from the antigen-binding regions of the heavy and light chains of monoclonal antibodies, transmembrane domains, and intracellular domains with signal transduction domains derived from CD3-ζ. Subsequent CARs generally follow a similar structural design, incorporating co-stimulatory signaling intracellular domains. Therefore, the CAR constructs used in this paper can include antigen-binding domains or extracellular domains, hinge domains, transmembrane domains, intracellular domains, and combinations thereof.

[0462] In some implementations, the extracellular domain is the scFv. The affinity of the scFv can predict the function of the CAR (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 arm to the CAR, so that two different scFvs can be expressed, recognizing two different antigens (Grada et al., Mol Ther Nucleic Acids, 2:e105(2013)) (called tandem CAR (TanCAR)). Tandem CARs can more effectively kill cancers that express low levels of each antigen alone, and can also reduce the risk of tumor immune escape caused by single antigen loss variants. Other extracellular domains include 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 (such as T1E peptide ligands), and so-called “universal extracellular domains” (such as the streptavidin extracellular domain designed to recognize targets contacted by biotin-labeled monoclonal antibodies, or FITC-specific single-chain antibody fragments (scFv) designed to recognize targets contacted by 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)).

[0463] In some implementations, the CAR includes a hinge region. While the extracellular domain is important for CAR specificity, the sequence connecting the extracellular domain and the transmembrane domain (the hinge region) can also affect CAR-T cell function by generating differences in CAR length and flexibility. For example, the hinge may include a CH2CH3 hinge or a fragment thereof derived from an immunoglobulin such as IgG1. For example, Hudecek et al. (Hudecek, et al., Clin Cancer Res, 19(12):3153-64(2013)) compared the effects of the CH2-CH3 hinge [229 amino acids (AA)], the CH3 hinge (119 AA) and the short hinge (12 AA) on the effect of T cell effector function expressing third-generation ROR1-specific CARs and found that T cells expressing the “short hinge” CAR had stronger anti-tumor activity. Other researchers found that the CH2-CH3 hinge weakened epitope recognition of first-generation CD30-specific CARs (Hombach, et al., GeneTher, 7:1067-1075(2000)).

[0464] A transmembrane domain typically exists between the hinge domain (or extracellular domain if there is no hinge domain) and the intracellular signaling domain. The most typical transmembrane domains originate from CD3-ζ, CD4, CD8, or CD28 molecules. Like the hinge domain, the transmembrane domain also affects the effector function of CAR-T cells.

[0465] Following antigen recognition, the intracellular domains of the CAR transmit activation and co-stimulatory signals to T cells. T cell activation depends on the phosphorylation of the cytoplasmic CD3-ζ domain of the TCR complex by the immunoreceptor tyrosine activation motif (ITAM) in the cytoplasmic domain (Irving et al., Cell, 64:891-901 (1991)). While most CAR intracellular domains include activation domains derived from CD3-ζ, some also include ITAM domains, such as the Fc receptor with an IgE-γ domain (Haynes et al., J Immunol., 166:182-187 (2001)).

[0466] The targeting specificity of CAR-expressing cells is determined by the antigen recognized by the antibody / extracellular domain. The disclosed compositions and methods can be used to manufacture constructs targeting any antigen and cells expressing the constructs. In immunotherapy, particularly cancer immunotherapy, many antigens and suitable extracellular domains for targeting antigens are well known. Unlike natural TCRs, most scFv-based CARs recognize target antigens expressed on the cell surface, rather than internal antigens processed and presented by the cell's MHC. However, the advantage of CARs over classic TCRs is that they can recognize structures other than protein epitopes, including carbohydrates and glycolipids. Dotti, et al., Immunol Rev. 2014 Jan; 257(1):.doi:10.1111 / imr.12131(35 pages), thereby increasing the potential target antigen library. Preferred targets include antigens expressed only on cancer cells or the surrounding matrix (Cheever et al., Clin Cancer Res., 15:5323-5337 (2009)), such as splice variants of the epidermal growth factor receptor (EGFRvIII) specific to glioma cells (Sampson et al., Semin Immunol., 20(5):267-75 (2008)). However, human antigens can also meet this requirement, and most target antigens are expressed at low levels on normal cells (e.g., GD2, CAIX, HER2) and / or expressed in a lineage-restricted manner (e.g., CD19, CD20).

[0467] Preferred targets and CAR targets are known in the art (e.g., see Dotti et al., Immunol Rev. 2014 Jan; 257(1): doi:10.1111 / imr.12131 (35 pages). For example, CAR targets for hematologic 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., 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., 3:95ra73 (2011), Brentjens, et al., Sci Transl Med., 5:177ra38 (2013), Grupp, et al., N Engl J Med (2013)); 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 cells) (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 cells) (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 cells) (Peinert, et al.,Gene Ther.,17:678-686(2010),Ritchie, et al.,Mol Ther.(2013)); NKG2D ligands (e.g., myeloid cells) (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)).

[0468] 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., cervical cancer) (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 cancer) (Nolan, et al., Clin Cancer Res., 5:3928-3941 (1999)); EGFRvIII (e.g., glioma) (Bullain, et al., J Neurooncol. (2009), Morgan, et al., HumGene Ther., 23:1043-1053 (2012)); EGP2 (e.g., tumor) (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 cancer) (Daly, et al., Cancer Gene Ther., 7:284-291 (2000); EphA2 (e.g., glioma, lung cancer) (Chow, et al., Mol Ther., 21:629-637 (2013)); ErbB2 (HER2) (e.g., breast cancer, lung cancer, prostate cancer, 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 GeneTher., 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 cancer, lung cancer, prostate cancer, glioma) (Davies, et al.,Mol Med.,18:565-576(2012));ErbB3 / 4 (e.g., breast cancer, ovarian cancer) (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 cancer) (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., Neopalasia., 2:449-459 (2000)); HMW-MAA (e.g., melanoma) (Burns, et al., Cancer Res., 70:3027-3033 (2010)); IL11RA (e.g.).Osteosarcoma (Huang, et al., Cancer Res., 72:271-281 (2012)); IL13RA2 (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 PractOncol., 6:53-58 (2009)); Lewis Y (e.g., breast / ovary / pancreas) (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 cancer, pancreatic cancer) (Lanitis, et al., Mol Ther., 20:633-643 (2012), Moon, et al., Clin Cancer Res., 17:4719-4730 (2011)); Mue1 (e.g., ovary, breast, prostate) (Wilkie, et al., J Immunol., 180:4901-4909 (2008)); NCAM (e.g., neuroblastoma, colorectal cancer) (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, pancreas) (Morgenroth et al., Prostate, 67:1121-1131 (2007), Katari et al., HPB, 13:643-650 (2011)); PSMA (e.g., prostate cancer) (Maher et al., Nat Biotechnol., 20:70-75 (2002), Gong, et al., Neoplasia., 1:123-127 (1999); TAG72 (e.g.).(Hombach, et al., Gastroenterology, 113:1163-1170 (1997), McGuinness, et al., Hum Gene Ther., 10:165-173 (1999)); VEGFR-2 (e.g., tumor vascular system) (J Clin Invest., 120:3953-3968 (2010), Niederman, et al., Proc Natl Acad Sci USA, 99:7009-7014 (2002)).

[0469] ii. Metabolic stability

[0470] In some embodiments, the metabolic stability of cells (such as CAR cells) can be improved by enabling them to produce limiting growth factors in vivo. In some embodiments, nucleic acid cargo encoding anti-apoptotic factors (such as BCL-XL) is transiently delivered into the cells. B-cell lymphoma-extra-large (Bcl-XL, or BCL2-like 1 subtype 1) is a transmembrane protein in mitochondria. It is a member of the Bcl-2 protein family and acts as a pro-survival protein in the intrinsic pathway of apoptosis, preventing the release of mitochondrial contents (such as cytochrome c) and thereby leading to caspase activation. The 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-xβ(bcl-x) gene, complete coding sequence (Human Bcl-x beta(bcl-x) gene, complete cds) (genomic nucleic acid sequence); ENA|Z23115|Z23115.1 Human bcl-XL mRNA (H. sapiens bcl-XL mRNA) (mRNA / cDNA nucleic acid sequence).

[0471] In some embodiments, the nuclear cargo encodes a proliferation-inducing factor, such as IL-2. The 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 (gene nucleic acid sequence); and ENA|V00564|V00564.1 human mRNA encoding interleukin-2 (IL-2) (mRNA / cDNA nucleic acid sequence).

[0472] However, the production of secreted IL-2 may produce unwanted side effects, namely, simultaneous stimulation of the proliferation of lymphoma and Treg cells and impaired formation of memory T cells (Zhang et al., Nature Medicine, 11:1238-1243 (2005)). Furthermore, the use of IL-2 in patients receiving tumor-infiltrating lymphocyte (TIL) therapy increases toxicity (Heemskerk et al., Human Gene Therapy, 19:496-510 (2008)). To avoid this possibility, in addition to IL-2, nucleic acid cargoes can also encode chimeric γc cytokine receptors (CγCRs), such as the chimeric γc cytokine receptor composed of interleukin-7 (IL-7) linked to IL-7Rα / CD127, which can confer exogenous cytokine-independent, intracellular STAT5 cytokine signaling (Hunter et al., Molecular Immunology, 56:1-11 (2013)). This design is modular, allowing the IL-2Rβ / CD122 cytoplasmic chain to exchange with the IL-7Rα / CD127 cytoplasmic chain to enhance Shc activity. This construct mimics wild-type IL-2 signaling in human CD8+ T cells (Hunter et al., Molecular Immunology, 56:1-11 (2013)), and therefore its function should be similar to that of IL-2 mRNA without producing unwanted side effects.

[0473] Alternatively, other anti-apoptotic molecules and cytokines can be used to maintain cell viability in its native state. Examples of such factors include, but are not limited to:

[0474] Myeloid leukemia 1 (MCL-1) (e.g., UniProtKB-Q07820(MCL1_HUMAN) (amino acid sequence); ENA|AF147742|AF147742.1 Human myeloid cell differentiation protein (MCL1) gene, promoter and complete coding sequence (genomic nucleic acid sequence); ENA|AF118124|AF118124.1 Human myeloid leukemia sequence 1 (MCL1) mRNA, complete coding sequence (mRNA / cDNA nucleic acid sequence)), which is an anti-apoptotic factor;

[0475] IL-7 (e.g., UniProtKB-P13232(IL7_HUMAN) (amino acid sequence); ENA|EF064721|EF064721.1 Human interleukin 7 (IL7) gene, complete coding sequence (genomic nucleic acid sequence); ENA|J04156|J04156.1 Human interleukin 7 (IL-7) mRNA, complete coding sequence (mRNA / cDNA nucleic acid sequence), which is very important for T cell survival and development, and IL-15 (e.g., UniProtKB-P40933(IL15_HUMAN) (amino acid sequence); ENA|X91233|X91233.1 Human interleukin 7 (IL-7) mRNA, complete coding sequence (mRNA / cDNA nucleic acid sequence).

[0476] IL-15 (e.g., UniProtKB-P40933(IL15_HUMAN) (amino acid sequence); ENA|X91233|X91233.1 human interleukin 15 gene (H. sapiens IL15 gene) (genomic nucleic acid sequence); ENA|U14407|U14407.1 human interleukin 15 gene (Human interleukin 15) (IL15) mRNA, complete coding sequence. (mRNA / cDNA nucleic acid sequence)) can promote the survival of T cells and NK cells (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 alone or in combination with BCL-XL, IL-2, and / or CγCR mRNAs. Therefore, in some embodiments, mRNAs encoding MCL-1, IL-7, IL-15, or combinations thereof are delivered to cells.

[0477] iii. Inhibitory CAR (iCAR)

[0478] In some embodiments, T-cell therapies are delivered to CAR cells. These therapies have demonstrated long-term efficacy and curative potential in treating certain cancers; however, their use can cause damage to non-cancerous tissues similar to graft-versus-host disease following donor lymphocyte infusion. Any of the disclosed compositions and methods can be used in combination with non-specific immunosuppression (such as high-dose corticosteroid therapy, which exerts cytotoxic or inhibitory effects on T cells to suppress the immune response), irreversible T-cell elimination (such as so-called suicide genetic engineering strategies), or combinations thereof. However, in some preferred embodiments, off-target effects can be reduced by introducing a construct encoding an inhibitory chimeric antigen receptor (iCAR) into CAR cells. By introducing an antigen-specific iCAR (inducible chimeric antigen receptor) into T cells, T cells with dual specificity for both tumor and non-target tissues can be restricted to targeting only the tumor, thereby protecting non-target tissues (Fedorov et al., Science Translational Medicine, 5:215ra172 (2013)). iCARs may include a surface antigen recognition domain that binds to a strong acute inhibitory signaling domain, which can limit T cell reactivity even when activating receptors (such as CARs) are also involved. In a preferred embodiment, the iCAR includes a single-stranded variable fragment (scFv) specifically targeting an inhibitory antigen that is fused via a transmembrane region to a signaling domain of an immunosuppressive receptor (such as CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGFβ receptor dominant-negative analogue, etc.) that specifically inhibits T cell function upon antigen recognition. Once CAR cells encounter cells that do not express the inhibitory antigen (such as cancer cells), iCAR-transduced T cells can produce a CAR-induced response to the CAR's target antigen. Fedorov et al., Science Translational Medicine, 5:215ra172 (2013) discussed the use of DNA iCARs with scFv specific to PSMA and inhibitory signaling domains of CTLA-4 or PD-1.

[0479] Design considerations included: PD-1 is a stronger inhibitor than CTLA-4; CTLA-4 exhibits cytoplasmic localization unless the Y165G mutant is used; and the expression level of iCAR is very important.

[0480] iCARs can be designed to target cell type-specific surface molecules. In some implementations, iCARs are designed to prevent the reactivity of T cells, NK cells, or other immune cells to certain tissues or cell types.

[0481] iv. Reduce endogenous inhibitory signals

[0482] In some embodiments, cells are contacted with nucleic acid cargo that can reprogram the cells to prevent the expression of one or more antigens. For example, in some embodiments, the nucleic acid cargo is interfering RNA or encoding interfering RNA that can block the expression of mRNA encoding antigens such as CTLA-4 or PD-1. This method can be used to prepare universal donor cells. The RNA used to alter allogeneic antigen expression can be used alone or in combination with RNA that causes target cell dedifferentiation.

[0483] Although the compositions and methods provided above utilize inhibitory signaling domains (e.g., CTLA-4 or PD-1 in artificial iCARs) to limit targeted / non-tumor cell toxicity, alternatively or optionally, by reducing the expression of endogenous inhibitory signals in CAR cells, CAR cells can be made resistant to inhibitory signals in the hostile tumor microenvironment, thereby improving the overall tumor effector efficiency of CAR cells.

[0484] CTLA-4 and PD-1 suppress T cells at different stages of activation and function. CTLA-4 modulates the T cell response to self-antigens, as gene knockout mice spontaneously develop organ damage due to highly active tissue-infiltrating T cells in the absence of specific antigen exposure (Tivol et al., Immunity 3:541-547 (1995); Waterhouse et al., Science, 270:985-988 (1995)). Interestingly, conditional gene knockout of CTLA-4 in Treg cells reproduces the effect of whole gene knockout, indicating that it functions normally in Treg cells (Wing et al., Science, 322:271-275 (2008)). In contrast, PD-L1 knockout mice are prone to autoimmune tendencies but do not spontaneously develop large amounts of inflammatory cell infiltration in normal organs, suggesting that its main physiological function is to mediate negative feedback control of ongoing tissue inflammation in an inducible manner (Dong et al., Immunity, 20:327-336 (2004)). In fact, according to the "adaptive resistance" hypothesis, most tumors upregulate PD-L1 in response to IFNγ; key cytokines released by effector T cells include 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)). Then, PD-L1 transmits inhibitory signals to T cells, reducing their proliferation, cytokine production, 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., NatImmunol, 12:492-499 (2011); Zou et al., Immunology, 8:467-477 (2008)). Furthermore, T cells send a reverse signal to cancer cells via B7-H1, inducing an anti-apoptotic effect, thereby counteracting Fas-L signaling (Azuma et al., Blood, 111:3635-3643 (2008)).

[0485] Given that the upregulation of B7-H1 in cancer cells and its expression are associated with cancer progression and poor clinical prognosis (Flies et al., Journal of Immunotherapy, 30:251-260 (2007); Nish imura et al., Immunity, 11:141-151 (1999); Wang et al., Curr Top Microbiol Immunol, 344:245-267 (2011)), antibodies antagonizing the PD-1 and CTLA-4 pathways have shown significant efficacy in solid tumors, especially melanoma. Combinations of these two pathways exhibit even stronger activity. The anti-CTLA-4 antibody, ipilimumab, primarily works by inhibiting Treg cells, increasing T cell infiltration into tumors, and enhancing intratumoral CD8+. + The ratio of CD8 cells to regulatory T cells (Tregs) (CD8) +Treg can improve the overall survival of patients with metastatic melanoma (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-Sai nt et al., Nature, 520:373-377 (2015)). The anti-PD-1 antibody nivolumab has shown 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)). Similar findings have been observed in early clinical trials of other solid tumors, including metastatic renal cell carcinoma, non-small cell lung cancer, and relapsed 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)). Since resistance to anti-CTLA-4 antibodies in mouse melanoma models is due to the upregulation of PD-L181, combination therapy with ipilimumab and nivolumab has shown further 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 the checkpoint inhibition pathway, it is thought that PD-1 / CTLA-4 inhibition will loosen the brakes, while chimeric antigen receptors will accelerate. Importantly, transient delivery technologies can be used to transiently release the brakes, preventing these cells from leading to future autoimmune diseases.

[0486] (1).CRISPRi

[0487] To avoid permanent genomic modifications and inactivation of repressive signals (such as PD-1 and CTLA-4), the dCAS9CRISPRi system can be used (Larson et al., Nat Protoc, 8:2180-2196 (2013)). Nucleic acids of sgRNAs encoding enzyme-inactivated dCAS9-KRAB repressive domains, fusion proteins, and repressive signaling proteins (such as CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, and TGFβ receptor dominant-negative analogs) can be co-delivered to CAR cells. The sgRNAs can be designed to target both the proximal promoter region and the coding region (non-template strand). An alternative approach is to utilize the single-component Cpf1CRISPR system, which uses smaller RNAs for electroporation and expression (Zetsche et al., Cell, doi:10.1016 / j.cell.2015.09.038 (2015)). Any of the aforementioned RNA components can also be encoded by DNA expression constructs, such as vectors, for example, plasmids. Therefore, RNA, DNA, or combinations thereof can all serve as nucleic acid cargo.

[0488] Although the broad inhibition of CTLA-4 by ipilimumab can lead to autoimmune sequelae, these side effects are believed to be reduced by limiting the loss of CAR cells and the transient nature of mRNA delivery. The inhibitory function will recover in time over time.

[0489] (2) Repressive RNA

[0490] Deliverable nucleic acid cargo to cells can be functional nucleic acids or peptides, or encode functional nucleic acids or peptides, designed to target and reduce or inhibit the expression or translation of inhibitory signaling molecule mRNAs; or reduce or inhibit the expression of inhibitory signaling molecule proteins, decrease their activity, or increase their degradation. Suitable technologies include, but are not limited to, antisense molecules, siRNAs, miRNAs, aptamers, ribozymes, triple-strand forming molecules, RNAi, etc. In some embodiments, the mRNA encodes an antagonist peptide that can reduce inhibitory signal transduction.

[0491] In some implementations, cargoes of functional RNAs suitable for reducing or silencing the expression of CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGF-β receptor dominant-negative analogues, or cargoes encoding these functional RNAs, may be delivered to cells alone or in combination.

[0492] In some embodiments, the cargo is RNA or DNA, encoding a polypeptide that may reduce bioavailability or act as an antagonist or other negative regulator or inhibitor of another protein in a dominant-negative analogue of CTLA-4, PD-1, LAG-3, 2B4 (CD244), BTLA (CD272), KIR, TIM-3, TGFβ receptor, or immunosuppressive pathway. This protein may be a paracrine, endocrine, or autocrine protein. It can regulate cells within the cell. It can be secreted and regulate expressing cells and / or other (e.g., neighboring) cells. It may be a transmembrane protein that regulates expressing cells and / or other cells. The protein may be a fusion protein, such as an Ig fusion protein.

[0493] v. Apoptosis-promoting factors

[0494] Compositions and methods for activating and reactivating apoptotic pathways are also provided. In some embodiments, the nucleic acid is a factor or agent encoding an intrinsic apoptotic pathway that activates, reactivates, or otherwise enhances or increases the intrinsic apoptotic pathway. Preferably, the factor can activate, reactivate, or otherwise enhance the intrinsic apoptotic pathway of cancer (e.g., tumor) cells; more preferably, the factor is specific to or targets cancer cells.

[0495] In some embodiments, cells delivered with anti-apoptotic or pro-proliferative factors (such as those discussed above or known in the art) are more resistant to or less sensitive to induced apoptosis than untreated cells. For example, pro-apoptotic factors can induce or increase apoptosis in untreated cells relative to treated T cells, and are preferably selective for cancer cells. This therapy can launch a two-pronged attack on cancer cells, one at the cellular level and the other at the molecular level.

[0496] Targeting members of the BCL-2 family can activate, reactivate, or otherwise enhance intrinsic apoptotic pathways. BCL-2 family members can be divided into three subgroups based on function and Bcl-2 homology (BH) domains: multi-domain anti-apoptotic proteins (such as BCL-2 or BCL-XL), multi-domain pro-apoptotic proteins (such as BAX and BAK), and pro-apoptotic proteins containing only the BH3 domain (such as BIM). The BH3-only subgroup, such as BIM, acts as a death sentinel distributed throughout the cell, ready to transmit various physiological and pathological signals of cellular damage to the core apoptotic mechanism located in the mitochondria (Danial et al., Cell, 116:205-219 (2004)).

[0497] In some implementations, the pro-apoptotic factor is a pro-apoptotic BH3-mimic. Various pro-apoptotic BH3-mimics can mimic the natural pro-apoptotic activity of BIM and manipulate multiple points in the apoptosis pathway. For example, BIM SAHB (a stable α-helix of the BCL-2 domain), ABT-737, and ABT-199 are pro-apoptotic BH3 mimics designed through structural studies of the interaction between the pro-apoptotic BH3-only helical domain and the hydrophobic groove formed by the convergence of the BH1, BH2, and BH3 domains of anti-apoptotic proteins (Oltersdorf et al., Nature, 435:677-681 (2005)).

[0498] 4. Target cells

[0499] In some embodiments, one or more specific cell types or tissues are target cells of the disclosed complex. Target cells can be in vitro, ex vivo, or in vivo (i.e., in vivo). The applications discussed herein can be performed in vitro, ex vivo, or in vivo. For in vitro applications, cells can be collected or isolated and processed during culture. Ex vivo-processed cells can be administered to a subject in need in a therapeutically effective amount. For in vivo applications, the cargo can be passively delivered to target cells, e.g., based on composition circulation, local delivery, etc., or can be actively targeted, e.g., using additional cell, tissue, or organ-specific targeting portions. Thus, in some embodiments, the cargo is delivered to target cells while excluding other cells. In some embodiments, the cargo is delivered to both target and non-target cells.

[0500] Researchers can select target cells based on the desired treatment and therapy, as well as the expected effects of the nucleic acid cargo. For example, when the nucleic acid cargo is designed to induce cell death, the target cells may be cancer cells; when the nucleic acid cargo is designed to induce genomic alterations, the target cells may be stem cells; and when the nucleic acid cargo encodes a chimeric antigen receptor, the target cells may be immune cells.

[0501] 4H2 infiltrates cells in a manner sensitive to dipyridamole, and the addition of GUO enhances this infiltration, suggesting that nucleoside transporter-dependent transport can be promoted by local nucleic acids.

[0502] In some implementations, nucleoside transporters are expressed on the plasma membrane of target cells. Nucleoside transporter expression is relatively common, but its abundance varies in different tissues and cell types. For example, ENT2 expression has been confirmed in the brain, heart, placenta, thymus, pancreas, prostate, and kidney (Griffiths et al., Biochem J, 1997.328(Pt 3): 739-43; Crawford et al., J Biol Chem, 1998.273(9): p.5288-93). Compared with other transporters, ENT2 is one of the transporters with the highest mRNA expression in skeletal muscle (Baldwin et al., Pflugers Arch, 2004.447(5): p.735-43; Govindarajan et al., Am J Physiol Regul Integr Comp Physiol, 2007.293(5): R1809-22). Therefore, in some implementations, the target cells are the brain, heart, placenta, thymus, pancreas, prostate, kidneys, or skeletal muscle.

[0503] Other non-limiting exemplary target cells will be discussed below.

[0504] i. Progenitor cells and stem cells

[0505] The cells can be hematopoietic progenitor cells or hematopoietic stem cells. In some implementations, particularly those related to gene editing and gene therapy, the target cells are CD34 cells. + Hematopoietic stem cells. Hematopoietic stem cells (HSCs), such as CD34... + The cell is a pluripotent stem cell, capable of producing all types of blood cells, including red blood cells.

[0506] Those skilled in the art can isolate and enrich stem cells. This isolation and enrichment of CD34... + Methods involving other cells are known in the art, for example, as disclosed in U.S. Patents 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 enrichment in hematopoietic progenitor cells and stem cells, “rich” means that the proportion of desired elements (such as hematopoietic progenitor cells and hematopoietic stem cells) is higher than that found in naturally derived cells. The cell composition may be enriched by at least one order of magnitude, preferably two or three orders of magnitude, more preferably 10, 100, 200 or 1000 orders of magnitude compared to cells of natural origin.

[0507] In the human body, CD34 +Cells can be recovered from umbilical cord blood, bone marrow, or from the bloodstream after cytokine mobilization following subcutaneous or intravenous injection of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF), and stem cell factor (SCF) into the donor. The amount injected subcutaneously or intravenously is sufficient to allow hematopoietic stem cells to enter the peripheral circulation from the bone marrow interstitium. Initially, bone marrow cells can be obtained from any suitable bone marrow source, such as the tibia, femur, spine, and other bone cavities. To isolate the bone marrow, the bone is flushed with a suitable solution, which will be a balanced salt solution readily supplemented with fetal bovine serum or other naturally occurring factors, combined with an acceptable low-concentration buffer, typically approximately 5 to 25 mM. Convenient buffers include Hepes buffer, phosphate buffer, lactate buffer, etc.

[0508] Cells can be screened using positive and negative selection techniques. Commercially available antibodies that bind to hematopoietic progenitor or hematopoietic stem cell surface antigens (such as CD34) can be used to select cells, using methods well known to those skilled in the art. For example, antibodies can be bound to magnetic beads, and the desired cell type can be recovered using an immunogenic procedure. Other techniques include the use of fluorescence-activated cell sorting (FACS). The CD34 antigen is present on progenitor cells in the hematopoietic system of non-leukemic individuals and is expressed on cell populations recognized by the monoclonal antibody My-10 (i.e., expressing the CD34 antigen), which can be used to isolate stem cells for bone marrow transplantation. My-10, deposited as HB-8483 at the American Center for Type Culture Collection (Rockville, Md.), is commercially available as an anti-HPCA 1. Additionally, negative selection can be performed using “specialized” cells differentiated from human bone marrow to select any desired cell marker, such as progenitor cells or stem cells, with CD34 being the most preferred. + Cells that can be characterized as CD3 - CD7 - CD8 - CD10 - CD14 - CD15 - CD19 - CD20 - CD33 - Class II HLA + and Thy-1 + Any one of them.

[0509] Once isolated, progenitor cells or stem cells can grow and proliferate in any suitable culture medium. For example, progenitor cells or stem cells can be grown in conditioned media derived from stromal cells, such as stromal cells associated with secretory factors obtained from bone marrow or liver, or in a medium containing cell surface factors that support stem cell proliferation. Hematopoietic cells in stromal cells can be removed by using appropriate monoclonal antibodies to eliminate unwanted cells.

[0510] The isolated cells are brought into in vitro contact with the antibody and nucleic acid cargo complex. The cells to which the cargo is delivered can be referred to as modified cells. The complex solution can be simply added to the cultured cells. It is desirable to synchronize the cells into the S phase. Methods for synchronizing cell culture, such as by blocking with thymidine, are known in the art (Zielke et al., Methods CellBiol., 8:107-121 (1974)).

[0511] Modified cells can be maintained or expanded in culture medium before administration to the subject. Culture conditions are generally known in the art, depending on the cell type. This is especially true for CD34 cells. + The maintenance conditions for hematopoietic progenitor cells have been well studied, and several suitable methods are available. A common method for expanding pluripotent hematopoietic cells in vitro is to culture purified progenitor cells or stem cells in the presence of early-acting cytokines such as interleukin-3. Studies have also shown that the addition of thrombopoietin (TPO), stem cell factor (SCF), and flt3 ligand (Flt-3L; i.e., the ligand of the flt-3L gene product) to the nutrient medium for maintaining hematopoietic progenitor cells in vitro facilitates the expansion of primitive (i.e., relatively undifferentiated) human hematopoietic progenitor cells in vitro, and these cells can be transplanted into SCID-hu mice (Luens et al., 1998, Blood 91:1206-1215). In other known methods, cells can be maintained in vitro in nutrient media (e.g., for minutes, hours, or 3, 6, 9, 13, or more days) containing mouse prolactin-like protein E (mPLP-E) or mouse prolactin-like protein F (mPIP-F; collectively referred to as mPLP-E / IF) (US Patent 6,261,841). Of course, other suitable cell culture and expansion methods can also be used. As described in US Patent 5,945,337, cells can also be cultured in serum-free media.

[0512] In another implementation, modified hematopoietic stem cells are differentiated into CD4+ cells in vitro using a specific combination of interleukins and growth factors. +Cell cultures are then administered to subjects using methods well-known in the art. The cells can be expanded in large quantities in vitro, and compared to the isolated primitive population of hematopoietic stem cells, the cells can be expanded in large quantities in vitro, preferably at least 5-fold, more preferably at least 10-fold, and even more preferably at least 20-fold.

[0513] In another implementation, the cells can be dedifferentiated somatic cells. These somatic cells can be reprogrammed into pluripotent stem cell-like cells, which can be induced to become hematopoietic progenitor cells. The hematopoietic progenitor cells can then be used with the aforementioned CD34... + The cells are processed using a composition. Representative somatic cells that can be reprogrammed include, but are not limited to, fibroblasts, adipocytes, and muscle cells. Hematopoietic progenitor cells derived from induced stem cell-like cells have been successfully cultured in mice (Hanna, J. et al. Science, 318:1920-1923 (2007)).

[0514] To generate hematopoietic progenitor cells from induced stem cell-like cells, somatic cells are required from a host. In a preferred embodiment, the somatic cells are autologous fibroblasts. These cells are cultured and transduced using vectors encoding transcription factors Oct4, Sox2, Klf4, and c-Myc. 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 cell-like cells. The cells are then screened for CD41 and c-kit markers (early hematopoietic progenitor cell markers) as well as myeloid and erythroid differentiation markers.

[0515] Modified hematopoietic stem cells or modified cells (including, for example, induced hematopoietic progenitor cells) are then introduced into the subject. Cell delivery can be performed using various methods, with preferred methods including intravenous infusion and direct injection into the periosteum, bone marrow, and / or subcutaneous tissue.

[0516] Subjects receiving modified cells may undergo bone marrow modulation to enhance cell transplantation. Recipients may receive radiation therapy or chemotherapy before cell use to promote cell transplantation. After administration, cells typically require a period of time before transplantation. Large-scale transplantation of hematopoietic stem cells or progenitor cells usually takes weeks to months.

[0517] To achieve significant preventative or therapeutic effects, a high proportion of modified hematopoietic stem cell transplantation may not be necessary. It is believed that transplanted cells will proliferate over time, increasing the proportion of modified cells. In some cases, it is believed that only a small number or a small proportion of modified hematopoietic stem cells need to be implanted to achieve preventative or therapeutic effects.

[0518] In a preferred embodiment, the cells to be administered to the subject will be autologous cells, such as cells derived from the subject or cells of the same genotype.

[0519] ii. Embryo

[0520] In some embodiments, the compositions and methods can be used to deliver cargo to embryonic cells in vitro. These methods typically involve contacting the embryo in vitro with an effective amount of antibody-cargo DNA to enhance cargo transduction to the embryo. The embryo can be a single-celled zygote, but can also be an embryo with 2, 4, 8, or 16 cells, including not only zygotes but also morula and blastocyst cells, processed with male and female gametes before and during fertilization. In some embodiments, the embryo is contacted with the composition during or after in vitro fertilization on days 0-6.

[0521] Contact can be achieved by adding the composition to the liquid culture medium in which the embryos are soaked. For example, the composition can be pipetted directly into the embryo culture medium and then absorbed by the embryos.

[0522] iii. Immune cells

[0523] In some implementations, the target cells are one or more types of immune cells. For example, different types of cells can be utilized or otherwise targeted for immunomodulation and as target cells for CAR-based therapies. Preferred targeted / engineered T cells may vary depending on the tumor and the target of the adoptive therapy. Effector T cells are generally preferred because they secrete high levels of effector cytokines and are skilled 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 potent CAR-mediated cytotoxicity. Using CD8+ T cells with CD4+ helper T cells results in an increase in suppressor T-reg cells and an inhibition of CD8+ T cell cytotoxicity. Since reprogrammed CD8+ T cells are pre-activated, they can act directly on tumor cells without activation in lymph nodes, thus CD4+ T cell support is not essential.

[0524] Furthermore, there is evidence that infusion of naive T cells (Rosenberg et al., Adv. Cancer Res., 25:323-388 (1977)) and central memory T cells (T cells) can reduce the risk of infection. CMTh17 cells (Berger et al., J. Clin. Invest., 118:294-305 (2008)), Th17 cells (Paulos et al., Sci. Transl. Med., 2:55-78 (2010)), and T stem cell memory cells (Gattinoni et al., Nat. Med., 17:1290-1297 (2012)) may all have certain advantages in some applications, for example, due to their high replication capacity. Tumor-infiltrating lymphocytes (TILs) also have certain advantages due to their antigen specificity and can be used in the delivery strategies disclosed herein.

[0525] Although sometimes referred to as CAR cells, CAR immune cells, and CART cells (or CAR T cells), it is understood that the CAR and other delivery strategies disclosed herein can also be implemented in other cell types, particularly different types of immune cells, including those discussed herein (e.g., lymphocytes, natural killer cells, dendritic cells, B cells, antigen-presenting cells, macrophages, etc.) and cells described elsewhere (e.g., see Barrett et al., Annu Rev Med., 65:333-347 (2014)).

[0526] iv. Cancer cells and tumors

[0527] In some implementations, the target cells are cancer cells. Such implementations provide treatment methods that can be used for cancer, including tumor therapy.

[0528] Goods that can be delivered to cancer cells include, but are not limited to, constructs expressing one or more pro-apoptotic factors, immunogenic factors, or tumor suppressor factors; gene editing compositions; repressive nucleic acids targeting oncogenes; and other strategies discussed herein and elsewhere. In some embodiments, the goods are mRNAs encoding pro-apoptotic factors or immunogenic factors that enhance the immune response against the cell. In other embodiments, the goods are siRNAs that reduce the expression of oncogenes or other oncogenic transcripts.

[0529] In mature animals, a balance is typically maintained between cell renewal and cell death in most organs and tissues. Various types of mature cells in the body have a limited lifespan; when these cells die, new cells are generated through the proliferation and differentiation of various stem cells. Under normal circumstances, the production of new cells is strictly regulated, and the number of any particular cell type remains constant. However, occasionally, cells that no longer respond to normal growth control mechanisms emerge. The cell clones produced by these cells can expand to a considerable size, forming tumors or new growths. Tumors that do not grow indefinitely and do not extensively invade surrounding healthy tissues are benign. Tumors that continue to grow and exhibit progressive invasiveness are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors can metastasize. In this process, small clusters of cancer cells detach from the tumor, invade the bloodstream or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. Thus, a primary tumor in one site can develop into a secondary tumor in another.

[0530] The compositions and methods described herein can be used to treat subjects with benign or malignant tumors, delay or inhibit tumor growth in the subject's body, reduce tumor growth or size, inhibit or reduce tumor metastasis, and / or inhibit or reduce symptoms associated with tumor occurrence or growth.

[0531] This article classifies treatable malignancies based on their embryonic origin from the tissue from which they originate. Carcinomas are tumors that arise from endoderm or ectoderm tissues, such as the epithelial lining of the skin or viscera and glands. The disclosed compositions are particularly effective in treating cancer. Sarcomas are less common 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. Leukemia proliferates as single cells, while lymphoma tends to grow as tumor masses. Malignant tumors can occur in multiple organs or tissues of the body, thus forming cancer.

[0532] The types of cancers that can be treated using the provided compositions and methods include, but are not limited to, angiomyocarcinomas (such as multiple myeloma), adenocarcinomas, and sarcomas, which may occur in the bone, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus.

[0533] In some embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. These compositions can also be used to treat metastatic tumors or tumors at multiple sites.

[0534] B. Methods for regulating immune responses

[0535] Methods for enhancing immune responses are provided. Immune responses can be increased in response to conditions such as cancer and infection. Therefore, methods for treating cancer and infection, as well as methods for vaccinating healthy and diseased subjects, are also provided. Immune responses are also involved in wound healing, so methods for promoting wound healing are also provided. Immune regulation is also involved in some autoimmune diseases, such as multiple sclerosis, so methods for promoting immune regulation in multiple sclerosis are also provided. Therefore, in some implementations, the subject has a wound or multiple sclerosis.

[0536] These methods typically involve administering an effective amount of 4H2 antibody to a subject in need to increase the activation of cGAS and / or another PRR (such as TLR7). In some embodiments, these compositions and methods can increase the activation of cGAS and / or another PRR (such as TLR7) through direct binding and 4H2 activation, or indirectly through simultaneous interactions between cGAS and / or other PPRs, 4H2, and cytoplasmic nucleic acids and / or GTP. Activated cGAS catalyzes the formation of cGAMP from the precursor molecules ATP and GTP, and cGAMP produced by cGAS promotes the nuclear translocation of NF-κB. Therefore, in some embodiments, the disclosed compositions increase the production of cGAMP and / or promote the nuclear translocation of NF-κB. Generally, these methods enhance the immune response by inducing or enhancing signal transduction through the cGAS / STING pathway.

[0537] In some embodiments, the compositions and methods include stimulating T cell proliferation, tumor angiogenesis and promoting tumor cell death and apoptosis, enhancing the release of tumor-associated antigens, improving antigen-specific IgG responses through mechanisms dependent on T helper cell 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, Vol. 20, pp. 657–674 (2019), the full text of which is incorporated herein by reference, which describes other results of enhanced cGAS / STING signaling transduction. In some embodiments, the compositions and methods include increasing the recruitment of tumor-infiltrating lymphocytes (TILs) to the tumor.

[0538] The results below also show that 4H2 interacts with TLR7, and this interaction is believed to be nucleic acid-dependent. The TLR family plays a crucial role in pathogen recognition and innate immune activation. TLRs recognize pathogen-associated molecular patterns (PAMPs) expressed on infectious pathogens and mediate the production of cytokines required for effective immune development. TLR7 is an intracellular pattern recognition receptor that recognizes single-stranded RNA in endosomes, a common feature of viral genomes internalized in macrophages and dendritic cells. For example, TLR7 recognizes single-stranded RNA from viruses such as HIV and HCV. TLR7 can also recognize GU-rich single-stranded RNA.

[0539] The results indicate that 4H2 can activate at least cGAS and TLR7, and may also activate other receptors that induce immune responses, adding another dimension to the use of 4H2 as an immunostimulant. Other immune receptors that may be activated by 4H2 include, but are not limited to, other PPRs, such as RIG-I-like receptors and other Toll-like receptors, including but not limited to TLR3, TLR8, and TLR9. Other receptors include, but are not limited to, those whose ligands are mentioned as goods and / or adjuvants.

[0540] In some embodiments, the method includes administering an effective amount of the 4H2 antibody to a subject in need, along with one or more adjunct drugs such as nucleic acid cargoes, immunostimulatory nucleic acids, vaccine components, immune checkpoint modulators, or combinations thereof. In some embodiments, the 4H2 antibody and the adjunct drug may be used in combination to provide a greater degree of enhancement of cGAS / STING and / or signaling of another immune receptor such as PPR (e.g., TLR7) than either agent alone. For example, in some embodiments, such as for cancer treatment, the enhanced activity is a stronger antitumor activity.

[0541] 4H2 antibodies and / or immune checkpoint modulators can be administered topically or systemically to subjects, or applied to or bound to devices.

[0542] The disclosed monotherapy and combination therapies and treatment regimens typically include methods for treating a disease or its symptoms, or for achieving desired physiological changes, including administering an effective amount of 4H2 antibody to animals (such as mammals, especially humans) to treat a disease (such as cancer or infection) or its symptoms, or to produce physiological changes.

[0543] When administered in combination with an adjunct drug, the 4H2 antibody and the adjunct drug can be administered together, as part of the same composition, or separately and independently at the same time or at different times (i.e., the administration of the 4H2 antibody and the immune checkpoint modulator is spaced some time apart). Therefore, the terms "combination" or "conjunction" refer to the simultaneous, synchronous, or sequential administration of two formulations. Combinations can be administered simultaneously (e.g., as a mixture), separately but simultaneously (e.g., administered to the same subject via different intravenous lines; one drug administered orally, and the other administered via infusion or injection, etc.), or sequentially (e.g., one drug administered first, followed by the second).

[0544] In some embodiments, the results obtained by combination are partly or entirely the sum of the results obtained by the individual components. In some embodiments, the effect obtained by combination is more additive than the effect obtained by using a single component alone. The effect achieved by combination exceeds the simple sum of the effects achieved by the individual components. In some embodiments, the effective amount of one or two agents used in combination is lower than the effective amount of each formulation when administered alone. In some embodiments, the dosage of one or two agents used in combination is a subtherapeutic dose when used alone.

[0545] The effectiveness of combination therapy or its individual agents can depend on the disease or condition being treated or its progression. For example, in some embodiments, combination therapy expands the range of treatable subjects (such as the type of cancer or infection) compared to using each agent alone. Therefore, in some embodiments, the effect of the combination on cancer or infection can be compared to the effect of the individual agents on cancer or infection.

[0546] Treatment regimens for monotherapy and combination therapy may include one or more administrations of the 4H2 antibody. Combination therapy regimens may include the administration of one or more additional medications.

[0547] In some embodiments, the 4H2 antibody and the adjuvant drug are administered sequentially, for example, in two or more different pharmaceutical compositions. In some embodiments, the 4H2 antibody is administered before the first administration of the adjuvant drug. In other embodiments, the adjuvant drug is administered before the first administration of the 4H2 antibody. For example, the 4H2 antibody and the adjuvant drug may be administered to the subject on the same day. Alternatively, the 4H2 antibody and the adjuvant drug may be administered to the subject on different days.

[0548] The 4H2 antibody may be administered at least 1, 2, 3, 5, 10, 15, 20, 24, or 30 hours or several days before or after administration of the adjuvant drug. Alternatively, the immune checkpoint modulator may be administered at least 1, 2, 3, 5, 10, 15, 20, 24, or 30 hours before or after administration of the 4H2 antibody. In some embodiments, the additive or more additive effect of the 4H2 antibody and the adjuvant drug is noticeable one, two, three, four, five, six, one week, or more after administration.

[0549] The dosing regimen or cycle of the medication can be completely or partially overlapping, or it can be sequential. For example, in some embodiments, all such administrations of the 4H2 antibody occur before or after the administration of the adjuvant medication. Alternatively, the administration of one or more doses of the 4H2 antibody can be staggered in time with the administration of the adjuvant medication to form a homogeneous or non-homogeneous course of treatment, wherein one or more doses of the 4H2 antibody are administered first, followed by one or more doses of the adjuvant medication, and finally one or more doses of the 4H2 antibody; or one or more doses of the adjuvant medication are administered first, followed by one or more doses of the 4H2 antibody, and finally one or more doses of the adjuvant medication; etc., all of which can be performed according to any schedule chosen or desired by the researcher or clinician administering the treatment.

[0550] The effective amount of each drug may be administered in the form of a single unit dose (e.g., as a dose unit) or a subtherapeutic dose administered over a finite time interval. Such a unit dose may 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, all of which are specifically taken into consideration.

[0551] 1. Cancer treatment

[0552] The therapies disclosed herein can be used to treat, reduce, and / or prevent cancer in a subject. Therefore, the compositions can be administered in effective amounts to treat, reduce, and / or prevent cancer in a subject. An effective amount for treating cancer or tumor, or a therapeutically effective amount, generally refers to a dose sufficient to reduce or prevent at least one symptom of cancer, or otherwise provide the desired pharmacological and / or physiological effect. Symptoms can be physical, such as tumor burden, or biological, such as reduced cancer cell proliferation or increased cancer cell death. In some embodiments, the dosage is effective in killing tumor cells or reducing or inhibiting tumor cell proliferation or metastasis. In some embodiments, the dosage is effective in reducing tumor burden. In some embodiments, an effective dosage is effective in reducing or preventing at least one cancer complication.

[0553] In mature animals, a balance is typically maintained between cell renewal and cell death in most organs and tissues. Various types of mature cells in the body have a limited lifespan; when these cells die, new cells are generated through the proliferation and differentiation of various stem cells. Under normal circumstances, the production of new cells is regulated, and the number of any particular type of cell remains constant. However, occasionally, cells that no longer respond to normal growth control mechanisms emerge. The clones produced by these cells can expand to a considerable size, forming tumors or growths. Tumors that do not grow indefinitely and do not extensively invade surrounding healthy tissues are benign. Tumors that grow continuously and gradually invade are malignant. The term cancer usually refers to a malignant tumor. In addition to uncontrolled growth, malignant tumors can metastasize. In this process, small clusters of cancer cells detach from the tumor, invade the bloodstream or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. Thus, a primary tumor in one site can develop into a secondary tumor in another.

[0554] The compositions and methods described herein can be used to treat subjects with benign or malignant tumors, delay or inhibit tumor growth in the subject's body, reduce tumor growth or size, inhibit or reduce tumor metastasis, and / or inhibit or reduce symptoms associated with tumor development or growth.

[0555] Treatable malignancies can be classified according to their embryonic origin from the tissue from which they originate. Carcinomas are tumors that arise from endoderm or ectoderm tissues, such as the epithelial lining of the skin or internal organs and glands. The disclosed compositions are particularly effective in treating cancer. Less common sarcomas originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemia and lymphoma are malignant tumors of hematopoietic cells in the bone marrow. Leukemia proliferates as single cells, while lymphoma tends to grow as masses. Malignant tumors can occur in multiple organs or tissues of the body, thus forming cancer.

[0556] The disclosed antigen-binding molecules can be used to treat cells that are growing out of control, invasive, or metastatic.

[0557] Cancer cells characterized by mutations in one or more Ras genes or mutations in genes encoding other components of the Ras / MAPK signaling pathway are particularly good targets for the disclosed compositions.

[0558] Cancer cell formation may be due to somatic gain-of-function mutations in the Ras gene, leading to activating mutations in the small GTPase Ras enzyme. Oncogenic mutations in the H-Ras, N-Ras, or K-Ras genes are most commonly associated with human malignancies. In some embodiments, cells express mutated forms of the small GTPase Ras family, such as K-Ras. In some embodiments, cells do not express the wild-type Ras gene.

[0559] Oncogenic mutations have also been found in other upstream or downstream components of the Ras intracellular signaling pathway, including cytoplasmic kinases and membrane RTKs (Ras / MAPK pathway).

[0560] Oncogenic mutations in the K-Ras gene can lead to constitutive activation of the exogenous Ras protein. Exemplary mutations include mutations in codons 12, 13, and / or 61 that result in any change to amino acid positions 12, 13, or 61 of the K-ras protein. This includes, for example, but not limited to, K-ras amino acid 12 (changing glycine to aspartic, cysteine, serine, threonine, arginine, or valine) and amino acids 13 and 61 (changing glutamine to lysine, arginine, leucine, or aspartic). Another way to describe K-Ras mutations is G12A, G12C, G12D, G12S, G12I, G12R, G12V, G13C, G13D, G13S, Q61L, and Q61R. Similarly, any changes in the content of amino acids at positions 12, 13, and 61 are considered exemplary mutations.

[0561] The composition can be used to treat representative but not limited cancers including hematologic and lymphatic system cancers (including leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, solitary plasmacytoma, and multiple myeloma), genitourinary system cancers (including prostate cancer, bladder cancer, kidney cancer, urethral cancer, penile cancer, and testicular cancer), nervous system cancers (including meningioma, glioma, glioblastoma, astrocytoma, oligodendroglioma, oligoastrocytoma, and ependymoma), and head and neck cancers (including oral cavity, nasal cavity, nasopharynx, and oropharynx). This disclosure relates to methods of treating breast cancer, ovarian cancer, colon cancer, prostate cancer, lung cancer, brain cancer, skin cancer, liver cancer, stomach cancer, pancreatic cancer, or leukemia. In some embodiments, this disclosure relates to methods of treating glioblastoma. In some embodiments, this disclosure relates to methods of treating glioblastoma.

[0562] Any of the disclosed methods can be further combined with radiotherapy, chemotherapy (such as antitumor drugs), or combinations thereof to treat any cancer, including carcinoma, glioma, sarcoma, or lymphoma. Examples of antitumor drugs that can bind to the disclosed antigen-binding molecules include, but are not limited to, alkylating agents (such as temozolomide, cisplatin, carboplatin, oxaliplatin, methylethylamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil, and ifosfamide), antimetabolites (such as fluorouracil, gemcitabine, methotrexate, cytarabine, fludarabine, and fluorouracil), certain antimitotic drugs, and vinca alkaloids, such as vinca... Vinpocetine, vinorelbine, vinorelbine and vindesine), anthracyclines (including doxorubicin, daunorubicin, pentorubicin, idarubicin and epirubicin, and actinomycins, such as actinomycin D), cytotoxic antibiotics (including mitomycin, procainamide and bleomycin), and topoisomerase inhibitors (including camptothecins such as irinotecan and topotecan, and derivatives of epipodophyllotoxin such as acridine, etoposide, etoposide phosphate and teniposide).

[0563] Strategies to combine STING immunotherapy with other immunomodulators are currently being explored. The antitumor effect of cGAMP via intratumoral injection into B16.F10 tumors was enhanced when combined with antibodies against programmed death protein-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4). (Demaria et al., Proc Natl Acad Sci USA (2015) 112(50): 15408-13.10.1073 / PNAS.1512832112). In other studies, CDN, when combined with anti-PD-1, elicited significantly stronger antitumor effects in squamous cell carcinoma and melanoma mouse models than monotherapy (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. combined STING-activated nanovaccines with anti-PD1 antibodies to generate long-term antitumor memory in a TC-1 tumor model with encouraging results (Luo et al., Nat Nanotechnol (2017) 12(7):648-54.10.1038 / nnano.2017.52). Therefore, particularly preferred methods for treating cancer include administering a combination of 4H2 antibodies and checkpoint modulators to subjects.

[0564] 2. Infection and viral transformation of cells

[0565] In some embodiments, the composition can be used to treat or prevent cells from being infected by bacteria or viruses (e.g., tumor viruses).

[0566] Therefore, the composition can be used to treat local or systemic infections.

[0567] Representative treatable infections include, but are not limited to, infections caused by microorganisms, including but not limited to Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caurolobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, and Hemophilus influenzae type b.B(HIB)), Histoplasma, Hyphomicrobium, Legionella, Leishmania, Leptospirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium (e.g., Tuberculosis), Mycoplasma, Myxococcus, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Protozoa Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia, Cryptococcus neoformans The following bacteria are listed: *Neoformans*, *Histoplasma capsulatum*, *Candida albicans*, *Candida tropicalis*, *Norcardia asteroides*, *Rickettsia ricketsii*, *Rickettsia typhi*, *Mycoplasma pneumoniae*, *Chlamydia psittaci*, *Chlamydia trachomatis*, *Plasmodium falciparum*, *Plasmodium vivax*, *Trypanosoma brucei*, and *Entamoeba histolytica*.Histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0568] Examples of viruses that may be affected by the disclosed compositions include human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-lymphovirus (HTLV), Kaposi's sarcoma-associated herpesvirus (HHV-8), Merkel cell polyomavirus, Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and human cytomegalovirus (CMV), including but not limited to infections caused by immunodeficiency viruses (such as HIV), human papillomavirus (such as HPV), herpes (such as HSV), encephalitis, influenza (such as influenza A virus), the common cold (such as human rhinovirus), coronaviruses (such as SARS-CoV-2), Zika virus, dengue virus, and vesicular stomatitis virus (VSV).

[0569] For example, the composition can be used topically to treat viral skin diseases such as herpes or shingles or genital warts. The composition can also be used to treat systemic viral diseases, including but not limited to HIV, influenza, the common cold, or encephalitis.

[0570] Other viral diseases that can be affected by the application of the composition include Colorado tick fever (caused by Coltivirus, an RNA virus), West Nile fever (encephalitis caused by flaviviruses that mainly occur in the Middle East and Africa), yellow fever, rabies (caused by many different neurotropic strains of the Rhabdoviridae family), viral hepatitis, gastroenteritis (viral) – acute viral gastroenteritis caused by Norwalk virus and Norwalk-like viruses, rotavirus, calicivirus, and astrovirus, poliomyelitis, influenza (flu) caused by orthomyxoviruses that can undergo frequent antigenic variation, measles (rubella), paramyxoviridae, mumps, respiratory syndromes caused by a variety of viruses collectively known as acute respiratory viruses, including viral pneumonia and acute respiratory syndromes (such as laryngotracheobronchitis, commonly known as "croup"), and respiratory diseases caused by respiratory syncytial virus (RSV, the most dangerous cause of respiratory infections in young children).

[0571] In some embodiments, the disclosed compositions are used to treat or prevent viral infection or the spread or worsening of viral infection. For example, in some embodiments, the compositions are used to treat or prevent viral infection or the spread or worsening of viral infection in a subject who has been exposed to a virus or is at risk of exposure to a virus (such as the virus discussed herein).

[0572] 3. Vaccination

[0573] The composition can be administered before, simultaneously with, or after vaccination. In one embodiment, the 4H2 antibody composition is administered simultaneously with the vaccine.

[0574] The disclosed compositions can be administered in combination with preventive or therapeutic vaccines, which can be used to initiate or enhance an immune response in a subject to an existing antigen (such as a tumor antigen from a cancer patient).

[0575] Based on principles well-known in the art, the expected outcomes of prophylactic, therapeutic, or desensitizing immune responses may vary depending on the disease. Similarly, immune responses against cancer, allergens, or infectious pathogens can either completely cure the disease, alleviate symptoms, or be one aspect of a holistic therapeutic intervention. For example, immune response stimulation against cancer can be combined with surgery, chemotherapy, radiation therapy, hormone therapy, and other immunological approaches to influence treatment outcomes.

[0576] STING agonists can enhance antitumor responses when combined with tumor vaccines. For example, CDN ligands formulated with cell-mediated cancer vaccines that produce granulocyte-macrophage colony-stimulating factor, known as STINGVAX, have shown strong in vivo efficacy in several established cancer models (Fu, et al., Sci Transl Med (2015) 7(283): 283ra52.10.1126 / scitranslmed.aaa4306). STING agonists can also elicit antitumor responses when combined with conventional chemotherapy drugs or radiotherapy (Xia, et al., Cancer Res (2016) 76(22): 6747-59.10.1158 / 0008-5472.CAN-16-1404; Baird, et al., Cancer Res(2016)76(1):50-61.10.1158 / 0008-5472.CAN-14-3619). Therefore, a particularly preferred method of treating subjects in need of this treatment includes administering to the subject a combination of one or more components of a 4H2 antibody and a vaccine. The vaccine can combat, for example, cancer or infectious pathogens.

[0577] 4. Wound healing

[0578] In some embodiments, the composition can be used to promote wound healing. cGAMP activation of STING can promote skin wound healing (Mizutani et al., J Dermatol Sci 202097(10:21-29)).

[0579] Representative wounds for which the composition can promote healing include skin wounds caused by trauma or surgery, eye wounds caused by trauma or surgery, and internal organ wounds caused by trauma or surgery.

[0580] For example, the composition can be applied topically to treat skin or eye wounds caused by trauma or surgery, or injected locally to treat skin, eye, or internal organ wounds caused by trauma or surgery.

[0581] 5. Immunomodulatory agents used to treat multiple sclerosis

[0582] In some embodiments, the composition can be used to promote immunomodulation to treat diseases of autoimmunity and / or immunomodulatory disorders, such as multiple sclerosis. Activation of STING via cGAMP can suppress the disease in a model of multiple sclerosis (Johnson et al., J Immunol 2021206(9):2015-28).

[0583] For example, the composition can be administered systemically to treat multiple sclerosis.

[0584] 6. Neurofibromatosis (NF)

[0585] Neurofibromatosis (NF) is a rare genetic disorder characterized by mole-like hamartomas or syndromes with neurological and cutaneous manifestations. It typically causes benign tumors of the nerves and proliferations in other parts of the body, including the skin.

[0586] Neurofibromatosis type 2 (NF2) is a disease characterized by the growth of non-cancerous tumors of the nervous system caused by mutations in the NF2 gene (encoding the Merlin protein). The most common tumor associated with NF2 is called a vestibular schwannoma. These tumors grow along the nerves that transmit information from the inner ear to the brain (the auditory nerve). Tumors that form on the thin membranes (meninges) covering the brain and spinal cord are also common in NF2. These tumors are called meningiomas. Tumors may also occur on other nerves or tissues in the brain or spinal cord of patients with this disease. In some implementations, the method involves delivering nucleic acids (such as mRNA) encoding NF2.

[0587] The results below indicate that 4H2 can mediate gene delivery to NF2 mRNA and affect NF2 tumors in vivo. Therefore, in some embodiments, the subject has neurofibromatosis, such as neurofibromatosis type 2. In some embodiments, the composition is used to treat subjects with schwannomas and / or meningiomas.

[0588] The invention can be further understood through the following numbered paragraphs:

[0589] 1. A composition comprising or consisting of the following components.

[0590] (a) A complete 4H2 monoclonal antibody or a cell-penetrating fragment thereof, optionally selected from monovalent, bivalent, or multivalent single-chain variable fragments (scFv), or bispecific antibody fragments; or its humanized form, chimeric form, or variant thereof; and

[0591] (b) Nucleic acid goods, including nucleic acids encoding polypeptides, functional nucleic acids, nucleic acids encoding functional nucleic acids, or combinations thereof.

[0592] 2. The composition described in paragraph 1, wherein (a) comprises

[0593] (i) A combination of the CDR of SEQ ID NO:5 and the CDR of SEQ ID NO:1;

[0594] (ii) Combinations of first, second, and third heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:6-8 and first, second, and third light chain CDRs comprising the amino acid sequences of SEQ ID NO:2-4;

[0595] (iii)(i) or (ii) humanized forms;

[0596] (iv) A heavy chain having at least 85% sequence identity with an amino acid sequence of SEQ ID NO:5 and a light chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1; or

[0597] (v)(iv) humanized forms.

[0598] 3. The composition described in paragraph 1 or 2, wherein (a) comprises epitope specificity that is the same as or different from that of monoclonal antibody 4H2.

[0599] 4. The compositi...

Claims

1. A composition comprising or consisting of the following components (a) A complete 4H2 monoclonal antibody or a cell-penetrating fragment thereof, optionally selected from monovalent, bivalent, or multivalent single-chain variable fragments (scFv), or bispecific antibody fragments; or its humanized form, chimeric form, or variant thereof; and (b) Nucleic acid goods, including nucleic acids encoding polypeptides, functional nucleic acids, nucleic acids encoding functional nucleic acids, or combinations thereof.

2. The composition according to claim 1, wherein (a) comprises: (i) A combination of the CDR of SEQ ID NO:5 and the CDR of SEQ ID NO:1; (ii) Combinations of first, second, and third heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:6-8 and first, second, and third light chain CDRs comprising the amino acid sequences of SEQ ID NO:2-4; (iii)(i) or (ii) humanized forms; (iv) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:5 and a light chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1; or (v)(iv) humanized forms.

3. The composition according to claim 1 or 2, wherein (a) comprises epitope specificity that is the same as or different from that of the monoclonal antibody 4H2.

4. The composition according to any one of claims 1-3, wherein (a) is a recombinant antibody having an antigen-binding site of monoclonal antibody 4H2.

5. A composition comprising: (a) Binding proteins, including: (i) A combination of the CDR of SEQ ID NO:5 and the CDR of SEQ ID NO:1; (ii) Combinations of first, second, and third heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:6-8 and first, second, and third light chain CDRs comprising the amino acid sequences of SEQ ID NO:2-4; (iii)(i) or (ii) humanized forms; (iv) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:5 and a light chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1; or (v)(iv) humanization forms, and (b) Nucleic acid cargo, including nucleic acids encoding polypeptides, functional nucleic acids, nucleic acids encoding functional nucleic acids, or combinations thereof.

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

7. The composition according to claim 6, wherein (a) it targets a specific cell type.

8. The composition according to any one of claims 1-7, wherein (a) and (b) are non-covalently linked or associated.

9. The composition according to any one of claims 1-8, wherein (a) and (b) are complexes.

10. The composition according to any one of claims 1-9, wherein (b) comprises DNA, RNA, PNA or other modified nucleic acids, or nucleic acid analogs, or combinations thereof.

11. The composition according to any one of claims 1-10, wherein (b) comprises mRNA.

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

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

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

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

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

17. The composition according to any one of claims 1-16, wherein (b) comprises a functional nucleic acid.

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

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

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

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

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

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

24. The composition according to any one of claims 1-23 further comprises vector DNA.

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

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

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

28. The composition according to claim 27, further comprising polymer nanoparticles encapsulating the (a) and (b) composites.

29. The composition of claim 28, wherein the targeting portion, cell-penetrating peptide, or combination thereof is associated with, connected to, conjugated to, or otherwise directly or indirectly attached to the nanoparticles.

30. A method of delivering nucleic acid cargo to cells, comprising contacting the cells with an effective amount of the composition of any one of claims 1-29.

31. The method of claim 30, wherein the contact occurs outside the body.

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

33. The method according to any one of claims 30-32, further comprising administering the cells to a desired subject.

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

35. The method of claim 30, wherein the contact occurs in vivo after administration to the desired subject.

36. The method according to any one of claims 33-35, wherein the subject suffers from a disease or condition.

37. The method of claim 36, wherein the disease or condition is a hereditary disease, cancer, or an infectious or contagious disease.

38. The method of claim 36 or 37, wherein (b) it is delivered in an effective amount to the cells of the subject to alleviate one or more symptoms of the subject’s disease or condition.

39. A method for preparing the composition of any one of claims 1-29, comprising culturing and / or mixing (a) and (b) at an appropriate temperature for a certain time prior to contact with cells to form a complex of (a) and (b).

40. A method for preparing the composition of any one of claims 1-29, 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°C.

41. The composition or method according to any one of claims 1-40, wherein the ratio of (a):(b) is between 1:3 and 5:1, and optionally, the ratio is 1:1 or 3:

1.

42. A method for increasing the activation of an immune receptor in the cells of a desired subject, comprising administering an effective amount of (a) an intact 4H2 monoclonal antibody or a cell-penetrating fragment thereof, optionally selected from monovalent, bivalent, or multivalent single-chain variable fragments (scFv), or bispecific antibody fragments; or humanized forms thereof, chimeric forms thereof, or variants thereof; optionally, wherein the immune receptor is cGAS or another pattern recognition receptor (PRR), optionally, a Toll-like receptor, optionally, TLR7.

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

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

45. The method according to any one of claims 42-44, wherein the subject has a wound that needs to heal.

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

47. The method according to any one of claims 42-46, further comprising administering an additional formulation to the subject (b).

48. The method of claim 47, wherein (b) is selected from nucleic acid cargoes, immunostimulatory nucleic acids, one or more vaccine components, immune checkpoint modulators that induce, increase or enhance immune responses, and combinations thereof.

49. A method for treating cancer or infection, comprising administering to a subject in need an effective amount of a combination of the following components: (a) A complete 4H2 monoclonal antibody or a cell-penetrating fragment thereof, optionally selected from monovalent, bivalent, or multivalent single-chain variable fragments (scFv) or bispecific antibody fragments; or humanized forms thereof, their chimeric forms, or variants thereof; and (b) Immune checkpoint modulators that can induce, increase or enhance immune responses.

50. The method according to any one of claims 48-49, wherein the immune checkpoint modulator induces an immune response against cancer or infection.

51. The method according to any one of claims 48-50, wherein the immune checkpoint modulator reduces immunosuppressive pathways.

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

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

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

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

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

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

58. A method for treating cancer or infection, comprising administering to a subject in need an effective amount of a combination of the following components (a) A complete 4H2 monoclonal antibody or a cell-penetrating fragment thereof, optionally selected from monovalent, bivalent, or multivalent single-chain variable fragments (scFv), or bispecific antibody fragments; or its humanized form, chimeric form, or variant thereof; and (b) Immunostimulatory nucleic acids.

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

60. A method of administering a vaccine to a subject, comprising injecting the subject with... (a) A complete 4H2 monoclonal antibody or its cell-penetrating fragment, optionally a monovalent, bivalent, or multivalent single-chain variable fragment (scFv) or bispecific antibody fragment; or its humanized form, chimeric form, or variant thereof; and (b) One or more vaccine components.

61. The method according to claim 48 or 60, wherein 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. The method of claim 61, wherein the antigen is derived from bacteria or a virus.

63. The method according to any one of claims 48-62, wherein the combination of applying (a) and (b) results in a greater reduction of one or more symptoms of cancer or infection than the sum of the effects of applying (a) or (b) in the absence of (a) or (b).

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

65. The method according to any one of claims 48-63, wherein the subject is given (b) 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 or 24 hours, 1, 2, 3, 4, 5, 6 or 7 days, 1, 2, 3 or 4 weeks, or any combination thereof, prior to the administration of (a) to the subject.

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

67. The method according to any one of claims 42-66 further includes surgery or radiation therapy.

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

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

70. The method according to claim 68 or 69, wherein (b) is the nucleic acid cargo, optionally, wherein the nucleic acid cargo consists of DNA, RNA, PNA, PMO, or other modified nucleic acids, or nucleic acid analogs, or combinations thereof.

71. The method according to claim 68 or 69, wherein (b) is not the nucleic acid cargo.

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

73. The method according to any one of claims 42-72, wherein (a) includes epitope specificity that is the same as or different from that of the monoclonal antibody 4H2.

74. The method according to any one of claims 42-73, wherein (a) is a recombinant antibody having an antigen-binding site of monoclonal antibody 4H2.

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

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

77. The method of claim 76, wherein (a) the target cell type is targeted.

78. A pharmaceutical composition comprising (a) and (b) as described in any one of claims 48-77 and a pharmaceutically acceptable excipient.

79. The pharmaceutical composition according to claim 78, comprising nucleic acid cargo.

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

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

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

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

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

85. A composition comprising (a) A dual-specific binding protein comprising: (i) The combination of the CDR of SEQ ID NO:5 and the CDR of SEQ ID NO:1; (ii) Combinations of first, second, and third heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:6-8 and first, second, and third light chain CDRs comprising the amino acid sequences of SEQ ID NO:2-4; (iii)(ai) or (aii) humanized forms; (iv) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:5 and a light chain comprising an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1; or (v)(iv) humanization forms, and Binding domains that bind to immune cell markers.

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

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

88. The composition according to any one of claims 85-87, comprising (b) Nucleic acid goods, including nucleic acids encoding polypeptides, functional nucleic acids, nucleic acids encoding functional nucleic acids, or combinations thereof.

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

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

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

Images