CN120897933A - Compositions and methods using cell-penetrating antibodies - Google Patents

Abstract

Compositions for delivering nucleic acid cargo to cells and methods of using the same are provided. The compositions generally comprise (a) a 4H2 monoclonal antibody or fragment thereof having antigen binding capacity, cell permeability, 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 (e.g., cGAS and TLR 7) in a subject's cells. These methods generally comprise administering to the subject an effective amount of a 4H2 antibody. The subject may be healthy, or may have a disease or disorder, such as cancer or infection.

Description

Compositions and methods using cell penetrating antibodies

Cross Reference to Related Applications

The present application claims the benefit and priority of U.S. provisional application Ser. No. 68/379,121 filed on day 10 and 11 of 2022 and U.S. provisional application Ser. No. 68/379,123 filed on day 10 and 11 of 2022, each of which is expressly incorporated herein in its entirety.

Reference sequence listing

The sequence listing is submitted in the form of a text file, with the file name "yu8475pct. Xml", created on 202310 month 11, size 27,711 bytes, according to 37c.f.r. ≡1.52 (e) (5), incorporated herein by reference.

Technical Field

The present invention relates generally to the field of intracellular delivery of nucleic acids, applications of which include, but are not limited to, in vitro, ex vivo and in vivo gene therapy and gene editing and/or enhancing immune responses, in particular through modulation of immune receptors, applications of which include, but are not limited to, treatment of cancer and infections and improved vaccination.

Background

Gene therapy

Gene therapy includes 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 complexity of production, limited packaging capacity, and unfavorable immunological properties, which limit the use of gene therapy, limiting the potential for prophylactic gene therapy (Seow and Wood, mol ter.17 (5): 767-777 (2009).

In vivo absorption and distribution of nucleotides in cells and tissues has been observed (Huang et al, FEBS Lett.,558 (1-3): 69-73 (2004)). Furthermore, studies by Nyce et al, for example, have shown that antisense Oligodeoxynucleotides (ODNs) bind to endogenous surfactants (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 cancer 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 techniques, in particular for in vivo applications. AAV9 found in 2003 is still a commonly used viral vector (Robbins, "gene therapy precursor" has called the field to be behind-the delivery technology is embarrassing ", stat,2019, 11).

Accordingly, it is an object of the present invention to provide compositions and methods of use thereof that improve the delivery of nucleic acids to cells.

Modulating immune responses

GMP-AMP (cGAMP) synthase (cGAS) is a cytoplasmic DNA sensor that activates the innate immune response by producing the second messenger cGAMP. In turn, cGAMP activates the aptamer 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 immunomodulation and Cancer progression 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.

For example, in tumor microenvironments, T cells, endothelial cells and fibroblasts produce type I IFN under stimulation by in vitro and in vivo STING agonists (Corrales et al, cell Rep (2015) 11 (7): 1018-30.10.1016/j.cellrep.2015.04.031). In contrast, most studies have shown that tumor cells inhibit activation of the STING pathway, potentially leading to immune evasion during canceration (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 induction of spontaneous anti-tumor T cell responses involving 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; wo, et al, immunol (2014) 41 (5): 830-42.10.1016/j.immunol.2014.10.017.) furthermore, host STING pathway is essential for Dendritic Cell (DC) -mediated efficient cross-priming of tumor-Ag specific cd8+ T cells (Woo et al, immunol (2014) 41 (5): 830-42.10.1016/j.immunol.2014.10.017; immunol.2014) 41 (5): 830-573-52.10.1016/j.2014.019.10.017). Based on these results, drugs have been explored to directly stimulate STING pathways to treat cancer.

In addition to cancer, the development of STING agonists has also been suggested for a number of different therapeutic purposes, including use as vaccine adjuvants and for chronic viral or bacterial infections.

With the increasing scope of clinical applications, modified compositions and methods for modulating cGAS-STING pathways and other immune response receptor signaling pathways are also becoming increasingly popular.

Accordingly, it is another object of the present disclosure to provide improved compositions and methods of use thereof to increase the activity of immune receptors such as cGAS and pattern recognition receptors (PPRs), including toll-like receptors (such as TLR 7).

Disclosure of Invention

The present invention provides compositions for delivering nucleic acid cargo into cells and methods of use thereof. The composition generally comprises (a) a 4H2 monoclonal antibody or cell penetrating fragment thereof, a monovalent, divalent, or multivalent single chain variable fragment (scFv), or a bispecific antibody fragment, or a humanized form thereof, or a variant thereof, and (b) a nucleic acid cargo comprising, for example, a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof. Elements (a) and (b) are typically non-covalently linked to form a complex. Exemplary 4H2 antibodies and fragments and fusion proteins thereof include those having (i) the CDR of SEQ ID NO. 1 (optionally SEQ ID NO. 2-4) in combination with the CDR of SEQ ID NO. 5 (optionally SEQ ID NO. 6-8), (ii) a combination of a first, second and third heavy chain CDR selected from SEQ ID NO. 1 (optionally SEQ ID NO. 2-4) with a first, second and third light chain CDR selected from SEQ ID NO. 5 (optionally SEQ ID NO. 5-8), (iii) a humanized version of (i) or (ii), (iv) a combination of a heavy chain having an amino acid sequence with at least 85% sequence identity to the amino acid sequence of SEQ ID NO. 5 and a light chain having an amino acid sequence with at least 85% sequence identity to SEQ ID NO. 1, (v) a humanized version of (iv).

In certain embodiments, the antibody or fragment or fusion protein may be bispecific, e.g., may include binding sequences that target a cell type, tissue or organ.

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

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, siRNA, microrna (miRNA), nucleic acid aptamer, ribozyme, RNAi, or an external guide sequence, or a nucleic acid construct encoding the same.

The cargo may 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 molecule of the cargo comprises or consists of a nucleic acid molecule between about 1 and about 25,000 nucleobases in length. The cargo may be single stranded nucleic acids, double stranded nucleic acids, or a combination thereof.

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

In addition, methods for delivering nucleic acid cargo into cells by contacting the cells with an effective amount of the complex alone or encapsulated in nanoparticles are provided. The contacting may occur in vitro, ex vivo, or in vivo. In certain embodiments, an effective amount of the ex vivo treated cells is administered to a subject in need thereof, e.g., in an effective amount to treat one or more symptoms of a disease or disorder.

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

Applications of the compositions and methods are also provided, including but not limited to gene therapy and CAR T cell manufacturing/formation/treatment.

Also provided are compositions and methods for enhancing the activation of cGAS and/or other immune receptors (e.g., pattern recognition receptors such as TLR 7) in cells 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), bispecific antibody fragments (diabodies), and the like. The antibody may be in its humanized form, its chimeric form or a variant thereof.

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

In certain embodiments, the subject has cancer or an infection. In certain embodiments, the subject is free of cancer. Thus, methods of treating cancer and infections in a subject are also provided. In certain embodiments, the subject is a healthy subject.

In certain embodiments, the compositions and/or methods comprise administering an additional drug to the subject. 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.

In a specific embodiment, a method of treating cancer or 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.

Immune checkpoint modulators are generally capable of inducing an immune response against cancer or infection. For example, immune checkpoint modulators may reduce immunosuppressive pathways, such as the PD-1 pathway. Thus, the modulator may be a PD-1 antagonist, a PD-1 ligand antagonist, or a CTLA4 antagonist. In certain embodiments, immune checkpoint modulators increase the immune activation pathway. The immune checkpoint modulator can be a small molecule, an antibody, a CAR-T cell, or an oncolytic virus.

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

In another specific embodiment, a 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 may include an antigen, a nucleic acid encoding an antigen, an adjuvant, a nucleic acid encoding an adjuvant, or a combination thereof. The antigen may be derived from, for example, a bacterium or a virus.

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

In certain embodiments, the 4H2 antibody 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 the additional drug. In other embodiments, the additional drug 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 the 4H2 antibody.

Any method may further employ a therapeutic agent or intervention such as a chemotherapeutic agent, an anti-infective agent, surgery, radiation therapy, or a combination thereof.

The nucleic acid cargo or nucleotide, nucleoside or nucleobase may increase cell penetration and/or activation of cGAS and/or another pattern recognition receptor (e.g., TLR 7) by the 4H2 antibody. Thus, any of the compositions and methods disclosed may further comprise a nucleic acid cargo or a nucleotide, nucleoside or nucleobase cargo. In certain embodiments, the nucleic acid cargo or nucleotide, nucleoside or nucleobase cargo is an additional drug. In certain embodiments, the nucleic acid cargo or nucleotide, nucleoside, or nucleobase cargo is not an additional drug (i.e., is administered in further combination with an additional drug). In preferred embodiments, the nucleic acid cargo or nucleotide, nucleoside or nucleobase cargo forms a complex with the 4H2 antibody. In a preferred embodiment, the nucleic acid cargo or nucleotide, nucleoside or nucleobase cargo (containing guanine or guanosine) forms a complex with the 4H2 antibody. The nucleic acid cargo may consist of DNA, RNA, PNA, phosphodiamide Morpholine Oligomers (PMOs) or other modified nucleic acids, nucleic acid analogs or modified nucleotides, nucleosides or nucleobase analogs or combinations thereof.

Drawings

FIGS. 1A-1C show that 4H2 is a cell penetrating anti-GUO autoantibody sensitive to DP. FIG. 1A is an image of lysates from Cal12T cells treated with 0-1mg/mL 4H2 for 24 hours, with actin primary antibody as loading control, with anti-mouse secondary antibodies to detect actin primary antibody and 4H2 (both mice), and analyzed by immunoblotting (western blot). The 4H2 HC and LC run at their expected Molecular Weights (MW), indicating that the antibodies did not significantly degrade after 24 hours of cell penetration. FIG. 1B shows images of total ERK1/2 and pERK1/2 immunoblots with Cal12T cell lysates treated with control medium, igG control or 4H2 for 24 hours. IgG controls had no effect on total ERK1/2 and pERK1/2, while 4H2 would decrease pERK1/2 but not total ERK1/2. FIG. 1C is a dot-matrix plot showing quantitative analysis of 4H2 fluorescence in Cal12T cells (cell penetration) with or without DP treatment by ImageJ.

Figures 2A-2D show that 4H2 penetrated glioma cells in a GUO-responsive manner and penetrated the BBB (blood brain barrier) Transwell model. FIGS. 2A-2C are graphs showing the effect of ADE or GUO addition to cell culture media on the efficiency of cell penetration through GSC, as assessed quantitatively by image J for DX1 or 4H2 fluorescent signals. ADE enhanced DX1 penetration (fig. 2A), but had no effect on 4H2 (fig. 2B). GUO significantly enhanced the cell penetration of 4H2 (FIG. 2C). FIG. 2D is a graph showing the results of a BBB Transwell model using hCMEC/D3 brain microvascular endothelial cells (BECs) and Normal Human Astrocytes (NHA) for assessing cross-barrier transport of 4H2 from the apical to the basolateral compartments. The results show that 4H2 can penetrate the barrier and that nucleoside transport inhibitor DP inhibits transport of 4H 2.

Figures 3A-3B show that 4H2 localizes to in situ brain tumors and prolongs survival of GBM models. Fig. 3A is a Kaplan-Meier survival graph of GSC-derived in situ GBM tumor mice treated with IgG control (n=4) or 4H2 (n=5). The median survival of the mice was increased by 66% compared to mice treated with the IgG control group (< 0.01, log-rank test) with a survival of 40% at the end of the study for the mice treated with 4H2 and 0% at the end of the study for the mice treated with the IgG control group. FIG. 3B is a Kaplan-Meier survival graph of GL 261-derived in situ GBM tumor mice treated with IgG control (6), 4H2 (6), anti-PD 1 (6), anti-PD1+IgG control (7) or anti-PD1+4H2 (7). The median survival increased by 32% compared to the IgG control group (p=0.03, log-rank test) with 4H2, and by 50% compared to the anti-pd1+igg control group with 4H2 in combination with anti-PD 1 (p=0.02, log-rank test). The 4H2 or 4h2+ anti-PD 1 alone gave 33% and 29% survival at the end of the study, respectively, while the survival for all other groups was 0%.

Fig. 4A is a bar graph of quantification of TUNEL staining with ImageJ, showing a relative 4.5±0.6 fold increase in TUNEL signaling in mice treated with 4H2 compared to IgG control group (P < 0.01). Fig. 4B is a bar graph showing relative CD8 cell counts in each High Power Field (HPF) following IgG control or 4H2 treatment of mice according to anti-CD 8 immunostaining of GBM brain tumor sections. 4H2 increased CD8 content in tumors by about 53%, relative counts for 4H2 treated mice were 1.53±0.15, while for IgG control treated mice were 1.00±0.04 (< 0.03). These data demonstrate the 4H2 mediated stimulation of T cell infiltration into GBM tumors. Figures 4C and 4D show that 4H2 did not improve survival in the immunodeficient in situ GBM model. Figures 4C and 4D show that 4H2 does not improve survival in the immunodeficient in situ GBM model. Kaplan-Meier survival plots of athymic nude mice with PPQ in situ GBM brain tumors were treated with either weekly (fig. 4C) or twice weekly (fig. 4D) IgG controls (n=4 and 6, respectively) or 4H2 (n=4 and 6, respectively) cycles. In this immunodeficiency model, 4H2 had no significant effect on median survival compared to IgG control group, indicating the importance of the functional immune system to 4H 2-affected survival.

FIGS. 5A-5D are immunoblot (western blot) images showing binding of 4H2 to cGAS. Protein G beads were used to separate antibody content and binding protein from IgG control or 4H2 treated Glioma Stem Cells (GSCs). The detection of G protein Ras and cGAS was performed on the western blot of import and protein G pulldown (pulldown). No IgG control or 4H2 binding to Ras was observed (fig. 5A), but 4H2 had a significant binding to cGAS with a greater binding signal than the background signal detected for the IgG control (fig. 5B). Purified cgas±nucleic acids were incubated with IgG controls or 4H2, and then the antibodies and binding proteins were pulled down with protein G. The presence of nucleic acid reduced the binding of 4H2 to cGAS, but did not affect the nonspecific binding of IgG controls to cGAS (fig. 5C). The western blot of anti-IgG confirmed that the IgG control and 4H2 content in the pull-down samples were equal (fig. 5D). FIGS. 5E and 5G are images of western blot, and FIG. 5F is a bar graph showing that 4H2 interacts with cGAS in a nucleic acid dependent manner. Purified recombinant cGAS was incubated with control IgG or 4H2 +/-nuclease (benzonase). Antibodies and binding proteins were then separated on protein G beads, cGAS were visualized with western blot, and quantified with ImageJ. In the absence of nuclease, the interaction of 4H2 with cGAS was shown by an approximately 6-fold increase in cGAS pulldown over IgG control (P < 0.001), whereas the addition of nuclease abrogated this interaction.

FIGS. 6A-6D show that 4H2 enhances cGAS activity. Fig. 6A is a line graph showing that 4H2 leads to a dose-dependent increase in cGAS activity. cGAS activity was detected by measuring the relative yields of ATP and GTP produced cGAMP in the presence of IgG control or 4H 2. FIG. 6B is an immunoblot image showing that 4H2 induces nuclear translocation of NF-kB in GSC. The cytoplasmic and nuclear content of GSC treated with IgG control or 4H2 was isolated and Western blot detection analysis was performed using NF-kB and Lamin B1 as loading controls. GSCs transfected with control or CGAS SIRNA were treated with IgG control or 4H 2. FIG. 6C is a CGAS WESTERN blot image demonstrating a successful knockout. FIG. 6D is a line graph showing the results of colony formation assays, demonstrating cGAS-dependent toxicity of 4H2 to GSC. FIG. 6E is a bar graph showing that 4H2 induces nuclear translocation of NF- κB. The cytoplasmic and nuclear contents of PPQ cells treated with IgG control or 4H2 were analyzed by immunoblotting (western blot) to detect NF- κB, and Lamin B1 was used as the loading control. The relative nuclear content of NF- κB was quantified using ImageJ. 4H2 increased the relative content of nuclear NF- κb by a factor of 2.2±0.2 (< 0.05). FIG. 6F is a bar graph showing survival as determined by colony formation assays in Cal12T lung cancer cells (D) transfected with control or CGAS SIRNA and treated with IgG control or 4H2, demonstrating the cGAS-dependent toxicity of 4H 2. P < 0.05).

FIGS. 7A-7B show 4H2 binding DNA and RNA. FIG. 7A is an image of the assessment of 4H2 binding to circular and linearized pcDNA3 plasmid DNA by 1% agarose EMSA. The 4H2, but not IgG control, shifted both forms of DNA consistent with binding. FIG. 7B is an image of the assessment of 4H2 binding to total DNA and mRNA by 1% agarose EMSA. The 4H2, but not the IgG control, caused consistent movement of both forms of RNA for binding.

Figures 8A-8B are bar graphs showing the delivery of DNA and mRNA by 4H2 to glioma cells. pGL4.13 (luc 2/SV 40) complexed with DX1 or 4H2 was added to U87 glioma cells and luciferase activity was detected after 24 hours (FIG. 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 detected after 24 hours (fig. 8B).

Figures 9A-9B show images of 4H2 mediating localized gene therapy in the Central Nervous System (CNS). 4H2/Cre mRNA was injected into the brains of Ai9 Cre reporter mice and Cre recombinase activity was assessed 24 hours later by RFP fluorescence. RFP signals were observed in a localized region of the injection trajectory (fig. 9A). RFP signal was assessed twenty-four hours after intraocular 4H2/Cre mRNA treatment Ai9 Cre report mice. The RFP signal observed in the retina demonstrated 4H 2-mediated retinal gene therapy (fig. 9B).

FIGS. 10A-10B are images showing in vivo delivery of 4H2 mRNA. Nude mice bearing H358 flank tumors received a single intratumoral injection of DX1 or 4H2 and Luc mRNA mixture (w/w=3). 6. After 24 and 72 hours, expression of Luc was assessed by IVIS. The 4H2/Luc mRNA successfully mediated Luc expression, while little signal was detected in DX1/Luc mRNA injected tumors (FIG. 10A). C57/BL6 mice were intramuscular injected with 4H2/Luc mRNA (left quadriceps w/w=3, right quadriceps w/w=1). After 6 hours and 24 hours, expression of Luc was assessed using IVIS. The 4H2/Luc mRNA successfully mediated expression of Luc (FIG. 10B).

FIGS. 11A-11B are a series of representative IVIS images (FIG. 11A) and corresponding histograms (FIG. 11B) showing luminescence in the HEI 193xenograph model expressing luciferase in untreated mice and mice treated with either 4H2, 4H2+NF2 DNA or 4H2+NF2 mRNA alone.

FIG. 12A is a schematic of the design of a 4H2-CD5 bispecific antibody. FIGS. 12B and 12C are a series of representative FACS plots (FIG. 12B) and corresponding histograms (FIG. 12C) showing the expression of DeRed tumor cells isolated from an Ai9 mouse model carrying MC38 tumors.

Fig. 13A and 13B are western blot images and corresponding graphs (determined by ImageJ) of glioma stem cell-like cells (GSCs) treated with IgG control or 4H2 and examined for TLR 7. FIG. 13C is an image of a western blot. Protein G beads pulled antibodies and binding proteins from GSC cleavage solution treated with IgG control or 4H2, followed by TLR7 immunoblot analysis. The blots represent two independent experiments.

Detailed Description

I. Definition of the definition

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, the single chain variable region and heavy chain variable region being linked by a linker arm such that the scFv forms the structure required for antigen binding (i.e., the VH and VL of a single polypeptide chain combine with each other to form an Fv). The VL and VH regions may be derived from the parent antibody, or may be synthesized chemically or recombinantly.

The term "variable region" as used herein is designed to distinguish such domains of immunoglobulins from domains that are widely shared by antibodies (e.g., fc domains of antibodies). The variable region includes a "hypervariable region" whose residues are responsible for antigen binding. Hypervariable regions include amino acid residues derived from the "complementarity determining regions" or "CDRs" (i.e., typically about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, about residues 27-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region; kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public health services, national institutes of health, bethesda, MD. (1991)) and/or residues derived from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region; chothia and Lesk,1987, J.Mol.biol.196:917).

The term "framework region" or "FR" residues as used herein refers to variable region residues other than the hypervariable region residues defined herein.

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 such immunoglobulin molecules, and human or humanized immunoglobulin molecules that bind to a target antigen.

The term "cell penetrating antibody" as used herein refers to an immunoglobulin, fragment thereof, variant thereof or 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 living mammalian cells and binds to guanosine. In certain embodiments, the antibody is transported into the cytoplasm of the cell without the aid of a carrier or conjugate. In other embodiments, the antibody is conjugated to a cell penetrating moiety (e.g., a cell penetrating peptide).

In addition to intact immunoglobulin molecules, the term "antibodies" also includes fragments, binding proteins and polymers of immunoglobulin molecules, chimeric antibodies, such as human antibodies or humanized antibodies, containing immunoglobulin specific types derived from more than one species, class or subclass of immunoglobulin sequences, and recombinant proteins containing at least immunoglobulin specific binding DNA. The antibodies may be tested for their expected activity using in vitro assays or the like as described herein, and then tested for therapeutic activity in vivo according to known clinical assays.

The term "variant" as used herein refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential characteristics. Typical variants of a polypeptide differ in amino acid sequence from another reference polypeptide. Generally, differences are limited, so the sequences of the reference polypeptides and variants are generally very similar, even identical in many regions. The amino acid sequences of the variant polypeptide and the reference polypeptide may differ by one or more modifications (e.g., substitutions, additions and/or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. Variants of the polypeptide may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally.

Modifications and alterations can be made to the polypeptide structures of the present disclosure, but molecules with similar characteristics to the polypeptide (e.g., conservative amino acid substitutions) can be obtained. For example, certain amino acids may be substituted for other amino acids in the sequence without significant loss of activity. Since the interactive capacity and nature of a polypeptide determines the biological functional activity of a polypeptide, certain amino acid sequence substitutions may be made in the polypeptide sequence, but a polypeptide with similar properties may still be obtained.

In making such a change, the hydropathic index of amino acids may be considered. The importance of the index of hydrophilic amino acids in conferring interactive biological functions on polypeptides is well known in the art. It is well known that certain amino acids may be substituted with other amino acids having similar hydropathic indices or scores, but still produce polypeptides having similar biological activities. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge characteristics. These indices are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9) and arginine (-4.5).

It is generally believed that the relative hydrophilicity of the amino acids determines the secondary structure of the resulting polypeptide, which in turn determines the interaction of the polypeptide with other molecules (e.g., enzymes, substrates, receptors, antibodies, antigens, and cofactors). It is well known that an amino acid may be substituted with another amino acid having a similar hydropathic index, but still a functionally equivalent polypeptide may be obtained. In this variation, substitution of amino acids having a hydropathic index within.+ -. 2 is preferred, amino acids within.+ -. 1 are particularly preferred, and amino acids within.+ -. 0.5 are even more particularly preferred.

Substitution of like amino acids may be made based on the hydrophilicity of the amino acids, particularly in the case where the resulting biologically equivalent polypeptide or peptide is used in an immunological example. The following are the hydrophilicity values assigned to the amino acid residues arginine (+3.0), lysine (+3.0), aspartic acid (+3.0.+ -. 1), glutamic acid (+3.0.+ -. 1), serine (+0.3), asparagine (+0.2), glutamic acid (+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 will be appreciated that an amino acid may be substituted with another amino acid having a similar hydrophilicity value, but that bioequivalent, in particular immunologically equivalent, polypeptides may still be obtained. In this variation, substitution of amino acids having a hydrophilicity value within.+ -. 2 is preferred, amino acids having a hydrophilicity value within.+ -. 1 are particularly preferred, and amino acids having a hydrophilicity value within.+ -. 0.5 are even more particularly preferred.

As noted above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account the various characteristics described above are well known to those of skill in the art and include (original residues: exemplary substitutions )：(Ala：Gly,Ser),(Arg：Lys),(Asn：Gln,His),(Asp：Glu,Cys,Ser),(Gln：Asn),(Glu：Asp),(Gly：Ala),(His：Asn,Gln),(Ile：Leu：Leu,Val)、(Leu：Ile,Val)、(Lys：Arg)、(Met：Leu,Tyr)、(Ser：Thr)、(Thr：Ser)、(Tip：Tyr)、(Tyr：Trp,Phe) and (Val: ile, leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of the polypeptides described above.

The term "percent (%) sequence identity" as used herein refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence after aligning the sequences and introducing gaps, as needed, to achieve the maximum percent sequence identity. The alignment for determining the percent sequence identity can be accomplished by various methods known to those skilled in the art, for example, using published computer software, such as BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR) software. Suitable parameters for measuring the alignment, including any algorithms required to achieve maximum alignment over the full length of the compared sequences, can be determined by known methods.

The term "specifically binds" as used herein refers to an antibody that binds to its cognate antigen (e.g., guanosine) without significant binding to other antigens. Under such conditions, to allow the antibody to specifically bind to the target, an antibody specific for the target must be selected. A variety of immunoassay methods can be used to select antibodies that have a specific immune response to a particular protein. For example, solid-phase ELISA immunoassays are commonly used to select monoclonal antibodies that have a specific immune response to a protein. See, e.g., harlow and Lane (1988) Antibodies, A Laboratory Manual, cold Spring Harbor Publications, new York, which describe immunoassay modes and conditions that can be used to determine specific immune responses. Preferably, the antibody "specifically binds" to an antigen with an affinity constant (Ka) for the second molecule of greater than about 10 ⁵mol^-1 (e.g., 10⁶mol^-1、10⁷mol^-1、10⁸mol^-1、10⁹mol^-1、10¹⁰mol^-1、10¹¹mol^-1 and 10 ¹²mol^-1 or higher).

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 in the population of antibodies are identical except for natural mutations that may be present in a small portion of the antibody molecule.

The term "subject" as used herein refers to any individual who is the subject of administration. The subject may be a vertebrate, for example a mammal. Thus, the subject may be a human. The term does not denote a particular age or sex.

The term "effective amount" as used herein means that the amount of the composition used is sufficient to ameliorate one or more causes or symptoms of the disease or disorder. Such improvements require only a reduction or change, and do not necessarily require elimination. The precise amount will vary depending on various factors such as subject-related variables (e.g., age, immune system health, etc.), the disease or condition being treated, as well as the route of administration and the pharmacokinetics of the agent being administered.

The term "pharmaceutically acceptable" as used herein refers to materials that do not adversely affect biology or other aspects, i.e., such materials can be administered to a subject without causing any adverse biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which they are included.

The term "carrier" or "excipient" as used herein refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient, in a formulation to which one or more active ingredients are bound. The choice of carrier or excipient is naturally to minimize degradation of the active ingredient and minimize any adverse side effects on the subject, as is well known to those skilled in the art.

The term "treatment" as used herein refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological state or condition. The term includes active therapies, i.e. therapies directed specifically to ameliorating a disease, pathological state or condition, as well as causal therapies, i.e. therapies directed to eliminating the etiology of the associated disease, pathological state or condition. Furthermore, the term also includes palliative treatment, i.e. treatment intended to alleviate symptoms rather than cure a disease, pathological state or condition, prophylactic treatment, i.e. treatment intended to minimize or partially or completely inhibit the development of the associated disease, pathological state or condition, and supportive treatment, i.e. treatment intended to supplement another specific therapy intended to ameliorate the associated disease, pathological state or condition.

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 coupling or attaching another molecule. As used herein, "directing" refers to preferentially attaching molecules to a selected cell or tissue type. This can be used to guide cellular material, molecules or drugs, as described below.

The term "inhibit" or "decrease" as used herein refers to decreasing activity, response, condition, disease or other biological parameter. This may include, but is not limited to, complete elimination of activity, response, condition or disease. This may also include, for example, a 10% reduction in activity, response, condition or disease compared to a natural or control level. Thus, the decrease may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any decrease in between, as compared to the native level or the control level.

The term "fusion protein" as used herein refers to a polypeptide formed by joining two or more polypeptides together by a peptide bond formed between the amino terminus of one polypeptide and the carboxy terminus of another polypeptide. Fusion proteins may be formed by chemical coupling of constituent polypeptides, or may be expressed as a single polypeptide by a nucleic acid sequence encoding a single, contiguous fusion protein. A single chain fusion protein is a fusion protein having a single continuous polypeptide backbone. Fusion proteins can be prepared using techniques conventional in molecular biology, by joining two genes in frame into a single nucleic acid sequence, and then expressing the nucleic acid in a suitable host cell, under conditions that result in the fusion protein.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

The term "about" is used to describe values above or below the stated value in a range of about +/-10%, in other embodiments the stated value may be above or below the stated value in a range of about +/-5%, in other embodiments the stated value may be above or below the stated value in a range of about +/-2%, in other embodiments the stated value may be above or below the stated value in a range of about +/-1%. The foregoing ranges are intended to be illustrative by context and are not meant to be further limiting.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. 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 invention and does not pose a limitation on the scope of the embodiments of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

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 the 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 may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed, and various modifications that can be made to various molecules, including the ligand, are discussed, each combination and permutation of the ligand, and the modifications that are possible, are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B and C and a class of molecules D, E and F are disclosed, and an example of a combination of molecules A-D is disclosed, then each molecule is considered individually and collectively even if each is not listed individually. Thus, in this example, each of the A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F combinations is specifically contemplated and should be considered disclosed in the publications A, B and C, D, E and F, and the example combinations A-D. Also, any subset or combination thereof is specifically contemplated and disclosed. Thus, for example, the subgroups of A-E, B-F and C-E are specifically contemplated and should be considered as disclosed A, B and C, D, E and F, and the example combinations A-D. In addition, each material, composition, component, etc. contemplated and disclosed above may or may not be specifically and independently included in any group, subgroup, list, collection, etc. of such materials.

These concepts apply to all aspects of the application, including but not limited to steps in methods of making and using the disclosed compositions. Thus, if various 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 that each such combination is specifically contemplated and should be considered disclosed.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples, or exemplary language (e.g., "such as") provided herein, are intended merely to better illuminate embodiments of the invention and do not pose a limitation on the scope of the embodiments of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

II composition

It was found that the 4H2 antibody aids in the delivery of nucleic acids across the plasma membrane into the cytoplasm. Thus, compositions and methods for enhancing delivery of nucleic acid constructs using 4H2 are provided. Typically, an effective amount of the 4H2 antibody is contacted with the nucleic acid that is desired to be delivered into the cell. Typically, contact will take a long enough time for the 4H2 and nucleic acid cargo to form a non-covalent complex. The complex is contacted with the cell for a sufficient period of time before the nucleic acid cargo is delivered into the cell. Nucleic acid cargo may accumulate in greater amounts, higher quality (e.g., more intact, more functional, etc.), or at a faster rate, or a combination thereof, than cells contacted with the nucleic acid cargo in the absence of the antibody. Because antibodies are the delivery means, the delivery system is typically non-viral.

Various cell penetrating anti-DNA autoantibodies were isolated from a mouse model of Systemic Lupus Erythematosus (SLE). While most of these antibodies penetrate the living nucleus, the anti-GUO autoantibody 4H2 is distinguished by its cytoplasmic localization. The epitope of 4H2 binding to GUO is consistent with the binding site of G protein, which is consistent with reports of anti-GUO autoantibody binding in serum of human Systemic Lupus Erythematosus (SLE) patients (Colburn et al Journal of Rheumatology, 993-97 (2003)):993-97 (2003). In addition, 4H2 is also able to penetrate and reduce cAMP concentration in cultured cells, which is consistent with interfering with G protein signaling (Colburn & Green CLIN CHIM ACTA 370:9-16 (2006)). The following results indicate that cytoplasmic penetration of 4H2 is associated with nucleoside transport, and that 4H2 binds to and mediates nucleic acid transport, binds to and enhances cGAS activity, thereby generating cGAS-dependent toxicity to tumor cells. The results also indicate that 4H2 can cause TLR7 activation (only the cleaved form of TLR7 is active) as shown by 4H2 inducing TLR 7. Furthermore, pulldown analysis showed that 4H2 binds to the cleaved form of TLR 7. Thus, compositions and methods for modulating cGAS and other pattern recognition receptors (e.g., TLR 7) are also provided.

A.4H2 antibody

Although generally referred to herein as "4H2", "4H2 antibodies" or "4H2 antibodies", it is understood that the phrases "4H2", "4H 2antibodies (4H 2 antibodies)" or "4H2 antibodies" disclosed herein include not only whole immunoglobulins, 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, unless otherwise indicated (e.g., experimental examples), and that the compositions and methods disclosed herein are expressly provided. Antibodies are also referred to herein as cell penetrating proteins and binding proteins.

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

Antibodies that may be used in the compositions and methods include any class of whole immunoglobulins (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least the antigen-binding variable regions of the antibodies. The variable regions of different antibodies differ in sequence and can be used for binding and specificity of each particular antibody for its particular antigen. However, the variability of antibody variable regions is not typically evenly distributed. It is usually concentrated in three segments of the light and heavy chain variable regions known as Complementarity Determining Regions (CDRs) or hypervariable regions. The part of the variable region where the degree of conservation is high is called a Frame (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, mostly in a β -sheet conformation, joined by three CDRs, forming loops linking the β -sheet conformation and in some cases forming part of the β -sheet conformation. The CDRs on each chain are held together by the FR regions and together with the CDRs on the other chain constitute the antigen binding site of the antibody. Thus, antibodies typically contain at least the CDRs required to maintain guanosine binding.

4H2 hybridomas have been previously generated from MRLmpj/lpr lupus mouse models. 4H2 is not localized to lysosomes or endosomes, where cargo molecules tend to be destroyed in other vehicles (e.g., TAT peptides). 4H2 is a cell penetrating lupus anti-guanosine antibody, which can reduce ERK and Akt phosphorylation in cells, is toxic to cancer cells carrying a series of small GTPase K-Ras mutations, and is not significantly toxic to cells carrying WT K-Ras. See published International applications WO 2015/134607 and WO 2017/218824, the entire contents of each of which are specifically incorporated by reference.

The 4H2 antibody is typically monoclonal 4H2 or a variant, derivative, fragment, fusion or humanized form thereof, which may bind to the same or a different epitope as 4H 2.

1. Antibody sequences

A.4H2 light chain variable region

The amino acid sequence of the kappa light chain variable region (VL) of mAb 4H2 is:

DIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSIISVETEDFGMYFCQQSNSWPLTFGAGTKLELK(SEQ ID NO:1).

The Complementarity Determining Regions (CDRs) are shown underlined and include RASQSISNYLH (SEQ ID NO: 2), CDR L2: YASQSIS (SEQ ID NO: 3), CDR L3: QQSNSWPLT (SEQ ID NO: 4).

B.4H2 heavy chain variable region

The amino acid sequence of the heavy chain variable region (VH) of mAb 4H2 is:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGRVNPSNGGISYNQKFKGKATLTVDKSLSTAYMQLNSLTSEDSAVYYCARGPYTMYYWGQGTSVTVSS(SEQ ID NO:5).

The Complementarity Determining Regions (CDRs) are underlined and include CDR H1: DYYMN (SEQ ID NO: 6), CDR H2: RVNPSNGGISYNQKFKG (SEQ ID NO: 7), CDR H3: GPYTMYY (SEQ ID NO: 8).

2. Forms of antibodies

Exemplary antibodies that may be used include any class of whole immunoglobulins (i.e., intact antibodies), fragments thereof, and synthetic proteins containing at least antibody antigen-binding variable regions. The variable regions of different antibodies differ in sequence and can be used for binding and specificity of each particular antibody to its particular antigen. However, the variability of antibody variable regions is not typically evenly distributed. It is usually concentrated in three segments of the light and heavy chain variable regions known as Complementarity Determining Regions (CDRs) or hypervariable regions. The part of the variable region where the degree of conservation is high is called a Frame (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, principally in a β -sheet conformation, joined by three CDRs, forming loops linking the β -sheet conformation and in some cases forming part of the β -sheet conformation. The CDRs on each chain are held together by the FR regions and together with the CDRs on the other chain constitute the antigen binding site of the antibody. Thus, an antibody may include CDR components required to penetrate cells and bind guanosine.

The antibody may be a humanized or chimeric antibody, or a fragment, variant or fusion protein thereof. Methods of humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically derived from an "import" variable region. Antibody humanization techniques typically involve the use of recombinant DNA techniques to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.

The 4H2 antibody may consist of an antibody fragment or fusion protein comprising one or more CDRs (e.g., CDRs of any of SEQ ID NOs: 1 and 5, e.g., SEQ ID NOs: 2-4 and 6-8, respectively) that are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence of the CDR of 4H2 or a variant or humanized version thereof. The percent identity of 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 CDRs of the preferred variable regions described above (e.g., SEQ ID NOs: 1 and 5) without any change, or with at most 0,1, 2, 3,4, or 5 changes per CDR (i.e., each CDR is independently selected), or with a total change of all CDRs.

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

Preferably, the antibody comprises heavy chain CDR1, CDR2 and CDR3 bound to light chain CDR1, CDR2 and CDR3.

Thus, in certain embodiments, the cell penetrating antibody comprises the CDRs of SEQ ID NOs 5 and 1 or the entire heavy and light chain variable regions, or a humanized version thereof.

Many non-human antibodies (e.g., antibodies derived from mice, rats, or rabbits) are naturally antigenic to humans and thus, when administered to humans, can elicit an adverse immune response. Thus, humanized 4H2 antibodies, antibody fragments and fusions are provided. Humanized antigen binding molecules can reduce the chance of an antibody or antibody fragment or scFv eliciting an adverse immune response when administered to humans.

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

Methods of humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically derived from an "import" variable region. Antibody humanization techniques typically involve the use of recombinant DNA techniques to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Humanization can be achieved essentially by replacing rodent CDRs or CDR sequences with corresponding sequences of human antibodies. Thus, a humanized version of a non-human antibody (or fragment thereof) is a chimeric antibody or fragment in which substantially less than the entire human variable region has been replaced with the corresponding sequence of a non-human species. Indeed, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues at similar sites in rodent antibodies.

To reduce antigenicity, it is important to select light human variable regions and heavy human variable regions for use in the manufacture of humanized antibodies. The variable region sequences of rodent antibodies were screened against an entire library of known human variable region sequences according to the "best fit" method. The human sequence closest to the rodent sequence is accepted as the human Framework (FR) of the humanized antibody. Another approach is to use a framework derived from consensus sequences of all human antibodies derived from specific subsets of light or heavy chains. The same framework may be used for a plurality of different humanized antibodies.

More importantly, antibodies are humanized while maintaining high affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies are preferably prepared by a method of analyzing a parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and humanized sequence. Three-dimensional immunoglobulin models are available and 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 shows, it is possible to analyze the possible role of residues in the function of the candidate immunoglobulin sequence, i.e. to analyze residues affecting the ability of the candidate immunoglobulin to bind antigen. By this means, FR residues can be selected and combined from consensus sequences and introduced sequences to obtain desired antibody properties, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in influencing antigen binding.

In addition, antibody fragments having biological activity are included. These fragments, whether attached to other sequences or not, include insertions, deletions, substitutions or other selective modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not significantly altered or compromised compared to the unmodified antibody or antibody fragment.

Techniques for producing specific single chain antibodies to the antigenic proteins of the present disclosure may also be adapted. Methods for producing single chain antibodies are well known to those skilled in the art. Single chain antibodies can reconstitute antigen binding sites on a single molecule by fusing the variable regions of the heavy and light chains together using a short peptide linker arm. In the developed single chain antibody variable fragments (scfvs), the C-terminus of one variable region is linked to the N-terminus of the other variable region by a 15 to 25 amino acid peptide or linker arm without significantly disrupting antigen binding or specificity of binding. The linker arm is selected to allow the heavy and light chains to be joined together in the correct conformational orientation.

The 4H2 antibodies may be modified to increase their therapeutic potential. For example, in certain embodiments, the cell penetrating 4H2 antibody is conjugated to another antibody specific for a second, e.g., therapeutic target, in the cytosol and/or nucleus of the target cell. For example, the cell penetrating 4H2 antibody can be a fusion protein comprising a 4H2 Fv and a single chain variable fragment of a monoclonal antibody that specifically binds to the second target. In other embodiments, the 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.

In certain embodiments, the second target is specific for a target cell type, tissue, organ, or the like. Thus, the second heavy chain and the second light chain can act as targeting moieties to target the complex to a target cell type, tissue, organ. In some embodiments, the second heavy and second light chains target hematopoietic stem cells, CD34 ⁺ cells, T cells, cancer cells, or infected cells or any other preferred cell type, e.g., by targeting a receptor or ligand expressed on the preferred cell type. In certain embodiments, the second heavy chain and the second light chain target thymus, spleen, or cancer cells.

In certain embodiments, particularly for in vivo targeting of T cells, e.g., for in vivo production of CAR T cells, immune cells or T cell markers, such as CD3, CD5, CD7, or CD8, may be targeted. For example, both anti-CD 8 antibodies and anti-CD 3 Fab fragments have been used to target T cells in vivo (Pfeiffer, et al, EMBO Mol Med.,10 (11) (2018). Pii: e9158.doi:10.15252/emmm.201809158, smith, et al, nat nanotechnol.,12 (8): 813-820 (2017), doi: 10.1038/nano.2017.57). Thus, in some embodiments, the 4H2 antibody or antigen binding fragment or fusion protein is a bispecific antibody moiety that can specifically bind to CD3, CD5, CD7, CD8 or another immune cell (e.g., T cell) marker or a marker of a specific tissue such as thymus, spleen or liver.

Exemplary fragments and fusions include, but are not limited to, single chain antibodies, single chain variable fragments (scFv), divalent 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).

For example, a bivalent single chain variable fragment (di-scFv) can be designed by ligating two scFv. This can be achieved by producing a single peptide chain with two VH regions and two VL regions, resulting in tandem scFv. scFv can also be designed as a linker peptide, but the linker peptide is too short, and the two variable regions cannot fold together (about 5 amino acids), forcing the scFv to dimerize. This type is known as bispecific antibody fragments (diabodies). Studies have shown that bispecific antibody fragments (diabodies) have a dissociation constant 40-fold lower than the corresponding scFv, meaning that their affinity for the target is much higher. Shorter linker arms (one or two amino acids) may lead to the formation of trimers (i.e., trispecific antibody fragments triabodies or tribodies). A tetravalent antibody fragment (teratbody) was also generated. They have even higher affinity for the target than bispecific antibody fragments (diabodies). In certain embodiments, the 4H2 antibody may comprise two or more linked 4H2 single chain variable fragments (e.g., 4H2 di-scFv, 4H2 tri-scFv) or conservative variants thereof. In certain embodiments, the 4H2 antibody is a bispecific antibody fragment (diabody) or a trispecific antibody fragment (triabody) (e.g., a 4H2 bispecific antibody fragment (4H 2 diabody), a 4H2 trispecific antibody fragment (triabody)).

In certain embodiments, the antibody is conjugated or fused to a cell penetrating moiety (e.g., a cell penetrating peptide) to facilitate entry of the antibody into the cell. Examples of cell penetrating peptides include, but are not limited to, polyarginine (e.g., R ₉), antennapedia sequence (ANTENNAPEDIA SEQUENCES), TAT, HIV-TAT, penetratin (PENETRATIN), antp-3A (Antp mutant), buforin II, cell penetrating peptide (Transportan), MAP (model amphiphilic peptide), K-FGF, ku70, prion, pVEC, pep-1, synB1, pep-7, HN-1, BGSC (biguanide-spermine-cholesterol), and BGTC (biguanide-triethylenetetramine-cholesterol). In other embodiments, the antibodies are modified by TransMabs ^TM techniques (InNexus biotech, inc., vancouver, BC)

The function of an antibody may be enhanced by coupling the antibody or fragment thereof to a therapeutic agent. The coupling of the antibody or antibody fragment to the therapeutic agent may be accomplished by making an immunoconjugate or fusion protein, or by linking the antibody or antibody fragment to a nucleic acid, such as DNA or RNA (e.g., siRNA), comprising the antibody or antibody fragment and the therapeutic agent.

Recombinant fusion proteins are proteins created by genetic engineering of fusion genes. This typically involves removing the stop codon from the cDNA sequence encoding the first protein and then adding the cDNA sequence of the second protein in frame by ligation or overlap extension PCR. The DNA sequence will then be expressed in the cell as a single protein. Such proteins may be designed to include the complete sequence of both original proteins, or may include only a portion of one of them. If both entities are proteins, a linker arm (or "spacer") is typically added to make the proteins more likely to fold independently and function in the desired manner.

In certain embodiments, the cell penetrating antibody is modified to alter its half-life. In certain embodiments, it is desirable to extend the half-life of the antibody so that it is present in the blood circulation or at the treatment site for a longer period of time. For example, it may be desirable to maintain antibody titers in the blood circulation or at the treatment site for a longer period of time. In other embodiments, the half-life of the 4H2 antibody is shortened to reduce potential side effects. The half-life of antibody fragments such as 4H2Fv may be shorter than the half-life of full-size antibodies. Other methods of altering half-life are known and may be used in the method. Antibodies are designed with Fc variants that can extend half-life, for example, xtend ^TM antibody half-life extension techniques (Xencor, monrovia, CA) can be used.

A. Connecting arm

The term "linker arm" as used herein includes, but is not limited to, a peptide linker arm. The peptide linker arm may be of any size so long as it does not interfere with the binding of the variable region to the epitope. In certain embodiments, the linker arm comprises one or more glycine and/or serine amino acid residues. Monovalent single chain antibody variable fragments (scFv), wherein the C-terminus of one variable region is typically linked to the N-terminus of the other variable region by a15 to 25 amino acid peptide or linker arm. The linker arm is selected to allow the heavy and light chains to be brought together in the correct conformational orientation. As described above, the linker arm of bispecific antibody fragments (diabodies), trispecific antibody fragments (triabodies), and the like, is typically shorter than the linker arm of monovalent scFv. Divalent, trivalent, and other multivalent scFv typically include three or more linker arms. The length and/or amino acid composition of the linker arms may be the same or different. Thus, the number of linker arms, the composition of the linker arms, and the length of the linker arms can be determined according to the desired titers of scFv known in the art. The linker arm can allow or drive the formation of bivalent, trivalent and other multivalent scfvs.

For example, the linker arm may comprise 4-8 amino acids. In a specific embodiment, the linker arm comprises amino acid sequence GQSSRSS (SEQ ID NO: 10). In another embodiment, the linker arm comprises 15-20 amino acids, e.g., 18 amino acids. In a specific embodiment, the linker arm comprises amino acid sequence GQSSRSSSGGGSSGGGS (SEQ ID NO: 11). Other flexible linker arms include, but are not limited to, 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) and (Gly ₄-Ser)₄ (SEQ ID NO: 15) and (Gly-Gly-Gly-Gly-Ser) ₃ (SEQ ID NO: 16).

Other exemplary linker arms include, for example, RADAAPGGGGSGGGGSGGGGS (SEQ ID NO: 17) and ASTKGPSVFPLAPLESSGS (SEQ ID NO: 18).

B. Exemplary 4H2 scFv sequences

Those of skill in the art will appreciate that exemplary fusion proteins or domains thereof may be used to construct the fusion proteins discussed in more detail above. For example, in some embodiments, the scFv comprises a scFv comprising a Vk variable region (SEQ ID NO:1 or a functional variant or fragment thereof) linked to a VH variable region (SEQ ID NO:5 or a functional variant or fragment thereof). In some embodiments, the di-scFv comprises a first scFv comprising a Vk variable region (SEQ ID NO:1 or a functional variant or fragment thereof), linked to a VH variable region (e.g., SEQ ID NO:5 or a functional variant or fragment thereof), linked to a second scFv comprising a Vk variable region (e.g., SEQ ID NO:1 or a functional variant or fragment thereof), linked to a VH variable region (e.g., SEQ ID NO:5 or a functional variant or fragment thereof). In some embodiments, the trivalent single chain variable fragment (tri-scFv) comprises a bivalent single chain variable fragment (di-scFv) linked to a third scFv domain comprising a Vk variable region (e.g., SEQ ID NO:1, or a functional variant or fragment thereof) linked to a VH variable region (e.g., SEQ ID NO:5, or a functional variant or fragment thereof).

For example, the Vk variable region may be linked to the VH variable region by, for example, a linker such as (GGGGS) ₃ (SEQ ID NO: 19) alone or in combination with (6 aa) of the linker and the light chain CH1 such as RADAAP (SEQ ID NO: 20). The scFv may be linked alone or in combination with a rotating sequence (e.g., LESSGS (SEQ ID NO: 22)) via a linker arm (e.g., 13 amino acids (e.g., ASTKGPSVFPLAP (SEQ ID NO: 21)) from human IgG CH 1. Other suitable linker arms are discussed above and are also known in the art.

In certain embodiments, the fusion protein comprises an additional domain. For example, in certain embodiments, the fusion protein includes a sequence that increases solubility. In certain embodiments, the fusion protein comprises one or more domains that enhance purification, isolation, capture, identification, isolation, etc., of the fusion protein. Exemplary domains include, for example, myc tags and/or His tags. Other substitutable domains and additional domains have been discussed in detail above.

Exemplary scFv molecules are also provided.

DIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSIISVETEDFGMYFCQQSNSWPLTFGAGTKLELKADAAPGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGRVNPSNGGISYNQKFKGKATLTVDKSLSTAYMQLNSLTSEDSAVYYCARGPY^TMYYWGQGTSVTVSSHHHHHH(SEQ ID NO:9)

Single underlined 4H2 VL sequences

Double underlined are linker arm sequences

Dashed underline 4H2 VH sequence

Wave underline His ₆ tag

For example, the scFv may comprise the C-terminus of the 4H2 VL sequence of SEQ ID NO:9 linked to the N-terminal sequence of the 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 the 4H2 VL of SEQ ID NO: 9. The linker arm of SEQ ID NO. 9 may be replaced with alternative linker arms, including but not limited to the alternative linker arms disclosed herein. Typically, the linker arm is about 10 to about 25 amino acids, typically including glycine. The His ₆ tag of SEQ ID NO. 9 may be replaced with another tag, moved to the N-terminus of the scFv or deleted entirely. In some embodiments, the 4H2 VL, 4H2 VH, or a combination thereof is a variant of the 4H2 VL and/or 4H2 VH of SEQ ID NO. 9 or a humanized form thereof. In certain embodiments, both the N-terminus and the C-terminus of the 4H2 VL and/or 4H2 VH domain are truncated compared to the 4H2 VL and/or 4H2 VH of SEQ ID NO. 9. The scFv may comprise the 3 CDRs of the 4H2 VL and/or 4H2 VH of SEQ ID NO 9 or a humanized form thereof. 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 to SEQ ID NO 9. In certain embodiments, the VL domain of an antibody or fragment or fusion thereof has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to 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 to the 4H2 VH domain of SEQ ID NO 9.

SEQ ID NO 9 and humanized forms thereof and variants thereof may be used in any of the compositions and methods disclosed herein. In certain embodiments, SEQ ID NO 9 or a humanized form thereof or variants thereof may be used in a method of treatment, such as the methods disclosed herein, without conjugation to a nanocarrier or therapeutic agent. Thus, in certain embodiments, SEQ ID NO 9 or a humanized form or variant thereof is a therapeutic agent only. In certain embodiments, SEQ ID NO 9 or a humanized form thereof or a variant thereof is not a therapeutic agent (e.g., is a targeting moiety only), or is one of two or more therapeutic agents.

C. exemplary 4H2 bispecific antibodies

An exemplary bispecific antibody is used in example 13 below. The antibody has the format shown in fig. 12A and heavy and light chain variable region sequences:

4H2 sequence

VL:

DIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSIISVETEDFGMYFCQQSNSWPLTFGAGTKLELK(SEQ ID NO:1)

VH:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGRVNPSNGGISYNQKFKGKATLTVDKSLSTAYMQLNSLTSEDSAVYYCARGPYTMYYWGQGTSVTVSS(SEQ ID NO:5)

CD5 sequence

VL:

NIVMTQSPSSLSASVGDRVTITCQASQDVGTAVAWYOQKPDQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTISSLOPEDIATYFCHQYNSYNTFGSGTKLEIK(SEQ ID NO:23)

VH:

QVTLKESGPVLVKPTETLTLTCTFSGFSLSTSGMGVGWIRQAPGKGLEWVAHIWWDDDVYYNPSLKSRLTITKDASKDQVSLKLSSVTAADTAVYYCVRRRATGTGFDYWGQGTLVTVSS(SEQ ID NO:24)

The antibody is exemplary only, 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 specifically provided. For example, in certain embodiments, chimeric or humanized bispecific antibodies having CDRs of SEQ ID NOs 1, 5, 23, and 24, or humanized versions thereof (e.g., 1,2,3 mutations per CDR or total, such as conservative substitutions) are provided having human heavy and light chain variable region frameworks and optionally constant domains.

The predicted CDRs for 4H2 have been indicated above by underlining. The predicted CDRs against CD5 are underlined above and are explicitly provided:

CDR L1:QASQDVGTAVA(SEQ ID NO:25);CDR L2:YWTSTRHT(SEQ ID NO:26);HQYNSYNT CDR L3:(SEQ ID NO:27)。

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

For example, a 4H2-CD5 bispecific antibody fragment may consist of an antibody fragment or fusion protein comprising a combination of one or more CDRs (e.g., any one of SEQ ID NOs: 1 and 5, such as SEQ ID NOs: 2-4 and 6-8, respectively) having 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 to the amino acid sequence of a CDR of 4H2 or a variant or humanized version thereof (e.g., any one of SEQ ID NOs: 23 and 24, such as SEQ ID NOs: 25-27 and 28-30, respectively) with an anti-CD 5 antibody fragment or fusion protein or variant thereof, or humanized version thereof. The percent identity of 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 CDRs of the preferred variable regions described above (e.g., SEQ ID NOs: 1 and 5 and/or 23 and 24) without any change, or with up to 0,1, 2, 3, 4, or 5 changes per CDR (i.e., each CDR independently selected) or all CDRs.

The 4H2-CD5 bispecific antibody may consist of an antibody fragment or fusion protein comprising an amino acid sequence of a variable heavy chain and/or a variable light chain having 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 to the amino acid sequence of a variable heavy chain and/or a variable light chain of 4H2 or a humanized version thereof (e.g., SEQ ID NOS: 5 and 1) and to the amino acid sequence of a variable heavy chain and/or a light chain of an anti-CD 5 or a humanized version thereof (e.g., SEQ ID NOS: 24 and 23).

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 of corresponding CDR regions of an anti-CD 5 antibody.

Thus, in certain embodiments, the cell penetrating bispecific antibody comprises the CDRs of SEQ ID NOs 5 and 1 and 24 and 23, or the entire heavy and light chain variable regions, or a humanized version thereof.

B. additional medicine

The following results indicate that extracellular nucleic acids can facilitate cell penetration by 4H 2. Furthermore, activation of cGAS by cytoplasmic DNA results in endogenous production of cyclic GMP-AMP, a unique second messenger that binds to interferon gene stimulatory factor (STING), resulting in activation of TANK binding kinase 1 (TBK 1) and IRF3, and thus transcription of the gene encoding type I interferon (PESIRIDIS and Fitzgerald, nature REVIEWS GENETICS, vol.20, pages 657-674 (2019)). The following experimental results indicate that 4H2 can activate cGAS and other pattern recognition receptors (PPRs), such as TLR7. This activation may be by direct binding and activation of 4H2 or indirect binding via simultaneous interaction between the immunoreceptor, 4H2 and cytoplasmic nucleic acid and/or GTP.

Thus, the disclosed compositions are useful for facilitating delivery of nucleic acid cargo. Additionally or alternatively, 4H2 antibodies may also be used to modulate immune responses with or without the aid of nucleic acid cargo. For example, in certain embodiments, the compositions and methods include nucleic acid and/or GTP (also referred to as nucleic acid cargo) to facilitate 4H2 cell penetration and/or activate cGAS and/or another PRR, such as TLR7.

In addition, STING agonists have been proposed for a number of different therapeutic purposes, including the treatment of cancer, infections and as vaccine adjuvants. See, e.g., PESIRIDIS and Fitzgerald, nature REVIEWS GENETICS, volume 20, pages 657-674 (2019), the entire contents of which are incorporated herein by reference. Thus, in certain embodiments, the disclosed compositions and methods include additional drugs that facilitate these applications. Non-limiting examples of additional drugs include, but are not limited to, additional STING agonists, vaccine compositions, and immune checkpoint inhibitors, each of which will be discussed in more detail below. In some embodiments, the additional agent is a nucleic acid (e.g., an immunostimulatory oligonucleotide, a nucleic acid encoding a vaccine component such as a peptide antigen, etc.). The additional agent for such nucleic acids may be a nucleic acid cargo or may be an additive or alternative to the subject alone.

Thus, any additional drug may be in the same or different mixture as the 4H2 antibody, and may be administered at the same or different times as the 4H2 antibody. In certain embodiments, for example, where the additional drug is a nucleic acid cargo, the additional drug and the 4H2 antibody are contacted and form a complex prior to administration to the subject. The interaction between the antibody and the nucleic acid cargo is non-covalent. In such embodiments, the complex may be administered to a subject. Although referred to as cargo, cargo nucleic acids may also be administered alone as disclosed herein and thus are not necessarily cargo of 4H2 antibodies under these conditions.

1. Goods (e.g. freight)

Nucleic acid cargo is also provided. As discussed in more detail below, the disclosed 4H2 antibodies can be used to deliver nucleic acid cargo to cells for any purpose. In particular embodiments, cargo may also be used to increase cell penetration of 4H2 antibodies and/or increase activation of cGAS and/or another PRR (e.g., TLR 7). As used in the nucleic acid delivery methods provided herein, 4H2 is typically contacted with a cell in which the nucleic acid cargo is complexed. The interaction between the antibody or binding protein and the nucleic acid cargo is non-covalent.

The nucleic acid cargo may be single-stranded or double-stranded, or may be a single nucleotide, or nucleobase, or a plurality of nucleotides, or nucleobases. In certain embodiments, the cargo is GTP, GDP, GMP, cGAMP or cGMP. The nucleic acid cargo may be or include DNA, RNA, nucleic acid analogs, or combinations thereof. As described below, nucleic acid analogs can be modified on the base moiety, the glycosyl moiety, or the phosphate backbone. For example, such modifications may improve the stability, hybridization, or solubility of the nucleic acid. 4H2 can bind to guanosine. Thus, the cargo typically comprises one or more guanine nucleobases, preferably one or more guanosine.

The nucleic acid cargo may be functional, i.e., is an agent or encodes an agent that is biologically active once delivered into the cell, or non-functional, simply to facilitate delivery of 4H2 to the cytoplasm and/or activate cGAS and/or another PRR (e.g., TLR 7). Exemplary cargo will be discussed in more detail below, but includes mRNA or DNA encoding a polypeptide of interest, including for example, expression constructs and vectors, inhibitory nucleic acids (e.g., siRNA), or nucleic acids encoding inhibitory nucleic acids, including for example, expression constructs and vectors, or non-coding RNA or DNA.

The disclosed compositions can include a plurality of individual nucleic acid cargo molecules. In certain embodiments, the composition comprises a plurality (e.g., 2,3, 4,5, 6, 7, 8, 9, 10, or more) of different nucleic acid molecules.

In certain embodiments, 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 in length.

In certain embodiments, for example, the cargo 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, or 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 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 inclusive.

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

It will be appreciated that in particular applications, the length of the nucleic acid cargo may be one or more discrete lengths, e.g., falling within one of the ranges described above (including the endpoints), with the specific values of each range being expressly disclosed. For example, the size may be as small as a single nucleotide or nucleobase. In an exemplary application, 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 mers can be used for antisense or splice switching. Slightly longer oligomers (e.g., 18 to 20 polymers) can be used for gene editing.

A. form of goods

The nucleic acid cargo is a nucleic acid and may be an isolated nucleic acid composition. As used herein, "isolated nucleic acid" refers to nucleic acid that is isolated from other nucleic acid molecules present in the genome of a mammal, including nucleic acid that is typically located on one or both sides of the nucleic acid in the genome of a mammal. The term "isolated" as used herein in reference to nucleic acids also includes combinations with any non-naturally occurring nucleic acid sequence, as such non-naturally occurring sequence is not found in nature nor is there a direct adjacent sequence in a naturally occurring genome.

An isolated nucleic acid may be, for example, a DNA molecule, provided that one of the nucleic acid sequences immediately flanking the DNA molecule in the natural genome is removed or deleted. Thus, isolated nucleic acids include, but are not limited to, DNA molecules that exist as separate molecules from other sequences (e.g., chemically synthesized nucleic acids or cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment), as well as recombinant DNA incorporated into vectors, autonomously replicating plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, or herpesviruses), or genomic DNA of prokaryotic or eukaryotic cells. In addition, the isolated nucleic acid may also include engineered nucleic acids, such as recombinant DNA molecules that are part of a hybridization or fusion nucleic acid. Nucleic acids of the hundreds to millions of other nucleic acids present in gel slices such as cDNA libraries or genomic libraries or restriction digests containing genomic DNA cannot be considered isolated nucleic acids.

Nucleic acid sequences encoding polypeptides include genomic sequences. Also disclosed are mRNA/cDNA sequences in which exons have been deleted. Other nucleic acid sequences encoding polypeptides, such as polypeptides including the amino acid sequences described above, and fragments and variants thereof, are also disclosed. The nucleic acid encoding the polypeptide may be optimized for expression in a selected expression host. The codons may be replaced with substitution codons encoding the same amino acid to account for differences in codon usage between the organism from which the nucleic acid sequence was derived and the expression host. In this way, the nucleic acid may be synthesized using codons preferred by the expression host.

The nucleic acid may be in sense or antisense orientation or may be complementary to a reference sequence encoding a polypeptide.

I. Carrier body

The cargo may be a vector, for example a vector encoding a polypeptide and/or a functional nucleic acid. The nucleic acid as described above may be inserted into a vector for expression in a cell. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus, or cosmid, into which another DNA segment may be inserted to effect replication of the inserted segment. The vector may 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 transcription and/or translation of another DNA sequence.

The nucleic acid in the vector may be operably linked to one or more expression control sequences. For example, control sequences may be integrated into the genetic construct such that expression control sequences are effective to control expression of the coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. Promoters are expression control sequences consisting of regions of DNA molecules, typically located within 100 nucleotides upstream of the transcription start point (typically near the start site of RNA polymerase II). In order for the coding sequence to be under the control of a promoter, the translation initiation site of the polypeptide translation reading frame must be located between 1 and 50 nucleotides downstream of the promoter. Enhancers have expression specificity in terms of time, location and level. Unlike promoters, enhancers can function at different locations from the transcription site.

Enhancers may also be located downstream of the transcription initiation site. An RNA polymerase is "operably linked" when it is capable of transcribing a coding sequence into mRNA, and under the control of an expression control sequence in a cell, and then translating the mRNA into the protein encoded by the coding sequence.

Suitable expression vectors include, but are not limited to, plasmids, cosmids, and viral vectors, such as vectors derived from phage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retroviruses, vaccine viruses, adenoviruses, and adenoviruses. A number of vectors and expression systems are commercially available from companies such as Novagen (Madison, wis.), clontech (Palo Alto, calif.), stratagene (La Jolla, calif.), and Invitrogen Life Technologies (Carlsbad, calif.).

In certain embodiments, the cargo is delivered into the cell and remains extrachromosomal. In certain embodiments, the cargo is introduced into a host cell and integrated into the genome of the host cell. As discussed in detail below, the compositions may be used in gene therapy methods. The gene therapy method may comprise introducing into the cell a polynucleotide that alters the genotype of the cell. The introduction of the polynucleotide may be corrected, replaced or otherwise alter the endogenous gene by gene recombination. The method may include introducing an entire alternate copy of the defective gene, a heterologous gene or a small nucleic acid molecule (e.g., an oligonucleotide). For example, the correction gene may be introduced into a host genome at a non-specific location.

In certain embodiments, the cargo is a carrier. 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 translation and/or transcription of the inserted coding sequence, which may be, for example, a polynucleotide of interest. The coding sequence may be operably linked to a promoter and/or enhancer to help control the expression of the desired gene product. Promoters used in biotechnology are of different types, depending on the type of control expected from gene expression. Generally, it can be classified into constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters and synthetic promoters.

For example, in certain embodiments, the polynucleotide of interest is operably linked to a promoter or other regulatory element known in the art. Thus, the cargo may be a vector, such as an expression vector. Engineered expression of polynucleotides in prokaryotic or eukaryotic systems may be performed by techniques generally known to those skilled in the art of recombinant expression. Expression vectors typically include one of the disclosed compositions under the control of one or more promoters. To "control" the coding sequence by a "promoter, the 5 'end of the translation initiation site of the reading frame is typically placed between about 1 and 50 nucleotides" downstream "(i.e., 3' end) of the selected promoter. An "upstream" promoter stimulates transcription of the inserted DNA, promoting expression of the encoded recombinant protein or functional nucleic acid. This is the meaning of "recombinant expression" herein.

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

Expression vectors for mammalian cells typically include an origin of replication (if necessary), a promoter located in front of the gene to be expressed, and any necessary ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. The origin of replication may be provided by constructing a vector, which includes exogenous origins of replication, such as may be derived from SV40 or other viral (e.g., polyoma, adenovirus, VSV, BPV) sources, and may also be provided by host cell chromosomal replication mechanisms. The latter is usually sufficient if the vector is integrated into the host cell chromosome.

The promoter may be derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a mammalian virus (e.g., adenovirus late promoter; vaccine virus 7.5K promoter). Furthermore, it is also possible, and may be desirable, to utilize promoter or control sequences that are typically associated with the desired gene sequence, provided that these control sequences are compatible with the host cell system.

A variety of viral-based expression systems can be utilized, for example, common promoters derived from polyoma virus, adenovirus 2, cytomegalovirus and Simian cavitation virus 40 (Simian vacuolating virus, SV40). Both the early and late promoters of SV40 virus are useful because both promoters can be readily obtained from the virus as fragments, including the SV40 viral origin of replication. Smaller or larger SV40 fragments may also be used, but need to include an about 250bp sequence extending from the HindIII site to the BglI site at the viral origin of replication.

In the case of using adenovirus as an expression vector, the coding sequence may be linked to adenovirus transcription/translation control complexes, such as late promoters and tripartite leader sequences. Such chimeric genes can then be inserted into the adenovirus genome by in vitro recombination or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., the E1 or E3 regions) renders the recombinant virus viable and expresses the protein in the infected host. Specific initiation signals may also be required for efficient translation of the disclosed compositions. These signals include the ATG initiation codon and adjacent sequences. In addition, it may be desirable to provide exogenous translational control signals, including the ATG initiation codon. Those of ordinary skill in the art will readily ascertain such a need and provide the necessary signals. It is well known that the initiation codon must be in frame (or in phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. The sources of these exogenous translational control signals and initiation codons are diverse, both natural and synthetic. The inclusion of appropriate transcription enhancer elements or transcription terminators may increase expression efficiency.

In eukaryotic expression, it is often also necessary to add an appropriate polyadenylation site to the transcriptional unit if the original cloned fragment does not include one. Typically, the polyadenylation site is located about 30 to 2000 nucleotides "downstream" of the protein termination site, i.e., a position prior to transcription termination.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the protein-encoding construct may be engineered. Instead of using an expression vector containing viral origin of replication, the host cell may be transformed with a vector controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers. After introduction of the exogenous DNA, the engineered cells can be grown in the enrichment medium for 1-2 days and then transferred to the selective medium. The selectable marker in the recombinant plasmid confers cell selection resistance, allowing the cell to stably integrate the plasmid into its chromosome and grow to form colonies which in turn can be cloned and expanded into cell lines.

ii.mRNA

The cargo may be mRNA

Chemical structures with improved stability and/or translation efficiency may also be used. For example, the RNA may have 5 'and 3' UTRs. For example, the 3' UTR may be more than 100 nucleotides in length. In certain embodiments, the 3' utr sequence is between 100 and 5000 nucleotides. In certain embodiments, the 5' utr is between 0 and 3000 nucleotides in length. The length of the 5 'and 3' UTR sequences to be added to the coding region may be varied by different methods including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one of ordinary skill in the art can modify the 5 'and 3' UTR lengths to achieve optimal translational efficiency following delivery of transcribed RNA.

The 5 'and 3' UTRs may be endogenous 5 'and 3' UTRs naturally occurring for the gene of interest. Alternatively, the endogenous UTR sequences of non-target genes may be added by incorporating UTR sequences in the forward and reverse primers or by any other modification of the template. The use of UTR sequences endogenous to non-target genes can be used to alter RNA stability and/or translation efficiency. For example, AU elements rich in the 3' UTR sequence are known to reduce mRNA stability. Thus, the 3' UTR may be selected or designed according to UTR characteristics well known in the art to enhance stability of transcribed RNA.

In certain embodiments, the 5' utr comprises a Kozak sequence of an endogenous gene. Alternatively, when a 5'UTR endogenous to a non-target gene is added by PCR as described above, a consensus Kozak sequence may be redesigned by adding the 5' UTR sequence. Kozak sequences may increase the translation efficiency of certain RNA transcripts, but it does not seem that all RNAs require Kozak sequences to achieve efficient translation. The requirements of many mRNAs for Kozak sequences are well known in the art. In other embodiments, the 5' utr may 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 hinder the degradation of mRNA by exonucleases.

In certain embodiments, the mRNA has a cap at the 5 'end and a poly (A) tail at the 3' end, or a combination thereof, determines ribosome binding, translation initiation, and mRNA stability in the cell.

The 5' cap provides stability to the RNA molecule. For example, the 5' cap may be m ⁷G(5')ppp(5')G,m⁷ G (5 ') ppp (5 ') A, G (5 ') ppp (5 ') G or G (5 ') ppp (5 ') A cap analogues, all of which are commercially available. 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' caps can be incorporated using techniques known in the art (Cougot et al, trends in biochem. Sci.,29:436-444 (2001); STEPINSKI et al, RNA,7:1468-95 (2001); elango et al, biochem. Res. Commun.,330:958-966 (2005)).

The RNA may also include an Internal Ribosome Entry Site (IRES) sequence. IRES sequences may be any viral, chromosomal, or artificially designed sequence that can initiate cap-independent ribosome binding to mRNA and facilitate initiation of translation.

In general, the length of the poly (A) tail is positively correlated with the stability of transcribed RNA. In one embodiment, the number of adenosines in the poly (a) tail is between 100 and 5000.

Reverse primers containing a poly T tail, such as a 100T tail (which may be 50-5000T in size), may be used during PCR, or any other method may be used after PCR, including but not limited to DNA ligation or in vitro recombination, to generate a poly A fragment. poly (a) tails also provide stability to RNA and reduce its degradation. The poly (A) tail of RNA may be further or alternatively extended by in vitro transcription using a poly (A) polymerase, such as E.coli poly A polymerase (E-PAP).

Furthermore, the attachment of different chemical groups at the 3' end may increase the stability of the mRNA. Such attachments may include modified/artificial nucleotides, aptamers, and other compounds. For example, a poly (A) polymerase can be used to incorporate an ATP analog into the poly (A) tail. ATP analogues may further improve RNA stability. Suitable ATP analogs include, but are not limited to cordiocipin and 8-azaadenosine.

B. Cargo order

I. Related polypeptides

The cargo may encode one or more proteins. The cargo may be a monocistronic or polycistronic polynucleotide. In certain embodiments, the polynucleotide is polygenic. For example, the polynucleotide may be, for example, an mRNA or an expression construct, such as a vector.

The cargo may encode one or more polypeptides of interest. The polypeptide may be any polypeptide. For example, the polypeptide encoded by the polynucleotide may be a polypeptide having a therapeutic or prophylactic effect on an organism, or a polypeptide useful for diagnosing a disease or disorder in an organism. For example, for the treatment of cancer, autoimmune diseases, parasites, viruses, bacteria, fungi or other infections, the polynucleotide to be expressed may encode a polypeptide which may act as a ligand or receptor for cells of the immune system, or may act to stimulate or inhibit the immune system of an organism.

In certain embodiments, the polynucleotide may supplement or replace a polynucleotide that is defective in an organism.

In specific embodiments, the polynucleotide encodes a dystrophin protein (Dystrophin), a dystrophin-related protein (Utrophin), or a combination thereof. Such compositions may be administered in an amount effective to treat muscular dystrophy, particularly in subjects with muscular dystrophy, such as subjects with duchenne muscular dystrophy.

In another embodiment, the polynucleotide encodes an antigen, e.g., an antigen useful in vaccine formulations and related methods. In a specific embodiment, the polynucleotide encodes a viral antigen, e.g., SARS-CoV-2 antigen. Thus, compositions and methods of use thereof for the prevention and treatment of SARS-CoV-2 virus and viral infections and diseases associated therewith, including COVID < 19 > are provided.

In certain embodiments, the polynucleotide comprises a selectable marker, e.g., a selectable marker that is effective in eukaryotic cells, such as a drug resistance selectable marker. Such selectable marker genes may encode factors necessary for survival or growth of the transformed host cell grown in the selective medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, kanamycin, gentamicin, bleomycin (Zeocin) or tetracycline, complement auxotrophs, or provide a critical nutritional component for deletion in the medium.

In working example 12 below, nucleic acid encoding wild-type Merlin (a protein that is mutated in type 2 neurofibromatosis), NF2, can reduce tumor growth. Thus, in certain embodiments, the nucleic acid encodes a wild-type or other compensatory variant of an oncogenic protein (e.g., merlin).

In certain embodiments, the polynucleotide comprises a reporter gene. A reporter gene is typically a gene that is not present or expressed in a 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 the Glucuronidase (GUS) gene and the GFP gene.

Functional nucleic acid

The cargo may be a functional nucleic acid or encode a functional nucleic acid. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding to a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following non-limiting categories of antisense molecules, siRNA, miRNA, aptamers, ribozymes, RNAi, external guide sequences, and cyclic dinucleotides, as described in detail below. The functional nucleic acid molecule may act as an effector, inhibitor, modulator, or stimulator of a particular activity possessed by the target molecule, or the functional nucleic acid molecule may possess entirely new activity independent of any other molecule.

The functional nucleic acid molecule may interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, the functional nucleic acid may interact with the mRNA or genomic DNA of the polypeptide of interest, 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 between the functional nucleic acid molecule and the target molecule, but rather on the formation of tertiary structures that allow specific recognition to occur.

Thus, the composition may include one or more functional nucleic acids that are intended to reduce expression of a gene or gene product thereof. For example, a functional nucleic acid or polypeptide may be designed to target and reduce or inhibit the expression or translation of mRNA, or reduce or inhibit the expression of a protein, reduce the activity of a protein, or increase protein degradation. In certain embodiments, the composition comprises a vector suitable for in vivo expression of the functional nucleic acid.

(1) Antisense sense

The functional nucleic acid may be an antisense molecule or encode an antisense molecule. Antisense molecules are designed to interact with a target nucleic acid molecule through canonical or non-canonical base pairing. The interaction of the antisense molecule with the target molecule is designed to facilitate the destruction of the target molecule by, for example, RNAse H-mediated RNA-DNA hybrid degradation. Or antisense molecules are designed to interrupt processing functions, such as transcription or replication, that typically occur on the target molecule. Antisense molecules can be designed based on the sequence of the target molecule. There are many ways in which antisense efficiency can be optimized by finding the most accessible regions in the target molecule. Typical methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. The dissociation constant (Kd) for the binding of the antisense molecule to the target molecule is preferably less than or equal to 10 ^-6、10^-8、10^-10 or 10 ^-12.

(2) RNA interference

In certain embodiments, the functional nucleic acid induces gene silencing by RNA interference. Gene expression can also be efficiently silenced in a highly specific manner by RNA interference (RNAi). This silencing was originally observed by 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 the dsRNA enters the cell, it is cleaved by the RNase III-like enzyme Dicer into a double-stranded small interfering RNA (siRNA) 21-23 nucleotides in length, which contains a2 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, commonly referred to as RNAi-induced silencing complex (RISC), which directs the siRNA into a target RNA sequence (Nykanen, et al (2001), cell, 107:309-21). The siRNA duplex will melt at some point while the antisense strand appears to remain bound to RISC and directs the degradation of the complementary mRNA sequence by a combination of endonucleases and exonucleases (Martinez et al (2002), cell, 110:563-74). However, the action or use of iRNA or siRNA is not limited to any type of mechanism.

Short interfering RNAs (sirnas) are double-stranded RNAs that induce sequence-specific post-transcriptional gene silencing, thereby reducing or even inhibiting gene expression. In one example, the siRNA triggers specific degradation of a homologous RNA molecule (e.g., mRNA) within a region of sequence identity between the siRNA and the target RNA. For example, WO 02/44321, which is incorporated herein by reference, discloses that siRNA can undergo sequence-specific degradation of target mRNA when paired with 3' overhanging bases.

In mammalian cells, sequence-specific gene silencing can be achieved using short, artificially synthesized double-stranded RNA that mimics siRNA produced by a dicer enzyme (Elbashir et al (2001), nature, 411:494498) (Ui-Tei et al (2000), FEBS Lett 479:79-82). The siRNA may be chemically synthesized or synthesized in vitro, or may be the result of processing short double-stranded hairpin-like RNAs (shrnas) into siRNA in a cell. Synthetic siRNAs are 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). Ambion can also be used for siRNAThe siRNA construction kit and other kits are synthesized in vitro.

A more common method for producing siRNA from vectors is by short hairpin RNAse (shRNA) transcription. Kits for producing vectors with shRNA are available, for example, the GENESUPPRESSOR ^TM construction kit of Imgenex and the BLOCK-IT ^TM inducible RNAi plasmid and lentiviral vector of Invitrogen.

In certain embodiments, the functional nucleic acid is siRNA, shRNA, miRNA. In certain embodiments, the composition comprises a vector expressing the functional nucleic acid.

(3) Aptamer

The functional nucleic acid may be an aptamer or encode an aptamer. An aptamer is a molecule that interacts with a target molecule, preferably in a specific manner. Typically, the aptamer is a small nucleic acid 15-50 bases in length that can be folded into defined secondary and tertiary structures, such as a stem loop or G-quadruplex. The aptamer can be combined with small molecules such as ATP and theophylline, and large molecules such as reverse transcriptase and thrombin. The aptamer can bind very tightly to target molecules with Kd less than 10 ^-12 M. Preferably the aptamer binds to a target molecule having a Kd of less than 10 ^-6、10^-8、10^-10 or 10 ^-12. The aptamer is capable of binding target molecules with extremely high specificity. For example, an aptamer that has been isolated that differs by more than a factor of 10,000 from the binding affinity between a target molecule and another molecule that differs only at one location. Preferably, the dissociation constant (Kd) of the aptamer to the target molecule is at least 10, 100, 1000, 10,000 or 100,000 times lower than the Kd of the background binding molecule. In the case of comparing molecules such as polypeptides, the background molecules are preferably different polypeptides.

(4) Ribozyme

The functional nucleic acid may be a ribozyme or encode a ribozyme. Ribozymes are nucleic acid molecules capable of catalyzing an intramolecular or intermolecular chemical reaction. Preferably, the ribozyme is capable of catalyzing intermolecular reactions. There are many different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions, all based on ribozymes found in natural systems, such as hammerhead ribozymes. Still other ribozymes are not found in natural systems, but are engineered to catalyze specific reactions from the head. Preferred ribozymes cleave RNA or DNA substrates, more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates by recognizing and binding to the target substrate and then cleaving. Such recognition is typically based primarily on canonical or non-canonical base pair interactions. Since recognition of the target substrate is based on the sequence of the target substrate, this property makes ribozymes the best candidate for targeted specific cleavage of nucleic acids.

(5) External boot sequence

The functional nucleic acid may be or encode an external guide sequence. An External Guide Sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex, and RNase P recognizes the complex and then cleaves the target molecule. EGS can be designed specifically for a selected RNA molecule. RNAse P can aid in processing of intracellular transport ribonucleic acids (tRNA). Bacterial RNAse P can cleave almost any RNA sequence by recruitment using an EGS that allows the target RNA, EGS complex, to mimic a natural tRNA substrate. Likewise, eukaryotic EGS/RNAse P-directed RNA cleavage can also be used to cleave a desired target within eukaryotic cells. How to make and use EGS molecules to facilitate cleavage of a variety of different target molecules, representative examples of which are well known in the art.

Methods of making and using vectors for expressing functional nucleic acids (e.g., antisense oligonucleotides, siRNA, shRNA, miRNA, EGSs, ribozymes, and aptamers) in vivo are known in the art.

(6) Cyclic dinucleotides

In certain embodiments, the 4H2 antibody is co-administered in combination with an immunostimulatory oligonucleotide. The immunostimulatory oligonucleotide may be a cargo and thus may be administered in combination with an antibody or may be administered alone. In certain embodiments, the immunostimulatory oligonucleotide is a cyclic dinucleotide.

The functional nucleic acid may be a cyclic dinucleotide or encode a cyclic dinucleotide. The cyclic dinucleotide binds directly to the STING adapter protein, producing 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, 3'-cGAMP, c-di-GMP, 2' -cGAMP, 2'3' -cGAM (PS) 2 (Rp/Sp), fluorinated 3'-cGAMP, fluorinated c-di-GMP, or 2'3'-c-di-GMP, c-di-AMP, c-di-GMP, caimap (CL 592), cAIMP difluoride (CL 614), cAIM (PS) 2 difluoride (Rp/Sp) (CL 656), fluorinated c-di-AMP, 2'3'-c-di-AM (PS) 2 (Rp, rp), fluorinated c-di-GMP, 2' di-c-di-GMP, and dmp.

(7) Immunostimulatory oligonucleotides

In certain embodiments, the immunostimulatory oligonucleotide is an oligonucleotide ligand or encodes an oligonucleotide ligand. Examples include, but are not limited to, pattern Recognition Receptor (PRR) ligands.

Examples of pattern recognition receptors include Toll-like family signaling molecules that play a role in the initiation of innate immune responses, while also affecting later and more antigen-specific adaptive immune responses. Thus, the oligonucleotides may act as ligands for Toll-like family signaling molecules, such as Toll-like receptor 9 (TLR 9). 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, an oligonucleotide sequence may include one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. "p" refers to the phosphodiester backbone of DNA, however, in certain embodiments, oligonucleotides comprising CG may have a modified backbone, such as a Phosphorothioate (PS) backbone.

In certain embodiments, the oligonucleotide may include more than one CG dinucleotide, which may be arranged consecutively or separated by an intervening nucleotide. The CpG motif may be located within the oligonucleotide sequence. Many nucleotide sequences stimulate TLR9, but the exact base sequences flanking CG dinucleotides vary in number and position. Many nucleotide sequences are capable of stimulating TLR9 by varying the number and location of CG dinucleotides, as well as by varying the exact base sequences flanking CG dinucleotides.

Generally, CG ODNs are classified according to their sequence, secondary structure, and impact on human Peripheral Blood Mononuclear Cells (PBMCs). These five classes are class A (type D), class B (type K), class C, class P and class S (Vollmer, J & Krieg, AM, advanced Drug DELIVERY REVIEWS (3): 195-204 (2009), incorporated herein by reference), respectively. CG ODNs stimulate the production of type I interferons (e.g., ifnα) and induce Dendritic Cell (DC) maturation. Certain classes of ODNs are also strong activators of Natural Killer (NK) cells through indirect cytokine signaling. Some classes of ODNs are also potent stimulators (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), of human B cell and monocyte maturation, each of which is incorporated herein by reference.

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

RIG-I (retinoic acid-induced protein 1, also known as Ddx 58) 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, which are critical for the antiviral response of the host.

RIG-I and MDA-5 sense the replication intermediate double-stranded RNA (dsRNA) of RNA virus and signal via mitochondrial antiviral signal protein MAVS (also known as IPS-1, VISA or cardiof), thereby producing type I interferons (IFN- α and IFN- β).

The viral RNA detected by RIG-I has uncapped 5' -diphosphate or triphosphate ends and a short blunt-ended double strand, both of which are essential features that aid in its differentiation from its own RNA. The characteristics of the MDA-5 physiological ligand are not yet fully defined. However, RIG-I and MDA-5 show a different dependence on the length of double stranded RNA (dsRNA) in that RIG-I selectively binds short dsRNA, whereas MDA-5 selectively binds long dsRNA. Consistent with this, RIG-I and MDA-5 bind to the synthetic dsRNA analog Poly (I: C) with different length preferences.

In some cases, RIG-I is also capable of indirectly sensing dsDNA. Viral dsDNA can be transcribed by RNA polymerase III into dsRNA with a 5' -triphosphate group. Thus, the synthetic analogue Poly (dA: dT) of type B DNA constitutes another RIG-I ligand.

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

In certain embodiments, the oligonucleotide contains a functional ligand for TLR3, TLR7, TLR8, TLR9 or RIG-I like receptor or a combination thereof.

Examples of immunostimulatory oligonucleotides and methods of making the same are known in the art and commercially available, e.g., see Bodera, p. Receptor PAT INFLAMM ALLERGY Drug discovery.5 (1): 87-93 (2011), incorporat, incorporated herein by reference.

C. Composition of goods

The disclosed nucleic acid cargo may be or include DNA or RNA nucleotides, which typically include a heterocyclic base (nucleobase), a sugar group attached to the heterocyclic base, and a phosphate group that esterifies the hydroxyl function of the sugar group. The main natural nucleotides include uracil, thymine, cytosine, adenine and guanine as heterocyclic bases, and ribose or deoxyribose linked by phosphodiester bonds.

In certain embodiments, the cargo comprises or consists of chemically modified nucleotide analogs that can improve stability, half-life, or specificity or affinity for the target receptor relative to DNA or RNA counterparts. Chemical modifications include chemical modifications of nucleobases, sugar molecules, nucleotide linkages, or combinations thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" refers to a nucleotide that has been chemically modified with respect to one or more components of a heterocyclic base, sugar group, or phosphate group. In certain embodiments, the modified nucleotide has a reduced charge compared to DNA or RNA of the same nucleobase sequence. For example, the oligonucleotide may be negatively, non-charged or positively charged.

Typically, nucleoside analogs support bases capable of forming hydrogen bonding with standard polynucleotide bases by Watson-Crick base pairing, wherein the manner in which the analog backbone presents the bases allows the oligonucleotide analog molecule to hydrogen bond with bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA) in a sequence-specific manner. In certain embodiments, the analog has a substantially uncharged phosphorus-containing backbone.

I. Heterocyclic base

Naturally occurring major nucleotides include heterocyclic bases such as uracil, thymine, cytosine, adenine and guanine. The cargo may include chemical modifications of its nucleobase composition. Chemical modification of the heterocyclic base or heterocyclic base analogue is effective to increase the affinity or stability of binding to the target sequence. 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-. Beta. -D-ribofuranosyl) pyridine (2-aminopyridine), and various pyrrolopyrimidine and pyrazolopyrimidine derivatives.

Modification of saccharides

Nucleotides with modified sugar groups or sugar-based analogues may also be included in the cargo molecule. 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 substituents are particularly preferred because they will protonate at neutral pH, thereby inhibiting charge repulsion between the TFO and the target duplex. This modification stabilizes the C3' -internal conformation of ribose or deoxyribose and also forms a bridging structure with the i-1 phosphate in the double-stranded purine chain.

In certain embodiments, the nucleic acid is a morpholino oligonucleotide. Morpholino oligonucleotides are typically composed of more than two morpholino monomers containing purine or pyrimidine base pairing moieties, which can bind to bases in a polynucleotide through base specific hydrogen bonds. The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structure and binding characteristics of morpholino oligomers are described in detail in U.S. Pat. nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063 and 5,506,337.

Important properties of morpholino-based subunits generally include the ability to be linked in an oligomeric form through a stable, uncharged backbone linkage, the ability to support nucleotide bases such as adenine, cytosine, guanine, thymidine, uracil or inosine, thereby enabling the resulting polymer to hybridize to target nucleic acids (including target RNAs) having complementary bases of high T _M, even to oligomers as short as 10-14 bases, the ability of the oligomer to be actively transported into mammalian cells, and the ability of the oligomer to resist RNAse degradation.

In certain embodiments, as described above, the oligonucleotides employ morpholino subunits containing base pairing moieties and are linked by uncharged linking arms. For example, the morpholine oligonucleotide may be a diamide phosphate morpholine oligomer.

Internucleotide linkages

Oligonucleotides are linked by internucleotide linkages, which refers to the chemical linkage between two nucleoside units. Modification of the phosphate backbone of a DNA or RNA oligonucleotide may increase the binding affinity or stability of the oligonucleotide or decrease the sensitivity of the oligonucleotide to digestion by nucleases. Cationic modifications, including but not limited to diethyl ethylenediamine (DEED) or Dimethylaminopropylamine (DMAP), may be particularly useful because of their ability to reduce electrostatic repulsion between the oligonucleotide and the target. Modification of the phosphate backbone also includes substitution of one of the non-bridging oxygen atoms in the phosphodiester bond with a sulfur atom. Such substitution will result in phosphorothioate internucleoside linkages substituted for phosphodiester linkages. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo.

Examples of charge-reduced modified nucleotides include modified internucleotide linkages, such as phospho-analogues with achiral and uncharged internucleotide linkages (e.g., sterchak, e.p. et al, organic. Chem.,52:4202, (1987)), and uncharged morpholino polymers with achiral intersubunit linkages (as described above, see, e.g., U.S. patent 5,034,506). Some internucleoside linked analogs include morpholino, acetal, and polyamide linked heterocycles.

In another embodiment, the cargo consists of locked nucleic acids. Locked Nucleic Acids (LNAs) are modified RNA nucleotides (see, e.g., braasch et al, chem. Biol.,8 (1): 1-7 (2001)). The hybrid formed by LNA and DNA is more stable than DNA/DNA hybrids, a property similar to Peptide Nucleic Acid (PNA)/DNA hybrids. Thus, LNA can be used like PNA molecules. In certain embodiments, the binding efficiency of the LNA may be improved by adding a positive charge. Commercial nucleic acid synthesizers and standard phosphoramidite chemistry methods can be used to manufacture LNA.

In certain embodiments, the cargo consists of peptide nucleic acids. Peptide Nucleic Acids (PNAs) are artificially synthesized DNA mimics in which the phosphate backbone of an oligonucleotide is entirely replaced by a repeating N- (2-aminoethyl) glycine unit, and the phosphodiester bond is typically replaced by a peptide bond. The various heterocyclic bases are linked to the backbone by methylene carbonyl linkages. Peptide Nucleic Acids (PNAs) maintain a heterocyclic base spacing similar to traditional DNA oligonucleotides, but belong to achiral and neutral charged molecules. Peptide nucleic acids consist of peptide nucleic acid monomers.

Other backbone modifications include alterations and modifications of peptides and amino acids. Therefore, the backbone component of an oligonucleotide such as PNA may be a peptide chain or a non-peptide chain. Examples include acetyl caps, amino spacers (e.g., 8-amino-3, 6-dioxaoctanoic acid) (referred to herein as an O-linker arm), amino acids (e.g., lysine) (particularly useful if PNA is desired to have a positive charge), and the like. Methods of chemical assembly of PNAs are well known. See, for example, U.S. Pat. nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.

Optionally, the cargo comprises one or more terminal residues, or is modified at one or both ends to increase stability and/or affinity of the oligonucleotide for its target. Common 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 with the propylamine groups end capped to prevent degradation. The 3 'or 5' capping procedure for oligonucleotides is well known in the art.

In certain embodiments, the nucleic acid may be single-stranded or double-stranded.

Fine tuning combination

The sequence of cargo can be modified based on these properties and the binding strength between cargo and 4H2 binding protein can be fine tuned.

The 4H2 antibody binds to guanosine, thus increasing the number of guanines and/or selecting the position of guanines in the polynucleotide sequence to increase the binding capacity of the antibody, create an antibody binding site, increase the number of antibodies bound to a single polynucleotide, and/or target the antibodies to specific positions of the polynucleotide. Additionally or alternatively, reducing the number of guanines in a polynucleotide and/or selecting for the location of guanine deletions in a polynucleotide sequence may also be used to increase binding of an antibody, reduce or remove binding sites for an antibody, reduce the number of antibodies bound to a single polynucleotide, and/or target binding of an antibody to alternative locations of a polynucleotide.

For example, any of the disclosed cargo can include or consist of guanine (G) (e.g., single G, dual 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 certain embodiments, synthetic non-coding sequences are added to cargo to increase or decrease binding to the 4H2 binding protein. Such sequences may be, but are not necessarily, at the 5 'or 3' end of the nucleic acid cargo. The cargo may be single-or double-stranded DNA or RNA.

Additionally or alternatively, these binding characteristics may also utilize codon optimization to increase binding bias (e.g., bias guanine) or decrease binding bias (e.g., bias adenine (a), thymine (T), cytosine (C), uracil (U), or inosine (I)) in the course of rationally designing the nucleic acid sequence of the cargo.

2. Vaccine formulations

Vaccines require a powerful immune response. The 4H2 antibodies described herein may be administered as a component of a vaccine to enhance the immune response associated therewith. In certain embodiments, the vaccine disclosed herein comprises an adjuvant of a 4H2 antibody, an antigen, and optionally other additional agents.

A. Antigens

The antigen may be a peptide, protein, polysaccharide, carbohydrate, lipid, nucleic acid, or a combination thereof. The antigen may be derived from a transformed cell such as a cancer cell or a leukemia cell, or may be the whole cell or an immunogenic component thereof. Suitable antigens are known in the art and are available from commercial government and scientific sources.

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

The antigens may be provided alone or in combination. In addition, the antigen may also be provided as a complex mixture of polypeptides or nucleic acids.

For example, the antigen may be, for example, a tumor antigen, or may be derived from an infectious pathogen or disease requiring vaccination, such as polio, tetanus, influenza (influenza), hepatitis b, hepatitis a, hepatitis c, rubella, hib, measles, pertussis (pertussis), pneumococcal disease, HIV, SAR-CoV-2, or any other infection and disease discussed in detail below.

I. Viral antigens

Viral antigens may be isolated from any virus, including, but not limited to, any of the viruses of the Arenaviridae (Arenaviridae), arterivirus (Arterivirus), astroviridae (Astroviridae), baculoviridae (Baculoviridae), badberg (Badnavirus), hamster viridae (Barnaviridae), birna viridae (Birnaviridae), legioviridae (Bromoviridae), bunyaviridae (Bunyaviridae), and Pogostemonis, Caliciviridae (CALICIVIRIDAE), hairy virus (Capillovirus), kara virus (Carlavirus), cauliflower mosaic virus (Caulimovirus), circoviridae (Circoviridae), rhabdoviridae (Closterovirus), bacillus viridae (Comoviridae), coronaviridae (Coronaviridae) (e.g., coronaviridae (e.g., severe Acute Respiratory Syndrome (SARS) virus), dorsal viridae (Corticoviridae), The family of vesicular viruses (Cystoviridae), the family of delta viruses (Deltavirus), the genus Caryophyllus (Dianthovirus), the genus otomosaic virus (Enamovirus), the family of Filoviridae (Filovidae) (e.g., marburg virus and Ebola virus strains (e.g., zaler, leston, kotada or Sudan)), the family of Flaviviridae (Flaviviridae), (e.g., hepatitis C virus, dengue virus type 1, dengue virus type 2, dengue virus type 3 and dengue virus type 4), the family of Hepadnaviridae (HEPADNAVIRIDAE), Herpesviridae (Herpesviridae) (e.g., human herpesviruses type 1, type 3, type 4, type 5, and type 6, and cytomegalovirus), hypoviridae (Hypoviridae), iridoviridae (Hypoviridae), lividae (LEVIVIRIDAE), lipopropviridae (Lipothrixviridae), picoviridae (Microviridae), orthomyxoviridae (Orthomyxoviridae) (e.g., influenza viruses type A, type B, and type C), papillomaviridae (Papovaviridae), Paramyxoviridae (Paramyxoviridae) (e.g., measles virus, mumps virus, and human respiratory syncytial virus), parvoviridae (Parvoviridae), picornaviridae (Picornaviridae) (e.g., polio virus, rhinovirus, hepatovirus, and foot and mouth disease virus), poxviridae (Poxviridae) (e.g., vaccinia virus and smallpox virus), reoviridae (Reoviridae) (e.g., rotavirus), retroviridae (retrovirae) (e.g., lentiviruses such as Human Immunodeficiency Virus (HIV) 1 and HIV 2), and the like, rhabdoviridae (Rhabdoviridae) (e.g., rabies, measles, respiratory syncytial, etc.), togaviridae (Togaviridae) (e.g., rubella, dengue, etc.), and whole viridae (Totiviridae). suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2 and dengue D1NS 3.

Viral antigens may be derived from specific strains or combinations of strains, such as SAR-CoV-2, papilloma virus (papilloma virus), herpes virus (herpes virus) (i.e., herpes simplex type 1 and type 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, EB virus (Epstein-Barr virus), rotavirus, rhinovirus, adenovirus, coxsackie virus, equine encephalitis, japanese encephalitis virus, yellow fever virus, split valley fever virus and lymphochoriomeningitis virus.

Bacterial antigens

Bacterial antigens may be derived from any bacteria including, but not limited to, actinomycetes (Actinomyces), candida (Anabaena), bacillus (Bacillus), bacteroides (bacilli), bdellovibrio (Bdellovibrio), pertussis bauter (Bordetella), borrelia burgdorferi (Borrelia), campylobacter (Campylobacter), campylobacter (Caulobacter), chlamydia (Chlamydia), viridae (Chlamydia), coloribacteria (Chromatium), clostridium (Clostridium), corynebacteria (Corynebacterium), cytophagy (Cytophaga), deinococcus (Deinococcus), escherichia coli (Escherichia), francissambucia (FRANCISELLA), halophil (Halobacterium), escherichia (Heliobacter), haemophilus influenzae (Haemophilus), haemophilus influenzae type B (Hemophilus influenza type B, HIB), microcosm (hyphobacterium), pneumophila (leiomycoti), leptospira (Leptospira), listeria (Listeria monocytogenes), neisseria (iii) and Neisseria (iii), actinomycetes (p.sp), actinomycetes (p.sp.sp.sp.sp.sp.sp. (p.35), actinomycetes (p.sp.sp.sp.sp.sp.sp.35), p.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.sp.35 (p.sp.sp.35), p.sp.sp.sp.sp.sp.sp.sp.sp.sp.35 (p.sp.sp.sp.35, m.sp.sp.sp.sp.sp.sp.sp.sp.35, p.sp.sp.sp.sp.sp.sp.sp.sp.sp., rickettsia (Rickettsia), salmonella (Salmonella), shigella (Shigella), spirochete (Spirillum), spirochete (Spir ° Chaeta), staphylococcus (Staphylococcus), streptococcus (Streptococcus), streptomyces (Streptomyces), sulfolobus (Sulfolobus), pyrogens (thermoplastia), thiobacillus (Thiobacillus), treponema pallidum (Treponema), vibrio cholerae (Vibrio), yersinia (Yersinia).

Parasite antigen

Antigens for parasites may be obtained from parasites such as, but not limited to, those derived from Cryptococcus neoformans (Cryptococcus neoformans), histoplasma capsulatum (Histoplasma capsulatum), candida albicans (Candida albicans), candida tropicalis (Candida tropicalis), nocardia astrotrichia (Nocardia asteroides), rickettsia (RICKETTSIA RICKETTSII), rickettsia typhosa (RICKETTSIA TYPHI), mycoplasma pneumoniae (Mycoplasma pneumoniae), chlamydia psittaci (CHLAMYDIAL PSITTACI), chlamydia trachomatis (CHLAMYDIAL TRACHOMATIS), plasmodium falciparum (Plasmodium falciparum), trypan brucella (Trypanosoma brucei), endomainan histolytica (Entamoeba histolytica), toxoplasma gondii (Toxoplasma gondii), trichomonas vaginalis (Trichomonas vaginalis) and Schistosoma mansoni (Schistosoma mansoni). Such antigens include sporozoite antigens, plasmodium antigens, such as all or part of circumsporozoite proteins, sporozoite surface proteins, liver stage antigens, top coat related proteins or merozoite surface proteins.

Tumor antigen

The antigen may be a tumor antigen, including a tumor-associated antigen or tumor-specific antigen, such AS, but not limited to, alphA-Actin-4, bcr-Abl fusion protein, casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO, mart2, mum-1, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RAR alpha 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, melan A (MART-I), gp100 (Pdel 17), tyrosinase 、TRP-1、TRP-2、MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、p15(58)、CEA、RAGE、NY-ESO(LAGE)、SCP-1、Hom/Mel-40、PRAME、p53、H-Ras、HER-2/neu、BCR-ABL、E2A-PRL、H4-RET、IGH-IGK、MYL-RAR、Epstein Barr virus antigen, NA, human papilloma virus (346) antigen and 43-F9, TLP 4-75, and the T-75, and the TPP-associated protein, tp-83, tp-type A, and the protein 52-72. Tumor antigens, such as BCG, may also be used as immunopotentiators for adjuvants.

B. Adjuvant

Alternatively, the vaccine described herein may include an adjuvant. Adjuvants may be, but are not limited to, one or more of oil emulsions (e.g., friedel adjuvants), saponin formulations, virosomes and virus-like particles, bacterial and microbial derivatives, immunostimulatory oligonucleotides, ADP-ribosylating toxins and detoxified derivatives thereof, alum, BCG, mineral-containing ingredients (e.g., mineral salts such as aluminum salts and calcium salts, hydroxides, phosphates, sulfates, etc.), bioadhesives and/or mucoadhesives, microparticles, liposomes, polyoxyethylene ether and polyoxyethylene ester formulations, polyphosphazenes, muramyl peptides, imidazoquinolinone compounds, and surface active components (e.g., lysolecithin, poloxamer polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

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, etc.), interferons (e.g., interferon-gamma), macrophage colony stimulating factor, and tumor necrosis factor. In addition to PD-1 antagonists, other co-stimulatory molecules may be administered, including other polypeptides of the B7 family. Such protein adjuvants may be provided in the form of full-length polypeptides or active fragments thereof, or in the form of RNA or DNA (e.g., plasmid DNA).

3. Immune checkpoint modulators

The 4H2 antibodies can be used in combination with immune checkpoint modulators.

Immune checkpoints may be stimulatory or inhibitory, and tumors may utilize these checkpoints to protect themselves from the immune system. Current approved checkpoint therapies may block inhibitory checkpoint receptors, but therapies studies to activate stimulatory checkpoints are also underway. Thus, immune checkpoint modulators may be modulators that block inhibitory checkpoints, or modulators that activate stimulatory checkpoints. In general, immune checkpoint modulators are modulators that may induce or otherwise activate or increase an immune response against a target cell (e.g., a cancer cell or an infected cell). Thus, in certain embodiments, the immune checkpoint modulator can be a Chimeric Antigen Receptor (CAR) -directed cell, such as a CAR-T cell. In another embodiment, the immune checkpoint modulator may be an oncolytic virus.

In preferred embodiments, immune checkpoint modulators may block inhibitory checkpoints. Thus, blocking negative feedback signaling to immune cells may enhance immune responses to tumors. Thus, in certain embodiments, an effective amount of an immune checkpoint modulator is administered to a subject to block an inhibitory checkpoint. Exemplary compounds are compounds that block or otherwise inhibit, for example, PD-1, PD-L1, or CTLA 4.

PD-1 antagonists

In certain embodiments, the active agent is a PD-1 antagonist. Activation of T cells is generally dependent on antigen specific signals of T Cell Receptors (TCRs) upon contact with antigen peptides presented by Major Histocompatibility Complex (MHC), the extent of this response being controlled by antigen independent positive and negative signals from various accessory stimulatory molecules. The latter is typically a member of the CD28/B7 family. In contrast, programmed death-1 (PD-1) is a member of the CD28 receptor family, which produces a negative immune response when induced on T cells. PD-1 contact with 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. patent 8,114,845, 8,609,089, and 8,709,416, and include compounds or agents that bind to and block the PD-1 ligand to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or compounds or agents that bind directly to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

In certain embodiments, the PD-1 receptor antagonist binds directly to the PD-1 receptor without triggering inhibitory signal transduction, while also binding to the ligand of the PD-1 receptor to reduce or inhibit ligand triggering signal transduction through the PD-1 receptor. By reducing the number and/or amount of ligands that bind to the PD-1 receptor and trigger inhibition of signaling, cells that are attenuated by the negative signal transmitted by PD-1 signaling may be less, and thus a more robust immune response may be achieved.

It is currently believed that the PD-1 signal is driven by binding to a PD-1 ligand (e.g., B7-H1 or B7-DC) and in close proximity to a peptide antigen presented by the Major Histocompatibility Complex (MHC) (see, e.g., freeman, proc. Natl. Acad. Sci. U.S.A., 105:10275-10276 (2008)). Thus, proteins, antibodies or small molecules that prevent co-binding of PD-1 and TCR on T cell membranes are also useful PD-1 antagonists.

In a preferred embodiment, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signaling by binding to the ligand of PD-1 or PD-1 itself, especially where co-binding of PD-1 to the TCR does not follow such binding, thereby not triggering inhibitory signaling through the PD-1 receptor. Other PD-1 antagonists include antibodies that bind to PD-1 or a ligand of PD-1, such as PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), as well as other antibodies.

Suitable anti-PD-1 antibodies include, but are not limited to, antibodies described in the following documents:

PCT/IL03/00425 (Hardy et al, WO/2003/099196)

PCT/JP2006/309606 (Korman et al, WO/2006/121168)

PCT/US2008/008925 (Li et al, WO/2009/014708)

PCT/JP03/08420 (Honjo et al, WO/2004/004771)

PCT/JP04/00549 (Honjo et al, WO/2004/072286)

PCT/IB2003/006304 (Collins et al, WO/2004/056875)

PCT/US2007/088851 (Ahmed et al, WO/2008/083174)

PCT/US2006/026046 (Korman et al, WO/2007/005874)

PCT/US2008/084923 (Terrett et al, WO/2009/073533)

Berger et al, clin.cancer Res., 14:3043251 (2008).

A specific example of an anti-PD-1 antibody is MDX-1106 (see Kosak, US20070166281 (published 7/19 2007), paragraph 42), which is a human anti-PD-1 antibody, preferably administered at a dose of 3 mg/kg.

Exemplary anti-B7-H1 antibodies include, but are not limited to, antibodies described in the following documents:

PCT/US06/022423 (WO/2006/133396,2006, 12, 14 days disclosure)

PCT/US07/088851 (WO/2008/083174,2008, 7, 10 days disclosure)

US2006/0110383 (published 25 th month of 2006)

A specific example of an anti-B7-H1 antibody is MDX-1105 (published on 11/2007/005874,2007, 1), which is a human anti-B7-H1 antibody.

For anti-B7-DC antibodies, see 7,411,051, 7,052,694, 7,390,888 and U.S. published application 2006/0099203.

The antibody may be a bispecific antibody, including an antibody that binds to a PD-1 receptor that is bridged by an antibody that binds to a PD-1 ligand (e.g., B7-H1). In certain embodiments, the PD-1 binding moiety may reduce or inhibit signal transduction through a PD-1 receptor.

Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants thereof, as well as active fragments of any of the above polypeptides, and fusion proteins incorporating any of the above polypeptides. In a preferred embodiment, the fusion protein comprises a soluble portion of B7-DC coupled to the Fc portion of an antibody (e.g., human IgG) and does not comprise all or part of the human B7-DC transmembrane region.

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

Other useful polypeptide PD-1 antagonists include polypeptides that bind to ligands of the PD-1 receptor. These polypeptides include PD-1 receptor proteins or soluble fragments thereof that bind to PD-1 ligands (e.g., B7-H1 or B7-DC) and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction. B7-H1 also binds to protein B7.1 (Butte et al, immunity, volume 27, pages 111-122, (2007)). These fragments also include the soluble ECD portion of PD-1 proteins, which includes mutations, such as A99L mutations, that increase binding to the natural ligand (Molnar et al, PNAS,105:10483-10488 (2008)). Also useful are B7-1 or soluble fragments thereof that bind to B7-H1 ligands and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction.

PD-1 and B7-H1 antisense nucleic acids (DNA and RNA) and siRNA molecules can also be PD-1 antagonists. Such antisense molecules can prevent the expression of PD-1 on T cells and the production of T cell ligands (e.g., B7-H1, PD-L1, and/or PD-L2). For example, siRNA (e.g., about 21 nucleotides in length, which is specific for a gene encoding PD-1 or a ligand encoding PD-1, and which is readily commercially available) is complexed with a carrier such as polyethylenimine (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, and reduces the expression of these receptors and ligands, thereby reducing inhibitory signal transduction in T cells, thereby activating T cells.

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

Pembrolizumab (original name MK-3475 or lambrolizumab, keystuda) was developed by merck, inc, and was first approved by the United states food and drug administration for the treatment of melanoma in 2014.

Nivolumab (Opdivo) was developed by Bai-Shi-Mei-Guibao, the first approval of the United states food and drug administration for the treatment of melanoma was obtained in 2014.

Pimelizumab, developed by CureTech company

AMP-224 developed by the company Gelanin Smith and MedImmune

AMP-514 developed by the company Gelanin Smith and MedImmune

PDR001 developed by North America

Cemiplimab developed by Regeneron and Sainophenanthrene Inc

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

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

-Avelumab (Bavencio) is a fully human IgG1 antibody developed by merck-snow lano and pyroxene. Avelumab have been approved by the FDA for the treatment of metastatic merck cell carcinoma. The phase III clinical trial of the medicine for treating gastric cancer fails.

-Durvalumab (Imfinzi) is a fully human IgG1 antibody developed by the company aslicon. Durvalumab have been FDA approved for the treatment of urothelial cancer and unresectable non-small cell lung cancer following chemotherapy.

BMS-936559 developed by Bai-Shi Mi Guibao Co

CK-301 developed by Checkpoint Therapeutics Co

See, e.g., iwai, et al, journal of Biomedical Science, (2017) 24:26,DOI 10.1186/s12929-017-0329-9.

CTLA4 antagonists

Other molecules that help mediate T cell effects in immune responses are also contemplated as active agents. For example, in certain embodiments, the molecule is a formulation that binds to an immune response-mediating molecule that is not PD-1. In a preferred embodiment, the molecule is an antagonist of CTLA4, e.g., an antagonistic anti-CTLA 4 antibody. Examples of anti-CTLA 4 antibodies are described in PCT/US2006/043690 (Fischkoff et al, WO/2007/056539).

Dosages of anti-PD-1, anti-B7-H1 and anti-CTLA 4 antibodies are known in the art and may range from 0.1 to 100mg/kg, preferably from 1 to 50mg/kg, more preferably from 10 to 20 mg/kg. For human subjects, a suitable dose is 5 to 15mg/kg, most preferably 10mg/kg of antibody (e.g., human anti-PD-1 antibody, such as MDX-1106).

Specific examples of CTLA antagonists include ipilimumab (Ipilimumab), also known as MDX-010 or MDX-101, human anti-CTLA 4 antibody, preferably administered at a dose of about 10mg/kg, and terlimumab (Tremelimumab), human anti-CTLA 4 antibody, preferably administered at a dose of about 15mg/kg. See also Sammartino et al, CLINICAL KIDNEY Journal,3 (2): 135-137 (2010), 12 months in 2009.

In other embodiments, the antagonist is a small molecule. A series 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). This small organic compound may be administered alone or in combination with an anti-CTLA 4 antibody to reduce inhibitory signal transduction to T cells.

C. Chimeric antigen receptor-directed cells

The modulator may be a chimeric antigen receptor-directing cell. The term "chimeric antigen receptor" or "CAR" as used herein refers to a group of polypeptides, typically two in the simplest embodiment, that when in an immune effector cell, provide the cell with specificity for a cancer cell and produce an intracellular signal. In certain embodiments, the CAR comprises 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"), including functional signaling domains derived from a stimulatory molecule and/or co-stimulatory molecule as defined below. In one embodiment, the stimulatory molecule is a zeta chain (zeta chain) associated with the T cell receptor complex ("zeta stimulatory domain"). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule (e.g., 4-1BB (i.e., CD 137), CD27, and/or CD 28). In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a functional signaling domain derived from a stimulatory molecule. In various embodiments, the CAR is a fusion protein of a single chain variable fragment (scFv) fused to a CD3-zeta transmembrane domain. However, other intracellular signaling domains, such as CD28, 41-BB, and Ox40, may also be used in various combinations to provide the desired intracellular signaling. In certain embodiments, a CAR disclosed herein comprises an extracellular binding domain.

The term "antigen binding domain" as used herein refers in the present disclosure to the portion of the CAR that specifically recognizes and binds to an antigen of interest. The antigen binding domain "may be derived from a binding protein disclosed herein, such as an antibody or fragment thereof. In certain embodiments, the "binding domain" is a single chain variable fragment (scFv). In certain embodiments, a "binding domain" comprises the complementarity determining regions of the binding proteins disclosed herein. In this embodiment, the CAR-directed cell may represent a 4H2 cell penetrating antibody that induces cGAS/STING signaling (assuming it penetrates cancer cells) or a combination thereof in combination 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-directed cells disclosed herein are administered with a cell penetrating 4H2 antibody disclosed herein.

The term "zeta" or "CD3-zeta" as used herein is used to define the protein provided by GenBan Acc.BAG36664.1, or equivalent residues derived from a non-human species, "zeta stimulating domain" or alternatively "CD3-zeta stimulating domain" is defined as an amino acid residue of the zeta chain cytoplasmic domain, or a functional derivative thereof, sufficient to functionally transmit the initial signal required for T cell activation.

The term "immune effector cell" as used herein refers to a cell that is involved in an immune response (e.g., promotes an immune effector response). 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 certain embodiments, the immune effector cell is heterologous. In certain embodiments, the immune effector cells are autologous. In some embodiments, the immune checkpoint modulator is a CAR-directed T cell (CAR-T cell). Exemplary CAR-T cells include Axicabtagene ciloleucel(KTE-C19,Axi-cel)、Tisagenlecleucel、Lisocabtagene Maraleucel(liso-cel;JCAR017).

Immune effector cells, such as T cells, are typically activated and expanded using methods previously described, for example, as described in U.S. patent 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;6,867,041. For example, a population of immune effector cells (e.g., T regulatory cells depleted) can be expanded by contact with a surface to which are attached agents that stimulate CD3 complex-associated signals and ligands that stimulate T cell surface costimulatory molecules.

D. Oncolytic viruses

The modulator may be an oncolytic virus. In the present disclosure, the term "oncolytic virus" refers to a virus that is capable of infecting cancer cells and reducing their growth. For example, oncolytic viruses can inhibit cell proliferation. In certain embodiments, the oncolytic virus may kill cancer cells. In certain embodiments, the oncolytic virus preferentially infects and inhibits growth of cancer cells compared to corresponding normal cells. In another embodiment, the oncolytic virus preferentially replicates in and inhibits growth of cancer cells compared to corresponding normal cells.

In certain embodiments, the oncolytic virus is capable of naturally infecting cancer cells and reducing 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 that naturally infect and reduce the growth of cancer cells are often used to target cancer cells with cellular aberrations that occur in cancer cells. For example, oncolytic viruses may utilize surface attachment receptors, activated oncogenes (e.g., ras, akt, p a 53), and/or Interferon (IFN) pathway defects.

In another embodiment, the oncolytic viruses encompassed by the present disclosure are engineered to infect and reduce the growth of cancer cells. Exemplary viruses suitable for use in such engineering include oncolytic DNA viruses such as adenovirus, herpes Simplex Virus (HSV) and vaccinia virus, and oncolytic RNA viruses such as lentivirus, reovirus, coxsackie virus, seikagavirus, poliovirus, measles virus, newcastle disease virus, vesicular Stomatitis Virus (VSV) and parvovirus such as rodent parvovirus H-1PV. In certain embodiments, the oncolytic virus comprises the backbone of the virus described above.

In certain embodiments, the tumor specificity of an oncolytic virus can be engineered to mutate or delete genes that are required for the virus to survive in normal cells but not in cancer cells. For example, oncolytic viruses can be engineered by mutating or deleting genes encoding thymidine kinases (enzymes required for nucleic acid metabolism). In this example, the virus relies on the expression of cellular thymidine kinase, which is highly expressed in proliferating cancer cells but inhibited in normal cells. In another example, oncolytic viruses are designed to include capsid proteins that bind to tumor-specific cell surface molecules. In certain embodiments, the bacteriophage protein is a fibrin, pentameric protein or hexameric protein. In another example, oncolytic viruses are engineered to include tumor-specific cell surface molecules for transduction of targeted cancer cells. Exemplary tumor-specific cell surface molecules can include integrins, members of the epidermal growth factor receptor family, proteoglycans, disialogangliosides, B7-H3, CA-125, epCAM, ICAM-1, DAF, A21, integrin- α2β1, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, CEA, tumor-associated glycoprotein, CD19, CD20, CD22, CD30, CD33, CD40, CD44, CD52, CD74, CD152, CD155, MUC1, tumor necrosis factor receptor, insulin-like growth factor receptor, folate receptor a, transmembrane glycoprotein NMB, C-C chemokine receptor, PSMA, RON-receptor, and cytotoxic T lymphocyte antigen 4.

Oncolytic viruses may have replication ability. In certain embodiments, the oncolytic virus is selectively replicable in cancer cells compared to corresponding normal cells.

Conditional replication can be achieved by, for example, inserting a tumor-specific promoter to drive expression of the critical gene. Such promoters may be determined based on differences in gene expression between the tumor and the corresponding surrounding tissue. Exemplary natural promoters include AFP, CCKAR, CEA, erbB2, cerb2, COX2, CXCR4, E2F1, HE4, LP, MUC1, PSA, survivin, TRP1, STAT3, hTERT, and Tyr. Exemplary composite promoters include AFP/hAFP, SV40/AFP, CEA/CEA, PSA/PSA, SV40/Tyr and Tyr/Tyr.

Various viruses may be engineered as in the examples above. For example, the oncolytic virus may be a modified HSV, lentivirus, baculovirus, retrovirus, adenovirus (AdV), adeno-associated virus (AAV) or a recombinant form, such as a recombinant adeno-associated virus (rAAV) or a derivative thereof, such as a self-complementing adeno-associated virus (scAAV) or a non-integrating adenovirus. The oncolytic virus may be a modified HSV oncolytic virus, and may be a modified lentivirus. Other example viruses include vaccine virus, vesicular Stomatitis Virus (VSV), measles virus, and maraba virus (maraba virus).

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

Exemplary oncolytic viruses include T-Vec (HSV-1; amgen), JX-594 (vaccine; sillajen), JX-594 (AdV; cold Genesys), reolysin (reovirus; oncolytics Biotech). Other examples of oncolytic viruses are disclosed in WO 2003/080083、WO 2005/086922、WO 2007/088229、WO 2008/110579、WO 2010/108931、WO 2010/128182、WO 2013/112942、WO 2013/116778、WO 2014/204814、WO 2015/077624 and WO 2015/166082, WO 2015/089280.

E. other immune checkpoint modulators

Targets for other immune checkpoints include, but are not limited to, ICOS, OX40, GITR, 4-1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B-H3, KIR, PARP, etc., and are targeted for cancer therapy alone or in combination with anti-PD-1, anti-PD-L1, and anti-CTLA compounds. See, for example Iwai et al, journal of Biomedical science.24 (1): 26.doi:10.1186/S12929-017-0329-9; donini, et al J Thorac dis.2018May;10 (Suppl 13): S1581-S1601.Doi:10.21037/jtd.2018.02.79. Thus, in some embodiments, the 4H2 antibody is administered in combination with a compound targeting ICOS, OX40, GITR, 4-1BB, CD40, CD27-CD70, LAG3, TIM-3, TIGIT, VISTA, B-H3, KIR, or PARP, alone or in combination with a compound targeting PD-1, PD-L1, and/or CTLA. In another embodiment, the immune checkpoint modulator is an antibody disclosed in WO 2016/013870.

C. Pharmaceutical composition

The present compositions may be combined with a pharmaceutically acceptable carrier for use in therapy.

The composition is preferably used for treatment in combination with a suitable pharmaceutically acceptable carrier. Such compositions comprise an effective amount of the composition and a pharmaceutically acceptable carrier or excipient.

The composition may be a formulation for topical (topically), topical (locally) or systemic administration in a suitable pharmaceutical carrier. Typical vectors and methods of preparation are disclosed in Remington's Pharmaceutical Sciences, 15 th edition, by E.W. Martin (Mark Publishing Company, 1975). The antibodies or complexes formed therefrom may also be encapsulated in suitable biocompatible particles formed from biodegradable or non-biodegradable polymers or proteins or liposomes in order to target cells. Such systems are well known to those skilled in the art. In certain embodiments, the antibody or complex formed therefrom is encapsulated in a nanoparticle.

The injectable preparation may be in unit dosage form such as ampules or multi-dose containers optionally with the addition of preservatives. The compositions may take the form of sterile aqueous or nonaqueous solutions, suspensions, emulsions, and the like, which in certain embodiments may be isotonic with the blood of the subject. Examples of nonaqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil (arachis oil), peanut oil (peanut oil), mineral oils, injectable organic esters such as ethyl oleate, or fixed oils including synthetic mono-or diglycerides. Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, 1, 3-butanediol, ringer's dextrose, dextrose and sodium chloride, emulsified ringer's solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements and electrolyte supplements (e.g., ringer's dextrose-based supplements). The material may be a solution, emulsion, or suspension (e.g., incorporated into particles, liposomes, or cells). Typically, an appropriate amount of a pharmaceutically acceptable salt will be used in the formulation to render the formulation isotonic. Trehalose may be added to the pharmaceutical composition, typically in an amount of 1-5%. The pH of the solution is preferably about 5 to about 8, preferably about 7 to about 7.5.

The pharmaceutical composition may include a carrier, a thickener, a diluent, a buffer, a preservative, and a surfactant. Carrier formulations can be found in Remington's Pharmaceutical Sciences, mack Publishing co., easton, pa. The various parameters for preparing and formulating the compositions can be readily determined by those skilled in the art without undue experimentation.

The compositions may also be formulated as aerosol formulations (i.e., they may be "nebulized") alone or in combination with other suitable ingredients, for administration by inhalation. The aerosol formulation may be placed in an acceptable pressurized propellant such as dichlorodifluoromethane, propane, nitrogen and air. For inhalation administration, the compounds may be administered in the form of an aerosol spray by a pressurized pack or nebulizer, using a suitable propellant.

In certain embodiments, the compounds include pharmaceutically acceptable carriers with formulation ingredients, such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, preservatives, solubilizers or stabilizers.

The nucleic acid may be conjugated with lipophilic groups such as cholesterol, lauric acid, and lithocholic acid derivatives having a C32 functional group to increase cellular absorptivity. For example, cholesterol has been shown to improve siRNA absorption and serum stability in vitro (Lorenz et al, biorg. Med. Chem. Lett.,14 (19): 4975-4977 (2004)) and in vivo (Sonschek et al, nature,432 (7014): 173-178 (2004)). Furthermore, studies have shown that steroid conjugated oligonucleotides bind to different lipoproteins in the blood, such as Low Density Lipoproteins (LDL), protecting their integrity and promoting biodistribution (Rump et al, biochem. Pharmacol,59 (11): 1407-1416 (2000)). Other groups that may be linked or conjugated to the above nucleic acids to increase cellular uptake include acridine derivatives, cross-linking agents such as psoralen derivatives, azidobenzoyl, proflavine and azidopropane, artificial endonucleases, metal complexes such as ethylenediamine tetraacetic acid-iron (II) (EDTA-Fe (II)) and porphyrin-iron (II), alkylating groups, nucleases such as alkaline phosphatase, terminal transferases, abzymes, cholesterol groups, lipophilic carriers, peptide conjugates, long chain alcohols, phosphates, radiolabels, nonradioactive labels, carbohydrates, and polylysine or other polyamines. U.S. patent 6,919,208 to Levy et al also describes methods of enhancing delivery. These pharmaceutical preparations can be manufactured in a manner known per se, for example by means of conventional mixing, dissolving, granulating, milling, emulsifying, encapsulating, entrapping or lyophilizing processes.

Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, or complexes formed therefrom, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Implantation includes insertion of 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 in an inhaler with an aerosol, either alone or attached to an absorbable carrier. For systemic administration, the composition is preferably encapsulated in liposomes.

Invasive devices (such as vascular catheters or urinary catheters) and interventional devices (such as stents having drug delivery capabilities and configured as stent-grafts or stent-grafts) may be used to deliver the compositions in a manner that is capable of tissue-specific uptake of the agent and/or nucleotide delivery systems.

The biodegradable implants may be used to deliver the formulation by diffusion or by degradation through a polymer matrix. In certain embodiments, administration of the formulation may be designed to be continuously exposed to the composition over a period of time (e.g., hours, days, weeks, months, or years). For example, this may be achieved by repeated administration of the formulation or a sustained or controlled release delivery system in which the composition is administered for a prolonged period without repeated administration.

Other suitable delivery systems include sustained release, delayed release, sustained release or controlled release delivery systems. In many cases, these systems can avoid repeated administration, providing more convenience to the subject and physician. Many types of delivery systems are available and are well known to those of ordinary skill in the art. For example, they include polymer-based systems such as polylactic and/or polyglycolic acid, polyanhydrides, polycaprolactone, copolyoxalate, polyesteramide, polyorthoester, polyhydroxybutyrate, and/or combinations thereof. Microcapsules of the above polymers containing nucleic acids are described, for example, in U.S. Pat. No. 5,075,109. Other examples also include lipid-based non-polymeric systems including sterols such as cholesterol, cholesterol esters, fatty acids or neutral fats such as mono-, di-and triglycerides, hydrogel release systems, liposome-based systems, phospholipid-based systems, silicone systems, peptide-based systems, wax coatings, compressed tablets using conventional binders and excipients, or partially fused implants. The formulation may be, for example, a microsphere, a hydrogel, a polymer reservoir, a cholesterol matrix, or a polymer system, etc. In certain embodiments, the system may achieve sustained or controlled release of the composition by controlling the diffusion or erosion/degradation rate of a formulation containing the antibody or complex formed therefrom, and the like.

The compositions may be formulated for pulmonary or mucosal administration. Administration may include delivery of the composition to the pulmonary, nasal, oral (sublingual, buccal), vaginal or rectal mucosa. The term aerosol as used herein refers to any formulation of a fine mist of particles, which may be a solution or suspension, whether or not it is generated using a propellant. Aerosols may be produced using standard techniques such as ultrasonic or high pressure treatment.

For administration via the upper respiratory tract, the formulations may be formulated as solutions, such as water or isotonic physiological saline, buffers or non-buffers, or as suspensions, for intranasal administration as drops or sprays. Preferably, these solutions or suspensions are isotonic with respect to nasal secretions and have about the same pH, for example from about pH 4.0 to about pH 7.4, or from about pH 6.0 to about pH 7.0. Buffers should be physiologically compatible, including phosphate buffers for simple example.

The composition may be delivered to the target cell using a particulate delivery vehicle. Nanoparticles generally refer to particles having diameters in the range of 500 nanometers to less than 0.5 nanometers, preferably diameters in the range of 50 to 500 nanometers, and more preferably diameters in the range of 50 to 300 nanometers. The cellular internalization of polymer particles is highly dependent on their size, and the efficiency with which the nano-polymer particles are internalized by cells is much higher than with micro-polymer particles. For example, desai et al showed that nanoparticles with a diameter of 100nm were absorbed approximately 2.5 times more by cultured Caco-2 cells than particles with a diameter of 1. Mu.M (Desai et al, pharm. Res.,14:1568-73 (1997)). The ability of the nanoparticle to diffuse deep into tissue in vivo is also greater.

In certain embodiments, the delivery vehicle is a dendrimer.

Examples of preferred biodegradable polymers include synthetic polymers degradable by hydrolysis, such as poly (hydroxy acids), such as polymers and copolymers of lactic acid and glycolic acid, other degradable polyesters, polyanhydrides, poly (ortho) esters, polyesters, polyurethanes, poly (butyric acid), poly (valeric acid), poly (caprolactone), poly (hydroxyalkanoate), poly (lactide-co-caprolactone) and poly (amine-co-ester) polymers, such as those described in Zhou et al Nature Materials,11:82-90 (2012) and WO 2013/082529, U.S. application publication 2014/0342003 and WO 2016/081621.

In some embodiments, particularly for in vivo targeting of T cells, e.g., for in vivo production of CAR T cells, immune cells or T cell markers, such as CD3, CD7, or CD8, or markers of target tissue, such as liver, may be targeted. For example, both anti-CD 8 antibodies and anti-CD 3 Fab fragments have been used to target T cells in vivo (Pfeiffer, et al, EMBO Mol Med.,10 (11) (2018). Pii: e9158.doi:10.15252/emmm.201809158, smith, et al, nat nanotechnol.,12 (8): 813-820 (2017), doi: 10.1038/nano.2017.57). Thus, in some embodiments, the particles or other delivery vehicle include a targeting moiety specific for CD3, CD7, CD8, or another immune cell (e.g., T cell) marker, or a marker of a specific tissue of thymus, spleen, or liver, among others. For example, the binding moiety may be an antibody or antigen binding fragment thereof.

The targeting moiety may be associated, linked, conjugated or otherwise attached directly or indirectly to the nanoparticle or other delivery vehicle. The targeting moiety may be a protein, peptide, nucleic acid molecule, sugar or polysaccharide that binds to a receptor or other molecule on the surface of the target cell. The specificity and affinity of binding to the graft can be modulated by the selection of the targeting moiety.

Examples of moieties include, for example, targeting moieties that can deliver molecules to specific cells, such as hematopoietic stem cells, CD34 ⁺ cells, T cells, or any other antibody of a preferred cell type, as well as receptors and ligands expressed on a preferred cell type. Preferably, the moiety targets hematopoietic stem cells. Examples of molecules targeting the extracellular matrix ("ECM") include glycosaminoglycans ("GAGs") and collagen. In one embodiment, the outer surface of the polymer particles may be modified to enhance the ability of the particles to interact with selected cells or tissues. Preferably, the adaptor element conjugated to the targeting moiety is inserted into the particle using the methods described above. However, in another embodiment, the outer surface of the polymer microparticle or nanoparticle having a carboxyl terminus may be linked to a targeting moiety having a free amine terminus.

Other useful ligands attached to polymeric microparticles and nanoparticles include Pathogen Associated Molecular Patterns (PAMPs). PAMPs target Toll-like receptors (TLRs) on the surface of cells or tissues or signal the interior of cells or tissues, potentially increasing uptake. PAMPs conjugated or co-encapsulated to the particle surface may include unmethylated CpG DNA (bacteria), double stranded RNA (viruses), lipopolysaccharide (bacteria), peptidoglycan (bacteria), lipoarabinomannan (bacteria), zymosan (yeast), protoplasmic lipoproteins, such as MALP-2 (bacteria), flagellin (bacteria), poly (inosinic acid-cytidylic acid) (bacteria), lipoteichoic acid (bacteria) or imidazoquinoline (synthesis).

In another embodiment, the outer surface of the particles may be treated with mannosamine, thereby mannosylating the outer surface of the particles. Such treatment allows the particles to bind to the target cells or tissues at mannose receptors on the surface of antigen presenting cells. In addition, surface conjugation to immunoglobulin molecules containing Fc moieties (targeting Fc receptors), heat shock protein molecules (HSP receptors), phosphatidylserine (scavenger receptors) and Lipopolysaccharide (LPS) is an additional receptor target on cells or tissues.

Lectins can be covalently attached to microparticles and nanoparticles, making them targeted to mucins and mucosal cell layers.

The choice of targeting moiety will depend on the method of administration of the nanoparticle composition and the cell or tissue to be targeted. The targeting moiety may generally increase the binding affinity of the particle to a cell or tissue, or target the nanoparticle to a specific tissue in an organ or a specific cell type in a tissue. In certain embodiments, the targeting moiety targets thymus, spleen, or cancer cells.

Any natural component of mucin is covalently attached to the microparticles in purified or partially purified form, which reduces the surface tension of the bead-gut interface and increases the solubility of the bead in the mucin layer. Attachment of polyamino acids containing additional pendant carboxylic acid side groups (e.g., polyaspartic acid and polyglutamic acid) can increase bioadhesion. Polyamino acids having a molecular weight in the range of 15,000 to 50,000kDa are used to generate chains of 120 to 425 amino acid residues attached to the surface of the particle. Polyamino chains can increase bioadhesion through chain entanglement in mucin chains and an increase in carboxyl charge.

III methods of use

A. Delivery of nucleic acids

Methods of enhancing delivery of nucleic acid constructs using 4H2 antibodies are provided. Typically, an effective amount of the 4H2 antibody is first contacted with the nucleic acid cargo that is desired to be delivered into the cell. For example, the nucleic acid cargo and the antibody may be mixed in solution for a time sufficient to form a complex of the nucleic acid cargo and the antibody. The mixture is then contacted with the cells. In other embodiments, the cargo and antibody are added to a solution containing or otherwise soaking the cells, forming a complex in the presence of the cells. The complex may be contacted with the cell in vitro, ex vivo, or in vivo. Thus, in certain embodiments, the complex solution is added to the cultured cells or injected into the animal to be treated. The treatment may be, for example, administration by simple intravenous injection, of a mixture of the antibody and nucleic acid cargo to a subject in need thereof.

The compositions and methods can include 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic acid constructs formed from RNA, DNA, PNA or other modified nucleic acids, or combinations thereof.

An effective or therapeutically effective amount of a composition may be a dose sufficient to treat, inhibit, or ameliorate one or more symptoms of a disease or disorder, or a dose that otherwise provides a desired pharmacological and/or physiological effect, e.g., reduces, inhibits, or reverses one or more pathophysiological mechanisms of a disease or disorder.

An effective amount may also be an amount effective to increase the rate, amount, and/or quality of delivery of the nucleic acid cargo relative to administration of the nucleic acid cargo in the absence of the antibody. The formulation of the composition is adapted to the mode of administration.

The pharmaceutically acceptable carrier will depend in part on the particular composition being administered and the particular method of administering the composition. Accordingly, there are a variety of suitable formulations for pharmaceutical compositions containing the complexes. The precise dosage will vary depending on various factors, such as subject dependent variables (e.g., age, immune system health, clinical symptoms, etc.).

The target cells may be administered or otherwise contacted once, twice or three times per day, once, twice, three times, four times, five times, six times, seven times per week, once, twice, three times, four times, five times, six times, seven times or eight times per month. For example, in certain embodiments, the composition is administered once every two or three days, or about 2 to about 4 times per week on average. Thus, in certain embodiments, the composition is administered as part of a dosage regimen comprising two or more monotherapy.

Dosage regimens include maintenance regimens (doses remain unchanged between two or more administrations), escalation regimens (dose increases between two or more administrations), degradation regimens (dose decreases between two or more administrations), or combinations thereof.

In certain embodiments, the first dose may be a low dose. The dosage may be continued to 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 certain embodiments, the dosage may be increased until the therapeutic effect is determined, preferably while not causing undesired toxicity or controlling it within acceptable limits. Next, the dose may be maintained or steadily reduced to a maintenance dose. These methods can be used to normalize, optimize or customize the dosage level, dosage frequency or duration of treatment.

Generally, the antibody and nucleic acid are mixed for a period of time, e.g., at room temperature, prior to administration, particularly in vivo. In certain embodiments, the time of complexation ranges, for example, from 1 minute to 30 minutes (inclusive), or from 10 minutes to 20 minutes (inclusive), with a preferred complexation time of about 15 minutes. Antibody doses range from 0.0001 mg to 1 mg (inclusive), with a preferred dose of about 0.1 mg. Nucleic acid doses range from 0.001 micrograms to 100 micrograms (inclusive), with a preferred dose of 10 micrograms. In the experiments below, the 4H2/mRNA ratios were 1:1w/w and 3:1w/w, although other ratios are also contemplated. In certain embodiments, the ratio of antibody to nucleic acid may 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 1:5w/w.

In certain embodiments, the RNA and/or DNA cargo is mixed with the vector DNA. The vector DNA may be, for example, plasmid DNA or low molecular weight DNA, such as DNA derived from salmon sperm. In certain embodiments, the vector DNA is non-coding DNA. The vector DNA may be single-stranded or double-stranded, or a combination thereof. In certain embodiments, the vector DNA consists of a nucleic acid of 1-10, 1-100, 1-1,000, or 1-10,000 nucleotides in length, or any subrange or integer or combination thereof. The vector DNA is typically not conjugated 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.

1. In vitro and ex vivo methods

For in vitro and ex vivo methods, the cells are typically contacted with the composition during culture. For ex vivo methods, cells can be isolated from a subject and contacted ex vivo with a composition to produce cells containing a cargo nucleic acid. In a preferred embodiment, the cells are isolated from the subject to be treated or from an isogenic host. The target cells may be removed from the subject prior to contact with the composition. The antibody and cargo may be contacted with the cells together or separately, or may be contacted with the cells as a preformed complex.

2. In vivo methods

In certain embodiments, the delivery of nucleic acid cargo to cells in vivo is used for gene editing and/or treating a disease or disorder in a subject. Compositions that generally include an antibody-nucleic acid cargo complex can be administered directly to a subject for in vivo treatment.

Generally, methods of administering compounds (including antibodies, oligonucleotides and related molecules) are well known in the art. In particular, routes of administration and formulations currently in use that have been used for nucleic acid therapy provide preferred routes of administration and formulations for the donor oligonucleotides described above. The composition is preferably injected or infused into an animal.

The compositions may be administered by a variety of routes including, but not limited to, intravenous injection, intraperitoneal injection, intra-amniotic injection, intramuscular injection, subcutaneous injection or topical (sublingual, rectal, intranasal, pulmonary, rectal mucosal and vaginal) and oral (sublingual, buccal).

In certain embodiments, the composition is formulated for pulmonary administration, such as intranasal administration or oral inhalation. Administration of the formulation may be accomplished by any acceptable method that allows the complex to reach its target. Depending on the disease being treated, administration may be topical (i.e., administration to a particular area, physiological system, tissue, organ or cell type) or systemic. Compositions and methods of in vivo delivery are also discussed in WO 2017/143042.

The methods may further comprise administering an effective amount of the antibody-nucleic acid complex composition to the embryo or fetus or pregnant mother thereof in vivo. In certain methods, the composition is delivered intrauterine by injection and/or infusion of the composition into a vein or artery, such as the yolk vein or umbilical vein, or into the amniotic sac of an embryo or fetus. See, e.g., RICCIARDI et al, nat Commun.2018, 6, 26, 9 (1): 2481.doi:10.1038/s41467-018-04894-2, and WO 2018/187493.

3. Application of

Nucleic acid cargo, such as mRNA, functional nucleic acid, DNA expression constructs, vectors, etc., encoding the polypeptide of interest or functional nucleic acid can be delivered into cells using 4H2 antibodies to express or inhibit the polypeptide in the cells. These compositions and methods may be used in a variety of different applications. Non-limiting examples include CRISPR and gRNA expression vectors +/-editing of DNA, delivery of large DNA (plasmids and expression vectors), gene replacement and gene therapy, delivery of DNA and/or RNA, e.g., for in vivo or ex vivo generation of CAR-T cells and simplified production of in vivo or ex vivo CAR-T cells, delivery of siRNA, delivery of mRNA, and the like. Exemplary applications related to gene therapy/gene editing and immunomodulation, in particular chimeric antigen receptor T cell production, are discussed below.

A. Gene therapy and gene editing

In certain embodiments, the compositions are useful for gene editing. For example, these methods are particularly useful for treating genetic defects, disorders, and diseases caused by single gene mutations, e.g., correcting genetic defects, disorders, and diseases caused by point mutations. If the target gene contains mutations that result in a genetic disorder, these methods can be used for mutation repair to restore the DNA sequence of the target gene to normal. The target sequence may be in the coding DNA sequence of the gene or in an intron. The target sequence may also be within a DNA sequence that regulates expression of the target gene, including a promoter or enhancer sequence.

In the methods described herein, cells contacted with the complex can be administered to a subject. The subject may have hemophilia, muscular dystrophy, globulinemia, cystic fibrosis, xeroderma pigmentosum or lysosomal storage disorder, or genetic or acquired diseases of the retina, eye, brain or spine, or coronary or other vascular diseases. In these embodiments, the genetic modification, gene replacement, gene addition, or combination thereof may be effective to reduce one or more symptoms of a disease or disorder in the subject.

In certain embodiments, the disclosed compositions are useful in retinal gene therapy. Hereditary retinal diseases (IRDs) are usually caused by single gene mutations, including, but not limited to, leber congenital vision-free disease (LCA), choroidemia (CHM), stargardt disease (Stargardt), retinal pigment degeneration (such as mutations in RHO, USH2A and RPGR), and X-linked retinal cleavage (XLRS). Different routes of administration may be used, including intravitreal, subretinal and suprachoroidal, and provide different biodistribution. See also Gupta et al, "GENE THERAPY for INHERITED RETINAL DISEASE," Review of Ophthalmology,2022, 5 month 10 day.

In certain embodiments, the disclosed compositions and methods are useful for inducing or enhancing repair of damaged endothelial cells, for example, upon revascularization. Thus, the disclosed compositions and methods are useful as an adjunct to cardiovascular surgery and other interventions. For example, revascularization is a therapeutic means by which occluded arterial or venous blood flow can be restored. The disclosed compositions and methods may be used in conjunction with such interventions to reduce the expression or biological activity of pro-inflammatory cytokines (e.g., IL-6, IL-8, and TNF-a), increase the growth and proliferation of endothelial cells, and/or reduce neointimal hyperplasia (e.g., smooth muscle cell growth, proliferation, migration, etc.).

In some embodiments, the disclosed compositions and methods comprise local delivery to a site or adjacent site in need of treatment. Such localized areas include, but are not limited to, the brain, ears, and skin, and such delivery may be useful in the treatment of diseases associated therewith.

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

1. Gene editing nucleases

Nucleic acid cargo comprises a nucleic acid encoding one or more elements that induce single or double strand breaks in the target cell genome, optionally, but preferably, cargo 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 a target gene.

(1) Broken chain inducing element

CRISPR/Cas

In certain embodiments, the nucleic acid cargo comprises one or more elements of a CRISPR/Cas-mediated genome editing composition, a nucleic acid encoding one or more elements of a CRISPR/Cas-mediated genome editing composition, or a combination thereof. As used herein, a CRISPR/Cas-mediated genome editing composition refers to the CRISPR system elements required for CRISPR/Cas-mediated genome editing in a mammalian subject. As discussed in detail below, CRISPR/Cas-mediated genome editing compositions generally include one or more nucleic acids encoding crRNA, tracrRNA (or a chimera thereof, also referred to as guide RNA or single guide RNA) and a Cas enzyme (such as Cas 9). The CRISPR/Cas-mediated genome editing composition can optionally include a donor polynucleotide that can be recombined into the genome of the target cell at or adjacent to the target site (e.g., single-or double-strand break site induced by Cas 9).

CRISPR/Cas systems have been engineered for gene editing (silencing, enhancing or altering specific genes) for eukaryotic organisms (see, e.g., cong, science,15:339 (6121): 819-823 (2013) and Jinek, etc., science,337 (6096): 816-21 (2012)). By transfecting cells with the desired elements (including Cas genes and specifically designed CRISPRs), the genome of the organism can be cleaved and modified at any desired location. WO 2013/176572 and WO 2014/018423, the entire contents of which are hereby incorporated by reference, describe in detail methods of preparing compositions for genome editing using a CRISPR/Cas system.

The delivery methods disclosed herein are applicable to a variety of variants of CRISPR/Cas systems.

In general, "CRISPR system" refers to a generic term for transcripts and other elements involved in expressing or directing CRISPR-associated ("Cas") gene activity, including sequences encoding Cas genes, tracr (transactivation CRISPR) sequences (e.g., tracrRNA or active moiety tracrRNA), tracr-mate sequences (including "direct repeat" and direct repeat of tracrRNA-treated portions in endogenous CRISPR systems), guide sequences (also referred to as "spacers" in endogenous CRISPR systems), or other sequences and transcripts derived from CRISPR loci. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer-direct repeat) may also be referred to as pre-crRNA (pre CRISPR RNA) prior to processing or crRNA after nuclease processing.

In certain embodiments, the tracrRNA and crRNA are joined and form a chimeric crRNA-tracrRNA hybrid, wherein the mature crRNA is fused to a portion of the tracrRNA by a synthetic stem loop to mimic the natural crRNA: tracrRNA duplex, as described in Cong, science,15:339 (6121): 819-823 (2013) and Jinek, etc., science,337 (6096): 816-21 (2012). The single fused crRNA-tracrRNA construct is also referred to herein as a guide RNA or gRNA (or single guide RNA (sgRNA)). In sgrnas, the crRNA portion can be defined as a "target sequence", while the tracrRNA is generally referred to as a "scaffold".

In certain embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In certain embodiments, one or more elements of the CRISPR system are derived from a particular organism, such as streptococcus pyogenes, that includes an endogenous CRISPR system.

In general, CRISPR systems are characterized by having elements that promote the formation of CRISPR complexes (also referred to as protospacers in endogenous CRISPR systems) at target sequence sites. In the case of CRISPR complex formation, "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, hybridization between the target sequence and the guide sequence facilitating CRISPR complex formation. The target sequence may be any polynucleotide, such as a DNA or RNA polynucleotide. In certain embodiments, the target sequence is located in the nucleus or cytoplasm.

In target nucleic acids, each protospacer is associated with a Protospacer Adjacent Motif (PAM) whose recognition is CRISPR system specific. In the streptococcus pyogenes CRISPR/Cas system, PAM is the nucleotide sequence NGG. In the streptococcus thermophilus CRISPR/Cas system, PAM is the nucleotide sequence NNAGAAW. the tracrRNA duplex directs Cas to a DNA target consisting of a protospacer and the necessary PAM through heteroduplex formed between the spacer of the crRNA and the protospacer DNA.

Typically, in endogenous CRISPR systems, the formation of a CRISPR complex (including a guide sequence that hybridizes to a target sequence and that is complexed with one or more Cas proteins) results in cleavage of one or both strands within or near the target sequence (e.g., within 1, 2,3, 4,5,6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). All or part of the tracr sequence may also form part of a CRISPR complex, for example by hybridization with all or part of the tracr mate sequence operably linked to the guide sequence.

Once the desired DNA target sequence is determined, there are many sources that can help researchers determine the appropriate target site. For example, a list of many public resources, including about 190,000 potential sgrnas generated by bioinformatics, can be used to target more than 40% of human exons, which can help researchers select target sites and design related sgrnas to affect gaps or double strand breaks at the sites. See also CRISPR. U-pseudo. Fr/, a tool that aims to help scientists find CRISPR targets in a variety of species and generate appropriate crRNA sequences.

In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the CRISPR system elements directs the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence may each be operably linked to different regulatory elements on different vectors. Or two or more elements expressed by the same or different regulatory elements may be combined in a single vector, any element of the CRISPR system not comprised in the first vector being provided by one or more additional vectors. The CRISPR system elements combined in a single vector may be arranged in any suitable orientation, for example one element located 5 '("upstream") or 3' ("downstream") of the second element. The coding sequences of one element may be located on the same or opposite strands of the coding sequence of a second element and in the same or opposite direction. In some embodiments, a single promoter drives expression of tracr encoding a CRISPR enzyme and one or more guide sequences, tracr mate sequences (optionally operably linked to guide sequences), and embedded into 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, the guide sequence, the tracr mate sequence, and the tracr sequence are operably linked to and expressed from the same promoter.

In some embodiments, the vector includes one or more insertion sites, such as restriction enzyme recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about or more than about 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 one or more vectors. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that upon insertion of the guide sequence into the insertion site, the guide sequence, upon expression, directs sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell. In certain embodiments, the vector comprises two or more insertion sites, each located between two tracer sequences, so that a guide sequence is inserted at each site. In such an arrangement, the two or more priming sequences can include two or more copies of a single priming sequence, two or more different priming sequences, or a combination thereof. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within a cell. For example, a single vector may include about or more than about 1,2,3, 4, 5,6, 7, 8, 9, 10, 15, 20 guide sequences. In some embodiments, about or more than about 1,2,3, 4, 5,6, 7, 8, 9, 10 such vectors containing a guide sequence may be provided and may be selectively delivered to a cell.

In some embodiments, the vector comprises a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme (e.g., cas protein). Non-limiting examples of Cas proteins include Casl, caslB, cas, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csnl and Csxl2)、CaslO、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, homologs thereof or modifications thereof in some embodiments, unmodified CRISPR enzymes have DNA cleavage activity, such as cas9. In some embodiments, CRISPR enzymes direct cleavage of one or both strands at a position within a target sequence, such as within the target sequence and/or within a complementary sequence to the target sequence.

In some embodiments, the vector-encoded CRISPR enzyme is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide comprising a target sequence. For example, substitution of aspartic acid to alanine in the RuvC I catalytic domain of streptococcus pyogenes Cas9 (D10A) converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves single strand). Other examples of mutations that make Cas9 a nickase 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 RuvC III) can be mutated to produce a mutated Cas9 that lacks substantially all DNA cleavage activity. In some embodiments, the D10A mutation binds to one or more of the H840A, N854A or N863A mutations, resulting in a Cas9 enzyme that lacks substantially all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to lack substantially 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 less relative to its non-mutant form.

In certain embodiments, the enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in a particular cell (e.g., eukaryotic cell). Eukaryotic cells may be cells of or derived from a particular organism, such as a mammal, including but not limited to, a human, mouse, rat, rabbit, dog, or non-human primate. 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 (e.g., about or more than about 1,2, 3,4,5, 10, 15, 20, 25, 50 or more codons) in the native sequence with a more or most frequently used codon in the host cell gene while maintaining the native amino acid sequence. Different species exhibit particular preferences for certain codons for a particular amino acid. Codon preference (the difference in codon usage between organisms) is generally related to the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on factors such as the nature of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule.

The dominance of the selected tRNA in the cell generally reflects the codons most commonly used in peptide synthesis. Thus, based on codon optimization, genes can be tailored for optimal gene expression in a particular organism. Codon usage tables can be found on websites such as "codon usage database" (Codon Usage Database), and these tables can be adjusted in a number of ways. See Nakamura, y., et al, nucleic acids res.,28:292 (2000). The specific sequences may also be codon optimized for expression in a specific host cell using a computer algorithm, such as Gene force (Aptagen; jacobus, pa.). In some embodiments, 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 codons most commonly used for a particular amino acid.

In certain embodiments, the vector-encoded CRISPR enzyme comprises one or more Nuclear Localization Sequences (NLS). When more than one NLS is present, each NLS may be selected independently of the other NLS, such that a single NLS may be present in more than one copy and/or combined with one or more other NLSs present in one or more copies. In certain embodiments, an NLS is considered near the N-or C-terminus when the nearest amino acid of the NLS is within about 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids from the N-or C-terminus along the polypeptide chain.

Generally, the one or more NLSs are of sufficient intensity to drive the CRISPR enzyme to accumulate in the nucleus of a eukaryotic cell in a detectable amount. In general, the intensity of the nuclear localization activity may be derived from the number of NLSs in the CRISPR enzyme, the particular NLS used, or a combination of these factors.

Accumulation within the nucleus may be detected using any suitable technique. For example, a detectable label can be fused to a CRISPR enzyme and the intracellular location can be visualized, e.g., in combination with a method of detecting nuclear location (e.g., nuclear-specific stain such as DAPI). The nuclei may also be isolated from the cells and the nuclear content analyzed by any suitable method for detecting proteins, such as immunohistochemistry, western blot (Western blot) or enzymatic activity assays. Accumulation in the nucleus can also be determined indirectly, e.g., by detecting an effect of CRISPR complex formation (e.g., detecting DNA cleavage or mutation on a target sequence, or detecting a change in gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control group not exposed to CRISPR enzyme or complex, or as compared to a control group exposed to CRISPR enzyme lacking one or more NLSs.

In certain embodiments, one or more elements of the CRISPR system are under the control of an inducible promoter, including an inducible Cas, such as Cas9.

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

While the specific details of the different engineered CRISPR systems may vary, the overall approach is similar. Researchers who have intentionally targeted DNA sequences using CRISPR techniques (which can be recognized using one of a variety of in-line tools) can insert short DNA fragments containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid comprises the target sequence (about 20 nucleotides), one form of the tracrRNA sequence (scaffold) and a suitable promoter and necessary elements for proper processing in eukaryotic cells. Such vectors are commercially available (see, e.g., addgene). Many systems rely on custom complementary oligonucleotides that are annealed to form double stranded DNA and then cloned into an sgRNA expression plasmid. The sgrnas are co-expressed in transfected cells with appropriate Cas enzymes derived from the same or different plasmids, resulting in single-or double-strand breaks (depending on Cas enzyme activity) at the desired target site.

(2) Zinc finger nucleases

In certain embodiments, the element that induces single-or double-strand breaks in the genome of the target cell is one or more nucleic acid constructs encoding Zinc Finger Nucleases (ZFNs). Thus, the nucleic acid cargo may encode a ZFN.

ZFNs are typically fusion proteins that include a DNA-binding domain derived from a zinc finger protein linked to a cleavage domain. The most common cleavage domain is the type IIS enzyme Fokl. Fok1 catalyzes double-strand cleavage of DNA, 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. For example, see U.S. Pat. Nos. 5,356,802, 5,436,150 and 5,487,994, and Li et al Proc, natl Acad Sci USA 89 (1992): 4275-4279, li et al Proc Natl Acad Sci USA 90:2764-2768 (1993), kim et al Proc Natl Acad Sci USA 91:883-887 (1994 a), kim et al J.biol. Chem.269:31,978-31,982 (1994 b), one or more of these enzymes (or enzyme functional fragments thereof) may be used as a source of cleavage domains. The DNA binding domain can in principle be designed to target any genomic location of interest, which can be a tandem array of Cys ₂His₂ zinc fingers, each of which typically recognizes three to four nucleotides in the target DNA sequence. The Cys ₂His₂ domain has the 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 ligating multiple zinc fingers together (variable number: three to six zinc fingers are used per monomer in published studies), ZFN pairs can be designed to bind to 18-36 nucleotide long genomic sequences.

Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. For example, rational design includes the use of 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 particular trimer or tetramer sequence. See, for example, U.S. Pat. Nos. 6,140,081, 6,453,242, 6,534,261, 6,610,512, 6,746,838, 6,866,997, 7,067,617, U.S. published application Nos. 2002/0165356, 2004/0197892, 2007/0154989, 2007/0213269, and International patent application publications WO 98/53059 and WO 2003/016496.

(3) Transcriptional activator-like effector protein nucleases

In certain embodiments, the element that induces a single-or double-strand break in the genome of the target cell is a nucleic acid construct or constructs encoding a transcription activator-like effector protein nuclease (TALEN). Thus, the nucleic acid cargo may encode a TALEN.

The overall structure of TALENs is similar to ZFNs, the main difference being that the DNA binding domain is derived from TAL effector proteins, i.e. transcription factors of plant pathogens. The DNA binding domain of TALENs is a tandem array of amino acid repeats, each of which is about 34 residues in length. These repeat sequences are very similar to each other and typically they differ primarily in two positions (amino acids 12 and 13, called repeat variable diradicals, or RVDs). Each RVD is assigned to preferentially bind one of four possible nucleotides, meaning that each TALEN repeat binds a single base pair, but NN RVDs are known to bind adenine in addition to guanine. The mechanisms of TAL effector DNA binding are less well understood than zinc finger proteins, but they appear to be simpler codes that may be very beneficial to the design of engineered nucleases. TALENs are also able to cleave in dimeric form, the target sequence is relatively long (the shortest target sequence reported so far can bind 13 nucleotides per monomer), and it appears that the requirement for the length of the gap between binding sites is less stringent than ZFNs. Monomeric and dimeric TALENs may include more than 10, more than 14, more than 20, or more than 24 repeats.

Cermak, et al, nucleic acids Res.1-11 (2011). U.S. published application 2011/0145940 discloses TAL effectors and methods of modifying DNA therewith. Miller et al Nature Biotechnol 29:143 (2011), report the preparation of TALENs for site-specific nuclease structures by ligation of TAL truncated variants to the catalytic domain of Fokl nuclease. The results indicate that TALENs are capable of inducing genetic modifications in immortalized human cells. General design principles for TALE binding domains can be found in e.g. WO 2011/072246.

Donor polynucleotide

The nuclease activity of the genome editing systems described herein can cleave target DNA, creating single-or double-strand breaks in the target DNA. The cell can repair double strand breaks in at least two ways, non-homologous end joining and homology directed repair. In non-homologous end joining (NHEJ), double strand breaks are repaired by direct ligation of the broken ends to each other. Thus, while some nucleic acid components may be lost, resulting in a deletion, no new nucleic acid components will be inserted at that site. In Homology Directed Repair (HDR), a donor polynucleotide having homology to a cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in transfer of genetic information from the donor polynucleotide to the target DNA. Thus, new nucleic acid components can be inserted/copied into the site.

Thus, in certain embodiments, the nucleic acid cargo is or includes a donor polynucleotide. Modification of target DNA by NHEJ and/or homology-directed repair can be used to induce gene correction, gene replacement, gene labeling, transgene insertion, nucleotide deletion, gene disruption, gene mutation, and the like.

Thus, cleavage of DNA by the genome editing composition can be used to delete a nucleic acid component from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence without the exogenously supplied donor polynucleotide. Or if the genome editing composition comprises a donor polynucleotide sequence that includes at least a fragment homologous to the target DNA sequence, these methods can be used to add a nucleic acid component to the target DNA sequence, i.e., insert or replace a nucleic acid component (e.g., a nucleic acid that "knocks in" to encode a protein, siRNA, miRNA, etc.), add a tag (e.g., 6xHis, fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), add a regulatory sequence (e.g., promoter, polyadenylation signal, internal Ribosome Entry Sequence (IRES), 2A peptide, start codon, stop codon, splicing signal, localization signal, etc.), modify a nucleic acid sequence (e.g., introduce mutations), etc. Thus, 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 labeling, and the like, as used in gene therapy.

In applications where it is desired to insert a polynucleotide sequence into a target DNA sequence, it is also desirable to provide the cell with a polynucleotide comprising a donor sequence to be inserted. The term "donor sequence" or "donor polynucleotide" or "donor oligonucleotide" refers to a nucleic acid sequence inserted into a cleavage site. The donor polynucleotide typically has sufficient homology to the genomic sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95% or 100% homology to the nucleotide sequence flanking the cleavage site, e.g., within about 50 bases or less, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the cleavage site, to support homology-directed repair between the cleavage site and the genomic sequence to which it has homology. The donor sequence is typically not identical to the genomic sequence it replaces. Conversely, a donor sequence may include at least one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the genomic sequence so long as sufficient homology exists to support homology directed repair. In certain embodiments, the donor sequence comprises a non-homologous sequence flanked by two homologous regions, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence into the target region.

B. Immunomodulation

CAR T cells

The disclosed compositions and methods are particularly useful for preparing lymphocytes that express an immune receptor, particularly a Chimeric Immune Receptor (CIR), such as a Chimeric Antigen Receptor (CAR). 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 engraft a selected specificity onto a cell. As discussed in detail below, cells modified according to the methods discussed can be used in various immunotherapies for the treatment of cancer, infection, inflammation, and autoimmune diseases.

In particularly preferred embodiments, mRNA or DNA encoding the chimeric antigen receptor cargo is delivered to immune cells, such as lymphocytes.

The cargo may be delivered to the immune cells in vivo, ex vivo, or in vitro. In preferred embodiments, the cargo is mRNA, which may reduce one or more of cost, ease of manufacture, and reduced side effects (e.g., cytokine storm, neurotoxicity, graft versus host disease, etc.). In particular embodiments, immune cells (e.g., T cells) are collected from a subject in need of 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 certain embodiments, the entire process takes 1 week or less, e.g., 1, 2, 3, 4, 5, 6, or 7 days, from the initial collection of cells to the return of cells to the subject. In particular embodiments, the process from initial collection of cells to return to the subject is completed within 1 or 2 days, or within 1 day, e.g., 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.

The design and development strategies for chimeric antigen receptors are reviewed in Dotti et al, immunol rev.2014january;257 (1): doi:10.1111/imr.12131 (page 35), the entire contents of which are specifically incorporated herein by reference, and 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 (4), eaton, et al, GENE THERAPY,9:527-35 (2002), barrett, et al, annu Rev med, 65:333-347 (2014), CARTELLIERI, et al, journal of Biomedicine and Biotechnology, volume 2010, page 956304,13, doi:10.1155/2010/956304, and U.S. published applications 201837120, 2012012015/007035, 2012015/2015, 201020704/1414.

The antigen binding properties of the CAR, combined with the lytic and self-renewing capabilities of the monoclonal antibodies, have 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 Major Histocompatibility Complex (MHC) to recognize and kill cancer cells. Thus, target cell recognition is not affected by some mechanism by which tumors evade MHC restricted T cell recognition, such as Human Leukocyte Antigen (HLA) class I molecules down-regulation and antigen processing defects.

Chimeric immune receptors were originally developed in the 80 th generation of the 20 th century, and initially included the variable region of monoclonal antibodies (antigen binding region) and the constant regions of the alpha and beta chains of the T Cell Receptor (TCR) (Kuwana et al Biochem Biophys Res Commun,149:960-968 (1987)). In 1993, this design was modified to incorporate the extracellular domain of a single chain variable fragment (scFv) derived from the heavy and light chain antigen binding regions of a monoclonal antibody, the transmembrane domain and the intracellular domain with the signaling domain derived from CD 3-zeta. Later CARs generally followed a similar structural design with co-stimulatory signaling intracellular domains. Thus, CAR constructs for use in the methods herein can include an antigen binding domain or extracellular domain, a hinge domain, a transmembrane domain, an intracellular domain, and combinations thereof.

In certain embodiments, the extracellular domain is an scFv. The affinity of scFv predicts the function of CAR (Hudecek et al, CLIN CANCER Res.,19 (12): 3153-64 (2013); chuelewski et al, J immunol.,173:7647-7653 (2004)). Antigen binding and subsequent activation can also be modified by adding flexible linker arms to the CAR, so that two different scFv can be expressed, recognizing two different antigens (Grada et al, mol Ther Nucleic Acids,2:e105 (2013)) (known as 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 IL13 ra 2 (Kahlon et al, CANCER RES,64:9160-9166 (2004)), brown et al, CLIN CANCER RES,18 (8): 2199-209 (2012), kong et al, CLIN CANCER RES,18:5949-5960 (2012), NKG2D ligands and CD70 receptors, peptide ligands (e.g. T1E peptide ligands), and so-called "universal extracellular domains" (e.g. streptavidin (avidin) extracellular domains designed to recognize targets contacted with biotin-labeled monoclonal antibodies, or FITC-specific single-chain antibody fragments (scFv) designed to recognize targets contacted with FITC-labeled monoclonal antibodies) (Zhang, et al, blood,106:1544-1551 (2005), barber et al, exp heat, 36:1318-1328 (2008), shaffer et al, 117:4-14 (2011), davies et al, mol 576, 18:4345, 2012), resp 2012, 2012-18:2012, 2012, 18:35-35, 2012, and so-18:2012 (2012).

In certain embodiments, the CAR comprises a hinge region. Although the extracellular domain is important for CAR specificity, the sequence (hinge region) connecting the extracellular domain and the transmembrane domain can also affect CAR-T cell function by creating differences in CAR length and flexibility. For example, the hinge may comprise a CH2CH3 hinge derived from an immunoglobulin (e.g., igG 1) or a fragment thereof. For example, hudecek et al (Hudecek, et al, CLIN CANCER RES,19 (12): 3153-64 (2013)) compared the effects of CH2-CH3 hinge [229 Amino Acids (AA) ], CH3 hinge (119 AA), and short hinge (12 AA) on T cell effector functions expressing third generation ROR 1-specific CARs, and found that T cells expressing "short hinge" CARs had stronger antitumor activity, while other researchers found that CH2-CH3 hinge attenuated epitope recognition of first generation CD 30-specific CARs (Hombach, et al, gene ter, 7:1067-1075 (2000)).

A transmembrane domain is typically present between the hinge domain (extracellular domain if there is no hinge domain) and the signal internal domain, most typically the transmembrane domain is derived from a CD3- ζ, CD4, CD8 or CD28 molecule. Like the hinge domain, the transmembrane domain also affects the effector function of CAR-T cells.

Following antigen recognition, the CAR intracellular domain transmits activation and costimulatory stimulation signals to T cells. Activation of T cells relies on phosphorylation of the cytoplasmic CD 3-zeta domain of the TCR complex by the Immunoreceptor Tyrosine Activation Motif (ITAM) in the cytoplasmic domain (Irving et al, cell,64:891-901 (1991)). Although most CAR intracellular domains include an activation domain derived from CD3- ζ, there are also some Fc receptors that include ITAM domains, such as the IgE- γ domain (Haynes et al, J immunol.,166:182-187 (2001)).

The targeting specificity of the CAR-expressing cells is determined by the antigen recognized by the antibody/extracellular domain. The disclosed compositions and methods can be used to make constructs and cells expressing constructs that target any antigen. In the context of immunotherapy, particularly cancer immunotherapy, many antigens and suitable extracellular domains suitable for targeting antigens are well known. Unlike native TCRs, most scFv-based CARs recognize cell surface expressed target antigens, rather than internal antigens processed and presented by the MHC of the cell, however, CARs have the advantage over classical TCRs in that they can recognize structures other than protein epitopes, including carbohydrates and glycolipids Dotti, et al, immunol rev.2014, month 1; 257 (1): doi:10.1111/imr.12131 (page 35), thereby increasing the pool of potential target antigens. Preferred targets include antigens expressed only on cancer cells or their surrounding matrix (Cheever et al, CLIN CANCER res.,15:5323-5337 (2009)), such as splice variants of epidermal growth factor receptor specific for glioma cells (EGFRvIII) (Sampson et al, semin immunol.,20 (5): 267-75 (2008)). However, human antigens also meet this requirement, and most target antigens are expressed at low levels on normal cells (e.g., GD2, CAIX, HER 2), and/or in a lineage restricted manner (e.g., CD19, CD 20).

Preferred targets and target-targeted CARs are known in the art (see, e.g., dotti et al, immunol Rev.2014, month 1; 257 (1): doi:10.1111/imr.12131 (page 35)). For example, CAR targets for hematological malignancies include, but are not limited to, CD19 (e.g., B cells) (Savoldo et al, J Clin invest, 121:1822-1826 (2011), cooper, et al, 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, 3:95raf73 (2011), brentjens, et al, SCI TRANSL Med, 5:177raf38 (2013), grupp, et al, N Engl J (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)); CD (e.g., marrow lines) (J.28, 678, et al). 161:2791-2797 (1998)), CD70 (e.g., B cells/T cells) (Shaffer, et al, blood,117:4304-4314 (2011)), CD123 (e.g., blood, marrow cells) (TETTAMANTI, et al, br J handto, 161:389-401 (2013)), kappa (e.g., B-cells) (Vera, et al, blood,108:3890-3897 (2006)), lewis Y (e.g., blood, marrow cells) (Peinert, et al, gene ter., 17:678-686 (2010), ritchie, et al, mol thor (2013)), NKG2D li gands (e.g., body, marrow cells) (Barber, et al, exp heat, 36:1318-8 (2008), lehner, et al, boss 7, 35, 35:3890-3897 (2006)), lewis Y (e.g., 35, set forth, etc.), and so forth (35:2012, set forth, 35.g., 35, 2012, set forth (2012, etc.), and so forth (35, set forth) of genes, 35:2012, set forth (2012, set forth in the order of claims), and so forth.

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) (polymers, 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 neurooomol (2009), morgan, et al, hum Gene 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, 51:417-423 (2002)); EGP40 (e.g., da, et al), CANCER GENE Thpatient colon Cancer (7:284-291) (e.g., ep), glioma 2, such as Ep) lung cancer) (Chow, et al, mol ter, 21:629-637 (2013)); erbB2 (HER 2) (e.g., breast, lung, prostate, cancer, Glioma) (Zhao, et al, J immunol, 183:5563-5574 (2009), morgan, et al, mol ter, 18:843-851 (2010), pinthus, et al, 114:1774-1781 (2004), teng, et al, hum Gene ter, 15:699-708 (2004), stancovski, et al, J immunol, 151:6577-6582 (1993), ahmed, et al, mol ter, 17:1779-1787 (2009), ahmed, et al, CLIN CANCER res, 16:474-485 (2010), moritz, et al, proc NATL ACAD SCI u.s.a.,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 ter., 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, E.G., B., Melanoma) (Schuberth, et al, gene Ther.,20:386-395 (2013)), FR-alpha (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-related fibroblasts) (Kakarla, et al, mol Ther. (2013)), FAR (e.g., rhabdomyosarcoma) (Gattenlohner, et al, cancer Res.,66:24-28 (2006)), neuroblastoma, GD2 (e.g., neuroblastoma, etc.), Sarcomas, melanomas) (Pule, et al, nat Med.,14:1264-1270 (2008), louis, et al, blood,118:6050-6056 (2011), rossig, et al, int J cancer, 94:228-236 (2001)); GD3 (e.g., melanoma), Lung Cancer) (Yun, et al, neoplasia, 2:449-459 (2000)); HMW-MAA (e.g., melanoma) (Burns, et al, cancer Res.,70:3027-3033 (2010)); IL11R alpha (e.g., osteosarcoma) (Huang, et al, cancer Res.,72:271-281 (2012)); IL13R alpha 2 (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., 18:599-5960 (2012), yaghoubi, et al, NAT CLIN PRACT Oncol.,6:53-58 (2009)), lewis Y (e.g., breast/ovary/pancreas) (Peinert, et al, gene Theer, 17:678-686 (2010), westwood, et al, prowo, et al, 24:1904, et al, kong, et al, CLIN CANCER Res. (1995, etc.), mead., 1905:1905, 1995, etc.), said 35:1905 (2012) Breast cancer, pancreatic cancer) (Lanitis, et al, mol ter., 20:633-643 (2012), moon, et al, CLIN CANCER res, 17:4719-4730 (2011)); mue (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 iigands (e.g., ovaries, sarcomas) (Barber, et al, exp heat, 36:1318-1328 (2008), lehner, et al, PLoS One,7:e31210 (2012), song, et al, gene ter, 24:295-305 (2013), speed, 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., colon cancer) (Hombach, et al, gastroenterology,113:1163-1170 (1997), mcGuinness, et al, hum Gene ter, 10:165-173 (1999)); VEGFR-2 (e.g., tumor vasculature) (J. In Invert, 120:3953-3968 (2010), 82, et al, proc 34 U.S. 2002-709).

Metabolic stability

In certain embodiments, the metabolic stability of a cell (e.g., CAR cell) can be increased by providing the cell (e.g., CAR cell) with the ability to produce in vivo limiting growth factors. In certain embodiments, a nucleic acid cargo encoding an anti-apoptotic factor (e.g., BCL-XL) is transiently delivered into cells. B cell lymphoma-extra large (Bcl-XL, or BCL 2-like subtype 1) is a transmembrane protein in mitochondria. It is a member of the Bcl-2 protein family, acting as a pro-survivin in the intrinsic pathway of apoptosis, preventing release of mitochondrial contents (e.g. cytochrome c), leading to caspase (caspase) activation. The amino acid and nucleic acid sequences encoding BCL-XL are known in the art and include, for example, uniProtKB-Q07817 (B2CL 1. Sup. HUMAN), the isomer Bcl-X (L) (identifier: Q07817-1) (amino acid sequence), the ENA|U72398|U72398.1 HUMAN Bcl-X beta (BCL-X) gene, the complete coding sequence (HUMAN Bcl-X beta (BCL-X) gene, complex cds) (genomic nucleic acid sequence), ENA|Z23115|Z23115.1 HUMAN BCL-XL mRNA (H.sapiens BCL-XL mRNA) (mRNA/cDNA nucleic acid sequence).

In certain embodiments, the nuclear cargo encodes a proliferation-inducing factor, such as IL-2. Amino acid and nucleic acid sequences encoding IL-2 are known in the art and include, for example, uniProtKB-P60568 (IL 2_HUMAN) (amino acid sequence), the ENA|X00695|X00695.1 HUMAN interleukin-2 (IL-2) gene and the 5' -flanking region (gene nucleic acid sequence), and ENA|V00564|V00564.1 HUMAN mRNA (mRNA/cDNA nucleic acid sequence) encoding interleukin-2 (IL-2).

However, the production of secreted IL-2 may produce unwanted side effects, i.e. stimulation of both lymphoma and Treg cell proliferation and impairment of memory T cell formation (Zhang et al, nature Medicine,11:1238-1243 (2005)). Furthermore, IL-2 use in patients receiving tumor-infiltrating lymphocyte (TIL) treatment increases toxicity (HEEMSKERK et al, human GENE THERAPY,19:496-510 (2008)). To avoid this possibility, in addition to IL-2, the nucleic acid cargo may encode a chimeric yc cytokine receptor (CγCR), e.g., a chimeric yc cytokine receptor consisting of interleukin-7 (IL-7) linked to IL-7Rα/CD127, which is capable of conferring an intracellular STAT5 cytokine signal independent of the extracellular cytokine (Hunter et al Molecular Immunology,56:1-11 (2013)). This design is modular, and the cytoplasmic chain of IL-2Rβ/CD122 can be exchanged with the cytoplasmic chain of IL-7Rα/CD127 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 should function similarly to IL-2mRNA without producing unwanted side effects.

In addition, or alternatively, other anti-apoptotic molecules and cytokines may be used to maintain cell viability in the natural state. Example factors include, but are not limited to:

Myeloid leukemia 1 (MCL-1) (e.g., uniProtKB-Q07820 (mcl1_human) (amino acid sequence), ena|af147742|af147742.1 HUMAN myeloid differentiation protein (MCL 1) (Homo sapiens myeloid cell differentiation protein (MCL 1) gene, promoter and complete coding sequence (genomic nucleic acid sequence), ena|af118124|af118124.1 HUMAN myeloid leukemia sequence 1 (MCL 1) (Homo sapiens myeloid cell leukemia sequence 1) mRNA, complete coding sequence (mRNA/cDNA nucleic acid sequence)), which is an anti-apoptotic factor;

IL-7 (e.g., uniProtKB-P13232 (IL 7_HUMAN) (amino acid sequence), the ENA|EF064721|EF064721.1 HUMAN interleukin 7 (Homo sapiens interleukin) (IL 7) gene, the complete coding sequence (genomic nucleic acid sequence), ENA|J04156|J04156.1 HUMAN interleukin 7 (Human interleukin 7) (IL-7) mRNA, the complete coding sequence (mRNA/cDNA nucleic acid sequence) which is important for T cell survival and development, and IL-15 (e.g., uniProtKB-P40933 (IL 15_HUMAN) (amino acid sequence), ENA|X91233|X91233.1 HUMAN interleukin 7 (IL-7) mRNA, the complete coding sequence (mRNA/cDNA nucleic acid sequence).

IL-15 (e.g., uniProtKB-P40933 (IL 15_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) (IL 15) mRNA, complete coding sequence (mRNA/cDNA nucleic acid sequence)) can promote T-cell and NK-cell survival (Opferman et al, nature,426:671-676 (2003); meazza et al, journal of Biomedicine & Biotechnology, 86920, doi:10.1155/2011/86920 (2011); michaud et al, journal of Immunotherapy,33:382-390 (2010)). These cytokine mRNAs can be used either alone or in combination with BCL-XL, IL-2 and/or CγCR mRNAs. Thus, in certain embodiments, mRNA encoding MCL-1, IL-7, IL-15, or a combination thereof is delivered to a cell.

Inhibitory CAR (iCAR)

In some embodiments, T cell therapies are delivered to CAR cells that have demonstrated long-term efficacy and cure potential for treating certain cancers, however, their use can cause damage to non-cancerous tissues similar to graft versus host disease that occurs following donor lymphocyte infusion. Any of the compositions and methods disclosed can be used in combination with nonspecific immunosuppression (e.g., high dose corticosteroid therapy that produces a cytostatic or cytotoxic effect on T cells to suppress immune responses), irreversible T cell elimination (e.g., so-called suicide genetic engineering strategies), or a combination thereof. However, in some preferred embodiments, off-target effects can be reduced by introducing a construct encoding an Inhibitory Chimeric Antigen Receptor (iCAR) in the CAR cell. By introducing antigen-specific icars (inducible chimeric antigen receptors) in T cells, T cells with dual specificity for tumor and non-target tissues can be restricted to targeting tumors, 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 powerful acute inhibitory signaling domain, which can limit T cell reactivity even if an activating receptor (e.g., a CAR) is involved at the same time. In a preferred embodiment, the iCAR comprises a single chain variable fragment (scFv) specific for an inhibitory antigen fused to a signal domain of an immunosuppressive receptor (e.g., CTLA-4, PD-1, LAG-3, 2B4 (CD 244), BTLA (CD 272), KIR, TIM-3, tgfβ receptor dominant negative analog, etc.) via a transmembrane region that specifically inhibits T cell function upon antigen recognition. Once CAR cells encounter cells that do not express inhibitory antigens (e.g., cancer cells), iCAR-transduced T cells can produce a CAR-induced response to the target antigen of the CAR. Fedorov et al, science Translational Medicine,5:215ra172 (2013)) discusses DNA iCAR using scFv specific for PSMA and inhibitory signaling domains of CTLA-4 or PD-1.

Design considerations include that PD-1 is a stronger inhibitor than CTLA-4, that CTLA-4 exhibits cytoplasmic localization unless the Y165G mutant is used, and that the level of expression of iCAR is very important.

Icars can be designed for cell type specific surface molecules. In certain embodiments, icars are designed to prevent T-cell, NK-cell or other immune cell reactivity to certain tissues or cell types.

Reducing endogenous inhibitory signals

In certain embodiments, the cells are contacted with a nucleic acid cargo, which can reprogram the cells to prevent expression of one or more antigens. For example, in certain embodiments, the nucleic acid cargo is or encodes an interfering RNA that can prevent expression of mRNA encoding an antigen such as CTLA-4 or PD-1. This method can be used to prepare universal donor cells. The RNA used to alter alloantigen expression may be used alone or in combination with RNA that causes dedifferentiation of the target cells.

While the compositions and methods provided above utilize inhibitory signal domains (e.g., CTLA-4 or PD-1 in artificial iCAR) to limit targeted/non-tumor cytotoxicity, additionally or alternatively, the CAR cells are rendered resistant to inhibition signals against the hostile tumor microenvironment by reducing expression of endogenous inhibition signals in the CAR cells, thereby increasing the overall tumor effector efficiency of the CAR cells.

CTLA-4 and PD-1 inhibit T cells at different stages of activation and function. CTLA-4 can modulate T cell responses to autoantigens because knockout mice spontaneously develop organ damage due to highly active tissue-infiltrating T cells without 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 reproduced 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 form massive inflammatory cell infiltration of normal organs, suggesting that their primary physiological function is to mediate negative feedback control of ongoing tissue inflammation in an inducible manner (Dong et al, immunity,20:327-336 (2004)). Indeed, according to the "adaptive resistance" hypothesis, most tumors up-regulate 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)). PD-L1 then transmits an inhibitory signal to the T cells, reducing their proliferation, cytokine and perforin production (Butte et al, immunity 27:111-122 (2007); chen et al, immunology 4:336-347 (2004); park et al, blood 116:1291-1298 (2010); wherry et al, nat Immunol 12:492-499 (2011); zou et al, immunology 8:467-477 (2008)). In addition, T cells send a reverse signal to cancer cells through B7-H1, inducing an anti-apoptotic effect, thus counteracting the Fas-L signal (Azuma et al, blood,111:3635-3643 (2008)). Azuma et al, blood,111:3635-3643 (2008).

Whereas up-regulation of B7-H1 by cancer cells and their expression are related to 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 that antagonize the PD-1 and CTLA-4 pathways show a significant effect in solid tumors, especially melanoma, and the combination of both shows a greater activity. anti-CTLA-4 antibodies, ipilimumab, improve overall survival in metastatic melanoma patients by mainly inhibiting Treg cells, increasing T cell infiltration into the tumor and increasing the ratio of CD8 ⁺ cells to regulatory T cells (Treg) in the tumor (CD 8 ⁺: treg) (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)). anti-PD-1 antibody nivolumab showed 30-40% overall response in metastatic melanoma (Robert et al, THE NEW ENGLAND Journal of Medicine,372:320-330 (2015); topalian et al, J Clin Oncol,32:1020-1030 (2014)), and similar findings were found in early clinical trials of other solid tumors, including metastatic renal carcinoma, non-small cell lung carcinoma, and recurrent Hodgkin's 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 the resistance of the mouse melanoma model to anti-CTLA-4 antibodies is due to the upregulation of PD-L181, the combination therapy of ipilimumab and nivolumab has shown further efficacy in both the mouse model and in 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 Ass℃iation for Cancer Research,16:6019-6028(2010))., in view of the importance of the checkpoint inhibition pathway, consider that PD-1/CTLA-4 inhibition will release the brake, whereas the chimeric antigen receptor will step on the accelerator. Importantly, the brake can be released instantaneously using transient delivery techniques so that these cells do not cause future autoimmune diseases.

(1).CRISPRi

To avoid permanent genome modification and inactivation of inhibitory signals such as PD-1 and CTLA-4, the dCAS CRISPRi system (Larson et al, nat Protoc,8:2180-2196 (2013)) may be utilized. Nucleic acids encoding enzyme-inactivated dCAS-KRAB inhibition domains, fusion proteins, and sgRNAs that inhibit signaling proteins (e.g., CTLA-4, PD-1, LAG-3, 2B4 (CD 244), BTLA (CD 272), KIR, TIM-3, TGF-beta receptor dominant-negative analogs, etc.) may be co-delivered to the CAR cells. sgrnas can be designed to target the proximal promoter region and coding region (non-template strand). An alternative approach is to use a single component Cpf1CRISPR system, which is a smaller RNA for electroporation and expression (Zetsche et al, cell, doi:10.1016/j.cell.2015.09.038 (2015)). Any of the above RNA components may also be encoded by a DNA expression construct, such as a vector, e.g., a plasmid. Thus, RNA, DNA, or a combination thereof can be used as a nucleic acid cargo.

While extensive inhibition of CTLA-4 by ipilimumab leads to autoimmune sequelae, these side effects are believed to be reduced by limiting the loss of CAR cells and the transience of mRNA delivery. Over time, the suppression function will resume in time.

(2) Inhibitory RNA

The nucleic acid cargo that can be delivered to the cell can be or encode a functional nucleic acid or polypeptide designed to target and reduce or inhibit the expression or translation of an inhibitory signal molecule mRNA, or reduce or inhibit the expression, reduce the activity, or increase the degradation of an inhibitory signal molecule protein. Suitable techniques include, but are not limited to, antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, and the like. In certain embodiments, the mRNA encodes an antagonist polypeptide that reduces inhibitory signaling.

In certain embodiments, a cargo of functional RNAs suitable for reducing or silencing expression of CTLA-4, PD-1, LAG-3, 2B4 (CD 244), BTLA (CD 272), KIR, TIM-3, TGF- β receptor dominant negative analogs, or the like, or a cargo encoding such functional RNAs, alone or in combination, may be delivered to a cell.

In certain embodiments, the cargo is RNA or DNA encoding a polypeptide that reduces bioavailability or acts as an antagonist or other negative modulator or inhibitor of CTLA-4, PD-1, LAG-3, 2B4 (CD 244), BTLA (CD 272), KIR, TIM-3, a dominant negative analog of the TGF-beta receptor, or another protein in the immunosuppressive pathway. Such a protein may be a paracrine, endocrine or autocrine protein. It can regulate cells in cells. It may be secreted and regulate the expressing cells and/or other (e.g., adjacent) cells. It may be a transmembrane protein that regulates expression of cells and/or other cells. The protein may be a fusion protein, for example an Ig fusion protein.

Pro-apoptotic factors

Compositions and methods for activating and reactivating apoptotic pathways are also provided. In certain embodiments, the nucleic acid is a factor or agent that activates, re-activates, or otherwise enhances or increases an intrinsic apoptotic pathway or encodes a factor or agent that activates, re-activates, or otherwise enhances or increases an intrinsic apoptotic pathway. Preferably, the agent activates, re-activates or otherwise enhances the intrinsic apoptotic pathway of the cancer (e.g., tumor) cell, more preferably, the agent is specific or targeted to the cancer cell.

In certain embodiments, the cells are more resistant or less sensitive to induced apoptosis than untreated cells after delivery of an anti-apoptotic or pro-proliferative factor (such as those discussed above or known in the art). For example, pro-apoptotic factors may induce or increase apoptosis in untreated cells relative to treated T cells, and are preferably selective for cancer cells. The therapy can be used for double-tube attack on cancer cells, wherein one is cell attack and the other is molecular attack.

By targeting BCL-2 family members, intrinsic apoptotic pathways may be activated, re-activated, or otherwise enhanced. BCL-2 family members can be divided into three subgroups based on function and BCL-2 homology (BH) domains, multi-domain anti-apoptotic proteins (e.g., BCL-2 or BCL-XL), multi-domain pro-apoptotic proteins (e.g., BAX and BAK), and pro-apoptotic proteins containing only BH3 domains (e.g., BIM). Members of the subgroup (BH 3-only) containing only the BH3 domain, such as BIM, are distributed throughout the cell as death sentries ready to transmit various physiological and pathological signals of cell damage to the core apoptotic mechanisms located at mitochondria (Danial et al, cell,116:205-219 (2004)).

In certain embodiments, the pro-apoptotic factor is a pro-apoptotic BH 3-mimetic. Various pro-apoptotic BH 3-mimics can mimic the natural pro-apoptotic activity of BIM and can manipulate multiple points of the apoptotic pathway. For example, BIM SAHB (stabilized alpha helix of the BCL-2 domain), ABT-737 and ABT-199 are pro-apoptotic BH3 mimics designed by structural studies of the interaction between the pro-apoptotic BH3-only helical domain and the hydrophobic groove formed by the confluence of BH1, BH2 and BH3 domains of anti-apoptotic proteins (Oltersdorf et al Nature,435:677-681 (2005)).

4. Target cells

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

The target cells can be selected by the researcher based on the desired treatment and therapy and the desired effect of the nucleic acid cargo. For example, the target cell may be a cancer cell when the nucleic acid cargo is intended to induce cell death, a stem cell when the nucleic acid cargo is intended to induce genomic changes, and an immune cell when the nucleic acid cargo encodes a chimeric antigen receptor.

4H2 infiltrated cells in a manner sensitive to dipyridamole, and this infiltration was enhanced by the addition of GUO, suggesting that nucleoside transporter dependent transport could be promoted by local nucleic acids.

In certain embodiments, the nucleoside transporter is expressed on the plasma membrane of the target cell. Nucleoside transporters are expressed relatively universally but in varying abundance in different tissues and cell types. For example, the expression of ENT2 in the brain, heart, placenta, thymus, pancreas, prostate and kidney has been demonstrated (Griffiths et al, biochem J1997.328 (Pt 3): pages 739-43, crawford et al, J Biol Chem 1998.273 (9): p.5288-93). ENT2 is one of the transport proteins with the highest mRNA expression in skeletal muscle compared to other transport proteins (Baldwin et al, pflugers Arch,2004.447 (5): p.735-43, govindarajan et al, am J Physiol Regul Integr Comp Physiol,2007.293 (5): pages R1809-22). Thus, in certain embodiments, the target cell is brain, heart, placenta, thymus, pancreas, prostate, kidney, or skeletal muscle.

Other non-limiting exemplary target cells are discussed below.

I. Progenitor and stem cells

The cells may be hematopoietic progenitor cells or hematopoietic stem cells. In certain embodiments, particularly those related to gene editing and gene therapy, the target cells are CD34 ⁺ hematopoietic stem cells. Hematopoietic Stem Cells (HSCs), such as CD34 ⁺ cells, are multipotent stem cells that can produce all blood cell types including erythrocytes.

The skilled artisan can isolate and enrich for stem cells. Such methods of isolating and enriching CD34 ⁺ and other cells are known in the art, for example, as disclosed in U.S. Pat. Nos. 4,965,204, 4,714,680, 5,061,620, 5,643,741, 5,677,136, 5,716,827, 5,750,397 and 5,759,793. As used herein in the context of being enriched for hematopoietic progenitor cells and stem cells, "enriched" means that the proportion of the desired element (e.g., hematopoietic progenitor cells and hematopoietic stem cells) is higher than in the natural source of the cell. 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.

In humans, CD34 ⁺ cells may be recovered from cord blood, bone marrow, or blood after cytokine mobilization of granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte colony-stimulating factor (GM-CSF), stem Cell Factor (SCF) by subcutaneous or intravenous injection into the donor in an amount sufficient to allow hematopoietic stem cells to enter the peripheral circulation from the bone marrow space. Initially, bone marrow cells may be obtained from any suitable bone marrow source, such as the tibia, femur, spine, and other bone cavities. To isolate bone marrow, the bone may be washed with a suitable solution, which will be a balanced salt solution, conveniently supplemented with fetal bovine serum or other naturally occurring factors, in combination with an acceptable low concentration buffer, typically about 5 to 25mM. Convenient buffers include Hepes buffer, phosphate buffer, lactate buffer, and the like.

Cells can be screened by positive and negative selection techniques. Cells can be selected using commercially available antibodies that bind to hematopoietic progenitor or hematopoietic stem cell surface antigens (e.g., CD 34), using methods well known to those skilled in the art. For example, antibodies can be conjugated to magnetic beads and the desired cell type recovered using an immunogenicity procedure. Other techniques include the use of Fluorescence Activated Cell Sorting (FACS). The CD34 antigen is expressed on progenitor cells in the hematopoietic system of non-leukemia individuals, on a cell population recognized by monoclonal antibody My-10 (i.e., expressing CD34 antigen), and can be used to isolate stem cells for bone marrow transplantation. My-10, deposited as HB-8483 with the American type culture Collection (Rockville, md.), is commercially available as anti-HPCA 1. In addition, negative selection can be performed using "dedicated" cells differentiated from human bone marrow to select for any desired cell markers. For example, progenitor or stem cells, most preferably CD34 ⁺ cells, can be characterized as either of the CD3^-、CD7^-、CD8^-、CD10^-、CD14^-、CD15^-、CD19^-、CD20^-、CD33^-、II class HLA ⁺ and Thy-1 ⁺.

After isolation of the progenitor or stem cells, they can be grown in any suitable medium. For example, progenitor or stem cells may be grown in conditioned medium from stromal cells, such as stromal cells associated with secreted factors obtained from bone marrow or liver, or in medium comprising cell surface factors that support proliferation of stem cells. The hematopoietic cells in the stromal cells can be removed by removing unwanted cells using a suitable monoclonal antibody.

The isolated cells are contacted ex vivo with an antibody and nucleic acid cargo complex. The cell into which the cargo is delivered may be referred to as a modified cell. The complex solution may simply be added to the cultured cells. It may be desirable to synchronize cells into S phase. Methods for simultaneous Cell culture, for example by double thymidine blocking, are known in the art (Zielke et al, methods Cell biol.,8:107-121 (1974)).

The modified cells may be maintained or expanded in culture prior to administration to a subject. Depending on the cell type, culture conditions are generally known in the art. In particular, the maintenance conditions of CD34 ⁺ have been well studied and several suitable methods have been chosen. A common method for expansion of pluripotent hematopoietic cells ex vivo is to culture purified progenitor 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., ligand of Flt-3L gene product) to nutrient media that maintains hematopoietic progenitor cells ex vivo helps to expand primitive (i.e., relatively undifferentiated) human hematopoietic progenitor cells in vitro, and that these cells can be transplanted in SCID-hu mice (Luens et al, 1998,Blood 91:1206-1215). In other known methods, cells can be maintained ex vivo in a nutrient medium (e.g., minutes, hours, or 3, 6, 9, 13, or more days) that includes mouse prolactin-like protein E (mPLP-E) or mouse prolactin-like protein F (mPIP-F; collectively mPLP-E/IF) (U.S. Pat. No. 6,261,841). Of course, other suitable cell culture and expansion methods may be used. Cells may also be cultured in serum-free medium as described in U.S. patent 5,945,337.

In another embodiment, the modified hematopoietic stem cells are differentiated ex vivo into a CD4 ⁺ cell culture using a specific interleukin and growth factor combination, and then administered to a subject using methods well known in the art. The cells may be expanded in vitro in large amounts, preferably at least 5-fold, more preferably at least 10-fold, even more preferably at least 20-fold, compared to the original population of isolated hematopoietic stem cells.

In another embodiment, the cells may be dedifferentiated somatic cells. Somatic cells can be reprogrammed into pluripotent stem cell-like cells, which can be induced into hematopoietic progenitor cells. Hematopoietic progenitor cells may then be treated with the compositions described above with respect to CD34 ⁺ cells. Representative somatic cells that can be reprogrammed include, but are not limited to, fibroblasts, adipocytes, and muscle cells. Hematopoietic progenitor cells from induced stem cell-like cells have been successfully cultured in mice (Hanna, J. Et al. Science,318:1920-1923 (2007)).

To produce hematopoietic progenitor cells from induced stem cell-like cells, somatic cells are obtained from the host. In a preferred embodiment, the somatic cells are autologous fibroblasts. These cells were cultured and transduced with vectors encoding Oct4, sox2, klf4 and c-Myc transcription factors. Culturing the transduced cells and screening 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. Cells were then screened for CD41 and c-kit markers (early hematopoietic progenitor markers) and for myeloid and erythroid differentiation markers.

The modified hematopoietic stem cells or modified cells (including, for example, induced hematopoietic progenitor cells, etc.) are then introduced into the subject. Delivery of cells may take a variety of forms, most preferably including intravenous administration by infusion, and direct injection into periosteum, bone marrow and/or subcutaneous injection.

The subject receiving the modified cells may undergo bone marrow modulation to enhance the transplantation of the cells. The recipient may receive radiation therapy or chemotherapy prior to use of the cells to facilitate transplantation of the cells. After administration, the cells typically take a period of time to transplant. The large number of hematopoietic stem or progenitor cells transplanted typically takes several weeks to months.

High proportions of modified hematopoietic stem cell transplantation may not be required to achieve significant prophylactic or therapeutic effects. It is believed that cells after transplantation will expand over time to increase the proportion of modified cells. It is believed that in some cases, only a small or small proportion of modified hematopoietic stem cells need to be implanted to achieve a prophylactic or therapeutic effect.

In a preferred embodiment, the cells to be administered to the subject will be autologous cells, e.g., cells derived from the subject, or cells of the same genotype.

Embryo

In certain embodiments, the compositions and methods can be used to deliver cargo to embryonic cells in vitro. These methods generally involve contacting the embryo with an effective amount of antibody-cargo DNA in vitro to enhance transduction of cargo into the embryo. The embryo may be a single-cell fertilized egg, but male and female gametes may also be treated before and during fertilization, and embryos with 2, 4, 8 or 16 cells, including not only fertilized eggs but also morula and blasts. In certain embodiments, the embryo is contacted with the composition during or after day 0-6 of in vitro fertilization.

The contacting may be by adding the composition to a liquid medium in which the embryo is immersed. For example, the composition may be directly pipetted into the embryo culture medium and then absorbed by the embryo.

Immune cells

In certain embodiments, the target cell is one or more types of immune cells. For example, different types of cells can be utilized or otherwise targeted for target cells of immunomodulation and CAR-based therapies. The preferred targeted/engineered T cells may vary depending on the tumor and the goals of adoptive therapy. Effector T cells are generally preferred because they secrete high levels of effector cytokines and are capable of being a skilled killer 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. The use of cd8+ T cells with cd4+ helper T cells results in an increase in suppressor T-reg cells and inhibits the cytotoxicity of cd8+ T cells. Since reprogrammed cd8+ T cells have been pre-activated, they can act directly on tumor cells without activation in the lymph nodes, and thus support of cd4+ T cells is not essential.

Furthermore, there is evidence that infusion of naive T cells (Rosenberg et al, adv. Cancer Res.,25:323-388 (1977)), central memory T cells (T _CM 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 have certain advantages in certain applications, for example, due to their high replicative capacity. Tumor Infiltrating Lymphocytes (TILs) also have certain advantages due to their antigen specificity and can be used in the delivery strategies disclosed herein.

While sometimes referred to as CAR cells, CAR immune cells, and CART cells (or CAR T cells), it is understood that the CARs 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 (see, e.g., barrett et al, annu Rev med.,65:333-347 (2014)).

Cancer cells and tumors

In certain embodiments, the target cell is a cancer cell. In such embodiments, methods of treatment are provided that are useful for cancer, including tumor treatment.

Goods that may be delivered to cancer cells include, but are not limited to, constructs that express one or more pro-apoptotic, immunogenic, or tumor suppressor factors, gene editing compositions, inhibitory nucleic acids that target oncogenes, and other strategies discussed herein and elsewhere. In certain embodiments, the cargo is an mRNA encoding a pro-apoptotic or immunogenic factor that increases the immune response against the cell. In other embodiments, the cargo is an expressed siRNA that reduces oncogenes or other oncogenic transcripts.

In mature animals, a balance is typically maintained between cell renewal and cell death in most organs and tissues. The mature cells in the body have a certain life span, and when the cells die, new cells are generated by proliferation and differentiation of various stem cells. Under normal conditions, the production of new cells is tightly regulated and the number of cells of any particular type remains unchanged. But occasionally cells that are no longer responsive to normal growth control mechanisms may also appear. The cell clones produced by these cells can be expanded to a considerable scale, forming tumors or neoplasms. Tumors that do not grow indefinitely and do not invade extensively healthy surrounding tissues are benign. Tumors continue to grow and are malignant in progressive invasiveness. The term cancer refers specifically to malignant tumors. In addition to uncontrolled growth, malignant tumors can also develop metastases. During this process, small clusters of cancer cells fall off the tumor, invade the blood or lymphatic vessels, and are carried to other tissues where they continue to proliferate. Thus, a primary tumor at one site will produce a secondary tumor at another site.

The compositions and methods described herein are useful for treating a subject having benign or malignant tumors, delaying or inhibiting tumor growth in a subject, decreasing tumor growth or size, inhibiting or reducing metastasis of a tumor, and/or inhibiting or reducing symptoms associated with tumorigenesis or growth.

Treatable malignancies are classified herein according to the embryonic origin of the tumor-derived tissue. Cancers are tumors arising from endodermal or ectodermal tissue, such as the skin or viscera and the epithelial lining of the gland. The disclosed compositions are particularly effective in treating cancer. Sarcomas occur less frequently and originate from mesodermal connective tissue such as bone, fat and cartilage. Leukemia and lymphoma are malignant tumors of bone marrow hematopoietic cells. Leukemia proliferates as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may occur in multiple organs or tissues of the body, thereby forming cancers.

Types of cancers that can be treated using the provided compositions and methods include, but are not limited to, vascular cancers (e.g., multiple myeloma), adenocarcinomas, and sarcomas, which may occur in bone, bladder, brain, breast, cervix, colorectal, esophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin, stomach, and uterus, among others.

In certain embodiments, the disclosed compositions are used to treat multiple cancer types simultaneously. These compositions can also be used to treat metastases or tumors at multiple sites.

B. Methods of modulating immune responses

Methods of enhancing an immune response are provided. The immune response may be increased against cancer, infection, and the like. Thus, methods of treating cancer and infections, and methods of vaccinating healthy and diseased subjects are also provided. Immune responses are also involved in wound healing and thus methods of promoting wound healing are also provided. Immunomodulation is also involved in some autoimmune diseases, such as multiple sclerosis, and thus methods for promoting immunomodulation in multiple sclerosis are also provided. Thus, in certain embodiments, the subject has a wound or multiple sclerosis.

These methods generally comprise administering to a subject in need thereof an effective amount of a 4H2 antibody to increase activation of cGAS and/or another PRR (e.g., TLR 7). In some embodiments, these compositions and methods can increase activation of cGAS and/or another PRR (e.g., TLR 7) by direct binding and 4H2 activation, or by indirect binding through simultaneous interaction between cGAS and/or other PPRs, 4H2 and cytoplasmic nucleic acid and/or GTP. Activated cGAS catalyzes the formation of cGAMP from the precursor molecules ATP and GTP, and cGAMP produced by cGAS promotes nuclear translocation of NF-kB. Thus, in certain embodiments, the disclosed compositions increase cGAMP production and/or promote NF-kB nuclear translocation. Typically, these methods are used to enhance immune responses by inducing or enhancing signaling through the cGAS/STING pathway.

In some embodiments, the compositions and methods include stimulating T cell proliferation, tumor vessel collapse and promoting tumor cell death and apoptosis, enhancing release of tumor-associated antigens, improving antigen-specific IgG responses by a mechanism that depends on T helper 1 (TH 1), TH2, and/or TH17 cell responses, reducing viral or bacterial load, reducing susceptibility to viruses or bacteria, or any combination thereof. See also Motwani and Fitzgerald, nature REVIEWS GENETICS, volume 20, pages 657-674 (2019), which are incorporated by reference herein in their entireties, wherein other results of enhancing cGAS/STING signaling are described. In some embodiments, the compositions and methods include increasing recruitment of Tumor Infiltrating Lymphocytes (TILs) to a tumor.

The results below also show that 4H2 interacts with TLR7 and is believed to proceed in a nucleic acid dependent manner. The TLR family plays an important 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 efficient immune development. TLR7 is an intracellular recognition receptor that recognizes single-stranded RNA in endosomes, a common feature of the viral genome for macrophage and dendritic cell internalization. For example, TLR7 recognizes single-stranded RNAs of viruses such as HIV and HCV. TLR7 can recognize GU-rich single stranded RNA.

The results indicate that 4H2 activates at least cGAS and TLR7, and perhaps other immune response-inducing receptors, which adds another layer 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, TLR9, and the like. Other receptors include, but are not limited to, those whose ligands are mentioned as cargo and/or adjuvants.

In certain embodiments, the methods comprise administering to a subject in need thereof an effective amount of a 4H2 antibody with one or more additional drugs such as nucleic acid cargo, immunostimulatory nucleic acids, vaccine components, immune checkpoint modulators, or a combination thereof. In certain embodiments, the 4H2 antibody and additional drug may be used in combination to provide a greater degree of enhancement of cGAS/STING and/or signaling by another immune receptor such as PPR (e.g., TLR 7) than either agent alone. For example, in certain embodiments, such as treating cancer, the enhanced activity is a greater anti-tumor activity.

The 4H2 antibody and/or immune checkpoint modulator may be administered locally or systemically to the subject, or may be coated or incorporated onto or into the device.

The disclosed monotherapy and combination therapies and treatment regimens generally comprise a method of treating a disease or a symptom thereof, or for effecting a desired physiological change, comprising administering to an animal (e.g., a mammal, particularly a human) an effective amount of a 4H2 antibody to treat the disease (e.g., cancer or infection) or a symptom thereof, or to produce a physiological change.

When administered in combination with an additional agent, the 4H2 antibody and the additional agent may be administered together, such 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 are at a time from each other). Thus, the term "combination" or "association" refers to the simultaneous, concurrent or sequential administration of two agents. The compositions may be administered simultaneously (e.g., as a mixture), separately but simultaneously (e.g., via different intravenous lines to the same subject; oral administration of one drug, infusion or injection of another drug, etc.), or sequentially (e.g., first to one drug and then to a second drug).

In certain embodiments, the combination of results obtained is partially or completely a superposition of the results obtained for the individual components. In certain embodiments, the effect achieved by the combined use is additive over the effect achieved by the individual components alone. The effect achieved by the combination exceeds the simple addition of the effects achieved by the individual components. In certain embodiments, the effective amount of one or both agents used in combination is less than the effective amount of each formulation when administered alone. In certain embodiments, the amount of one or both agents used in combination is a sub-therapeutic amount when used alone.

The effect of a combination therapy or individual agents thereof may depend on the disease or disorder to be treated or its progression. For example, in certain embodiments, combination therapy expands the treatable subject (e.g., type of cancer or infection) relative to each agent used alone. Thus, in certain embodiments, the effect of the combination on a cancer or infection may be compared to the effect of the agent alone on the cancer or infection.

Treatment regimens for monotherapy and combination therapy may comprise one or more administrations of the 4H2 antibody. The treatment regimen of the combination therapy may include administration of one or more additional drugs.

In certain embodiments, the 4H2 antibody and the additional drug are administered sequentially, e.g., in two or more different pharmaceutical compositions. In certain embodiments, the 4H2 antibody is administered prior to the first administration of the additional drug. In other embodiments, the additional drug is administered prior to the first administration of the 4H2 antibody. For example, the 4H2 antibody and the additional drug may be administered to the subject on the same day. Alternatively, the 4H2 antibody and the additional drug may be administered to the subject on different dates.

The 4H2 antibody may be administered at least 1, 2, 3, 5, 10, 15, 20, 24, or 30 hours or days before or after administration of the additional drug. Or 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 certain embodiments, the additive effect or effects that exceed the additive effect of the 4H2 antibody and the additional drug are clearly visible one, two, three, four, five, six, one or more weeks after administration.

The dosage regimen or cycle of the agents may overlap, either entirely or partially, or may be continuous. For example, in certain embodiments, all such administration of the 4H2 antibody occurs before or after administration of the additional drug. Or the administration of one or more doses of the 4H2 antibody may be staggered in time from the administration of the additional agent to form a uniform or non-uniform course of treatment, wherein the one or more doses of the 4H2 antibody are administered first, followed by the one or more doses of the additional agent, and finally the one or more doses of the 4H2 antibody are administered, or the one or more doses of the additional agent are administered first, followed by the one or more doses of the 4H2 antibody, and finally the one or more doses of the additional agent, etc., all of which may be performed according to any schedule selected or desired by the researcher or clinician performing the treatment.

The effective amount of each agent may be administered in a single unit dose (e.g., as a dosage unit) or as a sub-therapeutic dose administered over a limited time interval. Such unit doses may be administered daily for a limited 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 particularly contemplated.

1. Treatment of cancer

The therapies disclosed herein can be used to treat, reduce and/or prevent cancer in a subject. Thus, the composition can be administered in an effective amount to treat, reduce and/or prevent cancer in a subject. An effective amount or therapeutically effective amount for treating cancer or tumor generally refers to a dosage sufficient to alleviate or prevent at least one symptom of the cancer, or otherwise provide the desired pharmacological and/or physiological effect. Symptoms may be physical, such as tumor burden, or biological, such as reducing cancer cell proliferation or increasing cancer cell death. In certain embodiments, the amount is effective to kill tumor cells or reduce or inhibit proliferation or metastasis of tumor cells. In certain embodiments, the amount is effective to reduce tumor burden. In certain embodiments, an effective amount may reduce or prevent at least one cancer complication.

In mature animals, a balance is typically maintained between cell renewal and cell death in most organs and tissues. The mature cells in the body have a certain life span, and when the cells die, new cells are generated through proliferation and differentiation of various stem cells. Under normal conditions, the production of new cells is regulated and the number of cells of any particular type remains unchanged. But occasionally cells that are no longer responsive to normal growth control mechanisms may also appear. The clonal cells produced by these cells can be expanded to a substantial scale to form tumors or neoplasms. Tumors that do not grow indefinitely and do not widely invade healthy surrounding tissues are benign. Tumors that continue to grow and progressively invade are malignant. The term cancer generally refers to malignant tumors. In addition to uncontrolled growth, malignant tumors can also develop metastases. During this process, small clusters of cancer cells fall off the tumor, invade the blood or lymphatic vessels, and are carried to other tissues where they continue to proliferate. Thus, a primary tumor at one site will produce a secondary tumor at another site.

The compositions and methods described herein are useful for treating a subject having benign or malignant tumors, delaying or inhibiting tumor growth in a subject, decreasing tumor growth or size, inhibiting or reducing metastasis of a tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.

Treatable malignant tumors can be classified according to the embryonic origin of the tumor-derived tissue. Cancers are tumors arising from endodermal or ectodermal tissue, such as the skin or viscera and the epithelial lining of the gland. The disclosed compositions are particularly effective in treating cancer. Less frequently occurring sarcomas originate from mesodermal connective tissue such as bone, fat and cartilage. Leukemia and lymphoma are malignant tumors of bone marrow hematopoietic cells. Leukemia proliferates as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may occur in multiple organs or tissues of the body, thereby forming cancers.

The disclosed antigen binding molecules are useful for treating cells that have uncontrolled growth, invasion or metastasis.

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

The formation of cancer cells may be due to a somatic function-acquired mutation of the Ras gene, resulting in an activating mutation of the small gtpase Ras enzyme. Oncogenic mutations in H-Ras, N-Ras or K-Ras genes are most often associated with human malignancies. In certain embodiments, the cells express mutant forms of the small GTPase Ras family, such as K-Ras. In certain embodiments, the cell does not express a wild-type Ras gene.

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

Oncogenic mutations in the K-Ras gene can result in constitutive activation of the exogenous Ras protein. Exemplary mutations include mutations in codons 12, 13 and/or 61, which result in any change in amino acid 12, 13 or 61 of the K-ras protein. This includes, for example, but is not limited to, K-ras amino acid 12 (glycine to aspartic acid, cysteine, serine, threonine, arginine, or valine) and amino acids 13 and 61 (glutamine to lysine, arginine, leucine, or aspartic acid). Another method of describing K-Ras mutations is G12A, G12C, G12D, G S, G12I, G12R, G12V, G C, G13D, G13S, Q61L, Q61R. Also, any change in the amino acid content at positions 12, 13, 61 is considered an exemplary mutation.

Representative but non-limiting cancers for which the compositions are useful in treatment include cancers of the blood and lymphatic system (including leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, solitary plasma cell tumor, multiple myeloma), genitourinary system cancers (including prostate cancer, bladder cancer, kidney cancer, urinary tract cancer, penile cancer and testicular cancer), nervous system cancers (including meningioma, glioma, glioblastoma, astrocytoma, oligodendroglioma, oligodendroastrocytoma, ependymoma), head and neck cancers (including squamous cell carcinoma of the oral cavity, nasal cavity, nasopharyngeal cavity, oropharyngeal cavity, laryngeal and paranasal sinuses), lung cancers (including small cell and non-small cell lung cancer), gynaecological cancers (including cervical cancer, endometrial cancer, vaginal cancer, vulval cancer, ovarian cancer and fallopian tube cancer), gastrointestinal cancers (including gastric cancer, small bowel cancer, colorectal cancer, liver cancer, hepatobiliary tract cancer and pancreatic cancer), skin cancers (including melanoma, squamous cell carcinoma and basal cell carcinoma), breast cancers (including ductal cancer and lobular cancer and triple negative breast cancer (including squamous cell carcinoma) and myelema, medulloblastoma, blastoma. Thus, in some embodiments, the 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 blood cancer. In certain embodiments, the disclosure relates to treating glioblastoma.

Any of the methods disclosed can be further used in combination with radiation therapy, chemotherapy (e.g., antineoplastic agents), or a combination thereof, to treat any cancer, including carcinoma, glioma, sarcoma, or lymphoma. Examples of antineoplastic agents that may be conjugated to the disclosed antigen binding molecules include, but are not limited to, alkylating agents (such as temozolomide, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites (such as fluorouracil, gemcitabine, methotrexate, cytarabine, fludarabine and fluorouridine), certain antimitotics and vinca alkaloids (such as vincristine, vinblastine, vinorelbine and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin and epirubicin, and actinomycins (such as actinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin and bleomycin), and topoisomerase inhibitors (including camptothecins such as irinotecan and topotecan and etoposide, etoposide and etoposide.

Strategies are currently being explored to combine STING immunotherapy with other immunomodulators. The antitumor effect was enhanced when combined with anti-apoptotic protein-1 (PD-1) and anti-cytotoxic T lymphocyte-associated protein-4 (CTLA-4) antibodies by intratumoral injection (i.t.injection) of cGAMP into B16.F10 tumors. (Demaria et al, proc NATL ACAD SCI U S A (2015) 112 (50): 15408-13.10.1073/PNAS.1512832112). In other studies CDNs along with anti-PD-1 elicited much stronger anti-tumor effects than monotherapy in squamous cell carcinoma models as well as in murine models of melanoma (Gadkaree et al, head Neck (2017) 39 (6): 1086-94.10.1002/hed.24704; wang et al, proc NATL ACAD SCI U S A (2017) 114 (7): 1637-42.10.1073/PNAS.1621363114). Luo et al combined STING-activating nanovaccine with anti-PD 1 antibodies produced long-term anti-tumor memory in TC-1 tumor models with encouraging results (Luo et al Nat Nanotechnol (2017) 12 (7): 648-54.10.1038/nnano.2017.52). Thus, a particularly preferred method of treating cancer comprises administering to a subject a combination of a 4H2 antibody and a checkpoint modulator.

2. Infection and viral transformation of cells

In certain embodiments, the compositions are useful for treating or preventing infection of cells by bacteria or viruses (e.g., oncological viruses).

Thus, the compositions are useful for treating local or systemic infections.

Representative infections that may be treated include, but are not limited to, infections caused by microorganisms including, but not limited to, actinomycetes (Actinomyces), anabaena (Anabaena), bacillus, bacteroides (Bacteroides), bdellovibrio (Bdellovibrio), baume (Bordetella), borrelia (Borrelia), campylobacter (Campylobacter), bacillus (Caulobacter), chlamydia (Chlamydia), viridis (Chlorobium), Coloring bacteria (Chromatium), clostridium (Clostridium), corynebacterium (Corynebacterium), phagostimula bacteria (Cytophaga), deinococcus (Deinococcus), escherichia (Escherichia), franciscensis (FRANCISELLA), halophil (Halobacterium), helicobacter (Heliobacter), haemophilus (Haemophilus), haemophilus influenzae (Hemophilus influenza type B (HIB)), and a bacterial strain (c), Histoplasma (Histoplasma), comamonas (Hyphomicrobium), legionella (Legionella), leishmania (Leishmania), leishmania leptospira (Leptspirosis), listeria (Listeria), meningococcus (Meningococcus) A, B and C, methanobacillus (Methanobacillus), micrococcus (Micrococcus), mycobacterium (Myobacterium) (such as Bacillus Tuberculosis), Mycoplasma (Myxoplasma), (Myxococcus), myxococcus (Myxococcus), neisseria (Neisseria), nitrobacter (Nitrobacter), oscillatoria (Osciliatria), proviridae (Prochloron), proteus (Proteus), pseudomonas (Pseudomonas), rhodospirillum (Phodospirillum), rickettsia (Rickettsia), salmonella (Salmonella), shigella (Shigella), Helicobacter (Spirillum), spirochete (Spir DEG Chaeta), staphylococcus (Staphylococcus), streptococcus (Streptomyces), streptomyces (Streptomyces), sulfolobus (Sulfolobus), thermoplasma (thermoplastma), thiobacillus (Thiobacillus), and treponema (Treponema), vibrio (Vibrio), yersinia (Yersinia), cryptococcus neoformans (Cryptococcus neoformans), histoplasma capsulatum (Histoplasma capsulatum), candida albicans (Candida albicans), candida tropicalis (Candida tropicalis), nocardia stellate (n° Cardia asteroides), rickettsia (RICKETTSIA RICKETSII), rickettsia typhosa (RICKETTSIA TYPHI), mycoplasma pneumoniae (Mycoplasma pneumoniae), chlamydia psittaci (CHLAMYDIAL PSITTACI), and, Chlamydia trachomatis (CHLAMYDIAL TRACHOMATIS), plasmodium falciparum (Plasmodium falciparum), plasmodium vivax, trypanosoma brucei (Trypanosoma brucei), endomonas histolytica (Entamoeba histolytica), toxoplasma gondii (Toxoplasma gondii), trichomonas vaginalis (Trichomonas vaginalis), and Schistosoma mansoni (Schistosoma mansoni).

Exemplary viruses that may be affected by the disclosed compositions include Human Papilloma Virus (HPV), hepatitis B Virus (HBV), hepatitis C Virus (HCV), human T-lymphocyte virus (HTLV), kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell polyoma virus, epstein Barr Virus (EBV), human Immunodeficiency Virus (HIV), and human Cytomegalovirus (CMV), including but not limited to immunodeficiency virus (e.g., HIV), human papilloma virus (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., influenza a virus), common cold (e.g., human rhinovirus), coronavirus (e.g., SARS-CoV-2), zika virus, dengue virus, and Vesicular Stomatitis Virus (VSV).

For example, the composition may be used topically for the treatment of viral skin disorders such as herpes or shingles or genital warts. The composition may also be used to treat systemic viral diseases including, but not limited to, aids, influenza, common cold or encephalitis.

Other viral diseases that can be affected by administration of the composition include Colorado tick fever (caused by Kort virus (Coltivirus), RNA virus), west Nile fever (encephalitis, caused by flaviviruses that occur primarily in the middle east and Africa), yellow fever, rabies (caused by a number of different strains of the Rhabdoviridae family), viral hepatitis, gastroenteritis (viral) - -, acute viral gastroenteritis caused by Norwalk and Norwalk-like viruses, rotavirus, calicivirus and astrovirus, polio, influenza (influenza) caused by orthomyxoviruses that can undergo frequent antigenic variation, measles (rubella), paramyxoviridae, mumps, respiratory tract syndrome including viral pneumonia and acute respiratory tract syndrome (such as laryngotracheobronchitis, colloquially called "croup") caused by a variety of viruses commonly known as acute respiratory viruses, and respiratory diseases caused by respiratory syncytial virus (RSV, the most dangerous cause of respiratory infections in infants).

In certain embodiments, the disclosed compositions are used to treat or prevent a viral infection or spread or exacerbation of a viral infection. For example, in certain embodiments, the compositions are used to treat or prevent a viral infection or spread or exacerbation of a viral infection in a subject that has been exposed to a virus or is at risk of being exposed to a virus (as discussed herein).

3. Vaccination

The composition may be administered prior to, simultaneously with or after vaccination. In one embodiment, the 4H2 antibody composition is administered concurrently with the administration of the vaccine.

The disclosed compositions may be administered in combination with a prophylactic or therapeutic vaccine, which may be used to initiate or enhance the immune response of a subject to an existing antigen (e.g., a tumor antigen of a cancer patient).

The expected outcome of a prophylactic, therapeutic or desensitizing immune response may vary from disease to disease, according to principles well known in the art. Likewise, an immune response against cancer, an allergen, or an infectious agent may treat the disease entirely, may alleviate symptoms, or may be an aspect of overall therapeutic intervention against the disease. For example, immune response stimulation against cancer may be combined with surgery, chemotherapy, radiation therapy, hormonal therapy, and other immunological methods to affect the therapeutic effect.

STING agonists, when combined with tumor vaccines, enhance anti-tumor responses. For example, CDN ligands formulated with cell cancer vaccines that produce granulocyte-macrophage colony stimulating factor, referred to as STINGVAX, have shown strong in vivo efficacy in several established cancer models (Fu, et al SCI TRANSL MED (2015) 7 (283): 28r52.10.1126/scitranslmed.aa4306), STING agonists used in combination with conventional chemotherapeutics or radiotherapy can elicit an anti-tumor response (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.) thus, a particularly preferred method of treating a subject in need thereof involves administering to the subject a combination of 4H2 antibody and one or more components of the vaccine. The vaccine may be against, for example, cancer or infectious agents.

4. Wound healing

In certain embodiments, the compositions may be used to promote wound healing. Activation of STING by cGAMP promotes skin wound healing (Mizutani et al J Dermatol Sci 202097 (10:21-29)).

Representative wounds for which the composition may promote healing include skin wounds resulting from trauma or surgery, ocular wounds resulting from trauma or surgery, and visceral wounds resulting from trauma or surgery.

For example, the composition may be topically applied to treat a wound of the skin or eye caused by trauma or surgery, or topically injected to treat a wound of the skin or eye caused by trauma or surgery or an organ.

5. Immunomodulation for the treatment of multiple sclerosis

In some embodiments, the compositions are useful for promoting immunomodulation to treat autoimmune and/or immunomodulatory disorders, such as multiple sclerosis. Activation of STING by cGAMP inhibits disease in multiple sclerosis models (Johnson et al, J Immunol 2021206 (9): 2015-28).

For example, the composition may be administered systemically to treat multiple sclerosis.

6. Neurofibromatosis (NF)

Neurofibromatosis (NF) is a mole-like hamartoma or syndrome with neurological and cutaneous manifestations, a rare genetic disease that usually causes benign tumors of the nerve and hyperplasia of other parts of the body, including the skin.

Neurofibromatosis type 2 is a disease characterized by the growth of a non-cancerous tumor of the nervous system caused by mutations in the NF2 gene (encoding Merlin protein). The most common tumor associated with neurofibromatosis type 2 is known as vestibular schwannoma. These tumors grow along nerves (auditory nerves) that transmit information from the inner ear to the brain. Tumors that form on the membranes (meninges) covering the brain and spinal cord are also common in neurofibromatosis type 2. These tumors are called meningiomas. Tumors may also occur in other nerves or tissues of the brain or spinal cord of a patient with such a disease. In certain embodiments, the method comprises delivering a nucleic acid (e.g., mRNA) encoding NF 2.

The following results indicate that 4H2 can mediate the gene delivery to NF2 mRNA and have an effect on NF2 tumors in vivo. Thus, in some embodiments, the subject has a neurofibromatosis, e.g., neurofibromatosis type 2. In some embodiments, the compositions are used to treat a subject having a schwannoma and/or meningioma.

The invention may be further understood by the following numbered paragraphs:

1. A composition of matter comprising a blend of two or more of the above, comprising the following components or is composed of the following components

(A) An intact 4H2 monoclonal antibody or cell-penetrating fragment thereof, optionally selected from a monovalent, bivalent or multivalent single chain variable fragment (scFv), or bispecific antibody fragment, or a humanized form thereof, chimeric form thereof or variant thereof, and

(B) A nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof.

2. The composition of paragraph 1 wherein (a) comprises

(I) A combination of the CDR of SEQ ID NO.5 and the CDR of SEQ ID NO. 1;

(ii) A combination of first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 6-8, respectively, and first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 2-4, respectively;

(iii) A humanized form of (i) or (ii);

(iv) A combination of a heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 5 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO.1, or

(V) The humanized form of (iv).

3. The composition of paragraph 1 or 2, wherein (a) comprises the same or different epitope specificity as monoclonal antibody 4H 2.

4. The composition of any one of paragraphs 1-3, wherein (a) is a recombinant antibody having the antigen-binding site of monoclonal antibody 4H 2.

5. A composition comprising:

(a) A binding protein comprising:

(i) A combination of the CDR of SEQ ID NO.5 and the CDR of SEQ ID NO. 1;

(ii) A combination of first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 6-8, respectively, and first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 2-4, respectively;

(iii) A humanized form of (i) or (ii);

(iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 5 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO.1, or

(V) The humanized form of (iv)

And

(B) A nucleic acid cargo comprising a nucleic acid encoding a polypeptide, a functional nucleic acid, a nucleic acid encoding a functional nucleic acid, or a combination thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

24. The composition of any one of paragraphs 1-23, further comprising a vector DNA.

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

26. The composition of paragraph 24 or 25, wherein (b) consists of RNA.

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

28. The composition of paragraph 27 further comprising polymeric nanoparticles encapsulating the complexes of (a) and (b).

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

30. A method of delivering a nucleic acid cargo to a cell comprising contacting the cell with an effective amount of the composition of any one of paragraphs 1-29.

31. The method of paragraph 30, wherein the contacting occurs in vitro.

32. The method of paragraph 31, wherein the cells are hematopoietic stem cells or T cells.

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

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

35. The method of paragraph 30, wherein the contacting occurs in vivo after administration to a subject in need thereof.

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

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

38. The method of paragraph 36 or paragraph 37, wherein (b) is delivered into the cells of the subject in an amount effective to alleviate one or more symptoms of the disease or disorder in the subject.

39. A method of preparing the composition of any one of paragraphs 1-29, comprising incubating and/or mixing (a) and (b) at an appropriate temperature for an effective time to form a complex of (a) and (b) prior to contacting with a cell.

40. A method of preparing the composition of any one of paragraphs 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 ℃.

41. The composition or method of any of the preceding paragraphs, wherein the ratio of (a) to (b) is between 1:3 and 5:1, alternatively the ratio is 1:1 or 3:1.

42. A method of enhancing immune receptor activation in a cell of a subject in need thereof, comprising administering an effective amount of (a) an intact 4H2 monoclonal antibody or cell-penetrating fragment thereof, optionally selected from a monovalent, bivalent or multivalent single chain variable fragment (scFv), or bispecific antibody fragment, or a humanized form thereof, chimeric form thereof, or variant 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 paragraph 42, wherein the subject has cancer or an infection.

44. The method of paragraph 42 or 43, wherein the subject is free of cancer.

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

46. The method of any one of paragraphs 42-44, wherein the subject has an immune disorder, optionally wherein the immune disorder is multiple sclerosis.

47. The method of any one of paragraphs 42-46, further comprising administering to subject (b) an additional agent.

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

49. A method of treating cancer or infection comprising administering to a subject in need thereof an effective amount of a combination of

(A) An intact 4H2 monoclonal antibody or cell-penetrating fragment thereof, optionally selected from a monovalent, bivalent or multivalent single chain variable fragment (scFv) or bispecific antibody fragment, or a humanized form thereof, chimeric form thereof or variant thereof, and

(B) Immune checkpoint modulators that can induce, increase or enhance an immune response.

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

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

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

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

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

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

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

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

58. A method of treating cancer or infection comprising administering to a subject in need thereof an effective amount of a combination of:

(a) An intact 4H2 monoclonal antibody or cell-penetrating fragment thereof, optionally selected from a monovalent, bivalent or multivalent single chain variable fragment (scFv), or bispecific antibody fragment, or a humanized form thereof, chimeric form thereof or variant thereof, and

(B) Immunostimulatory nucleic acids.

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

60. A method of vaccinating a subject comprising injecting the subject with a vaccine

(A) An intact 4H2 monoclonal antibody or cell penetrating fragment thereof, optionally selected from a monovalent, bivalent or multivalent single chain variable fragment (scFv) or bispecific antibody fragment, or a humanized form thereof, chimeric form thereof or variant thereof, and

(B) One or more vaccine components.

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

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

63. The method of any one of paragraphs 48-62, wherein the combination of administration of (a) and (b) results in a more than additive reduction of one or more symptoms of the cancer or infection as compared to administration of (a) or (b) in the absence of (a) or (b).

64. The method of any one of paragraphs 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 to the subject.

65. The method of any one of paragraphs 48-63, wherein (b) 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 to the subject (a).

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

67. The method of any of paragraphs 42-66, further comprising surgery or radiation therapy.

68. The method of any one of paragraphs 42-67, further comprising a nucleic acid cargo.

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

70. The method of paragraph 68 or 69, wherein (b) is a nucleic acid cargo, optionally wherein the nucleic acid cargo consists of nucleic acid comprising DNA, RNA, PNA, PMO, or other modifications, or a nucleic acid analog, or a combination thereof.

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

72. The method of any one of paragraphs 42-71, wherein (a) comprises:

(i) The CDR of SEQ ID NO. 5 is combined with the CDR of SEQ ID NO. 1;

(ii) A combination of first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 6-8, respectively, and first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 2-4, respectively;

(iii) A humanized form of (i) or (ii);

(iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 5 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO.1, or

(V) The humanized form of (iv).

73. The method of any one of paragraphs 42-72, wherein (a) comprises the same or a different epitope specificity as monoclonal antibody 4H 2.

74. The method of any one of paragraphs 42-73, wherein (a) is a recombinant antibody having the antigen-binding site of monoclonal antibody 4H 2.

75. The method of any one of paragraphs 42-74, wherein (a) comprises:

(i) A combination of the CDR of SEQ ID NO.5 and the CDR of SEQ ID NO. 1;

(ii) A combination of first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 6-8, respectively, and first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 2-4, respectively;

(iii) A humanized form of (i) or (ii);

(iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 5 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO.1, or

(V) The humanized form of (iv).

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

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

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

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

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

81. The pharmaceutical composition of paragraph 79, wherein (b) is not a nucleic acid cargo.

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

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

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

85. A composition comprising:

(a) A bispecific binding protein comprising:

(i) A combination of the CDR of SEQ ID NO.5 and the CDR of SEQ ID NO. 1;

(ii) A combination of first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 6-8, respectively, and first, second and third light chain CDRs comprising the amino acid sequences of SEQ ID nos. 2-4, respectively;

(iii) A humanized form of (ai) or (aii);

(iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 5 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO.1, or

(V) The humanized form of (iv)

And

A binding domain that binds to an immune cell marker.

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

87. The composition of paragraph 86, wherein the binding domain that binds to CD5 comprises:

(vi) A combination of the CDR of SEQ ID NO. 24 and the CDR of SEQ ID NO. 23;

(vii) A combination of first, second and third light chain CDRs comprising amino acid sequences of SEQ ID NOs 25-27 and first, second and third light chain CDRs comprising amino acid sequences of SEQ ID NOs 28-30, respectively;

(viii) A humanized form of (iv) or (iiv);

(ix) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 24 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 23, or

(X) A humanized form of (ix).

88. The composition of any one of paragraphs 85-87, comprising:

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

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

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

91. A binding protein, optionally an antibody, comprising:

(i) A combination of the CDR of SEQ ID NO.24 and the CDR of SEQ ID NO. 23;

(ii) A combination of first, second and third light chain CDRs comprising amino acid sequences of SEQ ID NOs 25-27 and first, second and third light chain CDRs comprising amino acid sequences of SEQ ID NOs 28-30, respectively;

(iii) A humanized form of (i) or (ii);

(iv) A heavy chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 24 and a light chain comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO. 23, or

(V) The humanized form of (iv).

Examples

Autoantibodies against host DNA reactions lead to inflammation and type I interferon characteristics associated with Systemic Lupus Erythematosus (SLE) (Nehar-Belaid et al, nat Immunol 2 (9): 1094-1106 (2020), li, et al, clin Exp Immunol 159 (3): 281-291 (2010)). Some anti-DNA autoantibodies can penetrate living cells, avoid lysosomal degradation, and enter the nucleus or cytoplasm (Hansen et al, J Biol chem.282:20790-20793 (2007), noble et al, nat Rev Rheumatol (7): 429-34 (2016)), cGAS cytoplasmic nucleic acid sensors activate STING/interferon pathways in the core mechanisms of the innate immunity (Wan et al, front Immunol.13:826880 (2022)), it is currently unclear whether there is an interaction between cytoplasmic localized anti-DNA autoantibodies and cGAS activity.

Anti-DNA autoantibodies exhibit a variety of nucleic acid binding specificities, some of which recognize a variety of conformations and sequences of DNA (Shoenfeld et al, N Engl J Med.308 (8): 414-420 (1983)) and others of which exhibit a fine specificity for a single nucleoside (Weisbar et al ,Clin Immunol and Immunopathol.27:403-11(1983),Yee&Weisbart,Clin Immunol and Immunopathol.36:161-67(1985)). Guanosine (GUO) is the most immunogenic nucleoside, compared to other anti-DNA autoantibodies, anti-guanosine autoantibody titers correlate better with systemic Lupus erythematosus (Stollar & Borel, J Immunol 117:1308-1313 (1976); colburn et al, lupus 10:410-7 (2001); weisbar et al, clin immunopathol.27:403-11 (1983)), it is notable that anti-GUO autoantibodies in Systemic Lupus Erythematosus (SLE) patient serum are identical to epitopes on G proteins (Colburn et al, journal of Rheumatology (5): 993-97 (2003)), and hence, 60:9:37 (2006) are thought to interfere with signal transduction by cells of the anti-GR (2006: 35:9).

Various cell penetrating anti-DNA autoantibodies were isolated from a mouse model of systemic lupus erythematosus. Although most of these antibodies penetrate the nucleus of living cells, the anti-GUO autoantibody 4H2 is significantly different due to its cytoplasmic localization. The epitope on GUO bound by 4H2 is located at the G protein binding site, which coincides with the report of anti-GUO autoantibody binding in serum of human Systemic Lupus Erythematosus (SLE) patients (Colburn et al, journal of Rheumatology (5), 993-97 (2003)). In addition, 4H2 is also able to penetrate and reduce cAMP concentration in cultured cells, which is consistent with interfering with G protein signaling (Colburn & Green CLIN CHIM ACTA 370:9-16 (2006)). The following results indicate that cytoplasmic penetration of 4H2 is associated with nucleoside transport, and that 4H2 binds to and mediates nucleic acid transport, binds to and enhances cGAS activity, thereby causing cGAS-dependent toxicity to tumor cells.

Example 1 localization of 4H2 to the cytoplasm of cancer cells, avoidance of endosomes and lysosomes

Materials and methods

Hybridomas and cell lines

The 4H2 hybridoma was obtained by MTA calibrated to los Angeles, university California. Hybridoma maintenance and antibody purification from supernatant as described above (Noble et al Sci Rep 4:5958 (2014)). The IgG control was murine monoclonal IgG2a. The humanized, deimmunized and CDR-optimized di-scFv fragment of 3E10 was purified from CHO cell culture medium as previously described (Rattray et al, JCI Insight 6 (14): E145875 (2021)), designated Deoxymab-1 ("DX 1"), the entire contents of which are specifically incorporated herein by reference. Cal12T cells were obtained from Horizon Discovery Ltd (Cambridge, UK). hCMEC/D3 cells were purchased from MilliporeSigma (SCC 066). NHA is purchased from Lonza. Mouse GSC, which is isogenic to C57/BL6 mice, was purchased from MD Anderson cancer center. Cells were grown in RPMI 1640 medium supplemented with 10% fbs and stored at 37 ℃ per 5% co ₂.

Cell penetration and co-localization immunofluorescence assay

Live Cal12T cells were grown on glass coverslips and treated with control medium or medium containing 0.5mg/mL 4H2 for one hour. Cells were then washed and fixed, immunostained with Alexa Fluor 488 conjugated goat anti-mouse IgG antibody (CELL SIGNAL ING, danvers, mass.) and the position of 4H2 within the cells was detected as described previously (Chang et al, acta neuropathol Commun9:112 (2021)). For co-localization studies, fixed cells were also tested overnight with a separate rabbit primary antibody to detect endosomes (C45B 10 anti-EEA 1 antibody, CELL SIGNALING), lysosomes (D2D 11 anti-LAMP 1 antibody, CELL SIGNALING), endoplasmic reticulum (C81H 6 anti-PDI antibody, CELL SIGNALING), golgi (D2B 6N anti-RCAS 1 antibody, CELL SIGNALING), mitochondria (3E 11 anti-COX IV, cell Signaling), then washed with PBS, and incubated with Alexa Fluor 555 conjugated goat anti-rabbit IgG antibody (CELL SIGNALING) for one hour at room temperature. After the last series of washes, cells were treated with Prolong Gold anti-quench cappers (CELL SIGNALING) containing DAPI and imaged under bright field, DAPI, GFP or RFP filters using a EVOS fl digital fluorescence microscope (Advanced Microscopy Group, botull, WA). The fluorescence images were combined using ImageJ (NIH, bethesda, MD). In the DP study, cells were pretreated with control medium or medium containing 100mM DP (MilliporeSigma, D9766) for thirty minutes, then 1mg/mL 4H2 was added for one hour, and then cell penetration was assessed as described above. The 4H2 fluorescence intensity of at least 30 cells was quantified using ImageJ.

FITC labeling of 4H2 and live cell imaging

Purified 4H2 was labeled with FITC using PIERCE FITC antibody labeling kit (Thermo FISHER SCIENTIFIC, waltham, mass.). For the live cell penetration assay, cal12T and A549 cells were treated overnight with 0.5mg/mL of FITC-labeled 4H2, then 500nM MitoTracker Red FM (Thermo FISHER SCIENTIFIC) for 45 minutes, then 1 μg/mL Hoechst 33342 (Thermo FISHER SCIENTIFIC) for 15 minutes. The cells were then rinsed with PBS and imaged using a EVOS fl digital fluorescence microscope.

Western blotting (Western blotting)

Cells were treated overnight with medium containing the indicated amounts of IgG control or 4H2, then cell lysates were prepared for 4-15% sds-PAGE analysis and nitrocellulose transfer. Membranes blocked with 5% milk in TBST were incubated with the relevant primary antibody overnight at 4 ℃, and HRP conjugated anti-rabbit or anti-mouse IgG secondary antibody (CELL SIGNALING) after washing were incubated for one hour at room temperature. After washing again, the strip was tested with Lumiglo (Cell Signaling). Primary antibodies used included rabbit anti-p-ERK 1/2 (T202/Y204) (CELL SIGNALING), rabbit anti-pan-ERK 1/2 (CELL SIGNALING) or murine anti- β -actin (Ambion, austin, TX), NF-kB p65 rabbit antibodies (CELL SIGNALING, # 8242).

Statistical analysis

The chart is generated using GRAPHPAD PRISM 9.4.1 versions. The P-value of in vivo studies was determined by Student's t-test or log rank test (log-rank). Error bars represent SEM.

Results

Previous studies on 4H2 reported its penetration of lymphocytes and thyroid epithelial cells (13). The experiments were designed to test 4H2 penetration into solid tumor cancer cells. 4H2 purified from hybridoma supernatants migrated on SDS-PAGE as expected and penetrated cultured non-small cell lung cancer Cal12T cells, showing significant cytoplasmic localization. Western blot analysis of cell lysates 24 hours after 4H2 treatment with primary anti-actin and secondary anti-mouse IgG revealed that bands of both the 4H2 Heavy (HC) and Light (LC) chains appeared at the expected Molecular Weight (MW) positions (fig. 1A). This indicates that the antibody chains remain intact after 24 hours of cell penetration. Non-small cell lung cancer cells (A549 and Cal 12T) were treated with FITC-labeled 4H2 and counterstained with MitoTracker Red FM and Hoechst 33342 (ThermoFisher Scientific, waltham, mass.) to exclude the effects of fixation artifacts by live cell imaging. Superposition of FITC, hoechst and MitoTracker images showed no 4H2 FITC signal in the nucleus and confirmed overlap with MitoTracker, consistent with previous reports (Colburn & Green, CLIN CHIM ACTA 370:9-16 (2006)).

In vivo engulfment of the antibody and degradation in lysosomes is a common phenomenon, but 4H2 remains intact after 24 hours of cell penetration (fig. 1A). Fluorescence co-localization studies probed the localization of 4H2 within cells. Cal12T cells treated with Alexa Fluor 488 conjugated anti-mouse IgG antibody were immunostained for 4H2 and early endosomes (EEA 1), lysosomes (LAMP 1), golgi (RASC 1) endoplasmic reticulum (PDI) and mitochondria (COX IV) were detected with rabbit primary antibodies, followed by counterstaining with Alexa Fluor 555 conjugated anti-rabbit IgG antibody and DAPI nuclei (CELL SIGNALING Technology, danvers, mass.). 4H2 was not co-localized with any of the tested organelles (including endosomes or lysosomes).

Example 24H 2 activation of ERK1/2 is reduced

4H2 has been previously reported to reduce cAMP levels in rat epithelial cells, suggesting that it interferes with G-protein mediated signaling (Colburn & Green CLIN CHIM ACTA 370:9-16 (2006)). Measuring ERK1/2 phosphorylation downstream of Ras provides another method for assessing G protein activity. Lysates of Cal12T cells were treated with control medium or 1mg/mL IgG control or 4H2 and assayed for total ERK1/2 content and phosphorylated ERK1/2 content by western blot. 4H2 did not affect the total ERK1/2 content, but reduced its phosphorylation, while the IgG control had no effect on either total ERK1/2 content or pERK1/2 (FIG. 1B). These results are consistent with the interference of 4H2 with G protein signaling.

Example 3 penetration of 4H2 into living cells by nucleoside transporter dependent mechanisms

The lack of early degradation and avoidance of entry into the endosome/lysosome of 4H2 observed here suggests that the cell penetration mechanism of 4H2 is non-endocytosis mediated. Previous studies have shown that nuclear penetrating anti-DNA autoantibody 3E10, isolated from the same lupus model that produces 4H2, uses an Equilibrium Nucleoside Transporter (ENT) to cross the cell membrane and across the Blood Brain Barrier (BBB) (Hansen et al, J Biol chem.282:20790-20793 (2007), rattray, et al, JCI Insight 6 (14): E145875 (2021)). Preliminary data reported in the abstract of the conference show that nucleoside transporters also play a similar role in the uptake of 4H2 by Jurkat cells (Andersen et al, J invest med 57 (1): 168 (2009)). Experiments were designed to investigate the dependence of 4H2 transport on nucleoside transport by treating Cal12T cells with the transport inhibitor Dipyridamole (DP) and examine its effect on the subsequent 4H2 penetration cell efficiency. The penetration of 4H2 was reduced to 0.290.03 (P < 0.0001) with DP relative to no DP (the image in fig. 1C was quantified by ImageJ). This suggests that 4H2, like 3E10, is transported into cells using a mechanism that relies on nucleoside transporters.

Example 4 binding of 4H2 to RNA in cells

Both 3E10 and 4H2 are anti-DNA antibodies isolated from the same lupus model, and they both utilize nucleoside transport to penetrate cells. However, their localization pattern within the cell is different (3E 10: nucleus, 4H2: cytoplasm). It was initially thought that 4H2 simply cannot pass through the nuclear membrane and would bind to nuclear DNA if 4H2 were able to contact it. If correct, a comparison between 4H2 and 3E10 may help to further elucidate the mechanism by which 3E10 passes through the nuclear membrane. EO771 cells were pre-fixed with frozen ethanol to expose the cytoplasm and nuclear antigen, and the IgG controls, forms of IgG1 Deoxymab, 3E10 (also referred to herein as "Deoxymab-3" and "DX3" (Shirali et al ,DNA-targeting and cell-penetrating antibody-drug conjugate,bioRxiv.doi:doi.org/10.1101/2023.04.12.536500)), or 4H 2. The results show that control IgG binds very little to the cells while DX3 shows specific nuclear binding. However, the 4H2 signal is still predominantly present in the cytoplasm, although it can be exposed to nuclear content after pre-fixation in ethanol).

An important difference between the images of live EO771 cells treated with 4H2 and pre-fixed EO771 cells suggests that the localization of 4H2 in live cells is cytoplasmic rather than nuclear. No 4H2 signal was detected in the nuclei of living cells, but discrete 4H2 signal clusters were observed in the nuclei of fixed cells. The focal distribution region of these 4H2 signals in the nucleus is consistent with the results of 4H2 binding to nucleoli (Pederson et al Cold Spring Harb Perspect biol.3, a000638 (2011)). Binding of 4H2 to the cytoplasm and nucleolus increases the likelihood that 4H2 preferentially binds RNA over DNA in cells. Consistent with this, the binding pattern of anti-RNA IgG to pre-immobilized EO771 cells was very consistent with the observed 4H2 pattern. To confirm binding of 4H2 to RNA in the cells, EO771 cells were pre-fixed in frozen ethanol and incubated with IgG control or anti-RNA IgG antibodies (to block the RNA binding site) followed by incubation with FITC-labeled 4H 2. anti-RNA IgG (rather than the IgG control) interfered with the binding of 4H2-FITC to the cytoplasm and nucleolus, confirming that 4H2 bound to RNA in the cells. Based on these findings, 4H2 binding to RNA is thought to be responsible for its retention in the cytoplasm.

EXAMPLE 5GUO enhances cell penetration by 4H2

Extracellular DNA/nucleosides promote cell penetration by 3E10 (Weisbart, et al, sci Rep5:12022 (2015), chen, et al, oncotarget 7 (37): 59965-59975 (2016)), and promote localization into necrotic tumors, releasing DNA. This property, combined with its ability to cross the Blood Brain Barrier (BBB), constitutes the theoretical basis for the continued development of enhanced 3E10 fragments (e.g., DX 1) for brain tumor therapy (Rattray et al, JCI Insight 6 (14): E145875 (2021)). After recognizing that 4H2 employs a cell penetration mechanism similar to 3E10, experiments were designed to determine if the addition of nucleosides, particularly GUO based on known binding epitopes of 4H2, would enhance the cell penetration of 4H 2. Human U87 glioma and mouse glioma stem cell-like cells (GSCs) were treated with PBS control, igG control, DX1 or 4H2, and the penetrability of the antibodies was detected with immunofluorescence. DX1 is localized to the nuclei of U87 and GSC and 4H2 is localized to the cytoplasm. The control IgG had no significant penetration of any cells. DX1 penetration of GSC nuclei was enhanced to 1.7±0.01 (P < 0.0001) after addition of Adenosine (ADE) compared to DX1 penetration in Adenosine (ADE) deficient medium (fig. 2A). In contrast, ADE did not improve 4H2 penetration, with a penetration of 0.97±0.16 (ns) observed relative to the case without ADE (fig. 2B). However, the penetration of 4H2 increased to 3.2±0.3 (P < 0.0001) after addition of GUO compared to that when no GUO was added (fig. 2C). These findings are consistent with the cell penetration mechanism of 4H2 through nucleoside transporter dependence and GUO responsiveness.

Example 6 Transwell model for 4H2 crossing the BBB in a nucleoside transporter dependent manner

Materials and methods

Transwell model of BBB

The ability of 4H2 to cross the BBB Transwell model was tested using the previously described protocol (Rattray et al, JCI Insight 6 (14): e145875 (2021)). Briefly, hCMEC/D3 brain endothelial cells and Normal Human Astrocytes (NHA) were adhered to the apical and basal sides of cell culture inserts (MilliporeSigma 353095) coated with fibronectin (MilliporeSigma F1141) and poly L-lysine (MilliporeSigma P4832), respectively. The formation of a functional barrier was confirmed as previously described (REF) and the BBB model was treated with control buffer or 50mM DP for 30min, then with 5mM 4H 2.+ -. 50mM DP. The relative cross-barrier permeation of 4H2 at 15 and 30 minutes in the presence or absence of DP was assessed by ImageJ quantification of anti-mouse IgG dot blots on the basal side chamber contents.

Results

Nucleoside translocation facilitates localization across the BBB and brain tumors by 3E10 (Rattray et al, JCI Insight 6 (14): E145875 (2021)). In view of the similarity in cell penetration mechanisms, experiments were designed to determine if 4H2 could also cross the BBB and penetrate brain tumors. Consistent with this, 4H2 successfully passed the Transwell model of the BBB, moving from the apical chamber to the basal-side chamber. Treatment of the BBB with the nucleoside transport inhibitor Dipyridamole (DP) prior to use of 4H2 reduced antibody transport, indicating a crossover of nucleoside transport dependencies (fig. 2D).

Example 7 localization of 4H2 to in situ GBM, increased recruitment of TIL, prolonged in vivo survival time

Materials and methods

In situ GBM research

The study was performed according to the protocol approved by IACUC at the university of jelutong. In situ GBM tumors were established in female C57/BL6 mice of 5-6 weeks of age by stereospecific injection of 50,000 cells (GSC or GL261 engineered to express luciferase) using the method previously described (Rattray et al, JCI Insight 6 (14): e145875 (2021)). In GSC studies, mice confirmed by IVIS for tumor formation were randomized and received either weekly tail vein injection of IgG control (40 mg/kg tail vein) or 4H2 (40 mg/kg tail vein) for three weeks, and received the IgG control (40 mg/kg tail vein), 4H2 (40 mg/kg tail vein), anti-PD 1 (5 mg/kg IP) and combinations thereof as described in GL261 results. Mice were closely monitored throughout and after treatment, and mice at the endpoint of nervous system changes or weight loss were humanly euthanized. KAPLAN MEIER survival maps and median survival were generated using GRAPHPAD PRISM 9.4.1 th edition. Antibody localization studies were performed, tumor and normal tissues were collected 24 hours after a single intravenous injection of IgG control or 4H2 (40 mg/kg), fixed with 10% neutral buffered formalin, and paraffin embedded. The presence of antibodies was detected by Immunohistochemistry (IHC) with anti-mouse IgG-HRP (1:50) using the protocol previously described (Rattray et al, JCI Insight 6 (14): e145875 (2021)). The signal was visualized by counterstaining with DAB and methyl green.

Results

Mice confirmed by IVIS to have in situ GSC-derived brain tumors were injected via the tail vein with a single dose of IgG control or 40mg/kg of 4H 2. Tumor and normal tissues were collected after 24 hours and assessed for the presence of antibodies by IHC. After 4H2 treatment, significant antibody staining was detected in the cytoplasm of GBM tumor cells, whereas IgG control was not. No antibody staining was detected in normal brain tissue distant from the tumor after treatment of mice with IgG control or 4H 2. In normal tissues, localization of 4H2 was increased in the kidney compared to IgG control, but the two antibodies were similarly distributed in skeletal muscle.

Mice bearing in situ GSC-derived GBM tumors were randomized into IgG control groups (4, n=4) and 4H2 groups (5, n=5), injected with 40mg/kg weekly tail vein for three weeks, and monitored for toxicity and survival. No adverse reaction was observed. The median survival rate was improved by 66% for 4H2 compared to mice treated with IgG control group (< 0.01 for P, log rank test). In the group treated with 4H2, the survival rate at the completion of the study was 40% and the survival rate of the IgG control group was 0% (fig. 3A, table 1).

TABLE 1 median survival and survival at completion of GSC-derived in situ GBM tumor study in IgG control or 4H2 treated C57/BL6 mice

Treatment group	Median survival time (Tian)	Survival rate of 100 days%
			IgG	38.5	0
4H2	64	40

In the second GBM model, mice bearing GL261 in situ GBM tumors were randomized into IgG control (6), 4H2 (6), anti-PD 1 (6), anti-pd1+igg control (7) and anti-pd1+4h2 (7) once weekly for three weeks of treatment and monitored for toxicity and survival. No adverse reaction was observed. The median survival of 4H2 was improved by 32% compared to the IgG control group (×p=0.03, log rank test). When used in combination with anti-PD 1, 4H2 increased median survival by 50% compared to the anti-pd1+igg control group (×p=0.02, log rank test). Survival rates were 33% and 29% after study completion for the 4H2 or 4H2+ anti-PD 1 treatment group alone, respectively, while survival rates were 0% for the other groups (fig. 3B, table 2). These results demonstrate the efficacy of 4H2 as a single agent in combination with anti-PD 1 for the treatment of GL261 GBM tumors.

Table 2 median survival and percentage at study completion for GL 261-derived in situ GBM tumors of C57/BL6 mice treated with IgG control, 4H2 or anti-PD 1 combinations.

Treatment group	Median survival time (Tian)	Survival rate of 70 days%
			IgG	20.5	0
4H2	27	33
			Anti-PD 1 antibodies	26.5	0
Anti-PD 1 antibody+IgG	24	0
			Anti-PD 1 antibody +4H2	36	29

Mechanistically, the prolongation of survival by 4H2 may be the result of cGAS-mediated GBM tumor cell senescence/toxicity, cGAS-mediated immune response enhancement, or a combination of both. TUNEL and CD8+ T cell staining evaluations were performed on mice tumors (4 and 3 IgG control and 4H2 groups, respectively) meeting euthanasia criteria. Compared to tumors of IgG control treated mice, 4H2 treatment correlated with an increase in tumor TUNEL signaling and cd8+ T cell content by 4.5±0.6 and 1.5±0.2 fold (P < 0.05), respectively (fig. 4A, 4B). Treatment with 4H2 increased CD8 content in tumors by 53% compared to IgG control group, relative content of 1.53±0.15 (< 0.03) fig. 4B. This suggests that 4H2 leads to an increase in TIL and indicates the presence of immune-mediated components in the response to 4H 2.

To test the importance of the immune system in mediating 4H2 anti-tumor effects, athymic nude mice bearing PPQ GBM tumors intracranially were randomized, treated 1 or 2 times weekly with IgG control groups (4 and 6, respectively) or 4H2 groups (4 and 6, respectively), and survival was measured. No adverse reaction of 4H2 was observed. The median survival was not significantly improved by 4H2 compared to the IgG control group, and the survival at the end of the study was 0% for all groups (fig. 4C, 4D, tables 3, 4). These results indicate that the anti-tumor effect of 4H2 in vivo is dependent on T cells in the functional immune system.

TABLE 3 median survival and survival at the completion of the athymic nude mice study on PPQ in situ GBM tumors treated weekly with IgG control or 4H 2. Compared to the IgG control group, 4H2 did not change survival (p=ns, log-rank test).

Treatment group	Median survival time (Tian)	Survival rate of 20 days%
			IgG control	11.5	0
4H2	12	0

Table 4 median survival and survival at the completion of the athymic nude mice study on either the twice weekly IgG control or 4H2 treatment of PPQ in situ GBM tumors. Compared to the IgG control group, 4H2 did not change survival (p=ns, log rank test).

Treatment group	Median survival time (Tian)	Survival rate of 20 days%
			IgG control	8.5	0
4H2	12	0

Example 8 binding of 4H2 to cGAS in a nucleic acid dependent manner

Materials and methods

GSC pull-down test (pulldown)

After GSC treatment with 1mg/mL IgG control or 4H2 for one hour, the content was washed and collected using the NE-PER ^TM core and cytoplasmic protein extraction kit (Thermo Fisher, # 78833). 50mL of 50% protein G bead slurry was added to the 500mL final extraction volume and incubated at 4℃for 1.5 hours with rotation. The beads were washed and the remaining binding protein eluted with 40mL SDS loading buffer. Ras or cGAS was detected by western blotting with 1:1000 rabbit anti-murine cGAS (CELL SIGNAL ING # 31659) and 1:5000 goat anti-rabbit HRP secondary antibody (abcam ab 205718) and samples were analyzed.

CGAS binding studies

2 Mg of recombinant human cGAS (Cayman) were incubated with 8 mg of IgG control or 4H2 nucleic acid (1.25 mg of PvuII-digested pcDNA3 and 50mM GTP) in 500mL of PBS containing 0.01% Triton X-100 at 4℃for one hour with rotation. 50mL of 50% protein G bead slurry was added to the reaction and incubated at 4℃for one hour with rotation. The beads were removed, washed five times with PBS+0.05% Triton X100 and the binding proteins eluted with 50mL of mild elution buffer (fisher). Samples were subjected to western blot analysis with either rabbit anti-human cGAS monoclonal antibodies (CELL SIGNALING, # 15102) or HRP-conjugated horse anti-mouse IgG (CELL SIGNALING, # 7076).

Detection result

Cyclic GMP-AMP synthetases (cGAS) are cytoplasmic nucleic acid sensors that play a central role in innate immunity. When activated, cGAS produces cyclic GMP-AMP (cGAMP) to promote interferon gene Stimulator (STING) signaling and type I interferon response (18). With respect to the mechanisms by which 4H2 brings survival benefits in GBM models, the mainstream theory is mainly focused on both cell signaling pathway interference and immune response enhancement. Antibody pulldown analysis was performed after GSC treatment with IgG control or 4H2 to explore the interaction between 4H2 and Ras or cGAS. Ras is selected as a target based on its role in cell signaling as a key G protein, while cGAS is selected as a target based on its function as a cytoplasmic nucleic acid sensor that catalyzes GTP formation cGAMP. No 4H2-Ras interaction was observed (fig. 5A), but cGAS produced a stronger pulldown effect under the action of 4H2 compared to IgG control (fig. 5B). Binding of 4H2-cGAS was further assessed by incubating purified cGAS with IgG controls or 4H2 and testing for competitive inhibition of binding by added nucleic acid. Binding of 4H2 to cGAS was demonstrated and in the presence of nucleic acid, binding of 4H2 to cGAS was reduced, consistent with competitive inhibition. IgG control binding to background non-specific cGAS was not affected by the presence of nucleic acid (fig. 5c,5 d).

This result suggests that 4H2 may interact with cGAS in the cytoplasm by binding to intermediate nucleic acids (e.g., endogenous mRNA) or to nucleic acids that are transported into the cell after 4H2 binding. Purified recombinant cGAS was incubated with control IgG or 4H2 +/-nuclease Benzonase (Benzonase) to degrade any nucleic acid bound to 4H2 and the antibodies and their interacting proteins were isolated with protein G. The binding of 4H2 to purified cGAS was stronger than the protein G/IgG control, and nuclease addition reduced the interaction of 4H2 with cGAS, but did not reduce the binding of the non-specific protein G/IgG control to cGAS (FIGS. 5D-5F). These findings indicate that the 4H2-cGAS interface is dependent on the presence of nucleic acids.

Example 9 4H2 enhances the Activity of cGAS

Materials and methods

CGAS Activity assay

The effect of IgG control or 4H2 (0-160 mg) on the production of relative cGAMP was tested using a cGAS inhibitor screening test kit (# 701930, cayman) according to the manufacturer's instructions.

NF-kB test

GSC was treated with 1mg/ml IgG control IgG or 4H2 in growth medium (DMEM+10% FBS) for 18 hours. Cytoplasmic and nuclear protein fractions were extracted using NE-PER ^TM nuclear and cytoplasmic protein extraction kit (Thermo Fisher, # 78833) and evaluated using NF-kB (CELL SIGNALING, # 8242) western blot, lamin B1 as loading control.

CGAS knockout and colony formation assay

GSCs (glioma stem cells) cultured in 6-well plates were transfected with RNAiMax (Thermo Fisher) either with 100nM control siRNA or CGAS SIRNA (Dharmacon), and the efficiency of cGAS knockout was verified by Western blot two days after transfection. Cells were then treated with IgG control or 4H2 (0-1.6 mM) and the colony survival was assessed by colony formation experiments.

Results

The activated cGAS catalyzes the formation of cGAMP from the precursor molecules ATP and GTP. The effect of 4H2 on cGAS activity in vitro was assessed by measuring the relative cGAMP production of cGAS in the presence of IgG control or 4H 2. 4H2 increased the yield of cGAMP by 83% + -18 compared to the IgG control (FIG. 6A). cGAMP produced by cGAS promotes nuclear translocation through NF-kB, and experiments aimed at studying the effect of 4H2 on NF-kB nuclear content in IgG control group or GSC after 4H2 treatment by western blot. NF-kB nuclear content was increased in 4H2 treated cells compared to the IgG control (FIG. 6B). Finally, GSC was knocked out cGAS by siRNA as confirmed by western blot (fig. 6C). Control and cGAS knockdown GSCs were treated with IgG control or 4H2 (0-1.6 mM) and evaluated by colony formation assays. cGAS knockout significantly reduced GSC sensitivity to 4H2, suggesting cGAS-dependent toxicity (fig. 6D). Survival in control or cGAS knockdown PPQ cells treated with 1.6 μm 4H2 was 0.16±0.09 and 0.46±0.07 (P < 0.05), respectively (fig. 6D). Western blot of the cytoplasmic and nuclear contents of PPQ cells also showed a 2.2±0.2 fold increase in NF- κb nuclear content in 4H2 compared to IgG control treated cells (P < 0.05) (fig. 6E), consistent with 4H2 mediated cGAS activation.

Example 10 delivery of nucleic acids to cells by 4H2

Materials and methods

DNA binding assay

0.5Mg of circular or linearized pcDNA3 plasmid DNA (5.4 kb) was incubated with 10Mg of IgG control or 4H2 in 50ml of buffer (PBS or binding buffer with 10mM Mg or 1mM EDTA) for one hour at room temperature. Samples were evaluated by EMSA on 1% agarose gel stained with SYBR ^TM Green I (Thermo Fisher).

RNA binding assay

0.2Mg of total RNA or GFP mRNA was incubated with control IgG or 4H2 (0-4 mg) for one hour at room temperature. Samples were subjected to EMSA evaluation on 1% agarose gel stained with SYBR ^TM Green I (Thermo Fisher).

Detection result

CGAS is a cytoplasmic nucleic acid sensor that initiates an innate immune response. The findings above indicate that 4H2 interacts with cGAS and promotes its activity. The specific details of this interaction are not yet clear and may involve direct binding, or indirect binding by co-binding with a GUO-containing nucleic acid that is brought into the cytoplasm by 4H 2. With this possibility in mind, experiments were designed to explore the ability of 4H2 to bring exogenous DNA and RNA into cells. Binding of 4H2 to circular and linearized plasmid DNA and total DNA and mRNA has been confirmed by EMSA (fig. 7A, 7B). Luciferase expression plasmids mixed with 4H2 or DX1 were added to the cultured U87 glioma cells and luciferase signals were measured after 24 hours. Luciferase signal was minimal in cells treated with dx1+ plasmid, whereas the signal generated by 4h2+ plasmid was much stronger (fig. 8A). Luciferase mRNA was entrapped in lipid nanoparticles (MC 3-LNP) or mixed with DX1 or 4H2, added to cultured U87 glioma cells and luciferase signal was measured after 24 hours. 4H2+mRNA and mRNA-loaded MC3-LNPs produced similar luciferase signals, whereas DX1+ mRNA showed no apparent signal (FIG. 8B).

Example 11 4H2 mediated local Gene delivery in vivo

Materials and methods

Nucleic acid delivery assay

For DNA, pGL4.13 (luc 2/SV 40) plasmids were incubated with DX1 or 4H2 and added to the cultured U87 glioma cells. Luciferase activity was measured after 24 hours. For RNA, luc mRNA was incubated with DX1 or 4H2, or encapsulated into MC3-LNP lipid nanoparticles. Samples were added to the cultured U87 glioma cells. Luciferase activity was measured after 24 hours.

Delivery of mRNA to brain and retina

The study was performed following the protocol approved by the university of yards IACUC. Female Ai9 Cre reporter mice (jackson laboratories) 5-6 weeks old were treated with intracranial or intraocular injection of 4H2/Cre mRNA (w/w 3). Brains and eyeballs were collected twenty-four hours later and sectioned for immunofluorescence. The activity of the functional Cre recombinase was detected by visualizing RFP fluorescence.

Delivery of mRNA to tumors

The study was performed following the protocol approved by the university of yards IACUC. H358 tumors were produced by subcutaneous injection into the flank of 5-6 week old female nude mice using the protocol previously described (Chen et al Oncotarget (37): 59965-59975 (2016)). Caliper measurements were performed after tumor formation. When the tumor reached about 100 cubic millimeters, mice were subjected to intratumoral injection of a mixture of luciferase (Luc) mRNA and DX1 or 4H2 at a weight ratio (w/w) of 3. Luciferase signal was visualized by IVIS as previously described (Rattray et al, JCI Insight 6 (14): e145875 (2021)).

MRNA is delivered to skeletal muscle.

The study was performed following the protocol approved by the university of yards IACUC. Female C57/BL6 mice of 5-6 weeks of age were intramuscular injected with a mixture of luciferase (Luc) mRNA and DX1 or 4H2 at a weight ratio (w/w) of 3 (left quadriceps) or 1 (right quadriceps). Luciferase signal was visualized by IVI S as previously described (Rattray et al, JCI Insight 6 (14): e145875 (2021)).

Experimental results

Compared to the IgG control group, the systemically administered 4H2 showed increased localization of in situ brain tumors, but no increase in localization of normal brain (fig. 3A, 3B). This is believed to reflect that DNA/nucleosides released by necrotic GBM tumors promote tumor localization of 4H2 via nucleoside transporters. Nucleoside transporters are also expressed in normal brain (Chang et al Acta neuropathol Commun 9:112 (2021)), designed experiments to test whether direct injection of a mixture of 4H2 and nucleic acid would promote uptake to produce local gene expression. RFP was generated in tissues following Cre recombinase expression using engineered Ai9 mice to examine 4H2 mediated gene delivery in the brain. The 4h2+cre mRNA was injected into the mouse brain and RFP signal was measured after 24 hours. RFP signals reflecting Cre activity were detected along the injection trajectory in the brain (fig. 9A). In another experiment, ai9 mice were treated by intraocular injection of 4H2+Cre mRNA, visualization of RFP signal after 24 hours, showing strong Cre activity in the retina, consistent with the expression of known retinal nucleoside transporters (Dos Santos-Rodrigues et al Vitam Horm 98:487-523 (2015)), demonstrating 4H2 mediated retinal gene therapy (FIG. 9B).

Delivery of mRNA by 4H2 to extracranial tissues was evaluated in tumors and skeletal muscle where large numbers of expressed nucleoside transporters are known. Nude mice were treated with intratumoral injection of dx1+ luciferase mRNA or 4h2+ luciferase mRNA for subcutaneous H358 flank tumors and luciferase expression was monitored by continuous IVIS. Tumors treated with dx1+ luciferase mRNA did not detect a signal, but 4h2+ luciferase mRNA produced strong and durable signals in tumors at 6, 24, and 72 hours (fig. 10A). No significant signal was observed outside the tumor. In another experiment, after injection of 4H2+ luciferase mRNA into quadriceps in non-tumor bearing C57/BL6 mice, luciferase signals localized to the injected muscle were shown at 6 and 24 hours (FIG. 10B).

Example 12 4H2 as Gene delivery vehicle for the treatment of NF2

Mouse xenografts were established by seeding the sciatic nerve with HEI193 cells expressing luciferase. After 17 days, mice were imaged using IVIS. Mice were divided into 4 groups based on luciferase expression, and treated with physiological saline, 4H2 alone, 4H2 with NF2 cDNA plasmid, and direct injection of 4H2 mRNA, respectively. The second treatment was performed on day 42 after tumor inoculation. Tumor growth was monitored by IVIS. Fig. 11A shows representative In Vivo Imaging (IVIS) images, and fig. 11B is a graph of fluorescence intensity over time. 4h2+ dna and 4h2+ mrna reduced tumor growth compared to untreated controls and 4H2 alone, further demonstrating the ability of 4H2 to treat disease by delivering therapeutic nucleic acids.

Example 13 use of 4H2-CD5 bispecific antibody fragments for targeted delivery of genes to T cells

FIGS. 12A-12C illustrate the design and use of 4H2-CD5 bispecific antibody fragments for targeted gene delivery to T cells

4H2 sequence

VL:

DIVLTQSPATLSVTPGDRVSLSCRASQSISNYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSIISVETEDFGMYFCQQSNSWPLTFGAGTKLELK(SEQ ID NO:1)

VH:

EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGRVNPSNGGISYNQKFKGKATLTVDKSLSTAYMQLNSLTSEDSAVYYCARGPYTMYYWGQGTSVTVSS(SEQ ID NO:5)

CD5 sequence

VL:

NIVMTQSPSSLSASVGDRVTITCQASQDVGTAVAWYOQKPDQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTISSLOPEDIATYFCHQYNSYNTFGSGTKLEIK(SEQ ID NO:23)

VH:

QVTLKESGPVLVKPTETLTLTCTFSGFSLSTSGMGVGWIRQAPGKGLEWVAHIWWDDDVYYNPSLKSRLTITKDASKDQVSLKLSSVTAADTAVYYCVRRRATGTGFDYWGQGTLVTVSS(SEQ ID NO:24)

FIG. 12A is a schematic representation of the design of a 4H2-CD5 bispecific antibody fragment. The Ai9 mouse model can measure Cre recombinase delivery by detecting DeRed expression. The Ai9 mice carrying MC38 tumor were intratumorally injected with control, 4h2+cre mRNA or 4H2-CD5 bispecific antibody+cre mRNA. After 48 hours, mice were euthanized. Tumors are isolated and dissociated into single cells. Flow cytometry was used to analyze the expression of DeRed in T cells within tumors. T cells in tumors treated with 4H2-CD5+ Cre mRNA showed a higher DeRed signal compared to control or 4H2+ Cre mRNA. Representative FACS plots are shown in fig. 12B, and combinatorial analysis is shown in fig. 12C. These experiments demonstrate a 4H 2-based bispecific antibody fragment for use in a strategy for delivering nucleic acids to specific cells by cell penetration and nucleic acid binding properties of 4H2, in combination with homing specificity provided by a second antibody component (in this case CD5 for T cells).

Example 14 4H2 binding and activating toll-like receptor 7 (TLR 7)

TLR7 is an intracellular recognition receptor that recognizes RNA and initiates an innate immune response. TLR7 is particularly sensitive to GU-rich RNA reactions. 4H2 is an anti-G autoantibody. Experiments were aimed at determining whether 4H2 interacted with TLR7 in a nucleic acid dependent manner, similar to the results of 4H2-cGAS interactions described above.

Cell lysates of glioma stem cell-like cells (GSCs) treated with IgG control or 4H2 detected TLR7 by western blot. The band representing activation of TLR7 cleavage was significantly increased in cells treated with 4H 2. Representative blots are shown (fig. 13A) and the amount of cleaved TLR7 measured with ImageJ relative to IgG control is shown (fig. 13B). These results indicate that 4H2 can induce cleavage of TLR7.

Antibodies and binding proteins were pulled from IgG control or 4H2 treated GSC lysates with protein G beads and then western blot analysis with TLR7 (fig. 13C). In this assay, 4H2 (rather than the IgG control) has a strong binding force to cleaved TLR7, as demonstrated in this assay from its pulldown effect with 4H 2. The blots are representative of two independent experiments. These results indicate that 4H2 binds to cleaved TLR 7.

This adds another layer to the use of 4H2 as an immunostimulant, as 4H2 can activate cGAS and TLR7 simultaneously. TLR7 agonists have been sought for use in immunotherapy, but 4H2 can activate cGAS and TLR7 simultaneously, which makes it different from other agonists that can only activate TLR7 or cGAS but not both.

Summary

This study revealed previously unknown interactions between lupus anti-GUO autoantibodies and cGAS. In particular, the cell penetrating lupus anti-GUO autoantibody 4H2 can localize to the cytoplasm and avoid endosomes, bind to nucleic acids and deliver them to cells and tissues, activate cGAS, produce cGAS-dependent toxicity to glioma cells, and increase survival in the in situ GBM model. These findings provide an opportunity for the use of anti-GUO autoantibodies in biotechnology and suggest the possibility that such antibodies could contribute to cGAS activation and type I interferon characteristics associated with Systemic Lupus Erythematosus (SLE).

The inhibition of the nucleoside transport inhibitor DP and the enhancement of 4H2 penetration by free GUO indicate that cell penetration of 4H2 is dependent on the nucleoside transporter. In agreement with this, systemically administered 4H2 is able to localize in DNA/nucleoside releasing brain tumors, but not in normal brain tissue. However, when 4H2 is injected directly with mRNA as a complex, the gene is delivered to the normal brain. Likewise, 4H2/mRNA can produce localized gene expression in retina and extracranial target tissues (including tumors and skeletal muscle) following intraocular injection. Taken together, these findings correlate cell penetration of 4H2 with nucleoside transport and establish 4H2 as a non-covalent cytoplasmic delivery ligand for nucleic acids.

4H2 and 3E10 were isolated from the same lupus model, and it appears that they all have a cell-penetrating and BBB-traversing nucleoside transporter-dependent mechanism, but at the same time they are located in completely different cellular compartments. Both avoid endosomes and lysosomes, but 3E10 localizes to the nucleus, while 4H2 localizes to the cytoplasm. The reason for this difference is currently unknown, but this may explain why 4H2 is more capable of delivering functional mRNA to cells than 3E10, because translation of mRNA is performed in the cytoplasm rather than the nucleus.

The exact mechanism by which 4H2 activates cGAS and TLR7 is not known. Possible mechanisms include direct binding and activation of 4H2, or indirect binding through simultaneous interactions between cGAS and TLR7, 4H2, and cytoplasmic nucleic acids and/or GTP. In summary, the 4H2 nucleoside transporter-dependent cytoplasmic penetration method, which is capable of delivering nucleic acids to cells and activating cGAS and TLR7, is an attractive formulation for use as an immunostimulant in tumors, as a non-covalent cytoplasmic delivery ligand for nucleic acids in gene therapy, and for simultaneous gene delivery and activation of immune receptors (e.g., cGAS and TLR 7) in vaccine design to enhance responses.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All documents cited herein and the materials to which they refer are expressly incorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (91)

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, 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.