Drugs and methods for targeted delivery to cells

Abstract

This invention provides drugs and methods for the targeted delivery of payloads to cells. These drugs and methods are useful for delivering therapeutic or diagnostic agents to target cells. [Solution] In one embodiment, the present invention includes the step of administering RNA encoding a peptide or polypeptide (docketing compound) comprising a binding portion that binds to a target cell (primary targeting portion) and a further binding portion (secondary target) that binds to a drug comprising a payload (effector probe). After RNA expression, the primary targeting portion may bind to a target antigen such as a cancer antigen on a cancer cell, and then the secondary targeting portion contained in the effector probe may target the secondary target, thereby accurately delivering the "payload" to the target cell such as a cancer cell.

Description

[Technical Field]

[0001] The present invention relates to drugs and methods for targeted delivery of payloads to cells. The drugs and methods are useful for delivering therapeutic or diagnostic agents to target cells. In one embodiment, the present invention includes the step of administering RNA encoding a peptide or polypeptide (docketing compound) comprising a binding portion that binds to a target cell (primary targeting portion) and a further binding portion (secondary target) that binds to a drug comprising a payload (effector probe). After RNA expression, the primary targeting portion may bind to a target antigen such as a cancer antigen on a cancer cell, and then the secondary targeting portion contained in the effector probe may target the secondary target, thereby accurately delivering the “payload” to the target cell, such as a cancer cell. [Background technology]

[0002] In many areas of pharmacotherapy and diagnosis, it is desirable to selectively deliver therapeutic drugs (medicines) or diagnostic drugs (e.g., imaging agents) to specific sites or limited areas within the body of a target, such as a patient.

[0003] Active targeting of an organ or tissue can be achieved by directly or indirectly conjugating a desired active moiety (e.g., a cytotoxic compound) to a targeting construct that binds to the cell surface at the target site of interest. The targeting moiety used to target such a drug is typically a construct having affinity for a cell surface target, such as a membrane protein, and includes an antibody or antibody fragment. [Overview of the project] [Problems that the invention aims to solve]

[0004] The present invention relates to an approach using RNA-encoded docking compounds that label target cells, for example, by binding to a primary target (e.g., a cell surface antigen). The docking compound comprises a secondary target, which is ultimately targeted by a further compound, i.e., an effector probe, having a moiety that targets the secondary target. The effector probe comprises an effector moiety, e.g., a therapeutic and / or diagnostic compound, or a target moiety of a therapeutic and / or diagnostic compound, e.g., an effector cell. Thus, according to the present invention, RNA encoding a docking compound is administered. After the RNA is expressed, the docking compound may bind to target cells, for example, by binding to a primary target. An effector probe that binds to the secondary target on the docking compound via the secondary targeting moiety of the docking compound is added. A common example of a secondary target / secondary targeting moiety pair is an antibody / antigen system. The concepts described herein enable the use of a single effector compound to target a wide range of target cells, i.e., by using a single effector compound in combination with different docking compounds that target different primary targets and contain the same secondary target. The concept described herein is even more advantageous because primary targeting using a single docking compound can be performed in combination with different effector probes that target the same secondary target and contain different effector moieties. Therefore, the concept described herein enables sequential targeting of different effector moieties to target cells labeled with docking compounds. [Means for solving the problem]

[0005] In one aspect, the present invention is a method for targeted delivery of a payload to target cells, (i) Transfecting one or more cells with RNA encoding a peptide or polypeptide containing the first binding site; (ii) expressing a peptide or polypeptide in one or more cells such that the peptide or polypeptide associates with target cells and a first binding site is presented on the surface of the target cells; and (iii) Adding a payload that includes or is connected to a second coupling portion. The present invention relates to a method comprising a first bonding portion and a second bonding portion being bonded to each other.

[0006] In one embodiment, RNA is transfected into one or more cells by bringing them into contact with RNA-containing particles.

[0007] In one embodiment, the particles contain targeting molecules for targeting one or more cells.

[0008] In one embodiment, one or more cells include or consist of target cells.

[0009] In one embodiment, one or more cells express a peptide or polypeptide containing a first binding site, such that it remains associated with one or more cells.

[0010] In one embodiment, one or more cells are different from the target cells.

[0011] In one embodiment, one or more cells express a peptide or polypeptide containing a first binding site, such that it is secreted by one or more cells.

[0012] In one embodiment, one or more cells express a peptide or polypeptide containing a first binding site, such that it is released into the bloodstream.

[0013] In one embodiment, the peptide or polypeptide comprising the first binding site comprises a third binding site that binds to a target on a target cell.

[0014] In one embodiment, the target is a cell surface antigen.

[0015] In one embodiment, the first joint and the third joint are connected to each other. In another embodiment, the first joint and the third joint are covalently connected to each other.

[0016] In one embodiment, the first binding site is an antibody or antibody derivative. In another embodiment, the second binding site is a peptide tag.

[0017] In one embodiment, the first binding site is a peptide tag. In another embodiment, the second binding site is an antibody or antibody derivative.

[0018] In one embodiment, the third binding site is an antibody or an antibody derivative.

[0019] In one embodiment, the antibody derivative is an antibody fragment.

[0020] In one embodiment, the peptide or polypeptide is a bispecific antibody. In one embodiment, the bispecific antibody is a bispecific single-chain antibody.

[0021] In one embodiment, the second binding portion and the payload are covalently or noncovalently linked to each other. In one embodiment, the payload contains a pharmaceutically active drug. In one embodiment, the payload contains a diagnostic compound. In one embodiment, the payload contains a therapeutic compound. In one embodiment, the payload contains a carrier. In one embodiment, the carrier is a microparticle carrier. In one embodiment, the microparticle carrier contains lipid-based particles, polymer-based particles, or a mixture thereof. In one embodiment, the carrier incorporates a diagnostic compound. In one embodiment, the carrier incorporates a therapeutic compound. In one embodiment, the payload contains a fourth binding portion. In one embodiment, the fourth binding portion binds to a cell surface antigen. In one embodiment, the cell surface antigen to which the fourth binding portion binds is present on an immune cell.

[0022] In one embodiment, the target cells are present in the target.

[0023] In one embodiment, the method described herein is carried out in vivo.

[0024] In one embodiment, the method described herein applies to the subject, (i) RNA encoding a peptide or polypeptide containing a first binding site, or particles containing said RNA; and (ii) A payload containing or ligated to the second junction, or RNA encoding it. The process includes administering [the substance].

[0025] In one embodiment, the methods described herein are for diagnosing and / or treating a disease, wherein target cells express or may express disease-related antigens.

[0026] In one embodiment, the target cells are disease cells.

[0027] In one embodiment, the target is a tumor antigen. In another embodiment, the target cell is a tumor cell or cancer cell.

[0028] In one embodiment, the target cells are immune effector cells. In another embodiment, the target cells are T cells. In another embodiment, the target is an antigen characteristic of the immune effector cells.

[0029] In one embodiment, the method described herein is for delivering nucleic acids encoding antigen receptors to immune effector cells.

[0030] In one aspect, the present invention is a method for targeted delivery of a payload to target cells in an object, (i) RNA encoding a peptide or polypeptide containing the first binding site; and (ii) A payload containing or ligated to the second junction, or RNA encoding it. The process includes administering the drug to the target, The first binding site and the second binding site are bound to each other, and the peptide or polypeptide containing the first binding site further includes a third binding site that binds to a target on a target cell. Regarding the method.

[0031] In one embodiment, RNA is present in the particles when administered.

[0032] In one embodiment, after RNA administration, the peptide or polypeptide containing the first and third binding sites is expressed by one or more target cells. In one embodiment, one or more cells secrete the peptide or polypeptide containing the first and third binding sites. In one embodiment, one or more cells express the peptide or polypeptide containing the first and third binding sites so that it is released into the bloodstream.

[0033] In one embodiment, the present invention is a kit for targeted delivery of a payload to target cells, (i) RNA encoding a peptide or polypeptide containing the first binding site; and (ii) A payload containing or ligated to the second junction, or RNA encoding it. Includes, This relates to a kit in which the first and second bonding parts connect to each other.

[0034] In one embodiment, the peptide or polypeptide comprising the first binding site comprises a third binding site that binds to a target on a target cell.

[0035] In one embodiment, the target is a cell surface antigen.

[0036] In one embodiment, the first joint and the third joint are connected to each other. In another embodiment, the first joint and the third joint are covalently connected to each other.

[0037] In one embodiment, the first binding site is an antibody or antibody derivative. In another embodiment, the second binding site is a peptide tag.

[0038] In one embodiment, the first binding site is a peptide tag. In another embodiment, the second binding site is an antibody or antibody derivative.

[0039] In one embodiment, the third binding site is an antibody or an antibody derivative.

[0040] In one embodiment, the antibody derivative is an antibody fragment.

[0041] In one embodiment, the peptide or polypeptide is a bispecific antibody. In one embodiment, the bispecific antibody is a bispecific single-chain antibody.

[0042] In one embodiment, the second binding portion and the payload are covalently or noncovalently linked to each other. In one embodiment, the payload contains a pharmaceutically active drug. In one embodiment, the payload contains a diagnostic compound. In one embodiment, the payload contains a therapeutic compound. In one embodiment, the payload contains a carrier. In one embodiment, the carrier is a microparticle carrier. In one embodiment, the microparticle carrier contains lipid-based particles, polymer-based particles, or a mixture thereof. In one embodiment, the carrier incorporates a diagnostic compound. In one embodiment, the carrier incorporates a therapeutic compound. In one embodiment, the payload contains a fourth binding portion. In one embodiment, the fourth binding portion binds to a cell surface antigen. In one embodiment, the cell surface antigen to which the fourth binding portion binds is present on an immune cell.

[0043] In one embodiment, RNA is present within the particle.

[0044] In one embodiment, the present invention relates to a drug or composition described herein for use in the method described herein. [Brief explanation of the drawing]

[0045] [Figure 1A] Sequences of Anti-ALFA and Anti-CLDN6 Bispecific Constructs The figure shows five sequences associated with four anti-ALFA and anti-CLDN6 bispecific constructs. Figure 1A shows, from top to bottom, a) a variable fragment of the heavy chain of the anti-claudin 6 antibody linked at the C-terminus to anti-ALFA-VHH with a C-terminal His tag via a GS linker, b) anti-ALFA-VHH linked at the C-terminus to the variable fragment of the heavy chain of the anti-claudin 6 antibody with a C-terminal His tag via a GS linker, and c) the complete heavy chain of the anti-claudin 6 antibody linked at the C-terminus to anti-ALFA-VHH with a C-terminal His tag via a GS linker. All sequences contain an N-terminal secretory signal or reader signal. Each sequence element is highlighted in the figure. [Figure 1B] Sequences of Anti-ALFA and Anti-CLDN6 Bispecific Constructs The figure shows five sequences associated with four anti-ALFA and anti-CLDN6 bispecific constructs. Figure 1B shows d) anti-ALFA-VHH linked at the C-terminus to the full heavy chain of an anti-claudin 6 antibody with a C-terminal His tag via a GS linker, and e) the light chain of a variable fragment of the anti-claudin 6 antibody. All sequences contain an N-terminal secretory signal or leader signal. Each sequence element is highlighted in the figure. [Figure 2] Sequences of Anti-ALFA and Anti-CD3 Bispecific Constructs The figure shows four sequences associated with four anti-ALFA and anti-CD3 bispecific constructs. Figure 2 shows, from top to bottom, a) anti-ALFA-VHH C-terminus linked to anti-CD3-VHH(F04) with a C-terminal His tag via a GS linker, b) anti-CD3-VHH(F04) C-terminus linked to anti-ALFA-VHH with a C-terminal His tag via a GS linker, c) anti-ALFA-VHH C-terminus linked to a single-strand variable fragment (scFv) of anti-CD3 antibody (TR66) with a C-terminal His tag via a GS linker, and d) single-strand variable fragment (scFv) of anti-CD3 antibody (TR66) C-terminus linked to anti-ALFA-VHH with a C-terminal His tag via a GS linker. All sequences contain an N-terminal secretory signal. Each sequence element is highlighted in the figure. [Figure 3] Overview of Synthetic ALFA Pigment Peptide: Three different conjugation reactions were tested to conjugate the synthetic ALFA peptide with the fluorophores Cy5 or Alexa Fluor 680 (AF680): (i) copper-free click chemistry, (ii) cysteine ​​maleimide conjugation, and (iii) conjugation via a low molecular weight PEG3 spacer. Three different constructs of Cy5-bound ALFA peptide (i) were established by click chemistry: Cy5-DBCO-azide-ALFA-NH2, Cy5-azide-FCO-ALFA-OH, and Cy5-azide-FCO-ALFA-NH2. [Figure 4A] Binding analysis of bispecific anti-CLDN6 and anti-ALFA constructs. Figure 4A shows schematic diagrams of bispecific constructs targeting ALFA tag and CLDN6. [Figure 4B] Binding Analysis of Bispecific Anti-CLDN6 and Anti-ALFA Constructs: In Figure 4B, CLDN6-overexpressing cells and target-negative cells were incubated with Cy5-ALFA or ALFA-AF680 peptide in the absence (without RiboDocker) or in the presence of ALFA bispecific targeted CLDN6 constructs (based on IMAB027 Fab or IgG constructs) and analyzed by FACS. [Figure 5] Binding Analysis of Bispecific Anti-CD3 and Anti-ALFA Constructs: CD3-overexpressing cells and target-negative cells were incubated with Cy5-DBCO-ALFA-NH2 peptide (at different concentrations) in the absence (without RiboDocker) or in the presence of ALFA bispecifically targeted CD3 constructs (based on TR66 scFv or VHH against CD3) and analyzed by FACS. [Figure 6]Binding Analysis of Bispecific Anti-CD3 (Administered as RNA) and Anti-ALFA Constructs RiboDockers for CD3 and ALFA peptides were constructed by electroporation of HEK-293T-17 cells. Different concentrations (2.5 μg, 25 μg) of RNA encoding aALFA-VHH×aCD3-VHH(F04) or aCD3-VHH(F04)×aALFA-VHH were used for electroporation of HEK293T-17 cells. After a specified time point, the supernatant was collected and used for binding analysis by FACS. Thus, target overexpression cells and target negative cells were incubated with 100 μL of supernatant, followed by incubation with the Cy5-DBCO-ALFA-NH2 peptide. As a negative control, the Cy5-DBCO-ALFA-NH2 peptide was incubated with cells in the absence of RiboDocker but in the presence of anti-ALFA VHH. As positive controls, purified proteins of aALFA-VHH X aCD3-VHH(F04) or aCD3-VHH(F04) × aALFA-VHH at two different concentrations (100 nM and 500 nM) were used. [Figure 7] Modular Bispecific Antibodies for Cancer Treatment The figure includes a schematic diagram of a cancer treatment approach using modular bispecific antibodies. Here, a first construct carrying a first binding site specific to a tag and a second binding site to a tumor-associated antigen is provided in the form of coding RNA. The RNA is formulated as lipid nanoparticles and administered to the patient. The RNA is translated in vivo into a bispecific protein and released into the bloodstream. A second construct containing the tag and a third binding site is administered to the patient (e.g., in the form of coding RNA, formulated as lipid nanoparticles), released into the bloodstream, and binds to the first construct. Depending on the specificity of the third binding site, the complex recruits other effectors, such as immune cells that engage the tumor. [Figure 8]Modular CAR-T Cell Approach The figure includes a schematic diagram of a universal CAR-T approach based on modular interaction pairs, in the example of ALFA tag / NbALFA. Here, a common and readily producible CART cell is created that carries a tag-binding moiety (e.g., NbALFA VHH) on its surface (1). A second binding moiety, a so-called targeted ligand (TL), consisting of the ALFA tag fused to a tumor antigen-specific ligand (e.g., scFv, VHH, or Fab fragment), is administered to the patient as RNA formulated in lipid nanoparticles (2). The RNA is translated in vivo into a bispecific protein that is released into the bloodstream. After the targeted ligand accumulates in the tumor based on its specific binding to a particular tumor antigen, it is bound by NbALFA-CAR T cells, resulting in activation and specific lysis of tumor cells (3). By using different targeted ligands, different tumor antigens can be addressed sequentially or in parallel using the same CART cell product from the patient. [Figure 9]RiboDocker was constructed for targeting CD3 and ALFA peptides by electroporation of HEK293T-17 cells using RiboDocker to target ALFA peptide-presenting nanoparticles. 25 μg of RNA encoding aCD3-VHH(F04)×aALFA-VHH was used for electroporation of HEK293T-17 cells. After 48 hours, 15 μL of supernatant containing RiboDocker was collected and incubated with 15 μL of ALFA peptide-presenting nanoparticles (PLX = polyplexes with different N / P ratios) containing reporter genes (for luciferase and Thy1.1). Nanoparticles without ALFA peptide were used as a negative control. After incubation, 5 × 10⁵ CD3-expressing cells were incubated with 6 μL of RiboDocker-nanoparticle mixture. Purified protein of aCD3-VHH(F04)×aALFA-VHH at 1.25 μg / mL was used as a positive control. 400 μL of growth medium was added, 100 μL of the mixture was seeded onto white plates for luciferase assay, and 250 μL was used for FACS analysis to detect the Thy1.1 signal. N / P ratio: ratio of positively chargeable polymer amine (N=nitrogen) groups to negatively charged nucleic acid phosphate (P) groups. [Modes for carrying out the invention]

[0046] Array description The following table provides a list of specific sequences referenced herein.

[0047] [Table 1]

[0048] [Table 2-1]

[0049] [Table 2-2]

[0050] [Table 2-3]

[0051] [Table 2-4]

[0052] This disclosure is described in detail below, but it should be understood that this disclosure is not limited to the specific methods, protocols, and reagents described herein, and that they may differ. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of this disclosure, and that the scope of this disclosure is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0053] Preferably, the terms used herein are defined as those found in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0054] Unless otherwise indicated, the implementation of this disclosure will utilize conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0055] The elements of this disclosure are described below. These elements are listed along with specific embodiments, but it should be understood that they may be combined in any way and in any number to create further embodiments. The various examples and embodiments described should not be construed as limiting this disclosure to only the embodiments expressly described. This description should be understood as disclosing and encompassing embodiments that combine the expressly described embodiments with any number of disclosed elements. Furthermore, any rearrangement and combination of all described elements should be considered disclosed by this description unless specifically indicated in the context.

[0056] The term "about" means approximately or nearly, and in the context of the numbers or ranges described herein, in one embodiment, means ±20%, ±10%, ±5%, or ±3% of the listed or claimed numbers or ranges.

[0057] The terms “a” and “an” and “the” and similar references used in the context describing this disclosure (particularly in the context of the claims) should be construed to encompass both singular and plural unless otherwise specifically indicated herein or unless the contextual context clearly contradicts this. The enumeration of ranges of values ​​herein is intended simply as a way of concisely referring to each separate value belonging to that range individually. Unless otherwise specifically indicated herein, individual values ​​are incorporated herein as if they were individually enumerated herein. All methods described herein may be carried out in any suitable order unless otherwise specifically indicated herein or unless the contextual context clearly contradicts this. The use of any examples or illustrative language provided herein (e.g., “etc.”) is intended solely to better illustrate this disclosure and does not impose any limitation on the claims. No language herein should be construed to indicate any unclaimed elements essential to the practice of this disclosure.

[0058] Unless otherwise specified, the term “including” is used in the context of this Document to indicate that there may be additional members in addition to the members of the list introduced by “including.” However, the term “including” is intended to encompass the possibility that there may be no additional members, and for the purposes of this embodiment, “including” should be understood to mean “consisting of” or “essentially consisting of.”

[0059] Throughout this specification, several sources are referenced. Each source referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that this disclosure had no prior rights to such disclosure.

[0060] definition The following definitions are provided, applicable to all aspects of this disclosure. Unless otherwise indicated, the following terms have the meanings set forth below. Terms not defined have the meanings widely recognized in their respective art.

[0061] As used herein, terms such as “reduce,” “decrease,” “inhibit,” or “impair” preferably relate to the ability to produce an overall reduction or overall decrease of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or even higher levels. These terms include complete or essentially complete inhibition, i.e., reduction to zero or essentially zero.

[0062] Terms such as “increase,” “boost,” or “exceed” preferably relate to an increase or boost of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

[0063] According to this disclosure, the term “peptide” includes oligopeptides and polypeptides and refers to substances containing about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about 50, about 100 or about 150 consecutive amino acids linked to one another by peptide bonds. The terms “protein” or “polypeptide” refer to larger peptides, in particular peptides having at least about 150 amino acids, but the terms “peptide,” “protein,” and “polypeptide” are generally used as synonyms herein.

[0064] A “fragment” of an amino acid sequence (peptide or protein) refers to a sequence representing a portion of the amino acid sequence, i.e., a shortened amino acid sequence at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A N-terminal shortened fragment (C-terminal fragment) can also be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, insofar as it contains a start codon that acts to initiate translation. An amino acid sequence fragment contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. Preferably, an amino acid sequence fragment contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0065] In this specification, “variant” means an amino acid sequence that differs from the parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a natural or wild-type (WT) amino acid sequence, or a modified form of a wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, for example, 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0066] In this specification, “wild-type,” “WT,” or “natural” means an amino acid sequence found in nature, including allelic mutations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0067] For the purposes of this disclosure, “variants” of an amino acid sequence (peptide, protein, or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term “variant” includes all mutants, splice variants, post-translational modification variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, in particular those occurring in nature. The term “variant” also includes, in particular, fragments of an amino acid sequence.

[0068] Amino acid insertion mutants contain the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence mutants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, but random insertions are also possible with appropriate screening of the resulting product. Amino acid addition mutants contain amino-terminal and / or carboxyl-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion mutants are characterized by the removal of one or more amino acids from a sequence, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein. Amino acid deletion mutants containing deletions at the N-terminus and / or C-terminus of a protein are also called N-terminal and / or C-terminal cleavage mutants. Amino acid substitution mutants are characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. It is preferable to modify amino acid sequences at non-conserved positions between homologous proteins or peptides, and / or to substitute amino acids with other amino acids having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one of the families of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, alanine; Valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine; and Phenylanine, tyrosine.

[0069] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments for consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment, for example, using Align, with a standard setting, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extension 0.5.

[0070] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0071] The terms “% identical,” “identity%,” or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may, but not necessarily, be randomly distributed across the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences with respect to a segment or “comparison window” after optimal alignment to identify local regions of the corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm by Smith and Waterman, 1981, Ads App.Math.2, 482, the local homology algorithm by Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 88, 2444, or by using computer programs that employ the aforementioned algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percentage of identity between two sequences is determined using the BLASTN or BLASTP algorithm available on the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) a match / mismatch score set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low complexity regions used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to exist: 11, extended: 1; and (vi) a conditional composition score matrix adjustment.

[0072] The identity percentage is obtained by determining the number of identical positions in the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0073] In some embodiments, the degree of similarity or identity is given for regions that are at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the total length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments for consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the total length of the reference sequence.

[0074] Homologous amino acid sequences, according to this disclosure, exhibit identity of at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98%, or at least 99% of the amino acid residues.

[0075] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. Manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions, or deletions is described in detail, for example, Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as solid-phase synthesis and similar methods.

[0076] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a “functional fragment” or “functional variant.” The term “functional fragment” or “functional variant” of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., functionally equivalent. With respect to the sequence of a binder such as an antibody, one particular function is one or more binding activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term “functional fragment” or “functional variant” refers in particular to a variant molecule or sequence that includes an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, and still performs one or more functions of the parent molecule or sequence, for example, that can bind to a target molecule. In one embodiment, the modification of the amino acid sequence of the parent molecule or sequence does not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present; for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the binding of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0077] An amino acid sequence (peptide, protein, or polypeptide) "derived" from a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the original amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to that particular sequence or fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant or fragment of that particular sequence. For example, it will be understood by those skilled in the art that sequences suitable for use herein can be modified to differ from the naturally occurring sequences from which they are derived, while retaining the desired activity of the natural sequences.

[0078] Where used herein, “instructional materials” or “instructions” include publications, records, figures, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. Instructional materials for a kit of the present invention may, for example, be affixed to the container containing the compositions of the present invention, or shipped together with the container containing the compositions. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the instructional materials and the compositions be used in conjunction by the recipient.

[0079] "Isolated" means modified or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living animals are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0080] In the context of this invention, the term "recombinant" means "produced through genetic manipulation." Preferably, "recombinant products," such as recombinant nucleic acids, in the context of this invention do not exist in nature.

[0081] As used herein, the term “naturally occurring” refers to the fact that a substance can be found in nature. For example, a peptide or nucleic acid that is present in living organisms (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0082] As used herein, "physiological pH" refers to a pH of approximately 7.5.

[0083] The terms “genetic modification” or simply “modification” include the transfection of cells with nucleic acids. The term “transfection” relates to the introduction of nucleic acids, particularly RNA, into cells. For the purposes of this invention, the term “transfection” also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, and the cells may be present in a subject, e.g., a patient. Accordingly, according to this invention, the cells for the transfection of nucleic acids described herein may be present in vitro or in vivo, and for example, the cells may form an organ, tissue, and / or part of an organism of a patient. According to this invention, transfection may be transient or stable. In some applications of transfection, it is sufficient that the transfected genetic material is expressed only transiently. RNA can be transfected into cells to transiently express the protein it encodes. Since nucleic acids introduced in the process of transfection are not usually incorporated into the nuclear genome, the foreign nucleic acids are diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desirable that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, then stable transfection must occur. Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection. Generally, nucleic acids encoding docking compounds are transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

[0084] primary target The “primary target” used in this invention relates to a target that is detected, modulated, bound to, or otherwise addressed in therapeutic, diagnostic, and / or imaging methods, for example.

[0085] The primary target can be selected from any suitable target within the human or animal body, and may be a cell, pathogen, or parasite, or may be present on a cell, pathogen, or parasite.

[0086] According to certain embodiments, the primary target is a protein present on the surface of the target cell, such as a cell surface antigen or cell surface receptor. The primary target can be upregulated during disease, such as infection or cancer. In diseased tissue, the above markers can be obtained differently from those in healthy tissue, which can offer unique potential for early detection, specific diagnosis, and treatment, particularly for targeted therapy.

[0087] In some embodiments, the primary target or simply “target” is a tumor antigen. In the context of the present invention, the terms “tumor antigen” or “tumor-associated antigen” refer to proteins that are specifically expressed in a limited number of tissues and / or organs or at specific developmental stages under normal conditions. For example, a tumor antigen may be specifically expressed in gastric tissue, preferably gastric mucosa, reproductive organs, e.g., testes, trophoblast tissue, e.g., placenta, or germline cells under normal conditions, and may be expressed or abnormally expressed in one or more tumor or cancerous tissues. In this context, “limited number” means preferably three or fewer, more preferably two or fewer. Examples of tumor antigens in the context of the present invention include differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins specifically expressed in specific cell types at specific differentiation stages under normal conditions, cancer / testicular antigens, i.e., proteins specifically expressed in the testes and sometimes the placenta under normal conditions, and germline-specific antigens. In the context of the present invention, tumor antigens are preferably associated with the cell surface of cancer cells and preferably not expressed or rarely expressed in normal tissues. Preferably, tumor antigens or abnormal expression of tumor antigens identify cancer cells. In the context of the present invention, the tumor antigen expressed by cancer cells in a subject, for example, a patient suffering from cancer, is preferably an autologous protein in the subject. In preferred embodiments, the tumor antigen in the context of the present invention is specifically expressed in tissues or organs that are non-essential under normal conditions, i.e., tissues or organs that do not result in the death of the subject when damaged by the immune system, or in organs or structures of the body that are inaccessible or have little access to the immune system. Preferably, the amino acid sequence of the tumor antigen is identical for tumor antigens expressed in normal tissue and tumor antigens expressed in cancerous tissue.

[0088] Examples of tumor antigens include cell surface proteins of the claudin family such as p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, claudin 6, claudin 18.2 and claudin 12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAG Examples of preferred tumor antigens include E-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan A, MC1R, Myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, Proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, Survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, and WT. Particularly preferred tumor antigens include Claudin 18.2 (CLDN18.2) and Claudin 6 (CLDN6).

[0089] According to the present invention, the payload can be specifically delivered to a target, such as a target cell, by specifically delivering RNA encoding a doketing compound to the target cell and / or by providing the doketing compound with a portion that binds to a target, such as an antigen on the target cell.

[0090] Specific delivery of RNA encoding docking compounds to target cells can be achieved by using particles containing RNA and a targeting molecule that binds to a target, such as an antigen on the target cell.

[0091] Docking compounds According to the present invention, an RNA-encoded “docking compound” is used to form a bond, such as a non-covalent bond, between the docking compound and a primary target, such as a target cell or an antigen on a target cell. The docking compound may form a bond, such as a non-covalent or covalent bond, to an effector probe. The RNA-encoded docking compound is also referred to herein as “RiboDocker”.

[0092] In one embodiment, the doketing compound includes a “primary targeting moiety,” also referred to as a “target-binding moiety,” and in particular a “target-binding moiety on a target cell,” which can bind to a primary target of interest. The “primary targeting moiety” as used in this invention relates to the portion of the doketing compound that binds to a primary target. Such targeting moieties are typically portions that have affinity for cell surface targets (e.g., membrane receptors) or structural proteins (e.g., amyloid plaques). These portions may be any peptide or protein that binds to the primary target (e.g., an antibody or antibody fragment). Specific embodiments of primary targeting moieties suitable for use herein include cell surface antigen-binding peptides and antibodies. Other examples of primary targeting moieties are receptor-binding peptides or proteins.

[0093] The primary targeting portion preferably binds with high specificity and / or high affinity, and the binding to the primary target is preferably stable in the body.

[0094] To enable specific targeting of the primary targets listed above, the primary targeting portion of a docking compound may include, but is not limited to, antibodies, antibody fragments, such as Fab2, Fab, scFV, VHH domains, and other proteins or peptides.

[0095] According to certain embodiments of the present invention, the primary target is a receptor, and a suitable primary targeting moiety includes, but is not limited to, a ligand or part thereof of such receptor that still binds to the receptor, for example, a receptor-binding peptide in the case of a receptor-binding protein ligand.

[0096] Other examples of protein-based primary targeting moieties include interferons, such as alpha, beta, and gamma interferons, interleukins, and protein growth factors such as transforming growth factors (TGF) or platelet-derived growth factors (PDGF).

[0097] According to further specific embodiments of the present invention, the primary target and primary targeting portion are selected to result in specific or increased targeting of tissues or diseases such as cancer, inflammation, infection, cardiovascular disease, e.g., thrombosis, atherosclerotic lesions, hypoxic sites, e.g., stroke, tumors, cardiovascular disorders, brain disorders, apoptosis, angiogenesis, organs, and reporter genes / enzymes. This can be achieved by selecting a primary target having tissue, cell, or disease-specific expression. For example, a tumor antigen may be overexpressed in various tumor cell types but not expressed or expressed in smaller amounts in normal cells.

[0098] The doketing compound further includes a group that acts as part of the doketing compound, providing a “secondary target,” i.e., a binding partner for the effector probe containing the payload. The binding portion of the doketing compound that binds to the effector probe (the “secondary target”) and the binding portion of the effector probe that binds to the doketing compound (the “secondary targeting portion”) bind to each other.

[0099] According to one embodiment, the docking compound comprises a bispecific antibody. In one embodiment, the docking compound comprises a binding domain that binds to a primary target and a binding domain that binds to a secondary targeting portion on an effector probe. In one embodiment, the docking compound comprises an antibody fragment that binds to a primary target and an antibody fragment that binds to a secondary targeting portion on an effector probe. In one embodiment, at least one binding domain comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody. In one embodiment, at least one binding domain comprises a single-domain antibody such as VHH. In one embodiment, one binding domain comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody, and the other binding domain comprises a single-domain antibody such as VHH. In one embodiment, the binding domain that binds to the primary target comprises the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody. In one embodiment, the binding domain that binds to the secondary targeting portion on an effector probe comprises a single-domain antibody such as VHH.

[0100] In one embodiment, the docking compound comprises a Fab fragment of an antibody that binds to a primary target. In one embodiment, the Fab chain derived from the heavy chain is C-terminated via a GS linker to a VHH that binds to a secondary targeting region on the effector probe. In one embodiment, the VHH that binds to a secondary targeting region on the effector probe is C-terminated via a GS linker to a Fab chain derived from the heavy chain.

[0101] In one embodiment, the docking compound comprises a full-length antibody that binds to a primary target. In one embodiment, the heavy chain of the full-length antibody is C-terminated to a VHH that binds to a secondary targeting moiety on the effector probe via a GS linker. In one embodiment, the VHH that binds to a secondary targeting moiety on the effector probe is C-terminated to the heavy chain of the full-length antibody via a GS linker.

[0102] In one embodiment, the docking compound comprises a fusion protein including a binding domain that binds to a primary target and a binding domain that binds to a secondary targeting portion on an effector probe.

[0103] As used herein, the term “fusion protein” refers to a polypeptide or protein comprising two or more subunits. Preferably, a fusion protein is a translational fusion between two or more subunits. A translational fusion can be produced by genetically manipulating the coding nucleotide sequence of one subunit in a reading frame with the coding nucleotide sequence of a further subunit. The subunits may be interspersed by linkers.

[0104] In one embodiment, the docking compound comprises a single peptide chain. In one embodiment, the single peptide chain comprises an antibody fragment that binds to a primary target and an antibody fragment that binds to a secondary targeting portion on an effector probe. In one embodiment, the antibody fragment is VHH, scFv, or a mixture thereof. In a different embodiment, the docking compound comprises one of the following structures (from N-terminus to C-terminus): VHH (alpha secondary targeting portion on effector probe) - arbitrary linker - VHH (alpha primary target) VHH(alpha primary target) - arbitrary linker - VHH(alpha secondary targeting portion on effector probe) VHH (alpha secondary targeting portion on effector probe) - arbitrary linker - scFv (alpha primary target) scFv (alpha primary target) - arbitrary linker - VHH (alpha secondary targeting portion on effector probe) VHH (alpha primary target) - arbitrary linker - scFv (alpha secondary targeting portion on effector probe) scFv (alpha secondary targeting portion on effector probe) - arbitrary linker - VHH (alpha primary target) scFv(alpha secondary targeting portion on effector probe)-arbitrary linker-scFv(alpha primary target) scFv(alpha primary target) - arbitrary linker - scFv(alpha secondary targeting portion on effector probe)

[0105] In one embodiment, the docking compound includes a signal peptide, such as an N-terminal signal peptide, that enables the secretion of the docking compound from RNA-expressing cells.

[0106] Effector probe The effector probe includes a secondary targeting portion. The secondary targeting portion relates to a portion of the effector probe that forms a binding partner for an available secondary target contained in the docking compound. The effector probe further includes a portion referred to herein as the “effector portion” or “payload” that can induce, provide or bring about a desired diagnostic, imaging, and / or therapeutic effect. The secondary targeting portion and the effector portion may be covalently or noncovalently linked. For example, if the secondary targeting portion is an antigen receptor and the effector portion is a cell, the antigen receptor may be expressed on the surface of the cell and may be noncovalently linked to the cell.

[0107] In one embodiment, the effector probe may comprise a single peptide chain, where the secondary targeting portion comprises a peptide or protein (e.g., an antibody fragment or peptide tag) and the effector portion comprises a peptide or protein. In one embodiment, the effector probe comprises a fusion protein comprising the secondary targeting portion and the effector portion. In this embodiment, the effector probe may be administered as is or as RNA encoding the effector probe (similar to the administration of RNA encoding a docking compound).

[0108] In one embodiment, the secondary targeting portion comprises a peptide or protein (e.g., an antibody fragment or a peptide tag) and the effector portion comprises a compound that is not a peptide or protein, the secondary targeting portion may be chemically linked to the effector portion, for example, via a linker.

[0109] In one embodiment, the secondary target contained in the docking compound and the secondary targeting portion contained in the effector probe bind to each other under physiological conditions.

[0110] In one embodiment, the secondary target contained in the docking compound includes a peptide or protein, such as a peptide tag, and the secondary targeting portion contained in the effector probe includes a binder that binds to the peptide or protein, such as an antibody fragment.

[0111] In one embodiment, the secondary targeting portion included in the effector probe includes a peptide or protein, such as a peptide tag, and the secondary target included in the docking compound includes a binder that binds to the peptide or protein, such as an antibody fragment.

[0112] In one embodiment, the secondary target / secondary targeting partial system used herein includes an epitope tag / binding system.

[0113] In one embodiment, the epitope tag / binding system comprises an epitope tag containing the sequence SRLEEELRRRLTE, and the binding agent comprises a camelid VHH domain containing the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY. In one embodiment, the epitope tag / binding system comprises an epitope tag comprising the sequence SRLEEELRRRLTE, and the binding agent comprises a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQVTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence, or a fragment of the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the amino acid sequence.

[0114] As used herein, "epitope tag" refers to a stretch of amino acids to which an antibody or protein molecule having antibody-like function can bind.

[0115] In one embodiment, a covalent bond is formed after the docking compound is bound to the effector probe. In this embodiment, the secondary target / secondary targeting subsystem used herein includes a tag / catcher system that forms a covalent bond, such as a SpyTag / SpyCatcher that forms an isopeptide bond.

[0116] The SpyTag / SpyCatcher system is a technology for the irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher to form an intermolecular isopeptide bond between the pair. Using the tag / catcher pair, bioconjugation can be achieved between two recombinant proteins.

[0117] In one embodiment, the effector portion included in the effector probe includes a therapeutic portion or a diagnostic portion. In one embodiment, the effector portion included in the effector probe includes a target of the therapeutic portion or the diagnostic portion, such as an effector cell, such as an immune cell.

[0118] The effector portion may be, for example, a detectable label. As used herein, “detectable label” refers to a portion of the effector probe that enables detection of the probe when present, for example, in a cell, tissue, or organism. One type of detectable label envisioned in the context of the present invention is a contrast agent. Various types of detectable labels envisioned in the context of the present invention are described below.

[0119] Thus, according to certain embodiments of the present invention, the agents and methods of the present invention are used in imaging, particularly medical imaging. To identify a primary target, an imaging probe comprising one or more detectable labels is used as an effector probe. Specific examples of detectable labels of the imaging probe are contrast-providing moieties used in conventional imaging systems such as MRI-imaginable constructs, spin labels, optical labels, ultrasonic-responsive constructs, X-ray-responsive moieties, radionuclides, (bio)luminescence, and FRET-type dyes. Exemplary detectable labels contemplated within the context of the present invention include fluorescent molecules, such as autofluorescent molecules, molecules that fluoresce upon contact with a reagent, radiolabels; biotin, for example, detected via the binding of avidin to biotin; fluorescent tags, MRI imaging constructs containing paramagnetic metals, imaging reagents, and the like, but are not limited thereto. Radionuclides used in imaging are, for example, 3 H, 11 C, 13 N, 15 O, 18 F, 19 F, 51 Cr, 52 Fe, 52 Mn, 55 Co, 60 Cu, 61 Cu, 62 Zn, 62 Cu, 63 Zn, 64 Cu, 66 Ga, 67 Ga, 68 Ga, 70 As, 71 As, 72 As, 74 As, 75 Se, 75 Br, 76 Br, 77 Br, 80 Br, 82 Br, 82 Rb, 86 Y, 88 Y, 89 Sr, 89 Zr, 97 Ru, 99 Tc, 110In, 111 In, 113 In, 114 In, 117 Sn, 120 I, 122 Xe, 123 I, 124 I, 125 I, 166 Ho, 167 Tm, 169 Yb, 193 Pt, 195 Pt, 201 Tl, and 203 It may be an isotope selected from the group consisting of Pb. Other elements and isotopes used in medicine may also be applied to imaging in specific applications.

[0120] The MRI-imaging portion may be a paramagnetic ion or a superparamagnetic particle. The paramagnetic ions may be elements selected from the group consisting of Gd, Fe, Mn, Cr, Co, Ni, Cu, Pr, Nd, Yb, Tb, Dy, Ho, Er, Sm, Eu, Ti, Pa, La, Sc, V, Mo, Ru, Ce, Dy, and Tl.

[0121] The X-ray response region includes, but is not limited to, iodine, barium, and barium sulfate.

[0122] Furthermore, detectable labels envisioned within the context of the present invention also include peptides or polypeptides that can be detected by antibody binding, for example, by binding of a detectable labeled antibody. In one embodiment, the detectable label is: 18 F, 11 C or 123 These are small organic PET and SPECT labels such as I.

[0123] The effector portion may also be a therapeutic agent, such as a pharmaceutically active drug. Examples of pharmaceutically active drugs are known to those skilled in the art and are provided herein. The therapeutic probe may optionally include a detectable label.

[0124] Therefore, according to another embodiment, the agents and methods of the present invention are used for targeted therapy. This is achieved by using an effector probe comprising a secondary targeting moiety and one or more pharmaceutically active agents (e.g., drugs or radioisotopes for radiotherapy).

[0125] The effector portion may also include vesicles, liposomes, polymer capsules, or any other carrier filled or loaded with the diagnostic or therapeutic portion.

[0126] The term “pharmaceutically active agent” refers to any agent, such as a compound or cell, that is therapeutically effective when administered to an individual. The term “pharmaceutically active agent” further refers to any agent that alters, preferably cures, alleviates or partially cessates, the clinical symptoms of a given disease and its complications in a therapeutic intervention involving the administration of the agent.

[0127] In one embodiment, the pharmaceutically active agent includes pharmaceutically active RNA or pharmaceutically active peptide or protein.

[0128] "Pharmacologically active RNA" is RNA that encodes a pharmaceutically active peptide or protein, or is pharmaceutically active in itself, such as one or more RNAs with pharmaceutically active properties as described for pharmaceutically active proteins. For example, RNA may be one or more strands of RNA interference (RNAi). Such drugs include small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), or precursors of siRNAs or microRNA-like RNAs that target a target transcript, such as a transcript associated with the endogenous disease of interest.

[0129] A "pharmaceutically active peptide or protein" has a positive or beneficial effect on a subject's condition or disease state when administered to the subject in a therapeutically effective dose. Preferably, a pharmaceutically active peptide or protein has therapeutic or mitigating properties and may be administered to improve, reduce, alleviate, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" includes the entire protein or polypeptide and may also refer to its pharmaceutically active fragment. The term may also include pharmaceutically active analogs of peptides or proteins. The term "pharmaceutically active peptide or protein" includes peptides and proteins that are antigens, i.e., administration of the peptide or protein to a subject induces an immune response in the subject that may be therapeutic or partially or completely protective.

[0130] Examples of pharmaceutically active proteins include cytokines and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factors (G-CSFs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), erythropoietins, tumor necrosis factor (TNFs), interferons, integrins, adresins, seretins, homing receptors, T cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, immunologically active antigens, etc.) (e.g., bacterial antigens, parasitic antigens or viral antigens, allergens, autoantigens, antibodies), hormones (insulin, thyroid hormones, catecholamines, gonadotropins, trophotropic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthesis or degradation enzymes) Enzymes, steroid-producing enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, neuraminidases, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, etc.), transcription and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (collagen, fibroin, fibrinogen, elastin, tubulin, actin, myosin, etc.), blood proteins (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin granulocyte colony-stimulating factor (GCSF) or modified factor VIII, anticoagulant factors, etc.), etc., are examples of but are not limited to these.

[0131] In one embodiment, the pharmaceutically active protein is a cytokine involved in regulating lymphatic homeostasis, preferably a cytokine involved in, and preferably inducing or enhancing, T cell development, priming, expansion, differentiation, and / or survival. In one embodiment, the cytokine is an interleukin. In one embodiment, the pharmaceutically active protein according to the present invention is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0132] In one embodiment, the effector probe includes an effector moiety that is a compound useful for radiotherapy and / or chemotherapy. In another embodiment, the effector probe includes an effector moiety that is a chemotherapy compound.

[0133] Chemotherapy is a type of cancer treatment that typically uses one or more anticancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. The term chemotherapy has come to imply the nonspecific use of intracellular toxins to inhibit mitosis. This implied exclusion of more selective drugs that block extracellular signals (signaling). The development of therapies using specific molecules or gene targets that inhibit growth-promoting signals from classical endocrine hormones (primarily estrogen for breast cancer and androgens for prostate cancer) is now called hormone therapy. In contrast, other inhibitions of growth signals, such as those related to receptor tyrosine kinases, are referred to as targeted therapies.

[0134] Traditional chemotherapy agents are cytotoxic by interfering with cell division (mitosis), but cancer cells vary greatly in their sensitivity to these drugs. For the most part, chemotherapy can be thought of as a way to damage or stress cells, and if apoptosis is initiated, it can lead to cell death.

[0135] Examples of chemotherapeutic agents include alkylating agents, antimetabolites, antimicrotubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0136] Alkylating agents have the ability to alkylate many molecules, including proteins, RNA, and DNA. Subtypes of alkylating agents include nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and their derivatives, as well as non-classical alkylating agents. Nitrogen mustards include mechloretamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, and busulfan. Nitrosoureas include N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin. Tetrazines include dacarbazine, mitozolomide, and temozolomide. Aziridines include thiotepa, mitomycin, and diazicone (AZQ). Cisplatins and their derivatives include cisplatin, carboplatin, and oxaliplatin. These impair cellular function by forming covalent bonds with amino groups, carboxyl groups, sulfhydryl groups, and phosphate groups in biologically important molecules. Non-classical alkylating agents include procarbazine and hexamethylmelamine. In one particularly preferred embodiment, the alkylating agent is cyclophosphamide.

[0137] Antimetabolites are a group of molecules that interfere with DNA and RNA synthesis. Many of them have structures similar to the building blocks of DNA and RNA. Antimetabolites are similar to either nucleic acid bases or nucleosides, but have altered chemical groups. These drugs exert their effects by blocking enzymes necessary for DNA synthesis or by being incorporated into DNA or RNA. Subtypes of antimetabolites include folate antagonists, fluoropyrimidines, deoxynucleoside analogs, and thiopurines. Examples of folate antagonists include methotrexate and pemetrexed. Fluoropyrimidines include fluorouracil and capecitabine. Examples of deoxynucleoside analogs include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nerarabine, cladribine, clofarabine, and pentostatin. Examples of thiopurines include thioguanine and mercaptopurine.

[0138] Antimicrotubule agents inhibit cell division by interfering with microtubule function. Vinca alkaloids inhibit microtubule formation, while taxanes inhibit microtubule degradation. Examples of vinca alkaloids include vinorelbine, vindesine, and vinflunine. Examples of taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0139] Topoisomerase inhibitors are drugs that affect the activity of two enzymes: topoisomerase I and topoisomerase II, and include irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, melbaron, and acralubicin.

[0140] Cytotoxic antibiotics are a diverse group of drugs with various mechanisms of action. A common theme they share in their chemotropic applications is the disruption of cell division. The most important subgroups are anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, and acralubicin) and bleomycin; other notable examples include mitomycin C, mitoxantrone, and actinomycin.

[0141] In one embodiment, the effector probe includes an effector portion which is an effector cell. In this embodiment, the effector cell may express a peptide or protein, such as an antigen receptor, on its surface, which includes a secondary targeting portion. In one embodiment, the effector portion includes a target for the effector cell. In this embodiment, the effector cell may express a peptide or protein, such as an antigen receptor, on its surface which targets the effector portion on the effector probe.

[0142] Cells used in connection with the present invention, into which nucleic acids (DNA or RNA) encoding antigen receptors can be introduced, include, in particular, immunoeffector cells such as lytic cells, especially lymphoid cells, preferably T cells, and more preferably cytotoxic lymphocytes selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. When activated, each of these cytotoxic lymphocytes causes the destruction of target cells. For example, cytotoxic T cells cause the destruction of target cells by one or both of the following means: First, when activated, T cells release cytotoxic substances such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cell, and granzyme enters the cell, causing a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between the T cell and the target cell. The cells used in connection with the present invention are preferably autologous cells, but heterologous cells or allogeneic cells can also be used.

[0143] In the context of the present invention, the term "effector function" includes any function mediated by components of the immune system that result in inhibition of tumor growth and / or inhibition of tumor development, including, for example, the death of diseased cells such as tumor cells, or the suppression of tumor dissemination and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function is a helper T cell (CD4 + In the case of T cells, cytokine release and / or CD8 + This includes activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, elimination of cells, i.e., cells characterized by antigen expression, via apoptosis or perforin-mediated cytolysis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic death of target cells expressing the antigen.

[0144] In the context of the present invention, the terms “immune effector cell” or “immunoreactive cell” refer to cells that exert effector function during an immune response. In one embodiment, an “immune effector cell” can bind to an antigen, such as an antigen presented by a Dokketing compound as a secondary target, or by an effector probe as an effector moiety. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, an “immune effector cell” is a T cell, preferably CD4 + and / or CD8 + T cells, most preferably CD8 + These are T cells. According to the present invention, the term “immune effector cells” also includes cells that can mature into immune cells (such as T cells, particularly T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 +This includes hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells is analogous to clonal selection in the immune system when exposed to antigens.

[0145] In one embodiment, the immune effector cells are immune effector cells that express CAR.

[0146] The immune effector cells used in accordance with the present invention may express endogenous antigen receptors such as T cell receptors or B cell receptors, or they may lack the expression of endogenous antigen receptors.

[0147] "Lymphoid cells" are cells or precursor cells of such cells that, after optional and appropriate modification, for example, after the transfer of an antigen receptor such as a TCR or CAR, can generate an immune response, such as a cellular immune response, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells may be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express an antigen receptor on their cell surface. In one embodiment, lymphoid cells lack endogenous expression of the T cell receptor.

[0148] The terms "T cell" and "T lymphocyte" are used interchangeably herein, and T helper cell (CD4) + Cytotoxic T cells (CTL, CD8 + Includes T cells.

[0149] T cells belong to the group of white blood cells known as lymphocytes and play a central role in cellular immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of special receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ involved in the maturation of T cells. Several different subsets of T cells have been discovered, each with a different function.

[0150] Among its many functions, T helper cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface, hence the name CD4. + They are also known as T cells. Helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0151] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in graft rejection. These cells express the CD8 glycoprotein on their surface, therefore CD8 + They are also known as T cells. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0152] Regulatory T cells, or Tregs, are a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0153] As used herein, the term “naive T cell” refers to a mature T cell that has never encountered its congener antigens in the periphery, unlike activated T cells or memory T cells. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), absence of the activation markers CD25, CD44, or CD69, and absence of the memory CD45RO isoform.

[0154] As used herein, the term “memory T cells” refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their congener antigens. Upon a second encounter with the antigen, memory T cells can regenerate to initiate a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells are CD4 + or CD8 + It can be one of the following, and usually expresses CD45RO.

[0155] According to the present invention, the term "T cell" also includes cells that can mature into T cells upon appropriate stimulation.

[0156] Most T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor is produced from independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains called the α-TCR chain and the β-TCR chain. γδ T cells (gamma delta T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is far rarer than αβ T cells (2% of all T cells).

[0157] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus, where they expand through cell division to form a large population of immature thymocytes. The earliest thymocytes do not express CD4 or CD8, and are therefore double-negative (CD4). - CD8 - They are classified as ) cells. As development progresses, they become double-positive thymocytes (CD4 + CD8 + ) and ultimately single positive (CD4 + CD8 - or CD4 - CD8 + They mature into thymocytes and are then released from the thymus into peripheral tissues.

[0158] T cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or fractions of bone marrow or peripheral blood of a mammal, such as a patient, using a commercially available cell isolation system. Alternatively, T cells may originate from related or unrelated human, non-human animal, cell line, or culture. A sample containing T cells may be, for example, peripheral blood mononuclear cells (PBMCs).

[0159] As used herein, the terms “NK cells” or “natural killer cells” refer to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of T cell receptors. As provided herein, NK cells can also be differentiated from stem cells or progenitor cells.

[0160] Cells described herein, such as immune effector cells, can be genetically modified ex vivo / in vitro or in vivo in a subject being treated to express antigen receptors, such as chimeric antigen receptors (CARs) that bind to an antigen. In one embodiment, the modification to express an antigen receptor is performed ex vivo / in vitro. The modified cells can then be administered to the patient.

[0161] Adoptive cell transfer therapy using CAR-modified T cells expressing chimeric antigen receptors is a promising anti-cancer treatment because CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered to express CARs that specifically target antigens on the patient's tumor cells, and then injected back into the patient.

[0162] According to the present invention, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor," and relates to an artificial receptor comprising a single molecule or a complex of molecules that can recognize a target structure (e.g., an antigen), i.e., bind to the target structure (e.g., by binding of an antigen-binding domain to an antigen), and confer specificity to immune effector cells such as T cells expressing the CAR on the cell surface. Such cells do not necessarily require antigen processing and presentation for recognition of the target cell, but rather can preferably specifically recognize any antigen. Preferably, recognition of the target structure by the CAR results in activation of immune effector cells expressing the CAR. The CAR may comprise one or more protein units comprising one or more domains as described herein. The term "CAR" is not limited to T cell receptors.

[0163] CARs generally include a target-specific binding element, also called an antigen-binding moiety or antigen-binding domain, which is part of the extracellular domain of the CAR. Specifically, the CARs of the present invention target an antigen on a docking compound or effector probe.

[0164] In one embodiment, the antigen-binding domain includes a variable region (VH) of the heavy chain of an immunoglobulin having specificity for the antigen and a variable region (VL) of the light chain of an immunoglobulin having specificity for the antigen. In one embodiment, the immunoglobulin is an antibody. In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked by a peptide linker. Preferably, a portion of the antigen-binding portion in the CAR is scFv.

[0165] The CAR is designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain does not naturally associate with one of the domains in the CAR. In one embodiment, the transmembrane domain naturally associates with one of the domains in the CAR. In one embodiment, the transmembrane domain is modified by amino acid substitution to minimize interaction with other members of the receptor complex by avoiding binding of such domain to transmembrane domains of the same or different surface membrane proteins. The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-binding protein or transmembrane protein. The transmembrane domains particularly used in the present invention may originate from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 (i.e., including at least one of these transmembrane domains). Alternatively, the transmembrane domain may be synthetic, in which case it mainly contains hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0166] In some cases, the CAR of the present invention includes a hinge domain that forms a bond between the transmembrane domain and the extracellular domain.

[0167] The cytoplasmic domain or, otherwise, the intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is located. The term "effector function" refers to a specific function of the cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and instructs the cell to perform a specific function. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits effector function signals. Therefore, the term intracellular signaling domain means that it includes any truncated portion of an intracellular signaling domain sufficient to transmit effector function signals.

[0168] It is well known that signals generated solely through the TCR are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0169] In one embodiment, the CAR includes a primary cytoplasmic signaling sequence derived from CD3ζ. Furthermore, the cytoplasmic domain of the CAR may include a CD3ζ signaling domain combined with a co-stimulatory signaling region.

[0170] The identity of the co-stimulatory domain is limited only in that it has the ability to enhance cell proliferation and survival upon binding of the target moiety by the CAR. Suitable co-stimulatory domains include CD28, CD137 (4-1BB), which is a member of the tumor necrosis factor receptor (TNFR) superfamily, CD134 (OX40), which is a member of the receptor of the TNFR superfamily, and CD278 (ICOS), which is a CD28 superfamily co-stimulatory molecule expressed on activated T cells. Those skilled in the art will understand that sequence variants of these described co-stimulatory domains can be used without adversely affecting the present invention if they have the same or similar activity as the domain they model. Such variants have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived. In some embodiments of the present invention, the CAR construct comprises two co-stimulatory domains. Specific combinations include all possible permutations of the four described domains, and specific examples include CD28 + CD137 (4-1BB) and CD28 + CD134 (OX40).

[0171] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random order or in a specified order. Optionally, preferably a short oligopeptide linker or polypeptide linker of 2 to 10 amino acids in length can form a bond. A glycine-serine doublet provides a particularly suitable linker.

[0172] In one embodiment, the CAR comprises a signal peptide that directs the nascent protein into the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen-binding domain. In one embodiment, the signal peptide is derived from an immunoglobulin such as IgG.

[0173] CARs may contain the above domains together in the form of a fusion protein. Such fusion proteins generally include an antigen-binding domain linked from the N-terminus to the C-terminus, one or more costimulatory domains, and a signaling sequence. However, the CARs of the present invention are not limited to this arrangement, and other arrangements are also permitted, including a binding domain, a signaling domain, and one or more costimulatory domains. Since the binding domain must be able to freely bind to the antigen, the arrangement of the binding domain in a fusion protein is generally understood to be an arrangement in which the regional representation is achieved outside the cell. Similarly, since the costimulatory and signaling domains function to induce the activity and proliferation of cytotoxic lymphocytes, fusion proteins generally display these two domains inside the cell.

[0174] In one embodiment, the CAR molecule is i) Target antigen (e.g., epitope tag) binding domain; ii) Transmembrane domain; and iii) Intracellular domains including the 4-1BB co-stimulatory domain and the CD3ζ signaling domain Includes.

[0175] In one embodiment, the antigen-binding domain includes scFv. In one embodiment, the transmembrane domain includes the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19 , IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain of a protein selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGLl, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or functional variants thereof. In one embodiment, the transmembrane domain includes a CD8α transmembrane domain. In one embodiment, the antigen-binding domain is connected to the transmembrane domain by a hinge domain. In one embodiment, the hinge domain is a CD8α hinge domain.

[0176] In one embodiment, the CAR molecule of the present invention is i) Target antigen-binding domain; ii) CD8α hinge domain; iii) CD8α transmembrane domain; and iv) Intracellular domains including the 4-1BB co-stimulatory domain and the CD3ζ signaling domain Includes.

[0177] Various methods can be used to introduce antigen receptors, such as CAR constructs, into cells, such as T cells, to produce genetically modified cells that express the antigen receptor. Such methods include non-viral-based DNA transfection, non-viral-based RNA transfection (e.g., mRNA transfection), transposon-based systems, and virus-based systems. Non-viral-based DNA transfection carries a low risk of insertional mutagenesis. Transposon-based systems can integrate the transgene more efficiently than plasmids that do not contain the integration element. Virus-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to use to produce T cells and efficiently and persistently transduce them, and have been preliminaryly proven to be safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and persistently transduce T cells, but are more expensive to manufacture. They are also potentially safer than retrovirus-based systems.

[0178] In one embodiment, T cells or T cell precursors are transfected with a nucleic acid encoding an antigen receptor either ex vivo or in vivo. In one embodiment, a combination of ex vivo and in vivo transfection may be used. In one embodiment, the T cells or T cell precursors are derived from the subject being treated. In one embodiment of all aspects of the present invention, the T cells or T cell precursors are derived from a subject different from the subject being treated.

[0179] CAR T cells can be produced in vivo and therefore almost instantaneously using nanoparticles that target T cells. For example, poly(β-aminoester)-based nanoparticles can be coupled to an anti-CD3e F(ab) fragment to bind to CD3 on T cells. Upon binding to T cells, these nanoparticles are endocytized. Their contents, such as plasmid DNA encoding the antitumor antigen CAR, can be induced into the T cell nucleus because they contain peptides with microtubule-associated sequences (MTAS) and nuclear localization signals (NLS). Efficient integration of the CAR vector into the chromosome may be possible by including separate plasmids encoding transposons and highly active transposases adjacent to the CAR gene expression cassette. Such a system enabling in vivo production of CAR T cells after nanoparticle injection is described in Smith et al. (2017) Nat. Nanotechnol. 12:813-820.

[0180] Furthermore, human CD8 + CD19-CAR T cells can be directly generated in vivo using the lentiviral vector CD8-LV, which specifically targets cells (Pfeiffer A. et al., EMBO Mol. Med. Nov;10(11),2018,9158).

[0181] Another possibility is to intentionally position the CAR coding sequence at a specific locus using the CRISPR / Cas9 method. For example, the CAR could be knocked in and placed under the dynamic regulatory control of an endogenous promoter that would otherwise suppress TCR expression, while the existing T cell receptor (TCR) could be knocked out; see, for example, Eyquem et al. (2017) Nature 543:113-117.

[0182] In one embodiment, cells genetically modified to express an antigen receptor are stably or transiently transfected with the nucleic acid encoding the antigen receptor. Thus, the nucleic acid encoding the antigen receptor is either integrated into the cell's genome or not.

[0183] In one embodiment, cells genetically modified to express an antigen receptor are inactivated with respect to the expression of endogenous T cell receptor and / or endogenous HLA.

[0184] In one embodiment, the cells described herein may be autologous, allogeneic, or syngeneic to the target being treated. In one embodiment, the disclosure assumes the extraction of cells from a patient and subsequent re-delivery of the cells to the patient. In one embodiment, the disclosure does not assume the extraction of cells from a patient. In the latter case, all steps of genetic modification of the cells are performed in vivo.

[0185] The term "autologous" is used to describe something that originates from the same source. For example, "autotransplantation" refers to the transplantation of tissue or organs from the same source. Such a procedure is advantageous because it overcomes immunological barriers that would otherwise lead to rejection.

[0186] The term "homogenetic" is used to describe things that originate from different individuals of the same species. Two or more individuals are said to be homogeneous if they do not have identical genes at one or more gene loci.

[0187] The term "related" is used to describe individuals or tissues that have the same genotype, i.e., identical twins or animals of the same inbred lineage, or tissues derived from them.

[0188] The term "xenotransplant" is used to describe something consisting of multiple different elements. For example, transferring bone marrow from one individual to another constitutes xenotransplantation. Xenogenes are genes that originate from a source other than the target organism.

[0189] Binding part and drug The present disclosure describes binding moieties or binding agents such as antibodies or antibody derivatives. Further, the present disclosure describes bispecific or multispecific binding agents such as bispecific antibodies that include first and second binding domains, wherein the first binding domain is capable of binding to a primary target and the second binding domain is capable of binding to a secondary targeting moiety on an effector probe.

[0190] The term "epitope" refers to a part or fragment of a molecule or antigen that is recognized by a binding agent. For example, an epitope can be recognized by an antibody or any other binding protein. An epitope can include contiguous or non - contiguous portions of an antigen and can be about 5 to about 100, such as about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length. For example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes structural epitopes.

[0191] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low - molecular - weight chains and one pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins is well - characterized. See, e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically consists of a heavy - chain variable region (abbreviated herein as V H or VH) and a heavy - chain constant region (abbreviated herein as C H or CH). The heavy - chain constant region typically consists of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is very flexible. The disulfide bonds within the hinge region are part of the interaction between the two heavy chains within an IgG molecule. Each light chain typically consists of a light - chain variable region (abbreviated herein as VL (or abbreviated as VL) and light chain constant region (C in this specification) L The light chain constant region consists of a single domain, the CL. The VH and VL regions can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs) (or hypervariable regions where the morphology of sequencely and / or structurally defined loops can be hypervariable). Each VH and VL typically consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or inconsistent with the context, references to amino acid positions in constant regions in this invention follow EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May; 63(1): 78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). In general, CDRs as used herein are defined by Kabat.

[0192] As used herein, the term “amino acid corresponding to position…” refers to the amino acid position number in the human IgG1 heavy chain. The corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that “corresponds” to an amino acid or segment in another sequence is typically aligned with the other amino acid or segment using ALIGN, ClustalW, or a similar standard sequence alignment program, with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences or segments in a sequence, thereby determining the positions in a sequence corresponding to the amino acid positions according to the present invention, are considered to be well known in the art.

[0193] In the context of this invention, the term “antibody” (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof that has the ability to bind to an antigen, preferably specifically, an antigen. In one embodiment, binding occurs under typical physiological conditions having a significant half-life, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 3, 4, 5, 6, 7 days, etc., or any other relevant functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response related to antibody binding to an antigen). The variable regions of the heavy and light chains of the immunoglobulin molecule include a binding domain that interacts with the antigen. As used herein, the terms “antigen-binding region,” “binding region,” or “binding domain” refer to a region or domain that interacts with an antigen and typically includes both a VH region and a VL region. As used herein, the term antibody includes not only monospecific antibodies but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more different antigen-binding regions. The constant region of an antibody (Ab) can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, which is the first component in the classical pathway of complement activation. As stated above, as used herein, the term antibody includes antigen-binding fragments, i.e., fragments of an antibody that retain the ability to specifically bind to an antigen, and antibody derivatives, i.e., constructs derived from an antibody, unless otherwise specified or unless clearly inconsistent with the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) monovalent fragments consisting of Fab' or Fab fragments, VL, VH, CL, and CH1 domains, or monovalent antibodies described in International Publication No. 2007059782 (Genmab); (ii) bivalent fragments containing F(ab')2 fragments, two Fab fragments linked by disulfide crosslinks at the hinge region; (iii) Fd fragments essentially consisting of VH and CH1 domains; (iv) Fv fragments essentially consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments essentially consisting of the VH domain, also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 21(11): 484-90); (Ward et al., Nature 341, 544-546 (1989)); (vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by synthetic linkers that allow them to be produced as a single protein chain (known as a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)) by pairing the VL and VH regions using recombination. Such single-chain antibodies are encompassed by the term antibody unless otherwise specified or clearly indicated by the context. While such fragments generally fall within the scope of the meaning of antibody, they are unique features of the present invention, both collectively and independently, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention, as well as the bispecificity forms of such fragments, will be discussed further herein.The term antibody should be understood to include, unless otherwise specified, antibody-like polypeptides such as polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that possess the ability to specifically bind to an antigen, provided by any known technique such as enzymatic cleavage, peptide synthesis, and recombination.

[0194] The terms "single-stranded Fv" or "scFv" refer to antibodies in which the variable domains (VH and VL) of the heavy and light chains of a conventional double-stranded antibody are bound together to form a single chain. Optionally, a linker (usually a peptide) is inserted between the two chains to enable proper folding and the creation of an active binding site.

[0195] A single-domain antibody, also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. In one embodiment, the single-domain antibody is a variable domain (V) of a heavy chain antibody. H These are called VHH fragments. Like whole antibodies, single-domain antibodies can selectively bind to specific antigens. The first single-domain antibody was engineered from a heavy-chain antibody found in camelids. Cartilaginous fish also possess heavy-chain antibodies (IgNAR, "novel immunoglobulin antigen receptor"), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split dimeric variable domains from common immunoglobulin G (IgG) derived from humans or mice into monomers. Most research on single-domain antibodies is currently based on heavy-chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind to target epitopes.

[0196] Antibodies can have any isotype. As used herein, the term “isotype” refers to an immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by a heavy chain constant region gene. When a specific isotype, e.g., IgG1, is referred to herein, this term is used to indicate that the antibody is sequence-closer to that isotype, e.g., IgG1, than to other isotypes, although it is not limited to a specific isotype sequence, e.g., a specific IgG1 sequence. Thus, for example, the IgG1 antibody of the present invention may be a sequence variant of a naturally occurring IgG1 antibody, including a mutation in the constant region.

[0197] In various embodiments, the antibody is an IgG1 antibody, more specifically an IgG1 kappa or IgG1 lambda isotype (i.e., IgG1κ, IgG1λ), an IgG2a antibody (e.g., IgG2aκ, IgG2aλ), an IgG2b antibody (e.g., IgG2bκ, IgG2bλ), an IgG3 antibody (e.g., IgG3κ, IgG3λ), or an IgG4 antibody (e.g., IgG4κ, IgG4λ).

[0198] As used herein, the term “monoclonal antibody” refers to a preparation of an antibody molecule with a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting a single binding specificity, having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, which have a genome containing human heavy chain and light chain transgenes fused to immortalized cells.

[0199] As used herein, the term “chimeric antibody” refers to an antibody in which the variable region originates from a non-human species (e.g., a rodent) and the constant region originates from a different species, such as a human. Chimeric monoclonal antibodies for therapeutic use are developed to reduce antibody immunogenicity. As used in the context of chimeric antibodies, the term “variable region” or “variable domain” refers to the region containing the CDR and framework regions of both the heavy and light chains of an immunoglobulin. Chimeric antibodies can be prepared using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Chimeric antibodies may be genetically engineered or enzymatically engineered recombinant antibodies. The preparation of chimeric antibodies is within the knowledge of those skilled in the art, and therefore, the preparation of chimeric antibodies according to the present invention may be carried out by methods other than those described herein.

[0200] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody that includes a non-human variable domain modified to exhibit a high level of sequence homology to the human antibody constant domain and the human variable domain. This can be achieved by transplanting six non-human antibody complementarity-determining regions (CDRs) that together form an antigen-binding site into a homologous human acceptor framework region (FR) (see International Publication 92 / 22653 and European Patent No. 0629240). To completely reconstitute the binding affinity and specificity of the parental antibody, substitution of framework residues (reverse mutations) from the parental antibody (i.e., the non-human antibody) to the human framework region may be required. Structural homology modeling can help identify amino acid residues within the framework region that are important for the antibody’s binding properties. Thus, a humanized antibody may include a non-human CDR sequence, a human framework region mainly containing one or more amino acid reverse mutations into a non-human amino acid sequence, and a fully human constant domain. Optionally, further amino acid modifications, not necessarily reverse mutations, can be applied to obtain a humanized antibody with desirable characteristics such as affinity and biochemical properties.

[0201] As used herein, the term “human antibody” refers to an antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies may contain amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse or rat, has been transplanted into a human framework sequence. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While somatic hybridization procedures are preferred in principle, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of human antibody genes, can be used. The mouse is a suitable animal line for preparing hybridomas that secrete human monoclonal antibodies. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be produced, for example, using transgenic or transchromosomal mice or rats that carry a portion of the human immune system rather than a mouse or rat lineage. In one embodiment, therefore, human antibodies are obtained from transgenic animals such as mice or rats that carry human germline immunoglobulin sequences instead of animal immunoglobulin sequences.In such embodiments, the antibody is derived from a human germline immunoglobulin sequence introduced into an animal, but the final antibody sequence is the result of further modification of the human germline immunoglobulin sequence by somatic hypermutation and affinity maturation mediated by the endogenous animal antibody mechanism, see, for example, Mendez et al. 1997 Nat Genet. 15(2):146-56.

[0202] Where used herein, unless inconsistent with the context, the terms “Fab arm,” “joint arm,” or “arm” refer to a single heavy-chain-light-chain pair and are used herein interchangeably with “half-chain.”

[0203] When used in relation to antibodies, the term "full length" indicates that the antibody is not a fragment but contains all of the domains of a particular isotype that are typically found in nature for that isotype, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0204] As used herein, unless otherwise inconsistent with the context, the term “Fc region” refers to an antibody region consisting of two Fc sequences in the heavy chain of an immunoglobulin, wherein the Fc sequences include at least a hinge region, a CH2 domain, and a CH3 domain.

[0205] As used herein, the terms “to bind” or “to be able to bind” in relation to the binding of an antibody to a given antigen or epitope typically mean approximately 10 when determined using biolayer interferometry (BLI) or when determined using surface plasmon resonance (SPR) technique in a BIAcore 3000 instrument, for example, using the antigen as a ligand and the antibody as the analyte. -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even lower, K DIt is a binding with an affinity corresponding thereto. The antibody has an affinity at least 10-fold lower, for example at least 100-fold lower, for example at least 1,000-fold lower, for example at least 10,000-fold lower, for example at least 100,000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than a predetermined antigen or an antigen closely related thereto. K D binds to a predetermined antigen with an affinity corresponding thereto. The amount with a lower degree of affinity depends on the K D of the antibody. Since it depends on the K D of the antibody, when the K

[0206] of the antibody is very low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than the affinity for the non-specific antigen can be at least 10,000-fold.

[0207] The term "k d " (sec -1 ) used herein refers to the dissociation rate constant of a specific antibody-antigen interaction. The said value is also referred to as the k off value.

[0208] The term "K D " (M) used herein refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.

[0209] The present invention also contemplates an antibody comprising a functional variant of the VL region, VH region, or one or more CDRs of the antibody described herein. A functional variant of VL, VH, or CDR used in relation to an antibody still enables the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and / or specificity / selectivity of the "reference" or "parent" antibody, and in some cases, such an antibody may be associated with a higher affinity, selectivity and / or specificity than the parent antibody.

[0210] Exemplary variants include those that differ from the parent antibody sequence's VH and / or VL and / or CDR regions primarily through conservative substitutions; for example, up to 10 substitutions in a variant, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1, are conservative amino acid residue substitutions.

[0211] Functional variants of antibody sequences described herein, such as the VL region or VH region, or antibody sequences having a certain degree of homology or identity with antibody sequences described herein, such as the VL region or VH region, preferably include modifications or mutations of non-CDR sequences, but the CDR sequence preferably remains unchanged.

[0212] As used herein, the term “specificity” is intended to have the following meanings, unless otherwise inconsistent with the context: Two antibodies have “same specificity” if they bind to the same antigen and the same epitope.

[0213] The terms “competition” and “competition” can refer to competition between a first antibody and a second antibody against the same antigen. Alternatively, “competition” and “competition” can also refer to competition between an antibody and an endogenous ligand for the binding of the endogenous ligand to its corresponding receptor. If an antibody prevents the binding of an endogenous ligand to its receptor, such an antibody is said to block the endogenous interaction between the ligand and its receptor and is therefore competing with the endogenous ligand. Methods for testing antibody competition for binding to a target antigen are well known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be performed, for example, as ELISA or by flow cytometry. Alternatively, competition may be determined using biolayer interference.

[0214] Antibodies competing for binding to a target antigen can bind to different epitopes on the antigen, and since the epitopes are very close to each other, a first antibody binding to one epitope prevents a second antibody from binding to the other epitope. However, in other situations, two different antibodies can bind to the same epitope on the antigen and compete for binding in a competitive binding assay. Such antibodies that bind to the same epitope are considered to have the same specificity as herein. Thus, in one embodiment, antibodies that bind to the same epitope are considered to bind to the same amino acid on the target molecule. Whether antibodies bind to the same epitope on the target antigen can be determined by a standard alanine scanning experiment or antibody-antigen crystallization experiment known to those skilled in the art. Preferably, antibodies or binding domains that bind to different epitopes do not compete with each other for binding to their respective epitopes.

[0215] As described above, various forms of antibodies have been described in the Art. The binder of the present invention can, in principle, contain any isotype of antibody. The selection of isotype is typically guided by the need for a desired Fc-mediated effector function, e.g., ADCC induction, or an antibody lacking Fc-mediated effector function ("inactive" antibody). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region kappa or lambda may be used. The effector function of the antibody of the present invention can be varied for various therapeutic uses by isotype switching to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. In one embodiment, both heavy chains of the antibody of the present invention are the IgG1 isotype, e.g., IgG1κ. Optionally, the heavy chains may be modified with hinges and / or CH3 regions, as described elsewhere in this specification.

[0216] Preferably, each antigen-binding region or domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which comprises three CDR sequences, CDR1, CDR2, and CDR3, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. Furthermore, preferably, the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0217] In one embodiment, the binder includes a full-length antibody such as a full-length IgG1 antibody.

[0218] In other embodiments, the binder includes an antibody fragment such as Fab' or a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains, a monovalent antibody described in International Publication No. 2007059782 (Genmab), an F(ab')2 fragment, an Fd fragment, an Fv fragment, a dAb fragment, a camelid or nanobody, or an isolated complementarity-determining region (CDR).

[0219] In the context of the present invention, the term “binding agent” refers to any agent capable of binding to a desired antigen. In certain embodiments of the present invention, the binding agent is an antibody, an antibody fragment, or any other binding protein, or any combination thereof, or comprises them.

[0220] In the context of the present invention, the term “binding portion” refers to any portion, group, or domain capable of binding to a desired antigen. In certain embodiments of the present invention, the binding portion is an antibody, an antibody fragment, or any other binding protein, or any combination thereof, or includes them.

[0221] Naturally occurring antibodies are generally monospecific, meaning they bind to a single antigen. This invention describes conjugates, such as docking compounds, that bind to different epitopes on primary and secondary targeting moieties. Such conjugates are at least bispecific or multispecific, e.g., triplicate, quadruplicate, etc. Therefore, conjugates may comprise two or more antibodies or fragments thereof, as described herein. In particular, conjugates described herein may be artificial proteins composed of two different antibodies, an antibody and a fragment of a different antibody, and two different antibody fragments (the fragments of two different antibodies that form two binding domains).

[0222] According to the present invention, a bispecificity binder, in particular a bispecificity protein such as a bispecificity antibody, is a molecule that has two different binding specificities and is therefore capable of binding to two epitopes. In particular, the term “bispecificity antibody” as used herein refers to an antibody comprising two antigen-binding sites, namely a first binding site having affinity for a first epitope and a second binding site having binding affinity for a second epitope different from the first epitope.

[0223] In the context of this invention, the term "bispecificity" refers to a drug having two different antigen-binding domains that bind to different epitopes.

[0224] A "multispecific binding agent" is a molecule that possesses three or more different binding specificities.

[0225] Many different forms and uses of bispecific antibodies are known in the art and have been reviewed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97.

[0226] The bispecific antibodies according to the present invention are not limited to a specific bispecificity form or method for producing it.

[0227] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody specific to two different epitopes via two scFvs linked in tandem by an extra peptide linker, for example; and (iii) a dual variable domain antibody (DVD-Ig) in which each light chain and heavy chain contains two variable domains in tandem via short peptide bonds (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig®) Molecule, In: Antibody Engineering, Springer Berlin). Heidelberg (2010): (iv) two chemically linked bispecific (Fab') fragments; (v) a Tandab, a fusion of two single-stranded diabodies resulting in a tetravalent bispecific antibody having two binding sites for each target antigen; (vi) a flexibody, a combination of an scFv and a diabody resulting in a multivalent molecule; (vii) a so-called "dock-and-lock" molecule based on the "dimerization and docking domain" of protein kinase A, which, when applied to Fab, can produce a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) a so-called Scorpion molecule, for example, containing two scFvs fused to both ends of a human Fab arm; and (ix) a diabody.

[0228] The term "bispecific antibody" includes diabodies. A diabody is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between the two domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). Bispecific antibodies also include bispecific single-chain antibodies. The term "bispecific single-chain antibody" refers to a single polypeptide chain containing two binding domains. In particular, the terms “bispecific single-chain antibody” or “single-chain bispecific antibody” or related terms according to the present invention preferably refer to an antibody construct resulting from linking at least two antibody variable regions in a single polypeptide chain that lacks the constant and / or Fc portions present in complete immunoglobulin. For example, a bispecific single-chain antibody may be a construct having a total of two antibody variable regions, e.g., two VH regions, each capable of specifically binding to a distinct epitope and connected to one another via a short polypeptide spacer so that the two antibody variable regions separated by a spacer exist as a single continuous polypeptide chain. Another example of a bispecific single-chain antibody may be a single polypeptide chain having three antibody variable regions, where two antibody variable regions, e.g., one VH and one VL, can constitute an scFv, and the two antibody variable regions are connected to one another via a synthetic polypeptide linker, the latter often genetically engineered to have minimal immunogenicity while remaining maximally resistant to proteolysis. This scFv can specifically bind to a particular epitope and can be connected to a further antibody variable region, such as a VH region, which can bind to an epitope different from the one bound by the scFv. Yet another example of a bispecific single-chain antibody may be a single polypeptide chain having four antibody variable regions.Here, the first two antibody variable regions, for example, the VH region and the VL region, may form one scFv that can bind to one epitope, while the second VH region and VL region may form a second scFv that can bind to another epitope. Within a single continuous polypeptide chain, individual antibody variable regions of one specificity can be conveniently separated by a synthetic polypeptide linker, while each scFv can be conveniently separated by a short polypeptide spacer as described above. According to one embodiment, the first binding domain of a bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to one embodiment, the first binding domain of a bispecific antibody comprises two antibody variable domains, preferably scFvs, i.e., VH-VL or VL-VH. According to one embodiment, the second binding domain of a bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to one embodiment, the second binding domain of a bispecific antibody comprises two antibody variable domains, preferably scFvs, i.e., VH-VL or VL-VH. Therefore, in its smallest form, the total number of antibody variable regions in the bispecific antibody according to the present invention is only two. For example, such an antibody may contain two VH domains or two VHH domains. According to one embodiment, the first and second binding domains of the bispecific antibody each contain one antibody variable domain, preferably a VHH domain. According to another embodiment, the first and second binding domains of the bispecific antibody each contain two antibody variable domains, preferably scFv, i.e., VH-VL or VL-VH. In this embodiment, the binder preferably contains (i) the heavy chain variable domain (VH) of the first antibody, (ii) the light chain variable domain (VL) of the first antibody, (iii) the heavy chain variable domain (VH) of the second antibody, and (iv) the light chain variable domain (VL) of the second antibody.

[0229] In one embodiment, the bispecific molecule according to the present invention contains two Fab regions, each for a different epitope. In one embodiment, the molecule of the present invention is an antigen-binding fragment (Fab)2 complex. The Fab2 complex consists of two Fab fragments, one of which contains an Fv domain specific to one epitope, namely a VH domain and a VL domain, and the other Fab fragment contains an Fv domain specific to another epitope. Each of the Fab fragments may consist of two single strands of a VL-CL module and a VH-CH module. Alternatively, each of the individual Fab fragments may be arranged in a single strand, preferably VL-CL-CH-VH, and the individual variable domains and constant domains may be linked by a peptide linker.

[0230] In one embodiment, the binding agent according to the present invention includes various types of bivalent and trivalent single-stranded variable fragments (scFv), fusion proteins that mimic the variable domains of two antibodies. Bivalent single-stranded variable fragments (di-scFv, bi-scFv) can be manipulated by linking two scFv. This can be done by generating a single peptide chain having two VH regions and two VL regions to obtain a tandem scFv. The present invention also includes multispecific molecules containing three or more scFv-binding domains.

[0231] Another possibility is the creation of scFvs with linker peptides (approximately 5 amino acids) that are too short for the two variable regions to fold together, forcing the scFv to dimerize. This type is known as a diabody. Even shorter linkers (1 or 2 amino acids) result in the formation of trimers, so-called tribodies or tribodies. Tetrabodies have also been created. They exhibit even higher affinity for their targets than diabodies.

[0232] A particularly preferred example of a bispecific antibody fragment is a diabody (Kipriyanov, Int. J. Cancer 77(1998), 763-772), which is a small, bivalent bispecific antibody fragment. A diabody contains a heavy-chain variable domain (VH) and a light-chain variable domain (VL) on the same polypeptide chain (VH-VL), connected by a peptide linker that is too short to allow pairing between the two domains on the same chain. This forces pairing with a complementary domain on another chain, facilitating the assembly of a dimeric molecule with two functional antigen-binding sites.

[0233] In one embodiment, the bispecific or multispecific molecule according to the present invention comprises a variable domain (VH, VL) and a constant domain (C) of an immunoglobulin. In one embodiment, the bispecific molecule is a minibody comprising two single VH-VL-C chains, preferably connected to each other via the constant domain (C) of each chain. According to this embodiment, the corresponding variable heavy chain region (VH), the corresponding variable light chain region (VL), and the constant domain (C) are arranged in the order VH(epitope 1)-VL(epitope 1)-(C) and VH(epitope 2)-VL(epitope 2)-C from the N-terminus to the C-terminus, where C is preferably a CH3 domain, epitope 1 refers to a first epitope, and epitope 2 refers to a second epitope. Pairing of the constant domains results in the formation of the minibody.

[0234] In another embodiment, the bispecificity binder of the present invention is in the form of a bispecificity single-chain antibody construct, the construct comprising or comprising at least two binding domains. In one embodiment, each binding domain comprises one variable region ("VH region") derived from the antibody heavy chain, the VH region of the first binding domain specifically binding to epitope 1, and the VH region of the second binding domain specifically binding to epitope 2. The two binding domains are linked to each other by an optional short polypeptide spacer. Each binding domain may further comprise one variable region ("VL region") derived from the antibody light chain, and the VH and VL regions in the first and second binding domains, respectively, are linked to each other via a polypeptide linker of sufficient length to allow the VH and VL regions of the first binding domain and the VH and VL regions of the second binding domain to pair with each other.

[0235] In one embodiment, the binder described herein comprises an antibody containing a first binding domain, such as a full-length antibody. In one embodiment, the binder described herein comprises an antibody fragment such as scFv or VHH containing a second binding domain covalently linked to the antibody containing the first binding domain. In one embodiment, the binder comprises an antibody fragment such as scFv or VHH covalently linked to the N-terminus or C-terminus of the light or heavy chain of the antibody.

[0236] nucleic acid As used herein, the terms “polynucleotide” or “nucleic acid” are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0237] Nucleic acids may be included in vectors. As used herein, the term “vector” includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as retroviruses, adenoviruses or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). The vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of an operablely linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0238] In one embodiment of all aspects of the present invention, the RNA encoding the docking compound described herein is expressed in cells to be treated to provide the docking compound. If the docking compound comprises two or more polypeptide chains, the different polypeptide chains may be encoded by the same or different RNA molecules.

[0239] The nucleic acids described herein may be recombinant and / or isolated molecules.

[0240] In this disclosure, the term “RNA” refers to nucleic acid molecules containing ribonucleotide residues. In preferred embodiments, RNA comprises all or most of the ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA includes, but is not limited to, isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends (one or both) of RNA. In this disclosure, nucleotides in RNA are also construed to be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogues of naturally occurring RNA.

[0241] In certain embodiments of this disclosure, RNA is messenger RNA (mRNA) relating to an RNA transcript encoding a peptide or protein. As is established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemosynthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0242] In one embodiment, the RNA is in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for regulating transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0243] In certain embodiments of this disclosure, RNA is “replicon RNA” or simply “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or contains elements derived from ssRNA viruses, particularly positive-strand ssRNA viruses such as alphaviruses. Alphaviruses are a typical example of positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856 for a review of the alphavirus life cycle). The total genome length of many alphaviruses is typically in the range of 11,000–12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (forming the viral particle). Typically, there are two open reading frames (ORFs) within the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF (Order of Reference Frame) beginning near the 5' end of the genome, while the alphaviral structural proteins are found downstream of the first ORF and are encoded together by a second ORF extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol.87, pp.111-124). After infection, i.e., in the early stages of the viral life cycle, the (+) strand genomic RNA acts directly like messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or target organisms. A simple approach involves replacing an open reading frame encoding an alphavirus structural protein with an open reading frame encoding the protein of interest. Alphavirus-based trans replication systems rely on alphavirus nucleotide sequence elements on two distinct nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule can be replicated in trans by the replicase (hence the name trans replication system). Trans replication requires the presence of both of these nucleic acid molecules within a given host cell. The nucleic acid molecule that can be replicated in trans by the replicase must contain specific alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0244] In one embodiment, the RNA described herein may have a modified nucleoside. In some embodiments, the RNA comprises a modified nucleoside instead of at least one (e.g., all) uridines.

[0245] As used herein, the term "uracil" refers to one of the nucleic acid bases that may be present in RNA. The structure of uracil is:

[0246] [ka]

[0247] That is the case.

[0248] As used herein, the term "uridine" refers to one of the nucleosides that can be present in RNA. The structure of uridine is:

[0249] [ka]

[0250] That is the case.

[0251] UTP (uridine 5'-triphosphate) has the following structure:

[0252] [ka]

[0253] It has.

[0254] PseudoUTP (pseudouridine 5'-triphosphate) has the following structure:

[0255] [ka]

[0256] It has.

[0257] Pseudouridine is an example of a modified nucleoside, an isomer of uridine, in which uracil is bonded to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0258] Another exemplary modified nucleoside is N1-methylpsoiduridine (m1Ψ), which has the structure:

[0259] [ka]

[0260] It has.

[0261] N1-methylpsoid UTP has the following structure:

[0262] [ka]

[0263] It has.

[0264] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the structure:

[0265] [ka]

[0266] It has.

[0267] In some embodiments, one or more uridines in the RNA described herein are replaced with modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0268] In some embodiments, the RNA contains a modified nucleoside in place of at least one uridine. In some embodiments, the RNA contains a modified nucleoside in place of each uridine.

[0269] In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, the RNA may comprise two or more modified nucleosides, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and N1-methylpseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0270] In some embodiments, the modified nucleoside that replaces one or more, such as all, of the uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), methyl ester of uridine 5-oxyacetic acid (mcmo 5 U), 5-carboxymethyluridine (cm 5U), 1-carboxymethylpsoidouridine, 5-carboxyhydroxymethyluridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethylpsoidouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpsoiduridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpsoidouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopsoidouridine), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopsoiduridine (m 1 s 4 Ψ), 4-thio-1-methylpsoidouridine, 3-methylpsoidouridine (m 3 Ψ), 2-thio-1-methylpsoidouridine, 1-methyl-1-deazapsoidouridine, 2-thio-1-methyl-1-deazapsoidouridine, dihydrouridine(D), dihydropsoidouridine, 5,6-dihydrouridine, 5-methyldihydrouridine(m 5D) 2-Thiodihydrouridine, 2-Thiodihydropsoiduridine, 2-Methoxyuridine, 2-Methoxy-4-Thiouridine, 4-Methoxypsoiduridine, 4-Methoxy-2-Thiopsoiduridine, N1-Methylpsoiduridine, 3-(3-Amino-3-Carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpsoiduridine (Ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 It may be one or more of the following modified uridines known in the art: Um), 1-thiouridine, deoxythymidine, 2'-F-arauridine, 2'-F-uridine, 2'-OH-arauridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0271] In one embodiment, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in one embodiment, cytidine is partially or completely, preferably completely, replaced with 5-methylcytidine in the RNA. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methylpsoiduridine (m1ψ), and 5-methyluridine (m5U). In one embodiment, the RNA contains 5-methylcytidine and N1-methylpsoiduridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine instead of each cytidine and N1-methylpsoiduridine (m1ψ) instead of each uridine.

[0272] In some embodiments, the RNA according to this disclosure includes a 5' cap. In one embodiment, the RNA according to this disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified by a 5' cap analogue. The term “5' cap” refers to a structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA by a 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at position 7. Providing the RNA with a 5' cap or a 5' cap analogue can be achieved by in vitro transcription in which the 5' cap is co-transcribed onto the RNA strand, or by post-transcriptional attachment to the RNA using a capping enzyme.

[0273] In some embodiments, the building block cap for RNA is m2 7,3’-O Gppp(m1 2’-O )ApG(sometimes m2 7,3’O G(5')ppp(5')m 2’-O It is also called ApG, and it has the following structure:

[0274] [ka]

[0275] It has.

[0276] The following are RNA and m2 7,3’O G(5')ppp(5')m 2’-O This is an example of cap 1 RNA containing ApG.

[0277] [ka]

[0278] The following is another example of cap 1 RNA (without cap analogues).

[0279] [ka]

[0280] In some embodiments, RNA has the following structure:

[0281] [ka]

[0282] Cap analogues having anti-reverse caps (ARCA caps (m2 7,3’O It is modified with a "cap 0" structure using G(5')ppp(5')G).

[0283] The following are RNA and m2 7,3’O This is an example of cap 0 RNA containing G(5')ppp(5')G.

[0284] [ka]

[0285] In some embodiments, the "cap 0" structure is structure:

[0286] [ka]

[0287] Cap analog β-S-ARCA(m2 7,2’O It is generated using G(5')ppSp(5')G).

[0288] The following is β-S-ARCA(m2 7,2’O This is an example of cap 0 RNA containing G(5')ppSp(5')G) and RNA.

[0289] [ka]

[0290] The "D1" diastereomer of β-S-ARCA, or "β-S-ARCA(D1)", is a diastereomer of β-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of β-S-ARCA (β-S-ARCA(D2)) (see International Publication 2011 / 015347, incorporated herein by reference).

[0291] A particularly preferred cap is β-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O ) It's ApG.

[0292] In some embodiments, the RNA according to this disclosure includes a 5'-UTR and / or a 3'-UTR. The terms “untranslated region” or “UTR” refer to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region within an RNA molecule such as an mRNA molecule. Untranslated regions (UTRs) may be located on the 5' side (upstream) (5'-UTR) and / or the 3' side (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, if present, is located at the 5' end upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap (if present) and, for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end downstream of the stop codon of the protein-coding region, although the term “3'-UTR” preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of a poly(A) sequence (if present) and, for example, directly adjacent to the poly(A) sequence.

[0293] In some embodiments, the RNA includes a 5'-UTR containing the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 1.

[0294] In some embodiments, the RNA includes a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 2 or 3, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 2 or 3.

[0295] A particularly preferred 5'-UTR contains the nucleotide sequence of SEQ ID NO: 1. A particularly preferred 3'-UTR contains the nucleotide sequence of SEQ ID NO: 2 or 3.

[0296] In some embodiments, the RNA according to this disclosure includes a 3'-poly(A) sequence.

[0297] As used in the present invention, the terms “poly(A) sequence” or “polyA tail” typically refer to a continuous or discontinuous sequence of adenylate residues located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3'UTR of the RNA described herein. Continuous poly(A) sequences are characterized by consecutive adenylate residues. Continuous poly(A) sequences are typical in nature. The RNA disclosed herein may have a poly(A) sequence that has been ligated to the free 3' end of the RNA by template-independent RNA polymerase after transcription, or a poly(A) sequence encoded by DNA and transcribed by template-dependent RNA polymerase.

[0298] A poly(A) sequence of approximately 120 A nucleotides has been shown to have beneficial effects on RNA levels and protein levels translated from the open reading frame located upstream (5' end) of the poly(A) sequence in transfected eukaryotic cells (Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017).

[0299] The poly(A) sequence can be of any length. In some embodiments, the poly(A) sequence contains, essentially consists of, or comprises at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, “essentially consists of” means that most of the nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the total number of nucleotides in the poly(A) sequence, are A nucleotides, but the remaining nucleotides may be other nucleotides such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consists of" means that all nucleotides in a poly(A) sequence, i.e., 100% of the nucleotides in a poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.

[0300] In some embodiments, the poly(A) sequence is bound during RNA transcription, for example, during the preparation of in vitro transcription RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidylate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as the poly(A) cassette.

[0301] In some embodiments, poly(A) cassettes present in the coding strand of DNA are essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5–50, 10–30, or 10–20 nucleotides long. Such cassettes are disclosed in International Publication No. 2016 / 005324A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324A1 may be used in the present invention. Poly(A) cassettes, essentially composed of dA nucleotides but with four nucleotides (dA, dC, dG, and dT) evenly distributed and interrupted by a random sequence having, for example, a length of 5–50 nucleotides, demonstrate, at the DNA level, sustained proliferation of plasmid DNA in Escherichia coli (E. coli), and at the RNA level, still associated with beneficial properties relating to RNA stability and support for translational efficiency. As a result, in some embodiments, the poly(A) sequence contained in the RNA molecule described herein is essentially composed of A nucleotides but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such a random sequence may be 5–50, 10–30, or 10–20 nucleotides long.

[0302] In some embodiments, nucleotides other than A nucleotides are not adjacent to the poly(A) sequence at their 3' end; that is, the poly(A) sequence is not masked or followed at its 3' end by nucleotides other than A.

[0303] In some embodiments, the poly(A) sequence may contain at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence contains at least 100 nucleotides. In some embodiments, the poly(A) sequence contains approximately 150 nucleotides. In some embodiments, the poly(A) sequence contains approximately 120 nucleotides.

[0304] In some embodiments, the RNA includes a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 4.

[0305] A particularly preferred poly(A) sequence contains the nucleotide sequence of sequence number 4.

[0306] According to this disclosure, the docking compound is preferably administered as a single-stranded 5'-capped mRNA that, upon entering the target cells to which the RNA is administered, is translated into the respective protein. Preferably, the RNA contains structural elements (5' cap, 5'-UTR, 3'-UTR, poly(A) sequence) optimized for the maximum effectiveness of the RNA in terms of stability and translation efficiency.

[0307] In one embodiment, β-S-ARCA(D1) is used as a specific capping structure for the 5' end of RNA. In one embodiment, m2 7,3’-O Gppp(m1 2’-O ApG is used as a specific capping structure for the 5' end of RNA. In one embodiment, the 5'-UTR sequence is derived from human α-globin mRNA and optionally has a "Kozak sequence" optimized to enhance translation efficiency. In one embodiment, a combination of two sequence elements (FI elements) derived from "split amino-terminal enhancer" (AES) mRNA (referred to as F) and mitochondrial coding 12S ribosomal RNA (referred to as I) is placed between the coding sequence and the poly(A) sequence to ensure higher maximal protein levels and long-term mRNA persistence. These were identified by an ex vivo selection process of sequences that confer RNA stability and enhance total protein expression (see International Publication No. 2017 / 060314, incorporated herein by reference). In one embodiment, two re-repeat 3'-UTRs derived from human β-globin mRNA are placed between the coding sequence and the poly(A) sequence to ensure higher maximal protein levels and long-term mRNA persistence. In one embodiment, a poly(A) sequence, measured to be 110 nucleotides long, is used, consisting of a stretch of 30 adenosine residues followed by a sequence of 10 nucleotide linkers and another 70 adenosine residues. This poly(A) sequence is designed to enhance RNA stability and translation efficiency.

[0308] In one embodiment of all aspects of the present invention, the RNA encoding the docking compound is expressed in the target cells to be treated to provide the docking compound. In one embodiment of all aspects of the present invention, the RNA is transiently expressed in the target cells. In one embodiment of all aspects of the present invention, the RNA is in vitro transcription RNA. In one embodiment of all aspects of the present invention, the expression of the docking compound is in the extracellular space, i.e., the docking compound is secreted.

[0309] In the context of this disclosure, the term “transcription” refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins.

[0310] According to the present invention, the term “transcription” includes “in vitro transcription,” and the term “in vitro transcription” relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is applied to the production of the transcript. These cloning vectors are generally called transcription vectors and, according to the present invention, are encompassed in the term “vector.” According to the present invention, the RNA used in the present invention is preferably in vitro transcription RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0311] In relation to RNA, the terms "expression" or "translation" refer to the process in the ribosomes of a cell in which a strand of mRNA directs the assembly of amino acid sequences to make a peptide or protein.

[0312] In one embodiment, after administration of the RNA described herein, formulated, for example, as RNA lipid particles, at least a portion of the RNA is delivered to target cells for expression. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is translated by the target cells to produce the peptide or protein it encodes.

[0313] "Code" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties that arise therefrom, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and acting as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, if the transcription and translation of mRNA corresponding to a gene produce a protein in a cell or other biological system, that gene codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0314] In one embodiment, the RNA encoding the docketing compound administered according to the present invention is non-immunogenic.

[0315] As used herein, the term “non-immunogenic RNA” refers to RNA that, for example, does not induce an immune response when administered to a mammal, or induces a weaker response than that induced by the same RNA, which is only different in that it has not been modified and treated to make the non-immunogenic RNA non-RNA; that is, RNA that induces a weaker response than that induced by standard RNA (stdRNA). In a preferred embodiment, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating a modified nucleoside into the RNA that suppresses RNA-mediated activation of innate immune receptors and by removing double-stranded RNA (dsRNA).

[0316] Any modified nucleoside can be used to make non-immunogenic RNA non-immunogenic by incorporating the modified nucleoside, as long as it reduces or suppresses the immunogenicity of the RNA. Modified nucleosides that suppress RNA-mediated activation of innate immune receptors are particularly preferred. In one embodiment, the modified nucleoside comprises the substitution of one or more uridines by a nucleoside containing a modified nucleic acid base. In one embodiment, the modified nucleic acid base is modified uracil. In one embodiment, the nucleoside containing a modified nucleic acid base is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s) 2 U), 4-thio-uridine (s 4 U), 4-thiopsoiduridine, 2-thiopsoiduridine, 5-hydroxyuridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), Uridine 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-psoidouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 1-ethyl-psoidouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-psoidouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-psoidouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thio-psoidouridine, 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-psoidouridine (m 1 s 4 Ψ), 4-thio-1-methyl-psoidouridine, 3-methyl-psoidouridine (m 3 Ψ), 2-thio-1-methyl-psoidouridine, 1-methyl-1-deaz-psoidouridine, 2-thio-1-methyl-1-deaz-psoidouridine, dihydrouridine(D), dihydropsoidouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsoiduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psoiduridine, 4-methoxy-2-thiopsoiduridine, N1-methylpsoiduridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)psoidouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methylpsoiduridine (Ψm), 2-thio-2'-O-methyluridine (s 2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 The group is selected from Um), 1-thiouridine, deoxythymidine, 2'-F-ala-uridine, 2'-F-uridine, 2'-OH-ala-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine. In one particularly preferred embodiment, the nucleoside containing the modified nucleic acid base is pseudouridine (ψ), N1-methyl-psoidouridine (m1ψ), or 5-methyluridine (m5U), particularly N1-methyl-psoidouridine.

[0317] In one embodiment, the substitution of one or more uridines by a nucleoside containing a modified nucleic acid base includes substitutions of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridine.

[0318] During mRNA synthesis by in vitro transcription (IVT) using T7 RNA polymerase, a significant amount of abnormal products, including double-stranded RNA (dsRNA), are produced due to the enzyme's unusual activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to inhibition of protein synthesis. dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reversed-phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using E. coli RNase III can be used to specifically hydrolyze dsRNA rather than ssRNA, thereby removing dsRNA contaminants from the IVT RNA preparation. Furthermore, dsRNA can be separated from ssRNA by using cellulose material. In one embodiment, the RNA preparation is brought into contact with cellulose material to allow dsRNA to bind to the cellulose material, while ssRNA is separated from the cellulose material under conditions that do not allow ssRNA to bind to the cellulose material.

[0319] As used herein, the terms “remove” or “remove” refer to the characteristic of a population of a first substance, such as non-immunogenic RNA, being isolated from a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, a population of the first substance characterized by the removal of a population of the second substance has a measurably lower content of the second substance compared to an unseparated mixture of the first and second substances.

[0320] In one embodiment, the removal of dsRNA from non-immunogenic RNA includes the removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In one embodiment, the non-immunogenic RNA does not contain dsRNA or is essentially free of it. In some embodiments, the non-immunogenic RNA composition includes a purified preparation of single-stranded nucleoside-modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside-modified RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single-stranded nucleoside-modified RNA compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0321] In one embodiment, non-immunogenic RNA is translated more efficiently in cells than standard RNA having the same sequence. In one embodiment, translation is enhanced 2-fold compared to its unmodified counterpart. In one embodiment, translation is enhanced 3-fold. In one embodiment, translation is enhanced 4-fold. In one embodiment, translation is enhanced 5-fold. In one embodiment, translation is enhanced 6-fold. In one embodiment, translation is enhanced 7-fold. In one embodiment, translation is enhanced 8-fold. In one embodiment, translation is enhanced 9-fold. In one embodiment, translation is enhanced 10-fold. In one embodiment, translation is enhanced 15-fold. In one embodiment, translation is enhanced 20-fold. In one embodiment, translation is enhanced 50-fold. In one embodiment, translation is enhanced 100-fold. In one embodiment, translation is enhanced 200-fold. In one embodiment, translation is enhanced 500-fold. In one embodiment, translation is enhanced 1000-fold. In one embodiment, translation is enhanced 2000-fold. In one embodiment, the multiplier is 10 to 1000-fold. In one embodiment, the multiplier is 10 to 100-fold. In one embodiment, the multiplier is 10 to 200 times. In one embodiment, the multiplier is 10 to 300 times. In one embodiment, the multiplier is 10 to 500 times. In one embodiment, the multiplier is 20 to 1000 times. In one embodiment, the multiplier is 30 to 1000 times. In one embodiment, the multiplier is 50 to 1000 times. In one embodiment, the multiplier is 100 to 1000 times. In one embodiment, the multiplier is 200 to 1000 times. In one embodiment, the translation is augmented by any other significant amount or range of amounts.

[0322] In one embodiment, non-immunogenic RNA exhibits significantly lower innate immunogenicity than standard RNA with the same sequence. In one embodiment, non-immunogenic RNA exhibits a 2-fold lower innate immune response than its unmodified counterpart. In one embodiment, innate immunogenicity is reduced 3-fold. In one embodiment, innate immunogenicity is reduced 4-fold. In one embodiment, innate immunogenicity is reduced 5-fold. In one embodiment, innate immunogenicity is reduced 6-fold. In one embodiment, innate immunogenicity is reduced 7-fold. In one embodiment, innate immunogenicity is reduced 8-fold. In one embodiment, innate immunogenicity is reduced 9-fold. In one embodiment, innate immunogenicity is reduced 10-fold. In one embodiment, innate immunogenicity is reduced 15-fold. In one embodiment, innate immunogenicity is reduced 20-fold. In one embodiment, innate immunogenicity is reduced 50-fold. In one embodiment, innate immunogenicity is reduced 100-fold. In one embodiment, innate immunogenicity is reduced 200-fold. In one embodiment, innate immunogenicity is reduced 500-fold. In one embodiment, innate immunogenicity is reduced by 1000 times. In another embodiment, innate immunogenicity is reduced by 2000 times.

[0323] The term "significantly lower innate immunogenicity" refers to a detectable reduction in innate immunogenicity. In one embodiment, this term refers to a reduction that allows for the administration of an effective amount of non-immunogenic RNA without inducing a detectable innate immune response. In one embodiment, this term refers to a reduction that allows for repeated administration of non-immunogenic RNA without inducing an innate immune response sufficient to detectably reduce the production of proteins encoded by the non-immunogenic RNA. In one embodiment, the reduction allows for repeated administration of non-immunogenic RNA without inducing an innate immune response sufficient to eliminate the detectable production of proteins encoded by the non-immunogenic RNA.

[0324] "Immunogenicity" is the ability of foreign substances, such as RNA, to trigger an immune response in the body of a human or other animal. The innate immune system is a relatively nonspecific and immediate component of the immune system. It is one of the two main components of the vertebrate immune system, along with the adaptive immune system.

[0325] As used herein, “endogenous” means any substance produced from or within an organism, cell, tissue, or system.

[0326] As used herein, the term “exogenous” means any substance introduced from or produced outside of an organism, cell, tissue, or system.

[0327] As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence.

[0328] As used herein, the terms “linked,” “fused,” and “fused” are interchangeable. These terms refer to the combination of two or more elements, components, or domains.

[0329] Codon optimization / Increased G / C content In some embodiments, the amino acid sequence of the doketing compound described herein is encoded by a codon-optimized coding sequence and / or a coding sequence in which its G / C content is increased compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence regions of the wild-type coding sequence. In one embodiment, the codon optimization and / or increased G / C content preferably do not alter the sequence of the encoded amino acid sequence.

[0330] The term “codon-optimized” preferably refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage frequency of a host organism, without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of the present invention, the coding region is preferably codon-optimized for optimal expression in a target treated with the RNA molecule described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies of tRNA appearance in a cell. Therefore, the RNA sequence may be modified so that codons where frequently occurring tRNA is available are inserted in place of “rare codons.”

[0331] In some embodiments of the present invention, the guanosine / cytosine (G / C) content of the coding region of the RNA described herein is increased compared to the G / C content of the corresponding coding sequence of wild-type RNA, and the amino acid sequence encoded by the RNA is preferably unmodified compared to the amino acid sequence encoded by wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region being translated is important for the efficient translation of its mRNA. Sequences with increased G (guanosine) / C (cytosine) content are more stable than sequences with increased A (adenosine) / U (uracil) content. With respect to the fact that some codons encode exactly the same amino acids (so-called degeneracy of the genetic code), it is possible to determine the most preferred codon for stability (so-called alternative codon usage frequency). Depending on the amino acids encoded by the RNA, there are various possibilities for modification of the RNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by substituting these codons with other codons that encode the same amino acids but do not contain A and / or U or contain lower levels of A and / or U nucleotides.

[0332] In various embodiments, the G / C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more, compared to the G / C content of the coding region of wild-type RNA.

[0333] Nucleic acid containing particles Nucleic acids described herein, such as RNA encoding docking compounds, may be formulated and administered as particles.

[0334] In the context of this disclosure, the term “particle” refers to a structured entity formed by a molecule or molecular complex. In one embodiment, the term “particle” refers to a micro-sized or nano-sized structure, such as a micro-sized or nano-sized dense structure dispersed in a medium. In one embodiment, the particles are nucleic acid-containing particles, such as particles containing DNA, RNA, or mixtures thereof.

[0335] Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This leads to complex formation and the spontaneous formation of nucleic acid particles. In one embodiment, the nucleic acid particles are nanoparticles.

[0336] As used in this disclosure, “nanoparticles” refers to particles having an average diameter suitable for parenteral administration.

[0337] "Nucleic acid particles" can be used to deliver nucleic acids to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles may be formed from at least one cationic or ionizable lipid or lipid-like substance, at least one cationic polymer such as protamine, or a mixture thereof, and nucleic acids. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0338] While not intended to be bound by any particular theory, it is thought that cationic or ionizable lipids or lipid-like substances and / or cationic polymers, together with nucleic acids, form aggregates, and these aggregates result in colloidally stable particles.

[0339] In one embodiment, the particles described herein further comprise at least one lipid or lipid-like substance other than a cationic or cationically ionizable lipid or lipid-like substance, at least one polymer other than a cationic polymer, or a mixture thereof.

[0340] In some embodiments, nucleic acid particles comprise multiple types of nucleic acid molecules, and the molecular parameters of the nucleic acid molecules may be similar or different from one another, such as molar mass or fundamental structural elements such as molecular structure, capping, coding region, or other features.

[0341] In one embodiment, the nucleic acid particles described herein may have an average diameter in the range of about 30 nm to about 1000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm.

[0342] The nucleic acid particles described herein may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or less than or equal to about 0.2. For example, nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0343] For RNA lipid particles, the N / P ratio gives the ratio of nitrogen groups in the lipid to phosphate groups in the RNA. Since nitrogen atoms (pH-dependent) are typically positively charged and phosphate groups negatively charged, this correlates with the charge ratio. When charge equilibrium exists, the N / P ratio is pH-dependent. Because positively charged nanoparticles are considered favorable for transfection, lipid formulations are often formed with N / P ratios greater than 4 and up to 12. In this case, the RNA is considered to be completely bound to the nanoparticles.

[0344] The nucleic acid particles described herein can be prepared using a wide range of methods that may include the steps of obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain nucleic acid particles.

[0345] As used herein, the term “colloid” refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be called a colloid or colloidal suspension. The term “colloid” may refer only to the particles in the mixture and not to the entire suspension.

[0346] For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, conventionally used and appropriately adapted methods for preparing liposome vesicles are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolution of lipids in an organic solvent, (ii) drying of the resulting solution, and (iii) hydration of the dried lipids (using various aqueous media).

[0347] In the membrane hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of the flask. The obtained lipid film is then hydrated using a suitable aqueous medium to obtain a liposome dispersion. Further miniaturization steps may also be included.

[0348] Reverse-phase evaporation is an alternative to membrane hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary for homogenization of the system. Removal of the organic phase under reduced pressure yields a milky gel, which subsequently becomes a liposome suspension.

[0349] The term "ethanol injection technique" refers to a process in which a lipid-containing ethanol solution is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, such as the formation of lipid vesicles, including liposomes. Generally, the RNA lipoplex particles described herein are obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in one embodiment as follows: an ethanol solution containing lipids, such as cationic lipids and further lipids, is injected into an aqueous solution with stirring. In one embodiment, the RNA lipoplex particles described herein are obtained without an extrusion step.

[0350] The terms "extrusion" or "extrusion" refer to the creation of particles with a fixed cross-sectional profile. In particular, this refers to the miniaturization of particles so that they can pass through a filter with defined pores.

[0351] Other methods having the property of not containing organic solvents may also be used in accordance with this disclosure to prepare colloids.

[0352] LNPs typically consist of four components: ionizable cationic lipids, neutral lipids such as phospholipids, steroids such as cholesterol, and polymer conjugate lipids such as polyethylene glycol (PEG)-lipids. Each component plays a role in payload protection, enabling effective intracellular delivery. LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acids in an aqueous buffer.

[0353] The term "average diameter" is a so-called Z, which has the dimension of length. 平均This refers to the mean hydrodynamic diameter of a particle measured by dynamic laser light scattering (DLS), accompanied by data analysis using the so-called cumulant algorithm, which results in a dimensionless multidispersion index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here, the "mean diameter", "diameter", or "size" of a particle is this Z 平均 It is used synonymously with the value.

[0354] The "polydispersion index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, as mentioned in the definition of "average diameter." Under certain preconditions, this can be considered a measure of the size distribution of the nanoparticle aggregate.

[0355] It has been previously described that various types of nucleic acid-containing particles are suitable for the delivery of nucleic acids in particulate form (e.g., Kaczmarek, JCet al., 2017, Genome Medicine 9, 60). In the case of nonviral nucleic acid delivery vehicles, encapsulation of nucleic acids in nanoparticles can physically protect the nucleic acids from degradation and, depending on their specific chemical properties, can aid in cellular uptake and endosomal extrusion.

[0356] This disclosure describes particles comprising nucleic acids, at least one cationic or ionizable lipid or lipid-like substance, and / or at least one cationic polymer that associates with nucleic acids to form nucleic acid particles, and compositions comprising such particles. Nucleic acid particles may comprise nucleic acids complexed with the particles in various forms by non-covalent interactions. The particles described herein are not viral particles, in particular infectious viral particles; that is, they cannot virally infect cells.

[0357] Appropriate cationic or ionizable lipids or lipid-like substances and cationic polymers that form nucleic acid particles are included in the term “particle-forming components” or “particle-forming agents.” The term “particle-forming components” or “particle-forming agents” refers to any component that associates with nucleic acids to form nucleic acid particles. Such components include any component that may be part of a nucleic acid particle.

[0358] Cationic polymers Polymers are commonly used materials for nanoparticle-based delivery due to their high degree of chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change their protonation state in the pH range of 5.5–7.5, which is thought to lead to ionic imbalances resulting in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamines, protamines, and polyethyleneimines, as well as naturally occurring polymers such as chitosan, are all applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-aminoesters) are widely used in nucleic acid delivery, particularly due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0359] As used herein, “polymer” is given in its usual sense, namely, a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may all be identical, or, in some cases, multiple types of repeating units may exist within the polymer. In some cases, the polymer is biologically derived, i.e., a biomolecule such as a protein. In some cases, further parts, such as targeted parts as described herein, may also be present within the polymer.

[0360] When multiple types of repeating units are present within a polymer, that polymer is said to be a "copolymer." It should be understood that the polymers used herein may be copolymers. The repeating units forming a copolymer can be arranged in any way. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., one or more regions each containing a first repeating unit (e.g., a first block), and one or more regions each containing a second repeating unit (e.g., a second block), and so on. A block copolymer can have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0361] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, meaning the polymer can be chemically and / or biologically degraded in a physiological environment, such as inside the body.

[0362] In certain embodiments, the polymer may be a protamine or a polyalkyleneimine, particularly a protamine.

[0363] The term "protamine" refers to any of several relatively low molecular weight strongly basic proteins that are rich in arginine and found in the sperm cells of various animals (such as fish), often associating with DNA in place of somatic histones. In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, water-soluble, do not coagulate with heat, and primarily produce arginine upon hydrolysis. In purified form, they are used in long-acting insulin formulations to neutralize the anticoagulant effect of heparin.

[0364] As used herein, the term “protamine” is intended to include any protamine amino acid sequence and fragments thereof obtained from or derived from natural or biological sources, as well as polymeric forms of such amino acid sequence or fragments, and artificial, specifically designed for a particular purpose, unisolated (synthesized) polypeptides from natural or biological sources.

[0365] In one embodiment, the polyalkyleneimine includes polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. The preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75 × 10⁻⁶. 2 ~10 7 Da, preferably 1000-10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0366] According to this disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0367] The cationic polymers intended for use herein (including polycationic polymers) include any cationic polymers that can electrostatically bind to nucleic acids. In one embodiment, the cationic polymers intended for use herein include any cationic polymer to which nucleic acids can associate, for example, by forming a complex with nucleic acids or by forming vesicles in which nucleic acids are encapsulated or enclosed.

[0368] The particles described herein may also include polymers other than cationic polymers, namely non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0369] Lipids and lipid-like substances The terms “lipid” and “lipid-like substance” are broadly defined herein as molecules containing one or more hydrophobic moieties or groups, and optionally one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also often referred to as amphiphilic substances. Lipids are typically poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and various phases. One of these phases consists of a lipid bilayer, if they exist in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the presence of long-chain saturated and unsaturated aliphatic hydrocarbon groups, as well as nonpolar groups, including but not limited to those substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylic acid groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0370] As used herein, the term “amphiphilic” refers to a molecule having both a polar and a nonpolar moiety. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have either a formal positive charge or a formal negative charge. Alternatively, the polar moiety may have both a formal positive and a formal negative charge, and may be a zwitterion or an internal salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0371] The terms “lipid-like substance,” “lipid-like compound,” or “lipid-like molecule” refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, this term includes compounds that can form amphiphilic layers when present in vesicles, multilayer / monolayer liposomes, or membranes in an aqueous environment, and includes surfactants or synthetic compounds that have both hydrophilic and hydrophobic parts. Generally speaking, this term refers to molecules that have hydrophilic and hydrophobic parts with different structural arrangements, which may or may not be similar to the structure of lipids. Where used herein, the term “lipid” should be interpreted as encompassing both lipids and lipid-like substances unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context.

[0372] Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0373] In certain embodiments, amphiphilic compounds are lipids. The term “lipid” refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenolipids (derived from the condensation of isoprene subunits). The term “lipid” is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0374] Fatty acids, or fatty acid residues, are a diverse group of molecules consisting of hydrocarbon chains with carboxylic acid groups at their ends; this arrangement confers a polar, hydrophilic end and a water-insoluble, nonpolar, hydrophobic end to the molecule. Typically 4–24 carbon chains, they may be saturated or unsaturated and may be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, it can be either cis or trans geometric isomerized, which significantly affects the molecule's stereochemistry. A cis double bond results in the bending of the fatty acid chain, which is an effect that combines with more double bonds in the chain. Other major lipid classes in the fatty acid category are fatty acid esters and fatty acid amides.

[0375] Glycerolipids are composed of monosubstituted, disubstituted, and trisubstituted glycerols, the most well known being fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified by a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0376] Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that have a glycerol core attached to two fatty acid-derived "tails" by ester bonds and to a single "head" group by a phosphate ester bond. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0377] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base skeleton. The major sphingoid bases in mammals are generally referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives containing amide-linked fatty acids. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The major sphingophospholipid in mammals is sphingomyelin (ceramidephosphocholine), while insects primarily contain ceramidephosphoethanolamine, and fungi have phytoceramidephosphoinositol and mannose-containing head groups. Sphingoglycolipids are a diverse family of molecules composed of one or more sugar residues linked to a sphingoid base via glycosidic bonds. Examples of these include simple and complex sphingoglycolipids such as cerebrosides and gangliosides.

[0378] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelin.

[0379] Saccharolipids are compounds in which fatty acids are directly linked to a sugar backbone, forming a structure compatible with the membrane bilayer. In saccharolipids, monosaccharides replace the glycerol backbone found in glycerolipids and glycerophospholipids. The best-known saccharolipid is the acylated glucosamine precursor of lipid A component of the lipopolysaccharide in Gram-negative bacteria. A typical lipid A molecule is a glucosamine disaccharide derivatized by as many as seven fatty acid acyl chains. The minimum lipopolysaccharide required for growth in E. coli is Kdo2-lipid A, a hexaacylated glucosamine disaccharide glycosylated by two 3-deoxy-D-mann-octurosonic acid (Kdo) residues.

[0380] Polyketides are synthesized by polymerization of acetyl and propionyl subunits using classical enzymes, as well as repeating and multimodular enzymes that share mechanistic features with fatty acid synthases. They exhibit great structural diversity, encompassing numerous secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources. Many polyketides are cyclic molecules whose backbone is often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0381] According to this disclosure, lipids and lipid-like substances may be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in uncharged or neutral zwitterionic forms at a selected pH.

[0382] Cationic or ionizable lipids or lipid-like substances The nucleic acid particles described herein may comprise at least one cationic or cationically ionizable lipid or lipid-like substance as a particle-forming agent. The cationic or cationically ionizable lipid or lipid-like substance intended for use herein comprises any cationic or cationically ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acids. In one embodiment, the cationic or cationically ionizable lipid or lipid-like substance intended for use herein can associate with nucleic acids, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is encapsulated or enclosed.

[0383] As used herein, “cationic lipid” or “cationic lipid-like substance” refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids or lipid-like substances bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have lipophilic moieties such as sterols, acyl chains, diacyl chains or more, and the lipid head groups typically carry a positive charge.

[0384] In certain embodiments, cationic lipids or lipid-like substances have a net positive charge only at specific pH levels, particularly acidic pH levels, but at different, preferably higher, pH levels such as physiological pH, they preferably have no net positive charge, and preferably are chargeless, i.e., neutral. This ionizable behavior is thought to enhance efficacy by facilitating endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH levels.

[0385] For the purposes of this disclosure, such “cationically ionizable” lipids or lipid-like substances are included in the term “cationic lipids or lipid-like substances” unless otherwise inconsistent with the context.

[0386] In one embodiment, a cationic or cationically ionizable lipid or lipid-like substance comprises a head group containing at least one positively charged or protonable nitrogen atom (N).

[0387] Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-diacyloxy-3 -Dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioley Oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3- Beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadieneoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadieneoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3] -Dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminonium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2 -({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane-1-amine(octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammoniumpropane(DMDAP), 1,2-dipalmitoyl-3-dimethylammoniumpropane(DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamide)ethyl]-3,4-Di[oleyloxy]-benzamide (MVL5), 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amonium bromide (DMORIE), Di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azandiyl)dioctanoate (ATX), N,N-dimethyl- 2,3-Bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12 -5) Examples include, but are not limited to, 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (lipidoid C12-200).

[0388] In some embodiments, cationic lipids may constitute about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the particles.

[0389] Further lipids or lipid-like substances The particles described herein may also contain lipids or lipid-like substances other than cationic or cationically ionizable lipids or lipid-like substances, i.e., non-cationic lipids or lipid-like substances (including non-cationically ionizable lipids or lipid-like substances). Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances. By optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties such as cholesterol and lipids in addition to ionizable / cationic lipids or lipid-like substances, particle stability and the effectiveness of nucleic acid delivery can be enhanced.

[0390] Further lipids or lipid-like substances may be incorporated, which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the further lipids or lipid-like substances are noncationic lipids or lipid-like substances. Noncationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, “anionic lipid” refers to any lipid that is negatively charged at a selected pH. As used herein, “neutral lipid” refers to any of several lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the further lipids include one of the following neutral lipid components: (1) phospholipids; (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0391] Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), and dibehenoylphosphatidylcholine (D BPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Examples include Lyso PC and phosphatidylethanolamines, particularly diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids having various hydrophobic chains.

[0392] In certain preferred embodiments, the further lipids are DSPC, or DSPC and cholesterol.

[0393] In certain embodiments, the nucleic acid particles include both cationic lipids and further lipids.

[0394] In one embodiment, the particles described herein include polymer-conjugated lipids such as PEGylated lipids. The term "PEGylated lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art.

[0395] While we do not wish to be bound by theory, the amount of at least one cationic lipid compared to the amount of at least one further lipid can affect important nucleic acid particle properties such as charge, particle size, stability, tissue selectivity, and the biological activity of nucleic acids. Therefore, in some embodiments, the molar ratio of at least one cationic lipid to at least one further lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0396] In some embodiments, noncationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may constitute about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 0 mol% to about 70 mol%, about 0 mol% to about 60 mol%, or about 0 mol% to about 50 mol% of the total lipids present in the particles.

[0397] Lipoplex particles In certain embodiments of this disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0398] In the context of this disclosure, the term “RNA lipoplex particles” refers to particles containing lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in the complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and further lipids such as DOPE. In one embodiment, the RNA lipoplex particles are nanoparticles.

[0399] In certain embodiments, the RNA lipoplex particles contain both cationic lipids and further lipids. In exemplary embodiments, the cationic lipid is DOTMA and the further lipid is DOPE.

[0400] In some embodiments, the molar ratio of at least one cationic lipid to at least one further lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one further lipid is about 2:1.

[0401] In one embodiment, the RNA lipoplex particles described herein have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, RNA lipoplex particles have an average diameter of approximately 200 nm, approximately 225 nm, approximately 250 nm, approximately 275 nm, approximately 300 nm, approximately 325 nm, approximately 350 nm, approximately 375 nm, approximately 400 nm, approximately 425 nm, approximately 450 nm, approximately 475 nm, approximately 500 nm, approximately 525 nm, approximately 550 nm, approximately 575 nm, approximately 600 nm, approximately 625 nm, approximately 650 nm, approximately 700 nm, approximately 725 nm, approximately 750 nm, approximately 775 nm, approximately 800 nm, approximately 825 nm, approximately 850 nm, approximately 875 nm, approximately 900 nm, approximately 925 nm, approximately 950 nm, approximately 975 nm, or approximately 1000 nm. In one embodiment, RNA lipoplex particles have an average diameter in the range of approximately 250 nm to approximately 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0402] The RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for the delivery of RNA to target tissues after parenteral administration, particularly after intravenous administration. RNA lipoplex particles can be prepared using liposomes, which may be obtained by infusing an ethanol solution of lipids into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase contains, for example, about 5 mM of acetic acid. Liposomes may be used to prepare RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one further lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). In one embodiment, at least one further lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and at least one further lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0403] Lipid nanoparticles (LNPs) In one embodiment, nucleic acids such as RNA described herein are administered in the form of lipid nanoparticles (LNPs). LNPs may comprise any lipids capable of forming particles to which one or more nucleic acid molecules are bound or encapsulated.

[0404] In one embodiment, the LNP comprises one or more cationic lipids and one or more stabilized lipids. The stabilized lipids include neutral lipids and pegylated lipids.

[0405] In one embodiment, the LNP includes cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA encapsulated within or associated with lipid nanoparticles.

[0406] In one embodiment, the LNP contains 40-55 mol%, 40-50 mol%, 41-49 mol%, 41-48 mol%, 42-48 mol%, 43-48 mol%, 44-48 mol%, 45-48 mol%, 46-48 mol%, 47-48 mol%, or 47.2-47.8 mol% of cationic lipids. In one embodiment, the LNP contains approximately 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% of cationic lipids.

[0407] In one embodiment, neutral lipids are present at concentrations ranging from 5–15 mol%, 7–13 mol%, or 9–11 mol%. In another embodiment, neutral lipids are present at concentrations of approximately 9.5, 10, or 10.5 mol%.

[0408] In one embodiment, the steroid is present at concentrations ranging from 30–50 mol%, 35–45 mol%, or 38–43 mol%. In another embodiment, the steroid is present at concentrations of approximately 40, 41, 42, 43, 44, 45, or 46 mol%.

[0409] In one embodiment, the LNP contains 1-10 mol%, 1-5 mol%, or 1-2.5 mol% of polymer conjugate lipids.

[0410] In one embodiment, the LNP comprises 40-50 mol% cationic lipids; 5-15 mol% neutral lipids; 35-45 mol% steroids; 1-10 mol% polymer conjugate lipids; and RNA encapsulated within or associated with lipid nanoparticles.

[0411] In one embodiment, the mole percentage is determined based on the total moles of lipids present in the lipid nanoparticles.

[0412] In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the neutral lipid is DSPC.

[0413] In one embodiment, the steroid is cholesterol.

[0414] In one embodiment, the polymer conjugate lipid is a pegylated lipid. In one embodiment, the pegylated lipid has the following structure:

[0415] [ka]

[0416] or having a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, in the formula, R 12 and R 13 Each is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain may be interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. In some embodiments, R 12 and R 13 Each is independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In one embodiment, w has an average value in the range of 40 to 55. In one embodiment, the average w is approximately 45. In one embodiment, R 12 and R 13 Each of these is an independent, linear saturated alkyl chain containing approximately 14 carbon atoms, and w has an average value of approximately 45.

[0417] In some embodiments, the cationic lipid component of LNP is given by formula (III):

[0418] [ka]

[0419] The structure of, or a pharmaceutically acceptable salt thereof, tautomer, prodrug or stereoisomer thereof, wherein, L 1 or L 2 One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- and L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or direct bond; G 1 or G 2 These are, independently, unsubstituted C1-C 12 Alkylene or C1-C 12 It is an alkenylene; G 3 C1-C 24 Alkylene, C1-C 24 These are alkenylenes, C3-C8 cycloalkylenes, and C3-C8 cycloalkenylenes; R aH or C1-C 12 It is alkyl; R 1 and R 2 These are, independently, C6-C 24 Alkyl or C6-C 24 It is an alkenil; R 3 H, OR 5 , CN, -C(=O)OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 It is alkyl; R 5 is H or C1-C6 alkyl; and x is 0, 1, or 2.

[0420] In some of the aforementioned embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB):

[0421] [ka]

[0422] It has one of the following, in the formula, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 In each instance, independently, H, OH, or C1-C 24 It is alkyl; n is an integer in the range of 1 to 15.

[0423] In some of the aforementioned embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0424] In other embodiments of formula (III), the lipid has the following structure (IIIC) or (IIID):

[0425] [ka]

[0426] The formula has one such expression, where y and z are each independent integers in the range of 1 to 12.

[0427] In any of the aforementioned embodiments of formula (III), L 1 or L 2 One of them is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of them is -O(C=O)-. In any of the several different embodiments described above, L 1 and L 2 These are independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of these is -(C=O)O-.

[0428] In several different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF):

[0429] [ka]

[0430] It has one of the following.

[0431] In some of the aforementioned embodiments of formula (III), the lipid has the following structure: (IIIG), (IIIH), (IIII), or (IIIJ):

[0432] [ka]

[0433] Take one of them.

[0434] In some of the aforementioned embodiments of formula (III), n is an integer in the range of 2 to 12, for example, 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0435] In some other embodiments of the aforementioned embodiments of formula (III), y and z are each independently integers in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

[0436] In some of the aforementioned embodiments of formula (III), R 6 is H. In other embodiments of the above-described embodiment, R 6 is C1-C 24 It is alkyl. In other embodiments, R 6 It is OH.

[0437] In some embodiments of formula (III), G 3 G is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is a linear C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0438] In some other aforementioned embodiments of formula (III), R 1 Or R 2 or both are C6-C 24 It is an alkenyl. For example, in some embodiments, R 1 and R 2 Each of these has the following independent structure:

[0439] [ka]

[0440] It has, in the formula, R 7a and R 7b In each existence, independently, H or C1-C 12 Alkyl and as a is an integer between 2 and 12. In the formula, R 7a , R 7b and a are R 1 and R 2 Each of these is independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

[0441] In some of the aforementioned embodiments of formula (III), R 7a The presence of at least one of is H. For example, in some embodiments, R 7a In each existence, is H. In other different embodiments of the above embodiments, R 7b At least one of the elements is a C1-C8 alkyl group. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

[0442] In a different embodiment of formula (III), R 1 Or R 2 , or both, have the following structure:

[0443] [ka]

[0444] It has one of the following.

[0445] In some of the aforementioned embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 -OC(=O)R 4 Or -NHC(=O)R 4 In some embodiments, R 4 It is either methyl or ethyl.

[0446] In various different embodiments, the cationic lipid of formula (III) has one of the structures shown in the following table.

[0447] A representative compound of formula (III).

[0448] [ka]

[0449] [ka]

[0450] [ka]

[0451] [ka]

[0452] [ka]

[0453] [ka]

[0454] In some embodiments, the LNP comprises a lipid of formula (III), RNA, a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is ALC-0159. ALC-0159:

[0455] [ka]

[0456] In some embodiments, cationic lipids are present in LNPs in an amount of about 40 to about 50 mol%. In one embodiment, neutral lipids are present in LNPs in an amount of about 5 to about 15 mol%. In one embodiment, steroids are present in LNPs in an amount of about 35 to about 45 mol%. In one embodiment, pegylated lipids are present in LNPs in an amount of about 1 to about 10 mol%.

[0457] In some embodiments, the LNP contains compound III-3 in an amount of about 40 to about 50 mol%, DSPC in an amount of about 5 to about 15 mol%, cholesterol in an amount of about 35 to about 45 mol%, and ALC-0159 in an amount of about 1 to about 10 mol%.

[0458] In some embodiments, the LNP comprises about 47.5 mol% of compound III-3, about 10 mol% of DSPC, about 40.7 mol% of cholesterol, and about 1.8 mol% of ALC-0159.

[0459] The N / P value is preferably at least about 4. In some embodiments, the N / P value is in the range of 4-20, 4-12, 4-10, 4-8, or 5-7. In one embodiment, the N / P value is about 6.

[0460] Pharmaceutical composition The agents described herein, such as RNA encoding a docking compound or effector probe, may be administered in a pharmaceutical composition or drug, or in any suitable form of pharmaceutical composition.

[0461] In one embodiment of all aspects of the present invention, the components described herein, such as RNA encoding a docking compound or effector probe, may be administered in a pharmaceutical composition that may include a pharmaceutically acceptable carrier and optionally include one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for use in therapeutic or prophylactic treatment, for example, to treat or prevent a disease.

[0462] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0463] The pharmaceutical compositions described herein are generally applied in terms of "pharmaceutically effective amounts" and "pharmaceutically acceptable formulations."

[0464] The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interact with the action of the active ingredient in a pharmaceutical composition.

[0465] The terms “pharmaceutical effective dose” or “therapeutic effective dose” refer to the amount obtained alone or in combination with additional doses and / or drugs to achieve the desired response or effect. In the case of treating a particular disease, the desired response preferably relates to inhibiting the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression. The desired response in the treatment of a disease may also be the delay or prevention of the onset of the disease or condition. The effective dose of the compositions described herein depends on the individual parameters of the patient, including the condition being treated, the severity of the disease, age, physiological state, size and weight, the duration of treatment, the type of accompanying treatment (if any), the specific route of administration, and similar factors. Therefore, the dose administered of the compositions described herein may depend on such various parameters. If the patient’s response is insufficient with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0466] The pharmaceutical compositions of this disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical composition of this disclosure comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0467] Suitable preservatives for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0468] As used herein, the term “excipient” refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

[0469] The term “diluent” refers to a substance used to dilute and / or reduce a substance. Furthermore, the term “diluent” includes one or more fluids, liquids, or solid suspensions and / or mixtures. Examples of suitable diluents include ethanol, glycerol, and water.

[0470] The term "carrier" refers to a component that may be natural, synthetic, organic, or inorganic, to which the active ingredient is combined in order to facilitate, enhance, or enable the administration of the pharmaceutical composition. As used herein, carriers may be one or more suitable solid or liquid fillers, diluents, or encapsulants suitable for administration to a target. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of this disclosure comprises isotonic saline.

[0471] Pharmacopoeia-acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0472] The pharmaceutical carrier, excipient, or diluent may be selected in relation to the intended route of administration and standard pharmaceutical practices.

[0473] In one embodiment, the pharmaceutical composition described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration, including absorption through the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” refers to administration by any method other than through the gastrointestinal tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, for example, intravenous administration.

[0474] As used herein, the term “concurrent administration” means the process of administering different compounds or compositions, such as RNA encoding a docking compound and effector probes, to the same patient. These different compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially.

[0475] treatment The agents, compositions, and methods described herein can be used to treat subjects having diseases characterized by the presence of disease cells expressing an antigen (which may serve as a primary target). Particularly preferred diseases are cancers. For example, if the antigen is derived from a virus, the agents, compositions, and methods may be useful in treating viral diseases caused by the virus. If the antigen is a tumor antigen, the agents, compositions, and methods may be useful in treating cancers in which cancer cells express the tumor antigen.

[0476] The agents, compositions, and methods described herein may be used in the therapeutic or prophylactic treatment of various diseases. In one embodiment, the agents, compositions, and methods described herein are useful for the prophylactic and / or therapeutic treatment of antigen-related diseases.

[0477] The term “disease” refers to an abnormal condition affecting an individual’s body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by factors originating from external sources, such as infections, or by internal dysfunctions, such as autoimmune diseases. In humans, “disease” is often more broadly used to refer to any condition that causes pain, dysfunction, distress, social problems, or death in the affected individual, or similar problems in those in contact with the individual. In this broader sense, disease sometimes includes injury, helplessness, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes these may be considered distinct categories. Many diseases, and living with them, can alter one’s outlook on life and personality, so diseases usually affect individuals not only physically but also emotionally.

[0478] In this context, the terms “treatment,” “to treat,” or “therapeutic intervention” refer to the management and care of an individual aimed at combating a condition such as a disease or disorder. The term is intended to encompass all forms of treatment for a given condition in which an individual is afflicted, including the administration of therapeutically effective compounds to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition, to alleviate or reduce symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, and to prevent the condition. Prevention should be understood as the management and care of an individual aimed at combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0479] The term “therapeutic treatment” refers to any treatment that improves the health of an individual and / or extends (increases) their lifespan. Such treatment may eliminate a disease in an individual, stop or delay the onset of a disease in an individual, inhibit or delay the onset of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual that currently has or has previously had a disease.

[0480] The terms “preventive measures” or “preventive measures” refer to any treatment intended to prevent the development of disease in an individual. The terms “preventive measures” and “preventive measures” are used interchangeably herein.

[0481] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) that is susceptible to, but may or may not have, a disease or disorder (e.g., cancer). In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not indicate a specific age and therefore encompass adults, the elderly, children, and newborns. In embodiments of this disclosure, the “individual” or “subject” is a “patient.”

[0482] The term "patient" means an individual or subject for treatment, in particular an individual or subject that is afflicted.

[0483] In one embodiment of this disclosure, the objective is to deliver a pharmaceutically active agent (including compounds and cells) to disease cells expressing antigens, such as cancer cells expressing tumor antigens, in order to treat diseases such as cancer that involve cells expressing antigens such as tumor antigens.

[0484] The terms “antigen-related disease,” “antigen-expressing cell-related disease,” or similar terms refer to any disease related to an antigen, such as a disease characterized by the presence of an antigen. Antigen-related diseases may be infectious diseases, cancerous diseases, or simply cancer. As described above, the antigen may be a disease-related antigen such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In one embodiment, the antigen-related disease is preferably a disease involving cells that express an antigen on their cell surface.

[0485] The term “infectious disease” refers to any disease caused by microbial factors that can be transmitted from individual to individual or from organism to organism (e.g., the common cold). Infectious diseases are well known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases, which are caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases may include, for example, hepatitis, sexually transmitted infections (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0486] The terms “cancer disease” or “cancer” refer to or represent a physiological condition in an individual typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancer, hematological cancers, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, genital and reproductive cancers, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), neuroectodermal carcinoma, spinal axial tumors, gliomas, meningiomas, and pituitary adenomas. The term “cancer” as used in this disclosure also includes cancer metastases.

[0487] As used herein, the terms “solid tumor” or “solid carcinoma” refer to the occurrence of a cancerous mass, as is well known in the art, for example, in Harrison’s Principles of Internal Medicine, 14th edition. Preferably, the terms refer to cancer or carcinoma of body tissue other than blood, preferably other than blood, bone marrow, and lymphatic tissue. For example, but not limited to, solid tumors include cancers of the prostate, lung, colorectal tissue, bladder, oropharyngeal / laryngeal tissue, kidney, breast, endometrium, ovary, cervix, stomach, pancreas, brain, and central nervous system.

[0488] The references to documents and tests made herein are not intended to constitute an acknowledgment that any of the foregoing constitutes relevant prior art. All statements relating to the contents of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the contents of these documents.

[0489] The following description is provided to enable those skilled in the art to create and use various embodiments. Specific descriptions of apparatus, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but should be given a scope consistent with the claims. [Examples]

[0490] material and method Cells and cell cultures Human CLDN6 (CHO-K1-CLDN6), CHO-K1-mock, FlpIn-CHO-GFP, and human CD3-expressing cells (FlpIn-CHO-huCD3) were grown in Dulbecco's Modified Nutrient Mixture F-12 (DMEM / F-12) medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 600 μg / mL hygromycin B (for FlpIn-CHO-huCD3 and FlpIn-CHO-GFP) and 1 mg / mL geneticin (for CHO-K1-CLDN6). The cells were maintained at 37°C in a 5% CO2 humidified air atmosphere and subcultured every 48-72 hours. HEK297T-17 cells were grown in Dulbecco's Modified Nutrient Mixture (DMEM) medium supplemented with 10% FBS, maintained at 37°C in a 7.5% CO2 humidified air atmosphere, and subcultured every 48-72 hours. For large-scale production of bispecific proteins, the Invitrogen Freestyle® CHO-S cell line was used. Cells were cultured in disposable sterile polycarbonate Erlenmeyer flasks with vent caps (125 mL) at 120–135 rpm (Minitron Incubator shaker, Infors-HT) under standard humidified conditions (37°C and 8% CO2) using 15–25% of the nominal volume. In a protein-free medium for CHO cells (CDCHO medium, Invitrogen) supplemented with 1 × HT additive and 4 mM glutamine, cells were cultured at a density of approximately 1–1.5 × 10⁶. 6 When the viable cells / mL reached a certain level, the cells were typically subcultured every 48–72 hours.

[0491] To analyze translation efficiency, RNA was electroporated into HEK-293-T-17 cells, and the cell culture supernatant containing RiboDocker protein was analyzed by SDS-PAGE and Western blotting.

[0492] Construction and affinity purification of bispecific proteins Human codon-optimized Fab, scFv, IgG, or VHH sequences were synthesized by gene synthesis (TWIST Bioscience). Anti-CLDN6 (IMAB027)IgG (VH-CH1-3), Fab fragment (VH-CH1), anti-CD3 scFv (TR66), or anti-CD3 VHH (F04) constructs were fused to anti-ALFA VHH (NanoTag) using the (G4S1)1 peptide linker coding sequence, including the secretion signal sequence and 6xHis tag sequence (see sequences in Tables 1 and 2 and Figures 1 and 2). For recombinant expression of the bispecific constructs, CHO-S cells were electroporated under MaxCyte flow electroporation conditions using the manufacturer's protocol. The culture supernatant was collected from the CHO-S producing cell line, and the recombinant protein was purified using the Capturem His-tagged Purification Maxiprep Kit (TaKaRa). Quality was tested by SDS-PAGE, Coomassie brilliant blue staining, and Western blot analysis using peroxidase-conjugated monoclonal anti-6xHis tag antibody.

[0493] Electroporation of HEK293_T17 cells HEK-293_T17 cells were raised in X-Vivo medium (Biozym) in 8 × 10⁶ cells. 6 The concentration was adjusted to 1 / mL. 250 μL of cells were transferred to a 0.4 cm cuvette (VWR) and electroporated using 25 μL of RNA diluted in 10 mM HEPES / 0.1 mM EDTA (final RNA concentration of 0.1 mg / mL or 0.01 mg / mL), or HEPES / EDTA buffer alone as a simulated control. The electroporation conditions using an ECM830 instrument (BTX Harvard Aparatus) were 250 volts, 2 pulses, and 5 milliseconds. Subsequently, 750 μL of Expi293® medium (Gibco) was added to the cells, 2 × 10⁻¹⁶. 6A total of 1 mL of the solution containing the specified number of cells was added. These were seeded into the wells of a 12-well cell culture plate (Cellstar). The supernatant of the cell culture was collected after 48 hours, centrifuged (300xg, 10 minutes), and stored at 2-8°C until analysis by flow cytometry (FACS).

[0494] Synthesis of ALFA pigment peptides ALFA peptides were chemically synthesized and conjugated to either Cy5 or Alexa-Fluor-680 (AF680) dyes. Cy5 was conjugated by copper-free click chemistry using different strategies: In the first construct (Cy5-DBCO-azide-ALFA-NH2), Cy5-DBCO (dibenzocyclooctylene) was conjugated to a C-terminally amidated ALFA peptide with an azide moiety at the N-terminus. For the other constructs (Cy5-azide-FCO-ALFA-OH, Cy5-azide-FCO-ALFA-NH2), Cy5-azide was used in a click reaction with FCO (fluorocyclooctane) containing the Alfa peptide. AF680-conjugated peptides were prepared from Pepscan and JPT. In these cases, the fluorophores were conjugated via a short PEG3 spacer (Pepscan) or using an introduced N-terminal cysteine ​​residue that allows for maleimide-based conjugation to a sulfhydryl group. Different ALFA peptide fluorophore conjugates are shown in Figure 3.

[0495] FACS analysis The cells were placed in a 96-well microtita plate (round button) with a final density of 2 × 10⁶ 5The cells were seeded and incubated at 4°C for 1 hour in the presence of the indicated ALFA bispecific antibody. The cells were then washed with FACS buffer (1×PBS + 5 mL of 0.5 M EDTA + 10 mL of FBS) and incubated on ice for 30 minutes with ALFA-Cy5 or ALFA-AF680 peptide. After a further washing step with FACS buffer, the cells were analyzed using FACS Celesta (BD Biosciences). Cy5 and AF680 signals were detected using a 633 nM excitation laser line.

[0496] (Example 1) In vitro functional analysis of bispecific constructs The bispecific construct sequences shown in Figures 1 and 2 were synthesized by gene synthesis as described above. The ALFA peptide was chemically synthesized and conjugated to either Cy5 or Alexa-Fluor-680 as described above.

[0497] Figures 4–6 show FACS analysis of target (CLDN6 or CD3) overexpressing cells and target-negative cells (FlpIn-CHO-mock). In Figure 4, cells are analyzed in the presence of purified recombinant anti-CLDN6 anti-ALFA antibody and fluorescently labeled ALFA peptide. The following amounts / concentrations were applied: 1. 100nM aALFA×aCLDN6 VH(IMAB027)-CH1-H6 containing VL-CL(IMAB027) (ratio of aALFA×aCLDN6 VH(IMAB027)-CH1-H6 to VL-CL(IMAB027) = 1:1.5) 2. 100nM aCLDN6 VH(IMAB027)-CH1×aALFA-H6 containing VL-CL(IMAB027) (ratio of aCLDN6 VH(IMAB027)-CH1×aALFA-H6 to VL-CL(IMAB027) = 1:1.5) 3. 100nM aALFA×aCLDN6 VH(IMAB027)CH1-CH2-CH3-H6 containing VL-CL(IMAB027) (ratio of aALFA×aCLDN6 VH(IMAB027)CH1-CH2-CH3-H6 to VL-CL(IMAB027) = 1:2.5) 4. 100nM aCLDN6 VH(IMAB027)CH1-CH2-CH3×aALFA-H6 containing VL-CL(IMAB027) (ratio of aCLDN6 VH(IMAB027)CH1-CH2-CH3×aALFA-H6 to VL-CL(IMAB027) = 1:2.5) 1.~4.+VL-CL(IMAB027) a) 1μg / mL Cy5-DBCO-Azide-ALFA-NH2 b) 1μg / mL Cy5-Azide-FCO-ALFA-OH c) 1μg / mL Cy5-Azide-FCO-ALFA-NH2 d) or 1 μg / mL ALFA-AF680

[0498] Figure 5 shows the analysis of cells in the presence of purified recombinant anti-CD3-anti-ALFA antibody and the indicated amounts (1, 0.2, 0.04, 0.008, 0.0016, and 0.00032 μg / mL) of Cy5-DBCO-azide-ALFA-NH2 fluorescently labeled ALFA peptide. The following constructs and amounts / concentrations were used: 1.240nM aALFA×aCD3 VL-VH(TR66)-H6 2.156nM aCD3 VL-VH(TR66)×aALFA-H6 3.294nM aCD3 VHH(F04)×aALFA-H6 4.416nM aALFA×aCD3 VHH(F04)-H6

[0499] The FACS measurements in Figures 4 and 5 show an increase in the specific fluorescence intensity of the complexed (aALFA-ALFA peptide linked) constructs upon presentation of each cell surface target.

[0500] In Figure 6, cells treated with purified recombinant bispecific antibodies are analyzed in comparison to cells incubated with the supernatant of RNA-transfected HEK293T-17 cells expressing the same construct. Here, the experiment shows similar results for purified recombinant proteins and proteins in the cell supernatant after RNA electroporation.

[0501] (Example 2) Modular bispecific antibody for cancer. In this embodiment illustrated in Figure 7, anti-CLDN6 scFv, a tumor-specific ligand fused to an anti-ALFA VHH sequence, and anti-CD3 VHH, an anti-T cell-specific ligand fused to an ALFA tag, are administered to the patient in the form of two separate encoding RNAs encapsulated in lipid nanoparticles. The lipid nanoparticles are taken up by hepatocytes, which then express both bispecific fusion proteins and release them into the bloodstream. The anti-CLDN6x anti-ALFA-targeted ligand accumulates at tumor sites, and the anti-CD3-ALFA tag accumulates at T cell sites. Due to the high specificity of anti-ALFA VHH to the ALFA tag, both cell populations (tumor cells and T cells) connect with each other. This leads to CD3-mediated T cell activation and lysation of tumor cells. The described procedure is theoretically universally applicable to different tumor antigens and immune cell antigens.

[0502] (Example 3) Universal CART approach. In this embodiment illustrated in Figure 8, CAR-T cells are generated in which the tag-targeting binding portion is fused as a recognition domain to the hinge, transmembrane, and intracellular signaling domains of a chimeric antigen receptor. To reduce costs and enable rapid patient supply, allogeneic CART cells are preferred for this purpose, which can be generated by established methods such as genetic engineering for αβ T cell depletion. General CART cells are administered to the patient. As a second component, a tumor-specific targeting ligand, e.g., anti-CLDN6 scFv, is fused to the tag sequence and administered to the patient as RNA encapsulated in lipid nanoparticles. The lipid nanoparticles are taken up by hepatocytes, which then express a bispecific fusion protein and release it into the bloodstream. The targeting ligand accumulates at the tumor site, where general CAR-T cells can ultimately bind to it and mediate tumor cell death. The described procedure is theoretically universally applicable to different tumor antigens, enhances efficacy in principle, and also enables simultaneous targeting of several antigens by providing a mixture of RNAs encoding different targeting ligands. Another key advantage of this approach is the safe persistence of CAR-T cells in the absence of targeted ligands. This allows for the cessation of treatment without depleting CAR-T cells, provided that all tumor cells are eliminated and, in parallel, the option to continue treatment with the same or a different targeted ligand is provided in case of tumor recurrence.

[0503] (Example 4) RiboDocker for targeting ALFA peptide-presenting nanoparticles RiboDocker generation and quality control RiboDocker for CD3 and ALFA peptides was tested using 25 μg of RNA encoding aCD3-VHH(F04) × aALFA-VHH (in 25 μL of 10 mM Hepes and 0.1 mM EDTA) in a 2 × 10⁶ test. 6RiboDocker was generated by electroporation of HEK293T-17 cells (in 250 μL of X-Vivo15 medium). As a negative control (mock), cells were electroporated with RNA-free buffer. After 48 hours, the cell supernatant was collected and filtered. Analysis of SDS-PAGE and Western blotting confirmed the successful generation of RiboDocker, and flow cytometry analysis against CD3-expressing cell lines demonstrated its functionality.

[0504] Nanoparticle-RiboDocker experiment For the nanoparticle-RiboDocker experiment, 15 μL of RiboDocker supernatant was incubated with 15 μL of ALFA peptide-coated polyplex (PLX) at room temperature for 5 minutes. The PLX had an N / P ratio of 15 or 7.5 and was loaded with polynucleic acid encoding the reporter gene luciferase and Thy1.1. Nanoparticles without ALFA peptide were used as a negative control.

[0505] 5 x 10 5 100 μL of CD3-expressing Jurkat cells were incubated with 6 μL of RiboDocker-PLX mixture in a 96-deep-well plate at 37°C for 5 minutes. A simulated supernatant was used as a negative control, and 1.25 μg / mL of purified aCD3-VHH(F04)×aALFA-VHH protein was used as a positive control. 400 μL of growth medium (RPMI + 10% FBS) was added per well. For overnight culture, 100 μL of the mixture was seeded into a 96-well white flat plate for luciferase assay, and 250 μL was seeded into a 96-well round-bottom plate for FACS analysis (Thy1.1 detection). Overnight incubation was performed at 37°C and 5% CO2.

[0506] analysis To evaluate the success of RiboDocker generation, its aALFA-VHH-mediated binding to ALFA-coated PLX, subsequent particle internalization, and reporter gene expression, luciferase activity was measured by flow cytometry analysis via luminescence and Thy1.1 expression.

[0507] For the luciferase assay, 50 μL of Bright-Glo assay substrate (100 μL cell suspension) was added per well of a 96-well white flat plate and incubated in the dark at room temperature for 3 minutes. Luminescence was measured using a Tecan Reader.

[0508] Thy1.1-positive cells were stained with a-Thy1.1-AF647 (clone OX-7) for flow cytometry. Dead cells (eFlour450) + ) was excluded from the analysis. FACS data was used in Thy1.1 + Median fluorescence intensity (MFI) of cells and Thy1.1 + The comparison was made by multiplying by the cell proportion.

[0509] Figure 9 shows that RiboDocker promotes the specific uptake of ALFA-coated nanoparticles into target cells and their expression from nucleic acid cargo.

Claims

1. A method for targeted delivery of a payload to target cells, (i) Transfecting one or more cells with RNA encoding a peptide or polypeptide containing the first binding site; (ii) expressing the peptide or polypeptide in one or more cells such that the peptide or polypeptide associates with target cells and the first binding portion is presented on the surface of the target cells; and (iii) A step of adding a payload which includes or is connected to a second coupling portion. A method comprising the first bonding portion and the second bonding portion being bonded to each other.

2. The method according to claim 1, wherein the RNA is transfected into the one or more cells by bringing the one or more cells into contact with the RNA-containing particles.

3. The method according to claim 1 or 2, wherein the particles include a targeting molecule for targeting one or more cells.

4. The method according to any one of claims 1 to 3, wherein one or more cells include target cells or consist of target cells.

5. The method according to any one of claims 1 to 4, wherein one or more cells express the peptide or polypeptide containing the first binding site such that it remains associated with the one or more cells.

6. The method according to any one of claims 1 to 3, wherein one or more of the cells are different from the target cells.

7. The method according to any one of claims 1 to 6, wherein one or more cells express the peptide or polypeptide containing the first binding site such that it is secreted by the one or more cells.

8. The method according to any one of claims 1 to 3, 6, and 7, wherein one or more cells express the peptide or polypeptide containing the first binding site in such a manner that it is released into the bloodstream.

9. The method according to any one of claims 1 to 8, wherein the peptide or polypeptide comprising the first binding portion comprises a third binding portion that binds to a target on a target cell.

10. The method according to claim 9, wherein the target is a cell surface antigen.

11. The method according to claim 9 or 10, wherein the first connecting portion and the third connecting portion are connected to each other.

12. The method according to any one of claims 1 to 11, wherein the first connecting portion and the third connecting portion are covalently connected to each other.

13. The method according to any one of claims 1 to 12, wherein the first binding portion is an antibody or an antibody derivative.

14. The method according to any one of claims 1 to 13, wherein the second binding portion is a peptide tag.

15. The method according to any one of claims 1 to 12, wherein the first binding portion is a peptide tag.

16. The method according to any one of claims 1 to 12 and 15, wherein the second binding portion is an antibody or an antibody derivative.

17. The method according to any one of claims 9 to 16, wherein the third binding portion is an antibody or an antibody derivative.

18. The method according to any one of claims 13, 14, 16, and 17, wherein the antibody derivative is an antibody fragment.

19. The method according to any one of claims 1 to 14, 17, and 18, wherein the peptide or polypeptide is a bispecific antibody.

20. The method according to claim 19, wherein the bispecific antibody is a bispecific single-chain antibody.

21. The method according to any one of claims 1 to 20, wherein the second coupling portion and the payload are covalently or non-covalently connected to each other.

22. The method according to any one of claims 1 to 21, wherein the payload comprises a pharmaceutically active drug.

23. The method according to any one of claims 1 to 22, wherein the payload comprises a diagnostic compound.

24. The method according to any one of claims 1 to 23, wherein the payload comprises a therapeutic compound.

25. The method according to any one of claims 1 to 24, wherein the payload includes a carrier.

26. The method according to claim 25, wherein the carrier is a fine particle carrier.

27. The method according to claim 26, wherein the fine particle carrier comprises lipid-based particles, polymer-based particles, or a mixture thereof.

28. The method according to any one of claims 25 to 27, wherein the carrier incorporates a diagnostic compound.

29. The method according to any one of claims 25 to 28, wherein the carrier incorporates a therapeutic compound.

30. The method according to any one of claims 1 to 29, wherein the payload includes a fourth coupling portion.

31. The method according to claim 30, wherein the fourth binding portion binds to a cell surface antigen.

32. The method according to claim 31, wherein the cell surface antigen to which the fourth binding portion binds is present on an immune cell.

33. The method according to any one of claims 1 to 32, wherein the target cells are present in the target.

34. The method according to any one of claims 1 to 33, performed in vivo.

35. For the target, (i) the RNA or particles containing the RNA that encode a peptide or polypeptide including the first binding site; and (ii) The payload or RNA encoding the payload, which includes or is ligated to the second binding portion. The method according to any one of claims 1 to 34, comprising the step of administering.

36. The method according to any one of claims 1 to 35, for diagnosing and / or treating a disease, wherein the target cells express or may express an antigen associated with the disease.

37. The method according to any one of claims 1 to 36, wherein the target cells are disease cells.

38. The method according to any one of claims 9 to 37, wherein the target is a tumor antigen.

39. The method according to any one of claims 1 to 38, wherein the target cells are tumor cells or cancer cells.

40. The method according to any one of claims 1 to 39, wherein the target cell is an immune effector cell.

41. The method according to any one of claims 1 to 35 and 40, wherein the target cell is a T cell.

42. The method according to claim 40 or 41, wherein the target is an antigen characteristic of the immune effector cells.

43. The method according to any one of claims 1 to 35 and 40 to 42, for delivering a nucleic acid encoding an antigen receptor to the immune effector cells.

44. A method for targeted delivery of a payload to target cells in a subject, (i) RNA encoding a peptide or polypeptide containing the first binding site; and (ii) A payload containing or ligated to the second binding site, or RNA encoding it. The process includes administering the above to the subject, The first binding portion and the second binding portion are bound to each other, and the peptide or polypeptide containing the first binding portion further includes a third binding portion that binds to a target on a target cell. method.

45. The method according to claim 44, wherein the RNA is present in the particles when administered.

46. The method according to claim 44 or 45, wherein, after administration of the RNA, the peptide or polypeptide comprising the first binding site and the third binding site is expressed by one or more cells of the subject.

47. The method according to claim 46, wherein one or more cells secrete the peptide or polypeptide comprising the first binding portion and the third binding portion.

48. The method according to claim 46 or 47, wherein one or more cells express the peptide or polypeptide comprising the first binding site and the third binding site in such a manner that it is released into the bloodstream.

49. A kit for targeted delivery of payloads to target cells, (i) RNA encoding a peptide or polypeptide containing the first binding site; and (ii) A payload containing or ligated to the second binding site, or RNA encoding it. Includes, A kit in which the first connecting portion and the second connecting portion are connected to each other.

50. The kit according to claim 49, wherein the peptide or polypeptide comprising the first binding portion comprises a third binding portion that binds to a target on a target cell.

51. The kit according to claim 50, wherein the target is a cell surface antigen.

52. The kit according to claim 50 or 51, wherein the first connecting portion and the third connecting portion are connected to each other.

53. The kit according to any one of claims 50 to 52, wherein the first connecting portion and the third connecting portion are covalently connected to each other.

54. The kit according to any one of claims 49 to 53, wherein the first binding portion is an antibody or an antibody derivative.

55. The kit according to any one of claims 49 to 54, wherein the second binding portion is a peptide tag.

56. The kit according to any one of claims 49 to 53, wherein the first binding portion is a peptide tag.

57. The kit according to any one of claims 49 to 53 and 56, wherein the second binding portion is an antibody or an antibody derivative.

58. The kit according to any one of claims 50 to 57, wherein the third binding portion is an antibody or an antibody derivative.

59. The kit according to any one of claims 54, 55, 57, and 58, wherein the antibody derivative is an antibody fragment.

60. The kit according to any one of claims 49 to 55, 58, and 59, wherein the peptide or polypeptide is a bispecific antibody.

61. The kit according to claim 60, wherein the bispecific antibody is a bispecific single-chain antibody.

62. The kit according to any one of claims 49 to 61, wherein the second coupling portion and the payload are covalently or non-covalently connected to each other.

63. The kit according to any one of claims 49 to 62, wherein the payload contains a pharmaceutically active drug.

64. The kit according to any one of claims 49 to 63, wherein the payload comprises a diagnostic compound.

65. The kit according to any one of claims 49 to 64, wherein the payload comprises a therapeutic compound.

66. The kit according to any one of claims 49 to 65, wherein the payload includes a carrier.

67. The kit according to claim 66, wherein the carrier is a fine particle carrier.

68. The kit according to claim 67, wherein the fine particle carrier comprises lipid-based particles, polymer-based particles, or a mixture thereof.

69. The kit according to any one of claims 66 to 68, wherein the carrier incorporates a diagnostic compound.

70. The kit according to any one of claims 66 to 69, wherein the carrier incorporates a therapeutic compound.

71. The kit according to any one of claims 49 to 70, wherein the payload includes a fourth coupling portion.

72. The kit according to claim 71, wherein the fourth binding portion binds to a cell surface antigen.

73. The kit according to claim 72, wherein the cell surface antigen to which the fourth binding portion binds is present on the immune cell.

74. The kit according to any one of claims 49 to 73, wherein the RNA is present in the particles.

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