WO2024210160A1

WO2024210160A1 - Conjugation complex

Info

Publication number: WO2024210160A1
Application number: PCT/JP2024/013829
Authority: WO
Inventors: Satoru Matsumoto; Eriya Kenjo; Yutaka Nishimoto; Tomoya Takenaka; Yumiko Ishii; Joseph Sean HARRISON
Original assignee: Takeda Pharmaceutical Company Limited
Priority date: 2023-04-07
Filing date: 2024-04-03
Publication date: 2024-10-10
Also published as: AR132276A1; TW202449142A

Abstract

The invention provides a complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell, the ligand being added to the outer surface of the delivery vehicle, wherein the delivery vehicle is comprised of an anchor molecule, a first binding partner is covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.

Description

CONJUGATION COMPLEX

The present invention relates to a complex for delivering an active substance to a target cell, a method for producing the complex, a composition for delivering the active substance to the target cell comprising the complex, and the like. The present invention also relates to a method for activating and/or proliferating the target cell, and a method for delivering the active substance inside the target cell.

The research and development of a non-viral delivery system of a genetic material into mammalian cells is progressing rapidly especially in the field of cell therapy including cancer immunotherapy using T cells expressing chimeric antigen receptor (CAR) or T-cell receptor (TCR) derived from cancer antigen-specific killer T cell. Current CAR-T cell therapy, such as Kymriah (trade name) and Yescarta (trade name), which were approved in the U.S., generally includes producing CAR-T cells by introducing CAR genes into T cells collected from a patient ex vivo using virus vectors such as lentivirus vector or retrovirus vector, and administering the CAR-T cells to the patient. However, this method has the problem that the production cost becomes high due to the cost of cell culture and preparation of virus vectors because multiple steps are necessary over a long period of time such as activation/proliferation of T cells, preparation of virus vectors, gene transfer into T cells, and the like.

As a method for introducing CAR into T cells without using a virus vector, ex vivo or in vivo transfection of CAR into T cells has been reported which uses nanoparticles containing aggregates of CAR-encoding plasmid DNA and a cationic polymer that are coated with a non-cationic polymer conjugated with anti-CD3 antibody fragments (patent document 1, non-patent document 1), or a nanocarrier containing mesoporous silica encapsulating CAR-encoding DNA in the pores and coated with a lipid having a surface modified with an anti-CD3 antibody (patent document 2).

Apart therefrom, techniques have been reported for delivering siRNA to a target cell by encapsulating the siRNA of interest in "lipid nanoparticles (LNP)", which do not have an internal pore structure and are composed of a cationic lipid, a non-cationic helper lipid, and a ligand for delivery to the target cell. For example, ex vivo or in vivo transfection of siRNA for CD45 into T cells by using an anti-CD4 antibody fragment as a targeted ligand has been reported (patent document 3, non-patent document 2)

In addition, patent document 4 describes a cationic lipid for introducing an active ingredient such as a nucleic acid or the like into various cells including T cells, tissues and organs.

On the other hand, as a method for activating/proliferating T cells, a method for activating and/or proliferating cytotoxic immune cells using beads on which anti-CD3/CD28 antibody is immobilized or nano-sized matrix beads has been reported (patent documents 5 and 6).

We previously developed a technology based on lipid nanoparticle (LNP) to perform steps of activating/proliferating cytotoxic immune cells and of introducing a gene into T cells simultaneously in one pod (patent document 7).

However, the target specificity and transfection efficiency has not been satisfactory. In order to improve the target specificity of the LNP, chemical technology for linking the LNP with a ligand specific to a target cell needs to be re-examined. A dibenzocyclooctyne (DBCO) group and an azide group are known to allow copper-free click chemistry to be done with live cells, whole organisms, and non-living samples. Prior to the DBCO-azide reaction, the DBCO group must be conjugated to the ligand specific to the target cell. Except for the molar ratio of a DBCO derivative such as NHS-ester-DBCO to the ligand of at least 5: 1 to at least 20: 1 (patent document 8), however, there is no prior art which introduces finely controlled conjugation of DBCO to the ligand, such as the exact site of DBCO conjugation on the ligand molecule, or the number of DBCO conjugation per one ligand molecule.

Document List

Patent Documents

patent document 1: US 2017/0296676

patent document 2: US 2016/0145348

patent document 3: WO 2016/189532

patent document 4: WO 2016/021683

patent document 5: U.S. Pat. No. 6,352,694

patent document 6: US 2014/0087462

patent document 7: WO 2020/080475

patent document 8: WO2021/113519

Non-Patent Documents

non-patent document 1: Nature Nanotechnology 12, 813-820 (2017)

non-patent document 2: ACS Nano, 2015, 9(7), 6706-6716

An object of the present invention is to provide an agent for delivering an active substance to a target cell or tissue with enhanced target specificity and higher transfection efficiency, so that the target cell transduced by the complex has higher activity which the active substance is expected to show. Some of other objects of the present invention are to provide a method for producing the complex, and a method for activation and/or proliferating a target cell comprising a step of contacting the agent and a cell population or a tissue comprising the target cell. Surprisingly, the present inventors found that by finely controlling conjugation of the ligand, such as the exact site of conjugation on the ligand molecule, or the number of conjugations per one ligand molecule, it dramatically enhances not only the physicochemical characteristics of a complex but also target specificity and transfection efficiency especially for the complex comprising lipid nanoparticles.

The present inventors have conducted intensive studies in an attempt to achieve the above-mentioned object and succeeded in providing the agent for delivering an active substance to a target cell or tissue as a complex, comprising a delivery vehicle for the active substance and a ligand specific to a target cell. Furthermore, the present inventors have surprisingly found that the expression of payload genetic material in higher percentage of target cells in vitro and in vivo compared with the prior art agent can be efficiently achieved by arranging the ligand added to the outer surface of the delivery vehicle, wherein the complex is comprised of an anchor molecule, arranging a first binding partner as covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the first binding partner; arranging a second binding partner as covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and arranging the first binding partner as covalently bonded to the second binding partner, and completed the present invention. Alternatively, the expression of payload genetic material in higher percentage of target cells in vitro and in vivo compared with the prior art agent can be efficiently achieved by arranging the ligand added to the outer surface of the delivery vehicle, wherein the complex is comprised of an anchor molecule; a first binding partner is covalently bonded to the ligand by sortase recognition motif; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner. Alternatively, the expression of payload genetic material in higher percentage of target cells in vitro and in vivo compared with the prior art agent can be efficiently achieved by arranging the ligand added to the outer surface of the delivery vehicle, wherein the complex is comprised of an anchor molecule; a first binding partner is covalently bonded to a C-terminal of the ligand; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.

Accordingly, the present invention provides the followings.

[1] A complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell, the ligand being added to the outer surface of the delivery vehicle, wherein the delivery vehicle is comprised of an anchor molecule, wherein: a first binding partner is covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.

[1a] A complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell, the ligand being added to the outer surface of the delivery vehicle, wherein the delivery vehicle is comprised of an anchor molecule, wherein: a first binding partner is covalently bonded to the ligand by sortase recognition motif; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.

[2] A complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell, the ligand being added to the outer surface of the delivery vehicle,
wherein the delivery vehicle is comprised of an anchor molecule, a first binding partner is covalently bonded to a C-terminal of the ligand; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.

[3] The complex of [1], [1a] or [2], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[4] The complex of [3], wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.

[5] The complex of [4], wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.

[6] The complex of [4], wherein the second binding partner is a molecule comprised of an azide group.

[7] The complex of [1], [1a] or [2], wherein the target cell to which the active substance is delivered is an immune cell.

[8] The complex of [7], wherein the immune cell is a cytotoxic cell.

[9] The complex of [8], wherein the cytotoxic cell is a NK cell or a T cell.

[10] The complex of [8], wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD8, CD7, CD16 and CD56.

[11] The complex of [1], [1a] or [2], wherein the active substance is a nucleic acid.

[12] The complex of [11], wherein the nucleic acid is comprised of a nucleic acid encoding a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR).

[13] A medicament comprising the complex of [12].

[14] A method for producing the complex of [1],[1a] or [2], comprising: (1) a step of covalently bonding the first binding partner to the ligand at a ratio of one molecule of the ligand per one molecule of the first binding partner; (2) a step of covalently bonding the second binding partner to the anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner by a chemical reaction at a ratio of one molecule of the second binding partner per one molecule of the first binding partner.

[15] The method of [14], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[16] The method of [15], wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.

[17] The method of [16], wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.

[18] The method of [16], wherein the second binding partner is a molecule comprised of an azide group.

[19] The method of [14], wherein the target T cell to which the active substance is delivered is an immune cell.

[20] The method of [19], wherein the immune cell is a cytotoxic cell.

[21] The method of [20], wherein the cytotoxic cell is a NK cell or a T cell.

[22] The method of [20], wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD8, CD7, CD16 and CD56.

[23] The method of [14], wherein the active substance is a nucleic acid.

[24] A composition for delivering the active substance to the target cell comprising the complex of [1], [1a] or [2].

[25] The composition of [24], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[26] The composition of [25], wherein the first binding partner is a molecule comprised of an alkylene group, and wherein the second binding partner is a 1,3-dipolar molecule.

[27] The composition of [26], wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.

[28] The composition of [26], wherein the second binding partner is a molecule comprised of an azide group.

[29] The composition of [24], wherein the target cell to which the active substance is delivered is an immune cell.

[30] The composition of [29], wherein the immune cell is a cytotoxic cell.

[31] The composition of [30], wherein the cytotoxic cell is a NK cell or a T cell.

[32] The composition of [30], wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD8, CD7, CD16 and CD56.

[33] The composition of [24], wherein the active substance is a nucleic acid.

[34] The composition of [33], further comprising a culture medium or saline.

[35] A method for activating and/or proliferating the target cell, comprising: a step of contacting the complex of [1], [1a] or [2] and a cell population comprising the target cell, where the complex is comprised of at least one ligand specific to the target cell.

[36] The method of [35], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[37] The method of [36], wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.

[38] The method of [37], wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.

[39] The method of [37], wherein the second binding partner is a molecule comprised of an azide group.

[40] The method of [35], wherein the target cell to which the active substance is delivered is an immune cell.

[41] The method of [40], wherein the immune cell is a cytotoxic cell.

[42] The method of [41], wherein the cytotoxic cell is a NK cell or a T cell.

[43] The method of [41], wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD8, CD7, CD16 and CD56.

[44] The method of [41], wherein more than one ligand is added to the outer surface of the delivery vehicle.

[45] The method of [35], wherein the active substance is a nucleic acid.

[46] The method of [45], wherein the nucleic acid is comprised of a nucleic acid encoding a CAR and/or a TCR.

[47] The method of [35], wherein the step of contacting the complex and the cell population comprising the target cell is conducted ex vivo.

[48] A method for delivering the active substance inside the target cell, comprising: a step of contacting the complex of [1], [1a] or [2] and a cell population comprising the target cell, wherein the active substance does neither include any nucleic acid encoding a CAR nor a TCR.

[49] The method of [48], wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.

[50] The method of [49], wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.

[51] The method of [50], wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.

[52] The method of [50], wherein the second binding partner is a molecule comprised of an azide group.

[53] The method of [48], wherein the target cell to which the active substance is delivered is an immune cell.

[54] The method of [53], wherein the immune cell is a cytotoxic cell.

[55] The method of [54], wherein the cytotoxic cell is a NK cell or a T cell.

[56] The method of [55], wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD8, CD7, CD16 and CD56.

[57] The method of [54], wherein more than one ligand is added to the outer surface of the delivery vehicle.

[58] The method of [48], wherein the active substance is a nucleic acid.

[59] The method of [58], wherein the nucleic acid is comprised of a nucleic acid which inhibits the expression of a cytotoxic cell activation inhibitory factor, and/or a nucleic acid which encodes a cytotoxic cell activation promoting factor.

[60] The method of [48], wherein the step is conducted ex vivo.

[61] The target cell to which the active substance is delivered by the method of [48].

[62] A medicament comprising the target cell of [61].

[63] The medicament according to [62], further comprising a culture medium or saline.

According to the present invention, the complex for delivering an active substance is comprised of a delivery vehicle for the active substance and a ligand specific to a target cell. By arranging the first binding partner, which is covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand, or by arranging the first binding partner which is covalently bonded to the ligand by sortase recognition motif, or by arranging the first binding partner which is covalently bonded to a C-terminal of the ligand, and the second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle, the complex can express payload genetic material in higher percentage of target cells in vitro and in vivo compared with the prior art agent for cell therapy,

A biological sequence data described in the specification is presented in a standardized electronic format (a "Sequence Listing XML") as a separate part of the present specification. Namely, the Sequence Listing consists of a single amino acid sequence (SEQ ID NO.:1) of five amino acid residues representing a sortase recognition motif, or a consensus amino acid sequence which is recognized by sortase A (StrA) from Staphylococcus aureus. The amino acid sequence is: Leu-Pro-Xxx-Thr-Gly, or LPXTG, where Xxx or X is any amino acid and Gly or G cannot be a free carboxylate.

1. Complex of the present invention

In one aspect, the present invention provides a complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell,
the ligand being added to the outer surface of the delivery vehicle, wherein
the delivery vehicle is comprised of an anchor molecule,
a first binding partner is covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand;
a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and
the first binding partner is covalently bonded to the second binding partner. (hereinafter to be also referred to as "the complex of the present invention".
In another aspect, the present invention provides a complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell,
the ligand being added to the outer surface of the delivery vehicle,
the delivery vehicle is comprised of an anchor molecule, wherein: a first binding partner is covalently bonded to the ligand by sortase recognition motif; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.
In a further aspect, the present invention provides a complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell,
the ligand being added to the outer surface of the delivery vehicle,
the delivery vehicle is comprised of an anchor molecule, wherein: a first binding partner is covalently bonded to a C-terminal of the ligand; a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and the first binding partner is covalently bonded to the second binding partner.
In the present specification, the complex refers to a molecular assembly constituted of (a) a delivery vehicle for the active substance and (b) a ligand specific to a target cell. The complex serves as carrier of the active substance for specific delivery to the target cell.
The constituent elements (a) and (b) are explained below.
1. (a) Delivery vehicle for the active substance

A delivery vehicle for the active substance may be any molecular assembly which is comprised of an anchor molecule, which may function as a protective shield or vehicle for the active substance and to which the ligand is attached through the first and second binding partners. Examples of the delivery vehicle for the active substance include a lipid nanoparticle, a liposome, cationic polymers (e.g., polyethyleneimine, polylysine, polyornithine, chitosan, atelocollagen, protamine etc.), those in which a cationic polymer is encapsulated in liposomes, and the like. Alternatively, exosome, which is a component derived from living organisms, can also be used.

The anchor molecule is a lipid molecule which is covalently modified to comprise the second binding partner, a 1,3-dipole. The anchor molecule is amenable to covalent bonding with the ligand, which is conjugated to the first binding partner, a dipolarophile. The first and second binding partners are capable of making a specific chemical bond between each other in biologically safe manner, not only in vitro but also in vivo, without any adverse side reaction. The anchor molecule may be any one or more of a lipid molecule. The anchor molecule may be a non-cationic lipid. The "non-cationic lipid" means a lipid other than the cationic lipid, and is a lipid that does not have a net positive electric charge at a selected pH such as physiological pH and the like. Examples of the non-cationic lipid used in the lipid nanoparticle of the present invention include phospholipid, steroids, PEG lipid and the like.

The complex of the present invention is composed of a delivery vehicle and a ligand. The delivery vehicle is made by aggregation of anchor molecules and other lipid molecules, in which an active substance may be encapsulated and protected from the attack of enzyme or other chemical substance which degrades the active substance, such as protease when the active substance is a protein, and nuclease when the active substance is a nucleic acid. The delivery vehicle is preferably a lipid nanoparticle (LNP) having an interior lipid core, instead of lipid bilayer of cells in general and liposomes. Thus, the outer surface of the delivery vehicle is in aqueous phase and the inner core of the delivery vehicle is in less- or non-aqueous phase. When the active substance is hydrophilic, it is presumed that the hydrophilic active substance is encapsulated by the lipid with the hydrophilic portion of the lipid facing the hydrophilic active substance. By specifying that the ligand being added to the outer surface of the delivery vehicle, all of the ligand is exposed to the aqueous phase such as culture medium or saline and to the outer surface of the target cell, without losing any ligand buried in the delivery vehicle, so that the specific interaction between the ligand and target cell, or cell surface molecule of the target cell to be exact, promotes target-cell specific incorporation of the complex.

In the present application, an active substance loaded in the delivery vehicle of the present invention may be one or more selected drugs. In one embodiment the delivery vehicle contains a single drug component. In another embodiment, the delivery vehicle is loaded with multiple drug components. By "drug" as used herein is meant any therapeutic, prophylactic, or diagnostic compound or reagent that is contained within the delivery vehicle described herein. In one embodiment, the drug is a water-miscible compound. The active substance loaded in the delivery vehicle of the present invention may be any naturally occurred or synthetically generated molecule, including a low molecular-weight chemical compound with a molecular mass not exceeding 2,500 Dalton, a biopolymer with a molecular mass within the size limitation of the delivery vehicle such as a carbohydrate, a polypeptide, a protein, a nucleic acid or any derivative thereof.

The active substance loaded in the delivery vehicle of the present invention may be a nucleic acid including polynucleotide comprising deoxyribonucleotides, ribonucleotides and/or any of their non-natural analogues with modification in nucleobase, phosphodiester backbone and/or sugar moieties. When the active substance is a nucleic acid, it may encode a protein or an antisense polynucleotide which inhibits the expression of a protein.

In one embodiment of the present invention, the protein encoded in the nucleic acid loaded in the delivery vehicle may be any protein which exerts a therapeutic or prophylactic action in the delivered or transduced cell. For the delivery to an immune cell, preferably a cytotoxic cell, the protein encoded in the nucleic acid loaded in the delivery vehicle may be a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR). As the cytotoxic cell should be activated to kill a cell which is recognized by the CAR and/or TCR, the active substance serves as a therapeutic agent for cancer, or any other disease by activating the target cell to attack and kill pathologically changed cells such as malignant cells or cells which shed pathological deposits to cause fibrosis. Other than the immune cell, the target cell of the complex includes various somatic or germ line cells whose pathological change can be rescued by ectopic expression of a foreign gene or protein, such as cells of central and peripheral nervous system, sensory system, digestive system, respiratory system, urinary system, cardiovascular system, reproductive system, bone, muscle, skin and blood cells. The ligand for each of the various somatic and germ line cells can be identified using such internet resource as the CellMarker database (Xinxin Zhang, et al., Nucleic Acids Research, 2019, Vol. 47, Database issue D721-D728). In one embodiment, more than one ligand may be incorporated in the complex of the present invention.

The active substance loaded in the delivery vehicle of the present invention is neither limited to CAR nor TCR. When the active substance is a nucleic acid encoding a protein which is neither CAR nor TCR, the protein expressed in the target cell serves as a vaccine for infection or a replenishment therapy for a pathological loss or decrease of the protein. When the active substance is an antisense RNA or siRNA or other nucleic acid which inhibits the transcription and/or translation of a complementary RNA, the antisense RNA and others serves as an inhibitor to alleviate a pathological increase of the complementary RNA or its translated protein.

When the active substance is a genome editing system comprising a guide RNA and/or its associated site-specific nuclease or a nucleic acid encoding the nuclease, the guide RNA and/or the nuclease serves as a therapeutic agent to introduce genome editing modification in a gene of interest in the genome of the target cells. The guide RNA comprises a guide sequence which is complementary to the gene of interest. The nuclease associates with the guide RNA in the target cell, binds to the gene of interest at the complementary sequence in the genome of the target cell, and breaks a phosphodiester bond of the genomic DNA or cleave a chunk of DNA from the genomic DNA. Depending on the design of the guide RNA, the nuclease associated with the guide RNA may induce a single strand break or a double strand break of genomic DNA. The site-specific nuclease may be selected from the group consisting of meganucleases, Zinc finger nucleases (ZFNs), CRISPR/Cas9 proteins, CRISPR-Cpf1 proteins, and TAL effector nucleases (TALENs).

Thus, in one embodiment of the present invention, the active substance is the guide RNA and/or the site-specific nuclease as described in the above for the genome editing. Preferably, the active substance is the guide RNA and the site-specific nucleases derived from CRISPR system.

CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeat refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. The CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a memory of past exposures. Cas9 forms a complex with the 3' end of the sgRNA (also referred interchangeably herein as "gRNA"), and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5' end of the sgRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. This complex is directed to homologous loci of pathogen DNA via regions encoded within the crRNA, i.e., the protospacers, and protospacer-adjacent motifs (PAMs) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed sgRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.

Three classes of CRISPR systems (Types I, II, and III effector systems) are known. The Type II effector system carries out targeted DNA double-strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre-crRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9, forming a Cas9: crRNA-tracrRNA complex.

An engineered form of the Type II effector system of Streptococcus pyogenes was shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted "guide RNA" ("gRNA", also used interchangeably herein as a chimeric single guide RNA ("sgRNA")), which is a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. Provided herein are DNA targeting systems for use in genome editing and treating genetic diseases. The presently disclosed DNA targeting system can be designed to target any gene, including genes involved in a genetic disease, aging, tissue regeneration, or wound healing. The DNA targeting system includes a polynucleotide encoding a Cas9 protein or a Cas9 fusion protein and one or more gRNAs. In some embodiments, the polynucleotide encoding a Cas9 protein or a Cas9 fusion protein is an mRNA. The mRNA may be a modified mRNA. A modified mRNA may include one or more modifications selected from an N terminal NLS, a C terminal NLS, an HA Tag, and a uridine substitution. The Cas9 fusion protein may, for example, include a domain that has a different activity that what is endogenous to Cas9, such as a transactivation domain.

The target gene (e.g., any gene of interest) can be involved in differentiation of a cell or any other process in which activation of a gene can be desired, or can have a mutation such as a frameshift mutation or a nonsense mutation. If the target gene has a mutation that causes a premature stop codon, an aberrant splice acceptor site or an aberrant splice donor site, the DNA targeting system can be designed to recognize and bind a nucleotide sequence upstream or downstream from the premature stop codon, the aberrant splice acceptor site or the aberrant splice donor site. The DNA targeting system can also be used to disrupt normal gene splicing by targeting splice acceptors and donors to induce skipping of premature stop codons or restore a disrupted reading frame. The DNA targeting system may or may not mediate off-target changes to protein-coding regions of the genome. a. Cas9 Molecules and Cas9 Fusion Proteins.

The DNA targeting system of the invention comprises mRNA encoding a Cas9 protein or a Cas9 fusion protein. Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein can be from any bacterial or archaea species, including, but not limited to, Streptococcus pyogenes, Staphylococcus aureus (S. aureus), Acidovorax avenae, Actinobacillus pleuropneumonias, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter musteiae, llyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multodda, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae. In certain embodiments, the Cas9 molecule is a Streptococcus pyogenes Cas9 molecule (also referred herein as "SpCas9"). In certain embodiments, the Cas9 molecule is a Staphylococcus aureus Cas9 molecule (also referred herein as "SaCas9"). In some embodiments, the Cas9 molecule is a mutant Cas9 molecule. The Cas9 protein can be mutated so that the nuclease activity is inactivated. In some embodiments, the Cas9 molecule is a deactivated or inactivated Cas9 protein (dCas9 or iCas9), with no endonuclease activity. Exemplary mutations with reference to the S. pyogenes Cas9 sequence to inactivate the nuclease activity include: D10A, E762A, H840A, N854A, N863A and/or D986A. Exemplary mutations with reference to the S. aureus Cas9 sequence to inactivate the nuclease activity include D10A and N580A.

The mRNA encoding a Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. The synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein. In various embodiments of the invention there is limited or no humoral response that is cross reactive to Cas9 after administration to a subject.

"Repeat variable diresidue" or "RVD" as used interchangeably herein refers to a pair of adjacent amino acid residues within a DNA recognition motif (also known as "RVD module"), which includes 33-35 amino acids, of a TALE DNA-binding domain. The RVD determines the nucleotide specificity of the RVD module. RVD modules may be combined to produce an RVD array. The "RVD array length" as used herein refers to the number of RVD modules that corresponds to the length of the nucleotide sequence within the TALEN target region that is recognized by a TALEN, i.e., the binding region.
1. (a-1) A lipid nanoparticle

In one embodiment, the delivery vehicle for the active substance is a lipid nanoparticle. In the present specification, the "lipid nanoparticle (LNP)" means particles with an average diameter of less than 1 m and free of a small porous structure (e.g., mesoporous material) in a molecular assembly constituted of a cationic lipid and a non-cationic lipid.
1. (a-1.1) Cationic Lipid

In the present specification, the "cationic lipid" means a lipid that has a net positive charge in a low pH environment such as in physiological pH. The cationic lipids used in the lipid nanoparticle used in the present invention are not particularly limited. For example, cationic lipids and the like described in WO 2016/021683, WO 2015/011633, WO 2011/153493, WO 2013/126803, WO 2010/054401, WO 2010/042877, WO 2016/104580, WO 2015/005253, WO 2014/007398, WO 2017/117528, WO 2017/075531, WO 2017/00414, WO 2015/199952, US 2015/0239834, WO2019/131839, and the like can be mentioned.

Alternatively, the synthetic cationic lipids (e.g., K-E12, H-A12, Y-E12, G-O12, K-A12, R-A12, cKK-E12, cPK-E12, PK1K-E12, PK500-E12, cQK-E12, cKK-A12, KK-A12, PK-4K-E12, cWK-E12, PK500-O12, PK1K-O12, cYK-E12, cDK-E12, cSK-E12, cEK-E12, cMK-E12, cKK-O12, cIK-E12, cKK-E10, cKK-E14, and cKK-E16, preferably, cKK-E12, cKK-E14) described in Dong et al. (Proc Natl Acad Sci U S A. 2014 Apr. 15; 111(15):5753), and the synthetic cationic lipids (e.g., C14-98, C18-96, C14-113, C14-120, C14-120, C14-110, C16-96 and C12-200, preferably 014-110, C16-96 and C12-200) described in Love KT et al. (Proc Natl Acad Sci U S A. 2010 May 25; 107(21):9915) can be mentioned.

In one preferred embodiment, a cationic lipid represented by the following general formula and described in WO 2016/021683 can be mentioned.

wherein
W is the formula -NR1R2 or the formula -N+R3R4R5(Z-),
R1 and R2 are each independently a C1-4 alkyl group or a hydrogen atom,
R3, R4 and R5 are each independently a C1-4 alkyl group,
Z- is an anion,
X is an optionally substituted C1-6 alkylene group,
YA, YB and YC are each independently an optionally substituted methine group,
LA, LB and LC are each independently an optionally substituted methylene group or a bond, and
RA1, RA2, RB1, RB2, RC1 and RC2 are each independently an optionally substituted C4-10 alkyl group,
or a salt thereof.

Further detail of the cationic lipid is explained in WO2019/131770, the contents of which are incorporated in full herein by reference.

The ratio (mol%) of the cationic lipid to the total lipids present in the lipid nanoparticle of the present invention is, for example, about 10% to about 80%, preferably about 20% to about 70%, more preferably about 40% to about 60%; however, the ratio is not limited to these.
Only one kind of the above-mentioned cationic lipid may also be used or two or more kinds thereof may be used in combination. When multiple cationic lipids are used, the ratio of the whole cationic lipid is preferably as mentioned above.
1. (a-1.2) Non-cationic lipid

In the present specification, the "non-cationic lipid" means a lipid other than the cationic lipid, and is a lipid that does not have a net positive electric charge at a selected pH such as physiological pH and the like. Examples of the non-cationic lipid used in the lipid nanoparticle of the present invention include phospholipid, steroids, PEG lipid and the like.

When the active substance is nucleic acid such as polynucleotides encoding CAR or TCR, the phospholipid is not particularly limited, as long as it stably maintains nucleic acid and does not inhibit fusion with cell membranes (plasma membrane and organelle membrane). For example, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid, palmitoyloleoylphosphatidyl choline, lysophosphatidyl choline, lysophosphatidyl ethanolamine, dipalmitoylphosphatidyl choline, dioleoylphosphatidyl choline, distearoylphosphatidyl choline, dilinolenoylphosphatidyl choline and the like can be mentioned.

Preferred phospholipids include distearoylphosphatidyl choline (DSPC), dioleoylphosphatidyl choline (DOPC), dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidyl glycerol (DOPG), palmitoyloleoylphosphatidyl glycerol (POPG), dipalmitoylphosphatidyl glycerol (DPPG), dioleoyl-phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidyl choline (POPC), palmitoyloleoyl-phosphatidyl ethanolamine (POPE), and dioleoylphosphatidyl ethanolamine 4-(N-maleimide methyl)-cyclohexane-1-carboxylate (DOPE-mal), more preferably DOPC, DPPC, POPC, and DOPE.

The ratio (mol%) of the phospholipid to the total lipids present in the lipid nanoparticle of the present invention may be, for example, about 0% to about 90%, preferably about 5% to about 30%, more preferably about 8% to about 15%.
Only one kind of the above-mentioned phospholipid may be used or two or more kinds thereof may be used in combination. When multiple phospholipids are used, the ratio of the whole phospholipid is preferably as mentioned above.

As the steroids, cholesterol, 5α-cholestanol, 5β-coprostanol, cholesteryl-(2’-hydroxy)-ethylether, cholesteryl-(4’-hydroxy)-butylether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate can be mentioned, preferably cholesterol.

The ratio (mol%) of the steroid to the total lipids present in the lipid nanoparticle of the present invention when steroids are present may be, for example, about 10% to about 60%, preferably about 12% to about 58%, more preferably about 20% to about 55%.
Only one kind of the above-mentioned steroid may be used or two or more kinds thereof may be used in combination. When multiple steroids are used, the ratio of the whole steroid is preferably as mentioned above.

In the present specification, the "PEG lipid" means any complex of polyethylene glycol (PEG) and lipid. PEG lipid is not particularly limited, as long as it has an effect of suppressing aggregation of the lipid nanoparticles of the present invention. For example, PEG conjugated with dialkyloxypropyl (PEG-DAA), PEG conjugated with diacylglycerol (PEG-DAG) (e.g., SUNBRIGHT GM-020 or GS-020 (NOF CORPORATION)), PEG conjugated with phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated with ceramide (PEG-Cer), PEG conjugated with cholesterol (PEG-cholesterol), or derivatives thereof, or mixtures thereof, mPEG2000-1,2-Di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG), 1-[8’-(1,2-dimyristoyl-3-propanoxy)-carboxamide-3’,6-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethylene glycol) (2KPEG-DMG) and the like can be mentioned. Preferred PEG lipid includes PEG-DGA, PEG-DAA, PEG-PE, PEG-Cer, and a mixture of these, more preferably, a PEG-DAA conjugate selected from the group consisting of a PEG-didecyloxypropyl conjugate, a PEG-dilauryloxypropyl conjugate, a PEG-dimyristyloxypropyl conjugate, a PEG-dipalmityloxypropyl conjugate, a PEG-distearyloxypropyl conjugate, and mixtures thereof.

In addition to the methoxy group, the maleimide group, N-hydroxysuccinimidyl group and the like for binding the T cell targeting ligand described later can be used as the free end of PEG. For example, SUNBRIGHT DSPE-0201MA or SUNBRIGHT DSPE-0201MA (NOF) can be used as a PEG lipid having a functional group for binding a T cell-targeting ligand (sometimes to be referred to as "terminal reactive PEG lipid" in the present specification).

In one embodiment, the delivery vehicle having the anchor molecule (e.g., PEG lipid) covalently bonded to the second binding partner (e.g., azide) is prepared as follows. A mixture of lipids (Cationic lipid: DPPC: Cholesterol: SUNBRIGHT GS-020: DSPE-PEG (2000)-Azide=60: 10.6: 27: 1.4: 1, mol%) was dissolved in 90% EtOH/10% Acetate buffer (25 mM, pH4.0) to obtain a 14 mg/mL of lipid solution. As cationic lipid, the following compounds were used: 3-((4-(dimethylamino)butanoyl)oxy)-2,2-bis(((3- pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate (used in LNP targeting T cells) described in WO2016/021683; or 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyldidecanoate (used in LNP targeting NK cells) described in WO2020/032184.

The ratio (mol%) of the PEG lipid to the total lipids present in the lipid nanoparticle of the present invention may be, for example, about 0% to about 20%, preferably about 0.1% to about 5%, more preferably about 0.7% to about 2%.
The ratio (mol%) of the terminal reactive PEG lipid in the above-mentioned total PEG lipids is, for example, about 10% to about 100%, preferably about 20% to about 100%, more preferably about 30% to about 100%.
Only one kind of the above-mentioned PEG lipid may be used or two or more kinds thereof may be used in combination. When multiple PEG lipids are used, the ratio of the whole PEG lipid is preferably as mentioned above.
1. (a-2) Liposome

A liposome is referred to as a microscopic spherical particle formed by an outer lipid bilayer enclosing an inner pore structure or an aqueous compartment. As a delivery vehicle for the active substance, a liposome prepared by mixing various cationic lipids (e.g., DOTMA, DOTAP, DDAB, DMRIE etc.) developed as transfection reagents, and membrane-fused neutral lipids (e.g., DOPE, cholesterol etc.) that promote release from endosome are widely used. Liposomes in which functional molecules such as PEG, pH-responsive membrane fusion peptide, membrane permeation promoting peptide and the like are added to the surface of the liposome can also be used.

Due to their size and hydrophobic and hydrophilic character, liposome is a promising system for drug delivery. Liposome properties differ considerably with lipid composition, surface charge, size, and the method of preparation. Furthermore, the choice of bilayer components determines the ‘rigidity’ or ‘fluidity’ and the charge of the bilayer. For instance, unsaturated phosphatidylcholine species from natural sources (egg or soybean phosphatidylcholine) give much more permeable and less stable bilayers, whereas the saturated phospholipids with long acyl chains (for example, dipalmitoyl phosphatidylcholine) form a rigid, rather impermeable bilayer structure. Liposomes useful herein can be prepared using techniques known in the art. See, Akbarzadeh et al, Nanoscale Res Lett. Feb 2013; 8(1): 102, which is incorporated herein by reference. In one embodiment, the liposome comprises cholesterol. It has been observed that the amount of cholesterol in the liposome composition can affect the delivery of the liposome. Thus, the amount of cholesterol may be varied. In one embodiment the amount of cholesterol in the liposome is about 10 to 50% by lipid film composition. In one embodiment, the cholesterol content of the liposome is at about 25% (moles cholesterol / total moles of lipid). In one embodiment, the cholesterol content of the liposome is about 40% (moles cholesterol / total moles of lipid). In one embodiment, the cholesterol content of the liposome is at least 25% (moles cholesterol / total moles of lipid). In one embodiment, the cholesterol content of the liposome is at least 40% (moles cholesterol / total moles of lipid).

An exemplar delivery vehicle for the active substance of the present invention is a lipid nanoparticle.

1. (b) A ligand specific to a target cell
The ligand capable of targeting the complex of the present invention to the target cell is not particularly limited, as long as it can specifically recognize surface molecules that are specifically or highly expressed in the target cell. In embodiments, the ligand comprises a protein or peptide. In embodiments with an immune cell as the target cell, it includes those containing one or more antigen binding domains of antibodies against CD3, CD4, CD7, CD8, CD16, CD28 or CD56, and more preferably, it includes those containing antigen binding domains of anti-CD3 antibody, anti-CD16 antibody, anti-CD28 antibody and/or anti-CD56 antibody. A particularly preferable example for in vivo delivery to cytotoxic T cells is one containing only the antigen-binding domain of an anti-CD3 antibody. A particularly preferable example for in vivo delivery to NK cells is one containing only the antigen-binding domain of an anti-CD56 antibody or one containing only the antigen-binding domain of an anti-CD16 antibody. Here, the "antigen-binding domain" is synonymous with the antigen-binding domain that constitutes the above-mentioned CAR. However, since CAR needs to be prepared as a nucleic acid encoding same, restrictions occur and single-chain antibodies are generally used in many cases. Because the antigen-binding domain as a T cell targeting ligand is contained as a protein in the complex of the present invention, not only single-chain antibodies, but also any other antibody fragments, such as intact antibody molecules, Fab, F(ab’)2, Fab’, Fv, reduced antibody (rIgG), dsFv, scFv, diabody, triabody, HCAb, VHH and the like, can also be used preferably. Fab or Fab’ without an Fc moiety is preferably used. Fab or Fab’ is preferable especially for delivery to the target immune cell in vivo. For the complex of the present invention, the ligand for target cell needs to be conjugated with a molecule comprised of dipolarophile, such as DBCO, using a transpeptidase. Thus, the ligand for the target cell is an immunoglobulin such as IgG or its antigen binding domain which is produced by the recombinant DNA technology as a ligand fusion protein in order to incorporate oligopeptide corresponding to the sortase recognition motif as mentioned in the above.

When the immune cell targeting ligand is an intact antibody molecule, commercially available anti- CD3, CD4, CD7, CD8, CD16, CD28 or CD56 antibodies, etc. can be used, or the ligand can be isolated from the culture of the cells producing the antibody. On the other hand, when the ligand is any one of the aforementioned antigen-binding domains (antibody fragment), the nucleic acid encoding the antigen-binding domain, such as anti- CD3, CD4, CD7, CD8, CD16, CD28 or CD56 antibodies, etc., is isolated in the same way as in the nucleic acid encoding the antigen-binding domain constituting the said CAR is obtained, and the antigen-binding domain can be recombinantly produced using the same.

In the complex of the present invention, the immune cell-targeting ligand binds to the outer surface of the delivery vehicle by first preparing the first binding partner covalently bonded to the ligand and the second binding partner covalently bonded to an anchor molecule in the delivery vehicle; and then the first binding partner is covalently bonded to the second binding partner.

In some embodiments, the covalent bond between the first binding partner and the second binding partner is generated using a Click chemical reaction between a molecule comprised of a dipolarophile and a 1,3-dipole. The safety of the complex for delivering an active substance is assured because the chemical reaction does not involve copper as catalyst. In the present invention, a molecule comprised of a dipolarophile is selected to be covalently bonded to the ligand and a 1, 3-dipole is selected to be covalently bonded to an anchor molecule. The anchor molecules are aggregated together with other lipid molecules to constitute the delivery vehicle. We found this combination is preferable to the reverse combination of a 1, 3-dipole (an azide) covalently bonded to the ligand and a molecule comprised of a dipolarophile (a dibenzocyclooctyne (DBCO) group) covalently bonded to an anchor molecule, because the complex with the reverse combination exhibited a relatively higher polydispersity index which was not determined as adequate for a pharmaceutical formulation. While not wishing to be bound by theory, the relatively higher polydispersity index is probably due to hydrophobic interactions between the delivery vehicle having an aggregate of anchor molecule and other lipid molecules and the bulky hydrophobic DBCO group.

The covalent bonding of the first binding partner and the ligand is carried out at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand; or covalent bonding of the first binding partner and the ligand is carried out by sortase recognition motif; or the first binding partner is covalently bonded to the carboxy terminus (C-terminal) of the ligand. The ratio of molecules of the first binding partner per one molecule of the ligand is appropriately selected based on the first binding partner and the ligand. The ratio may be 1, 2, 3 or 4, preferably 1 or 2, more preferably 1. The ratio is preferably 1 when the ligand is Fab. The ratio is preferably 2 or 4 when the ligand is intact antibody.

It is possible to design the exact number and sites on the ligand of the covalent bond between the first binding partner and the ligand, because an exact site on the ligand to conjugate the first binding partner, or a molecule comprised of a dipolarophile is defined by preparing the fusion protein of the ligand, or ligand fusion protein, which incorporates an amino acid sequence known as a sortase recognition motif at the carboxy terminus, mixing the ligand fusion protein with a derivative of the molecule comprised of a dipolarophile and treating them with a sortase to conjugate the DBCO.

Most used sortase for protein modification is the sortase A (StrA) from Staphylococcus aureus which mediates a transpeptidase reaction by recognizing a motif of: Leu-Pro-Xxx-Thr-Gly (LPXTG, where X is any amino acid and Gly cannot be a free carboxylate; recited in the present sequence listing as SEQ ID NO: 1) near the carboxy terminus of a fusion protein and its counterpart molecule which must have a free glycine residue at its amino terminus. In the present invention where a ligand is conjugated with a molecule comprised of a dipolarophile such as DBCO, the counterpart molecule may be any derivative of DBCO which has a glycine at its amino terminus, for example, 5-(Glycylglycyl-beta-alanyl)-11,12-dihydro-5,6-dihydrodibenzo[b,f]azocin. By the transpeptidase reaction, the last glycine of the sortase recognition motif of the ligand protein is exchanged with the glycine at the amino terminus of the DBCO derivative, resulting a ligand fusion protein whose carboxy terminus is conjugated with DBCO. In the transpeptidase reaction of the present invention, sortase other than StrA of Staphylococcus aureus may be used, such as StrB of Staphylococcus aureus, which recognizes a different recognition motif with five amino acids. The sortase enzymes and their recognition motifs are found in, for example, Jacobitz, AW, et al., Adv Protein Chem Struct Biol. 2017; 109: 223-264. Purified recombinant sortase enzyme for conjugating DBCO group to a fusion protein is commercially available from many suppliers, such as Funakoshi Co., Ltd. (Tokyo).

The covalent bonding of the second binding partner and the anchor molecule is carried out by a known method. In one embodiment, azide as the second binding partner and the anchor molecule may be covalently bonded by nucleophilic addition reaction of azide reagent to the activated anchor molecule. In one embodiment, the anchor molecule and molecule conjugated with azide may be covalently bonded through linker structures such as amide, ester, carbamate, carbonate, but not limited to. The anchor molecule bonded to the second binding partner may be used to make a delivery vehicle by aggregating the molecules with other lipid molecules.

For the complex of the present invention, the target cell to which the active substance is delivered may be an immune cell. An immune cell encompasses all type of cells involved in immunological function of human or other mammalian cells, namely, T cell, which is responsible for cellular immunity among acquired immunity, NK cell, NKT cell, monocyte, macrophage, dendritic cell, and the like which is responsible for innate immunity, and NKT cell which is T cell having the properties of NK cell.

The complex of the present invention can be produced, for example, by the method described in US9,404,127. The delivery vehicle, which is comprised of an anchor molecule, is incubated with an azide to immobilize the azide on the delivery vehicle. The mixing ratio (molar ratio) of cationic lipid, phospholipid, cholesterol, PEG lipid and azide is, for example, 40 to 60:0 to 20:0 to 50:0 to 5: 1, but the ratio is not limited thereto. The above-mentioned mixing can be conducted using a pipette, a micro fluid mixing system (e.g., Asia microfluidic system (Syrris)) or Nanoassemblr (Precision Nanosystems)). The obtained lipid particles may be subject to purification by gel filtration, dialysis, or sterile filtration. The concentration of the total lipid component in the organic solvent solution is preferably 0.5 to 100 mg/mL.

As the organic solvent, for example, methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, tert-butanol, acetone, acetonitrile, N, N-dimethylformamide, dimethylsulfoxide, or a mixture thereof can be recited. The organic solvent may contain 0 to 20% of water or a buffer solution. As the buffer solution, acidic buffer solutions (e.g., acetate buffer solution, citrate buffer solution) or neutral buffer solutions (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, (HEPES) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, a phosphate buffer solution, phosphate buffered saline (PBS)) can be recited.

When a delivery vehicle dispersion is produced as described above, the dispersion containing the above-mentioned lipids can be produced by adding an active substance to water or buffer solution. Addition of the active substance in a manner to render the concentration thereof the active ingredient in water or a buffer solution 0.05 to 2.0 mg/mL is preferable.

When the complex further contains a ligand specific to a target cell, it can be produced by first preparing a ligand fusion protein incorporating the sortase recognition motif near the carboxy terminus, then incubating the ligand fusion protein with a derivative of the molecule comprised of a dipolarophile, so that the molecule comprised of a dipolarophile is covalently attached to the carboxy terminus of the ligand fusion protein.

The ligand fusion protein conjugated with the molecule comprised of a dipolarophile and the delivery vehicle which is conjugated with azide group are mixed so that molar concentration ratio of the ligand fusion protein to azide is in the range of 1:20, 1:10 or 1:20 to 2:1 (ligand fusion protein: azide) for the complex targeting mouse T cell; 1:100, 1:20 or 1:100 to 2:1 (ligand fusion protein: azide) for the complex targeting human T cell; and 1:40, 1:20 or 1:40 to 2:1 (ligand fusion protein: azide) for human NK cell. The delivery vehicle may encapsulate an active substance. The reactions can be carried out under a mild condition, such as for 24 hours at 25°C. The complex may be stored at -80°C.

When more than one ligand, or two or more different ligands are incorporated in the complex of the present invention, the two or more ligands are separately conjugated with the molecule comprised of a dipolarophile, and mixed with the delivery vehicle having the anchor molecule which is covalently bonded with azide group, at a desired mixing ratio of 1:1 to 1:10.

A physicochemical characteristic of the complex can be determined from any aspects that person skills in the art. Physicochemical characteristic item includes particle size, zeta-potential, polydispersity index (PDI). PDI is defined as the standard deviation (σ) of the particle diameter distribution divided by the mean particle diameter. PDI is used to estimate the average uniformity of a particle solution, and larger PDI values correspond to a larger size distribution in the particle sample. PDI of the complex may be less than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, preferably, less than 0.5, 0.4, 0.3, 0.2, 0.1, more preferably, less than 0.2 ,0.1.

2. Medicament of the present invention

The present invention provides a medicament comprising the complex of the present invention. The medicament comprising the complex of the present invention can be used for the prophylaxis or treatment of various diseases expressing the active substance delivered by the complex of the present invention, and can be administered, for example, to mammals (e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey, human), preferably human. Therefore, in one embodiment of the present invention, the medicament of the present invention for use for the prophylaxis or treatment of tumors expressing an antigen recognized by the complex, or the ligand to be precise, of the present invention is provided. In addition, the medicament comprising the complex of the present invention can be used for the prophylaxis or treatment of genetic or epigenetic disorder caused by the lack or decrease of expression of a gene by promoting the expression of the gene especially in blood, blood vessel, internal organs including but not limiting, digestive and/or respiratory tract and organs, peripheral and/or central nervous system, sensory organs, muscle, bone cartilage, skin and other body organs and tissues.

The dosage of the medicament of the present invention comprising the complex of the present invention is, for example, in the range of 0.001 mg to 10 mg as the amount of the active substance, per 1 kg body weight per dose. For example, when administered to a human patient, the dosage is in the range of 0.0001 to 50 mg for a patient weighing 60 kg. The above-mentioned dosage is an example, and the dosage can be appropriately selected according to the type of active substance to be used, administration route, age, weight, symptoms, etc. of the subject of administration or patient.

By administration to a mammal (e.g., human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey), preferably, human), the medicament of the present invention comprising the complex of the present invention can introduce the active substance, and when the active substance is a nucleic acid, can induce the expression of the protein encoded in the nucleic acid in the target cell in the body of the animal. When the active substance is CAR or exogenous TCR, and the target cell is the in vivo immunocyte or cytotoxic cell, the target cell specifically recognizes cancer cells and the like expressing surface antigen targeted by CAR or exogenous TCR and kills the diseased cells, thereby demonstrating a prophylactic or therapeutic effect against the disease.

The present invention provides a medicament comprising the target cell which is delivered by the complex of the present invention, and expressing the active substance. The medicament comprising the target cell of the present invention can be used for the prophylaxis or treatment of various diseases, and can be administered, for example, to mammals (e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey, human), preferably human by transplanting the medicament comprising the target cell of the present invention. Therefore, in one embodiment of the present invention, the medicament of the present invention for use for the prophylaxis or treatment of tumors expressing an antigen recognized by the complex, or the ligand to be precise, of the present invention is provided. In addition, the medicament comprising the target cell of the present invention can be used for the prophylaxis or treatment of genetic or epigenetic disorder caused by the lack or decrease of a particular cell by transplanting the medicament comprising the target cell of the present invention especially in blood, blood vessel, internal organs including but not limiting, digestive and/or respiratory tract and organs, peripheral and/or central nervous system, sensory organs, muscle, bone cartilage, skin and other body organs and tissues.

By administration to a mammal (e.g., human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkey), preferably, human), the medicament of the present invention comprising the target cell of the present invention can function in the body of the animal. When the active substance is CAR or exogenous TCR, and the target cell is the in vivo immunocyte or cytotoxic cell, the target cell specifically recognizes cancer cells and the like expressing surface antigen targeted by CAR or exogenous TCR and kills the diseased cells, thereby demonstrating a prophylactic or therapeutic effect against the disease.

A medicament containing the target cell of the present invention as an active ingredient is preferably administered parenterally to the subject. Parenteral methods of administration include intravenous, arterial, intramuscular, intraperitoneal, and subcutaneous administrations. While the dosage is selected according to the condition, body weight, age, etc. of the subject, administration is performed to a subject of 60 kg body weight to generally achieve 1x10⁶-1x10¹⁰ cells, preferably 1x10⁷-1x10⁹ cells, more preferably 5x10⁷-5x10⁸ cells, per dose. The medicament may be administered as a single dose or in multiple doses. The inventive medicament containing the target cell of the present invention as an active ingredient may be in a known form suitable for parenteral administration such as injection or infusion agent. The medicament may contain a pharmaceutically acceptable excipient as appropriate. The pharmaceutically acceptable excipient includes those described above. The medicament may contain saline, phosphate-buffered saline (PBS), medium, etc. to stably maintain the cells. The medium is not particularly limited, and examples thereof include, but are not limited to, RPMI, AIM-V, X-VIVO10, and the like. In addition, a pharmaceutically acceptable carrier (e.g., human serum albumin), preservative, and the like may be added to the medicament for the purpose of stabilization.

3. Method for producing the complex of the present invention

The present invention provides a method for producing the complex of the present invention, comprising: (1) a step of covalently bonding the first binding partner to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand; (2) a step of covalently bonding the second binding partner to the anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner by a chemical reaction at a ratio of one molecule of the second binding partner per one molecule of the first binding partner. Alternatively, the present invention provides a method for producing the complex of the present invention, comprising: (1) a step of covalently bonding the first binding partner to the ligand by sortase recognition motif; (2) a step of covalently bonding the second binding partner to the anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner by a chemical reaction at a ratio of one molecule of the second binding partner per one molecule of the first binding partner. Alternatively, the present invention provides a method for producing the complex of the present invention, comprising: (1) a step of covalently bonding the first binding partner to a C-terminal of the ligand; (2) a step of covalently bonding the second binding partner to the anchor molecule; and (3) a step of bonding the first binding partner to the second binding partner by a chemical reaction at a ratio of one molecule of the second binding partner per one molecule of the first binding partner.
The step (1) of covalently bonding the first binding partner to the ligand can be conducted by producing and purifying a fusion protein of the ligand with sortase recognition motif peptide ("ligand fusion protein") using the conventional recombinant DNA technology, followed by conjugation of the ligand fusion protein with a molecule comprised of dipolarophile using a sortase, a transpeptidase, followed by purification using conventional protein purification technology such as gel filtration or affinity chromatography.
The step (2) of covalently bonding the second binding partner to the anchor molecule is conducted by a known method. In one embodiment, azide as the second binding partner and the anchor molecule may be covalently bonded by nucleophilic addition reaction of azide reagent to the activated anchor molecule. In one embodiment, the anchor molecule and molecule conjugated with azide may be covalently bonded through linker structures such as amide, ester, carbamate, carbonate, but not limited to. The anchor molecule bonded to the second binding partner may be used to make a delivery vehicle by aggregating the molecules with other lipid molecules.
The step (3) of bonding the first binding partner with the second binding partner is conducted by reacting the target fusion protein conjugate of the molecule comprised of dipolarophile with the azidated lipid nanoparticle, or the delivery vehicle for 24 hours, at 25°C, for example.

4. Composition of the present invention

The present invention provides a composition comprising the complex of the present invention. The composition comprising the complex of the present invention can be used for delivery of the active substance inside the target cell, not only for medical use such as for the prophylaxis or treatment of various diseases, but also any non-medical use, such as experimental tool for labeling the target cell, or tool for veterinary medicine or food production. The composition comprising the complex of the present invention may also comprise a culture medium or saline.

5. Method for activating and/or proliferating the target cell of the present invention

In one aspect, the present invention provides a method for activating and/or proliferating the target cell, comprising: a step of contacting the complex of the present invention and a cell population comprising the target cell, where the complex is comprised of at least one ligand specific to the target cell.

In another aspect, the target cell may be an immune cell. As used herein, the "immune cell" is not particularly limited, as long as it is a cell capable of damaging the target cell (pathogenic cell) such as cancer cell and the like by some action mechanism (i.e., immune effector cell). Examples thereof include T cells that are responsible for cellular immunity among acquired immunities, NK cell, monocyte, macrophage, dendritic cell, etc. that are responsible for innate immunity, and NKT cells that are T cells with properties of NK cells. In one preferred embodiment, the immune cell may be a T cell. T cell collected from a living organism is also referred to as "ex vivo T cell" in the present specification.

In another preferred embodiment, the immune cell may be responsible for innate immunity such as NK cell, macrophage, dendritic cell, and the like. T cells are considered to be at considerable risk of causing GVHD by allogeneic (allo) transplantation even if HLA type matches, whereas allo-NK cells, etc. are considered not to cause GVHD. Therefore, the preparation of various HLA-type allo ex vivo immune cells permits use off-the-shelf. CAR-NK cell is described in, for example, US2016/0096892, Mol Ther. 25(8): 1769-1781 (2017) and the like, and CAR-dendritic cell, CAR-macrophage and the like are described in, for example, WO 2017/019848, eLIFE. 2018 e36688 and the like.

In another aspect, the present invention provides a composition for inducing the expression of a CAR or exogenous TCR containing the lipid nanoparticle of the present invention.

6. Method for delivering the active substance inside the target cell of the present invention

The present invention provides a method for delivering the active substance inside the target cell, comprising a step of contacting the complex of the present invention and a cell population comprising the target cell, wherein the active substance does neither include any nucleic acid encoding a CAR nor a TCR. The present invention may comprise a nucleic acid which inhibits the expression of a cytotoxic cell activation inhibitory factor, and/or a nucleic acid which encodes a cytotoxic cell activation promoting factor. The present invention may be conducted ex vivo or in vivo.

Unless otherwise indicated, wordings "about” or " approximately," when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value or within ±10 percent of the indicated value, whichever is greater.

1. Analytical Conditions
1.1 Evaluation of LNP Physical Properties

Physical properties of LNP were determined by DLS, RiboGreen assay and HPLC.

1.1.1 Size Measurements

Particle size and PDI were determined with Zetasizer Nano ZS (Malvern Panalytical).

1.1.2 Measurements of Nucleic Acid Concentration.

LNP was dissolved in 0.5% Triton X-100 and mRNA concentration was quantitatively determined with Quant-it^TM RiboGreen RNA Assay Kit (Thermo Fisher Scientific). mRNA concentration without Triton X-100 is measured as the concentration of mRNA which was not encapsulated in LNP. From these measurements, percentage of encapsulated mRNA was calculated.

1.1.3 Measurements of Antibody Concentration

HPLC analysis was carried out as below, and the concentration of the antibody bound to LNP was quantitatively determined. Concentration of the antibody not bound to LNP (residual antibody) was also determined. From these measurements, percentage of antibody remaining relative to the concentration of antibody added was calculated.
Measuring Instrument: Thermo Scientific Dionex UltiMate 3000
Moving Phase A: 0.2% Trifluoroacetic acid (TFA) in water
Moving Phase B: 0.2%TFA in acetonitrile
Detector: UV280 nm
Column: Agilent PLRP-S, 5um, 1000A, 2.1 mm x 50 mm

2. Preparation of LNP
2.1 Preparation of Fab-DBCO

Fab-DBCO was prepared by the following two steps. Molecular weight and DBCO/Antibody ratio (DAR) were determined by LC/MS and protein concentration was determined by BCA method. The results of physical property evaluation were shown in Table 1.
(1) Preparation of Fab having LPETGG-His6 at C-terminal
Plasmid pMG2.2 vectors encoding various genes for Fab were introduced into CHOZN cells using an electroporation instrument (Maxcyte), and the cells were cultured for six to eight days using EX-CELL Advanced CHO Feed 1 (with glucose). Then, LPETGG-His-tagged Fab was prepared by purification with cOmplete Ni column and Superdex 200.
(2) Preparation of Fab-DBCO
LPETGG-His-tagged Fab was mixed with CaCl₂ and 5-(Glycylglycyl-beta-alanyl)-11,12-dihydro-5,6-dihydrodibenzo[b,f]azocin, and then Sortase A treatment (Protein Science, 89, 15.3.1-15.3.19) was conducted. Reaction at room temperature for four hours followed by purification using Ni column and dialysis was conducted to obtain Fab-DBCO.

List of Fab-DBCO

*DAR: DBCO/Antibody ratio

2.2 Preparation of Azide-LNP

A mixture of lipids (Cationic lipid: DPPC: Cholesterol: SUNBRIGHT GS-020: DSPE-PEG (2000): Azide = 60: 10.6: 27: 1.4: 1, mol%) was dissolved in 90% EtOH/10% Acetate buffer (25 mM, pH4.0) to obtain a 14 mg/mL of lipid solution. As cationic lipid, the following compounds were used: 3-((4-(dimethylamino)butanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate (hereinafter to be also referred to as "Compound A"; used in LNP targeting T cells) described in WO2016/021683; or 2-(((4,5-dibutylnonanoyl)oxy)methyl)-2-(((5-(dimethylamino)pentanoyl)oxy)methyl)propane-1,3-diyldidecanoate (hereinafter to be also referred to as "Compound B"; used in LNP targeting NK cells) described in WO2020/032184. mRNA encoding CD19-targeting CAR having CD28 and CD3ε as intracellular signal transduction domain was dissolved in 10 mM 2-Morpholinoethanesulfonic acid (MES) buffer pH 5.5 to obtain 0.3 mg/mL nucleic acid solution. The above lipid solution and nucleic acid solution were mixed at a flow rate ratio of 3 mL/min: 6 mL/min (lipid solution: nucleic acid solution) using Nanoassemblr ^TM (Precision Nanosystems) to obtain a dispersion comprising the compositions. The above dispersed liquid was dialyzed against water for one hour at room temperature and against PBS for 24 hours at 4°C using Slide-A-Lyzer Dialysis (MWCO: 20 k, Thermo Fisher Scientific). Then, the dispersion was concentrated by ultrafiltration using Amicon Ultra (MWCO: 30K, Merck) and filtrated using a 0.2 µm syringe filter. The mRNA concentration was adjusted at the final concentration of 350 µg/mL with 20 % sucrose in PBS, and stored at 4°C.
2.3 Binding Reaction of Fab-DBCO and Azide-LNP

Each of various Fab-DBCO solutions was mixed with the LNP dispersion so that molar concentration of Fab-DBCO to azide was 1/20 or 1/10 for LNP targeting mouse T cell; 1/100 or 1/20 for LNP targeting human T cell; and 1/20 for LNP targeting human NK cell. The reactions were carried out for 24 hours at 25°C and stored at -80°C. Results of physical property evaluation are shown in TABLEs 2, 3 and 4.
2.4 Preparation of MAL-LNP
2.4.1 Preparation of Maleimide-LNP-CD19CAR

A mixture of lipids (Cationic lipid: DPPC: Cholesterol: SUNBRIGHT GS-020: DSPE-PEG-Maleimide (MW 2,000) = 60 :10.6 :27 :1.4 :1, molar ratio) was dissolved in 90% EtOH/10% acetate buffer (25 mM, pH 4.0) to obtain 14 mg/mL lipid solution. Compound A was used as cationic lipid. mRNA encoding CD19-targeting CAR having CD28 and CD3ε as intracellular signal transduction domain was dissolved in 10 mM MES buffer pH 5.5 to obtain 0.3 mg/mL nucleic acid solution. The above lipid solution and nucleic acid solution were mixed using Nanoassemblr^TM (Precision Nanosystems) at a flow rate of 3 mL/min: 6 mL/min to obtain a dispersion comprising the compositions. The above dispersed liquid was dialyzed against water for one hour at room temperature and against PBS for 24 hours at 4°C using Slide-A-Lyzer Dialysis (MWCO: 20 k, Thermo Fisher Scientific). Then, the dispersion was concentrated by ultrafiltration using Amicon Ultra (MWCO: 30K, Merck) and filtered using a 0.2 μm syringe filter. The mRNA concentration was adjusted at the final concentration of 350 μg/mL with 20 % sucrose in PBS, and stored at 4°C.
2.4.2 Reduction of F(ab') ₂

Anti mouse CD3 F(ab’)₂ solution (Bio X Cell) was mixed with PBS and 2-mercaptoethylamine hydrochloride (2-MEA) to adjust concentrations of F(ab’)₂ and 2-MEA at 1.5 mg/mL and 50 mM, respectively. After mixing, reaction was carried out for 90 minutes at 40°C under light shielding condition. 2-MEA was removed from the reaction mix by purification repeated three times using Zeba spin desalting column (MWCO 7K, Thermo Fisher Scientific) to obtain Fab’ solution. Concentrations of protein and thiol group were determined by absorbance at 280 nm and fluorescent colorimetric reaction with N-(7-Dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM), respectively. Protein and thiol group concentrations of obtained anti-mouse CD3 Fab’ were determined as 1.1 mg/mL and 34.3 μM, respectively.
2.4.3 Binding Reaction of Fab’ and Maleimide-LNP

Anti mouse CD3 Fab’ solution was mixed with Maleimide-LNP dispersion such that the molar concentration of reduced antibody was 1/20 of that of maleimide, and allowed to stand at room temperature for one hour. Thereafter, the mixture was stand still at 4°C for 18 hours and stored at -80°C. Results of physical property evaluation are shown in Table 2.

Properties of LNP targeting mouse T cell

Properties of LNP targeting human T cell

Properties of LNP targeting human NK cell

3. In vitro Expression of CAR in mouse T cell using LNP targeting mouse T cell.
3.1 Experimental Design

Spleens were harvested from C57BL/6NJcl mice and red blood cells were lysed using ACK lysing buffer (Lonza) to obtain mouse splenocytes. Mouse splenocytes were used for testing CD3-MAL-LNPs and T cells separated from the mouse splenocytes were used for testing CD3-DBCO-LNPs. Obtained cells were dispersed in RPMI 1640 medium supplemented with 10 % FBS, 1 % Penicillin-Streptomycin, 50 μM 2-Mercaptoethanol, 25 mM HEPES and 100 U/mL rhIL-2. The cells were inoculated to 24 well plates at 3 x 10⁶ cells/well. LNP were added to the wells so that the final concentration of mRNA is 3 μg/mL. The cells were cultured for 48 hours at 37°C. The cells were treated with labeled antibody reagents for markers (CD45, Live/Dead, CD90.2, B220, CD4, CD8, CD19-PE) and analyzed the ratio of CD19CAR positive cells in CD90.2 positive T cells using LSRFortessa flow cytometer (BD Biosciences). Table 5 shows the ratio of CAR positive cells.

3.2 Results

As shown in Table 5, CD3-DBCO-LNP showed higher CAR expression in vitro compared with CD3-MAL-LNP.

Ratio of CD19CAR positive cells in vitro in mouse spleen T cells

4. Animal Experiments (1) In vivo CAR expression in mouse T cell using LNP targeting mouse T cell
4.1 Experimental Design

10 mL/kg of LNP containing RNA at 0.375 mg/kg or 1 mg/kg was administered to C57BL/6NJcl mice into the tail veins twice with a 24 hr interval. Spleens were harvested after the administration. Following isolating immune cells from the spleen, the cells were treated with labeled antibody reagents for markers (CD45, CD90.2, CD4, CD8, Live/Dead, CD19-PE) and analyzed the ratio of CD19CAR positive cells in CD90.2 positive T cells using LSRFortessa flow cytometer (BD Biosciences). Table 6 shows the ratio of CAR positive cells.
4.2 Results

CD3-DBCO-LNP demonstrated higher CAR expression compared with CD3-MAL-LNP, even though the dose of the former is lower than the latter.

Ratio of CD19CAR positive cells in vivo in mouse spleen T cells

5. Experiments with Cultured Cells (1) In vitro CAR Expression in human T Cells using LNP targeting human T cell
5.1 Experimental Design

T cells were isolated from PBMC (Hemacare) using EasySep^TM Human T Cell Isolation Kit (STEMCELL Technologies). T cells were dispersed in X-VIVO15 (Lonza) medium supplemented with IL-2 (10 ng/mL, Miltenyi). Following inoculation of the T cells to 24 well plates at 1 x 10⁶ cells/well, the medium comprising LNP was added to the final mRNA concentration of 2 μg/mL. After culturing for 72 hours at 37°C, cells were treated with labeled antibody reagents for markers (CD45, Live/Dead, CD3, CD8, CD4, CD19-PE) and analyzed the ratio of CAR positive cells in CD3 positive T cells, CD4 positive T cells and CD8 positive T cells using LSRFortessa flow cytometer (BD Biosciences). Table 7 shows the ratio of CAR positive cells.

5.2 Results
CD3-DBCO-LNP induced high CAR expression in all of CD3 positive T cells, CD4 positive T cells and CD8 positive T cells. CD8-DBCO-LNP induced CAR expression selectively in CD8 positive T cells, demonstrating CAR expressing specific to target T cells.

Ratio of CD19 CAR positive cells in vitro in human T cells

5.2 In vitro CAR Expression in human NK cells using LNP targeting human NK cells
5.2.1 Experimental Design

NK cells were isolated from PBMC (Hemacare) using EasySep^TM Human NK Cell Isolation Kit (STEMCELL Technologies). NK cells were cultured in NK MACS (Trade Mark) medium (Miltenyi) supplemented with 5% AB serum (Sigma-Aldrich), 167 ng/mL Human IL-2 IS (Miltenyi) and 28 ng/mL Human IL-15 (Miltenyi). Following inoculation of the T cells to 24 well plates at 1 x 10⁶ cells/well, the medium comprising LNP was added to the final mRNA concentration of 2 μg/mL. 3 days after adding the LNP, cells were treated with labeled antibody reagents for markers and analyzed the ratio of CAR positive cells in CD56 positive NK cells, using LSRFortessa flow cytometer (BD Biosciences). Table 8 shows the ratio of CAR positive cells.
5.2.2 Results

All LNP targeting human NK cells induced CAR expression in NK cells.

Ratio of CD19CAR positive cells in vitro in human NK cells

6. Animal experiments (2)
6.1 In vivo CAR Expression in human T cells using LNP targeting human T cells
6.1.1 Experimental Design

T cells were isolated from human PBMC (Hemacare) using EasySep^TM Human T Cell Isolation Kit (STEMCELL Technologies). T cells were cultured for four days in 37°C in 5% CO₂ atomosphere in TheraPEAK^TM X-VIVO^TM 15 Serum-free Hematopoietic Cell Medium (Lonza) medium supplemented with Human IL-2 IS (Miltenyi) and T Cell TransAct (Miltenyi). Then, the T cells were suspended in PBS and NSG mice (The Jackson Laboratory) were implanted with the cells at 1 x 10⁷ cells/mouse. LNP was administered into tail veins repeatedly at the dose of 0.375 mg/kg. 48 hours after the administration of LNP, lungs were harvested. Cells were isolated using Dri Tumor & Tissue Dissociation Reagent (BD), the cells were treated with labeled antibody reagents for markers (CD45, Live/Dead, CD3, CD4, CD8) and analyzed the ratio of CD19CAR positive cells in T cells, CD4 positive T cells and CD8 positive T cells using LSRFortessa flow cytometer (BD Biosciences). Table 9 shows the ratio of CAR positive cells.
6.1.2 Results

CD3-DBCO-LNP induced high CAR expression in all of CD3 positive T cells, CD4 positive T cells and CD8 positive T cells. CD8-DBCO-LNP induced CAR expression selectively in CD8 positive T cells, demonstrating CAR expression specific to target T cells.

Ratio of in vivo CD19CAR positive cells in human T cells isolated from the lung

6.2 In vivo CAR Expression in human NK cells using LNP targeting human NK cells
6.2.1 Experimental Design

NK cells were isolated from human PBMC (Hemacare) using EasySep^TM Human NK Cell Isolation Kit (STEMCELL Technologies). Cells were cultured in NK MACS (Trade Mark) medium (Miltenyi) supplemented with 5% AB serum (Sigma-Aldrich), 167 ng/mL Human IL-2 IS (Miltenyi) and 28 ng/mL Human IL-15 (Miltenyi) to obtain cells for implantation. Then, the cells were suspended in PBS and NOG-hIL15-Tg mice (In-Vivo Science Inc.) were implanted with the cells at 1 x 10⁷ cells/mouse. LNP was administered into tail veins repeatedly at the dose of 0.8 mg/kg. 48 hours after the administration of LNP, lungs were harvested. Cells were isolated using Dri Tumor & Tissue Dissociation Reagent (BD), and the cells were treated with labeled antibody reagents for markers (CD45, Live/Dead, CD56, CD19CAR) and analyzed the ratio of CD19CAR positive cells in CD56 positive NK cells using LSRFortessa flow cytometer (BD Biosciences). Table 9 shows the ratio of CAR positive cells. Table 10 shows the ratio of CAR positive cells.

6.2.2 Results

All LNPs targeting human NK cells induced CAR expression in NK cells.

Ratio of in vivo CD19CAR positive cells in human NK cells isolated from the lungs

7. Animal experiments (3)
7.1 Efficacy of LNP targeting human T cells in tumor-bearing mice
7.1.1 Experimental Design

NALM6-Luc tumor cells were suspended in PBS and implanted from the tail veins of NSG mice (The Jackson Laboratory) at 0.5 x 10⁶ cells/mouse. Two days after tumor implantation, volume of tumor was determined by luminescent imaging using in vivo imaging instrument (IVIS LUMINA II) after abdominal administration of luciferin. The tumor-bearing mice were sorted into groups of five animals using EXSUS according to the tumor volume, and T cells were implanted to the animals. From Day 3 of T cell implantation, LNP was thawed at room temperature, prepared by diluting to the predetermined concentration with PBS, water, or PBS-20% sucrose and repeatedly administered into tail veins at the dose of 0.375 mg/kg. Nine days after the first administration, tumor volumes were determined using IVIS. Table 11 shows the results of tumor volume determination using luminescence intensity as an indicator.
7.1.2 Results

CD3-DBCO-LNP and CD8-DBCO-LNP were demonstrated to have anti-tumor effects.

Determination of tumor volume in NALM6-Luc mice using luminescence intensity as an indicator (average ± standard error)

7.2 Efficacy of LNP targeting human NK cells in tumor-bearing mice
7.2.1 Experimental Design

NALM6-Luc tumor cells, which express luciferase, were suspended in PBS and implanted from the tail veins of NOG-hIL-15 Tg mice (InVivo science) at 0.5 x 10⁶ cells/mouse. After the tumor was engrafted, luciferin was abdominally administrated and then volume of tumor was determined by luminescent imaging using in vivo imaging instrument (IVIS LUMINA II). The tumor-bearing mice were sorted into groups of five animals using an allocation software according to the tumor volume, and NK cells were implanted to the animals. On the day of LNP administration, LNP was thawed at room temperature, prepared by diluting to the predetermined concentration with PBS, water, or PBS-20% sucrose and repeatedly administered into tail veins at the dose of 0.375 mg/kg or 0.8 mg/kg. Tables 12 and 13 show the results of tumor volume determination using luminescence intensity as an indicator.
7.2.2 Results

CD16-DBCO-LNP and CD7-DBCO-LNP were demonstrated to have anti-tumor effects.

Determination of tumor volume in animals administered with CD16-DBCO-LNP using luminescence intensity as an indicator (average ± standard error)

Determination of tumor volume in animals administered with CD7-DBCO-LNP using luminescence intensity as an indicator (average ± standard error)

As used in this specification and the appended claims, singular articles such as “a,” “an,” and “the,” may refer to a single object or to a plurality of objects unless the context clearly indicates otherwise. Thus, for example, reference to a composition containing “a compound” may include a single compound or two or more compounds. The above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. Therefore, the scope of the invention should be determined with reference to the appended claims and includes the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references cited in the disclosure, including patents, patent applications and publications, are herein incorporated by reference in their entirety and for all purposes.

Claims

A complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell,
the ligand being added to the outer surface of the delivery vehicle,
wherein the delivery vehicle is comprised of an anchor molecule,
a first binding partner is covalently bonded to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the ligand;
a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and
the first binding partner is covalently bonded to the second binding partner.
A complex for delivering an active substance, comprising a delivery vehicle for the active substance and a ligand specific to a target cell,
the ligand being added to the outer surface of the delivery vehicle,
wherein the delivery vehicle is comprised of an anchor molecule,
a first binding partner is covalently bonded to a C-terminal of the ligand;
a second binding partner is covalently bonded to the anchor molecule whereby the second binding partner is immobilized to the delivery vehicle; and
the first binding partner is covalently bonded to the second binding partner.
The complex according to claim 1 or 2, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.
The complex according to claim3, wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.
The complex according to claim 4, wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.
The complex according to claim 4, wherein the second binding partner is a molecule comprised of an azide group.
The complex according to claim 1, wherein the target cell to which the active substance is delivered is an immune cell.
The complex according to claim 7, wherein the immune cell is a cytotoxic cell.
The complex according to claim8, wherein the cytotoxic cell is a NK cell or a T cell.
The complex according to claim 8, wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD7, CD16, CD8 and CD56.
The complex according to claim 1, wherein the active substance is a nucleic acid.
The complex according to claim 11, wherein the nucleic acid is comprised of a nucleic acid encoding a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR).
A medicament comprising the complex according to claim 12.
A method for producing the complex according to claim 1, comprising:
(1) a step of covalently bonding the first binding partner to the ligand at a ratio of 1-4 molecules of the first binding partner per one molecule of the first binding partner;
(2) a step of covalently bonding the second binding partner to the anchor molecule; and
(3) a step of bonding the first binding partner to the second binding partner by a chemical reaction at a ratio of one molecule of the second binding partner per one molecule of the first binding partner.
The method according to claim 14, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.
The method according to claim 15, wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.
The method according to claim 16, wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.
The method according to claim 16, wherein the second binding partner is a molecule comprised of an azide group.
The method according to claim 14, wherein the target cell to which the active substance is delivered is an immune cell.
The method according to claim19, wherein the immune cell is a cytotoxic cell.
The method according to claim 20, wherein the cytotoxic cell is a NK cell or a T cell.
The method according to claim 20, wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD7, CD16, CD8 and CD56.
The method according to claim 14, wherein the active substance is a nucleic acid.
A composition for delivering the active substance to the target cell comprising the complex according to claim 1.
The composition according to claim 24, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.
The composition according to claim 25, wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.
The composition according to claim 26, wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.
The composition according to claim 26, wherein the second binding partner is a molecule comprised of an azide group.
The composition according to claim 24, wherein the target cell to which the active substance is delivered is an immune cell.
The composition according to claim 29, wherein the immune cell is a cytotoxic cell.
The composition according to claim 30, wherein the cytotoxic cell is a NK cell or a T cell.
The composition according to claim 30, wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD7, CD16, CD8 and CD56.
The composition according to claim 24, wherein the active substance is a nucleic acid.
The composition according to claim 33, further comprising a culture medium saline, or buffer.
A method for activating and/or proliferating the target cell, comprising:
a step of contacting the complex according to claim 1 and a cell population comprising the target cell, where the complex is comprised of at least one ligand specific to the target cell.
The method according to claim 35, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.
The method according to claim 36, wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.
The method according to claim 37, wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.
The method according to claim 37, wherein the second binding partner is a molecule comprised of an azide group.
The method according to claim 35, wherein the target cell to which the active substance is delivered is an immune cell.
The method according to claim 40, wherein the immune cell is a cytotoxic cell.
The method according to claim 41, wherein the cytotoxic cell is a NK cell or a T cell.
The method according to claim 41, wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD7, CD16, CD8 and CD56.
The method according to claim 41, wherein more than one ligand is added to the outer surface of the delivery vehicle.
The method according to claim35, wherein the active substance is a nucleic acid.
The method according to claim 45, wherein the nucleic acid is comprised of a nucleic acid encoding a CAR and/or a TCR.
The method according to claim 35, wherein the step of contacting the complex and the cell population comprising the target cell is conducted ex vivo.
A method for delivering the active substance inside the target cell, comprising:
a step of contacting the complex according to claim 1 and a cell population comprising the target cell, wherein the active substance does neither include any nucleic acid encoding a CAR nor a TCR.
The method according to claim 48, wherein the delivery vehicle for the active substance is a lipid nanoparticle or a liposome.
The method according to claim 49, wherein the first binding partner is a molecule comprised of a dipolarophile, and wherein the second binding partner is a molecule comprised of a 1,3-dipole.
The method according to claim 50, wherein the first binding partner is a molecule comprised of a dibenzocyclooctyne (DBCO) group.
The method according to claim 50, wherein the second binding partner is a molecule comprised of an azide group.
The method according to claim 48, wherein the target cell to which the active substance is delivered is an immune cell.
The method according to claim 53, wherein the immune cell is a cytotoxic cell.
The method according to claim 54, wherein the cytotoxic cell is a NK cell or a T cell.
The method according to claim 55, wherein the ligand specific to the cytotoxic cell is comprised of at least one antibody, or antigen-binding fragment thereof, selected from the group consisting of CD3, CD7, CD16, CD8 and CD56.
The method according to claim 54, wherein more than one ligand is added to the outer surface of the delivery vehicle.
The method according to claim 48, wherein the active substance is a nucleic acid.
The method according to claim 58, wherein the nucleic acid is comprised of a nucleic acid which inhibits the expression of a cytotoxic cell activation inhibitory factor, and/or a nucleic acid which encodes a cytotoxic cell activation promoting factor.
The method according to claim 48, wherein the step is conducted ex vivo.
The target cell to which the active substance is delivered by the method according to claim 48.
A medicament comprising the target cell according to claim 61.
The medicament according to claim 62, further comprising a culture medium or saline.