CD117 antibody and use thereof

Abstract

The present invention relates to a CD117 antibody and a delivery vector using same, such as lipid nanoparticles (LNPs). Further provided in the present invention are a method for delivering a bioactive substance to CD117+ cells by using the delivery vector and a method for preparing such a lipid nanoparticle conjugate.

Description

CD117 antibodies and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to CD117 antibodies, and nanoparticles conjugated to the antibodies, wherein the nanoparticles comprise a therapeutic or prophylactic agent (including, for example, nucleic acids, such as mRNA). BACKGROUND

[0002] Improvement of symptoms of related diseases (e.g., sickle cell disease (SCD), thalassemia disease) is enabled by editing (e.g., targeted cleavage or targeted base modification) of, for example, hematopoietic stem cells (HSCs).

[0003] There is also a need for targeted specific delivery systems for, for example, HSCs. CD117, also known as tyrosine-protein kinase (c-Kit), is a cytokine receptor expressed on hematopoietic stem cells and some other types of cells. Upon binding to stem cell factor (SCF), the tyrosine kinase activity of CD117 is activated, leading to phosphorylation and activation of downstream molecules, which can serve as a target for treatment, diagnosis, and targeting of HSCs. SUMMARY

[0004] The present disclosure relates to monoclonal antibodies having high binding activity to human or non-human primate CD117 and uses thereof.

[0005] Certain anti-CD117 antibodies disclosed herein are capable of exhibiting high binding activity to CD117+ cells without inhibiting ligand-induced c-Kit phosphorylation. These characteristics indicate that the anti-CD117 antibodies disclosed herein are suitable for use in directing delivery of cargo (e.g., gene editing systems) to CD117+ cells with less impact on cell activity.

[0006] Accordingly, provided herein are monoclonal antibodies capable of binding to CD117 (e.g., human or non-human primate CD117) and carriers (e.g., lipid nanoparticles) conjugated with the above antibodies for delivering biologically active substances to CD117+ cells (i.e., cells expressing CD117).

[0007] Also provided herein are nucleic acids comprising nucleotide sequences encoding anti-CD117 antibodies, vectors and host cells comprising these nucleic acid sequences, and methods of making lipid nanoparticle (LNP) conjugates comprising anti-CD117 antibodies.

[0008] In one aspect, the present disclosure provides an anti-CD117 antibody or antigen binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein:

[0009] H-CDR1 comprises GYTX1X2SX3X4MH (SEQ ID NO. 89), wherein X1is selected from L, F; X2is selected from T, S; X3is selected from Y, H; X4is selected from Q, W, N;

[0010] H-CDR2 comprises VIYX5X6X7GX8TSYX9X 10 KX 11 K (SEQ ID NO. 90), wherein X5is selected from S, N; X6is selected from G, A, D, Y; X7is selected from N, E, R; X8is selected from D, E; X9is selected from N, Q; X 10 is selected from Q, N; X 11 is selected from F, Y;

[0011] H-CDR3 comprises ERDX 12 RFGX 13 (SEQ ID NO. 91), wherein X 12 is selected from T, E; X 13 is selected from N, F;

[0012] The light chain variable region comprises light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3; wherein,

[0013] L-CDR1 comprises RX a X b EX c X d DX e YX f X g X h FX i H (SEQ ID NO. 92), wherein X a is selected from A, V; X b is selected from S, T; X c is selected from S, N; X d is selected from V, A; X e is selected from I, L; X f is selected from G, Y; X g is selected from N, E; X h is selected from S, K; X i is selected from M, I;

[0014] L-CDR2 comprises LX j SX k LEX l (SEQ ID NO. 93), wherein X j is selected from A, V; X k is selected from N, R; X l is selected from S, T;

[0015] L-CDR3 comprises QQNX m X n X o PX p X q (SEQ ID NO. 94), wherein X m is selected from N, E; X n is selected from E, F; X o is selected from D, Y; X p is selected from Y, R; X q is selected from T, S.

[0016] In yet another aspect, the present disclosure provides a lipid nanoparticle conjugate comprising

[0017] a lipid nanoparticle (LNP), and

[0018] an antibody or antigen-binding fragment thereof that binds CD117, wherein the antibody or antigen-binding fragment thereof is conjugated on the surface of the LNP, the antibody or antigen-binding fragment thereof that binds CD117 being as described in the present disclosure.

[0019] In yet another aspect, the present disclosure provides a method for delivering a biologically active substance to a cell, the method comprising:

[0020] contacting a lipid nanoparticle conjugate of the present disclosure with a CD117 expressing cell to deliver a biologically active substance contained in the lipid nanoparticle conjugate to the CD117+ cell.

[0021] In yet another aspect, the present disclosure provides a method of editing a gene in a CD117 expressing cell, comprising: contacting a lipid nanoparticle conjugate of the present disclosure with a CD117+ cell to allow delivery of a biologically active agent contained in the lipid nanoparticle conjugate to the CD117+ cell, the biologically active agent being a gene editing composition / system that edits a target gene in the CD117+ cell.

[0022] In yet another aspect, the present disclosure provides a method of making a lipid nanoparticle conjugate, the method comprising: contacting an anti-CD117 antibody or antigen-binding fragment thereof of the present disclosure with an LNP to allow conjugation of the anti-CD117 antibody or antigen-binding fragment thereof to the surface of the LNP, thereby producing the lipid nanoparticle conjugate.

[0023] In yet another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding an anti-CD117 antibody or antigen-binding fragment thereof of the present disclosure.

[0024] In yet another aspect, the present disclosure provides a vector comprising a nucleic acid as in the present disclosure.

[0025] In yet another aspect, the present disclosure provides a host cell comprising the vector of the present disclosure.

[0026] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject the conjugate of the present disclosure.

[0027] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the conjugate of the present disclosure.

[0028] In yet another aspect, the present disclosure provides a method of treating or ameliorating a disease or disorder, comprising administering to a subject an effective amount of the conjugate or the pharmaceutical composition of the present disclosure or administering the conjugate or the pharmaceutical composition to a cell of the subject.

[0029] It should be understood that, in the scope of the present disclosure, all combinations between the above-mentioned technical features of the present disclosure and the technical features specifically described hereinafter (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 shows conjugate Pro2, Pro3, Pro4, Pro5, Pro6, Pro7, Pro8, Pro9, Pro10, Pro11 antibody-LNP / unconjugated antibody-LNP in vitro cell transfection luciferase assay statistics.

[0031] Figure 2 shows conjugate Pro12, Pro14, Pro15, Pro16, Pro17, Pro19, Pro20, Pro21, Pro22, Pro23 antibody-LNP / unconjugated antibody-LNP in vitro cell transfection luciferase assay statistics. DETAILED DESCRIPTION

[0032] Provided herein is a new anti-CD117 antibody, which is suitable for therapeutic use, and nanoparticles conjugated to the antibody, as well as methods of using the antibody-conjugated nanoparticles and preferred therapeutic uses of the antibody-conjugated nanoparticles.

[0033] TERMS

[0034] Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following descriptions of terms will apply and, where appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of a term in this section conflicts with the description of that term in any document incorporated herein by reference, the description of the term as set forth below shall prevail.

[0035] Unless otherwise defined in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0036] As used herein, including the appended claims, the singular forms "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0037] The term "or" is used in its inclusive sense (i.e., "and / or") unless the context clearly indicates otherwise.

[0038] The term "lipid" refers to a group of organic compounds that includes, but is not limited to, fatty acid esters, and is generally characterized by poor solubility in water but solubility in many nonpolar organic solvents. Although lipids generally have weak water solubility, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and are soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be classified into at least three categories: (1) "simple lipids" including fats and oils, and waxes; (2) "compound lipids" including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derived lipids" such as sterol-like compounds. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound" or simply "lipid-like" refers to a lipid-like compound (e.g., an amphiphilic compound having lipid-like physical properties).

[0039] The term "ionizable lipid" has its ordinary meaning in the art and can refer to a lipid that comprises one or more charged moieties. In some embodiments, an ionizable lipid can carry a positive or negative charge. For example, an ionizable lipid can carry a positive charge at lower pH, in which case it can be referred to as a "cationic lipid." In certain embodiments, the positive charge in a cationic lipid arises from the presence of a quaternary nitrogen atom. In certain embodiments, a cationic lipid comprises a zwitterionic lipid that carries a positive charge in the environment of its intended use (e.g., at physiological pH).

[0040] The term "polyethylene glycol (PEG) lipid molecule" refers to a molecule that comprises both a lipid moiety and a polymeric moiety (PEG).

[0041] The term "neutral lipid" encompasses any lipid molecule that exists in uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH value range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment for which the predetermined lipid use is intended, such as physiological pH values.

[0042] The term "genome editing vector" refers to a nucleic acid molecule encoding a component of a genome editing system, such as but not limited to a CRISPR / Cas protein (e.g. Cas9, Cas12, Cas13, IscB, etc.), a base editor or a prime editor, and any associated necessary components, such as appropriate guide RNA (gRNA).

[0043] The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts optionally specified, e.g., a biologically active molecule as provided herein, such as mRNA.

[0044] The term "nucleic acid" refers to a polymer of nucleotides of any length, and includes, for example, DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, or modified nucleotides or bases, and / or their analogs, or any substrate which can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. A nucleic acid can be in single- or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimetics, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, which generally, but not necessarily, is less than about 200 nucleotides in length. The term "oligonucleotide" is not mutually exclusive with "polynucleotide." The above description regarding polynucleotides applies equally and fully to oligonucleotides. Unless otherwise indicated, the left-hand end of any single-stranded polynucleotide sequence is the 5' terminus; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nucleotides to an nascent RNA transcript is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript and which are located 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA transcript and which are located 3' to the 3' end of the RNA transcript are referred to as "downstream sequences."

[0045] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues linked by peptide bonds. That is, a description of a polypeptide applies equally to a description of a protein, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more of the amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). As used herein, the term encompasses amino acid chains of any length, including full-length proteins (e.g., antigens).

[0046] The term "antigen" refers to a substance capable of being recognized by a subject's immune system, including the adaptive immune system, and capable of triggering an immune response, including an antigen-specific immune response, after the subject is exposed to the antigen. In certain embodiments, an antigen is a protein associated with a diseased cell, such as a cell infected with a pathogenic agent or a neoplastic cell (e.g., a tumor-associated antigen (TAA)).

[0047] An "epitope" is a site on the surface of an antigen molecule to which a single antibody molecule binds, such as a local region of the surface of an antigen that is capable of binding to one or more antigen binding regions of an antibody, and which has antigenic or immunogenic activity in an animal, such as in a mammal (e.g., in a human), capable of eliciting an immune response. An epitope that has immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope that has antigenic activity is a portion of a polypeptide that is bound by an antibody as determined by any method well known in the art, including, for example, by immunoassay. An antigenic epitope does not necessarily have immunogenicity. Epitopes are usually composed of chemical groups on the surface of a molecule, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody epitope can be a linear epitope or a conformational epitope. A linear epitope is formed by a contiguous amino acid sequence in a protein. A conformational epitope is formed by amino acids that are not contiguous in the protein sequence but come together when the protein folds into its three-dimensional structure. Induced epitopes are formed when the three-dimensional structure of a protein is changed, such as after activation or binding by another protein or ligand. In certain embodiments, an epitope is a three-dimensional surface characteristic of a polypeptide. In other embodiments, an epitope is a linear characteristic of a polypeptide. Generally, an antigen has several or many different epitopes, and can react with many different antibodies.

[0048] The term "antibody" is intended to include the polypeptide product of a B cell that is within the range of immunoglobulin polypeptides, which is capable of binding to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, where each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region containing about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd ed., 1997). In particular embodiments, the specific molecular antigen can be bound by an antibody provided herein, including a polypeptide, a fragment thereof, or an epitope. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above, by which is meant a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments include single chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing an antigen binding site (e.g., one or more CDRs of an antibody). Such antibody fragments can be found in, e.g., Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd ed., 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) of immunoglobulin molecule.

[0049] Generally, a basic antibody structural unit comprises a tetramer, each tetramer including two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain can define a constant region primarily responsible for effector functions. Generally, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as alpha, delta, epsilon, gamma, or mu, and define the antibody's isotype as IgA, IgD, IgE, IgG, and IgM, respectively. Within light chains and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain further including a "D" region of about 10 or more amino acids.

[0050] The variable regions of each light / heavy (VL / VH) pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are generally identical. Generally, the variable domains of both the heavy and light chains contain three hypervariable regions, also called "complementarity determining regions" (CDRs), which are located within relatively conserved framework regions (FRs). The CDRs are generally aligned with the framework regions such that specific epitopes can be bound. Generally, both light and heavy chain variable domains comprise FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4), from N-terminus to C-terminus. The location of CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, and AbM (see, e.g., Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997)). Definitions of antigen binding sites are also described in Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., in Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996). In the combined Kabat and Chothia numbering scheme, in some embodiments, CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.The hypervariable region comprises amino acid residues from "CDRs" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (defining the CDR regions of antibodies according to sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (defining the CDR regions of antibodies according to structure).

[0051] The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0052] The term "genetic vaccine" refers to a therapeutic or prophylactic composition comprising at least one nucleic acid molecule encoding an antigen associated with a disease of interest (e.g., an infectious disease or a neoplastic disease). Administration of the vaccine to a subject ("vaccination") allows for production of the encoded peptide or protein, thereby eliciting an immune response in the subject against the disease of interest. In certain embodiments, the immune response includes an adaptive immune response, such as the production of antibodies against the encoded antigen, and / or the activation and proliferation of immune cells capable of specifically eliminating diseased cells expressing the antigen. In certain embodiments, the immune response further includes an innate immune response. According to the present disclosure, the vaccine can be administered to the subject prior to or after the onset of clinical symptoms of the disease of interest. In some embodiments, vaccination of a healthy or asymptomatic subject renders the vaccinated subject immune or less susceptible to the development of the disease of interest. In some embodiments, vaccination of a subject displaying symptoms of the disease ameliorates the disease condition or treats the disease in the vaccinated subject.

[0053] The term "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues of an antibody of the disclosure that binds CD117, or an antigen-binding portion thereof, can be replaced with other amino acid residues from the same side chain family.

[0054] The term "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a lipid nanoparticle composition described herein) that is present outside of the body into the body of a patient, such as transmucosally, intradermally, intravenously, intramuscularly, and / or any other physical delivery method described herein or known in the art. When treating a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom thereof. When preventing a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom thereof.

[0055] The term "targeted delivery" or the verb form "target" refers to a process that facilitates the arrival of a delivered agent (e.g., a therapeutically effective payload molecule in a lipid nanoparticle composition described herein) to a particular organ, tissue, cell, and / or intracellular compartment (referred to as a target location) compared to delivery to any other organ, tissue, cell, or intracellular compartment (referred to as a non-target location). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of a delivered agent in a target cell population to the concentration of the delivered agent at a non-target cell population following systemic administration.

[0056] The term“biologically active substance” refers to a prophylactic or therapeutic agent, the“prophylactic” referring to any agent that can entirely or partially inhibit the development, recurrence, onset, or spread of a disease and / or its associated symptoms in a subject, and the“therapeutic” referring to any agent that can be used to treat, prevent, or alleviate a disease, disorder, or condition, including any agent that is used to treat, prevent, or alleviate one or more symptoms of a disease, disorder, or condition and / or its associated symptoms.

[0057] The term“therapies / therapy” refers to any regimen, method, and / or agent that can be used to prevent, manage, treat, and / or ameliorate a disease, disorder, or condition. In certain embodiments, the term“therapies / therapy” refers to a biological therapy, a supportive therapy, and / or other therapy known to one of skill in the art, such as a medical practitioner, to be useful in preventing, managing, treating, and / or ameliorating a disease, disorder, or condition.

[0058] The term“encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of the sequences of nucleotides in biologically- or otherwise- synthesized molecules, such as rRNA, tRNA, and mRNA, or of specific sequences of amino acids and the biological properties resulting therefrom. Thus, if a mRNA corresponding to a gene is transcribed and translated, it produces a protein, the gene encodes the protein. The code chain, whose sequence of nucleotides is identical to the sequence of mRNA and which is usually provided in sequence listings, and the non-coding chain, which serves as a template for transcription of the gene or cDNA, can both be referred to as encoding the protein or other products of the gene or cDNA.

[0059] The term“expression vector” refers to a vector that comprises a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. Expression vectors comprise sufficient cis-acting elements for gene expression; additional elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained within liposomes), RNA, and viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus) into which a recombinant polynucleotide can be

[0060] The term“operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0061] The terms“patient,”“subject,”“individual,” and the like are used interchangeably herein and refer to any animal or cell thereof, whether in vitro or in situ, suitable for use in the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0062] As used herein, endonuclease refers to a protein or polypeptide that is capable of catalyzing (e.g., cleaving) an internal region of a nucleic acid (e.g., DNA or RNA), where the DNA binding activity is sequence specific and depends on the sequence of a guide RNA. Exemplary endonucleases include Cas cleavases / nickases and their inactive forms (“dCas DNA binders”). As used herein,“Cas nuclease” encompasses Cas cleavases, Cas nickases, and dCas enzymes. Cas cleavases / nickases and dCas enzymes.

[0063] In some embodiments, the endonuclease comprises one or more mutations that ablate nuclease activity. In various embodiments, the endonuclease is a Cas9 protein or a variant thereof. In some embodiments, the endonuclease is a Cas9 nickase, a Cas12a nickase, or a Cas12i nickase. In some embodiments, the endonuclease is selected from Cas9, Cas12e, Cas12d, Cas12a, Cas12b1, Cas12b2, Cas13a, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas12f (Cas14), Cas12f1, Cas12j (Cas ), Cas13a, Cas13b, Cas13d, and Argonaute, and optionally has nickase activity such that only one strand is cleaved.

[0064] As used herein, the term "guide RNA," "guide RNA," or "gRNA" generally refers to a short ribonucleic acid capable of interacting with (e.g., binding to) an endonuclease and binding or hybridizing to a target genomic site or region. In some embodiments, the gRNA is a single-molecule guide RNA (sgRNA). In some embodiments, the gRNA can comprise a spacer extension region. In some embodiments, the gRNA can comprise a tracrRNA extension region. In some embodiments, the gRNA is single-stranded. In some embodiments, the gRNA comprises naturally occurring nucleotides. In some embodiments, the gRNA is a chemically modified gRNA. In some embodiments, the chemically modified gRNA is a gRNA comprising at least one nucleotide with a chemical modification (e.g., a 2'-0-methyl sugar modification). In some embodiments, the chemically modified gRNA comprises a modified nucleic acid backbone. In some embodiments, the chemically modified gRNA comprises 2'-0-methyl-thiophosphate residues. In some embodiments, the gRNA can be pre-complexed with an endonuclease.

[0065] As used herein, the term "exogenous" as used herein refers to a polynucleotide sequence that originates from outside the recipient cell or organism, is assembled from polynucleotide sequences outside the recipient cell or organism, or is integrated into the recipient genome at a location different from where it is found in nature. An exogenous polynucleotide sequence can comprise a gene sequence, can comprise a coding sequence (CDS) of a gene, can comprise coding sequences from more than one gene, can comprise a promoter sequence, an enhancer sequence, and / or other regulatory elements, can comprise a ribosomal skip sequence, and / or can comprise an artificial sequence. An exogenous polynucleotide can be codon-optimized to ensure efficient translation in the recipient cell or organism.

[0066] As used herein, a functionalized PEG lipid (polyethylene glycol lipid) refers to a PEG lipid in which the polyethylene glycol moiety has been derivatized with a chemically reactive group that can be used to conjugate a targeting moiety (e.g., an antibody) to the PEG lipid. The functionalized PEG lipid can be reacted with a targeting moiety (e.g., an antibody) after LNP formation, thereby conjugating the targeting moiety to the PEG moiety of the lipid, and the conjugated targeting moiety can serve as a targeting moiety for the LNP.

[0067] Anti-CD117 antibodies (antibodies that bind CD117)

[0068] According to one aspect of the present disclosure, there is provided an antibody or an antigen binding portion thereof that binds to CD117, which binds to cell surface receptor CD117 (also known as c-kit).

[0069] In some embodiments, the anti-CD117 antibody comprises a heavy chain variable region and a light chain variable region, the CDRs sequences of which are obtained according to numbering methods well known in the art, including but not limited to Kabat, Chothia, IMGT, AbM and Contact.

[0070] In some embodiments, the CDRs are defined according to the IMGT scheme.

[0071] In some embodiments, the anti-CD117 antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region,

[0072] The heavy chain variable region comprises heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein:

[0073] H-CDR1 comprises GYTX1X2SX3X4MH (SEQ ID NO. 89), wherein X1 is selected from L, F; X2 is selected from T, S; X3 is selected from Y, H; X4 is selected from Q, W, N;

[0074] H-CDR2 comprises VIYX5X6X7GX8TSYX9X 10 KX 11 K (SEQ ID NO. 90), wherein X5 is selected from S, N; X6 is selected from G, A, D, Y; X7 is selected from N, E, R; X8 is selected from D, E; X9 is selected from N, Q; X 10 is selected from Q, N; X 11 is selected from F, Y;

[0075] H-CDR3 comprises ERDX 12 RFGX 13 (SEQ ID NO. 91), wherein X 12 is selected from T, E; X 13 is selected from N, F;

[0076] The light chain variable region comprises light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3; wherein,

[0077] L-CDR1 comprises RX a X b EX c X d DX e YX f X g Xh FX i H (SEQ ID NO. 92), wherein X a is selected from A, V; X b is selected from S, T; X c is selected from S, N; X d is selected from V, A; X e is selected from I, L; X f is selected from G, Y; X g is selected from N, E; X h is selected from S, K; X i is selected from M, I;

[0078] L-CDR2 comprises LX j SX k LEX l (SEQ ID NO. 93), wherein X j is selected from A, V; X k is selected from N, R; X l is selected from S, T;

[0079] L-CDR3 comprises QQNX m X n X o PX p X q (SEQ ID NO. 94), wherein X m is selected from N, E; X n is selected from E, F; X o is selected from D, Y; X p is selected from Y, R; X q is selected from T, S.

[0080] Any one of the antibody amino acid sequences of the present disclosure also includes a derivative sequence that optionally has at least one amino acid added, deleted, modified and / or substituted, and is capable of retaining CD117 binding affinity.

[0081] In a preferred embodiment, the number of amino acids added, deleted, modified and / or substituted is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0082] In a preferred embodiment, H-CDR1 is GYTX1X2SX3X4MH, H-CDR2 is VIYX5X6X7GX8TSYX9X 10 KX 11 K, H-CDR3 is ERDX 12 RFGX 13 and L-CDR1 is RX a X b EX c Xd DX e YX f X g X h FX i H, L-CDR2 is LX j SX k LEX l , and L-CDR3 is QQNX m X n X o PX p X q wherein X1-X 13 , X a -X q is selected from the group consisting of:

[0083] X1is L or F, X2is T or S, X3is Y or H, X4is Q, W or N, X5is S or N, X6is G, A, D or Y, X7is N, E or R, X8is D or E, X9is N or Q, X 10 is Q or N, X 11 is F or Y, X 12 is T or E, X 13 is N or F, X a is A or V, X b is S or T, X c is S or N, X d is V or A, X e is I or L, X f is G or Y, X g is N or E, X h is S or K, X i is M or I, X j is A or V, X k is N or R, X l is S or T, X m is N or E, X n is E or F, X o is D or Y, X p is Y or R, X q is T or S.

[0084] In a preferred embodiment, X1-X 13 , X a -X q is selected from the group consisting of:

[0085] X1is F, X2is T, X3is, X4is Y, X5is N, X6is G, X7is N, X8is D, X9is Q, X 10 is Q, X 11 is Y, X 12 is T, X 13 is N, Xa is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is A, X k is N, X l is S, X m is N, X n is E, X o is D, X p is Y, X q is T, or

[0086] X1is F, X2is T, X3is Y, X4is N, X5is N, X6is D, X7is E, X8is D, X9is Q, X 10 is Q, X 11 is Y, X 12 is T, X 13 is N, X a is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is A, X k is N, X l is S, X m is N, X n is E, X o is D, X p is Y, X q is T, or

[0087] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 is Q, X 11 is F, X 12 is T, X 13 is N, X a is V, X b is T, X c is N, X d is A, X e is L, X f is G, X g is E, X h is S, Xi is I, X j is A, X k is N, X l is S, X m is N, X n is E, X o is D, X p is Y, X q is T, or

[0088] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 is Q, X 11 is F, X 12 is T, X 13 is N, X a is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is V, X k is N, X l is T, X m is N, X n is E, X o is D, X p is Y, X q is T, or

[0089] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 is Q, X 11 is F, X 12 is T, X 13 is N, X a is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is A, X k is N, X l is S, X m is E, X n is E, X o is D, X p is Y, Xq S.

[0090] In another preferred embodiment, X1-X 13 , X a , X q is selected from the group consisting of:

[0091] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is A, X7is N, X8is E, X9is N, X 10 is N, X 11 is F, X 12 is T, X 13 is N, X a is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is A, X k is N, X l is S, X m is N, X n is E, X o is D, X p is Y, X q is T, or

[0092] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is A, X7is N, X8is E, X9is N, X 10 is N, X 11 is F, X 12 is T, X 13 is N, X a is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is A, X k is N, X l is S, X m is N, X n is E, X o is D, X p is Y, X q is T, or

[0093] X1 is F, X2 is T, X3 is Y, X4 is N, X5 is N, X6 is Y, X7 is R, X8 is D, X9 is Q, X 10 For Q, X 11 Let Y and X be the numbers. 12 Let T, X 13 Let N, X a Let A and X be the two numbers. b Let S and X be the values ​​of S and X. c Let S and X be the values ​​of S and X. d Let V, X e For I, X f For G, X g Let N, X h Let S and X be the values ​​of S and X. i Let M and X be the numbers. j Let A and X be the two numbers. k Let N, X l Let S and X be the values ​​of S and X. m Let N, X n For E, X o For D, X p Let Y and X be the numbers. q For T, or

[0094] X1 is F, X2 is T, X3 is Y, X4 is N, X5 is S, X6 is G, X7 is N, X8 is D, X9 is N, X 10 For Q, X 11 Let F, X 12 Let T, X 13 Let N, X a Let V, X b Let T, X c Let N, X d Let A and X be the two numbers. e Let L and X be the numbers. f Let Y and X be the numbers. g For E, X h Let K, X i For I, X j Let A and X be the two numbers. k Let N, X l Let S and X be the values ​​of S and X. m Let N, X n For E, X o For D, X p Let Y and X be the numbers. q For T, or

[0095] X1 is F, X2 is T, X3 is Y, X4 is N, X5 is S, X6 is G, X7 is N, X8 is D, X9 is N, X 10 For Q, X 11 Let F, X 12 Let T, X 13 Let N, X a Let A and X be the two numbers. bS, X c S, X d V, X e I, X f G, X g N, X h S, X i M, X j V, X k R, X l T, X m N, X n E, X o D, X p Y, X q T, or

[0096] In another preferred embodiment, X1-X 13 , X a -X q is selected from the group consisting of:

[0097] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 Q, X 11 F, X 12 T, X 13 N, X a A, X b S, X c S, X d V, X e I, X f G, X g N, X h S, X i M, X j A, X k N, X l S, X m N, X n E, X o D, X p Y, X q T, or

[0098] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 Q, X 11 F, X 12 T, X 13 N, X a A, X b S, X c N, X d V, X eL, X f G, X g N, X h S, X i I, X j A, X k N, X l S, X m N, X n E, X o D, X p Y, X q T, or

[0099] X1is F, X2is T, X3is Y, X4is N, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 Q, X 11 F, X 12 T, X 13 N, X a A, X b S, X c S, X d V, X e I, X f G, X g N, X h S, X i M, X j A, X k N, X l S, X m N, X n F, X o Y, X p R, X q T, or

[0100] X1is L, X2is T, X3is Y, X4is Q, X5is S, X6is A, X7is N, X8is E, X9is N, X 10 N, X 11 F, X 12 T, X 13 N, X a A, X b S, X c N, X d V, X e L, X f G, X g N, X h S, X i I, X j A, X k N, X l S, X m N, Xn is F, X o is Y, X p is R, X q is T, or

[0101] In another preferred embodiment, X1-X 13 , X a -X q is selected from the group consisting of:

[0102] X1is L, X2is T, X3is Y, X4is Q, X5is S, X6is G, X7is N, X8is D, X9is N, X 10 is Q, X 11 is F, X 12 is T, X 13 is N, X a is A, X b is S, X c is S, X d is V, X e is I, X f is G, X g is N, X h is S, X i is M, X j is A, X k is N, X l is S, X m is N, X n is E, X o is D, X p is Y, X q is T.

[0103] In a preferred embodiment, the heavy chain variable region comprises heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein,

[0104] H-CDR1 is selected from:

[0105] (a) GYTLTSYQMH (SEQ ID NO. 63);

[0106] (b) GYTLSSHWMH (SEQ ID NO. 64);

[0107] (c) GYTFTSYNMH (SEQ ID NO. 65);

[0108] H-CDR2 is selected from:

[0109] (d) VIYSGNGDTSYNQKFK (SEQ ID NO. 66);

[0110] (e) VIYSANGETSYNNKFK (SEQ ID NO. 68);

[0111] (f) VIYNGNGDTSYQQKYK (SEQ ID NO. 69);

[0112] (g) VIYNDEGDTSYQQKYK (SEQ ID NO. 70);

[0113] (h) VIYNYRGDTSYQQKYK (SEQ ID NO. 71);

[0114] H-CDR3 is selected from:

[0115] (i) ERDTRFGN (SEQ ID NO. 72);

[0116] (j) ERDERFGF (SEQ ID NO. 76);

[0117] and

[0118] the light chain variable region comprises light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3; wherein,

[0119] L-CDR1 is selected from:

[0120] (k) RASESVDIYGNSFMH (SEQ ID NO. 77);

[0121] (l) RASENVDLYGNSFIH (SEQ ID NO. 78); (m) RVTENADLYGESFIH (SEQ ID NO. 80);

[0122] (n) RVTENADLYYEKFIH (SEQ ID NO. 81);

[0123] L-CDR2 is selected from:

[0124] (o) LASNLES (SEQ ID NO. 82);

[0125] (p) LVSNLET (SEQ ID NO. 83);

[0126] (q) LVSRLET (SEQ ID NO. 84);

[0127] L-CDR3 is selected from:

[0128] (r) QQNNEDPYT (SEQ ID NO. 85);

[0129] (s)QQNNFYPRT(SEQ ID NO.87);

[0130] (t)QQNEEDPYS(SEQ ID NO.88).

[0131] In some embodiments, the heavy chain variable region of the anti-CD117 antibody provided in this disclosure includes a region corresponding to SEQ ID NO. 1, 3, 7, 13, 19, 41, 43, 45, 47, 51, 53, 55, 57, 59 or 61.

[0132] The amino acid sequence from position 1 to position 117 in any amino acid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, but the anti-CD117 antibody containing this sequence retains the ability to bind CD117.

[0133] In some embodiments, the heavy chain variable region of the anti-CD117 antibody provided in this disclosure includes substitutions (e.g., conserved modifications), insertions, or deletions relative to amino acid positions 1-117 of any of the amino acid sequences in SEQ ID NO. 1, 3, 7, 13, 19, 41, 43, 45, 47, 51, 53, 55, 57, 59, or 61), but the anti-CD117 antibody containing this sequence retains its ability to bind CD117. In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR).

[0134] In some embodiments, the light chain variable region of the anti-CD117 antibody provided in this disclosure comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence at positions 1-111 of any one of the amino acid sequences of SEQ ID NO. 2, 4, 8, 14, 20, 42, 44, 46, 48, 52, 54, 56, 58, 60, or 62, but the anti-CD117 antibody containing this sequence retains the ability to bind CD117.

[0135] In certain embodiments, the light chain variable region of an anti-CD117 antibody provided by the present disclosure comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% substitution (e.g., conservative modifications), insertions or deletions relative to the reference sequence in the amino acid sequence of any one of the reference sequences (SEQ ID NO. 2, 4, 8, 14, 20, 42, 44, 46, 48, 52, 54, 56, 58, 60, or 62) from amino acid positions 1-111, but an anti-CD117 antibody comprising that sequence retains the ability to bind to CD117. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0136] In some embodiments, the CDRs of the CD117 antibody are as shown in Table 5.

[0137] In some embodiments, the heavy chain variable region of the antibody further comprises a framework region of human origin, and / or the light chain variable region of the antibody further comprises a framework region of human origin.

[0138] In some embodiments, the heavy chain variable region of the antibody further comprises a framework region of murine origin, and / or the light chain variable region of the antibody further comprises a framework region of murine origin.

[0139] In some embodiments, the heavy chain constant region of the antibody is of human origin, and / or the light chain constant region of the antibody is of human origin.

[0140] In some embodiments, the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

[0141] In some embodiments, the antibody is a diabody, or a single chain antibody.

[0142] In some embodiments, the antibody is an antibody full-length protein, or an antigen binding fragment.

[0143] In some embodiments, the antibody is a bispecific antibody, or a multispecific antibody.

[0144] In some embodiments, the antigen binding fragment comprises a Fab fragment, a F(ab’)2 fragment, a Fv fragment, a scFv.

[0145] Production of anti-CD117 antibodies

[0146] Antibodies of the present disclosure can be prepared by any antibody production technique known in the art. In certain embodiments, one or more individual components of the antibody (e.g., heavy and light chains) are derived from a chimeric, humanized, or fully human antibody. Methods of making such antibodies are well known in the art. For example, one or more heavy and / or light chains of an antibody of the present disclosure can be prepared using VELOCIMMUNE™ technology. Antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. Mouse constant regions are replaced with desired human constant regions to produce fully human-derived heavy and / or light chains that can be incorporated into the antibody. In some embodiments, antibodies can be generated by administering an immunogen to a mouse, optionally in combination with an adjuvant. Following immunization, spleen cells are harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines are screened and selected to identify cell lines that produce artemin-specific antibodies. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. 2007 / 0280945 or WO2016077666, both of which are incorporated by reference herein in their entireties.

[0147] Anti-CD117 antibody structure

[0148] In some embodiments, the antibodies provided by the present disclosure are full-length antibodies.

[0149] In some embodiments, the antibodies provided by the present disclosure are antibody fragments. For example, the antibody fragment can be a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. A "Fab" is a monovalent fragment consisting of a VL, VH, CL, and CH1 domain. A "Fab fragment" can be produced by papain cleavage of an antibody. A "Fab'" contains the VL, CL, and VH and CH1, and also contains the region between the CH1 and CH2 domains, so that a disulfide bond can be formed between the two heavy chains of two Fab' fragments, to form an F(ab')2 molecule. A "Fab'-SH" is a Fab' fragment in which the cysteine residue of the constant region has a free thiol group. An "F(ab')2" comprises a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region.

[0150] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody. Diabodies are antibody fragments with two antigen-binding sites, comprising the VHand VLjoined by a short linker that does not allow pairing between the two domains on the same chain. This forces the domains to pair with the complementary domains of another chain and creates two antigen- binding sites that can be identical or different

[0151] In another embodiment, the antibody fragment is a single chain Fab fragment, a "single chain Fab fragment" or "scFab" is a polypeptide consisting of a VH, a CH1, a VL, a CL and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In one embodiment, the linker is a polypeptide of at least 30 amino acids. In another embodiment, the linker is a polypeptide of between 32 and 50 amino acids. The single chain Fab fragments are stabilized via the native disulfide bond between CL and CH1. In addition, these single chain Fab molecules can be further stabilized by the creation of an interchain disulfide bond by the insertion of a cysteine residue.

[0152] In some embodiments, the antibody fragment is a single chain variable fragment (scFv), as used herein, an "scFv" is a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are linked in tandem by a short, flexible peptide linker, capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specifically indicated otherwise, scFv herein can have the VL and VH variable regions in either order, e.g., an scFv can comprise VL-linker-VH or can comprise VH-linker-VL, with respect to the N- and C-termini of the polypeptide.

[0153] In some embodiments, the antibody fragment is an Fd fragment consisting of a VH and a CH1 domain.

[0154] In some embodiments, the antibody fragment is an Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0155] In some embodiments, the antibody fragment is a dsFv, a dsFv is obtained by linking a polypeptide in which one amino acid residue in each of the VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on the three-dimensional structure prediction of an antibody according to a known method (Protein Engineering. 7: 697 (1994)).

[0156] In some embodiments, the antibody fragment is a single domain antibody, a single domain antibody is an antibody fragment comprising the entire or partial heavy chain variable domain or the entire or partial light chain variable domain of an antibody.

[0157] In some embodiments, the antibody fragment is a domain antibody (dAb); see, e.g., U.S. Patent No. 6,248,516. Domain antibodies (dAbs) are functional binding domains of antibodies, which correspond to the variable region of either the heavy (VH) or light (VL) chain of a human antibody. dAbs have a molecular weight of about 13 kDa, or are less than one-tenth the size of a whole antibody. dAbs are well expressed in a variety of hosts, including bacterial, yeast, and mammalian cell systems. Furthermore, dAbs are highly stable and retain activity even when subjected to harsh conditions, such as lyophilization or heat denaturation.

[0158] In some embodiments, the antibodies provided herein are chimeric antibodies. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.

[0159] In some embodiments, the antibodies are humanized antibodies. Typically, non- human antibodies are humanized by grafting to reduce immunogenicity in humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable regions in which CDRs or portions thereof are derived from a non-human antibody, while FRs or portions thereof are derived from a human antibody. Optionally, a humanized antibody will also comprise a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody can be replaced with corresponding residues from a non-human antibody (e.g., the antibody providing the CDR sequences).

[0160] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

[0161] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light chain or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions generated using a design strategy (see, e.g., U.S. Patent No. 6,187,287; and U.S. Patent No. 6,582,804).

[0162] In some embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, multispecific antibodies have three or more binding specificities. In certain embodiments, one of the binding specificities is for CD117, while the other specificity is for any other antigen. In certain embodiments, bispecific antibodies can bind to two (or more) different epitopes of CD117. Multispecific (e.g., bispecific) antibodies can also be used to localize cells of interest. Multispecific antibodies can be prepared in a full-length antibody or antibody fragment.

[0163] Nanolipid particles (LNPs)

[0164] As used herein, the term “lipid nanoparticle” or “LNP” refers to a particle having a size in the nanometer (nm) scale (e.g., 1 nm to 1000 nm) that comprises one or more types of lipid molecules. The LNPs provided herein can further comprise at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule that is partially or completely encapsulated inside a lipid shell. In particular, in some embodiments, where the payload is a negatively charged molecule (e.g., an mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. It is contemplated that the cationic lipid can interact with the negatively charged payload molecule and facilitate payload incorporation and / or encapsulation into the LNP during LNP formation. Other lipids that can form part of the LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as sterols, PEG lipids, and various zwitterionic lipids.

[0165] In some embodiments, the nanoparticle compositions provided herein can comprise one or more charged or ionizable lipids. It is contemplated that certain charged or zwitterionic lipid compositions of the nanoparticle compositions resemble lipid components in cell membranes, thereby improving cellular uptake of the nanoparticles. In some embodiments, the ionizable lipids can be positively charged or negatively charged. For example, the ionizable lipid can be positively charged at lower pH, in which case it can be referred to as a "cationic lipid." Exemplary charged or ionizable lipids that can form part of the nanoparticle compositions of the present disclosure include, but are not limited to, 3-(Dodecylamino)-N1,N1,4- Tridodecyl-1-piperazinethanamine (KL10), N1-[2-(Dodecylamino)ethyl]-N1,N4,N4- Tridodecyl-1,4-piperazinediamine (KL22), 14,25-Tricosyl-15,18,21,24- Tetraazaoctacosa- (KL25), 1,2-Dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), Heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-Diolinoxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3β)-Cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-Cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2R)), (2S)-2-({8-[(3β)-Cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA(2S)), (12Z,15Z)-N,N-dimethyl-2-nonyldocosan-1-amine, N,N-dimethyl-1-{((1S,2R)-2-octylcyclopropyl}heptadecan-8-amine, 11-({8-[(2-butyloctyl)oxy]-8-oxoctyl}amino)-10-(3-ethyl-6-oxo-3,7-diazahexadecan-7-yl)-11-oxo- undecanoic acid-2-butyloctyl ester.Additional exemplary charged or ionizable lipids that can form part of the nanoparticle compositions of the present application include those described in Sabnis et al. “A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates,” Molecular Therapy Vol. 26 No 6, 2018, the entire contents of which are incorporated herein by reference.

[0166] In some embodiments, suitable ionizable lipids include N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2- dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero- 3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristyl-sn-glycero-3-ethylphosphocholine (14:1); N1-[2- ((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylformamido)ethyl]- 3,4-bis[oxy-2-(oleoyloxy)]-benzamide (MVL5); dioctadecylamino-glycylcarbamide (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); SAINT-2, N-methyl-4-(dioleyl) methylpyridinium; 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); di-alkylated amino acids (DILA2) (e.g., C18:1-norArg-C16); dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3- ethylphosphocholine (POEPC); 1,2-dimyristyl-sn-glycero-3-ethylphosphocholine (MOEPC); (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1); (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G); and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1- ammonium chloride (DOTAPen). Cationic lipids with head groups that are charged at physiological pH, such as primary amines (e.g., DODAG N',N'-dioctadecyl-N-4,8-diaza-10-aminodecanoyl glycylamide) and guanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol (BGSC), bis-guanidinium-trisaminoethylamine-cholesterol (BGTC), PONA, and (R)-5- guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G)) are also suitable. Another suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1).In certain embodiments, the cationic lipid is a specific enantiomer or racemic form, and includes various salt forms (e.g., chloride or sulfate) of the cationic lipids as above. For example, in some embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-C1) or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate). In some embodiments, the cationic lipid is an ionizable cationic lipid such as, for example, dioctadecyldimethylammonium bromide (DDAB); 1,2-dilinoleyl- oxy-3-dimethylaminopropane (DLinDMA); 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]- dioxolane (DLin-KC2-DMA); heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA); 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP); 1,2- dioleyloxy-3-dimethylaminopropane (DODMA); and morpholinocholesterol (Mo-CHOL). In certain embodiments, the lipid nanoparticle includes a combination of two or more cationic lipids (e.g., two or more cationic lipids as above).

[0167] In some embodiments, the ionizable lipid is a compound according to any of the formulas of 01-I, 02-I, 02-I-1, 02-I-2, 02-I-3, 02-I-4, 02-I-5, 02-I-6, 02-I-7, 02-I-8, 02-I-9, 02-I-10, 02-I-11, 02-I-12, 02-I-13, 02-I-14, 03-I, 03-I-1, 03-I-2, 04-I, 04-I-1, 04-I-2, 04-I-1-3, 04-I-4, 04-II, 04-II-1, 04-II-2, 04-II-3, and subformulae thereof.

[0168] In one embodiment, the ionizable lipid contained in the lipid nanoparticle (LNP), pharmaceutical composition described herein is an ionizable lipid described in International Patent Publication No. WO2024152512, which is incorporated herein by reference in its entirety.

[0169] In one embodiment, the cationic lipid is a compound of formula (01-I):

[0170] wherein:

[0171] G1 C; 1-6 alkylene;

[0172] R 1 , R 2 each independently C 1-6 alkyl;

[0173] optionally, R 1 , R 2 and the N to which they are attached together form a 3-8 membered heterocyclyl, or, R 1 , R 2 any of R 1 and any of the carbon atoms in G

[0174] L 1 is selected from a heteroatom O, N, S or is absent;

[0175] R 3 , R 4 , R 5 , R 6 , R 7 each independently hydrogen, C 1-8 alkyl or -O-C 2-12 alkylene;

[0176] a, b are each independently selected from an integer from 1-10;

[0177] L 2 is selected from a heteroatom O, N, S or is absent;

[0178] R 8 , R 9 , R 10 , R 11 , R 12 each independently hydrogen or C 1-8 alkyl;

[0179] c, d are each independently selected from an integer from 1-10;

[0180] L 3 is selected from a heteroatom O, N, S or is absent;

[0181] R 13 , R 14 , R 15 , R 16 , R 17 each independently hydrogen, C 1-8 alkyl or -O-C 2-12 alkylene;

[0182] e, f are each independently selected from an integer from 1 to 10.

[0183] According to some embodiments of the present disclosure, in Formula 01-I, the G 1 is methylene, ethylene, propylene, butylene, or pentylene.

[0184] According to some embodiments of the present disclosure, in Formula 01-I, R 1 , R 2 are each independently methyl, ethyl, or propyl; or, R 1 , R 2 together with the N to which they are attached form a 5- or 6-membered heterocyclyl group. According to some other embodiments of the present disclosure, in Formula I, R 1 , R 2 one of R1, R2is directly connected to any carbon atom in G1to form a 5- or 6-membered heterocyclyl group, and the other of R1, R2is methyl, ethyl, or propyl.

[0185] According to some embodiments of the present disclosure, in Formula 01-I, G 1 , R 1 , R 2 correspond to the groups shown in any one of Compounds 1-35, respectively.

[0186] According to some embodiments, in Formula 01-I,

[0187] L 1 is or is absent;

[0188] R 3 , R 4 , R 5 at least two of R 6 , R 7 are not hydrogen;

[0189] a is selected from an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10);

[0190] b is 2, 3, 4, 5, 6, 7, 8, or 9.

[0191] According to some embodiments of the present disclosure, in Formula 01-I, L 1 , R 3 , R 4 , R 5 , R 6 , R 7 , a, b correspond to the groups or values shown in any one of Compounds 1-35, respectively.

[0192] ​According to some specific implementation schemes of this disclosure, in formula 01-I,

[0193] L 2 for It may not exist; preferably, it does not exist.

[0194] R 8 R 9 R 10 R 11 R 12 Each is independently hydrogen;

[0195] c and d are each independently selected from integers from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0196] According to some specific implementation schemes of this disclosure, the sum of c and d is 4, 5, 6, 7, 8, 9, 10.

[0197] According to some specific implementation schemes of this disclosure, L 2 It does not exist; the sum of c and d is 5, 6, 7, 8, 9, or 10.

[0198] According to some specific implementation schemes of this disclosure, in formula 01-I, L 2 R 8 R 9 R 10 R 11 R 12 c and d correspond to the groups or values ​​shown in any of compounds 1 to 35.

[0199] According to some specific implementation schemes of this disclosure, in formula 01-I,

[0200] L 3 for

[0201] R 13 R 14 Each is hydrogen; R 15 R 16 R 17 At least neither of them is hydrogen;

[0202] e is 2, 3, 4, 5, 6, 7, 8, or 9;

[0203] f is an integer selected from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

[0204] According to a specific embodiment of this disclosure, in formula 01-I, L 3 R 13 R 14 R 15R 16 R 17 e, f correspond to the groups or values shown in any one of compounds 1 - compound 35, respectively.

[0205] In the present disclosure, when the values of a, b, c, d, e, f are not 1, the corresponding plurality of R 3 R 4 R 6 R 7 R 8 R 9 R 10 R 11 R 13 R 14 R 15 R 17 may each independently be the same or different.

[0206] According to one specific embodiment of the present disclosure, the compound is selected from one or more of compounds 1 to 35 shown in Table 1.

[0207] Table 1

[0208] In some embodiments, the ionizable lipids contained in the lipid nanoparticles (LNPs), pharmaceutical compositions described herein are the ionizable lipids described in International Patent Publication No. PCT / CN2024 / 082316, which is incorporated herein by reference in its entirety.

[0209] In one embodiment, the cationic lipid is a compound of formula 02-I:

[0210] wherein:

[0211] G 1 is C 1-6 alkylene;

[0212] R 1 , R 2 are each independently C 1-6 alkyl or C 1-6 alkoxy; 0 to 3 H in the alkyl or alkoxy groups are optionally further substituted by halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl or hydroxyl-substituted C 1-6 alkyl;

[0213] Optionally, R 1 , R2 with the attached N together form a 3-8 membered heterocyclyl, or, R 1 , R 2 any of G 1 any of the carbon atoms is directly attached to form a 3-8 membered heterocyclyl; said heterocyclyl has 0 to 3 ring C optionally further substituted with N, O, or S; said heterocyclyl has 0 to 3 H optionally further substituted with halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkyl, or hydroxyl-substituted C 1-6 alkyl;

[0214] L 1 selected from alkynylene, a heteroatom O, N, S, or nothing; h, i, k are selected from an integer between 1-10; j is selected from 0 or 1;

[0215] R 3 , R 4 , R 5 , R 6 , R 7 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12 alkoxy; said alkyl, alkenyl, alkynyl, or alkoxy has 0 to 3 H optionally further substituted with halogen, hydroxyl, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, C 1-12 alkoxy, halogen-substituted C 1-12 alkyl, or hydroxyl-substituted C 1-12 alkyl;

[0216] a, b are each independently selected from an integer between 0-10;

[0217] L 2 selected from alkynylene, a heteroatom O, N, S, or nothing; h, i, k are selected from an integer between 1-10; j is selected from 0 or 1;

[0218] R 8 , R 9 , R 10 , R 11 , R 12 are each independently hydrogen, C 1-12 alkyl, C 1-12 alkenyl, C 1-12 alkynyl, or C 1-12alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C

[0219] each of c, d is independently selected from an integer from 0 to 10;

[0220] L 3 , L 4 each independently selected from alkynylene, a heteroatom O, N, S or nothing; l, n are selected from an integer from 1 to 10; m is selected from 0 or 1;

[0221] R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 each independently is hydrogen, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C 1-12 alkyl, C

[0222] each of e, f, g is independently selected from an integer from 0 to 10.

[0223] In some embodiments, in formula 02-I, the G 1 is methylene, ethylene, propylene, butylene, pentylene or hexylene. The propylene, butylene, pentylene or hexylene can be straight-chained or branched.

[0224] In some embodiments, in formula 02-I, the G 1 is methylene, ethylene, propylene, butylene or pentylene.

[0225] In some embodiments, the compounds are as shown in formula 02-I-1, formula 02-I-2, formula 02-I-3, formula 02-I-4, formula 02-I-5, respectively.

[0226] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-7 or the compound 02-14. (Herein, when a substituent is described as corresponding to a group represented by a certain compound, it means that the substituent corresponds to the group represented by the certain compound in the position of the substituent in a certain formula (e.g., Formula 02-I). For example, G 1 is a group represented by the compound 02-7, it means that G 1 is propylene between the leftmost N and S in the structure of the compound 02-7, i.e., a group represented by the middle line portion of the structure.

[0227] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-11.

[0228] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-13, the compound 02-12, or the compound 02-6.

[0229] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-9 or the compound 02-1.

[0230] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-5.

[0231] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-2.

[0232] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-8.

[0233] In some embodiments, in Formula 02-I, the G 1 is a group represented by the compound 02-3, the compound 02-4, or the compound 02-10.

[0234] In some embodiments, in Formula 02-I, the G 1 is a group represented by any one of the compounds 02-15 to 02-31.

[0235] In some embodiments, in Formula 02-I, R 1 , R 2Each alkyl or alkoxy group is independently methyl, ethyl, propyl, butyl, pentyl, hexyl, methoxy, ethoxy, propoxy, butoxy, pentoxy, or hexoxy; wherein the alkyl or alkoxy group has 0, 1, 2, or 3 H atoms, optionally further converted to halogen, hydroxyl, or C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 alkyl or hydroxy substituted C 1-6 Alkyl groups are substituted.

[0236] In some implementations, in formula 02-I, R 1 R 2 Each is independently methyl, ethyl, propyl, or butyl, wherein the methyl, ethyl, propyl, or butyl group has 0, 1, or 2 H atoms, optionally further converted by a halogen, hydroxyl group, or C. 1-3 Alkyl-substituted; or, R 1 R 2 Together with the attached N, it forms a 5- or 6-membered heterocyclic group, wherein 0, 1, or 2 ring Cs in the heterocyclic group are optionally further substituted with N, O, or S, and 0, 1, or 2 Hs in the heterocyclic group are optionally further substituted with halogens, hydroxyl groups, or C. 1-3 Alkyl groups are substituted.

[0237] In some implementations, in formula 02-I, optionally, R 1 R 2 Together with the connected N, they form 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic groups, or, R 1 R 2 Any of the two G 1 Any carbon atom in the group is directly bonded to form a 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, or 8-membered heterocyclic group; 0, 1, 2, or 3 ring Cs in the heterocyclic group may be further replaced by N, O, or S; 0, 1, 2, or 3 Hs in the heterocyclic group may be further replaced by halogens, hydroxyl groups, or C. 1-6 Alkyl, C 1-6 alkoxy- or halogen-substituted C 1-6 alkyl or hydroxy substituted C 1-6 Alkyl groups are substituted.

[0238] According to other specific embodiments of this disclosure, in formula 02-I, R 1 R 2 One of the middle and G 1 Any carbon atom in R can be directly bonded to form a 5- or 6-membered heterocyclic group. 1 R 2 The other is methyl, ethyl, propyl, or butyl, wherein the methyl, ethyl, propyl, or butyl group has 0, 1, or 2 H atoms, optionally further converted by a halogen, hydroxyl group, or C atom. 1-3substituted with 0, 1, or 2 alkyl groups. The heterocyclyl group has 0 or 1 or 2 rings optionally further substituted with N, O, or S, and the heterocyclyl group has 0 or 1 or 2 H optionally further substituted with halogen, hydroxyl, or C 1-3 substituted with 0, 1, or 2 alkyl groups. The heterocyclyl group has 0 or 1 or 2 rings optionally further substituted with N, O, or S, and the heterocyclyl group has 0 or 1 or 2 H optionally further substituted with halogen, hydroxyl, or C

[0239] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-7 or compound 02-14.

[0240] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-11.

[0241] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0242] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-9 or compound 02-1.

[0243] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-5.

[0244] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-2.

[0245] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-8.

[0246] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0247] In some embodiments, in Formula 02-I, the R 1 is a group represented by the corresponding compound 02-15 to compound 02-31.

[0248] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-7 or compound 02-14.

[0249] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-11.

[0250] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0251] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-9 or compound 02-1.

[0252] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-5.

[0253] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-2.

[0254] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-8.

[0255] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0256] In some embodiments, in Formula 02-I, the R 2 is a group represented by the corresponding any one of compounds 02-15 to 02-31.

[0257] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups represented by the corresponding compound 02-7 or compound 02-14, respectively. (Herein, when describing two or more substituents as corresponding to a group represented by a certain compound, it means that the two or more substituents correspond to the group represented by the certain compound in the position of the two or more substituents in a certain formula (e.g., Formula 02-I). For example, R 1 , R 2 are groups represented by the corresponding compound 02-7, respectively, means that R 1 , R 2 each independently is ethyl on the leftmost N of the structural formula of compound 02-7, i.e., a group represented by the following structural formula.

[0258] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups represented by the corresponding compound 02-11, respectively.

[0259] ​In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in Compound 02-13, Compound 02-12, or Compound 6, respectively.

[0260] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in Compound 02-9 or Compound 02-1, respectively.

[0261] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in Compound 02-5.

[0262] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in Compound 02-2.

[0263] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in Compound 02-8.

[0264] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in Compound 02-3, Compound 02-4, or Compound 02-10, respectively.

[0265] In some embodiments, in Formula 02-I, the R 1 , R 2 are groups corresponding to those shown in any one of Compounds 02-15 to 02-31.

[0266] In some embodiments, in Formula 02-I, the above-mentioned G 1 , R 1 , R 2 may be freely combined to form corresponding compounds.

[0267] In some embodiments, in Formula 02-I, G 1 , R 1 , R 2 are groups corresponding to those shown in any one of Compounds 02-1 to 02-31, respectively.

[0268] In some embodiments, the compounds are shown in Formula 02-I-6, Formula 02-I-7, Formula 02-I-8, Formula 02-I-9, respectively.

[0269] In formulae 02-I-6, 02-I-7, 02-I-8, 02-I-9, each substituent is described according to any of the schemes herein.

[0270] In some embodiments, in formula 02-I,

[0271] L 1 is alkynylene, O or nothing; h, i, k are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; j is selected from 0 or 1.

[0272] Herein, unless specifically stated, in formula 02-I, when a certain group is a specific structure, the remaining groups can be defined according to the definition of each group in formula 02-I. For example, when L 1 is in formula 02-I, the remaining groups can be defined according to any of the schemes herein.

[0273] In some embodiments, in formula 02-I, R 3 , R 4 , R 5 are not hydrogen, and R 6 , R 7 are hydrogen.

[0274] In some embodiments, in formula 02-I, a is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0275] In some embodiments, in formula 02-I, b is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0276] In some embodiments, in formula 02-I, preferably at least one or both of a, b is not 0.

[0277] In some embodiments, in formula 02-I, the L 1 is a group as shown in corresponding compound 02-7 or compound 02-14.

[0278] In some embodiments, in formula 02-I, the L 1 is a group as shown in corresponding compound 02-11.

[0279] In some embodiments, in formula 02-I, the L 1 is a group as shown in corresponding compound 02-13, compound 02-12 or compound 02-6.

[0280] In some embodiments, in formula 02-I, the L 1a group represented by the corresponding compound 02-9 or compound 02-1.

[0281] In some embodiments, in Formula 02-I, the L 1 a group represented by the corresponding compound 02-5.

[0282] In some embodiments, in Formula 02-I, the L 1 a group represented by the corresponding compound 02-2.

[0283] In some embodiments, in Formula 02-I, the L 1 a group represented by the corresponding compound 02-8.

[0284] In some embodiments, in Formula 02-I, the L 1 a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0285] In some embodiments, in Formula 02-I, the L 1 a group represented by the corresponding compound 02-15 to compound 02-31.

[0286] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-7 or compound 02-14.

[0287] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-11.

[0288] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0289] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-9 or compound 02-1.

[0290] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-5.

[0291] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-2.

[0292] In some embodiments, in Formula 02-I, the R 3 a group represented by the corresponding compound 02-8.

[0293] In some embodiments, in Formula 02-I, the R 3 is a group represented by any one of Compound 02-3, Compound 02-4, or Compound 02-10.

[0294] In some embodiments, in Formula 02-I, the R 3 is a group represented by any one of Compound 02-15 to Compound 02-31.

[0295] In some embodiments, in Formula 02-I, the R 4 is a group represented by Compound 02-7 or Compound 02-14.

[0296] In some embodiments, in Formula 02-I, the R 4 is a group represented by Compound 02-11.

[0297] In some embodiments, in Formula 02-I, the R 4 is a group represented by any one of Compound 02-13, Compound 02-12, or Compound 02-6.

[0298] In some embodiments, in Formula 02-I, the R 4 is a group represented by Compound 02-9 or Compound 02-1.

[0299] In some embodiments, in Formula 02-I, the R 4 is a group represented by Compound 02-5.

[0300] In some embodiments, in Formula 02-I, the R 4 is a group represented by Compound 02-2.

[0301] In some embodiments, in Formula 02-I, the R 4 is a group represented by Compound 02-8.

[0302] In some embodiments, in Formula 02-I, the R 4 is a group represented by any one of Compound 02-3, Compound 02-4, or Compound 02-10.

[0303] In some embodiments, in Formula 02-I, the R 4 is a group represented by any one of Compound 02-15 to Compound 02-31.

[0304] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups represented by Compound 02-7 or Compound 02-14, respectively.

[0305] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-11, respectively.

[0306] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-13, compound 02-12, or compound 02-6, respectively.

[0307] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-9 or compound 02-1, respectively.

[0308] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-5, respectively.

[0309] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-2, respectively.

[0310] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-8, respectively.

[0311] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in compound 02-3, compound 02-4, or compound 02-10, respectively.

[0312] In some embodiments, in Formula 02-I, the R 3 , R 4 are groups corresponding to those shown in any one of compounds 02-15 to 02-31, respectively.

[0313] In each of the above specific embodiments, a can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and R 3 , R 4 are groups corresponding to those shown in any one of compounds 02-1 to 02-31, respectively.

[0314] In some embodiments, in Formula 02-I, the R 5a group represented by the compound 02-7 or the compound 02-14.

[0315] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-11.

[0316] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-13, the compound 02-12, or the compound 02-6.

[0317] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-9 or the compound 02-1.

[0318] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-5.

[0319] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-2.

[0320] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-8.

[0321] In some embodiments, in Formula 02-I, the R 5 a group represented by the compound 02-3, the compound 02-4, or the compound 02-10.

[0322] In some embodiments, in Formula 02-I, the R 5 a group represented by any one of the compound 02-15 to the compound 02-31.

[0323] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups represented by the compound 02-7 or the compound 02-14, respectively.

[0324] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups represented by the compound 02-11.

[0325] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5R

[0326] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups corresponding to those shown for Compound 02-9 or Compound 02-1, respectively.

[0327] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups corresponding to those shown for Compound 02-5.

[0328] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups corresponding to those shown for Compound 02-2.

[0329] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups corresponding to those shown for Compound 02-8.

[0330] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups corresponding to those shown for Compound 02-3, Compound 02-4, or Compound 02-10, respectively.

[0331] In some embodiments, in Formula 02-I, the R 3 , R 4 , R 5 are groups corresponding to those shown for any one of Compounds 02-15 to 02-31.

[0332] In each of the above specific embodiments, a can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. 3 , R 4 , R 5 are groups corresponding to those shown for any one of Compounds 02-1 to 02-31, respectively.

[0333] In some embodiments, in Formula 02-I, the R 6 is a group corresponding to that shown for Compound 02-7 or Compound 02-14.

[0334] In some embodiments, in Formula 02-I, the R6 a group represented by the corresponding compound 02-11.

[0335] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0336] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-9 or compound 02-1.

[0337] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-5.

[0338] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-2.

[0339] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-8.

[0340] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0341] In some embodiments, in Formula 02-I, the R 6 a group represented by the corresponding compound 02-15 to any one of compounds 02-31.

[0342] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-7 or compound 02-14.

[0343] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-11.

[0344] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0345] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-9 or compound 02-1.

[0346] In some embodiments, in Formula 02-I, the R 7a group represented by the corresponding compound 02-5.

[0347] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-2.

[0348] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-8.

[0349] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0350] In some embodiments, in Formula 02-I, the R 7 a group represented by the corresponding compound 02-15 to any one of compounds 02-31.

[0351] In some embodiments, in Formula 02-I, the R 6 , R 7 are groups represented by the corresponding compound 02-7 or compound 02-14, respectively.

[0352] In some embodiments, in Formula 02-I, the R 6 , R 7 are groups represented by the corresponding compound 02-11.

[0353] In some embodiments, in Formula 02-I, the R 6 , R 7 are groups represented by the corresponding compound 02-13, compound 02-12, or compound 02-6, respectively.

[0354] In some embodiments, in Formula 02-I, the R 6 , R 7 are groups represented by the corresponding compound 02-9 or compound 02-1, respectively.

[0355] In some embodiments, in Formula 02-I, the R 6 , R 7 are groups represented by the corresponding compound 02-5.

[0356] In some embodiments, in Formula 02-I, the R 6 , R 7 are groups represented by the corresponding compound 02-2.

[0357] In some embodiments, in Formula 02-I, the R 6 , R 7are groups corresponding to compound 02-8, respectively.

[0358] In some embodiments, in formula 02-I, the R 6 , R 7 are groups corresponding to compound 02-3, compound 02-4, or compound 02-10, respectively.

[0359] In some embodiments, in formula 02-I, the R 6 , R 7 are groups corresponding to any one of compound 02-15 to compound 02-31, respectively.

[0360] In each of the above specific embodiments, b can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. 6 , R 7 are groups or values corresponding to any one of compound 02-1 to compound 02-31, respectively.

[0361] In some embodiments, in formula 02-I, the R 6 , R 7 , R 5 are groups corresponding to compound 02-7 or compound 02-14, respectively.

[0362] In some embodiments, in formula 02-I, the R 6 , R 7 , R 5 are groups corresponding to compound 02-11, respectively.

[0363] In some embodiments, in formula 02-I, the R 6 , R 7 , R 5 are groups corresponding to compound 02-13, compound 02-12, or compound 02-6, respectively.

[0364] In some embodiments, in formula 02-I, the R 6 , R 7 , R 5 are groups corresponding to compound 02-9 or compound 02-1, respectively.

[0365] In some embodiments, in formula 02-I, the R 6 , R 7 , R 5 are groups corresponding to compound 02-5, respectively.

[0366] In some embodiments, in formula 02-I, the R 6 , R7 R 5 R

[0367] In some embodiments, in Formula 02-I, the R 6 R 7 R 5 R

[0368] In some embodiments, in Formula 02-I, the R 6 R 7 R 5 R

[0369] In some embodiments, in Formula 02-I, the R 6 R 7 R 5 R

[0370] In each of the above specific embodiments, b can be independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, b is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 6 R 7 R 5 R

[0371] In some embodiments, in Formula 02-I, the above L 1 R 3 R 4 R 5 R 6 R 7 a, b can be freely combined to form the corresponding compounds.

[0372] In some embodiments, in Formula 02-I, L 1 R 3 R 4 R 5 R 6 R 7 a, b are independently the groups or values corresponding to any one of compounds 02-1 to 02-31.

[0373] In some embodiments, in Formula 02-I,

[0374] L 2 is h is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0375] R 8 , R 9 each independently is hydrogen, R 10 , R 11 , R 12 at least two of which are not hydrogen;

[0376] c, d are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably at least one or none of c, d is 0.

[0377] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-7 or compound 02-14.

[0378] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-11.

[0379] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-13, compound 02-12 or compound 02-6.

[0380] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-9 or compound 02-1.

[0381] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-5.

[0382] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-2.

[0383] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-8.

[0384] In some embodiments, in formula 02-I, the L 2 is a group as depicted for compound 02-3, compound 02-4 or compound 02-10.

[0385] In some embodiments, in formula 02-I, the L 2 is a group as depicted for any one of compound 02-15 to compound 02-31.

[0386] In some embodiments, in formula 02-I, the R 10a group represented by the compound 02-7 or the compound 02-14.

[0387] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-11.

[0388] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-13, the compound 02-12, or the compound 02-6.

[0389] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-9 or the compound 02-1.

[0390] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-5.

[0391] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-2.

[0392] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-8.

[0393] In some embodiments, in Formula 02-I, the R 10 a group represented by the compound 02-3, the compound 02-4, or the compound 02-10.

[0394] In some embodiments, in Formula 02-I, the R 10 a group represented by any one of the compound 02-15 to the compound 02-31.

[0395] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-7 or the compound 02-14.

[0396] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-11.

[0397] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-13, the compound 02-12, or the compound 02-6.

[0398] In some embodiments, in Formula 02-I, the R 11a group represented by the compound 02-9 or the compound 02-1.

[0399] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-5.

[0400] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-2.

[0401] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-8.

[0402] In some embodiments, in Formula 02-I, the R 11 a group represented by the compound 02-3, the compound 02-4 or the compound 02-10.

[0403] In some embodiments, in Formula 02-I, the R 11 a group represented by any one of the compound 02-15 to the compound 02-31.

[0404] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups represented by the compound 02-7 or the compound 02-14, respectively.

[0405] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups represented by the compound 02-11.

[0406] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups represented by the compound 02-13, the compound 02-12 or the compound 02-6, respectively.

[0407] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups represented by the compound 02-9 or the compound 02-1.

[0408] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups represented by the compound 02-5.

[0409] In some embodiments, in Formula 02-I, the R 10 , R 11R

[0410] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups corresponding to Compound 02-8, respectively.

[0411] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups corresponding to Compound 02-3, Compound 02-4, or Compound 02-10, respectively.

[0412] In some embodiments, in Formula 02-I, the R 10 , R 11 are groups corresponding to any one of Compound 02-15 to Compound 02-31, respectively.

[0413] In each of the above specific embodiments, d can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. 10 , R 11 are groups or values corresponding to any one of Compound 02-1 to Compound 02-31, respectively.

[0414] In some embodiments, in Formula 02-I, the R 12 is a group corresponding to Compound 02-7 or Compound 02-14.

[0415] In some embodiments, in Formula 02-I, the R 12 is a group corresponding to Compound 02-11.

[0416] In some embodiments, in Formula 02-I, the R 12 is a group corresponding to Compound 02-13, Compound 02-12, or Compound 02-6.

[0417] In some embodiments, in Formula 02-I, the R 12 is a group corresponding to Compound 02-9 or Compound 02-1.

[0418] In some embodiments, in Formula 02-I, the R 12 is a group corresponding to Compound 02-5.

[0419] In some embodiments, in Formula 02-I, the R 12 is a group corresponding to Compound 02-2.

[0420] In some embodiments, in Formula 02-I, the R 12a group represented by the corresponding compound 02-8.

[0421] In some embodiments, in Formula 02-I, the R 12 a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0422] In some embodiments, in Formula 02-I, the R 12 a group represented by the corresponding any one of compound 02-15 to compound 02-31.

[0423] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-7 or compound 02-14, respectively.

[0424] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-11.

[0425] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-13, compound 02-12, or compound 02-6, respectively.

[0426] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-9 or compound 02-1, respectively.

[0427] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-5.

[0428] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-2.

[0429] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups represented by the corresponding compound 02-8.

[0430] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups as shown in the corresponding compound 02-3, compound 02-4, or compound 02-10, respectively.

[0431] In some embodiments, in Formula 02-I, the R 10 , R 11 , R 12 are groups as shown in any one of the corresponding compound 02-15 to compound 02-31, respectively.

[0432] In each of the above specific embodiments, d can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. 10 , R 11 , R 12 are groups or values as shown in any one of the corresponding compound 02-1 to compound 02-31, respectively.

[0433] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-7 or compound 02-14.

[0434] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-11.

[0435] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0436] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-9 or compound 02-1.

[0437] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-5.

[0438] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-2.

[0439] In some embodiments, in Formula 02-I, the R 8 is a group as shown in the corresponding compound 02-8.

[0440] In some embodiments, in Formula 02-I, the R 8a group represented by any one of compounds 02-15 to 02-31.

[0441] In some embodiments, in Formula 02-I, the R 8 a group represented by any one of compounds 02-15 to 02-31.

[0442] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0443] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0444] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0445] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0446] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0447] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0448] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0449] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0450] In some embodiments, in Formula 02-I, the R 9 a group represented by any one of compounds 02-15 to 02-31.

[0451] In some embodiments, in Formula 02-I, the R 8 , R 9 a group represented by any one of compounds 02-15 to 02-31.

[0452] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-11, respectively.

[0453] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-13, compound 02-12, or compound 02-6, respectively.

[0454] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-9 or compound 02-1, respectively.

[0455] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-5, respectively.

[0456] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-2, respectively.

[0457] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-8, respectively.

[0458] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in compound 02-3, compound 02-4, or compound 02-10, respectively.

[0459] In some embodiments, in Formula 02-I, the R 8 , R 9 are groups corresponding to those shown in any one of compounds 02-15 to 02-31, respectively.

[0460] In each of the above specific embodiments, c can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. 8 , R 9 are groups or values corresponding to those shown in any one of compounds 02-1 to 02-31, respectively.

[0461] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12R

[0462] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-11.

[0463] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-13, Compound 02-12, or Compound 02-6.

[0464] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-9 or Compound 02-1.

[0465] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-5.

[0466] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-2.

[0467] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-8.

[0468] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to Compound 02-3, Compound 02-4, or Compound 02-10.

[0469] In some embodiments, in Formula 02-I, the R 8 , R 9 , R 12 are each a group corresponding to any one of Compound 02-15 to Compound 02-31.

[0470] In each of the above embodiments, c can be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. Preferably, c is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively, and R 6 , R 7 , R 5 are as shown in any one of Compounds 02-1 to 02-31, respectively.

[0471] In some embodiments, in Formula 02-I, the above L 2 , R 8 , R 9 , R 10 , R 11 , R 12 , c, d can be freely combined to form the corresponding compounds.

[0472] In some embodiments, in Formula 02-I, L 2 , R 8 , R 9 , R 10 , R 11 , R 12 , c, d are as shown in any one of Compounds 02-1 to 02-31, respectively.

[0473] In some embodiments, in Formula 02-I,

[0474] L 3 , L 4 are each independently selected from alkynylene, O, or absent;

[0475] R 13 , R 14 are each hydrogen; R 15 , R 16 , R 17 , R 18 , R 19 at least two of which are not hydrogen; preferably, R 16 , R 18 , R 19 at least two of which are not hydrogen;

[0476] e, f, g are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0477] In some embodiments, the compounds are as shown in Formula 02-I-10, Formula 02-I-11, Formula 02-I-12, Formula 02-I-13, Formula 02-I-14, respectively.

[0478] In some embodiments, in Formula 02-I, L is

[0479] In some embodiments, in Formula 02-I, L is

[0480] L 3 is alkynylene, O, or absent;

[0481] R 13 , R 14 each is hydrogen; R 15 , R 16 , R 17 at least two are not hydrogen;

[0482] e, f each is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0483] In some embodiments, in Formula 02-I, L is

[0484] L 4 is alkynylene, O, or absent;

[0485] R 13 , R 14 each is hydrogen; R 15 , R 16 , R 17 , R 18 , R 19 at least two are not hydrogen;

[0486] e, f, g each is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0487] In some embodiments, in Formula 02-I, L is 3 is a group shown for corresponding compound 02-7 or compound 02-14.

[0488] In some embodiments, in Formula 02-I, L is 3 is a group shown for corresponding compound 02-11.

[0489] In some embodiments, in Formula 02-I, L is 3 is a group shown for corresponding compound 02-13, compound 02-12, or compound 02-6.

[0490] In some embodiments, in Formula 02-I, L is 3 is a group shown for corresponding compound 02-9 or compound 02-1.

[0491] In some embodiments, in Formula 02-I, the L is 3 a group represented by the corresponding compound 02-5.

[0492] In some embodiments, in Formula 02-I, the L is 3 a group represented by the corresponding compound 02-2.

[0493] In some embodiments, in Formula 02-I, the L is 3 a group represented by the corresponding compound 02-8.

[0494] In some embodiments, in Formula 02-I, the L is 3 a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0495] In some embodiments, in Formula 02-I, the L is 3 a group represented by any one of the corresponding compound 02-15 to compound 02-31.

[0496] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-7 or compound 02-14.

[0497] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-11.

[0498] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0499] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-9 or compound 02-1.

[0500] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-5.

[0501] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-2.

[0502] In some embodiments, in Formula 02-I, the L is 4 a group represented by the corresponding compound 02-8.

[0503] In some embodiments, in Formula 02-I, the L is 4a group represented by any one of compounds 02-15 to 02-31.

[0504] In some embodiments, in formula 02-I, the L 4 a group represented by any one of compounds 02-15 to 02-31.

[0505] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-7 or compound 02-14, respectively.

[0506] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-11.

[0507] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-13, compound 02-12, or compound 02-6, respectively.

[0508] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-9 or compound 02-1, respectively.

[0509] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-5.

[0510] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-2.

[0511] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-8.

[0512] In some embodiments, in formula 02-I, the L 3 , L 4 are groups represented by compound 02-3, compound 02-4, or compound 02-10.

[0513] In some embodiments, in formula 02-I, the L 3 , L 4is a group corresponding to that shown for any one of compounds 02-15 to 02-31.

[0514] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-7 or compound 02-14.

[0515] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-11.

[0516] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-13, compound 02-12, or compound 02-6.

[0517] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-9 or compound 02-1.

[0518] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-5.

[0519] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-2.

[0520] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-8.

[0521] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for compound 02-3, compound 02-4, or compound 02-10.

[0522] In some embodiments, in Formula 02-I, the R 13 is a group corresponding to that shown for any one of compounds 02-15 to 02-31.

[0523] In some embodiments, in Formula 02-I, the R 14 is a group corresponding to that shown for compound 02-7 or compound 02-14.

[0524] In some embodiments, in Formula 02-I, the R 14 is a group corresponding to that shown for compound 02-11.

[0525] In some embodiments, in Formula 02-I, the R 14a group represented by any one of compounds 02-1 to 02-31.

[0526] In some embodiments, in formula 02-I, the R 14 a group represented by compound 02-9 or compound 02-1.

[0527] In some embodiments, in formula 02-I, the R 14 a group represented by compound 02-5.

[0528] In some embodiments, in formula 02-I, the R 14 a group represented by compound 02-2.

[0529] In some embodiments, in formula 02-I, the R 14 a group represented by compound 02-8.

[0530] In some embodiments, in formula 02-I, the R 14 a group represented by compound 02-3, compound 02-4, or compound 02-10.

[0531] In some embodiments, in formula 02-I, the R 14 a group represented by any one of compounds 02-15 to 02-31.

[0532] In some embodiments, in formula 02-I, the above-mentioned R 13 , R 14 may be combined at will to form a compound.

[0533] In some embodiments, in formula 02-I, the above-mentioned R 13 , R 14 respectively a group represented by any one of compounds 02-1 to 02-31.

[0534] In some embodiments, in formula 02-I, the R 15 a group represented by compound 02-7 or compound 02-14.

[0535] In some embodiments, in formula 02-I, the R 15 a group represented by compound 02-11.

[0536] In some embodiments, in formula 02-I, the R 15 a group represented by compound 02-13, compound 02-12, or compound 02-6.

[0537] In some embodiments, in Formula 02-I, the R 15 is a group represented by the corresponding compound 02-9 or compound 02-1.

[0538] In some embodiments, in Formula 02-I, the R 15 is a group represented by the corresponding compound 02-5.

[0539] In some embodiments, in Formula 02-I, the R 15 is a group represented by the corresponding compound 02-2.

[0540] In some embodiments, in Formula 02-I, the R 15 is a group represented by the corresponding compound 02-8.

[0541] In some embodiments, in Formula 02-I, the R 15 is a group represented by the corresponding compound 02-3, compound 02-4, or compound 02-10.

[0542] In some embodiments, in Formula 02-I, the R 15 is a group represented by the corresponding compound 02-15 to compound 02-31.

[0543] In some embodiments, in Formula 02-I, the R 17 is a group represented by the corresponding compound 02-7 or compound 02-14.

[0544] In some embodiments, in Formula 02-I, the R 17 is a group represented by the corresponding compound 02-11.

[0545] In some embodiments, in Formula 02-I, the R 17 is a group represented by the corresponding compound 02-13, compound 02-12, or compound 02-6.

[0546] In some embodiments, in Formula 02-I, the R 17 is a group represented by the corresponding compound 02-9 or compound 02-1.

[0547] In some embodiments, in Formula 02-I, the R 17 is a group represented by the corresponding compound 02-5.

[0548] In some embodiments, in Formula 02-I, the R 17 is a group represented by the corresponding compound 02-2.

[0549] In some embodiments, in Formula 02-I, the R 17a group represented by any one of compounds 02-1 to compound 02-31.

[0550] In some embodiments, in formula 02-I, the R 17 a group represented by any one of compounds 02-3, compound 02-4, or compound 02-10.

[0551] In some embodiments, in formula 02-I, the R 17 a group represented by any one of compounds 02-15 to compound 02-31.

[0552] In some embodiments, in formula 02-I, the above-mentioned R 15 , R 17 may be combined at will to form a compound.

[0553] In some embodiments, in formula 02-I, the above-mentioned R 15 , R 17 respectively a group represented by any one of compounds 02-1 to compound 02-31.

[0554] In some embodiments, in formula 02-I, the R 19 a group represented by any one of compounds 02-7 or compound 02-14.

[0555] In some embodiments, in formula 02-I, the R 19 a group represented by compound 02-11.

[0556] In some embodiments, in formula 02-I, the R 19 a group represented by any one of compounds 02-13, compound 02-12, or compound 02-6.

[0557] In some embodiments, in formula 02-I, the R 19 a group represented by any one of compounds 02-9 or compound 02-1.

[0558] In some embodiments, in formula 02-I, the R 19 a group represented by compound 02-5.

[0559] In some embodiments, in formula 02-I, the R 19 a group represented by compound 02-2.

[0560] In some embodiments, in formula 02-I, the R 19 a group represented by compound 02-8.

[0561] In some embodiments, in formula 02-I, the R 19a group represented by any one of compounds 02-15 to 02-31.

[0562] In some embodiments, in formula 02-I, the R 19 a group represented by any one of compounds 02-15 to 02-31.

[0563] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-7 or 02-14.

[0564] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-11.

[0565] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-13, 02-12 or 02-6.

[0566] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-9 or 02-1.

[0567] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-5.

[0568] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-2.

[0569] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-8.

[0570] In some embodiments, in formula 02-I, the R 18 a group represented by compound 02-3, 02-4 or 02-10.

[0571] In some embodiments, in formula 02-I, the R 18 a group represented by any one of compounds 02-15 to 02-31.

[0572] In some embodiments, in formula 02-I, the above-mentioned R 19 , R 18 may be combined at will to form a compound.

[0573] In some embodiments, in formula 02-I, the above-mentioned R 19, R 18 corresponding to the group shown in any one of compounds 02-1 to 02-31.

[0574] In some embodiments, in formula 02-I, the aforementioned L 3 , L 4 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 may be arbitrarily combined, wherein e, f, g may each independently be selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0575] In some embodiments, in formula 02-I, the aforementioned L 3 , L 4 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , e, f, g may be arbitrarily combined to form a compound.

[0576] In some embodiments, in formula 02-I, L 3 , L 4 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , e, f, g correspond to the group shown in any one of compounds 02-1 to 02-31 or take values.

[0577] In the present disclosure, when the values of a, b, c, d, e, f are greater than 1, the corresponding multiple R 3 , multiple R 4 , multiple R 6 , multiple R 7 , multiple R 8 , multiple R 9 , multiple R 10 , multiple R 11 , multiple R 13 , multiple R 14 , multiple R 15 , multiple R 17 may each independently be the same or different.

[0578] In some embodiments, the compound is selected from one or more of compounds 02-1 to 02-31 shown in Table 2.

[0579] Table 2

[0580] In some embodiments, the ionizable lipid in the lipid nanoparticle of the present disclosure is 11-({8-[(2-butyloctyl)oxy]-8-oxopentadecyl}amino)-10-(3-ethyl-6-oxo-3,7- diazahexadecan-7-yl)-11-oxopentadecanoic acid-2-butyloctyl ester

[0581] In some embodiments, the ionizable lipid contained in the lipid nanoparticle (LNP), pharmaceutical composition described herein is the ionizable lipid described in International Patent Publication No. PCT / CN2023 / 116607, which is incorporated herein by reference in its entirety.

[0582] In one embodiment, the ionizable lipid is the compound of 03-I:

[0583] wherein,

[0584] R1and R2are each independently selected from H, C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl, or C1-C 10 haloalkyl; or

[0585] R1and R2together with the N to which they are attached form a 4-10 membered heterocyclic group containing 1-3 heteroatoms selected from N, S, and O;

[0586] G1is -(CH2)n- wherein n is a positive integer from 1-15, preferably a positive integer from 1-10, more preferably a positive integer from 1-6;

[0587] R3is selected from H, substituted or unsubstituted C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, C3-C 20 cycloalkyl, or or R a -L a -R b wherein "substituted" means substituted with one or more substituents selected from the group consisting of halogen, C1-C 15 alkyl, C2-C 15alkenyl, C2-C 15 alkynyl, O-(C2-C 15 alkyl, C1-C 15 haloalkyl, C1-C 15 haloalkoxy, -OH, -CN, -NH2, Ra is -(CH2)s, s is a positive integer from 1 to 15, preferably a positive integer from 1 to 10, L a is selected from -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, preferably -(C=O)O; R b is selected from C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0588] R4 is -(CH2)m-, wherein m is a positive integer from 1 to 20, preferably a positive integer from 1 to 15; more preferably a positive integer from 1 to 10;

[0589] L1 is selected from -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, preferably -(C=O)O, -O(C=O)-;

[0590] R5 is selected from C1-C 30 alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl;

[0591] R6 is -(CH2)q-, wherein q is a positive integer from 1 to 20, preferably a positive integer from 1 to 15; more preferably a positive integer from 1 to 10;

[0592] L2 is selected from -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, preferably -(C=O)O, -O(C=O)-;

[0593] R7 is selected from substituted or unsubstituted C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20Alkyne group, wherein "substitution" refers to a substitution having one or more (preferably one or two) substituents selected from the group consisting of halogens, C1-C... 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, O-(C2-C 15 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 15 Haloalkyl, C1-C 15 Halogenated alkoxy groups, -OH, -CN, -NH2.

[0594] In one implementation, R7 is Where R c It is -(CH2)p-, where p is a positive integer from 1 to 15, preferably a positive integer from 1 to 10; more preferably a positive integer from 1 to 6; R d and R e Each is independently selected from C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, or R d and R e Together with their intermediate atoms, they form an optionally substituted ring: Where x is selected from 1 or 2.

[0595] In one embodiment, R1 and R2 are each independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl or C1-C6 haloalkyl.

[0596] In one embodiment, R1 and R2 together with the N that connects them form a 5-10 membered heterocyclic group containing 1-2 heteroatoms selected from N and O.

[0597] In one embodiment, R1 and R2 together with the N that connects them form a 5-7 membered heterocyclic group containing 1-2 heteroatoms selected from N and O.

[0598] In one embodiment, R3 is selected from substituted or unsubstituted C1-C. 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, C3-C 15 Cycloalkyl, wherein "substituted" is a substitution having one or more substituents selected from the group consisting of: halogens, C1-C... 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, O-(C2-C 10 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 10 Haloalkyl, C1-C10 Halogenated alkoxy groups, -OH, -CN, -NH2.

[0599] In one embodiment, R3 is selected from substituted or unsubstituted C1-C. 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, C3-C 10 Cycloalkyl, wherein “substitution” means substitution having one or more substituents selected from the group consisting of: halogens, C1-C... 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, O-(C2-C 10 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 10 Haloalkyl, C1-C 10 Halogenated alkoxy groups, -OH, -CN, -NH2.

[0600] In one implementation, R b Selected from C1-C 15 Alkyl, C2-C 15 alkenyl, C2-C 15 Alkyne group.

[0601] In one implementation, R5 is selected from C1-C 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group.

[0602] In one implementation, R5 is selected from C2-C 12 alkenyl, C2-C 12 Alkyne group.

[0603] In one implementation, R7 is selected from C1-C 12 Alkyl, C2-C 12 alkenyl, C2-C 12 Alkyne group.

[0604] In one implementation, R7 is selected from substituted C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group, wherein "substitution" is a substitution having one or more substituents selected from the group consisting of: halogen, C1-C 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, O-(C2-C 10 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 10 Haloalkyl, C1-C 10Halogenated alkoxy groups, -OH, -CN, -NH2.

[0605] In one embodiment, R7 is selected from substituted C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl groups, wherein "substituted" is a substitution having one or more substituents selected from the group consisting of: halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and C2-C6 alkenyl groups. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, O-(C2-C 10 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 10 Haloalkyl, C1-C 10 Halogenated alkoxy groups, -OH, -CN, -NH2.

[0606] In one embodiment, the ionizable lipid is a compound of formula 03-I-1 or formula 03-I-2:

[0607] In equation 03-I-1,

[0608] The definitions of R1-R6, G1, and L1 are as described above;

[0609] R7 is selected from substituted or unsubstituted C1-C. 20 Alkyl, C2-C 20 alkenyl, C2-C 20 Alkyne group, wherein "substitution" is substitution having one or more (preferably one or two) substituents selected from the group consisting of: halogens, C1-C... 15 Alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl group, O-(C2-C 15 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 15 Haloalkyl, C1-C 15 Halogenated alkoxy groups, -OH, -CN, -NH2.

[0610] In one implementation, R7 is selected from substituted C1-C. 10 Alkyl, C2-C 10 alkenyl, C2-C 10 Alkyne group, wherein "substitution" is substitution having one or more (preferably one or two) substituents selected from the group consisting of: halogens, C1-C... 10 Alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl group, O-(C2-C 10 Hydrocarbon groups (including alkyl, alkenyl, or ynyl groups), C1-C 10 Haloalkyl, C1-C 10haloalkoxy, -OH, -CN, -NH2.

[0611] wherein in formula 03-I-2,

[0612] R1-R6, G1, L1 are defined as above;

[0613] R7 is selected from C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl.

[0614] In one embodiment, R7 is selected from C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl.

[0615] In some embodiments, the ionizable lipid is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0616] Table 3

[0617] In some embodiments, the ionizable lipid contained in the lipid nanoparticle (LNP), pharmaceutical composition described herein is an ionizable lipid described in International Patent Publication No. PCT / CN2024 / 104304, which is incorporated herein by reference in its entirety.

[0618] In one embodiment, the ionizable lipid is a 04-I compound:

[0619] wherein

[0620] R1 is null or selected from -OH and R a (R b )N-, wherein R a and R b are each independently H, C1-C 10 alkyl or C1-C 10 haloalkyl;

[0621] R2 is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0622] R3 is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl or R c-(CH2)n-, wherein n is a positive integer from 1 to 20, preferably from 1 to 14, more preferably from 1 to 10;

[0623] Rc is selected from the following structures: wherein, represents a bond;

[0624] L1, L2, L3, L4, L5are nothing or are each independently selected from the group consisting of:

[0625] X is nothing or -CH- or N;

[0626] R4is nothing or C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0627] R5is nothing or C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0628] R6is C1-C 30 alkyl, C2-C 30 alkenyl, C2-C 30 alkynyl;

[0629] R7is C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl;

[0630] R8, R9are nothing or are each independently C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl, wherein Rhis O or S;

[0631] R 10 , R 11 are each independently C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 alkyl, -R h -C2-C 14alkyl, C2-C6alkenyl, C2-C6alkynyl, -R h -C2-C 14 alkynyl, wherein R h is O or S.

[0632] In one embodiment, R a and R b are each independently H, C1-C6alkyl or C1-C6haloalkyl.

[0633] In one embodiment, R2is selected from -(CH2)m-, wherein m is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, more preferably a positive integer from 1 to 6.

[0634] In one embodiment, R3is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0635] In one embodiment, R3is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl.

[0636] In one embodiment, R6is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl.

[0637] In one embodiment, R2is C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl.

[0638] In one embodiment, R2, R7are each independently C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0639] In one embodiment, R4, R5are each independently -(CH2)q-CH3, wherein q is selected from a positive integer from 1 to 20, preferably a positive integer from 1 to 12, more preferably a positive integer from 1 to 8.

[0640] In one embodiment, R4, R5are each independently of the formula -R d -R e -structure;

[0641] wherein R d is nothing or is selected from the following functional groups: -(CH2)-(CH=CH)-;

[0642] R e-(CH2)p-CH3, wherein p is selected from the group consisting of positive integers from 0 to 20, preferably from 0 to 12, more preferably from 0 to 8.

[0643] In one embodiment, R8, R9are each independently -(CH2)q-CH3, wherein q is selected from the group consisting of positive integers from 1 to 14, preferably from 1 to 12, more preferably from 1 to 8.

[0644] In one embodiment, R7is -(CH2)q-CH3, wherein q is selected from the group consisting of positive integers from 1 to 14, preferably from 1 to 12, more preferably from 1 to 8.

[0645] In one embodiment, R6has the structure of -R f -R g

[0646] wherein R f is selected from the group consisting of the following functional groups: -(CH2)s1-(CH=CH)-CH2-(CH=CH)-(CH2)s2-CH3, s1 is a positive integer from 1 to 14, preferably from 1 to 10, s2 is a positive integer from 1 to 8, preferably from 1 to 6;

[0647] R g is -(CH2)m-, wherein m is a positive integer from 1 to 14, preferably from 1 to 10, more preferably from 1 to 6.

[0648] In one embodiment, R 10 , R 11 are each independently C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl or -R h -C1-C 10 alkyl, -R h -C2-C 10 alkenyl, -R h -C2-C 10 alkynyl, wherein R h is O or S.

[0649] In one embodiment, R 11 is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl.

[0650] In one embodiment, R8, R9are each independently C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl or -R h -C1-C 10 ​alkyl, -R h -C2-C 10 alkenyl, -R h -C2-C 10 alkynyl, wherein R h is O or S.

[0651] In one embodiment, R9is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl.

[0652] In one embodiment, X is nothing or -CH-.

[0653] In one embodiment, the ionizable lipid is a compound of formula 04-I-1, 04-I-2, 04-I-3, or 04-I-4:

[0654] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0655] wherein, in formula 04-I-1,

[0656] R1-R2, R6-R7, R 11 , L2-L5 are as defined above;

[0657] R3is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl.

[0658] In one embodiment, R3is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0659] In one embodiment, R3is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl.

[0660] wherein, in formula 04-I-2,

[0661] R1-R2, R6-R7, R 10 -R 11 , L1-L5 are as defined above;

[0662] R3is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl, or R c -(CH2)n- wherein n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, R c are as defined above;

[0663] R5is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0664] R8, R9are each independently C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl.

[0665] R5is C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl.

[0666] R5is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0667] R3is C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl.

[0668] R3is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0669] wherein, in formula 04-I-3,

[0670] R1-R3, R6, R7, R 10 , R 11 , L1-L5 are defined as above;

[0671] R4is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0672] R5is C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0673] R8, R9are each independently C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl.

[0674] R4is C1-C14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl.

[0675] In one embodiment, R4is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0676] In one embodiment, R5is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl.

[0677] In one embodiment, R8, R9are each independently C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0678] In one embodiment, R9is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl.

[0679] wherein, in formula 04-I-4,

[0680] R1-R7, R 10 -R 11 , L1-L3, L5are defined as above.

[0681] In some embodiments, the ionizable lipid is a compound of formula 04-II:

[0682] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0683] wherein,

[0684] R1is selected from the group consisting of -OH and R a (R b )N-, wherein R a and R b are each independently H, C1-C6alkyl, or C1-C6haloalkyl;

[0685] R3is C1-C 15 alkyl, C2-C 15 alkenyl, C2-C 15 alkynyl or R c -(CH2)n-, wherein n is a positive integer from 1 to 20, preferably a positive integer from 1 to 14, more preferably a positive integer from 1 to 10, more preferably a positive integer from 1 to 6;

[0686] R c is selected from the group consisting of structures as shown below: wherein represents a bond;

[0687] L1, L2, L3, L4, L5are nothing or are each independently selected from the group consisting of:

[0688] R4is nothing or C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0689] R5is nothing or C1-C 20 alkyl, C2-C 20 alkenyl, C2-C 20 alkynyl;

[0690] R8, R9are each independently C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl or -R h -C1-C 14 alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl, wherein R h is O or S;

[0691] R 10 , R 11 are each independently C1-C 14 alkyl, C2-C 14 alkenyl, C2-C 14 alkynyl, or -R h -C1-C 14 alkyl, -R h -C2-C 14 alkenyl, -R h -C2-C 14 alkynyl, wherein R h is O or S;

[0692] r is 2 or 3;

[0693] u is a positive integer from 1 to 10;

[0694] t is a positive integer from 1 to 10;

[0695] Further, when R1is selected from R a (R b )N-, r is 2.

[0696] In one embodiment, R3is C1-C 10 alkyl, C2-C 10alkyl, C2-C 10 alkenyl, C2-C

[0697] In one embodiment, R4is absent or C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0698] In one embodiment, R5is absent or C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0699] In one embodiment, R8is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0700] In one embodiment, R9is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0701] In one embodiment, R 10 is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0702] In one embodiment, R 11 is C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10 alkynyl.

[0703] In one embodiment, u is a positive integer from 3 to 6, preferably a positive integer from 3 to 5, more preferably 4.

[0704] In one embodiment, t is a positive integer from 3 to 6, preferably a positive integer from 3 to 5, more preferably 4.

[0705] In one embodiment, L1, R4and R5are absent.

[0706] In one embodiment, L1, L2, L3, L4, L5are each independently selected from the group consisting of:

[0707] In one embodiment, the ionizable lipid is a compound of formula 04-II-1, 04-II-2 or 04-II-3:

[0708] wherein

[0709] In Formula 04-II-1, R3, r, R8-R 11 , L2-L5 are as defined above;

[0710] In Formula 04-II-2, r, R3-R5, R8-R 11 , L1-L5 are as defined above;

[0711] In Formula 04-II-3, R a , R b , R3-R5, R8-R 11 , r, L1-L5 are as defined above.

[0712] In one embodiment, the ionizable lipid is a compound in Table 4, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0713] Table 4

[0714] In some embodiments, the ionizable lipid is selected from the compounds listed in any one of Tables 1-4.

[0715] In some embodiments, the lipid component of the nanoparticle composition can include one or more structural lipids. It is contemplated that the structural lipids can stabilize the amphipathic structure of the nanoparticle, for example, but not limited to, the lipid bilayer structure of the nanoparticle. Exemplary structural lipids that can be used in conjunction with the present disclosure include, but are not limited to, cholesterol, non-steroidal sterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatin, ursolic acid, a-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof. In some embodiments, the lipid nanoparticle provided herein comprises a steroid or steroid analog. In some embodiments, the steroid or steroid analog is cholesterol.

[0716] In some embodiments, the lipid component of the nanoparticle composition can include one or more phospholipids, such as one or more (poly)unsaturated lipids. It is contemplated that the phospholipids can assemble into one or more lipid bilayer structures. Exemplary phospholipids that can form part of the nanoparticle composition include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1- oleyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChems PC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2- diarachidoyl-sn-glycero-3-phosphocholine, 1,2-docosahexaoyl-sn-glycero-3-phosphocholine, 1,2- diphytanyloyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinoleoyl 1-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn- glycero-3-phosphoethanolamine, 1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine, 1,2- docosahexaoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1- glycerol) sodium salt (DOPG), and sphingomyelin. In certain embodiments, the nanoparticle composition includes DSPC. In certain embodiments, the nanoparticle composition includes DOPE. In some embodiments, the nanoparticle composition includes DSPC and DOPE.

[0717] Exemplary neutral lipids also include dipalmitoylphosphatidylglycerol glycerol (DPPG), palmitoyloleo yl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l carboxylate (DOPE-mal), dipalmitoylphosphatidyl phosphatidylethanolamine (DPPE), distearoyl-phosphatidylethanolamine (DSPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). In one embodiment, the neutral lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In one embodiment, the neutral lipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

[0718] In some embodiments, the lipid component of the nanoparticle composition can include one or more PEG lipids (polyethylene glycol lipids). It is contemplated that the PEG lipid component in the nanoparticle composition can improve colloidal stability and / or reduce protein adsorption of the nanoparticle. Exemplary cationic lipids that can be used in conjunction with the present disclosure include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0719] In some embodiments, the lipid component of the nanoparticle composition comprises a functionalized PEG lipid comprising one or more fatty acid tails, no shorter than C 16 and no longer than C 20 for straight chain fatty acids. For branched chain fatty acids, no shorter than C 14 and no longer than C 20 fatty acid tails are acceptable. In some embodiments, the fatty acid tail is C 16 . In some embodiments, the fatty acid tail is C 18In certain embodiments, the functionalized PEG lipid comprises a dipalmitoyl lipid. In some embodiments, the functionalized PEG lipid comprises a distearoyl lipid. The fatty acid tail serves as a means of anchoring the PEG lipid in the LNP to reduce or eliminate shedding of the PEG lipid from the LNP. This is a useful property for both PEG lipids and / or functionalized PEG lipids, but is of greater significance for functionalized PEG lipids as it will link the targeting moiety (e.g., antibody), and if the PEG lipid (along with the conjugated binding moiety) sheds from the LNP, the targeting function of the LNP can be compromised.

[0720] Any suitable chemical method can be used to couple the targeting moiety to the PEG portion of the PEG lipid, including Michael addition reactions (see Parhiz et al., Journal of Controlled Release, 291 : 106-115, 2018) and click chemistry (see Kolb et al., Angewandte Chemie International Edition, 40(11): 2004-2021, 2001; and Evans, Australian Journal of Chemistry, 60(6): 384-395, 2007), among others. Reagents for such reactions include PEG lipid-maleimide, PEG lipid-cysteine, PEG lipid-alkyne, PEG lipid-DBCO (dibenzocyclooctyne), and PEG lipid-azide. Conjugation / coupling reactions utilize PEG lipid-bromomaleimide, PEG lipid-alkyl amide, PEG lipid-alkyne amide, and PEG lipid-alkyne. On the binding moiety side of the reaction, existing cysteine thiol groups can be used, or the protein can be derivatized by, for example, adding a thio-containing carboxylic acid to the epsilon amino group of lysine to react with the maleimide, bromomaleimide, alkyl amide, or alkyne amide. Alternatively, an alkyne can be added to a thiol or epsilon amino group of lysine to participate in a click chemistry reaction.

[0721] In some embodiments, the molar ratio of ionizable lipid, neutral lipid, sterol, PEG lipid, functionalized PEG lipid in the lipid nanoparticle is (20-60):(5-30):(25-45):(0.1-5):(0.1-15).

[0722] In some embodiments, the functionalized PEG lipid is conjugated to a targeting moiety (e.g., an antibody). In some embodiments, the targeting moiety is an antibody or antigen binding portion of the disclosure that targets CD117.

[0723] Lipid nanoparticle conjugate

[0724] In one aspect, the present disclosure provides a lipid nanoparticle conjugate comprising a conjugate of a targeting moiety and a lipid nanoparticle (LNP) encapsulating a biologically active substance, wherein the targeting moiety is conjugated to the lipid nanoparticle (LNP) via a linker moiety.

[0725] In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof. In some embodiments, the targeting moiety is an antibody, a Fab fragment, a ScFv, a VHH domain antibody, a nanobody, or a single domain antibody. In some embodiments, the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (e.g., a DNA or RNA aptamer), a carbohydrate, a vitamin, or N-acetylgalactosamine (GalNac). Conjugation of the targeting moiety to the LNP results in a targeted LNP. In some embodiments, the targeting moiety is a ligand that binds to a receptor on the surface of a cell. In some embodiments, the ligand can be a natural ligand for the receptor. In some embodiments, the ligand can be a synthetic ligand for the receptor. In some embodiments, the targeting moiety is a small molecule, e.g., a small molecule ligand for a cell surface receptor. In some embodiments, the targeting moiety is a peptide or polypeptide, e.g., a peptide or polypeptide ligand for a cell surface receptor. In some embodiments, the peptide or polypeptide is linear, or is cyclic. In some embodiments, the targeting moiety is a cytokine, e.g., such that the targeting cell group binds to a cytokine receptor on the surface of a cell. Conjugation of the targeting moiety to the LNP results in a targeted LNP.

[0726] In some embodiments, the targeting moiety is selected from an anti-CD117 antibody or antigen-binding fragment thereof of the present disclosure.

[0727] In some embodiments, the conjugate is used to treat a genetic disease or disorder, including sickle cell disease, hemoglobinopathy, hemophilia, thalassemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, inherited metabolic defects, Klinefelter syndrome, Marfan syndrome, methylmalonic academia, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, skin cancer, spinal muscular atrophy, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, and Wilson disease.

[0728] Generation of lipid nanoparticle conjugates

[0729] Methods that enable conjugation of antibodies to LNPs can be used, exemplary conjugation methods can include, but are not limited to, covalent bonds, electrostatic interactions, and hydrophobic ("van der Waals") interactions. In one embodiment, the conjugation is reversible conjugation. In another embodiment, the conjugation is irreversible conjugation.

[0730] In some embodiments, conjugation comprises a covalent bond between at least one of a functionalized PEG lipid and a targeting moiety (e.g., an antibody). The term “functionalized PEG lipid” refers to a molecule comprising a lipid moiety and a PEG moiety that is activated by functionalization of the PEG lipid with a first coupling group. In one embodiment, the functionalized PEG lipid comprises a first coupling group that is capable of reacting with a second coupling group. In one embodiment, the functionalized PEG lipid is a functionalized PEG lipid (a PEGylated lipid). In one embodiment, the first coupling group is bound to the lipid moiety of the PEG lipid. In another embodiment, the first coupling group is bound to the PEG moiety of the PEGylated lipid. In one embodiment, the second functional group is covalently linked to the targeting domain.

[0731] The first coupling group and the second coupling group can be any functional group known to one of skill in the art that together form a covalent bond, for example, under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or the second coupling group is selected from the group consisting of a maleimide, an N-hydroxysuccinimide (NHS) ester, a carbodiimide, a hydrazide, a pentafluorophenyl (PFP) ester, a phosphine, a hydroxymethyl phosphine, a psoralen, an imidoester, a pyridyl disulfide, an isocyanate, a vinyl sulfone, an alpha-haloacetyl, an aryl azide, an acyl azide, an alkyl azide, a diazirine, a benzophenone, an epoxide, a carbonate, an anhydride, a sulfonyl chloride, a cyclooctyne, an aldehyde, and a thiol. In some embodiments, the first coupling group or the second coupling group is selected from the group consisting of a free amine (-NH2), a free thiol (-SH), a free hydroxyl (-OH), a carboxylate, a hydrazide, and an alkoxyamine. In some embodiments, the first coupling group is a thiol-reactive functional group, for example, a maleimide, a pyridyl disulfide, or a haloacetyl. In one embodiment, the first coupling group is a maleimide.

[0732] In one embodiment, the second coupling group is a thiol. The thiol can be attached to the targeting moiety (e.g., an antibody) using any method known to one of skill in the art. In one embodiment, the thiol is present on a free cysteine residue. In one embodiment, the thiol is exposed by reduction of a disulfide bond for the targeting moiety, for example, by reaction with 2-mercaptoethylamine. In one embodiment, the thiol is installed by a chemical reaction, such as the reaction between a free amine and 2-iminothiolane or N-succinimidyl S-acetylthioacetate (SATA).

[0733] In some embodiments, the PEG lipid and targeting moiety are functionalized with groups used in "click" chemistry. Bio-orthogonal click chemistry includes reactions between functional groups with 1,3-dipoles (e.g., azides, nitrile oxides, nitrone, isocyanides) and linkers (with olefin or alkyne electrophiles). Exemplary electrophiles include any strained cycloalkenes and cycloalkynes known to one of skill in the art, including but not limited to cyclooctyne, dibenzocyclooctyne, monofluorinated cyclooctyne, difluorinated cyclooctyne, and diaryl azacyclooctynone.

[0734] In some embodiments, a sortase-mediated approach can be employed to conjugate the anti-CD117 antibody to a lipid nanoparticle (LNP) to functionalize the antibody, i.e., to couple a functional group to the antibody, which can form a covalent bond to a moiety on the LNP through a chemical reaction.

[0735] In some embodiments, the conjugation of the antibody to the LNP is performed using the methods of the Examples.

[0736] Payloads for delivery

[0737] The lipid nanoparticles of the present disclosure can carry cargo to be delivered to target cells. In some embodiments, the cargo can be a therapeutic agent, e.g., a peptide, polypeptide, protein, nucleic acid, small molecule, etc., or can produce a therapeutic agent, e.g., an expression cassette for expressing a therapeutic agent. Alternatively, the cargo can be a diagnostic agent.

[0738] In some embodiments, the payload can comprise one or more components of a gene editing system. As used herein, a gene editing system refers to a composition comprising protein components and optional nucleic acid components required for making genetic edits at a target genetic locus by design. Any of the cargos disclosed herein can be loaded into the lipid nanoparticles (e.g., by encapsulation or attachment) by conventional methods.

[0739] In some embodiments, the prophylactic or therapeutic agent is selected from an RNA, DNA, antisense nucleic acid, aptamer, nuclease, immunostimulatory nucleic acid, or PNA component;

[0740] In some embodiments, the antisense nucleic acid is an antisense oligonucleic acid;

[0741] In some embodiments, the RNA comprises one or more of an mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, microRNA inhibitor, microRNA activator, or shRNA;

[0742] In some embodiments, the DNA comprises a plasmid;

[0743] In some embodiments, the nucleic acid component encodes a gene editing composition / system comprising a nuclease or a nucleic acid encoding a nuclease, and an RNA guide or a nucleic acid encoding an RNA guide;

[0744] In some embodiments, the nucleic acid component is mRNA;

[0745] In some embodiments, the nuclease comprises a nuclease mRNA;

[0746] In some embodiments, the nuclease is selected from Cas9, Cas12, Cas13, IscB, TnpB, IsrB, and homologs thereof.

[0747] In some embodiments, the biologically active substance comprises at least one mRNA encoding an antigen or fragment or epitope thereof.

[0748] In some embodiments, non-canonical nucleobases are contained in the nucleic acid molecule. In some embodiments, canonical nucleobases (e.g., adenine, guanine, uracil, thymine, and cytosine) in the nucleotides can be modified or replaced to provide one or more functional analogs of the nucleotides. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications, including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused or open rings, oxidation, and / or reduction.

[0749] In some embodiments, the non-canonical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil), 3-methyluracil (m3U), 5-methoxyuracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thiouracil (mcm5s2U), 5-aminomethyl-2-thiouracil (nm5s2U), 5-methylaminomethyluracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm55s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio- dihydrouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-uridine, 4- methoxy-2-thio-uridine, N1-methyl-uridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl) pseudouridine (acp3y), 5-(isopentenylaminomethyl)uracil (m5U), 5-(isopentenylaminomethyl)-2-thio-uracil (m5s2U), 5,2'-O-dimethyl-uridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoyloxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5-[3-(1-E- propenylamino)]uracil.

[0750] In some embodiments, the non-canonical nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-azacytosine, 6-azacytosine, pseudisocytidine, 3-methylcytosine (m3C), N4-acetylcytosine (ac4C), 5-formylcytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudisocytidine, pyrrolocytosine, pyrroloisocytidine, 2-thiocytosine (s2C), 2-thio-5-methylcytosine, 4-thiopseudisocytidine, 4-thio-l-methyl-pseudisocytidine, 4-thio-l-methyl-l-deaza-pseudisocytidine, l-methyl-l-deaza-pseudisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudisocytidine, 4-methoxy-l-methyl-pseudisocytidine, lysidine (k2C), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thiocytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine.

[0751] In some embodiments, the non-canonical nucleobase is a modified adenine, exemplary nucleobases and nucleosides that substitute for adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl-adenine (g6A), N6- threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl- adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2’-O-dimethyl-adenosine (m6Am), N6,N6,2’-O-trimethyl-adenosine (m62Am), 1,2’-O-dimethyl-adenosine (mlAm), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino- pentaoxaninyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6- hydroxymethyl-adenine.

[0752] In some embodiments, the non-canonical nucleobase is a modified guanine, exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1- methyl-inosine (ml), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), iso- wyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OH yW), undermodified hydroxywybutosine (OH yW*), 7-deaza-guanine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQl), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (mlG), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (mlGm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mlm), 1-thio-guanine, and O-6-methyl-guanine.

[0753] In some embodiments, the non-canonical nucleobases of the functional nucleotide analogs can independently be a purine, a pyrimidine, a purine analog, or a pyrimidine analog. Illustratively, the non-canonical nucleobases can be a modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, the non-canonical nucleobases can also include, for example, naturally occurring and synthetic derivatives of the bases, including pyrazolo[3,4-d]pyrimidine; 5-methylcytosine (5-me-C); 5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2- aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2- thiocytosine; 5-propynyluracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, especially 8-bromo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; deazaguanine, 7-deazaguanine, 3-deazaguanine; deazade- adenine, 7-deazadeadenine, 3-deazadeadenine; pyrazolo[3,4-d]pyrimidine; imidazo[l,5-a]l,3,5-triazinone; 9-deazapurine; imidazo[4,5-d]pyrazine; thiazolo[4,5-d]pyrimidine; pyrazin-2-one; 1,2,4-triazine; pyridazine; or 1,3,5-triazine.

[0754] Methods of treatment

[0755] Diseases or conditions

[0756] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject comprising delivering a therapeutic agent for treating the disease or condition. In some embodiments, the present disclosure provides a method for targeted delivery of a therapeutic agent for treating a disease or condition in a subject comprising delivering a therapeutic agent for treating the disease or condition, wherein the delivery vehicle comprises a moiety that targets CD117+ cells. Exemplarily, CD117+ cells include hematopoietic cells, mast cells, germ cells, stromal cells, tumor cells. In some embodiments, the subject is a human patient having a genetic disease associated with a target gene. Preferably, the genetic disease includes sickle cell disease, hemoglobinopathy, hemophilia, thalassemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington disease, inherited metabolic defects, Klinefelter syndrome, Marfan syndrome, methylmalonic academia, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, skin cancer, spinal muscular atrophy, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, and Wilson disease.

[0757] Pharmaceutical composition

[0758] Any of the anti-CD117 antibody conjugated LNPs disclosed herein can be mixed with one or more pharmaceutically acceptable carriers, diluents or excipients to form a pharmaceutical composition. The amount of carrier can be that amount effective to carry cargo, useful to achieve the intended therapeutic or diagnostic purpose, e.g., editing a genetic target site and / or treating an associated disease, disorder or condition in a patient in need thereof. As used herein and unless otherwise indicated, the term “pharmaceutically acceptable carrier, diluent or excipient” includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening, diluting, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsor approved by the United States Food and Drug Administration for use in humans or domestic animals.

[0759] In some embodiments, the LNPs can be administered in solution. In some embodiments, the LNPs can be formulated into suitable pharmaceutical compositions for administration by any desired route of administration. Exemplary routes of administration include, for example, but are not limited to, topical, oral, or parenteral routes. Depending on the intended mode of administration, the composition used will take the form of solid, semi-solid, or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, and the like, preferably unit dosage forms suitable for single administration of precise dosages. The pharmaceutical compositions can comprise the particles, pharmaceutically acceptable excipients, and optionally can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and the like. In some embodiments, the administration is topical. In some embodiments, the administration is systemic.

[0760] In some embodiments, injectable preparations for parenteral administration can be prepared as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like. Aqueous injection suspensions can also contain substances that increase viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Optionally, the suspension can also contain stabilizers. The parenteral preparation can be in unit dosage form or multi-dose sealed containers, such as ampules and vials, and can be stored in freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. In certain embodiments, parenteral administration includes intravenous administration.

[0761] In some embodiments, for oral administration, a pharmaceutically acceptable, non-toxic composition can be formed by incorporating any of the commonly used excipients, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions include solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations and the like. Suitable formulations for oral administration can consist of liquid solutions, such as effective amounts of the compounds dissolved in diluents, such as water, saline or orange juice; sachets, tablets and lozenges, each containing a predetermined amount of the active ingredient, as solids or granules; powders; suspensions; and suitable emulsions. Liquid formulations can include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol and polyethylene alcohols, and can also include pharmaceutically acceptable surfactants, suspending agents or emulsifying agents.

[0762] The application is further described in connection with the following specific examples. It is to be understood that these examples are merely by way of illustration and are not intended to limit the scope of the application. Methods of experimentation not specifically noted in the following examples were generally conducted in accordance with routine procedures as set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as otherwise described in the literature, or as otherwise described by the manufacturer of any particular kit or reagent. Percentages and parts are by weight unless otherwise specified.

[0763] Unless otherwise specified, the materials and reagents used in the examples are commercially available.

[0764] The application is further described in connection with the following experimental examples. These examples are provided for illustration purposes only and are not intended to be limiting unless otherwise specified. Thus, the present application should in no way be interpreted as being limited to the following examples, but rather should be interpreted to encompass any and all variations which become evident as a result of the teaching provided herein.

[0765] Example 1 Synthesis of ionizable lipid compound 11-({8-[(2-butyloctyl)oxy]-8- oxopentadecyl}amino)-10-(3-ethyl-6-oxopentadecyl)-11-oxo-undecanoic acid-2- butyloctyl ester

[0766] The synthesis method is as follows:

[0767] The synthesis steps are as follows:

[0768] Step one: Synthesis of compound 1-1:

[0769] Compound 1-2 (2.69 g, 13.44 mmol, 1.5 eq), EDCI (2.58 g, 13.44 mmol, 1.5 eq), DMAP (0.55 g, 4.48 mmol, 0.5 eq) and diisopropyl ethylamine (3.47 g, 26.88 mmol, 3.0 eq) were added into 25 ml of dichloromethane, after stirring at room temperature for 0.5 h, alcohol 1-1 (2.00 g, 8.96 mmol, 1.0 eq) was added, the reaction solution was stirred at room temperature for 15.5 h, then extracted with dichloromethane, the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatography to obtain compound 1-3 (2.10 g, yield 57.8%).

[0770] Step two: synthesis of compound 1:

[0771] Compound 1-4 (200.0 mg, 2.66 mmol, 1.0 eq), 1-3 (2.70 g, 6.65 mmol, 2.5 eq), potassium carbonate (1.10 g, 7.98 mmol, 3.0 eq), potassium iodide (0.22 g, 1.33 mmol, 0.5 eq) were added into 20 ml of acetonitrile. After stirring at 80 degrees Celsius for 16 h, the solvent was removed by reduced pressure distillation, diluted with 50 ml of water, extracted with 20 ml of ethyl acetate for three times, the combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and column chromatography to obtain compound 1 (1.25 g, yield 64.8%).

[0772] Example 2 Screening of anti-CD117 antibodies

[0773] 1. Anti-CD117 antibody design

[0774] A plurality of antibodies targeting CD117 were designed by introducing mutations through gene synthesis, and were named as Pro2, Pro3, Pro4, Pro5, Pro6, Pro7, Pro8, Pro9, Pro10, Pro11, Pro12, Pro13, Pro14, Pro15, Pro16, Pro17, Pro18, Pro19, Pro20, Pro21, Pro22, Pro23, and the antibody sequences (the bold and underlined part is the CDR region, which is determined according to the Kabat numbering system) are as follows:

[0775] Pro2

[0776] Heavy chain (H) amino acid sequence:

[0777] The horizontal line part is CDR1, CDR2, CDR3 in turn, and the 1-117th amino acid in SEQ ID NO. 1 is the heavy chain variable region (VH).

[0778] Light chain (L) amino acid sequence:

[0779] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 2 are the variable region of the light chain (VL).

[0780] Pro 1

[0781] Heavy chain (H) amino acid sequence:

[0782] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 3 are the variable region of the heavy chain (VH).

[0783] Light chain (L) amino acid sequence:

[0784] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 4 are the variable region of the light chain (VL).

[0785] Pro 2

[0786] Heavy chain (H) amino acid sequence:

[0787] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 5 are the variable region of the heavy chain (VH).

[0788] Light chain (L) amino acid sequence:

[0789] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 6 are the variable region of the light chain (VL).

[0790] Pro 3

[0791] Heavy chain (H) amino acid sequence:

[0792] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 7 are the variable region of the heavy chain (VH).

[0793] Light chain (L) amino acid sequence:

[0794] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 8 are the variable region of the light chain (VL).

[0795] Pro6

[0796] Heavy chain (H) amino acid sequence:

[0797] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 9 are the variable region of the heavy chain (VH).

[0798] Light chain (L) amino acid sequence:

[0799] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 10 are the variable region of the light chain (VL).

[0800] Pro7

[0801] Heavy chain (H) amino acid sequence:

[0802] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 119 amino acids of SEQ ID NO. 11 are the variable region of the heavy chain (VH).

[0803] Light chain (L) amino acid sequence:

[0804] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 12 are the variable region of the light chain (VL).

[0805] Pro8

[0806] Heavy chain (H) amino acid sequence:

[0807] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 13 are the variable region of the heavy chain (VH).

[0808] Light chain (L) amino acid sequence:

[0809] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 14 are the variable region of the light chain (VL).

[0810] Pro9

[0811] Heavy chain (H) amino acid sequence:

[0812] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 15 are the variable region of heavy chain (VH).

[0813] Light chain (L) amino acid sequence:

[0814] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 16 are the variable region of light chain (VL).

[0815] Pro10

[0816] Heavy chain (H) amino acid sequence:

[0817] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 17 are the variable region of heavy chain (VH).

[0818] Light chain (L) amino acid sequence:

[0819] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 18 are the variable region of light chain (VL).

[0820] Pro11

[0821] Heavy chain (H) amino acid sequence:

[0822] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 19 are the variable region of heavy chain (VH).

[0823] Light chain (L) amino acid sequence:

[0824] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 20 are the variable region of light chain (VL).

[0825] Pro12

[0826] Heavy chain (H) amino acid sequence:

[0827] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 41 are the heavy chain variable region (VH).

[0828] Light chain (L) amino acid sequence:

[0829] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 42 are the heavy chain variable region (VL).

[0830] pro14

[0831] Heavy chain (H) amino acid sequence:

[0832] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 43 are the heavy chain variable region (VH).

[0833] Light chain (L) amino acid sequence:

[0834] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 44 are the heavy chain variable region (VL).

[0835] pro15

[0836] Heavy chain (H) amino acid sequence:

[0837] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 45 are the heavy chain variable region (VH).

[0838] Light chain (L) amino acid sequence:

[0839] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 46 are the heavy chain variable region (VL).

[0840] pro16

[0841] Heavy chain (H) amino acid sequence:

[0842] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 47 are the heavy chain variable region (VH).

[0843] Light chain (L) amino acid sequence:

[0844] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 48 are the variable region of the light chain (VL).

[0845] pro17

[0846] Heavy chain (H) amino acid sequence:

[0847] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 49 are the variable region of the heavy chain (VH).

[0848] Light chain (L) amino acid sequence:

[0849] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 50 are the variable region of the light chain (VL).

[0850] pro18

[0851] Heavy chain (H) amino acid sequence:

[0852] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 51 are the variable region of the heavy chain (VH).

[0853] Light chain (L) amino acid sequence:

[0854] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 52 are the variable region of the light chain (VL).

[0855] pro19

[0856] Heavy chain (H) amino acid sequence:

[0857] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 53 are the variable region of the heavy chain (VH).

[0858] Light chain (L) amino acid sequence:

[0859] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 54 are the variable region of the light chain (VL).

[0860] pro20

[0861] Heavy chain (H) amino acid sequence:

[0862] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 55 are the variable region of the heavy chain (VH).

[0863] Light chain (L) amino acid sequence:

[0864] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 56 are the variable region of the light chain (VL).

[0865] pro21

[0866] Heavy chain (H) amino acid sequence:

[0867] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 57 are the variable region of the heavy chain (VH).

[0868] Light chain (L) amino acid sequence:

[0869] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 58 are the variable region of the light chain (VL).

[0870] pro22

[0871] Heavy chain (H) amino acid sequence:

[0872] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 117 amino acids of SEQ ID NO. 59 are the variable region of the heavy chain (VH).

[0873] Light chain (L) amino acid sequence:

[0874] The underlined portions are CDR1, CDR2, CDR3, respectively, and the first 111 amino acids of SEQ ID NO. 60 are the variable region of the light chain (VL).

[0875] pro23

[0876] Heavy chain (H) amino acid sequence:

[0877] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-117th amino acids in SEQ ID NO. 61 are the variable region of heavy chain (VH).

[0878] Light chain (L) amino acid sequence:

[0879] The underlined parts are CDR1, CDR2, CDR3, respectively, and the 1st-111th amino acids in SEQ ID NO. 62 are the variable region of light chain (VL).

[0880] The CDR sequences of each antibody molecule are shown in Table 5 below (determined according to the Kabat numbering system).

[0881] Table 5

[0882] The human codon-optimized coding sequences of each antibody molecule are shown in Table 6 below:

[0883] Table 6

[0884] 2. Construction and expression of anti-CD117 antibodies

[0885] Humanized antibodies were expressed and purified in CHO cells (purchased from ATCC). First, the DNA sequences of the heavy and light chains of the Pro2-11 antibody molecules were inserted into the same pCHO1.0 plasmid (Invitrogen), with the heavy chain upstream of the light chain, and the DNA sequences of the heavy and light chains of each antibody molecule are shown in Table 7 below.

[0886] Table 7

[0887] The constructed pCHO1.0 plasmids were then transfected into a CHO cell line (Life Technology) using PEI, and after culture, the cell supernatant was collected and purified using Protein A to make the purity of the antibodies ≥95%.

[0888] Example 3 Preparation of CD117-LNP targeting CD117 cells

[0889] 1. Dissolve ionizable lipid compound 11-({8-[(2-butyloctyl)oxy]-8-oxo- octanoyl}amino)-10-(3-ethyl-6-oxo-3,7-diazahexadecan-7-yl)-11-oxo- undecanoic acid-2-butyloctyl ester with DSPC, cholesterol, PEG-DMG and DSPE-PEG-Mal in anhydrous ethanol according to the molar ratio of 50:10:38:1.5:0.5. Mix the solution of the lipid carrier with the buffer of Luc mRNA (Nanjing Kings River, SC2325) at 1:3 (volume / volume) (where the mass ratio of total lipid and Luc mRNA is 40:1), and obtain nucleic acid lipid nanoparticles by microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12 ml / min. The obtained nucleic acid lipid nanoparticles are immediately diluted by 40 times the volume in 1x DPBS buffer. The diluted nucleic acid lipid nanoparticle solution is concentrated to the desired volume by a speed-vac centrifuge tube and stored at 4°C.

[0890] 2. The Pro2, Pro3, Pro4, Pro5, Pro6, Pro7, Pro8, Pro9, Pro10, Pro11, Pro12, Pro14, Pro15, Pro16, Pro17, Pro19, Pro20, Pro21, Pro22, Pro23 antibodies obtained in Example 2 are respectively conjugated with the LNP obtained in Step 1, as follows:

[0891] Add 5 equivalents of TCEP-containing PBS solution (5 mM) to the antibody solution, and after gently shaking at 37°C for 1 hour, add the obtained antibody solution dropwise to the LNP solution (10 equivalents relative to DSPE-PEG-Mal). After gently shaking the reaction solution at 25°C for 1 hour, load it onto a SEC column (Sepharose CL-4B) and elute it with PBS solution (1x). Collect the LNP-containing fraction and concentrate it by ultrafiltration to the desired concentration (1 mg / ml).

[0892] For ease of comparison, the applicant also designed a comparative group LNP, which was prepared as follows:

[0893] Ionizable liposome 11-({8-[(2-butyloctyl)oxy]-8-oxopropyl}amino)-10-(3-ethyl-6- oxopropyl-3,7-diazahexadecan-7-yl)-11-oxopropylundecanoic acid-2-butyloctyl ester / DSPC / Cholesterol / PEG-bound lipid PEG-DMG was used to prepare the lipid carrier at a molar ratio of 50:10:38.5:1.5. The ethanol solution of the lipid carrier was mixed with the buffer of Luc mRNA mRNA at a ratio of 1:3 (volume / volume) (where the mass ratio of total lipid and mRNA was 40:1), and nucleic acid lipid nanoparticles, i.e., LNP controls, were obtained by a microfluidic nanomedicine manufacturing system (NanoAssemblr Ignite, Canada) at a flow rate of 12 mL / min.

[0894] Example 4 CD117-LNP in vivo cell-targeted delivery

[0895] 1. Take the CD34+ cells (Beijing Nuowei Biological, 903410) from the liquid nitrogen tank and place them in a 37°C water bath. After the cells are thawed, transfer them to a biological safety cabinet. Prepare a 15ml centrifuge tube, add 5ml X vivo 15 medium (Gibco), then transfer all the thawed cells to the 15ml centrifuge tube, centrifuge at 400g for 8 minutes at room temperature. After centrifugation, remove the supernatant, then add complete medium (purchased from GIBCO company) to resuspend the cells and evenly distribute 100,000 cells per well in a 96-well plate, and incubate in a 37°C cell incubator overnight.

[0896] 2. Add each antibody-LNP prepared in Example 2 and LNP control to the prepared CD34+ cells at a dose of 1ug Luc mRNA / well, then transfer to a 37°C cell incubator for further culture for 16h.

[0897] 3. After 16 hours of transfection, add 100ul X vivo 15 medium to each LNP-transfected well, mix well by blowing, then transfer to an 8-tube, and draw 10ul of cell suspension from the 8-tube, and use a cell counter to count. The other cells were centrifuged at 400g for 8 minutes, the supernatant was removed, and 100ul DPBS was added to resuspend the cells, which were again centrifuged at 400g for 8 minutes. After removing the supernatant, 100ul lysis buffer (Bi Yun Tian, RG009M) was added and mixed well by blowing. Then take 40ul of cell lysate to the enzyme-labeled plate, and add 40ul of luciferase detection reagent (Bi Yun Tian, RG009M) to each sample in the enzyme-labeled plate, and use an enzyme-labeled instrument to detect luciferase. The detection results are shown in Figures 1 and 2.

[0898] As shown in FIG. 1, the CD34+ cells treated with Pro2-LNP, Pro5-LNP, Pro3-LNP, Pro8-LNP, and Pro11-LNP have a significant level of luciferase activity, and the CD34+ cells treated with other antibody-conjugated LNPs and the LNP control (LNP control in the figure) have no detectable luciferase activity, which indicates that the LNPs conjugated with Pro2, Pro5, Pro3, Pro8, and Pro11 significantly improve the targeted delivery effect on CD117+ cells, and in particular, Pro2-LNP and Pro5-LNP show the best targeted delivery effect and have potential application prospects.

[0899] FIG. 2 shows that the CD34+ cells treated with Pro14-LNP, Pro15-LNP, Pro16-LNP, Pro19-LNP, Pro20-LNP, Pro21-LNP, Pro22-LNP, and Pro23-LNP have a significant level of luciferase activity, which indicates that the LNPs conjugated with Pro14, Pro15, Pro16, Pro19, Pro20, Pro21, Pro22, and Pro23 significantly improve the targeted delivery effect on CD117+ cells, and in particular, Pro14, Pro15, Pro19, Pro21, and Pro23 show relatively better targeted delivery effect.

[0900] Example 4: LNP delivery of other ionizable lipids

[0901] The LNP formulations encapsulating Luc mRNA were prepared by replacing the ionizable lipids with other ionizable lipids (03-1, 03-4, 03-7, 03-9, 03-10, 03-11, 03-12, 03-13, and 03-14) according to the method described in Examples 2-3, and the targeted delivery effect was evaluated, as shown in Table 8.

[0902] Table 8: LNP delivery effect using other ionizable lipids

[0903] As shown in Table 8, the LNP formulations using the above ionizable lipids all show significant delivery effect, and the LNP control has no obvious effect.

[0904] It is to be understood that the technical solutions of the present application are described with specific examples, but those skilled in the art can understand that the present application should not be limited thereto. The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical application, or technical improvement in the market, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

[0905] Sequence information:

Claims

An antibody or antigen-binding fragment thereof that binds CD117, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein: H-CDR1 comprises GYTX1X2SX3X4MH (SEQ ID NO. 89), wherein X1 is selected from L, F; X2 is selected from T, S; X3 is selected from Y, H; X4 is selected from Q, W, N; H-CDR2 comprises VIYX5X6X7GX8TSYX9X 10 KX 11 K (SEQ ID NO. 90), wherein X5 is selected from S, N; X6 is selected from G, A, D, Y; X7 is selected from N, E, R; X8 is selected from D, E; X9 is selected from N, Q; X 10 selected from Q, N; X 11 selected from F, Y; H-CDR3 comprises ERDX 12 RFGX 13 (SEQ ID NO. 91), wherein X 12 is selected from T, E; X 13 is selected from N, F; the light chain variable region comprises light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3; wherein, L-CDR1 comprises RX a X b EX c X d DX e YX f X g X h FX i H (SEQ ID NO. 92), wherein X a is selected from A, V; X b is selected from S, T; X c is selected from S, N; X d is selected from V, A; X e is selected from I, L; X f is selected from G, Y; X g is selected from N, E; X h is selected from S, K; X i is selected from M, I; L-CDR2 comprises LX j SX k LEX l (SEQ ID NO. 93), wherein X j is selected from A, V; X k is selected from N, R; X l is selected from S, T; L-CDR3 comprises QQNX m X n X o PX p X q (SEQ ID NO. 94), wherein X m is selected from N, E; X n is selected from E, F; X o is selected from D, Y; X p is selected from Y, R; X q is selected from T, S. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3, wherein, H-CDR1 is selected from: (a) GYTLTSYQMH (SEQ ID NO. 63); (b) GYTLSSHWMH (SEQ ID NO. 64); (c) GYTFTSYNMH (SEQ ID NO. 65); H-CDR2 is selected from: (d) VIYSGNGDTSYNQKFK (SEQ ID NO. 66); (e) VIYSANGETSYNNKFK (SEQ ID NO. 68); (f) VIYNGNGDTSYQQKYK (SEQ ID NO. 69); (g) VIYNDEGDTSYQQKYK (SEQ ID NO. 70); (h) VIYNYRGDTSYQQKYK (SEQ ID NO. 71); H-CDR3 is selected from: (i) ERDTRFGN (SEQ ID NO. 72); (j) ERDERFGF (SEQ ID NO. 76); wherein any one of the above amino acid sequences further comprises a derivative sequence optionally with addition, deletion, modification and / or substitution of at least one amino acid, and which is capable of retaining CD117 binding affinity. The antibody or antigen-binding fragment thereof of claim 1 or 2, the light chain variable region comprises light chain complementarity determining regions L-CDR1, L-CDR2, L-CDR3; wherein, L-CDR1 is selected from: (k) RASESVDIYGNSFMH (SEQ ID NO. 77); (l) RASENVDLYGNSFIH (SEQ ID NO. 78); (m) RVTENADLYGESFIH (SEQ ID NO. 80); (n) RVTENADLYYEKFIH (SEQ ID NO. 81); L-CDR2 is selected from: (o) LASNLES (SEQ ID NO. 82); (p) LVSNLET (SEQ ID NO. 83); (q) LVSRLET (SEQ ID NO. 84); L-CDR3 is selected from: (r) QQNNEDPYT (SEQ ID NO. 85); (s) QQNNFYPRT (SEQ ID NO. 87); (t) QQNEEDPYS (SEQ ID NO. 88). The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence at positions 1-117 of any one of the amino acid sequences of SEQ ID NO. 1, 3, 7, 13, 19, 41, 43, 45, 47, 51, 53, 55, 57, 59, or 61, but an anti-CD117 antibody comprising the sequence retains the ability to bind CD117; The light chain variable region comprises a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence at positions 1-111 of any one of the amino acid sequences of SEQ ID NO. 2, 4, 8, 14, 20, 42, 44, 46, 48, 52, 54, 56, 58, 60, or 62, but an anti-CD117 antibody comprising the sequence retains the ability to bind CD117. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody heavy chain variable region further comprises: a heavy chain FR region of a human IgGl, IgG2, IgG3, or IgG4, or a variant thereof, preferably, a heavy chain FR region of a human IgGl, IgG2, or IgG4, more preferably, a heavy chain FR region of a human IgGl or IgG2. The antibody or antigen-binding fragment thereof of any of the preceding claims, characterized in that The antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof of any one of the preceding claims, which comprises a conjugation functional group attached to a single domain antibody fragment. The antibody or antigen-binding fragment thereof of any of the preceding claims, characterized in that The functional group is an azido group, a dibenzocyclooctyne group (DBCO), biotin, streptavidin, or a thiol group. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the antibody or antigen-binding fragment thereof that binds CD117 of any one of claims 1-8. An expression vector, characterized in that, The expression vector contains the nucleotide molecule of claim 9. A host cell, characterized in that, The host cell contains the expression vector of claim 10. A method of preparing an antibody or antigen binding fragment thereof that binds CD117 as defined in any one of claims 1-8, characterized in that, The method comprises the steps of: a) culturing the host cell of claim 11 under expression conditions, thereby expressing the antibody or antigen-binding fragment thereof that binds CD117; b) isolating and purifying the antibody or antigen-binding fragment thereof that binds CD117 of a). A lipid nanoparticle conjugate characterized in that, comprises (1) a lipid nanoparticle (LNP), and (2) an antibody or antigen-binding fragment thereof that binds CD117, wherein the antibody or antigen-binding fragment thereof is conjugated on the surface of the LNP, the antibody or antigen-binding fragment thereof that binds CD117 is of any one of claims 1-8. The lipid nanoparticle conjugate of claim 13, wherein The antibody or antigen-binding fragment thereof is conjugated to a polyethylene glycol (PEG) lipid comprised in the LNP. The lipid nanoparticle conjugate of any one of claims 13-14, wherein The antibody or antigen-binding fragment thereof is covalently conjugated to the LNP. The lipid nanoparticle conjugate of claim 13, further comprising a biologically active substance encapsulated by or attached to the LNP; Preferably, the biologically active substance comprises a therapeutic agent, a prophylactic agent, or a diagnostic agent. The lipid nanoparticle conjugate of claim 16, wherein The biologically active substance comprises a prophylactic or therapeutic agent, preferably the prophylactic or therapeutic agent is selected from an RNA, a DNA, an antisense nucleic acid, an aptamer, a nuclease, an immunostimulatory nucleic acid, or a PNA component; Preferably, the antisense nucleic acid is an antisense oligonucleic acid; Preferably, the RNA comprises one or more of an mRNA, an rRNA, a circRNA, an siRNA, an saRNA, a tRNA, a snRNA, an antagomir, a microRNA inhibitor, a microRNA activator, or an shRNA; Preferably, the DNA comprises a plasmid; Preferably, the nucleic acid component encodes a gene editing composition / system comprising a nuclease or a nucleic acid encoding a nuclease, and an RNA guide or a nucleic acid encoding an RNA guide; Preferably, the nucleic acid component is an mRNA; Preferably, the nuclease comprises a nuclease mRNA; Preferably, the nuclease is selected from Cas9, Cas12, Cas13, IscB, TnpB, IsrB, and homologs thereof; Preferably, the biologically active substance comprises at least one mRNA encoding an antigen or a fragment or epitope thereof. The lipid nanoparticle conjugate of claim 13, wherein, The lipid nanoparticle (LNP) comprises: an ionizable lipid, a neutral lipid, a sterol, a PEG lipid, a functionalized PEG lipid; Preferably, the ionizable lipid is a compound according to any of the formulae of 01-I, 02-I, 02-I-1, 02-I-2, 02-I-3, 02-I-4, 02-I-5, 02-I-6, 02-I-7, 02-I-8, 02-I-9, 02-I-10, 02-I-11, 02-I-12, 02-I-13, 02-I-14, 03-I, 03-I-1, 03-I-2, 04-I, 04-I-1, 04-I-2, 04-I-1-3, 04-I-4, 04-II, 04-II-1, 04-II-2, 04-II-3, and subformulae thereof, or wherein the ionizable lipid is selected from the compounds listed in any of Tables 1 to 4; Preferably, the molar ratio of ionizable lipid, neutral lipid, sterol, PEG lipid, functionalized PEG lipid is (20-65):(5-25):(25-55):(0.01-5):(0.1-15); Preferably, the molar ratio between ionizable lipid, neutral lipid, structural lipid, functionalized PEG lipid, and non-functional PEG lipid is (35-50):(10-16):(38.5-46.5):(0.15-2.2):(0.3-2.2). A method for delivering a biologically active substance to a cell, characterized in that, comprises: an ionizable lipid, a neutral lipid, a sterol, a PEG lipid, a functionalized PEG lipid; contacting the lipid nanoparticle conjugate of any one of claims 13-18 with a cell expressing CD117 to deliver the biologically active agent contained in the lipid nanoparticle conjugate to the cell, preferably, the contacting occurs in vivo, ex vivo, or in vitro. The method of claim 19, wherein the cell comprises a hematopoietic stem cell, a mast cell, a germ cell, a stromal cell, a tumor cell. The method of claim 19, wherein the contacting step is by administering the lipid nanoparticle conjugate to a subject in need thereof to deliver the biologically active agent to a cell of the subject. A method of editing an intracellular gene expressed by a CD117 cell, characterized by, Comprising: contacting the lipid nanoparticle conjugate of any one of claims 13-18 with the cell to allow delivery of the biologically active agent contained in the lipid nanoparticle conjugate to the cell, the biologically active agent being a gene editing composition / system that edits a target gene in the cell, preferably, the method is an in vivo, ex vivo, or in vitro editing method. The method of claim 22, wherein the cell comprises a hematopoietic cell, a mast cell, a germ cell, a stromal cell, a tumor cell. The method of claim 22, wherein the contacting step is by administering the lipid nanoparticle conjugate to a subject in need thereof to deliver the biologically active agent to a CD117+ cell of the subject. The method of claim 24, wherein the subject is a human patient having a genetic disease associated with the target gene; Preferably, the genetic disease comprises sickle cell disease, a hemoglobinopathy, hemophilia, thalassemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, inherited metabolic defects, Klinefelter syndrome, Marfan syndrome, methylmalonic academia, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, skin cancer, spinal muscular atrophy, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, and Wilson disease. A method of preparing a lipid nanoparticle conjugate, characterized in that, The method comprises contacting the antibody or antigen-binding fragment thereof of any one of claims 1-8 with a LNP to allow conjugation of the antibody or antigen-binding fragment thereof to the surface of the LNP, thereby producing the lipid nanoparticle conjugate. The method of claim 26, wherein the antibody is conjugated to a PEG moiety of a PEG-lipid. The method of claim 26, the conjugate is prepared by a method comprising: (1) modifying an antibody heavy chain coding sequence to introduce a covalently conjugatable amino acid residue at or near the C-terminus of the heavy chain constant region; (2) preparing a nanoparticle comprising one or more PEG groups; and conjugating the nanoparticle to the antibody via a site-specific linker, wherein the amino acid residue located at or near the C-terminus of the heavy chain constant region is covalently bonded to one of the one or more PEG groups of the nanoparticle. The method of claim 28 wherein The conjugatable amino acid residue comprises a cysteine residue, a lysine residue, or a glutamine residue. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8 or the conjugate of any one of claims 13-18 in the manufacture of a medicament for the treatment of a disease, Preferably, the disease comprises a genetic disease or disorder, preferably comprises sickle cell disease, hemoglobinopathy, hemophilia, thalassemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, chronic myeloid leukemia or chronic lymphocytic leukemia, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, inherited metabolic defects, Klinefelter syndrome, Marfan syndrome, methylmalonic academia, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, skin cancer, spinal muscular atrophy, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, and Wilson disease. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds CD117 of any one of claims 1-8 and / or the conjugate of any one of claims 13-18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof that binds CD117 of any one of claims 1-8 and / or the conjugate of any one of claims 13-18. A method of treating or ameliorating a disease or disorder, characterized by, The method comprises administering to the subject an effective amount of the antibody or antigen-binding fragment thereof that binds CD117 of any one of claims 1-8, the conjugate of any one of claims 13-18, or the pharmaceutical composition of claim 31 ; or administering to the cells of the subject the antibody or antigen-binding fragment thereof that binds CD117 of any one of claims 1-8, the conjugate of any one of claims 13-18, or the pharmaceutical composition of claim 31. The method of claim 32, wherein the cells comprise stem cells, preferably the stem cells are selected from the group consisting of somatic stem cells, hematopoietic stem cells, and / or mesenchymal stem cells. The method of claim 32, wherein the conjugate is administered in a manner selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and / or intramuscular.

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