Ionizable cationic lipids and lipid nanoparticles, and methods for their synthesis and use

Abstract

Provided are ionizable cationic lipids and lipid nanoparticles for delivering nucleic acids to cells (e.g., immune cells), and methods of making and using such lipids and targeted lipid nanoparticles.

Description

【Technical Field】 【0001】 Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Application No. 63 / 350,404, filed on June 8, 2022, the disclosure of which is hereby incorporated by reference in its entirety. 【0002】 Reference to Electronic Sequence Listing The contents of the electronic sequence listing (183952034240seqlist.xml; size: 168,364 bytes; and creation date: June 1, 2023) are hereby incorporated by reference in their entirety. 【0003】 The present invention provides ionizable cationic lipids and lipid nanoparticles for delivering nucleic acids to immune cells, and methods of making and using such lipids and targeted lipid nanoparticles. 【Background Art】 【0004】 In recent years, several treatment modalities have been developed that involve delivering one or more nucleic acids. Examples of treatment modalities include, for example, gene therapy in which a gene of interest in the form of deoxyribonucleic acid (DNA) is introduced into a cell and then expressed to produce a gene product, such as a protein, to treat a disorder caused by or associated with a deficiency or absence of the gene product. In this approach, the gene is transcribed into messenger ribonucleic acid (mRNA), and then the mRNA is translated to produce the gene product. In another approach, instead of the gene of interest, mRNA can be delivered to the cell. The resulting expression product can correct a deficiency or absence of a specific protein in a subject (e.g., a protein deficiency that occurs in certain forms of cystic fibrosis or lysosomal storage disorders), or can be used to regulate cell function, for example, to initiate an immune response in a subject or to reprogram immune cells to regulate the immune response when not (e.g., as a therapeutic agent for treating cancer or as a prophylactic vaccine for preventing or minimizing the risk or severity of a microbial or viral infection). 【0005】 However, the delivery of mRNA to cells for translation within the cell has been difficult due to various factors such as nuclease degradation of the mRNA before entry into the cell and then after introduction into the cell but before translation. 【0006】 RNA can be delivered to a subject using different delivery vehicles, for example, based on cationic polymers or lipids that form nanoparticles with the RNA. The nanoparticles are intended to protect the RNA from degradation, enable delivery of the RNA to the target site, and facilitate cellular uptake and processing by the target cells. For delivery efficiency, in addition to the molecular composition, parameters such as particle size, charge, or grafting with molecular moieties such as polyethylene glycol (PEG) or ligands play a role. Grafting with PEG is thought to reduce serum interactions, increase serum stability, and increase circulation time, which can be useful for certain targeting approaches. 【0007】 Compared with DNA delivery technologies used in specific gene therapies, mRNA-based gene therapies have many excellent features, such as ease of manipulation, rapid and transient expression, and adaptive convertibility without mutagenesis. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION 【0008】 However, the delivery of therapeutic RNA into cells is difficult from the viewpoints of the relative instability of RNA and its low cell permeability. Therefore, there is a need to develop methods and compositions for promoting the delivery of RNA, such as mRNA, into cells. MEANS FOR SOLVING THE PROBLEMS 【0009】 The present invention provides an ionizable cationic lipid, a lipid-immune cell targeting group conjugate, and a lipid nanoparticle composition containing such an ionizable cationic lipid and / or a lipid immune cell (e.g., T cell) targeting group conjugate, a medical kit containing such a lipid and / or conjugate, and methods for producing and using such a lipid and conjugate. 【0010】 The lipid nanoparticle compositions provided herein may further comprise an RNA, such as a nucleic acid such as messenger RNA or mRNA. The lipid nanoparticle compositions can be used for the delivery of mRNA to cells (e.g., immune cells such as T cells) in a subject. Messenger RNA-based gene therapy requires efficient delivery of mRNA to circulating cells in the plasma (e.g., immune cells such as T cells or NK cells) or cells in a given tissue. The main challenges associated with efficient mRNA delivery to achieve robust levels of protein expression include: (a) the ability to protect the mRNA payload from common serum nucleases upon administration to a subject; (b) the ability to specifically target mRNA delivery to a target cell (e.g., T cell) population, thereby maximizing protein expression in the target cell population; and (c) the ability to deliver the mRNA payload to the cytosolic compartment of the cell (e.g., T cell) to the maximum extent for translation into cytosolic proteins. 【0011】 The present invention provides an ionizable cationic lipid for producing a lipid nanoparticle composition that facilitates the delivery of a payload (e.g., a nucleic acid such as DNA or RNA such as mRNA) disposed therein to a cell, such as a mammalian cell, such as an immune cell. The lipid enables intracellular delivery of a nucleic acid, such as mRNA, to the cytosolic compartment of the target cell type and is designed to rapidly degrade into non-toxic components. These complex functionalities are achieved by the interaction between the chemical nature and geometric shape of the ionizable lipid head group, the hydrophobic "acyl tail" group, and the linker connecting the head group and the acyl tail group in the ionizable cationic lipid. 【0012】 In one aspect, the present invention provides a compound of formula (I): 【Chemical formula】 or a salt thereof. In some embodiments, R 1 , R 2 , and R 3 are each independently a bond or C 1~3 alkylene. In some embodiments, R1A , R 2A , and R 3A are each independently a bond or C 1~10 alkylene. In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 are each independently H, C 1~20 alkyl, C 1~20 alkenyl, -(CH2) 0~10 C(O)OR a1 , or -(CH2) 0~10 OC(O)R a2 . In some embodiments, R a1 and R a2 are each independently C 1~20 alkyl or C 1~20 alkenyl. In some embodiments, R 3B is 【Chemical formula】 . In some embodiments, R 3B1 is C 1~6 alkylene. In some embodiments, R 3B2 and R 3B3 are each independently H or C 1~6 alkyl. In some embodiments, R 3B2 and R 3B3 are each independently H, unsubstituted C 1~6 alkyl, or C 1~6 alkyl substituted with one or more substituents independently selected from the group consisting of -OH and -O-(C 1~6 alkyl). 【0013】 Also provided herein are lipid nanoparticles (LNPs) comprising a lipid blend comprising an ionizable cationic lipid and / or a lipid-immunocyte targeting group conjugate (e.g., a lipid-T cell targeting group conjugate) provided herein. 【0014】 In another aspect, provided herein is a method of delivering a nucleic acid to an immune cell (e.g., a T cell), the method comprising exposing an immune cell to an LNP described herein that contains a nucleic acid under conditions that allow the nucleic acid to enter the immune cell. 【0015】 In another aspect, provided herein is a method of delivering a nucleic acid to an immune cell (e.g., a T cell) of a subject in need thereof, the method comprising administering to the subject a composition comprising an LNP described herein that contains a nucleic acid, thereby delivering the nucleic acid to the immune cell. 【0016】 In another aspect, provided herein is a method of targeting delivery of a nucleic acid (e.g., mRNA) to an immune cell (e.g., a T cell) in a subject, the method comprising administering to the subject an LNP described herein that contains a nucleic acid to facilitate targeted delivery of the nucleic acid to the immune cell. 【0017】 In one aspect, provided herein is a lipid nanoparticle (LNP) comprising a lipid blend for targeted delivery of a nucleic acid to an immune cell. In some embodiments, the lipid blend comprises a lipid-immune cell targeting group conjugate comprising a compound of formula (II): [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the lipid blend comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid comprises an ionizable cationic lipid described herein. In some embodiments, the LNP comprises a nucleic acid disposed therein. 【0018】 In some embodiments, the immune cell targeting moiety comprises an antibody that binds to a T cell antigen. In some embodiments, the T cell antigen is CD3, CD4, CD7, or CD8, or a combination thereof (e.g., both CD3 and CD8, both CD4 and CD8, or both CD7 and CD8). In some embodiments, the immune cell targeting moiety comprises an antibody that binds to a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD7, CD8, or CD56, or a combination thereof (e.g., both CD7 and CD8). In some embodiments, the antibody is a human antibody or a humanized antibody. 【0019】 In some embodiments, the immune cell targeting moiety is covalently linked to a lipid in a lipid blend via a polyethylene glycol (PEG)-containing linker. In some embodiments, the lipid covalently linked to the immune cell targeting moiety via the PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl glycerol (DPG), or ceramide. In some embodiments, the PEG is PEG 2000. 【0020】 In some embodiments, the lipid-immunocyte targeting group conjugate is present in the lipid blend in the range of 0.002 to 0.2 mole percent. In some embodiments, the lipid blend comprises one or more structural lipids (e.g., sterol), neutral phospholipids, and free PEG-lipids. In some embodiments, the ionizable cationic lipid is present in the lipid blend in the range of 40 to 60 mole percent. In some embodiments, the sterol is present in the lipid blend in the range of 30 to 50 mole percent. In some embodiments, the sterol is present in the lipid blend in the range of 20 to 70 mole percent. In some embodiments, the sterol is cholesterol. 【0021】 In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM). In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1 to 15 mole percent, such as about 5 to 15 mole percent. 【0022】 In some embodiments, the free PEG-lipid is selected from the group consisting of PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DSG), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof. In some embodiments, the free PEG-lipid comprises a diacylphosphatidylethanolamine containing dipalmitoyl (C16) chains or distearoyl (C18) chains. In some embodiments, the free PEG-lipid is a mixture of two or more unique free PEG-lipids. In some embodiments, the free PEG-lipid is present in the lipid blend in the range of 1 to 4 mole percent, such as about 1 to 2 mole percent, or about 2 to 4 mole percent, or about 1.5 mole percent. In some embodiments, the free PEG-lipid comprises a lipid that is the same as or different from the lipid in the lipid-immunocyte targeting group conjugate. 【0023】 In some embodiments, the LNP has an average diameter in the range of 50 to 200 nm. In some embodiments, the LNP has an average diameter of about 100 nm. In some embodiments, the LNP has a polydispersity index in the range of 0.05 to 1. In some embodiments, the LNP has a zeta potential of about +5 mV to about +50 mV at pH 5, such as about +10 mV to about +30 mV at pH 5. In some embodiments, the LNP has a zeta potential of about -10 mV to about +10 mV at pH 7.4. 【0024】 In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, siRNA, gRNA (guide RNA), circRNA (circular RNA), ribozyme, decoy RNA, or microRNA. In some embodiments, the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine. In some embodiments, the mRNA encodes a polypeptide capable of regulating the immune response in immune cells. In some embodiments, the mRNA encodes a polypeptide capable of reprogramming immune cells. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or chimeric antigen receptor (CAR). In some embodiments, the CAR is TTR-023 anti-CD20 (Leu-16). In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the mRNA encoding the CAR comprises 25 polynucleotide sequences. TTR-023 anti-CD20 (Leu-16) CAR sequence (including leader) (SEQ ID NO: 24): 【Chemical formula】 Corresponding nucleic acid sequence (SEQ ID NO: 25): 【Chemical formula】 【0025】 In some embodiments, the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain such as a nanobody. In some embodiments, the immune cell targeting moiety comprises a Fab, F(ab’)2, Fab’-SH, Fv, or scFv fragment. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to knock out one or more native interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment with a C233S substitution according to Kabat numbering, and / or a light chain fragment with a C214S substitution according to Kabat numbering. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to introduce one or more embedded interchain disulfide bonds. For example, in some embodiments, the Fab antibody comprises a heavy chain fragment with an F174C substitution according to Kabat numbering, and / or a light chain fragment with an S176C substitution according to Kabat numbering. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to knock out one or more native interchain disulfide bonds and introduce one or more embedded interchain disulfide bonds. In some embodiments, the immune cell targeting moiety comprises a Fab comprising a cysteine at the C-terminus of the heavy or light chain fragment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and the immune cell targeting moiety further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain by an amino acid linker.In some embodiments, the Fab antibody is a DS Fab (Fab having wild-type (native) inter-chain disulfide bonds), a NoDS Fab (Fab in which native disulfide bonds are knocked out, e.g., a Fab having a C233S substitution in the heavy chain and / or a C214S substitution in the light chain according to the Kabat numbering), a bDS Fab (Fab having no native disulfide bonds and having non-native inter-chain embedded disulfide bonds introduced, e.g., a Fab having F174C and C233S in the heavy chain and / or S176C and C214S substitutions in the light chain according to the Kabat numbering), or a bDS Fab-ScFv (bDS Fab linked to ScFv via a linker such as (G4S)x), as demonstrated in FIG. 31. 【0026】 In some embodiments, the immune cell targeting moiety comprises an immunoglobulin single variable domain such as a nanobody. In some embodiments, the immunoglobulin single variable domain comprises a cysteine at the C-terminus. In some embodiments, the nanobody further comprises a spacer comprising one or more amino acids between the V HH domain and the C-terminal cysteine. In some embodiments, the immune cell targeting moiety comprises two or more V HH domains. In some embodiments, the two or more V HH domains are linked by an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first V HH domain linked to the antibody CH1 domain and a second V HH domain linked to the antibody light chain constant domain, and the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds. In some embodiments, the immune cell targeting moiety is a V HHcomprising a domain, wherein the antibody CH1 domain is linked to the antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions according to Kabat numbering, and / or the light chain constant domain comprises S176C and C214S substitutions. In some embodiments, the antibody is ScFv, V HH , 2xV HH , V HH -CH1 / null Vk, or V HH 1-CH1 / V HH -2-Nb bDS as demonstrated in Figure 31. 【0027】 In some embodiments, the immunocyte targeting moiety comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the immunocyte targeting moiety comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody is an antibody described in the examples. 【0028】 In some embodiments, the immunocyte targeting moiety is (a) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3, (b) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 4 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 5, (c) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 7, (d) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 8 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 9, (e) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 10 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 11, (f) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 12 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 13, (g) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 14 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 15, (h) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 16 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 17, (i) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 18 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 19, (j) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 20 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 21, or (k) A Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 22 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 23. 【0029】 In some embodiments, the immunocyte targeting moiety comprises a Fab, F(ab’)2, Fab’-SH, Fv, or scFv fragment. In some embodiments, the immunocyte targeting moiety comprises a Fab engineered to knockout the native interchain disulfide bond at the C-terminus. In some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution according to Kabat numbering. In some embodiments, the immunocyte targeting moiety comprises a Fab having a non-native interchain disulfide bond (e.g., an engineered buried interchain disulfide bond). In some embodiments, the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment according to Kabat numbering. In some embodiments, the immunocyte targeting moiety comprises a Fab comprising a cysteine at the C-terminus of the heavy or light chain fragment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine. 【0030】 In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain comprises a cysteine at the C-terminus. In some embodiments, the immunoglobulin single variable domain comprises a VHH domain and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine. In some embodiments, the immune cell targeting group comprises two or more VHH domains. In some embodiments, the two or more V HH domains are linked by an amino acid linker. In some embodiments, the immune cell targeting group comprises a first V HH domain linked to the antibody CH1 domain and a second V HH domain linked to the antibody light chain constant domain. In some embodiments, the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds. In some embodiments, the immune cell targeting group comprises a VHH domain linked to the antibody CH1 domain. In some embodiments, the antibody CH1 domain is linked to the antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions according to Kabat numbering, and the light chain constant domain comprises S176C and C214S substitutions. 【0031】 In some embodiments, the immune cell targeting group comprises a Fab comprising (a) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3; or (b) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 7. 【0032】 In another aspect, provided herein are methods for targeting the delivery of nucleic acids to immune cells of interest. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising a compound of formula (II): [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. 【0033】 In some embodiments, one aspect of the disclosure relates to an LNP disclosed herein or a pharmaceutical composition containing the same for use in a method for targeting the delivery of nucleic acids to immune cells of a subject. Such methods can be for the treatment of a disease or disorder disclosed below. In some embodiments, the methods disclosed herein can include contacting the immune cells of the subject with lipid nanoparticles (LNPs) in vitro or ex vivo. In some embodiments, the LNP is the LNP described herein in the present disclosure. 【0034】 In some embodiments, methods are provided for expressing a polypeptide of interest in target immune cells of interest. In some embodiments, the method comprises contacting immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure of formula (II): [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid encoding a polypeptide. In some embodiments, one aspect of the disclosure relates to an LNP disclosed herein or a pharmaceutical composition containing the same for use in a method for expressing a polypeptide of interest in target immune cells of interest. Such methods can be for the treatment of diseases or disorders disclosed below. In some embodiments, the methods disclosed herein can comprise contacting immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo. 【0035】 In some aspects, methods are provided for modulating the cellular function of a target immune cell. In some embodiments, the method includes administering lipid nanoparticles (LNPs) to a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure of formula (II): [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid encoding a polypeptide for modulating the cellular function of an immune cell. In some embodiments, one aspect of the disclosure relates to an LNP disclosed herein or a pharmaceutical composition containing the same for use in a method for modulating the cellular function of a target immune cell in a subject. Such methods can be for the treatment of a disease or disorder disclosed below. In some embodiments, the methods disclosed herein can include contacting immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo. 【0036】 In some embodiments, modulating the cellular function includes reprogramming the immune cell to initiate an immune response. In some embodiments, modulating the cellular function includes modulating the antigen specificity of the immune cell. 【0037】 In some embodiments, methods are provided for treating, ameliorating, or preventing symptoms of a disorder or disease in a subject in need thereof. For example, in any of the embodiments described herein regarding methods of treating, ameliorating, and / or preventing symptoms of a disorder or disease by administration of the LNPs of the present invention, the disclosure is also intended to be construed as providing, for example, LNPs for use in such methods of treating, ameliorating, and / or preventing symptoms of a disorder or disease. In some embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP) for delivering a nucleic acid to immune cells of the subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure of formula (II): [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. 【0038】 In some embodiments, the nucleic acid modulates the immune response of immune cells and thus treats or ameliorates the symptoms. In some embodiments, one aspect of the disclosure relates to an LNP disclosed herein or a pharmaceutical composition containing the same for use in a method of treating, ameliorating, or preventing symptoms of a disorder or disease in a subject in need thereof. The disease or disorder can be as disclosed below. In some embodiments, the methods disclosed herein can comprise contacting immune cells of the subject with a lipid nanoparticle (LNP) in vitro or ex vivo. 【0039】 In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer. In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for treating or preventing infection by a pathogen. In some embodiments, the Fab antibody comprises a heavy chain fragment containing an F174C substitution according to Kabat numbering and / or a light chain fragment containing an S176C substitution according to Kabat numbering. 【0040】 In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or less of non-immune cells are transfected by the LNP. In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or less of unwanted immune cells that are not intended to be the destination of delivery are transfected by the LNP. In some embodiments, the half-life of the nucleic acid delivered to immune cells by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or longer than the half-life of the nucleic acid delivered to immune cells by a reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP. 【0041】 In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the immune cells intended to be the destination of delivery are transfected by the LNP. 【0042】 In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 5%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or higher than the expression level of the nucleic acid in the same immune cells delivered by the reference LNP. 【0043】 In one aspect, a lipid nanoparticle (LNP) for delivering a nucleic acid to NK cells of a subject is provided. The LNP comprises (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD56. 【0044】 In one aspect, a lipid nanoparticle (LNP) for delivering a nucleic acid to immune cells of a subject is provided. The LNP comprises (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD7 or CD8, and the free PEG lipid is DMG-PEG or DPG-PEG. 【0045】 In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to target immune cells. The LNPs include (a) an ionizable cationic lipid, (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. In some embodiments, the immune cell targeting group includes an antibody, and the antibody is a Fab or an immunoglobulin single variable domain. In some embodiments, the Fab is engineered to knock out the native interchain disulfide at the C-terminus. In some embodiments, the Fab has an embedded interchain disulfide. In some embodiments, the antibody is an immunoglobulin single-chain variable (ISV) domain, and the ISV domain is a nanobody® ISV. In some embodiments, the free PEG lipid includes a PEG having a molecular weight of at least 2000 daltons. In some embodiments, the PEG has a molecular weight of about 3000 - 5000 daltons. In some embodiments, the Fab is an anti-CD3 antibody, and the free PEG lipid in the LNP includes a PEG having a molecular weight of about 2000 daltons. In some embodiments, the Fab is an anti-CD4 antibody, and the free PEG lipid in the LNP includes a PEG having a molecular weight of about 3000 - 3500 daltons. 【0046】 In some embodiments, the free PEG-lipid includes diacyl phosphatidylethanolamine, dialkyl phosphatidylethanolamine, diacyl glycerol, ceramide, dialkyl glycerol, or dialkylacetamide. In some embodiments, the alkyl chain is myristic acid, palmitic acid, oleic acid, linoleic acid, or stearic acid. In some embodiments, the free PEG-lipid is DMG-PEG. In some embodiments, the free PEG-lipid is DPG-PEG. 【0047】 In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. The LNP comprises (a) an ionizable cationic lipid, (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD3 and an antibody that binds to CD11a or CD18. 【0048】 In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. The LNP comprises (a) an ionizable cationic lipid, (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD7 and an antibody that binds to CD8. 【0049】 In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to two different types of immune cells of a subject. The LNP comprises (a) an ionizable cationic lipid, (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. 【0050】 In some embodiments, the immune cell targeting group comprises a bispecific targeting moiety. In some embodiments, the bispecific targeting moiety binds to two different types of immune cells. In some embodiments, the two different types of immune cells are CD4+ T cells and CD8+ T cells. In some embodiments, the bispecific targeting moiety is a bispecific antibody. In some embodiments, the bispecific antibody is a Fab-ScFv. 【0051】 In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both CD4+ and CD8+ T cells of a subject. The LNP comprises (a) an ionizable cationic lipid, (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid. In some embodiments, the immune cell targeting group comprises a single antibody that binds to CD3 or CD7. 【0052】 Lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject, comprising (a) an ionizable cationic lipid, (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) a sterol or other structural lipid; (d) a neutral phospholipid; (e) a free polyethylene glycol (PEG) lipid, and (f) a nucleic acid, wherein the immune cell targeting group comprises a Fab lacking native interchain disulfide bonds, are further provided. In some embodiments, the Fab is engineered to replace one or both cysteines on the native constant light chain and native constant heavy chain that form native interchain disulfide bonds, thereby removing the native interchain disulfide bond in the Fab. 【0053】 An immunoglobulin single variable domain (ISVD) that binds to human CD8 is also provided. In some embodiments, the ISVD comprises three complementarity determining domains CDR1, CDR2, and CDR3, (a) CDR1 comprises GSTFSDYG (SEQ ID NO: 100), (b) CDR2 comprises IDWNGEHT (SEQ ID NO: 101), (c) CDR3 comprises AADALPYTVRKYNY (SEQ ID NO: 102). 【0054】 In some embodiments, the ISVD is humanized. 【0055】 In some embodiments, the ISVD comprises, consists of, or consists essentially of SEQ ID NO: 77. 【0056】 Also provided are polypeptides comprising GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101), and AADALPYTVRKYNY (SEQ ID NO: 102). 【0057】 In some embodiments, the polypeptide comprises an ISVD as described herein. 【0058】 In some embodiments, the polypeptide further comprises a second binding moiety that binds to CD8 or another distinct target. In some embodiments, the second binding moiety is also an ISVD. 【0059】 In some embodiments, the polypeptide further comprises a detectable marker or a therapeutic agent. 【0060】 Also provided are compositions comprising an ISVD or a polypeptide as described herein. 【0061】 Further provided are pharmaceutical compositions comprising an ISVD or a polypeptide as described herein and a pharmaceutically acceptable carrier. 【0062】 Further provided is a method of treating a disease or disorder associated with CD8 in a subject, the method comprising administering to the subject a pharmaceutical composition as described herein. 【0063】 In some embodiments, the disease is cancer. In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer. 【0064】 In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for treating or preventing infection by a pathogen. In some embodiments, the ionizable cationic lipid is an ionizable cationic lipid as disclosed herein, such as those in Table 1. 【0065】 In some embodiments, the immune cell targeting group comprises an antibody that binds to a T cell antigen. In some embodiments, the T cell antigen is CD3, CD8, or both CD3 and CD8. In some embodiments, the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD56. In some embodiments, the antibody is a human antibody or a humanized antibody. 【0066】 In some embodiments, the immune cell targeting group is covalently associated with a lipid in a lipid blend via a polyethylene glycol (PEG)-containing linker. In some embodiments, the lipid covalently associated with the immune cell targeting group via the PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl glycerol (DPG), or ceramide. In some embodiments, the PEG is PEG 2000. In some embodiments, the PEG is PEG 3400. 【0067】 In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002 to 0.2 mole percent. In some embodiments, the ionizable cationic lipid is present in the lipid blend in the range of 40 to 60 mole percent. 【0068】 In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is present in the lipid blend in the range of 30 to 50 mole percent. In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM). 【0069】 In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1 to 15 mole percent, such as about 5 to 15 mole percent, or about 5 to 10 mole percent. 【0070】 In some embodiments, the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG-lipid can be PEG-dioleoyl glycerol (PEG-DOG), PEG-dimyristoyl glycerol (PEG-DMG), PEG-dipalmitoyl glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoyl glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid. 【0071】 In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylethanolamine containing a dipalmitoyl (C16) chain or a distearoyl (C18) chain. In some embodiments, the free PEG-lipid is present in the lipid blend at about 0.1 to 4 mole percent, such as 0.5 to 2.5 mole percent or about 1 to 2 mole percent. In some embodiments, the free PEG-lipid is present in the lipid blend at about 1.5 mole percent. In some embodiments, the free PEG-lipid comprises a lipid that is the same as or different from the lipid in the lipid-immunocyte targeting group conjugate. 【0072】 In some embodiments, the LNPs have an average diameter in the range of 50 to 200 nm. In some embodiments, the LNPs have an average diameter of about 100 nm. In some embodiments, the LNPs have a polydispersity index in the range of 0.05 to 1. In some embodiments, the LNPs have a zeta potential of about +5 mV to about +50 mV at pH 5, such as about +10 mV to about +30 mV at pH 5. In some embodiments, the LNPs have a zeta potential of about -10 mV to about +10 mV at pH 7.4. 【0073】 In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, siRNA, gRNA (guide RNA), circRNA (circular RNA), ribozyme, decoy RNA, or microRNA. In some embodiments, the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine. In some embodiments, the mRNA encodes a polypeptide capable of regulating the immune response in immune cells. In some embodiments, the mRNA encodes a polypeptide capable of reprogramming immune cells. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR). 【0074】 In some embodiments, the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain. In some embodiments, the immune cell targeting moiety comprises an antibody fragment selected from the group consisting of Fab, F(ab’)2, Fab’-SH, Fv, and scFv fragments. In some embodiments, the immune cell targeting moiety comprises a Fab containing one or more interchain disulfide bonds. In some embodiments, the Fab comprises a heavy chain fragment containing F174C and C233S substitutions and a light chain fragment containing S176C and C214S substitutions according to the Kabat numbering. In some embodiments, the immune cell targeting moiety comprises a Fab containing a cysteine at the C-terminus of the heavy or light chain fragment. 【0075】 In some embodiments, the Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain. In some embodiments, the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds. In some embodiments, the immune cell targeting moiety further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain by an amino acid linker. 【0076】 In some embodiments, the immune cell targeting moiety comprises an immunoglobulin single variable domain. In some embodiments, the immunoglobulin single variable domain comprises a cysteine at the C-terminus. In some embodiments, the immunoglobulin single variable domain comprises a VHH domain and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine. In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked by an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain. In some embodiments, the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds. In some embodiments, the immune cell targeting moiety comprises a VHH domain linked to an antibody CH1 domain. In some embodiments, the antibody CH1 domain is linked to the antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions according to Kabat numbering, and the light chain constant domain comprises S176C and C214S substitutions. 【0077】 In some embodiments, the immune cell targeting moiety is (a) A heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3, (b) It comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 7. 【0078】 In some embodiments, 5% or less of non-immune cells are transfected by the LNP. In some embodiments, the half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by the reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP. In some embodiments, at least 10% of the immune cells are transfected by the LNP. In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by the reference LNP. 【0079】 In some aspects, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell is a NK cell. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD56. 【0080】 In some embodiments, lipid nanoparticles (LNPs) for delivering nucleic acids to target immune cells are provided herein. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD7 or CD8. In some embodiments, the free PEG lipid is DMG-PEG or DPG-PEG. 【0081】 In some embodiments, lipid nanoparticles (LNPs) for delivering nucleic acids to target immune cells are provided. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody. In some embodiments, the antibody is a Fab or an immunoglobulin single variable domain. 【0082】 In some embodiments, the Fab is engineered to knock out the native inter-chain disulfide at the C-terminus. In some embodiments, the Fab comprises a heavy chain fragment containing a C233S substitution and a light chain fragment containing a C214S substitution. In some embodiments, the Fab comprises a non-native inter-chain disulfide. In some embodiments, the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment. In some embodiments, the antibody is an immunoglobulin single-chain variable (ISV) domain, and the ISV domain is a nanobody® ISV. In some embodiments, the free PEG lipid comprises a PEG having a molecular weight of at least 2000 daltons. In some embodiments, the PEG has a molecular weight of about 3000 to 5000 daltons. In some embodiments, the antibody is a Fab. In some embodiments, the Fab binds to CD3, and the free PEG lipid in the LNP comprises a PEG having a molecular weight of about 2000 daltons. In some embodiments, the Fab is an anti-CD4 antibody, and the free PEG lipid in the LNP comprises a PEG having a molecular weight of about 3000 to 3500 daltons. 【0083】 In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked by an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to the antibody CH1 domain and a second VHH domain linked to the antibody light chain constant domain. 【0084】 In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to target immune cells. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. 【0085】 In some embodiments, the LNP binds to CD3 and also binds to CD11a or CD18. In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds to CD3. In some embodiments, the second conjugate comprises an antibody that binds to CD11a or CD18. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds to both CD3 and CD11a. In some embodiments, the conjugate comprises a bispecific antibody that binds to both CD3 and CD18. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or a Fab-ScFv. In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to target immune cells. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the LNP binds to CD7 and CD8 of immune cells. 【0086】 In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds to CD7 and a second conjugate that binds to CD8. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds to CD7 and CD8. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or a Fab-ScFv. 【0087】 In some aspects, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to two different types of immune cells of a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the LNP binds to a first antigen on the surface of a first type of immune cell and also binds to a second antigen on the surface of a second type of immune cell. In some embodiments, the two different types of immune cells are CD4+ T cells and CD8+ T cells. In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises a first antibody that binds to a first antigen of a first type of immune cell, and the second conjugate comprises a second antibody that binds to a second antigen of a second type of immune cell. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate comprises a bispecific antibody. In some embodiments, the bispecific antibody binds to both a first antigen on a first type of immune cell and a second antigen on a second type of immune cell. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or a Fab-ScFv. 【0088】 In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both CD4+ and CD8+ T cells of interest. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting moiety]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting moiety comprises a single antibody that binds to CD3 or CD7. 【0089】 In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both T cells and NK cells of interest. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting moiety]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD8. 【0090】 In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both target T cells and NK cells. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or another structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group binds to both (i) CD3 and CD56; (ii) CD8 and CD56; or (iii) CD7 and CD56. 【0091】 In some embodiments, the immune cell targeting group is covalently linked to a lipid in the lipid blend via a polyethylene glycol (PEG)-containing linker. In some embodiments, the lipid covalently linked to the immune cell targeting group via the PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), dialkylacetamide, or ceramide. In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002 to 0.2 mole percent. In some embodiments, the lipid blend further comprises one or more structural lipids (e.g., sterol), neutral phospholipids, and free PEG-lipids. 【0092】 In some embodiments, the ionizable cationic lipid is present in the lipid blend in the range of 40 to 60 mole percent. 【0093】 In some embodiments, the sterol is present in the lipid blend in the range of 30 to 50 mole percent. In some embodiments, the sterol is cholesterol. 【0094】 In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1 to 15 mole percent, such as about 5 to 15 mole percent. 【0095】 In some embodiments, the free PEG-lipid is selected from the group consisting of PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG) 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG) 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DSG), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof. In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylethanolamine containing a dipalmitoyl (C16) chain or a distearoyl (C18) chain. In some embodiments, the free PEG-lipid is present in the lipid blend in the range of 0.1 to 4 mole percent, such as about 0.5 to 2.5 mole percent. In some embodiments, the free PEG-lipid is about 1.5 mole percent. In some embodiments, the free PEG-lipid comprises a lipid that is the same as or different from the lipid in the lipid-immunocyte targeting group conjugate. 【0096】 In some embodiments, the LNP has an average diameter in the range of 50 to 200 nm. In some embodiments, the LNP has an average diameter of about 100 nm. In some embodiments, the LNP has a polydispersity index in the range of 0.05 to 1. In some embodiments, the LNP has a zeta potential of about -5 mV to 50 mV at pH 5, such as about +10 mV to about +30 mV at pH 5. In some embodiments, the LNP has a zeta potential of about -10 mV to about +10 mV at pH 7.4. 【0097】 In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA. In some embodiments, the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine. In some embodiments, the mRNA encodes a polypeptide that can regulate an immune response in immune cells. In some embodiments, the mRNA encodes a polypeptide that can reprogram immune cells. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR). 【0098】 In some aspects, lipid nanoparticles (LNPs) are provided for delivering a nucleic acid to immune cells of a subject. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having the structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the LNP comprises a sterol or other structural lipid. In some embodiments, the LNP comprises a neutral phospholipid. In some embodiments, the LNP comprises a free polyethylene glycol (PEG) lipid. In some embodiments, the LNP comprises a nucleic acid. 【0099】 In some embodiments, the immune cell targeting group comprises a Fab lacking native interchain disulfide bonds. In some embodiments, the Fab is engineered to replace one or both cysteines on the native constant light chain and the native constant heavy chain that form the native interchain disulfide, thereby removing the native interchain disulfide bond in the Fab. 【0100】 In some embodiments, methods are provided for targeting the delivery of nucleic acids to target immune cells of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs) provided herein. In some embodiments, the method is for targeting NK cells. In some embodiments, the immune cell targeting moiety binds to CD56. In some embodiments, the method is for targeting both T cells and NK cells simultaneously. In some embodiments, the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD8. In some embodiments, the method is for targeting both CD4+ T cells and CD8+ T cells simultaneously. In some embodiments, the immune cell targeting moiety includes a polypeptide that binds to CD3 or CD7. 【0101】 In some embodiments, methods are provided for expressing a polypeptide of interest in target immune cells of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs) provided herein. In some embodiments, methods are provided for modulating the cellular function of target immune cells of a subject. In some embodiments, the method includes administering to the subject lipid nanoparticles (LNPs) provided herein. 【0102】 In some embodiments, methods are provided for treating, ameliorating, or preventing a disorder or a symptom of a disease in a subject in need of treatment, amelioration, or prevention of the symptoms of the disease. In some embodiments, the method includes administering to the subject lipid nanoparticles (LNPs) provided herein. 【0103】 In some embodiments, an immunoglobulin single variable domain (ISVD) that binds to human CD8 is provided. In some embodiments, the ISVD comprises three complementarity determining domains CDR1, CDR2, and CDR3. In some embodiments, CDR1 comprises GSTFSDYG (SEQ ID NO: 100). In some embodiments, CDR2 comprises IDWNGEHT (SEQ ID NO: 101). In some embodiments, CDR3 comprises AADALPYTVRKYNY (SEQ ID NO: 102). In some embodiments, the ISVD is humanized. In some embodiments, the ISVD comprises SEQ ID NO: 77. 【0104】 In some embodiments, polypeptides comprising GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101), and AADALPYTVRKYNY (SEQ ID NO: 102) are provided. In another embodiment, polypeptides comprising the ISVDs provided herein are provided. In some embodiments, the polypeptide comprises a second binding moiety. In some embodiments, the second binding moiety binds to CD8 or another different target. In some embodiments, the second binding moiety is also an ISVD. In some embodiments, the polypeptide comprises a detectable marker. In some embodiments, the polypeptide comprises a therapeutic agent. 【0105】 In some embodiments, compositions comprising the ISVDs provided herein or the polypeptides provided herein are provided. 【0106】 In some embodiments, pharmaceutical compositions are provided comprising the ISVDs provided herein or the polypeptides provided herein and a pharmaceutically acceptable carrier. 【0107】 In some embodiments, methods of treating a disease or disorder associated with CD8 in a subject are provided. In some embodiments, the method comprises administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer. 【0108】 Various aspects and embodiments of the present invention will be described in further detail below. 【Brief Description of the Drawings】 【0109】 【Figure 1】 Figure 1 shows the proton NMR spectrum of intermediate 13-11. 【Figure 2A】 shows the proton NMR spectrum of intermediate 13-11a. 【Figure 2B】 shows the proton NMR spectrum of intermediate 13-11b. 【Figure 2C】 Figure 2C shows the LC-ELSD of intermediate 13-11b. 【Figure 3A】 Figure 3A shows the proton NMR spectrum of intermediate 13-10. 【Figure 3B】 Figure 3B shows the LC-CAD chromatogram of intermediate 13-10. 【Figure 4A-1】 Figure 4A-1 shows the proton NMR spectrum of lipid 1. 【Figure 4A-2】 Figure 4A-2 shows the LC-CAD chromatogram of lipid 1. 【Figure 4B-1】 Figure 4B-1 shows the proton NMR spectrum of lipid 3. 【Figure 4B-2】 Figure 4B-2 shows the LC-CAD chromatogram of lipid 3. 【Figure 4C-1】 Figure 4C-1 shows the proton NMR spectrum of lipid 4. 【Figure 4C-2】 Figure 4C-2 shows the LC-CAD chromatogram L of lipid 4. 【Figure 4D-1】 Figure 4D-1 shows the proton NMR spectrum of lipid 5A. 【Figure 4D-2】 Figure 4D-2 shows the LC-CAD chromatogram of lipid 5A. 【Figure 4E-1】 Figure 4E-1 shows the proton NMR spectrum of lipid 6. 【Figure 4E-2】 Figure 4E-2 shows the LC-CAD chromatogram of lipid 6. 【Figure 4F-1】Figure 4F-1 shows the proton NMR spectrum of lipid 7. 【Figure 4F-2】 Figure 4F-2 shows the LC-CAD chromatogram of lipid 7. 【Figure 4G-1】 Figure 4G-1 shows the proton NMR spectrum of lipid 2. 【Figure 4G-2】 Figure 4G-2 shows the LC-CAD chromatogram of lipid 2. 【Figure 4H-1】 Figure 4H-1 shows the proton NMR spectrum of lipid 8. 【Figure 4H-2】 Figure 4H-2 shows the LC-CAD chromatogram of lipid 8. 【Figure 4I-1】 Figure 4I-1 shows the proton NMR spectrum of lipid 9. 【Figure 4I-2】 Figure 4I-2 shows the LC-CAD chromatogram of lipid 9. 【Figure 4J-1】 Figure 4J-1 shows the proton NMR spectrum of lipid 10A. 【Figure 4J-2】 Figure 4J-2 shows the LC-CAD chromatogram of lipid 10A. 【Figure 4K-1】 Figure 4K-1 shows the proton NMR spectrum of lipid 11A. 【Figure 4K-2】 Figure 4K-2 shows the LC-CAD chromatogram of lipid 11A. 【Figure 4L-1】 Figure 4L-1 shows the proton NMR spectrum of lipid 12. 【Figure 4L-2】 Figure 4L-2 shows the LC-CAD chromatogram of lipid 12. 【Figure 4M-1】 Figure 4M-1 shows the proton NMR spectrum of lipid 13. 【Figure 4M-2】 Figure 4M-2 shows the LC-CAD chromatogram of lipid 13. 【Figure 4N-1】 Figure 4N-1 shows the proton NMR spectrum of lipid 15. 【Figure 4N-2】 Figure 4N-2 shows the LC-CAD chromatogram of lipid 15. 【Figure 4O-1】Figure 4O-1 shows the proton NMR spectrum of lipid 16. 【Figure 4O-2】 Figure 4O-2 shows the LC-CAD of lipid 16. 【Figure 4P-1】 Figure 4P-1 shows the proton NMR spectrum of lipid 19. 【Figure 4P-2】 Figure 4P-2 shows the LC-ELSD chromatogram of lipid 19. 【Figure 4Q-1】 Figure 4Q-1 shows the proton NMR spectrum of lipid 20. 【Figure 4Q-2】 Figure 4Q-2 shows the LC-ELSD chromatogram of lipid 20. 【Figure 4R-1】 Figure 4R-1 shows the proton NMR spectrum of lipid 31. 【Figure 4R-2】 Figure 4R-2 shows the LC-CAD chromatogram of lipid 31. 【Figure 4S-1】 Figure 4S-1 shows the proton NMR spectrum of lipid 32. 【Figure 4S-2】 Figure 4S-2 shows the LC-CAD chromatogram of lipid 32. 【Figure 4T-1】 Figure 4T-1 shows the proton NMR spectrum of lipid 33. 【Figure 4T-2】 Figure 4T-2 shows the LC-CAD chromatogram of lipid 33. 【Figure 4U-1】 Figure 4U-1 shows the proton NMR spectrum of lipid 34. 【Figure 4U-2】 Figure 4U-2 shows the LC-CAD chromatogram of lipid 34. 【Figure 4V-1】 Figure 4V-1 shows the proton NMR spectrum of lipid 14A. 【Figure 4V-2】 Figure 4V-2 shows the LC-CAD chromatogram of lipid 14A. 【Figure 4W-1】 Figure 4W-1 shows the proton NMR spectrum of lipid 17A. 【Figure 4W-2】 Figure 4W-2 shows the LC-CAD chromatogram of lipid 17A. 【Figure 4X-1】Figure 4X-1 shows the proton NMR spectrum of lipid 18A. 【Figure 4X-2】 Figure 4X-2 shows the LC-CAD chromatogram of lipid 18A. 【Figure 4Y-1】 Figure 4Y-1 shows the proton NMR spectrum of lipid 21A. 【Figure 4Y-2】 Figure 4Y-2 shows the LC-CAD chromatogram of lipid 21A. 【Figure 4Z-1】 Figure 4Z-1 shows the proton NMR spectrum of lipid 22. 【Figure 4Z-2】 Figure 4Z-2 shows the LC-CAD chromatogram of lipid 22. 【Figure 4AA-1】 Figure 4AA-1 shows the proton NMR spectrum of lipid 23A. 【Figure 4AA-2】 Figure 4AA-2 shows the LC-CAD chromatogram of lipid 23A. 【Figure 4AC-1】 Figure 4AC-1 shows the proton NMR spectrum of lipid 25A. 【Figure 4AC-2】 Figure 4AC-2 shows the LC-CAD chromatogram of lipid 25A. 【Figure 4AE-1】 Figure 4AE-1 shows the proton NMR spectrum of lipid 27. 【Figure 4AE-2】 Figure 4AE-2 shows the LC-CAD chromatogram of lipid 27. 【Figure 4AF-1】 Figure 4AF-1 shows the proton NMR spectrum of lipid 28. 【Figure 4AF-2】 Figure 4AF-2 shows the LC-CAD chromatogram of lipid 28. 【Figure 4AG-1】 Figure 4AG-1 shows the proton NMR spectrum of lipid 29. 【Figure 4AG-2】 Figure 4AG-2 shows the LC-CAD chromatogram of lipid 29. 【Figure 4AH-1】 Figure 4AH-1 shows the proton NMR spectrum of lipid 37A. 【Figure 4AH-2】 Figure 4AH-2 shows the LC-CAD chromatogram of lipid 37A. 【Figure 4AI-1】Figure 4AI-1 shows the proton NMR spectrum of lipid 19A. 【Figure 4AI-2】 Figure 4AI-2 shows the LC-CAD chromatogram of lipid 19A. 【Figure 4AJ-1】 Figure 4AJ-1 shows the proton NMR spectrum of lipid 20A. 【Figure 4AJ-2】 Figure 4AJ-2 shows the LC-CAD chromatogram of lipid 20A. 【Figure 5A】 Figure 5A shows the diameter (DLS, nm) of LNPs based on lipids 1-8 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze-thaw (-80°C). 【Figure 5B】 Figure 5B shows the polydispersity (DLS) of LNPs based on lipids 1-8 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze-thaw (-80°C). 【Figure 5C】 Figure 5C shows the charge (zeta potential, DLS) of LNPs based on lipids 1-8 in pH 5.5 MBS and pH 7.4 HBS. 【Figure 5D】 Figure 5D shows the % RNA recovery and dye-accessible RNA in LNPs based on lipids 1-8. 【Figure 6A】 Figure 6A shows the diameter (DLS, nm) of LNPs based on lipids 9, 10, 11, and 15 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze-thaw (-80°C). 【Figure 6B】 Figure 6B shows the polydispersity (DLS) of LNPs based on lipids 9, 10, 11, and 15 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze-thaw (-80°C). 【Figure 6C】 Figure 6C shows the charge (zeta potential, DLS) of LNPs based on lipids 9, 10, 11, and 15 in pH 5.5 MBS and pH 7.4 HBS. 【Figure 6D】 Figure 6D shows the % RNA recovery and dye-accessible RNA in LNPs based on lipids 9, 10, 11, and 15. 【Figure 7A】 Figure 7A shows the diameter (DLS, nm) of LNPs based on lipids 31 - 34 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze - thaw (-80 °C). 【Figure 7B】 Figure 7B shows the polydispersity (DLS) of LNPs based on lipids 31 - 34 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze - thaw (-80 °C). 【Figure 7C】 Figure 7C shows the charge (zeta potential, DLS) of LNPs based on lipids 31 - 34 in pH 5.5 MBS and pH 7.4 HBS. 【Figure 7D】 Figure 7D shows the % RNA recovery and dye - accessible RNA in LNPs based on lipids 31 - 34. 【Figure 8A】 Figure 8A shows the diameter (DLS, nm) of LNPs based on lipids 1, 3, 4, 5, 9 and 15 in pH 7.4 HBS and pH 6.5 MBS after insertion of αCD8 antibody conjugates TRX - 2 and T8. 【Figure 8B】 Figure 8B shows the polydispersity (DLS) of LNPs based on lipids 1, 3, 4, 5, 9 and 15 in pH 7.4 HBS and pH 6.5 MBS after insertion of αCD8 antibody conjugates TRX - 2 and T8. 【Figure 9A】 Figure 9A shows the diameter (DLS, nm) of LNPs based on lipids 1, 8, 9, 10, 11, and 15 in pH 7.4 HBS and pH 6.5 MBS after insertion of αCD8 antibody conjugates TRX - 2 and T8. 【Figure 9B】 Figure 9B shows the polydispersity (DLS) of LNPs based on lipids 1, 8, 9, 10, 11, and 15 in pH 7.4 HBS and pH 6.5 MBS after insertion of αCD8 antibody conjugates TRX - 2 and T8. 【Figure 10A】 Figure 10A shows the diameter (DLS, nm) of LNPs based on lipids 3, 4, 33, and 34 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze - thaw (-80 °C). 【Figure 10B】Figure 10B shows the polydispersity (DLS) of LNPs based on lipids 3, 4, 33, and 34 in pH 7.4 HBS and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze-thaw (-80 °C). 【Figure 10C】 Figure 10C shows the charge (zeta potential, DLS) of LNPs based on lipids 3, 4, 33, and 34 in pH 5.5 MBS, pH 7.4 HBS, and pH 6.5 MBS after antibody (αCD3, hSP34) insertion and freeze-thaw (-80 °C). 【Figure 10D】 Figure 10D shows the % RNA recovery and dye-accessible RNA in LNPs based on lipids 3, 4, 33, and 34. 【Figure 11A-11B】 Figure 11A shows GFP expression in primary human T cells transfected with αCD8 (hsp34)-targeted LNPs based on ALC-0315, DLin-MC3-DMA, lipid 3, lipid 6, and lipid 7 and stored at 4 °C. % GFP+ T cells at 24 hours. Figure 11B shows GFP expression in primary human T cells transfected with αCD3 (hsp34)-targeted LNPs based on ALC-0315, DLin-MC3-DMA, lipid 3, lipid 6, and lipid 7 after one freeze-thaw cycle (-80 °C storage). % GFP+ T cells at 24 hours. 【Figure 11C-11D】 Figure 11C shows GFP expression in primary human T cells transfected with αCD3 (hsp34)-targeted LNPs based on ALC-0315, DLin-MC3-DMA, lipid 3, lipid 6, and lipid 7 and stored at 4 °C. GFP MFI in live T cells at 24 hours. Figure 11D shows GFP expression in primary human T cells transfected with αCD3 (hsp34)-targeted LNPs based on ALC-0315, DLin-MC3-DMA, lipid 3, lipid 6, and lipid 7 after one freeze-thaw cycle (-80 °C storage). GFP MFI in live T cells at 24 hours. 【Figure 11E】Figure 11E shows the % viable T cells transfected by αCD3(hsp34)-targeted LNPs based on ALC-0315, DLin-MC3-DMA, Lipid 3, Lipid 6, and Lipid 7 after one freeze-thaw cycle (-80 °C storage). % viable T cells at 24 hours. 【Figure 12A-12B】 Figure 12A shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, and Lipid 4 and stored at 4 °C. % GFP+ T cells at 24 hours. Figure 12B shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, and Lipid 4 after one freeze-thaw cycle (-80 °C storage). % GFP+ T cells at 24 hours. 【Figure 12C-12D】 Figure 12C shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, and Lipid 4 and stored at 4 °C. GFP MFI in viable T cells at 24 hours. Figure 12D shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, and Lipid 4 after one freeze-thaw cycle (-80 °C storage). GFP MFI in viable T cells at 24 hours. 【Figure 12E】 Figure 12E shows the % viable T cells transfected with αCD3(hsp34)-targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, and Lipid 4 after one freeze-thaw cycle (-80 °C storage). % viable T cells at 24 hours. 【Figure 13A-13B】Figure 13A shows GFP expression in primary human T cells transfected with targeted LNPs based on DLin-KC2-DMA (stored at -80°C), Lipid 1 (stored at 4°C), Lipid 3 (stored at 4°C), and Lipid 5 (stored at 4°C). % GFP+ T cells. Figure 13B shows GFP expression in primary human T cells transfected with targeted LNPs based on DLin-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycles (stored at -80°C). % GFP+ T cells. 【Figure 13C-13D】 Figure 13C shows GFP MFI in live T cells, indicating GFP expression in primary human T cells transfected with targeted LNPs based on DLin-KC2-DMA (stored at -80°C), Lipid 1 (stored at 4°C), Lipid 3 (stored at 4°C), and Lipid 5 (stored at 4°C). Figure 13D shows GFP expression in primary human T cells transfected with targeted LNPs based on DLin-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycles (stored at -80°C). GFP MFI in live T cells. 【Figure 13E】 Figure 13E shows % live T cells transfected with targeted LNPs based on DLin-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 stored at -80°C. 【Figure 14A-14B】 Figure 14A shows GFP expression in primary human T cells transfected with αCD3(hSP34)-targeted LNPs based on DLin-KC2-DMA, Lipid 1 (stored at 4°C), Lipid 8 (stored at 4°C), and Lipid 8 (stored at -80°C). % GFP+ T cells. Figure 14B shows GFP expression in primary human T cells transfected with αCD3(hSP34)-targeted LNPs based on DLin-KC2-DMA, Lipid 1 (stored at 4°C), Lipid 8 (stored at 4°C), and Lipid 8 (stored at -80°C). GFP MFI in live T cells. 【Figure 14C】 Figure 14C shows % live cells with targeted LNPs based on DLin-KC2-DMA, Lipid 1 (stored at 4°C), Lipid 8 (stored at 4°C), and Lipid 8 (stored at -80°C). 【Figure 15A-15B】Figure 15A shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80 °C), lipid 8 (stored at 4 °C), lipid 9 (stored at 4 °C), and lipid 10 (stored at 4 °C). % GFP+ T cells. Figure 15B shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80 °C), lipid 8 (stored at -80 °C), and lipid 10 (stored at -80 °C). % GFP+ T cells. 【Figure 15C-15D】 Figure 15C shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80 °C), lipid 8 (stored at 4 °C), lipid 9 (stored at 4 °C), and lipid 10 (stored at 4 °C). GFP MFI in live T cells. Figure 15D shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80 °C), lipid 8 (stored at -80 °C), and lipid 10 (stored at -80 °C). GFP MFI in live T cells. 【Figure 15E-15F】 Figure 15E shows % live T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80 °C), lipid 8 (stored at 4 °C), lipid 9 (stored at 4 °C), and lipid 10 (stored at 4 °C). % live T cells. Figure 15F shows the % of live T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80 °C), lipid 8 (stored at -80 °C), and lipid 10 (stored at -80 °C). % live T cells. 【Figure 16A-16B】Figure 16A shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 15 (stored at 4°C). % GFP+ T cells. Figure 16B shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 15 (stored at -80°C). % GFP+ T cells. 【Figure 16C-16D】 Figure 16C shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 15 (stored at 4°C). GFP MFI in live T cells. Figure 16D shows GFP expression in primary human T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 15 (stored at -80°C). GFP MFI in live T cells. 【Figure 16E】 Figure 16E shows % live T cells transfected with αCD3(hsp34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 15 (stored at -80°C). 【Figure 17A-17B】 Figure 17A shows GFP expression in primary human T cells transfected with αCD8(TRX2)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15, compared to the corresponding non-targeted parental LNPs. % GFP+ T cells. Figure 17B shows GFP expression in primary human T cells transfected with αCD8(TRX2)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15, compared to the corresponding non-targeted parental LNPs. GFP MFI in live T cells. 【Figure 17C-17D】Figure 17C shows % +Dil T cells by αCD8(TRX2)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15, and is compared with the corresponding non-targeted parental LNPs. Figure 17D shows Dil MFI in live T cells by αCD8(TRX2)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15, and is compared with the corresponding non-targeted parental LNPs. 【Figure 17E】 Figure 17E shows % live T cells transfected with αCD8(TRX2)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15 and compared with the corresponding non-targeted parental LNPs. 【Figure 18A-18B】 Figure 18A shows GFP expression in primary human T cells transfected with αCD8(T8)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15 and compared with the corresponding non-targeted parental LNPs. % GFP+ T cells. Figure 18B shows GFP expression in primary human T cells transfected with αCD8(T8)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15 and compared with the corresponding non-targeted parental LNPs. GFP MFI in live T cells. 【Figure 18C-18D】 Figure 18C shows % +Dil T cells by αCD8(T8)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15, and is compared with the corresponding non-targeted parental LNPs. Figure 18D shows Dil MFI in live T cells by αCD8(T8)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15, and is compared with the corresponding non-targeted parental LNPs. 【Figure 18E】 Figure 18E shows % live T cells transfected with αCD8(T8)-targeted LNPs based on lipid 3, lipid 4, lipid 9, and lipid 15 and compared with the corresponding non-targeted parental LNPs. 【Figure 19A-19B】Figure 19A shows GFP expression in primary human T cells transfected with targeted LNPs based on αCD3(hSP34), DLin-KC2-DMA, lipid 2, lipid 3, lipid 31, and lipid 32 and stored at 4°C. % GFP+ T cells. Figure 19B shows GFP expression in primary human T cells transfected with targeted LNPs based on αCD3(hSP34), DLin-KC2-DMA, lipid 2, lipid 3, lipid 31, and lipid 32 and stored at 4°C. GFP MFI in live T cells. 【Figure 19C】 Figure 19C shows % live T cells with αCD3(hSP34)-targeted LNPs based on DLin-KC2-DMA, lipid 2, lipid 3, lipid 31, and lipid 32 stored at 4°C. 【Figure 20A-20B】 Figure 20A shows GFP expression in primary human T cells transfected with αCD3(hSP34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at 4°C), lipid 33 (stored at 4°C), lipid 34 (stored at 4°C), or transfected with αCD8(muOKT8)-targeted LNPs based on lipid 33 (stored at 4°C) and lipid 34 (stored at 4°C). % GFP+ T cells. Figure 20B shows GFP expression in primary human T cells transfected with αCD3(hSP34)-targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at -80°C), lipid 33 (stored at -80°C), and lipid 34 (stored at -80°C). % GFP+ T cells. 【Figure 20C-20D】Figure 20C shows GFP expression in primary human T cells transfected with αCD3 (hSP34)-targeting LNPs based on DLin-KC2-DMA (stored at -80°C), Lipid 3 (stored at -80°C), Lipid 33 (stored at 4°C), Lipid 34 (stored at 4°C), or transfected with αCD8 (muOKT8)-targeting LNPs based on Lipid 33 (stored at 4°C) and Lipid 34 (stored at 4°C). GFP MFI in live T cells. Figure 20D shows GFP expression in primary human T cells transfected with αCD3 (hSP34)-targeting LNPs based on DLin-KC2-DMA (stored at -80°C), Lipid 3 (stored at -80°C), Lipid 33 (stored at -80°C), Lipid 34 (stored at -80°C). GFP MFI in live T cells. 【Figure 20E】 Figure 20E shows the percentage of live T cells transfected with αCD3 (hSP34)-targeting LNPs based on DLin-KC2-DMA (stored at -80°C), Lipid 3 (stored at -80°C), Lipid 33 (stored at -80°C), Lipid 34 (stored at -80°C), or transfected with αCD8 (muOKT8)-targeting LNPs based on Lipid 33 (stored at 4°C) and Lipid 34 (stored at 4°C). 【Figure 21A-21B】 Figure 21A shows the percentage of αCD20 (TTR-023) CAR+ T cells transfected with αCD3 (hSP34)-targeting LNPs based on Lipid 3 (stored at 4°C), Lipid 4 (stored at 4°C), Lipid 9 (stored at 4°C), and Lipid 33 (stored at 4°C), as indicated by the %M1 value. Figure 21B shows the percentage of αCD20 (TTR-023) CAR+ T cells transfected with αCD3 (hSP34)-targeting LNPs based on Lipid 3 (stored at -80°C), Lipid 4 (stored at -80°C), Lipid 9 (stored at -80°C), Lipid 33 (stored at -80°C), as indicated by the %M1 value. 【Figure 21C-21D】Figure 21C shows the %αCD20 (TTR-023) CAR MFI in T cells transfected with αCD3 (hSP34)-targeted LNP based on lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 33 (stored at 4°C). Figure 21D shows the %αCD3 (hSP34) CAR MFI in T cells transfected with αCD3 (hSP34)-targeted LNP based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C). 【Figure 21E-21F】 Figure 21E shows the % viable T cells transfected with αCD3 (hSP34)-targeted LNP based on lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 33 (stored at 4°C). Figure 21F shows the % viable T cells transfected with αCD3 (hSP34)-targeted LNP based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C). 【Figure 22A-22B】 Figure 22A shows the %αCD20 (TTR-023) CAR+ T cells (CD8 population) with αCD8 (T8)-targeted LNP based on lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 33 (stored at 4°C), as indicated by the CD4-%M1 value. Figure 22B shows the αCD20 (TTR-023) CAR MFI in T cells (CD8 population) with αCD8 (T8)-targeted LNP based on lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 33 (stored at 4°C), as indicated by the CD4-M1 MFI value. 【Figure 22C-22D】Figure 22C shows the αCD20 (TTR-023) CAR levels in CD4+ T cells transfected with αCD8 (T8) -targeted LNPs based on lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 33 (stored at 4°C), as indicated by the M1% value. Figure 22D shows the αCD20 (TTR-023) CAR levels in CD4+ T cells transfected with αCD8 (T8) -targeted LNPs based on DLin-KC2-DMA (stored at -80°C), lipid 3 (stored at -80°C), lipid 33 (stored at -80°C), and lipid 34 (stored at -80°C), as indicated by the M1 MFI value. 【Figure 22E】 Figure 22E shows the % viable T cells (CD4 / CD8 population) transfected with αCD8 (T8) -targeted LNPs based on lipid 3 (stored at 4°C), lipid 4 (stored at 4°C), lipid 9 (stored at 4°C), and lipid 33 (stored at 4°C). 【Figure 23A-23B】 Figure 23A shows the % αCD20 (TTR-023) CAR+ T cells (CD8 population) transfected with αCD8 (T8) -targeted LNPs based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C) after one freeze-thaw cycle, as indicated by the CD4-%M1 value. Figure 23B shows the αCD20 (TTR-023) CAR MFI in T cells (CD8 population) transfected with αCD8 (T8) -targeted LNPs based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C) after one freeze-thaw cycle, as indicated by the CD4-M1 MFI value. 【Figure 23C-23D】Figure 23C shows the αCD20 (TTR-023) CAR levels in CD4+ T cells transfected with αCD8 (T8) -targeted LNPs based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C), as indicated by the CD4+%M1 value. Figure 23D shows the αCD20 (TTR-023) CAR levels in CD4+ T cells transfected with αCD8 (T8) -targeted LNPs based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C), as indicated by the CD4+ M1 MFI value. 【Figure 23E】 Figure 23E shows the % viable T cells transfected with αCD8 (T8) LNPs based on lipid 3 (stored at -80°C), lipid 4 (stored at -80°C), lipid 9 (stored at -80°C), and lipid 33 (stored at -80°C). 【Figure 24A-24B】 Figure 24A shows the GFP expression in CD8+ T cells transfected with αCD3 (hSP34) -targeted or αCD8 (TRX2) -targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by GFP expression. Figure 24B shows the GFP expression in CD8+ T cells transfected with αCD3 (hSP34) -targeted or αCD8 (TRX2) -targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by GFP MFI. 【Figure 24C-24D】Figure 24C shows GFP expression in CD4+ T cells transfected with αCD3(hSP34)-targeted or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by GFP expression. Figure 24D shows GFP expression in CD4+ T cells transfected with αCD3(hSP34)-targeted or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by GFP MFI. 【Figure 24E-24F】 Figure 24E shows % Dil+ CD8+ T cells transfected with αCD3(hSP34)-targeted or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by %Dil+ T cells. Figure 24F shows Dil MFI in CD8+ T cells transfected with αCD3(hSP34)-targeted or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by Dil MFI. 【Figure 24G-24H】 Figure 24G shows % Dil+ CD4+ T cells transfected with αCD3(hSP34)-targeted or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by %Dil+ T cells. Figure 24H shows Dil MFI in CD4+ T cells transfected with αCD3(hSP34)-targeted or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to vector control (mutOKT8) and non-transfected. Shown by Dil MFI. 【Figure 25A - 25B】Figure 25A shows GFP expression in NK cells in whole blood samples transfected with αCD3(hSP34)-targeted LNPs or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by %GFP+ NK cells. Figure 25B shows GFP expression in NK cells in whole blood samples transfected with αCD3(hSP34)-targeted LNPs or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by GFP MFI. 【Figure 25C - 25D】 Figure 25C shows GFP expression in granulocytes in whole blood samples transfected with αCD3(hSP34)-targeted LNPs or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by %GFP+ granulocytes. Figure 25D shows GFP expression in granulocytes in whole blood samples transfected with αCD3(hSP34)-targeted LNPs or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by GFP MFI. 【Figure 25E - 25F】 Figure 25E shows GFP expression in B cells in whole blood samples transfected with αCD3(hSP34)-targeted LNPs or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by %GFP+ B cells. Figure 25F shows GFP expression in B cells in whole blood samples transfected with αCD3(hSP34)-targeted LNPs or αCD8(TRX2)-targeted LNPs based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by GFP MFI. 【Figure 26A - 26B】 Figure 26A shows the LNP binding to NK cells in whole blood samples transfected with αCD3(hSP34)-targeted LNP or αCD8(TRX2)-targeted LNP based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by %Dil+ NK cells. Figure 26B shows the LNP binding to NK cells in whole blood samples transfected with αCD3(hSP34)-targeted LNP or αCD8(TRX2)-targeted LNP based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by Dil MFI. 【Figure 26C - 26D】 Figure 26C shows the LNP binding to granulocytes in whole blood samples transfected with αCD3(hSP34)-targeted LNP or αCD8(TRX2)-targeted LNP based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by %Dil+ granulocytes. Figure 26D shows the LNP binding to granulocytes in whole blood samples transfected with αCD3(hSP34)-targeted LNP or αCD8(TRX2)-targeted LNP based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by Dil MFI. 【Figure 26E - 26F】 Figure 26E shows the LNP binding to B cells in whole blood samples transfected with αCD3(hSP34)-targeted LNP or αCD8(TRX2)-targeted LNP based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by %Dil+ B cells. Figure 26F shows the LNP binding to B cells in whole blood samples transfected with αCD3(hSP34)-targeted LNP or αCD8(TRX2)-targeted LNP based on lipid 9, lipid 15, or DLin-KC3-DMA, compared to unbound control (mutOKT8) and non-transfected control. Shown by Dil MFI. 【Figure 27A - 27B】 Figure 27A shows the % viable T cells 24 hours after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2)CAR or mCherry based on lipid 9 or DLin-KC3-DMA. Figure 27B shows the % of CD8 (CD4-) T cells expressing M1(TRR-023)CAR after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2)CAR or mCherry based on lipid 9 or DLin-KC3-DMA. 【Figure 27C - 27D】 Figure 27C shows the mean fluorescence intensity (MFI) of M1(TTR-023) expression in CD8 (CD4-) T cells transfected with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2)CAR or mCherry based on lipid 9 or DLin-KC3-DMA. Figure 27D shows the % of CD8 (CD4-) T cells expressing mCherry after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2)CAR or mCherry based on lipid 9 or DLin-KC3-DMA. 【Figure 27E - 27F】 Figure 27E shows the mean fluorescence intensity (MFI) of mCherry expression in CD8 (CD4-) T cells transfected with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2)CAR or mCherry based on lipid 9 or DLin-KC3-DMA. Figure 27F shows the % of CD4+ T cells with M1(TTR-023)CAR expression after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2)CAR or mCherry based on lipid 9 or DLin-KC3-DMA. 【Figure 27G - 27H】Figure 27G shows the mean fluorescence intensity (MFI) of M1 (TTR-023) expression in CD4+ T cells after transfection with αCD20 (TTR-023) -targeted LNP expressing αCD8 (TRX2) CAR or mCherry based on lipid 9 or DLin-KC3-DMA. Figure 27H shows the percentage of CD4+ T cells expressing mCherry after transfection with αCD20 (TTR-023) -targeted LNP expressing αCD8 (TRX2) CAR or mCherry based on lipid 9 or DLin-KC3-DMA. 【Figure 27I】 Figure 27I shows the percentage of dead Raji cells in the Raji (B cell) co-culture experiment with CAR-T cells generated using αCD8 (TRX2) -targeted LNP expressing αCD20 (TTR-023) CAR or mCherry based on lipid 9 or DLin-KC3-DMA. 【Figure 28A - 28B】 Figure 28A shows the percentage of dead Raji cells in the Raji (B cell) co-culture experiment with CAR-T cells generated using αCD8 (TRX2) -targeted LNP expressing αCD20 (TTR-023) CAR or mCherry based on lipid 9 or DLin-KC3-DMA at effector:target ratios of 1:1, 4:1, and 8:1. Figure 28B shows the percentage of live CD8 (CD4) T cells in the Raji (B cell) co-culture experiment with CAR-T cells generated using αCD8 (TRX2) -targeted LNP expressing αCD20 (TTR-023) CAR or mCherry based on lipid 9 or DLin-KC3-DMA at effector:target ratios of 1:1, 4:1, and 8:1. 【Figure 28C】 Figure 28C shows the percentage of live CD4+ T cells in the Raji (B cell) co-culture experiment with CAR-T cells generated using αCD8 (TRX2) -targeted LNP expressing αCD20 (TTR-023) CAR or mCherry based on lipid 9 or DLin-KC3-DMA at effector:target ratios of 1:1, 4:1, and 8:1. 【Figure 29A - 29B】Figure 29A shows the % viable T cells 24 hours after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2) CAR or mCherry based on lipid 15 or DLin-KC3-DMA. Figure 29B shows the % of CD8 (CD4-) T cells expressing M1 (TRR-023 CAR) after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2) CAR or mCherry based on lipid 15 or DLin-KC3-DMA. 【Figure 29C - 29D】 Figure 29C shows the mean fluorescence intensity (MFI) of M1 (TTR-023 CAR) expression in CD8 (CD4-) T cells transfected with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2) CAR or mCherry based on lipid 15 or DLin-KC3-DMA. Figure 29D shows the % of CD8 (CD4-) T cells expressing mCherry after transfection with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2) CAR or mCherry based on lipid 15 or DLin-KC3-DMA. 【Figure 29E】 Figure 29E shows the mean fluorescence intensity (MFI) of mCherry expression in CD8 (CD4-) T cells transfected with αCD20 (TTR-023)-targeted LNPs expressing αCD8(TRX2) CAR or mCherry based on lipid 15 or DLin-KC3-DMA. 【Figure 30A - 30B】Figure 30A shows the % dead Raji cells in a Raji (B cell) co - culture experiment with CAR - T cells generated using αCD8(TRX2) - targeted LNPs expressing αCD20 (TTR - 023) CAR or mCherry, based on lipid 15 or DLin - KC3 - DMA, at effector:target ratios of 0.31:1, 1:1, 3.16:1, 10:1, and 31.6:1. Figure 30B shows the % live CD8(CD4 - ) T cells in a Raji (B cell) co - culture experiment with CAR - T cells generated using αCD8(TRX2) - targeted LNPs expressing αCD20 (TTR - 023) CAR or mCherry, based on lipid 15 or DLin - KC3 - DMA, at effector:target ratios of 0.31:1, 1:1, 3.16:1, 10:1, and 31.6:1 【Figure 30C】 Figure 30C shows the % live CD4 + T cells in a Raji (B cell) co - culture experiment with CAR - T cells generated using αCD8(TRX2) - targeted LNPs expressing αCD20 (TTR - 023) CAR or mCherry, based on lipid 15 or DLin - KC3 - DMA, at effector:target ratios of 0.31:1, 1:1, 3.16:1, 10:1, and 31.6:1 【Figure 31】 Figure 31 shows the structures of various Fabs, VHHs (Nbs), ScFvs, Fab - ScFv, and Fab - VHH hybrids. 【Figure 32A - 32B】 Figure 32A shows GFP expression in T cells transfected with αCD3 - targeted LNPs based on lipid 9, lipid 10, lipid 13, lipid 15, or DLin - KC2 - DMA and stored either at 4°C or after freeze - thaw (-80°C storage), as indicated by the % GFP + T cells. Figure 32B shows GFP expression in T cells transfected with αCD3 - targeted LNPs based on lipid 9, lipid 10, lipid 13, lipid 15, or DLin - KC2 - DMA and stored either at 4°C or after freeze - thaw (-80°C storage), as indicated by the GFP MFI. 【Figure 32C - 32D】Figure 32C shows %Dil+ T cells transfected with αCD3-targeted LNPs based on lipid 9, lipid 10, lipid 13, lipid 15, or DLin-KC2-DMA and stored either at 4°C or after freeze-thaw (-80°C storage). Figure 32D shows Dil MFI of Dil%MFI transfected with αCD3-targeted LNPs based on lipid 9, lipid 10, lipid 13, lipid 15, or DLin-KC2-DMA and stored either at 4°C or after freeze-thaw (-80°C storage). 【Figure 32E】 Figure 32E shows % viable T cells transfected with αCD3-targeted LNPs based on lipid 9, lipid 10, lipid 13, lipid 15, or DLin-KC2-DMA and stored either at 4°C or after freeze-thaw (-80°C storage). 【Figure 33A - 33B】 Figures 33A - 33C show % GFP+ T cells (CD4 and CD8 populations) in blood (Figure 33A), spleen (Figure 33B), and liver (Figure 33C) samples (analyzed for additional cell types of interest per legend) 24 hours after injection of GFP RNA using LNP formulations of lipid 15, DLin-KC3-DMA, and lipid 9 and α-CD8 targeting with the TRX-2 antibody. 【Figure 33C】 Ibid. 【Figure 34A - 34B】 Figures 34A - 34C show % DiI+ T cells (CD4 and CD8 populations) in blood (Figure 34A), spleen (Figure 34B), and liver (Figure 34C) samples (analyzed for additional cell types of interest per legend) 24 hours after injection of GFP RNA using LNP formulations of lipid 15 (DiI-dye labeled), DLin-KC3-DMA (no DiI-dye labeling), and lipid 9 LNP (DiI-dye labeled) and α-CD8 targeting with the TRX-2 antibody. 【Figure 34C】 Ibid. 【Figure 35A】 Figure 35A shows % GFP expression in CD8 T cells with α-CD8 and α-CD3 targets and α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid 15. 【Figure 35B】Figure 35B shows the mean fluorescence intensity (MFI) of GFP expression in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 35C】 Figure 35C shows the % GFP expression in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 35D】 Figure 35D shows the mean fluorescence intensity (MFI) of GFP expression in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 35E】 Figure 35E shows the % GFP expression in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 35F】 Figure 35F shows the mean fluorescence intensity (MFI) of GFP expression in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 35G】 Figure 35G shows the % GFP expression in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 35H】 Figure 35H shows the mean fluorescence intensity (MFI) of GFP expression in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 36A】 Figure 36A shows %DiI+ in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 36B】 Figure 36B shows DiI MFI in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 36C】 Figure 36C shows %Dil+ in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 36D】 Figure 36D shows %Dil+ in CD8 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 36E】 Figure 36E shows %DiI+ in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 36F】 Figure 36F shows DiI MFI in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid 15 together with α-CD8 and α-CD3 targets. 【Figure 36G】 Figure 36G shows %Dil+ in CD4 T cells having α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and non-binding (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA together with α-CD8 and α-CD3 targets. 【Figure 36H】Figure 36H shows the DiI MFI in CD4 T cells with α-CD2, α-CD4, α-CD7, α-CD28, α-TCR, and unbound (mutant OKT8) targeted LNPs in lipid DLin-KC3-DMA along with α-CD8 and α-CD3 targets. 【Figure 37A】 Figure 37A shows lipids 10, 15, 16, 24A, 26, and ALC-0315, which are the diameters (DLS, nm) of lipid-targeted LNPs (αCD8) before and after freeze-thaw. 【Figure 37B】 Figure 37B shows lipids 10, 15, 16, 24A, 26, and ALC-0315, which are the polydispersities (DLS) of lipid-targeted LNPs (αCD8) before and after freeze-thaw. 【Figure 37C】 Figure 37C shows the zeta potentials (mV) of lipid LNPs of lipids 10, 15, 16, 24A, 26, and ALC-0315 in pH 5.5 MES and pH 7.4 HBS. 【Figure 37D】 Figure 37D shows the total lipid RNA content (ug / mL) and the % of dye-accessible RNA of lipids 10, 15, 16, 24A, 26, and ALC-0315. 【Figure 38A】 Figure 38A shows the % GFP+ T cells in lipid 10, 15, 16, 24A, 26, and ALC-0315 LNP transfection. 【Figure 38B】 Figure 38B shows the GFP-MFI of T cells in lipid 10, 15, 16, 24A, 26, and ALC-0315 LNP transfection. 【Figure 38C】 Figure 38C shows the % DiI+ T cells in lipid 10, 15, 16, 24A, 26, and ALC-0315 LNP transfection. 【Figure 38D】 Figure 38D shows the DiI-MFI of T cells in lipid 10, 15, 16, 24A, 26, and ALC-0315 LNP transfection. 【Figure 38E】 Figure 38E shows the % live T cells in lipid 10, 15, 16, 24A, 26, and ALC-0315 LNP transfection. 【Figure 39A - 39D】Figure 39A shows the charge (zeta potential, DLS) of GFP and BiTE LNPs based on DLin-KC3-DMA in pH 5.5 MBS and pH 7.4 HBS before antibody insertion. Figure 39B shows the diameter (DLS, nm) of GFP and BiTE LNPs based on DLin-KC3-DMA and the polydispersity (DLS) of GFP and BiTE LNPs based on DLin-KC3-DMA before antibody insertion. Figure 39C shows the % RNA recovery and dye-accessible RNA in GFP and BiTE LNPs based on DLin-KC3-DMA before antibody insertion. Figure 39D shows the diameter Z-average size (DLS, nm) of BiTE LNPs based on DLin-KC3-DMA and the polydispersity (DLS) of BiTE LNPs based on DLin-KC3-DMA after antibody (αCD3, 500A2 and αCD8, YTS156.7.7) insertion. 【Figure 40A - 40C】 Figure 40A shows the % of live primary mouse T cells transfected by electroporation with DLin-KC3-DMA-based, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) -targeted LNPs. % live T cells at 24 hours. Figure 40B shows DiI LNP association in CD4 primary mouse T cells transfected by electroporation with DLin-KC3-DMA-based, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) -targeted LNPs. % DiI+ live T cells at 24 hours. Figure 40C shows DiI LNP association in CD8 primary mouse T cells transfected by electroporation with DLin-KC3-DMA-based, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) -targeted LNPs. % DiI+ live T cells at 24 hours. 【Figure 40D - 40E】Figure 40D shows DiI LNP association, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) targeting LNPs based on DLin-KC3-DMA in CD4 primary mouse T cells transfected by electroporation. DiI MFI in live T cells at 24 hours. Figure 40E shows DiI LNP association, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) targeting LNPs based on DLin-KC3-DMA in CD8 primary mouse T cells transfected by electroporation. DiI MFI in live T cells at 24 hours. 【Figure 40F - 40I】 Figure 40F shows GFP LNP transfection in CD4 primary mouse T cells, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) targeting LNPs based on DLin-KC3-DMA transfected by electroporation. % GFP+ live T cells at 24 hours. Figure 40G shows GFP LNP transfection in CD8 primary mouse T cells, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) targeting LNPs based on DLin-KC3-DMA transfected by electroporation. % GFP+ live T cells at 24 hours. Figure 40H shows GFP LNP transfection in CD4 primary mouse T cells, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) targeting LNPs based on DLin-KC3-DMA transfected by electroporation. GFP MFI in live T cells at 24 hours. Figure 40I shows GFP LNP transfection in CD8 primary mouse T cells, αCD4 (GK1.5), αCD3 (500A2) and / or αCD8 (YTS156.7.7) targeting LNPs based on DLin-KC3-DMA transfected by electroporation. GFP MFI in live T cells at 24 hours. 【Figure 41A - 41D】Figure 41A shows DiI LNP association in primary mouse T cells transfected with αCD8(2.43, YTS156.7.7, or YTS169.4.2.1)-targeted LNP (inserted with 5, 15, or 30 Fab / LNP) based on DLin-KC3-DMA. % DiI+ live T cells at 24 hours. Figure 41B shows GFP LNP transfection in primary mouse T cells transfected with αCD8(2.43, YTS156.7.7, or YTS169.4.2.1)-targeted LNP (inserted with 5, 15, or 30 Fab / LNP) based on DLin-KC3-DMA. % GFP+ live T cells at 24 hours. Figure 41C shows DiI LNP association based on DLin-KC3-DMA in primary mouse T cells transfected with αCD3(2C11, 500A2, or KT3) or αTCR(H57)-targeted LNP (inserted with 5, 15, or 30 Fab / LNP). % DiI+ live T cells at 24 hours. Figure 41D shows GFP LNP transfection based on DLin-KC3-DMA in primary mouse T cells transfected with αCD3(2C11, 500A2, or KT3) or αTCR(H57)-targeted LNP (inserted with 5, 15, or 30 Fab / LNP). % GFP+ live T cells at 24 hours. 【Figure 41E - 41H】 Figure 41E shows DiI LNP association in primary mouse T cells transfected with αCD4(GK1.5v1)-targeted LNP (inserted with 2.5, 5, 15, or 30 Fab / LNP) based on DLin-KC3-DMA. % DiI+ live T cells at 24 hours. Figure 41F shows GFP LNP transfection in primary mouse T cells transfected with αCD4(GK1.5v1)-targeted LNP (inserted with 2.5, 5, 15, or 30 Fab / LNP) based on DLin-KC3-DMA. % GFP+ live T cells at 24 hours. Figure 41G shows % DiI in mouse T cells transfected with various α-CD3 / α-CD8-targeted DLIN-KC3-DMA LNPs. Figure 41H shows % GFP+ mouse T cells transfected with various α-CD3 / α-CD8-targeted DLIN-KC3-DMA LNPs. 【Figure 42A - 42B】 Figure 42A shows CD69 expression in primary mouse T cells transfected with αCD8 (YTS156.7.7) and / or αCD3 (500A2) -targeted LNPs based on DLin-KC3-DMA. % CD69+ live T cells at 24 hours. Figure 42B shows IFN-γ secretion from primary mouse T cells transfected with αCD8 (YTS156.7.7) and / or αCD3 (500A2) -targeted LNPs based on DLin-KC3-DMA. Concentration of IFN-γ in the supernatant (pg / mL) at 24 hours. 【Figure 42C - 42D】 Figure 42C shows TNF-alpha secretion based on DLin-KC3-DMA from primary mouse T cells transfected with αCD8 (YTS156.7.7) and / or αCD3 (500A2) -targeted LNPs. Concentration of TNF-alpha in the supernatant (pg / mL) at 24 hours. Figure 42D shows the phenotypes of primary mouse T cells transfected with αCD8 (YTS156.7.7) and / or αCD3 (500A2) -targeted LNPs based on DLin-KC3-DMA. CM, central memory; EM, effector memory; SCM, T memory stem cell-like; cells were phenotyped 24 hours after transfection. 【Figure 42E】 Figure 42E shows the gene expression levels of genes related to activation in primary mouse T cells transfected with αCD8 (YTS156.7.7) and / or αCD3 (500A2) -targeted LNPs based on DLin-KC3-DMA. Gene expression was evaluated 24 hours after transfection. 【Figure 43A - 43D】Figure 43A shows the DiI LNP association of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % DiI+ of CD8+ T cells in blood 24 hours after intravenous injection. Figure 43B shows the DiI LNP association of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % DiI+ of CD4+ T cells in blood 24 hours after intravenous injection. Figure 43C shows the mCherry LNP expression of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % mCherry+ of CD8+ T cells in blood 24 hours after intravenous injection. Figure 43D shows the mCherry LNP expression of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % mCherry+ of CD4+ T cells in blood 24 hours after intravenous injection. 【Figure 43E - 43H】Figure 43E shows the DiI LNP association of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % DiI+ of CD8+ T cells in the spleen 24 hours after intravenous injection. Figure 43F shows the DiI LNP association of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % DiI+ of CD4+ T cells in the spleen 24 hours after intravenous injection. Figure 43G shows the mCherry LNP expression of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % mCherry+ of CD8+ T cells in the spleen 24 hours after intravenous injection. Figure 43H shows the mCherry LNP expression of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) targeting LNPs based on DLin-KC3-DMA. % mCherry+ of CD4+ T cells in the spleen 24 hours after intravenous injection. 【Figure 43I - 43L】Figure 43I shows the DiI LNP association of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) target LNPs based on DLin-KC3-DMA. %DiI+ of CD8+ T cells in the liver 24 hours after intravenous injection. Figure 43J shows the DiI LNP association of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) target LNPs based on DLin-KC3-DMA. %DiI+ of CD4+ T cells in the liver 24 hours after intravenous injection. Figure 43K shows the mCherry LNP expression of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) target LNPs based on DLin-KC3-DMA. %mCherry+ of CD8+ T cells in the liver 24 hours after intravenous injection. Figure 43L shows the mCherry LNP expression of primary mouse T cells treated in vivo with αCD4 (GK1.5v1), αCD8 (YTS156.7.7) and / or αCD3 (500A2) target LNPs based on DLin-KC3-DMA. %mCherry+ of CD4+ T cells in the liver 24 hours after intravenous injection. 【Figure 44A - 44D】Figure 44A shows the percentage of dead CT26 cells over time (0 - 120 hours) in a CT26 (EphA2+ cell line) co-culture experiment with BiTE-secreting mouse T cells generated using αCD8 (YTS156.7.7)-targeted LNP or BiTE-secreting mouse T cells expressing Fluc based on DLin-KC3-DMA. Figure 44B shows the percentage of dead CT26 cells over time (0 - 120 hours) in a CT26 (EphA2+ cell line) co-culture experiment with BiTE-secreting mouse T cells generated using LNP targeting αCD8 (YTS156.7.7) and αCD3 (500A2), or LNP expressing Fluc based on DLin-KC3-DMA. Figure 44C shows the percentage of dead CT26 cells over time (0 - 120 hours) in a CT26 (EphA2+ cell line) co-culture experiment with BiTE-secreting mouse T cells generated using αCD4 (GK1.5v1)-targeted LNP or BiTE-secreting mouse T cells expressing Fluc based on DLin-KC3-DMA. Figure 44D shows the percentage of dead CT26 cells over time (0 - 120 hours) in a CT26 (EphA2+ cell line) co-culture experiment with BiTE-secreting mouse T cells generated using αCD8 (YTS156.7.7)-targeted LNP or BiTE-secreting mouse T cells expressing Fluc based on DLin-KC3-DMA, in r500A2, recombinant 500A2 / EphA2 BiTE protein. Fluc, firefly luciferase. 【Figure 45A - 45B】 Figure 45A shows the tumor inoculation and LNP dosing regimen for the efficacy test. Figure 45B shows the efficacy (survival curve) based on DLin-KC3-DMA of mice treated with αCD8 (YTS156.7.7) and αCD3 (500A2)-targeted LNP secreting BiTE. 【Figure 45C - 45D】 Figure 45C shows the tumor growth of mice treated with αCD8 (YTS156.7.7) and αCD3 (500A2)-targeted LNP secreting BiTE based on DLin-KC3-DMA. Figure 45D shows the tumor growth of mice treated with αCD8 (YTS156.7.7) and αCD3 (500A2)-targeted LNP expressing a non-BiTE protein based on DLin-KC3-DMA. 【Figure 45E - 45H】 Figure 45E shows the tumor growth of mice treated with the recombinant BiTE protein. Figure 45F shows the tumor growth of mice treated with the PD-1 Ab. Figure 45G shows the tumor growth of mice treated with the vehicle. Figure 45H shows the relative body weight (%) relative to the baseline of mice treated with the αCD8 (YTS156.7.7) and αCD3 (500A2) targeting LNPs, expressing the BiTE protein based on DLin-KC3-DMA. 【Figure 46A - 46D】 Figure 46A shows the charge (zeta potential, DLS) of mCherry and CAR LNPs based on lipid 15 in pH 5.5 MBS and pH 7.4 HBS before antibody insertion. Figure 46B shows the diameter (DLS, nm) of mCherry and CAR LNPs based on lipid 15 and the polydispersity (DLS) of mCherry and CAR LNPs based on lipid 15 before antibody insertion. Figure 46C shows the % RNA recovery and dye-accessible RNA in mCherry and CAR LNPs based on lipid 15 before antibody insertion. Figure 46D shows the diameter (DLS, nm) of mCherry and CAR LNPs based on lipid 15 and the polydispersity (DLS) of mCherry and CAR LNPs based on lipid 15 after antibody (αCD8, TRX2 and / or αCD8, ibalizumab) insertion. 【Figure 47A - 47B】 Figure 47A shows the % viable cells of CD3+, CD4+ and CD8+ primary human T cells transfected with lipid 15-based αCD4 (ibalizumab) and / or αCD8 (TRX2) target LNPs. % viable T cells at 24 hours. Figure 47B shows the CAR expression based on lipid 15 at 24 hours after transfection in CD3+, CD4+ and CD8+ primary human T cells transfected with αCD4 (ibalizumab) and / or αCD8 (TRX2) target LNPs. CAR MFI in viable T cells at 24 hours. 【Figure 47C - 47D】Figure 47C shows CAR expression based on Lipid 15 24 hours after transfection in CD3+, CD4+ and CD8+ primary human T cells transfected with αCD4 (ibalizumab) and / or αCD8 (TRX2) targeting LNPs. % CAR+ live T cells at 24 hours. Figure 47D shows mCherry expression based on Lipid 15 24 hours after transfection in CD3+, CD4+ and CD8+ primary human T cells transfected with αCD4 (ibalizumab) and / or αCD8 (TRX2) targeting LNPs. mCherry MFI in live T cells at 24 hours. 【Figure 47E】 Figure 47E shows mCherry expression based on Lipid 15 24 hours after transfection in CD3+, CD4+ and CD8+ primary human T cells transfected with αCD4 (ibalizumab) and / or αCD8 (TRX2) targeting LNPs. % mCherry+ in live T cells at 24 hours. MODE FOR CARRYING OUT THE INVENTION 【0110】 The present invention provides an ionizable cationic lipid, a lipid-immune cell targeting group conjugate, and a lipid nanoparticle composition comprising such an ionizable cationic lipid and / or a lipid immune cell (e.g., T cell) targeting group conjugate, a medical kit containing such a lipid and / or conjugate, and methods of making and using such lipids and conjugates. 【0111】 The practice of the present invention uses conventional techniques of organic chemistry, pharmacology, cell biology, and biochemistry, unless otherwise specified. Such techniques are described in the literature, for example, “Comprehensive Organic Synthesis” (B.M. Trost & I. Fleming, eds., 1991 - 1992); “Current protocols in molecular biology” (F.M. Ausubel et al., eds., 1987, and periodic updates); and “Current protocols in immunology” (J.E. Coligan et al., eds., 1991), the entire contents of which are incorporated herein by reference. Various aspects of the present invention are described in the following sections, but aspects of the invention described in one particular section are not limited to any particular section. 【0112】 I. Definitions To facilitate understanding of the present invention, several terms and phrases are defined below. 【0113】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meanings in the fields of chemistry and biology. Chemical structures and formulas described herein should be interpreted according to standard rules of chemical valence known in the art of chemistry. Further, when a chemical group is a diradical, for example, the chemical group can be attached to its adjacent atoms in the remainder of the structure in one or both orientations; for example, it is understood that -OC(O)- is interchangeable with -C(O)O-, or -OC(S)- is interchangeable with -C(S)O-. 【0114】 As used herein, the terms "a" and "an" mean "one or more," and include the plural unless the context is inappropriate. In some embodiments, "one or more" is 1 or 2. In some embodiments, "one or more" is 1, 2, or 3. In some embodiments, "one or more" is 1, 2, 3, or 4. In some embodiments, "one or more" is 1, 2, 3, 4, or 5. In some embodiments, "one or more" is 1, 2, 3, 4, 5, or more. 【0115】 As used herein, the term "alkyl" herein refers to a saturated straight-chain or branched hydrocarbon having 1 to 12, 1 to 10, or 1 to 6 carbon atoms, such as a straight-chain or branched group of C1-C 12 alkyl, C1-C 10 alkyl, or C1-C6 alkyl, and is optionally substituted. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc. 【0116】 The term "alkylene" refers to a diradical of an alkyl group. In some embodiments, alkylene is optionally substituted. An exemplary alkylene group is -CH2CH2-. 【0117】 The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc. 【0118】 "Alkenyl" refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one carbon-carbon double bond. The group may be in either the cis or trans configuration (Z or E configuration) around the double bond. Examples of alkenyl groups include ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl) and butenyl (e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl), but are not limited thereto. 【0119】 "Alkynyl" refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl, propynyl (e.g., prop-1-yn-1-yl, prop-2-yn-1-yl) and butynyl (e.g., but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl), but are not limited thereto. 【0120】 The term "oxo" is recognized in the art and refers to an "=O" substituent. For example, cyclopentane substituted with an oxo group is cyclopentanone. 【0121】 The term "morpholinyl" refers to an optionally substituted substituent having the following structure: 【Chemical formula】 【0122】 The term "piperidinyl" refers to a substituent having the following structure, which is optionally substituted: [Chemical formula] 【0123】 In general, the term "substituted", whether or not preceded by the term "optionally", means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and where two or more positions in any given structure may be substituted with two or more substituents selected from a particular group, the substituents may be the same or different at each position. Combinations of substituents contemplated in the present invention preferably result in the formation of stable or chemically feasible compounds. In some embodiments, "optionally substituted" is equivalent to "unsubstituted or substituted". In some embodiments, "optionally substituted" indicates that the designated atom or group is optionally substituted with one or more substituents independently selected from any of the substituents provided herein. In some embodiments, any substituent may be selected from the group consisting of: C 1~6 alkyl, cyano, halogen, -O-C 1~6 alkyl, C 1-6 haloalkyl, C 3~7 cycloalkyl, 3-7 membered heterocyclyl, 5-6 membered heteroaryl and phenyl. In some embodiments, any substituent may be alkyl, cyano, halogen, halo, azide, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl. In some embodiments, any substituent is -OR s1 , -NR s2 R s3, -C(O)R s4 , -C(O)OR s5 , C(O)NR s6 R s7 , -OC(O)R s8 , -OC(O)OR s9 , -OC(O)NR s10 R 11 , -NR s12 C(O)R s13 , or -NR s14 C(O)OR s15 wherein, R s1 , R s2 , R s3 , R s4 , R s5 , R s6 , R s7 , R s8 , R s9 , R s10 , R s11 , R s12 , R s13 , R s14 , and R s15 are each independently H, C 1~6 alkyl, C 3~10 cycloalkyl, C 6~14 aryl, 5- to 10-membered heteroaryl or 3- to 10-membered heterocyclyl, each optionally substituted. 【0124】 The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc. 【0125】 The term "cycloalkyl" as used herein refers to, for example, "C 4~8The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, bridged cyclic (e.g., adamantyl), or spirocyclic hydrocarbon group having 3 to 12, 3 to 10, 3 to 8, 4 to 8, or 4 to 6 carbon atoms. In some embodiments, the cycloalkyl is optionally substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane. Unless otherwise specified, the cycloalkyl group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl. In certain embodiments, the cycloalkyl group is unsubstituted, i.e., non-substituted. 【0126】 The terms "heterocyclyl" and "heterocyclic group" are recognized in the art and refer to a saturated, partially unsaturated, or aromatic 3- to 10-membered ring structure, or a 3- to 7-membered ring, the ring structure of which contains 1 to 4 heteroatoms such as nitrogen, oxygen, and sulfur. In some embodiments, the heterocyclyl is optionally substituted. The number of ring atoms of the heterocyclyl group is C x ~C xIt can be specified using nomenclature, and x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing 1 to 4 heteroatoms such as nitrogen, oxygen, and sulfur. The name "C3-C7" indicates that the heterocyclic ring contains a total of 3 to 7 ring atoms, including any heteroatom occupying a ring atom position. An example of a C3 heterocyclyl is aziridinyl. The heterocycle can be, for example, a monocyclic system, a bicyclic system, or other polycyclic systems (e.g., fused, spiro, bridged bicyclic). The heterocycle may be condensed with one or more aryl rings, partially unsaturated rings, or saturated rings. Examples of heterocyclyl groups include biotinyl, chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-oneyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl, lactone, lactam such as azetidinone, pyrrolidinone, sultam, sultone, etc. Unless otherwise specified, the heterocyclic ring is optionally substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amide, amidino, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, oxo, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamide, sulfonyl, and thiocarbonyl. In certain embodiments, the heterocyclyl group is unsubstituted, i.e., non-substituted. 【0127】 The term "aryl" is recognized in the art and refers to a carbocyclic aromatic group. In some embodiments, the aryl is optionally substituted. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term "aryl" includes polycyclic ring systems having two or more carbocyclic rings (the rings are "fused rings") where two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. Unless otherwise specified, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, carboxylic acid, -C(O)alkyl, CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, etc. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is unsubstituted, i.e., non-substituted. In certain embodiments, the aryl group has a 6- to 10-membered ring structure. In some embodiments, the aryl group is C6-C 14 aryl. 【0128】 The term "heteroaryl" is recognized in the art and refers to an aromatic group containing at least one ring heteroatom. In some embodiments, the heteroaryl is optionally substituted. In certain cases, the heteroaryl group contains one, two, three, or four ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, and pyrimidinyl, among others. Unless otherwise specified, the heteroaryl can be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, carboxylic acid, -C(O)alkyl, CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF3, -CN, etc. The term "heteroaryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings (the rings are "fused rings"), and at least one of the rings is heteroaromatic, and for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. In certain embodiments, the heteroaryl is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the heteroaryl is unsubstituted, i.e., non-substituted. In certain embodiments, the heteroaryl group has a 5- to 10-membered ring structure, or a 5- to 6-membered ring structure, and the ring structure contains one, two, three, or four heteroatoms such as nitrogen, oxygen, and sulfur. 【0129】 The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines, for example, of the general formula -N(R 10 )(R 11 (wherein R 10 and R 11Each independently represents hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, aralkyl, or (CH2) m -R 12 refers to a moiety represented by), or R 10 and R 11 together with the N atom to which they are attached complete a heterocyclic ring having 4 to 8 atoms in the ring structure, and R 12 represents aryl, cycloalkyl, cycloalkenyl, heterocyclic ring or polycyclic ring, and m is an integer in the range of 0 or 1 to 8. In certain embodiments, R 10 and R 11 each independently represents hydrogen, alkyl, alkenyl or -(CH2) m -R 12 represented by). 【0130】 The terms "alkoxyl" or "alkoxy" are recognized in the art and refer to an alkyl group as defined above to which an oxygen radical is attached. In some embodiments, the alkoxyl is optionally substituted. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, etc. "Ether" is two hydrocarbons linked by a covalent bond through oxygen. Thus, the substituents of the alkyl that form an ether with the alkyl are -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH2) m -R 12 (wherein m and R 12 are as described above) and can be an alkoxyl or similar to an alkoxyl. The term "haloalkoxyl" refers to an alkoxyl group substituted with at least one halogen. For example, O-CH2F, -O-CHF2, -O-CF3, etc. In certain embodiments, the haloalkoxyl is an alkoxyl group substituted with at least one fluorine group. In certain embodiments, the haloalkoxyl is an alkoxyl group substituted with 1 to 6, 1 to 5, 1 to 4, 2 to 4, or 3 fluorine groups. 【0131】 The symbol " 【Chemical Structure】 " indicates the attachment point. 【0132】 The compounds of the present disclosure may contain one or more chiral centers and / or double bonds and, accordingly, may exist as stereoisomers such as geometric isomers, enantiomers or diastereomers. As used herein, the term "stereoisomers" consists of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols "R" or "S" depending on the arrangement of the substituents around the stereogenic carbon atoms. The present invention encompasses the various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated "(±)" in the nomenclature, but those skilled in the art will recognize that the structure may imply a chiral center. It is understood that a chemical structure, such as a graphical representation of a general chemical structure, encompasses all stereoisomeric forms of the specified compound unless otherwise indicated. 【0133】 Individual stereoisomers of the compounds of the present invention can be prepared synthetically from commercially available starting materials containing asymmetric or stereogenic centers or by the preparation of a racemic mixture followed by a resolution method well known to those skilled in the art. These resolution methods are exemplified by (1) the attachment of a chiral auxiliary to a mixture of enantiomers, the recrystallization or chromatographic separation of the resulting mixture of diastereomers, and the liberation of the optically pure product from the auxiliary, (2) salt formation using an optically active resolving agent, or (3) direct separation of a mixture of optical enantiomers on a chiral chromatography column. Mixtures of stereoisomers can also be resolved into their component stereoisomers by well-known methods such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Furthermore, enantiomers can be separated using supercritical fluid chromatography (SFC) techniques described in the literature. Additionally, stereoisomers can be obtained from stereoisomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthesis methods. 【0134】 Geometric isomers may also be present in the compounds of the present invention. The symbol " 【Chem.】 " indicates a bond that can be a single, double, or triple bond as described herein. The present invention encompasses various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration, and the terms "Z" and "E" are used according to IUPAC standards. Unless otherwise specified, structures depicting a double bond include both the "E" isomer and the "Z" isomer. 【0135】 Alternatively, substituents around a carbon-carbon double bond can be referred to as "cis" or "trans", where "cis" represents substituents on the same side of the double bond and "trans" represents substituents on opposite sides of the double bond. The configuration of substituents around a carbocyclic ring is referred to as "cis" or "trans". The term "cis" represents substituents on the same side of the plane of the ring, and the term "trans" represents substituents on opposite sides of the plane of the ring. A mixture of compounds in which the substituents are arranged on both the same and opposite sides of the plane of the ring is called "cis / trans". 【0136】 The present invention also encompasses isotopically labeled compounds of the invention that are identical to those recited herein, except that one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F, and 36 Cl. 【0137】 Certain isotopically labeled disclosed compounds (e.g., those labeled with 3H and 14C) are useful in compound and / or substrate tissue distribution assays. Tritium labeling (i.e., 3H) and carbon-14 (i.e., 14C) isotopes are particularly preferred due to the ease of their preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., 2H) can confer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or decreased required dosage), and may thus be preferred in some situations. The isotopically labeled compounds of the present invention can generally be prepared by using isotopically labeled reagents in place of non-isotopically labeled reagents, according to procedures similar to those disclosed in the examples herein, for example. 【0138】 As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods of the present invention. Such organisms are preferably mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), more preferably humans. 【0139】 As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, rendering the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo. 【0140】 As used herein, the term "pharmaceutically acceptable excipient" refers to any of the standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions, etc.), and various types of wetting agents. The composition can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Remington’s The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. 【0141】 As is known to those skilled in the art, the "salts" of the compounds of the present invention can be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids such as oxalic acid, although not pharmaceutically acceptable per se, can be used in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts. 【0142】 Examples of bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of the formula NW4 where W is C 1~4 an alkyl + and the like, but are not limited thereto. 【0143】 Examples of salts include, but are not limited to: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, flucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, oxalates, palmitates, pectates, persulfates, phenylpropionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, tosylates, undecanoates, etc. Other examples of salts include anions of the compounds of the present invention formulated with suitable cations such as Na + , NH4 + , NW4 + (wherein W is C 1~4 an alkyl group), etc. 【0144】 The abbreviations used in this specification are: diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); tetrabutylammonium iodide (TBAI); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 9-fluorenylmethoxycarbonyl (Fmoc), tetrabutyldimethylsilyl chloride (TBDMSCl), hydrogen fluoride (HF), phenyl (Ph), bis(trimethylsilyl)amine (HMDS), dimethylformamide (DMF); methylene chloride (DCM); tetrahydrofuran (THF); high performance liquid chromatography (HPLC); mass spectrometry (MS), evaporative light scattering detector (ELSD), electrospray (ES)); nuclear magnetic resonance spectroscopy (NMR). 【0145】 As used herein, the term "effective amount" refers to an amount of a compound (e.g., a nucleic acid, e.g., mRNA) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or route of administration. The term "effective amount" can be considered to include a therapeutically and / or prophylactically effective amount of a compound. 【0146】 As used herein, the phrase "therapeutically effective amount" means an amount of a compound (e.g., a nucleic acid, e.g., mRNA), material or composition that, at a reasonable benefit / risk ratio applicable to any medical treatment, is effective to produce some desired therapeutic effect in at least a subpopulation of cells of a mammal, such as a human or subject (e.g., a human subject). 【0147】 As used herein, the expression "prophylactically effective amount" means an amount of a compound (e.g., a nucleic acid, e.g., mRNA), material or composition comprising a compound (e.g., a nucleic acid, e.g., mRNA) that is effective to produce some desired prophylactic effect in at least a subpopulation of cells in a mammal, e.g., a human or subject (e.g., a human subject), by reducing, minimizing or eliminating the risk of developing a condition, or by reducing or minimizing the severity of a condition at a reasonable benefit / risk ratio applicable to any medical treatment. 【0148】 As used herein, the terms "treat", "treating" and "treatment" include any effect that results in the improvement of a condition, disease, disorder, etc., or the improvement of its symptoms, e.g., alleviation, reduction, modulation, amelioration or elimination. 【0149】 The phrase "pharmaceutically acceptable" as used herein is used to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. 【0150】 In this application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. 【0151】 Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether explicit or implicit herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, when referring to a particular compound, that compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless otherwise understood from the context. In other words, within this application, the embodiments are described and depicted in a manner that enables a clear and concise application to be written and drawn, but it is intended and will be understood that the embodiments can be variously combined or separated without departing from the present teachings and invention. For example, it will be understood that all features described and illustrated herein can be applicable to all aspects of the invention described and illustrated herein. 【0152】 The expression "at least one of" should be understood to individually include each of the recited objects following the expression, and various combinations of two or more of the recited objects, unless otherwise understood from the context and use. The expression "and / or" relating to three or more recited objects should be understood to have the same meaning unless otherwise understood from the context. 【0153】 The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains", or "containing", including their grammatical equivalents, should generally be understood as non-limiting and without limitation, for example, excluding additional elements or steps not recited, unless otherwise specified or understood from the context. 【0154】 When the term "about" is used in front of a quantitative value, the present invention includes the specific quantitative value itself as well, unless otherwise specified. As used herein, the term "about" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred. 【0155】 As used herein, unless otherwise specified, the term "antibody" means any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. This term is understood to encompass intact antibodies (e.g., intact monoclonal antibodies), or fragments thereof, such as the Fc fragment of an antibody (e.g., the Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody that includes an intact antibody, an antigen-binding fragment, or a modified or engineered Fc fragment (e.g., an antigen-binding fragment of a monoclonal antibody). Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). This term also encompasses immunoglobulin single variable domains, such as nanobodies (e.g., V HH )). 【0156】 As used herein, (i.e., X is a particular antigen) or "anti-X antibody" is an antibody that specifically recognizes antigen X. 【0157】 As used herein, "embedded interchain disulfide bond" or "interchain embedded disulfide bond" refers to a disulfide bond on a polypeptide that is not readily accessible to a water-soluble reducing agent or is effectively "embedded" in a hydrophobic region of the polypeptide, such that it is not available for both reduction and conjugation with other hydrophilic PEGs. Embedded interchain disulfide bonds are further described in International Publication No. WO 2017 / 096361 A1, which is incorporated herein by reference in its entirety. 【0158】 As used herein, in some embodiments, the specificity of targeted delivery by LNP is defined by the ratio between the % of the desired immune cell type that receives the delivered nucleic acid (e.g., on-target delivery) and the % of an undesired immune cell type that receives the delivered nucleic acid (e.g., off-target delivery), where the latter does not mean the destination of delivery. For example, when more desirable immune cells receive the delivered nucleic acid, the specificity is higher, while less desirable immune cells do not receive the delivered nucleic acid. The specificity of targeted delivery by LNP can also be defined as the ratio of the amount of nucleic acid delivered to the desired immune cells (e.g., on-target delivery) and the amount of nucleic acid delivered to the undesired immune cells (e.g., off-target delivery). The specificity of delivery can be determined using any suitable method. As a non-limiting example, the expression level of the nucleic acid in the desired immune cell type can be measured and compared to the expression levels in different immune cell types that are not meant to be the destination of delivery. 【0159】 As used herein, in some embodiments, the reference LNP is an LNP that does not have an immune cell targeting moiety but is otherwise the same as the LNP being tested. In some other embodiments, the reference LNP is an LNP that has a different ionizable cationic lipid but is otherwise the same as the LNP being tested. In some embodiments, the reference LNP contains D-Lin-MC3-DMA as an ionizable cationic lipid that is different from the ionizable cationic lipid in the LNP being tested but is otherwise the same as the LNP being tested. 【0160】 As used herein, a humanized antibody is an antibody that is wholly or partially of non-human origin and whose protein sequence has been modified, for example, to replace certain amino acids occurring at corresponding positions in the framework regions of the VH and VL domains in the sequence of an antibody from a human, so as to avoid or minimize the immune response in humans. For example, the variable domains of a non-human antibody of interest may be combined with the constant domains of a human antibody using genetic engineering techniques. The constant domains of a humanized antibody are most often human CH and CL domains. 【0161】 As used herein, the term "structural lipid" also refers to lipids containing sterols and sterol moieties. 【0162】 It should be understood that, as long as the present invention remains operable, the order of steps or the order for performing a particular operation is not important. Further, two or more steps or operations may be performed simultaneously. 【0163】 Throughout various places in this specification, substituents are disclosed in groups or ranges. It is specifically intended that the description include every and all individual and partial combinations of the members of such groups and ranges. For example, the term "C 1~6 alkyl" is specifically intended to individually disclose C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl. As another example, integers in the range of 0 to 40 are specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and integers in the range of 1 to 20 are specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. 【0164】 Any and all examples, or exemplary language, such as "such as" or "including" used herein are merely intended to better illustrate the invention and do not limit the scope of the invention unless claimed. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention. 【0165】 Throughout this specification, when compositions and kits are described as having, including, or containing certain components, or when processes and methods are described as having, including, or containing certain steps, it is also contemplated that there are compositions and kits of the invention consisting essentially of, or consisting of, the recited components, and processes and methods of the invention consisting essentially of, or consisting of, the recited process steps. 【0166】 As a general matter, compositions specifying percentages are by weight unless otherwise indicated. Further, when variables are not accompanied by definitions, the previous definitions of the variables control. 【0167】 Immunoglobulin single variable domain In some embodiments, the immune cell targeting moiety of the LNPs described herein includes an immunoglobulin single variable domain such as a nanobody. 【0168】 The term "immunoglobulin single variable domain" (ISV) is used interchangeably with "single variable domain" and defines an immunoglobulin molecule in which the antigen-binding site is present on a single immunoglobulin domain. This distinguishes the immunoglobulin single variable domain from "conventional" immunoglobulins (e.g., monoclonal antibodies) or their fragments (e.g., Fab, Fab’, F(ab’)2, scFv, di-scFv), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and the light chain variable domain (VL ) interact to form an antigen-binding site. In this case, both the V H and the V L complementarity-determining regions (CDRs) contribute to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site. In view of the above definition, conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD, or IgE molecules known in the art), or Fv fragments such as Fab, F(ab’)2 fragments, disulfide-bonded Fv, or scFv fragments, or bispecific antibodies derived from such conventional four-chain antibodies (all known in the art) are not normally regarded as immunoglobulin single variable domains, as in these cases, binding to each epitope of the antigen usually occurs not by a single (single) immunoglobulin domain but by a pair of (related) immunoglobulin domains such as light and heavy chain variable domains, i.e., the V H -V L pair of immunoglobulin domains that bind jointly to the epitope of each antigen. 【0169】 In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single V H , a single V HH or a single V L domain. Thus, the antigen-binding site of an immunoglobulin single variable domain is formed by three or fewer CDRs. 【0170】 Therefore, a single variable domain can be a light chain variable domain sequence (e.g., V L -sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., V H -sequence or V HHIt can be an array or a suitable fragment thereof. 【0171】 An immunoglobulin single variable domain (ISV) can be, for example, a heavy chain ISV, such as a camelized V H or a humanized V HH containing V H , V HH and can be. In one embodiment, this is a camelized V H or a humanized V HH containing V HH . The heavy chain ISV can be derived from a conventional 4-chain antibody or from a heavy chain antibody. 【0172】 For example, an immunoglobulin single variable domain can be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), a Nanobody® ISV (as defined herein and including, but not limited to, V HH ), another single variable domain, or any suitable fragment of any of these. 【0173】 In particular, an immunoglobulin single variable domain can be a Nanobody® ISV (e.g., a humanized V HH or a camelized V H containing V HH ) or a suitable fragment thereof. [Note: Nanobody® is a registered trademark of Ablynx N.V.] 【0174】 "V HH domain" refers to V HH s, V HH antigen fragments, and V HH also known as antibodies and originally of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al. 1993 (Nature 363:446-448). The term "V HH domain" refers to these variable domains as the heavy chain variable domains present in a conventional 4-chain antibody (herein "V Hfrom what is referred to as the "domain") and the light chain variable domain present in conventional four-chain antibodies (referred to herein as "V L domain"). For further explanation of V HH see the review article by Muyldermans 2001 (Review in Molecular Biotechnology 74:277-302). 【0175】 The terms "dAb’s" and "domain antibodies" are described, for example, in Ward et al. 1989 (Nature 341:544), Holt et al. 2003 (Trends Biotechnol. 21:484), and also in, for example, WO 2004 / 068820 pamphlet, WO 2006 / 030220 pamphlet, WO 2006 / 003388 pamphlet and other published patent applications of Domantis Ltd. It should also be noted that although not very preferred in the context of the present invention because it is not of mammalian origin, a single variable domain can be derived from certain species of sharks (e.g., the so-called "IgNAR domain", see for example WO 2005 / 18629 pamphlet). 【0176】 Typically, the production of immunoglobulins involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naïve, immune or synthetic libraries, for example by phage display. 【0177】 The generation of immunoglobulin sequences such as VHHs is widely described in various published documents, including WO 94 / 04678 pamphlet, Hamers-Casterman et al. 1993 (Nature 363:446-448) and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods, camelids are immunized with a target antigen, which elicits an immune response against the target antigen. The repertoire of VHHs obtained from the immunization is further screened for VHHs that bind to the target antigen. 【0178】 In these examples, the generation of antibodies requires an antigen purified for immunization and / or screening. The antigen can be from natural sources or purified during recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such light sources. 【0179】 Immunoglobulin sequences of different origins, including murine, rat, rabbit, bovine, human, and camelid immunoglobulin sequences, can be used herein. Also, in the methods described herein, fully human, humanized, or chimeric sequences can be used. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as those described by Ward et al. 1989 (Nature 341:544), WO 94 / 04678 pamphlet, and Davis and Riechmann (1994, Febs Lett., 339:285-290; and 1996, Prot. Eng., 9:531-537) can be used herein. Furthermore, SVs are fused to form multivalent and / or multispecific constructs (one or more V HHFor polyvalent and multispecific polypeptides containing domains and their preparation, reference is made to Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10.7346 - 7350) and also to, for example, WO 1996 / 34103 pamphlet and WO 1999 / 23221 pamphlet). 【0180】 "Humanized V HH " corresponds to the amino acid sequence of a naturally occurring V HH domain, but is "humanized", i.e., one or more amino acid residues in the amino acid sequence of said naturally occurring V HH sequence (and in particular in the framework sequence) are replaced with one or more (e.g., as described above) of the amino acid residues occurring at the corresponding positions within the V H domain from a conventional human four - chain antibody. This can be carried out by methods known per se, which will be apparent to those skilled in the art based on the prior art (e.g., WO 2008 / 020079 pamphlet). Further, such humanized V HH can be obtained by any traditional method known per se, and thus it should be noted that the polypeptides obtained using polypeptides containing a naturally occurring VHH domain as starting material are not strictly limited thereto. 【0181】 "Camelized V H " corresponds to the amino acid sequence of a naturally occurring V H domain, but is "camelized", i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring V H domain from a conventional four - chain antibody are replaced with the V HHIt comprises an amino acid sequence substituted with one or more of the amino acid residues occurring at the corresponding positions in the domain. As will be apparent to those skilled in the art, this can be carried out in a manner well known per se, for example, based on the descriptions of the prior art (e.g., Davies and Riechman 1994, FEBS 339:285; 1995, Biotechnol.13:475; 1996, Prot.Eng.9:531; and Riechman 1999, J.Immunol.Methods 231:25). Such "camelization" substitutions, as defined herein, are at the V H -V L interface and / or inserted at the positions of the amino acids forming and / or present in the so-called camelid hallmark residues (see, for example, WO 94 / 04678 pamphlet and Davies and Riechmann (1994 and 1996) supra). In one embodiment, the camelized V H used as a starting material or starting point for generating or designing is the V H sequence derived from mammals, such as the V H sequence derived from humans, such as the V H sequence, such as the V H 3 sequence. However, it should be noted that such camelized V H can be obtained by any suitable method known per se, and thus is not strictly limited to polypeptides obtained using polypeptides containing the naturally occurring V H domain as a starting material. 【0182】 The structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively. These framework regions are interrupted by three "complementary determining regions" ("CDRs"), which are referred to in the art and herein as "complementary determining region 1" ("CDR1"), "complementary determining region 2" ("CDR2"), and "complementary determining region 3" ("CDR3"), respectively. 【0183】 In such immunoglobulin sequences, the framework region can be any suitable framework region sequence, and examples of suitable framework sequences will be apparent to those skilled in the art based on, for example, standard handbooks and the further disclosure and prior art described herein. 【0184】 The framework sequence is an immunoglobulin framework sequence or a framework sequence (suitable combination thereof) derived from an immunoglobulin framework sequence (e.g., by humanization or camelization). For example, the framework sequence can be a framework sequence derived from a light chain variable domain (e.g., V L -sequence) and / or a heavy chain variable domain (e.g., V H -sequence or V HH sequence). In a particular embodiment, the framework sequence is a framework sequence derived from a V HH -sequence (wherein the framework sequence is optionally partially or fully humanized), or a camelized (as defined herein) conventional V H sequence. 【0185】 In particular, the framework sequence present in the ISV sequences described herein can include one or more hallmark residues (as defined herein), and the ISV sequences can be, for example, humanized V HH or camelized VH V containing HH may include Nanobody® ISV. Non-limiting examples of such frameworks (suitable combinations thereof) will be apparent from the following disclosure herein. 【0186】 V H domain and V HH The total number of amino acid residues in the domain and the V domain will typically be in the range of 110-120, and often 112-115. However, it should be noted that shorter and longer sequences may also be suitable for the purposes described herein. 【0187】 However, it should be noted that the ISV described herein is not limited with respect to the origin of the ISV sequence (or the nucleotide sequence used to express it), nor is it limited with respect to the method by which the ISV sequence or nucleotide sequence is generated or obtained. Thus, the ISV sequence may be a naturally occurring sequence (from any suitable species), a synthetic or semi-synthetic sequence. In one specific but non-limiting embodiment, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, which includes, but is not limited to, "humanized" (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V HH sequences), "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized V H sequences), as well as affinity maturation (starting, for example, from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, binding of fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and ISVs obtained by techniques such as these and other well-known immunoglobulin sequence engineering techniques among those skilled in the art, or any suitable combination of any of the foregoing. 【0188】 Similarly, the nucleotide sequences can be naturally-occurring nucleotide sequences or synthetic or semi-synthetic sequences, for example, sequences isolated from a suitable naturally-occurring template by PCR (such as DNA or RNA isolated from cells), nucleotide sequences isolated from libraries (and in particular expression libraries), nucleotide sequences prepared by introducing mutations into naturally-occurring nucleotide sequences (using any suitable technique known per se such as mismatch PCR), nucleotide sequences prepared by PCR using overlapping primers, or nucleotide sequences prepared using DNA synthesis techniques known per se. 【0189】 Generally, nanobody® ISVs (in particular, (partially) humanized V HH sequences and camelized V H sequences containing V HH sequences) can be characterized by the presence of one or more "hallmark residues" (as further described herein) in one or more framework sequences (as described herein). Thus, generally, a nanobody® ISV can be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 each refer to framework regions 1 to 4, CDR1 to CDR3 each refer to complementarity-determining regions 1 to 3, and one or more of the hallmark residues are as further defined herein). 【0190】 In particular, a nanobody® ISV can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein FR1 to FR4 each refer to framework regions 1 to 4, CDR1 to CDR3 each refer to complementarity-determining regions 1 to 3, and the framework sequences are as further defined herein). 【0191】 More specifically, the Nanobody® ISV can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (where FR1 to FR4 refer to framework regions 1 to 4 respectively, CDR1 to CDR3 refer to complementarity-determining regions 1 to 3 respectively, and one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, 108 according to Kabat numbering are selected from the hallmark residues shown in Table A below). 【0192】 【Table 1】 【0193】 【Table 2】 【0194】 In one embodiment, the immunoglobulin single variable domain has specific amino acid substitutions within a framework region effective to prevent or reduce the binding of so-called "existing antibodies" to the polypeptide. (i) The amino acid residue at position 112 is either K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is either K or Q; (iv) In each of the cases of (i) to (iii), an ISV in which the amino acid at position 11 is preferably V is described in WO 2015 / 173325 pamphlet. 【0195】 polypeptide An immunoglobulin single variable domain can comprise or consist essentially of one or more (at least one) immunoglobulin single variable domains and can optionally further comprise one or more additional amino acid sequences (all optionally linked via one or more suitable linkers) and can form part of a protein or polypeptide. The term "immunoglobulin single variable domain" can also encompass such polypeptides. One or more immunoglobulin single variable domains can be used as binding units in such proteins or polypeptides that can optionally contain one or more additional amino acids so as to each provide a monovalent, multivalent or multispecific polypeptide of the invention (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is made to Conrath et al. 2001 (J. Biol. Chem. 276:7346), and also to, for example, WO 96 / 34103, WO 99 / 23221 and WO 2010 / 115998). 【0196】 As outlined above, a polypeptide can comprise or consist essentially of one immunoglobulin single variable domain. Such a polypeptide is also referred to herein as a monovalent polypeptide. 【0197】 The term "multivalent" indicates the presence of multiple ISVs in a polypeptide. In one embodiment, the polypeptide is "bivalent", i.e., comprises or consists of two ISVs. In one embodiment, the polypeptide is "trivalent", i.e., comprises or consists of three ISVs. In another embodiment, the polypeptide is "tetravalent", i.e., comprises or consists of four ISVDs. Thus, the polypeptide can be "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", "heptavalent", "octavalent", "nonavalent", etc., i.e., the polypeptide comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, etc. ISVs, respectively. 【0198】 In one embodiment, the multivalent ISV polypeptide is trivalent. In another embodiment, the multivalent ISV polypeptide is tetravalent. In yet another embodiment, the multivalent ISV polypeptide is pentavalent. In one embodiment, the multivalent ISV polypeptide can also be multispecific. The term "multispecific" refers to binding to multiple different target molecules (also referred to as antigens). Thus, the multivalent ISV polypeptide can be "bispecific", "trispecific", "tetraspecific", etc., i.e., it can bind to 2, 3, 4, etc. different target molecules respectively. 【0199】 For example, the polypeptide may be bispecific and trivalent, for example, a polypeptide comprising or consisting of 3 ISVs, where 2 ISVs bind to a first target and 1 ISV binds to a second target different from the first target. In another example, the polypeptide may be trispecific and tetravalent, for example, a polypeptide comprising or consisting of 4 ISVs, where 1 ISV binds to a first target, 2 ISVs bind to a second target different from the first target, and 1 ISV binds to a third target different from the first and second targets. In yet another example, the polypeptide can be trispecific and pentavalent, such as a polypeptide comprising or consisting of 5 ISVs, where 2 ISVs bind to a first target, 2 ISVs bind to a second target different from the first target, and 1 ISV binds to a third target different from the first and second targets. 【0200】 In one embodiment, the multivalent ISV polypeptide can also be multiparatopic. The term "multiparatopic" means binding to multiple different epitopes on the same target molecule (also referred to as an antigen). Thus, the multivalent ISV polypeptide can be "biparatopic", "triparatopic", etc., i.e., it can bind to 2, 3, etc. different epitopes on the same target molecule respectively. 【0201】 In another aspect, the polypeptide of the invention comprising or consisting essentially of one or more immunoglobulin single variable domains (or suitable fragments thereof) may further comprise one or more other groups, residues, moieties or linking units. Such additional groups, residues, moieties, linking units or amino acid sequences may or may not provide additional functionality to the immunoglobulin single variable domain (and / or the polypeptide in which it is present) and may or may not modify the properties of the immunoglobulin single variable domain. 【0202】 For example, such additional groups, residues, moieties or linking units may be one or more additional amino acids such that the compound, construct or polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, the one or more other groups, residues, moieties or linking units are immunoglobulins. Even more preferably, the one or more other groups, residues, moieties or linking units are selected from the group consisting of domain antibodies, amino acids suitable for use as domain antibodies, single domain antibodies, amino acids suitable for use as single domain antibodies, "dAbs", amino acids suitable for use as dAbs, or nanobodies. 【0203】 Alternatively, such groups, residues, moieties or linking units may be chemical groups, residues, moieties which may or may not themselves be biologically and / or pharmacologically active. For example, without limitation, such groups may be linked to one or more immunoglobulin single variable domains so as to provide a "derivative" of the immunoglobulin single variable domain. 【0204】 In another embodiment, the additional residue(s) may be effective to prevent or reduce the binding of so-called "pre-existing antibodies" to the polypeptide. For this purpose, the polypeptide and the construct may include a C-terminal extension (X)n (SEQ ID NO: 150), where n can be from 1 to 10, preferably from 1 to 5, such as 1, 2, 3, 4 or 5 (preferably 1 or 2, such as 1), and each X is independently selected from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I), preferably independently selected (preferably naturally occurring) amino acid residues, for which reference is made to WO 2012 / 175741 pamphlet. Thus, the polypeptide may further include a C-terminal extension (X)n (SEQ ID NO: 151), where n is from 1 to 5, such as 1, 2, 3, 4 or 5, and X is a naturally occurring amino acid, preferably free of cysteine. 【0205】 In the above polypeptide, one or more immunoglobulin single variable domains and one or more groups, residues, moieties or binding units may be linked to each other directly and / or via one or more suitable linkers or spacers. For example, when one or more groups, residues, moieties or binding units are amino acids, the linker may be an amino acid, and as a result, the resulting polypeptide is a fusion protein or a fusion polypeptide. 【0206】 As used herein, the term "linker" means a peptide that fuses two or more ISVs together into a single molecule. The use of linkers to connect two or more (poly)peptides is well known in the art. Another exemplary peptide linker is shown in Table B. One class of frequently used peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues and typically have a GGGGS (SEQ ID NO: 154) motif (e.g., the formula (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 152), where n can be 1, 2, 3, 4, 5, 6, 7 or more) and include one or more repeats of such a peptide motif. Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 157), the 15GS linker (n = 3), and the 35GS linker (n = 7). See, for example, Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10):1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27(10):325-330). 【0207】 [Table 3] 【0208】 [Table 4] 【0209】 In one aspect, the disclosure also relates to such amino acid sequences and / or nanobodies that can bind to and / or are directed against CD8, including CDR sequences, as further defined herein in general, suitable fragments thereof, and polypeptides comprising or consisting essentially of one or more of such nanobodies and / or suitable fragments. In some aspects, the disclosure relates to a nanobody having SEQ ID NO: 77. In particular, the disclosure in some specific aspects provides the following: I) An amino acid sequence that is against CD8 and has at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with SEQ ID NO: 77; II) An amino acid sequence that cross-blocks the binding of the amino acid sequence of SEQ ID NO: 77 to CD8 and / or competes with at least the binding of the amino acid sequence of SEQ ID NO: 77 to CD8; 【0210】 Such an amino acid sequence can be as further described herein (for example, it can be a nanobody); and such an amino acid sequence (which can be further described herein), in particular the bispecific (or multispecific) polypeptide described herein, and a polypeptide of the present disclosure comprising one or more of such an amino acid sequence and a nucleic acid sequence encoding such a polypeptide and such a polypeptide. Such amino acid sequences and polypeptides do not include naturally occurring ligands. 【0211】 In some embodiments, CD8 is derived from a mammal such as a human. In one specific but non-limiting embodiment, the present disclosure provides an amino acid sequence against CD8, which a) has the amino acid sequence of SEQ ID NO: 77; b) has an amino acid sequence having at least 80% amino acid identity with SEQ ID NO: 77, or c) has an amino acid sequence having 3, 2 or 1 amino acid difference from SEQ ID NO: 77; or any suitable combination thereof. 【0212】 In some embodiments, a nanobody against CD8 consisting of four framework regions (FR1 to FR4 respectively) and three complementarity-determining regions (CDR1 to CDR3 respectively) is disclosed. In some embodiments, in such a nanobody, (I) CDR1 contains or consists essentially of the amino acid sequence of GSTFSDYG (SEQ ID NO: 100), or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with GSTFSDYG (SEQ ID NO: 100), wherein (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared with GSTFSDYG (SEQ ID NO: 100); and / or selected from the group consisting of amino acid sequences having only 2 or 1 amino acid difference from GSTFSDYG (SEQ ID NO: 100), any amino acid substitution is a conservative amino acid substitution; and / or the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared with GSTFSDYG (SEQ ID NO: 100). (II) CDR2 comprises or consists essentially of the amino acid sequence of IDWNGEHT (SEQ ID NO: 101), or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with IDWNGEHT (SEQ ID NO: 101), wherein (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared with IDWNGEHT (SEQ ID NO: 101); and / or selected from the group consisting of amino acid sequences having only 2 or 1 amino acid difference from IDWNGEHT (SEQ ID NO: 101), any amino acid substitution is a conservative amino acid substitution; and / or the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared with IDWNGEHT (SEQ ID NO: 101). (III) CDR3 comprises or consists essentially of the amino acid sequence of AADALPYTVRKYNY (SEQ ID NO: 102), or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with AADALPYTVRKYNY (SEQ ID NO: 102), wherein (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) the amino acid sequence contains only amino acid substitutions and does not contain any amino acid deletions or insertions as compared with AADALPYTVRKYNY (SEQ ID NO: 102); and / or selected from the group consisting of amino acid sequences having only 2 or 1 amino acid difference from AADALPYTVRKYNY (SEQ ID NO: 102), any amino acid substitution is a conservative amino acid substitution; and / or the amino acid sequence contains only amino acid substitutions and does not contain any amino acid deletions or insertions as compared with AADALPYTVRKYNY (SEQ ID NO: 102). The CD8 nanobody disclosed herein may include one, two, or all three of the CDRs explicitly listed above. In some embodiments, the CD8 nanobody CDR1: GSTFSDYG (SEQ ID NO: 100) based on IMGT designation; CDR2: IDWNGEHT (SEQ ID NO: 101) based on IMGT designation; and CDR3: including AADALPYTVRKYNY (SEQ ID NO: 102) based on IMGT designation. 【0213】 In the nanobodies of the present disclosure that include the above combinations of CDRs, each CDR can be replaced with a CDR selected from the group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the above CDRs, (1) any amino acid substitution is preferably a conservative amino acid substitution; and / or (2) the amino acid sequence preferably contains only amino acid substitutions and does not contain any amino acid deletions or insertions as compared with the above amino acid sequence; and / or selected from the group consisting of amino acid sequences having only 3, 2 or 1 "amino acid difference" with one of the recited CDRs of the above amino acid sequence (as shown in the previous paragraph), (1) Any amino acid substitution is preferably a conservative amino acid substitution; and / or (2) The amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions as compared to the above amino acid sequence. 【0214】 In one embodiment, the CD8 nanobody is BDSn: An anti-CD8 BDSn Nb sequence (CDR1, CDR2, CDR3 underlined based on IMGT notation): 【Chemical formula】 (SEQ ID NO: 77); 【0215】 In some embodiments, the CD8 nanobodies of the present disclosure are 10 -5 ~10 -12 mol / liter (M) or less, preferably 10 -7 ~10-12 mol / liter (M) or less, more preferably 10 -8 ~10 -12 mol / liter (M) dissociation constant (KD) and / or at least 107 M-1, preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , for example at least 10 12 M -1 association constant (KA) to bind to CD8, in particular, bind with a KD of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, for example less than 500 μM. The KD and KA values of the nanobodies of the present disclosure for vWF can be determined in a manner known per se, for example using the assays described herein. More generally, the nanobodies described herein preferably have a dissociation constant for vWF as described in this paragraph. 【0216】 In general, it should be noted that the term nanobody as used herein is not limited to a particular biological source or a particular preparation method in its broadest sense. For example, as will be discussed in more detail below, nanobodies can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) expressing the nucleotide sequence encoding a naturally occurring VHH domain; (3) "humanizing" a naturally occurring VHH domain (described below) or expressing a nucleic acid encoding such a humanized VHH domain; (4) "camelizing" (described below) a naturally occurring VH domain from any animal species, particularly mammalian species such as humans, or expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" the "domain antibody" or "Dab" described by Ward et al (supra), or expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) preparing a nucleic acid encoding a nanobody using nucleic acid synthesis techniques and expressing the resulting nucleic acid; and / or (8) by any combination of the above. Suitable methods and techniques for carrying out the above will be apparent to those skilled in the art based on the disclosure herein and include, for example, the methods and techniques described in more detail below. 【0217】 In some embodiments, the CD8 nanobodies of the present disclosure do not have an amino acid sequence that is exactly the same (i.e., to the extent of 100% sequence identity) as the amino acid sequence of a naturally occurring VH domain, such as the amino acid sequence of a naturally occurring VH domain from a mammalian, particularly human, source. 【0218】 One class of the CD8 nanobodies of the present disclosure corresponds to the amino acid sequence of a naturally occurring VHH domain, but is "humanized", i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence are replaced by one or more amino acid residues present at corresponding positions in the VH domain from a conventional four-chain antibody of human origin (e.g., as shown above), and includes nanobodies having the amino acid sequence. Such humanized CD8 nanobodies of the present disclosure can be obtained by any suitable method known per se (i.e., as shown in points (1) to (8) above), and thus it should be noted that they are not strictly limited to polypeptides obtained using a polypeptide containing a naturally occurring VHH domain as a starting material. 【0219】 Another class of the CD8 nanobodies of the present disclosure has an amino acid sequence corresponding to the amino acid sequence of a "camelized" naturally occurring VH domain, i.e., one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain are replaced by one or more amino acid residues present at corresponding positions in the VHH domain of a heavy-chain antibody from a conventional four-chain antibody, and includes nanobodies. This can be done by methods known per se, which will be apparent to those skilled in the art based on, for example, the following further description. See also WO 94 / 04678 pamphlet. Such camelization can occur preferentially at the VH-VL interface and at amino acid positions present in so-called camelid characteristic residues, as also mentioned below (see also WO 94 / 04678 pamphlet). In some embodiments, the VH domain or sequence used as a starting material or starting point for generating or designing camelized nanobodies is a VH sequence derived from a mammal, such as a human VH sequence. Such camelized nanobodies of the present disclosure can be obtained by any suitable method known per se, and thus it should be noted that they are not strictly limited to polypeptides obtained using a polypeptide containing a naturally occurring VH domain as a starting material. 【0220】 For example, both "humanization" and "camelization" provide nucleotide sequences encoding such naturally occurring VHH domains or VH domains, respectively, and then, in a manner known per se, one or more codons in the nucleotide sequence are changed such that the new nucleotide sequences each encode a humanized or camelized nanobody of the present disclosure, and then the nucleotide sequences thus obtained are expressed in a manner known per se so as to provide the desired nanobody. Alternatively, based on the amino acid sequences of the naturally occurring VHH domains or VH domains, respectively, the amino acid sequences of the desired humanized or camelized nanobodies of the present disclosure can be designed and then newly synthesized using techniques known per se for peptide synthesis. Also, based on the amino acid sequences or nucleotide sequences of the naturally occurring VHH domains or VH domains, respectively, nucleotide sequences encoding the desired humanized or camelized nanobodies can be designed and then de novo synthesized using nucleic acid synthesis techniques known per se, and thereafter, the nucleotide sequences thus obtained can be expressed in a manner known per se so as to provide the desired nanobody. 【0221】 Starting from a naturally occurring VH domain (amino acid sequence) or preferably a VHH domain (amino acid sequence) and / or nucleotide sequence and / or nucleic acid sequence encoding the same, other suitable methods and techniques for obtaining nanobodies and / or nucleotide sequences and / or nucleic acids encoding the same will be apparent to those skilled in the art and may include, for example, combining one or more amino acid sequences and / or nucleotide sequences derived from a naturally occurring VH domain (such as one or more FRs and / or CDRs, etc.) with one or more amino acid sequences and / or nucleotide sequences derived from a naturally occurring VHH domain (such as one or more FRs or CDRs, etc.) in a suitable manner to provide a nanobody (nucleotide sequence or nucleic acid encoding the same). Also provided are compounds and constructs, particularly proteins and polypeptides, comprising or consisting essentially of at least one such amino acid sequence and / or nanobody (or suitable fragment thereof) of the present disclosure and optionally further comprising one or more other groups, residues, moieties or linking units. In some embodiments, such additional groups, residues, moieties, linking units or amino acid sequences may or may not provide additional functionality to the amino acid sequence and / or nanobody (and / or the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence and / or nanobody. 【0222】 The present disclosure also encompasses any polypeptide of the present disclosure that is glycosylated at one or more amino acid positions, depending on the hot spot typically used to express the polypeptide. The polypeptide can comprise the amino acid sequence of a CD8 nanobody of the present disclosure that is fused to at least one additional amino acid sequence at its amino terminus, its carboxy terminus, or both its amino terminus and its carboxy terminus. Such additional amino acid sequences may include at least one additional nanobody in order to provide a polypeptide comprising at least two, such as three, four or five nanobodies, which nanobodies are optionally linked via one or more linker sequences (defined herein). A polypeptide comprising a CD8 nanobody of the present disclosure and one or more other nanobodies is a multivalent polypeptide. In a multivalent polypeptide, two or more nanobodies may be the same or different. For example, two or more nanobodies in a multivalent polypeptide may be · directed against the same antigen, i.e., the same portion or epitope of the antigen, or against two or more different portions or epitopes of the antigen; and / or: · directed against different antigens; · or directed against combinations thereof. Thus, a bivalent polypeptide, for example: · may comprise two identical nanobodies; · may comprise a first nanobody directed against a first portion or epitope of an antigen and a second nanobody directed against the same portion or epitope of the antigen or against another portion or epitope of the antigen; or may comprise a first nanobody directed against a first antigen and a second nanobody directed against a second antigen different from the first antigen; Whereas, a trivalent polypeptide of the invention, for example: · may comprise three identical or different nanobodies directed against the same or different portions or epitopes of the same antigen; · It may include two identical or different nanobodies directed to the same or different parts or epitopes on the first antigen and a third nanobody directed to a second antigen different from the first antigen; or · It may include a first nanobody directed to the first antigen, a second nanobody directed to a second antigen different from the first antigen, and a third nanobody directed to a third antigen different from the first and second antigens. 【0223】 The CD8 nanobodies and polypeptides disclosed herein can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and / or therapeutic purposes (e.g., as gene therapy). For this purpose, the nucleotide sequence encoding the CD8 nanobody or polypeptide disclosed herein can be inserted into a cell or tissue in any suitable manner, for example by such means (e.g., the use of liposomes), or by a suitable gene therapy vector (e.g., derived from a retrovirus (e.g., adenovirus, etc.) or parvovirus (e.g., adeno-associated virus, etc.)), and then introduced into the cell or tissue. As will be apparent to those skilled in the art, such gene therapy can be carried out in vivo and / or in situ within the body of a patient by administering the nucleic acid of the present invention or a suitable gene therapy vector encoding the same to the patient or to a specific cell or specific tissue or organ of the patient. Alternatively, suitable cells (such as explanted lymphocytes, bone marrow aspirates or tissue biopsies, etc., which are often taken from the body of the patient to be treated) can be treated in vitro with the nucleotide sequence of the present invention and then appropriately (re)introduced into the patient's body.All of these can be carried out using gene therapy vectors, techniques and delivery systems well known to those skilled in the art, such as those described in Culver, K.W., “Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.), Giordano, Nature F Medicine 2(1996), 534 - 539; Schaper, Circ.Res. 79(1996), 911 - 919; Anderson, Science 256(1992), 808 - 813; Verma, Nature 389(1994), 239; Isner, Lancet 348(1996), 370 - 374; Muhlhauser, Circ.Res. 77(1995), 1077 - 1086; Onodera, Blood 91;(1998), 30 - 36; Verma, Gene Ther. 5(1998), 692 - 699; Nabel, Ann.N.Y.Acad.Sci.:811(1997), 289 - 292; Verzeletti, Hum.Gene Ther. 9(1998), 2243 - 51; Wang, Nature Medicine 2(1996), 714 - 716; International Publication No. 94 / 29469 pamphlet; International Publication No. 97 / 00957 pamphlet, U.S. Patent No. 5,580,859 specification; 1 U.S. Patent No. 5,589,5466 specification; or Schaper, Current Opinion in Biotechnology 7(1996), 635 - 640. For example, in - situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850 - 1859(2003)) and of diabodies (Blanco et al., J.Immunol, 171, 1070 - 1077(2003)) is described in the art. 【0224】 Accordingly, provided are also nucleic acid sequences encoding the CD8 nanobodies described herein, as well as expression constructs and host cells comprising the nucleic acid sequences. 【0225】 Also disclosed are methods of using the CD8 nanobodies and polypeptides of the present disclosure. 【0226】 In some embodiments, as described herein, a polypeptide comprising a CD8 nanobody can be used in the lipid nanoparticles of the present disclosure for delivering nucleic acids to immune cells. In some embodiments, the CD8 nanobody and polypeptide of the present disclosure can be used to treat a condition or disease in a subject in need thereof. In some embodiments, such conditions or diseases include, but are not limited to, cancer, infectious diseases, immune disorders, autoimmune diseases. 【0227】 In some embodiments, a polypeptide comprising a CD8 nanobody can be used as an imaging agent. In some embodiments, the imaging agent enables the detection of human CD8, a specific biomarker found on the surface of a subset of T cells for the imaging diagnosis of the immune system. Imaging of CD8 enables in vivo detection of T cell localization. Changes in T cell localization can reflect the progression of the immune response and can occur over time as a result of various therapeutic treatments or disease states. In some embodiments, it is used for imaging T cell localization for immunotherapy. 【0228】 Furthermore, CD8 plays a role in activating an important downstream signaling pathway that functions to eliminate viral pathogens and provide immunity against tumors. CD8-positive T cells can recognize short peptides presented within the MHC I protein of antigen-presenting cells. In some embodiments, a polypeptide comprising a CD8 nanobody can enhance signal transduction via the T cell receptor and enhance the ability of a subject to eliminate viral pathogens and respond to tumor antigens. Thus, in some embodiments, the antigen-binding construct provided herein can be an agonist and can activate the CD8 target. 【0229】 II. Ionizable Cationic Lipids Provided herein are ionizable cationic lipids that can be used to generate lipid nanoparticle compositions for facilitating delivery of a payload (e.g., a nucleic acid, such as DNA or RNA, such as mRNA) disposed therein into cells, such as mammalian cells, such as immune cells. The ionizable cationic lipids enable intracellular delivery of nucleic acids, such as mRNA, into the cytosolic compartment of the target cell type and are designed to rapidly degrade into non-toxic components. The complex functionality of the ionizable cationic lipids is facilitated by the interaction between the chemical nature and geometric shape of the ionizable lipid headgroup, the hydrophobic “acyl tail” group, and the linker connecting the headgroup and the acyl tail group. Typically, the pK a of the ionizable amine headgroup is designed to be in the range of 6-8, such as 6.2-7.4 or 6.7-7.2, such that it remains strongly cationic (e.g., pH 4 - pH 5.5) under acidic formulation conditions, neutral or slightly anionic (7.4) at physiological pH, and cationic (e.g., pH 5.5 - pH 7) in early and late endosomal compartments. The acyl tail groups play an important role in membrane destabilization by fusion of the lipid nanoparticles with the endosomal membrane and structural perturbation. The three-dimensional structure of the acyl tail (determined by its length as well as degree and site of unsaturation) is thought to play a role in membrane fusion and thus promotion of lipid nanoparticle endosomal escape (an important requirement for cytosolic delivery of the nucleic acid payload), along with the relative sizes of the headgroup and tail group. The linker connecting the headgroup and the acyl tail group is designed to be cleaved by physiologically predominant enzymes (e.g., esterases, or proteases) or acid-catalyzed hydrolysis. 【0230】 In one aspect, the invention provides a compound of formula (I): 【Chemical formula】 or a salt thereof, wherein R 1 , R 2 and R 3 are each independently a bond or C 1~3 alkylene, R 1A , R 2A , and R 3A each independently represents a bond or C 1~10 is alkylene, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 are independently H, C 1~20 Alkyl, C 1~20 Alkenyl, -(CH2) 0~10 C(O)OR a1 , or -(CH2) 0~10 O.C.(O)R a2 and R a1 and R a2 are each independently 1~20 Alkyl or C 1~20 alkenyl, R 3B teeth, [ka] and R 3B1 is C 1~6 is alkylene, R 3B2 and R 3B3 are each independently H or C 1~6 It is an alkyl. 【0231】 In one aspect, the present invention provides a compound of formula (IA): [ka] or a salt thereof, wherein R 1 , R 2 , and R 3 each independently represents a bond or C 1~3 is alkylene, R 1A , R 2A , and R3A are each independently a bond or C 1~10 alkylene, R 1A1 、R 1A2 、R 1A3 、R 2A1 、R 2A2 、R 2A3 、R 3A1 、R 3A2 、and R 3A3 are each independently H, C 1~20 alkyl, C 1~20 alkenyl, -(CH2) 0~10 C(O)OR a1 、or -(CH2) 0~10 OC(O)R a2 wherein, R a1 and R a2 are each independently C 1~20 alkyl or C 1~20 alkenyl, R 3B is, 【Chemical formula】 wherein, R 3B1 is C 1~6 alkylene, R 3B2 and R 3B3 are each independently H, unsubstituted C 1~6 alkyl, or C 1~6 alkyl substituted with one or more substituents independently selected from the group consisting of -OH and -O-(C 1~6 alkyl). 【0232】 Any variable element or substituent provided herein is unsubstituted or substituted with one or more substituents. In some embodiments, any variable element or substituent provided herein is optionally substituted. In some embodiments, any variable element or substituent provided herein is -OR s1 、-NR s2 R s3 、-C(O)R s4 、-C(O)ORs5 , C(O)NR s6 R s7 , -OC(O)R s8 , -OC(O)OR s9 , -OC(O)NR s10 R 11 , -NR s12 C(O)R s13 , and -NR s14 C(O)OR s15 (wherein R s1 , R s2 , R s3 , R s4 , R s5 , R s6 , R s7 , R s8 , R s9 , R s10 , R s11 , R s12 , R s13 , R s14 , and R s15 is each independently H, C 1~6 alkyl, C 3~10 cycloalkyl, C 6-14 aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl, each of which is optionally substituted) and is optionally substituted with one or more substituents independently selected from the group consisting of. 【0233】 In some embodiments, R 1 , R 2 , and R 3 are each independently a bond or C 1~3 alkylene. In some embodiments, R 1 , R 2 , and R 3 are each independently a bond or methylene. In some embodiments, R 1 and R 2 are each methylene, and R 3 is a bond. In some embodiments, R 1 , R 2 , and R 3 are each methylene. In some embodiments, R 1 , R 2 , and R3 is, independently of each other, unsubstituted or substituted. In some embodiments, R 1 , R 2 , and R 3 are unsubstituted. 【0234】 In some embodiments, R 1A , R 2A , and R 3A are, independently of each other, a bond or C 1~10 alkylene. In some embodiments, R 1A , R 2A , and R 3A are, independently of each other, a bond or -(CH2) 1~10 -. In some embodiments, R 1A and R 2A are, independently of each other, a bond, -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, or -(CH2)8-. In some embodiments, R 1A and R 2A are each a bond, each -CH2-, each -(CH2)2-, each -(CH2)3-, each -(CH2)4-, each -(CH2)5-, each -(CH2)6-, each -(CH2)7-, or each -(CH2)8-. In some embodiments, R 1A and R 2A are, independently of each other, a bond, -(CH2)2-, -(CH2)4-, -(CH2)6-, -(CH2)7-, or -(CH2)8-. In some embodiments, R 1A and R 2A are each a bond, each -(CH2)2-, each -(CH2)4-, each -(CH2)6-, each -(CH2)7-, or each -(CH2)8-. In some embodiments, R 3A is a bond, -CH2-, -(CH2)2-, or -(CH2)7-. In some embodiments, R 1A , R 2A , and R 3Ais, independently of each other, unsubstituted or substituted. In some embodiments, R 1A , R 2A , and R 3A are unsubstituted. 【0235】 In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 are, independently of each other, H, C 1~20 alkyl, C 1~20 alkenyl, -(CH2) 0~10 C(O)OR a1 , or -(CH2) 0~10 OC(O)R a2 . In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 are, independently of each other, H, C 1~15 alkyl, -CH=CH-(C 1~15 alkyl), -CH=CH-CH2-CH=CH-(C 1~10 alkyl), -(CH2) 0~4 C(O)OCH(C 1~10 alkyl)(C 1~15 alkyl), -(CH2) 0~4 OC(O)CH(C 1~10 alkyl)(C 1~15 alkyl), -(CH2) 0~4 C(O)OCH2(C 1~15 alkyl), or -(CH2) 0~4 OC(O)CH2(C 1~15 alkyl). In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3, R 3A1 , R 3A2 , R 3A3 , R 1 , R 2 , R 3 , R 1A , R 2A , and R 3A is, independently of one another, unsubstituted or substituted. In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , R 3A3 , R 1 , R 2 , R 3 , R 1A , R 2A , and R 3A is unsubstituted. In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 is, independently of one another, unsubstituted or substituted. In some embodiments, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 is unsubstituted. In some embodiments, R 1 , R 2 , R 3 , R 1A , R 2A , and R 3A is, independently of one another, unsubstituted or substituted. In some embodiments, R 1 , R 2 , R 3 , R 1A , R 2A , and R 3Ais unsubstituted in each case. In some embodiments, R 1 , R 2 , and R 3 are each unsubstituted. 【0236】 In some embodiments, R 3B1 is unsubstituted. In some embodiments, R 3B1 is not substituted with oxo. 【0237】 In some embodiments, R 1A1 and R 2A1 are each independently -CH=CH-(C 1~15 alkyl), -CH=CH-CH2-CH=CH-(C 1~10 alkyl), -(CH2) 0~4 C(O)OCH(C 1~10 alkyl)(C 1~15 alkyl), or -(CH2) 0~4 OC(O)CH(C 1~10 alkyl)(C 1~15 alkyl), and R 1A2 , R 1A3 , R 2A2 , and R 2A3 are each H. In some embodiments, R 1A1 and R 2A1 are each -CH=CH-(C 1~15 alkyl), -CH=CH-CH2-CH=CH-(C 1~10 alkyl), -(CH2) 0~4 C(O)OCH(C 1~10 alkyl)(C 1~15 alkyl), or -(CH2) 0~4 OC(O)CH(C 1~10 alkyl)(C 1~15 alkyl), and R 1A2 , R 1A3 , R 2A2 , and R 2A3 are each H. In some embodiments, R 1A1 and R 2A1 are each 【Chemical Formula】 It is. In some embodiments, R 1A1 and R 2A1 are each 【Chemical formula】 It is. In some embodiments, R 1A2 , R 1A3 , R 2A2 , and R 2A3 are each H. 【0238】 In some embodiments, R 1A1 and R 2A1 are each C 1~15 alkyl, R 1A2 and R 2A2 are each C 1~15 alkyl, R 1A3 and R 2A3 are each H. In some embodiments, R 1A1 and R 2A1 are each 【Chemical formula】 It is. In some embodiments, R 1A2 and R 2A2 are each 【Chemical formula】 It is. In some embodiments, R 1A3 and R 2A3 are each H. In some embodiments, R 1A and R 2A are each a bond. 【0239】 In some embodiments, R 1A1 and R 2A1 are each -(CH2) 0~4 OC(O)CH2(C 1~15 alkyl), R 2A1 and R 2A2 are each -(CH2) 0~4 C(O)OCH2(C 1~15 alkyl), R 1A3and R 2A3 are each H. In some embodiments, R 1A1 and R 2A1 are each 【Chemical formula】 and R 2A1 and R 2A2 are each 【Chemical formula】 In some embodiments, R 1A3 and R 2A3 are each H. In some embodiments, R 1A and R 2A are each a bond. 【0240】 In some embodiments, R 1A1 and R 2A1 are each -C(O)OCH2(C 1~15 alkyl), and R 1A2 and R 2A2 are each -(CH2) 0~4 C(O)OCH2(C 1~15 alkyl), and R 1A3 and R 2A3 are each H. In some embodiments, R 1A1 and R 2A1 are each 【Chemical formula】 and R 1A2 and R 2A2 are each 【Chemical formula】 In some embodiments, R 1A1 and R 2A1 are each 【Chemical formula】 and R 2A1 and R 2A2 are each [Chemical formula] It is. In some embodiments, R 1A3 and R 2A3 are each H. In some embodiments, R 1A and R 2A are each a bond. 【0241】 In some embodiments, R 3A1 , R 3A2 , and R 3A3 are each independently H, C 1~15 alkyl, -(CH2) 0~4 C(O)OCH(C 1~5 alkyl)(C 1~10 alkyl), -(CH2) 0~4 OC(O)CH(C 1~5 alkyl)(C 1~10 alkyl), -(CH2) 0~4 C(O)OCH2(C 1~10 alkyl), or -(CH2) 0~4 OC(O)CH2(C 1~10 alkyl). 【0242】 In some embodiments, R 3A1 and R 3A2 are each independently C 1~15 alkyl, and R 3A3 is H. In some embodiments, R 3A1 and R 3A2 are each independently ethyl, propyl, butyl, pentyl, hexyl, or heptyl. In some embodiments, R 3A1 and R 3A2 are each independently ethyl, [Chemical formula] It is. In some embodiments, R 3A3 is H. In some embodiments, R 3A is a bond. 【0243】 In some embodiments, R 3A1 is C 1~15 alkyl, and R 3A2 and R 3A3 are each H. In some embodiments, R 3A1 is [Chemical formula] In some embodiments, R 3A2 and R 3A3 are each H. In some embodiments, R 3A is a bond. 【0244】 In some embodiments, R 3A1 is -C(O)OCH(C 1~5 alkyl)(C 1~10 alkyl), and R 3A2 and R 3A3 are each H. In some embodiments, R 3A1 is [Chemical formula] In some embodiments, R 3A1 is [Chemical formula] In some embodiments, R 3A is ethylene or -(CH2)2-. In some embodiments, R 3A2 and R 3A3 are each H. 【0245】 In some embodiments, R 3A1 is -(CH2) 0~4 OC(O)CH2(C 1~10 alkyl), R 3A2 is -(CH2) 0~4 (O)OCH2(C 1~10 alkyl), and R 3A3 is H. In some embodiments, R 3A1 is [Chemical formula] and R 3A2 is 【Chem.】 is. In some embodiments, R 3A3 is H. In some embodiments, R 3A is a bond. 【0246】 In some embodiments, R 3A1 is -(CH2) 0~4 C(O)OCH2(C 1~10 alkyl), and R 3A2 is -(CH2) 0~4 C(O)OCH2(C 1~10 alkyl), and R 3A3 is H. In some embodiments, R 3A1 is 【Chem.】 is, and R 3A2 is 【Chem.】 is. In some embodiments, R 3A3 is H. In some embodiments, R 3A is a bond. 【0247】 In some embodiments, R 3A1 , R 3A2 , and R 3A3 are each H. 【0248】 R a1 and R a2 are each independently C 1~20 alkyl or C 1~20 alkenyl. In some embodiments, R a1 and R a2 are each independently -(CH2) 0~15 CH3 or -CH(C 1~10 alkyl)(C 1~15is (alkyl). In some embodiments, R a1 and R a2 are each independently 【Chemical formula】 and each of them is optionally substituted. In some embodiments, R a1 and R a2 are each independently unsubstituted or substituted. In some embodiments, R a1 and R a2 are unsubstituted. 【0249】 In some embodiments, R 3B is 【Chemical formula】 In some embodiments, R 3B is H. In some embodiments, R 3B is unsubstituted or substituted. In some embodiments, R 3B is unsubstituted. 【0250】 In some embodiments, R 3B1 is C 1~6 alkylene. In some embodiments, R 3B1 is ethylene or propylene. In some embodiments, R 3B1 is unsubstituted or substituted. In some embodiments, R 3B1 is optionally substituted. 【0251】 In some embodiments, R 3B2 and R 3B3 are each independently optionally substituted. In some embodiments, R 3B2 and R 3B3 are each independently H or C 1~6 alkyl optionally substituted with one or more substituents independently selected from the group consisting of -OH and -O-(C 1~6 alkyl). In some embodiments, R3B2 and R 3B3 is, independently of one another, H, or -OR s1 , -NR s2 R s3 , -C(O)R s4 , -C(O)OR s5 , C(O)NR s6 R s7 , -OC(O)R s8 , -OC(O)OR s9 , -OC(O)NR s10 R 11 , -NR s12 C(O)R s13 , and -NR s14 C(O)OR s15 is one or more optionally substituted C 1~6 alkyl selected from the group consisting of s1 R s2 R s3 R s4 R s5 R s6 R s7 R s8 R s9 R s10 R s11 R s12 R s13 R s14 , and R s15 is, independently of one another, H, C 1~6 alkyl, C 3~10 cycloalkyl, C 6~14 aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl, each of which is optionally substituted. In some embodiments, R 3B2 and R 3B3 are, independently of one another, H, methyl, ethyl, propyl, butyl or pentyl, each of which is optionally substituted with one or more substituents independently selected from the group consisting of -OH and -O-(C 1~6 alkyl). In some embodiments, R 3B2 and R 3B3 are, independently of one another, methyl or ethyl, each optionally substituted with one or more -OH. In some embodiments, R 3B2 and R3B3 is each methyl or each ethyl, and each may be optionally substituted with one or more -OH. In some embodiments, R 3B2 and R 3B3 are each unsubstituted methyl. 【0252】 In some embodiments, 【Chemical formula】 is each optionally substituted, 【Chemical formula】 is as follows. 【0253】 In one aspect, the present invention provides a compound represented by formula (Ia): 【Chemical formula】 or a salt thereof, wherein R 1A , R 2A , R 3A , R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , R 3A3 , R 3B1 , R 3B2 , and R 3B3 are as defined for formula (I) or any variation or embodiment thereof. 【0254】 In one aspect, the present invention provides a compound represented by formula (Ib): 【Chemical formula】 or a salt thereof, wherein R 1A , R 2A , R 3A , R 1A1 , R 1A2 , R 1A3 , R 2A1 , R2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 is as defined for formula (I) or any variation or embodiment thereof. 【0255】 III. Lipid-Immune Cell Targeting Group Conjugates As discussed herein, LNPs can target specific cell types, such as immune cells, such as T cells, B cells, or natural killer (NK) cells. This can be achieved by using one or more of the lipids described herein. Additionally, targeting can be enhanced by including a targeting group on a surface of the LNP particle that is accessible to the solvent. For example, the targeting group can include a member of a specific binding pair, such as an antibody-antigen pair, a ligand-receptor pair, etc. In certain embodiments, the targeting group is an antibody. Targeting can be performed, for example, by using the lipid-immune cell targeting group conjugates described herein. 【0256】 Optionally, the targeting moiety is an antibody fragment that does not contain an Fc component. Previous attempts to target circulating immune cells with LNPs have used full antibodies (WO 2016 / 189532 pamphlet). Liposomes or lipid-based particles with conjugated full antibodies are removed more rapidly from circulation due to Fc binding, reducing their likelihood of reaching the target cells of interest (Harding et al. (1997) Biochim Biophys. Acta 1327, 181-192; Sapra et al. (2004) Clin Cancer Res 10, 1100-1111; Aragnol et al., (1986) Proc Natl Acad Sci USA 83, 2699-2703). Liposomes targeted with antibody fragments retain their long-circulation properties, such as liposomes targeted against EGFR (Mamot et al., (2005) Cancer Res 65, 11631-11638), ErbB2 (Park et al. (2002) Clin Cancer Res 8, 1172-1181), or EphA2 (Kamoun et al., 2019 Nat. Biomed. Eng 3, 264-280). Furthermore, lipid-based carriers allow for nearly quantitative incorporation of their separate post-production antibody conjugation, compared to the very inefficient insertion when binding full IgG (Ishida et al. (1999) FEBS Lett. 460, 129-133), using a micelle insertion process (Nellis et al. (2005) Biotechnol Prog 21, 221-232), or the need to complete conjugation directly on intact LNPs (WO 2016 / 189532 pamphlet). It is also possible to directly conjugate scFv, Fab, or VHH fragments to activating PEG-lipids to create insertable conjugates. 【0257】 In some embodiments, PEG-(lipid) is equivalent to (lipid)-PEG. 【0258】 In certain embodiments, the targeting moiety can be a surface-bound antibody or a surface-bound antigen-binding fragment thereof that can enable modulation of cell targeting specificity. This is particularly useful because highly specific antibodies can be generated against the epitope of interest for the desired targeting site. In one embodiment, multiple different antibodies can be incorporated into the LNP and presented on the surface of the LNP, with each antibody binding to a different epitope on the same antigen or different epitopes on different antigens. Such an approach can increase the binding activity and specificity of the targeting interaction to specific target cells. 【0259】 The targeting agent or combination of targeting agents can be selected based on the desired localization, function, or structural characteristics of a given target cell. For example, to target a T cell, T cell population, or T cell subset, one or more antibodies, antigen-binding fragments, or antigen-binding derivatives thereof that target the T cell via a T cell surface antigen, etc. may be selected. Exemplary T cell surface antigens include, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD39, CD69, CD103, CD137, CD45, T cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, GL7, TLR2, TLR4, TLR5, and IL-15 receptor, but are not limited thereto. To target an NK cell or NK cell population, one or more antibodies, antigen-binding fragments, or antigen-binding derivatives thereof that target the NK cell via an NK cell surface antigen, for example, may be selected. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, CD337 / NKP30. To target a B cell or B cell population, one or more antibodies, antigen-binding fragments, or antigen-binding derivatives thereof that target the B cell via a B cell antigen, etc. may be selected. Exemplary B cell antigens include CD19 of all B cells except plasma cells, CD19, CD25, and CD30 of activated B cells, CD27, CD38, CD78, CD138, and CD319 of plasma cells, CD20, CD27, CD40, CD80, and PDL-2 of memory cells, Notch2, CD1, CD21, and CD27 of marginal zone B cells, CD21, CD22, and CD23 of follicular B cells, and CD1, CD5, CD21, CD24, and TLR4 of regulatory B cells, but are not limited thereto. 【0260】 In certain embodiments, targeting can be effected, for example, by using a lipid-immune cell targeting group conjugate as described herein. Exemplary lipid-immune cell targeting group conjugates are of formula (II), [lipid]-[any linker]-[immune cell targeting group, e.g., a T cell targeting molecule, e.g., an anti-CD2 antibody, anti-CD3 antibody, anti-CD7 antibody or anti-CD8 antibody] (Formula II) and may include compounds of. 【0261】 In some embodiments, the immune cell targeting group is a polypeptide and the lipid is conjugated either at the N-terminus, C-terminus, or somewhere in the central portion of the polypeptide. 【0262】 In certain embodiments, the targeting group or targeting molecule is a T cell targeting agent, e.g., an antibody, that binds to a T cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor. In certain embodiments, the T cell antigen can be CD2 and the targeting group can be, for example, an anti-CD2 antibody. In certain embodiments, the T cell antigen can be CD3 and the targeting group can be, for example, an anti-CD3 antibody. In certain embodiments, the T cell antigen can be CD4 and the targeting group can be, for example, an anti-CD4 antibody. In certain embodiments, the T cell antigen can be CD5 and the targeting group can be, for example, an anti-CD5 antibody. In certain embodiments, the T cell antigen can be CD7 and the targeting group can be, for example, an anti-CD7 antibody. In certain embodiments, the T cell antigen can be CD8 and the targeting group can be, for example, an anti-CD8 antibody. In certain embodiments, the T cell antigen can be TCRβ and the targeting group can be, for example, an anti-TCRβ antibody. In some embodiments, the antibody is a human antibody or a humanized antibody. 【0263】 Exemplary CD2 binders can be antibodies selected from the group consisting of 9.6 (https: / / academic.oup.com / intimm / article / 10 / 12 / 1863 / 744536), 9-1 (https: / / academic.oup.com / intimm / article / 10 / 12 / 1863 / 744536), TS2 / 18.1.1 (ATCC HB-195), Lo-CD2b (ATCC PTA-802), Lo-CD2a / BTI-322 (U.S. Patent No. 6,849,258 B1), Sipilzumab / MEDI-507 (U.S. Patent No. 6,849,258 B1 / en), 35.1 (ATCC HB-222), OKT11 (ATCC CRL-8027), RPA-2.1 (PCT Application Publication WO 2020 / 023559 A1 Pamphlet), AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 (R&D Systems), MAB1856 (R&D Systems), PAB30359 (Abnova Corporation), 10299-1 (Abnova Corporation), and binding fragments thereof. In certain embodiments, the binder is the heavy chain variable domain (V H ) and the light chain variable domain (V L) and includes. In certain embodiments, the binder includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, which are determined under any other CDR determination method known in the art for the V H and V L sequences. It is determined under any other CDR determination method known in the art. 【0264】 Exemplary CD2 binders can also be selected from antibodies or antibody fragments using the CDRs of clone 9.6, 9-1, TS2 / 18.1.1, Lo-CD2b, Lo-CD2a, BTI-322, sipuleucel-T, 35.1, OKT11, RPA-2.1, SQB-3.21, LT2, TS1 / 8, UT329, 4F22, OX-34, UQ2 / 42, MU3, U7.4, NFN-76, or MOM-181-4-F(E). 【0265】 Exemplary CD3 binders (CD3γ / δ / ε, CD3γ, CD3δ, CD3γ / ε, CD3δ / ε, or CD3ε) can be antibodies selected from the group consisting of MEM-57 (CD3γ / δ / ε, Enzo Life Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Abnova Corporation), CD3-12 (CD3ε, Cell Signaling Technology), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, Novus Biologicals), 16669-1-AP (CD3δ, Invitrogen), and antigen-binding fragments thereof. In certain embodiments, the binder is the V H domain and V LIt includes domains. In certain embodiments, the binder includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, which are according to Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., see Chothia C & Lesk A M, (1987), J.MOL.BIOL. 196:901-917), MacCallum (see MacCallum R M et al., (1996) J.MOL.BIOL. 262:732-745), or MEM-57 (CD3γ / δ / ε, Enzo Life Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Abnova Corporation), CD3-12 (CD3ε, Cell Signaling Technology), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, Novus Biologicals) and 16669-1-AP (CD3δ, Invitrogen) and are determined under any other CDR determination method known in the art for the V H sequence and V L sequence determined under any other CDR determination method known in the art. 【0266】 Exemplary CD3 binders can also be selected from antibodies or antibody fragments using the CDRs of clone hsp34, OKT-3, UCHT1, 38.1, HIT3a, RFT8, SK7, BC3, SP34-2, HU291, TRX4, Katumaxomab, Teplizumab, 3-106, 3-114, 3-148, 3-190, 3-271, 3-550, 4-10, 4-48, H2C, F12Q, I2C, SP7, 3F3A1, CD3-12, 301, RIV9, JB38-29, JE17-74, GT0013, 4E2, 7A4, 4D10A6, SPV-T3b, M2AB, ICO-90, 30A1 or Hu38E4.v1 (U.S. Patent Application Publication No. 20200299409A1), REGN5458 (U.S. Patent Application Publication No. 20200024356A1), blinatumomab (https: / / go.drugbank.com / drugs / DB09052 / polypeptide_sequences.fasta). In some embodiments, the conjugate comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 2 or 3. 【0267】 Exemplary CD4 binders can be antibodies selected from the group consisting of ibalizumab (https: / / www.genome.jp / dbget-bin / www_bget?D09575), AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), CAL4 (Abcam) and antigen-binding fragments thereof. In certain embodiments, the binder is the V H domain and V LIt includes domains. In certain embodiments, the binder includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, which are determined under any other CDR determination method known in the art of the V and V sequences of antibodies selected from the group consisting of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., see Chothia C & Lesk A M, (1987), J.MOL.BIOL.196:901-917), MacCallum (see MacCallum R M et al., (1996) J.MOL.BIOL.262:732-745), or AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abcam). H and V L sequences are determined under any other CDR determination method known in the art of the relevant technical field. 【0268】 Exemplary CD4 binders can also be selected from antibodies or antibody fragments using the CDRs of clone ibalizumab, OKT4, RPA-T4, S3.5, SK3, N1UG0, RIV6, OTI18E3, MEM-241, B486A1, RFT-4g, 7E14, MDX.2, MEM-115, MEM-16, ICO-86, Edu-2, or ibalizumab. 【0269】 Exemplary CD5 binders can be antibodies selected from the group consisting of He3, MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Abcam), CD5 / 54 / F6 (Abcam), 65152 (Proteintech), and antigen-binding fragments thereof. In some embodiments, the binder is the V H domain and V L thereof. In certain embodiments, the binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, which are determined under Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., see Chothia C & Lesk A M, (1987), J. MOL. BIOL. 196:901-917), MacCallum (see MacCallum R M et al., (1996) J. MOL. BIOL. 262:732-745), or any other CDR determination method known in the art for the V H and V L sequences of antibodies selected from the group consisting of MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Abcam), CD5 / 54 / F6 (Abcam), and 65152 (Proteintech). 【0270】 Exemplary CD5 binders can also be selected from antibodies or antibody fragments using the CDRs of clones of zolimomab, 5D7, L17F12, and UCHT2, 1D8, 3I21, 4H10, 8J23, 5O4, 4H2, 5G2, 8G8, 6M4, 2E3, 4E24, 4F10, 7J9, 7P9, 8E24, 6L18, 7H7, 1E7, 8J21, 7I11, 8M9, 1P21, 2H11, 3M22, 5M6, 5H8, 7I19, 1A2, 8E15, 8C10, 3P16, 4F3, 5M24, 5O24, 7B16, 1E8, 2H16, BLa1, 1804, DK23, Cris1, MEM-32, H65, 4C7, OX-19, Leu-1, 53-7.3, 4H8E6, T101, EP2952, D-9, H-3, HK231, N-20, Y2 / 178, H-300, CD5 / 54 / F6, Q-20, CC17, MOM-18539-S(P), or MOM-18885-S(P). 【0271】 Exemplary CD7 binders can be antibodies selected from the group consisting of MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), NBP2-38440 (Novus Biologicals), and antigen-binding fragments thereof. In certain embodiments, the binder is the V H domain and V LIt includes. In certain embodiments, the binder includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, which are determined under any other CDR determination method known in the art of the V H and V L sequences of antibodies selected from the group consisting of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., see Chothia C & Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (see MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745), or MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), and NBP2-38440 (Novus Biologicals). 【0272】 Exemplary CD7 binders can also be selected from antibodies or antibody fragments using the CDRs of clone TH-69, 3Afl1, T3-3A1, 124-1D1, 3A1f, CD7-6B7, or VHH6. 【0273】 Exemplary CD8 (CD8α, CD8α / α, CD8α / β, or CD8β) binders can be antibodies selected from the group consisting of 2.43 (Invitrogen), Du CD8-1 (CD8α, Invitrogen), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abcam), C8 / 144B (CD8α, Novus Biologicals), YTS105.18 (CD8α, Novus Biologicals), TRX2 (https: / / patents.justia.com / patent / 20170198045), and antigen-binding fragments thereof. In certain embodiments, the binder is the V H domain and V LIt includes a domain. In certain embodiments, the binder includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, which are determined under any other CDR determination method known in the art of the V H and V L sequences of the antibodies selected from the group consisting of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., see Chothia C & Lesk A M, (1987), J.MOL.BIOL.196:901-917), MacCallum (see MacCallum R M et al., (1996) J.MOL.BIOL.262:732-745), or 2.43 (Invitrogen), Du CD8-1 (CD8α, Invitrogen), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abcam), C8 / 144B (CD8α, Novus Biologicals), and YTS105.18 (CD8α, Novus Biologicals). 【0274】 Exemplary CD8 binders can also be selected from antibodies or antibody fragments using the CDRs of clone OKT-8, 51.1, S6F1, TRX2, and UCHT4, SP16, 3B5, C8-144B, HIT8a, RAVB3, LT8, 17D8, MEM-31, MEM-87, RIV11, DK-25, YTC141.1HL, or YTC182.20. In some embodiments, the conjugate includes a Fab that includes a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 7. 【0275】 Exemplary CD137 binders can be selected from antibodies or antibody fragments using the CDRs of clone 4B4-1, P566, or urelumab. Exemplary CD28 binders can be selected from antibodies or antibody fragments using the CDRs of clone TAB08. Exemplary CD45 binders can be selected from antibodies or antibody fragments using the CDRs of clone BC8, 9.4, 4B2, Tu116, or GAP8.3. Exemplary CD18 binders can be selected from antibodies or antibody fragments using the CDRs of clone 1B4, TS1 / 18, MEM-48, YFC118-3, TA-4, MEM-148, or R3-3, 24. Exemplary CD11a binders can be selected from antibodies or antibody fragments using the CDRs of clone MHM24 or efalizumab. Exemplary IL-2 receptor binders can be selected from the group consisting of antibodies or antibody fragments using the CDRs of clone YTH 906.9HL, IL2R.1, BC96, B-B10, 216, MEM-181, ITYV, MEM-140, ICO-105, daclizumab, or IL2 or fragments of IL2. Exemplary IL-15R binders can be selected from the group consisting of antibodies or antibody fragments using the CDRs of clone JM7A4 or OTI3D5, or IL15 or fragments of IL15. Exemplary TLR2 binders can be selected from antibodies or antibody fragments using the CDRs of clone JM22-41, TL2.1, 11G7, or TLR2.45. Exemplary TLR4 binders can be selected from antibodies or antibody fragments using the CDRs of clone HTA125 or 76B357-1. Exemplary TLR5 binders can be selected from antibodies or antibody fragments using the CDRs of clone 85B152-5 or 9D759-2. Exemplary GL7 binders can be selected from antibodies or antibody fragments using the CDRs of clone GL7. 【0276】 Exemplary PD1 binders can be selected from antibodies or antibody fragments using the CDRs of clone MIH4, J116, J150, OTIB11, OTI17B10, OTI3A1 or OTI16D4. Further, exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136; 8,779,105; 8,008,449; 8,741,295; 9,205,148; 9,181,342; 9,102,728; 9,102,727; 8,952,136; 8,927,697; 8,900,587; 8,735,553 and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135; 7,943,743; 9,175,082; 8,741,295; 8,552,154 and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab and BMS 936559 (Bristol Myers Squibb Co.). 【0277】 Exemplary CTLA-4 binders can be selected from antibodies or antibody fragments using the CDRs of clone ER4.7G.11 [7G11], OTI9G4, OTI9F3, OTI3A5, A3.4H2.H12, 14D3, OTI3A12, OTI1A11, OTI1E8, OTI3B11, OTI3D2, OTI10C8, OTI2E9, OTI6F1, OTI7D3, OTI85B, OTI12C6. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International Publication Pamphlets of International Application (PCT) No. 98 / 42752, No. 00 / 37504, and No. 01 / 14424, and European Patent No. 1212422B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab. 【0278】 Exemplary TCRβ binders can be antibodies selected from the group consisting of H57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα / β, Abcam), E6Z3S (TRBC1 / TCRβ, Cell Signaling Technology), and antigen-binding fragments thereof. In certain embodiments, the binder is the V H domain and V LIt includes. In certain embodiments, the binder includes heavy chain CDR1, CDR2 and CDR3, and light chain CDR1, CDR2 and CDR3, which are determined under any other CDR determination method known in the art of the V H and V L sequences. 【0279】 Exemplary CD137 binders can be selected from antibodies or antibody fragments using the CDRs of clone 4B4-1, P566 or urelumab. 【0280】 In some embodiments, the immune cell targeting moiety comprises an antibody selected from the group consisting of Fab, F(ab’)2, Fab’-SH, Fv, and scFv fragments. In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the immune cell targeting moiety comprises a Fab or an immunoglobulin single variable domain, such as a nanobody. In some embodiments, the immune cell targeting moiety comprises a Fab that does not contain a native interchain disulfide bond. For example, in some embodiments, the Fab comprises a heavy chain fragment containing a C233S substitution and / or a light chain fragment containing a C214S substitution, according to the Kabat numbering. In some embodiments, the immune cell targeting moiety comprises a Fab that contains one or more non-native interchain disulfide bonds. In some embodiments, the interchain disulfide bond is between two non-native cysteine residues on the light chain fragment and the heavy chain fragment, respectively. For example, in some embodiments, the Fab comprises a heavy chain fragment containing an F174C substitution and / or a light chain fragment containing an S176C substitution, according to the Kabat numbering. In some embodiments, the Fab comprises a heavy chain fragment containing F174C and C233S substitutions and a light chain fragment containing S176C and / or C214S substitutions, according to the Kabat numbering. In some embodiments, the immune cell targeting moiety comprises a C-terminal cysteine residue. In some embodiments, the immune cell targeting moiety comprises a Fab that contains a cysteine at the C-terminus of the heavy chain or light chain fragment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain of the Fab and the C-terminal cysteine. For example, in some embodiments, the Fab comprises two or more amino acids derived from the antibody hinge region (e.g., a partial hinge sequence) between the C-terminus of the Fab and the C-terminal cysteine. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and the immune cell targeting moiety further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain by an amino acid linker.In some embodiments, the Fab antibody is DS Fab, NoDS Fab, bDS Fab, bDS Fab-ScFv, as demonstrated in FIG. 47. 【0281】 In some embodiments, the immune cell targeting moiety comprises an immunoglobulin single variable domain such as a nanobody (e.g., V HH ). In some embodiments, the nanobody comprises a cysteine at the C-terminus. In some embodiments, the nanobody further comprises a spacer comprising one or more amino acids between the V HH domain and the C-terminal cysteine. In some embodiments, the spacer comprises one or more glycine residues, e.g., two glycine residues. In some embodiments, the immune cell targeting moiety comprises two or more V HH domains. In some embodiments, the two or more V HH domains are linked by an amino acid linker. In some embodiments, the amino acid linker comprises one or more glycine and / or serine residues (e.g., one or more repeats of the sequence GGGGS). In some embodiments, the immune cell targeting moiety comprises a first V HH domain linked to the antibody CH1 domain and a second V HH domain linked to the antibody light chain constant domain, and the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds (e.g., interchain disulfide bonds). In some embodiments, the immune cell targeting moiety comprises a V HH domain linked to the antibody CH1 domain, and the antibody CH1 domain is linked to the antibody light chain constant domain by one or more disulfide bonds. In some embodiments, the CH1 domain comprises F174C and C233S substitutions according to Kabat numbering, and the light chain constant domain comprises S176C and C214S substitutions. In some embodiments, the antibody is ScFv, V HH , 2xV HH , V HH -CH1 / null Vk, or V HH 1-CH1 / V HH -2-Nb bDS, as demonstrated in FIG. 31. 【0282】 The exemplary targeting moiety may have the amino acid sequences shown below. Anti-CD3 hSP34-Fab sequence: hSP34 heavy chain (HC) sequence (SEQ ID NO: 1): 【Chem.】 hSP34-mlam light chain (LC) sequence (mouse lambda) (SEQ ID NO: 2): 【Chem.】 SP34-hlam LC (human lambda) (SEQ ID NO: 3): 【Chem.】 Anti-CD3 Hu291-Fab sequence: Hu291 HC (SEQ ID NO: 4): 【Chem.】 Hu 291 LC (SEQ ID NO: 5): 【Chem.】 Anti-CD8 TRX2-Fab sequence: TRX2 HC (SEQ ID NO: 6): 【Chem.】 TRX2 LC (SEQ ID NO: 7): 【Chem.】 Anti-CD8 OKT8-Fab sequence: OKT8 HC (SEQ ID NO: 8): 【Chem.】 OKT8 LC (SEQ ID NO: 9): 【Chem.】 Anti-CD4 ibalizumab-Fab sequence: Ibalizumab HC (SEQ ID NO: 10): 【Chemical Structure】 Ibalizumab LC (SEQ ID NO: 11): 【Chemical Structure】 Anti-CD5 He3-Fab Sequence: He3 HC (SEQ ID NO: 12): 【Chemical Structure】 He3 LC (SEQ ID NO: 13): 【Chemical Structure】 Anti-CD7 TH-69-Fab Sequence: TH-69 HC (SEQ ID NO: 14): 【Chemical Structure】 TH-69 LC (SEQ ID NO: 15): 【Chemical Structure】 Anti-CD2 TS2 / 18.1-Fab Sequence: TS2 / 18.1 HC (SEQ ID NO: 16): 【Chemical Structure】 TS2 / 18.1 LC (SEQ ID NO: 17): 【Chemical Structure】 Anti-CD2 9.6-Fab Sequence: 9.6 HC (SEQ ID NO: 18): 【Chemical Structure】 9.6 LC (SEQ ID NO: 19): 【Chemical Structure】 Anti-CD2 9-1-Fab Sequence: 9-1 HC (SEQ ID NO: 20): 【Chem.】 9-1 LC (SEQ ID NO: 21): 【Chem.】 mutOKT8-Fab sequence: mutOKT8 HC (SEQ ID NO: 22): 【Chem.】 mutOKT8 LC (SEQ ID NO: 23): 【Chem.】 Anti-CD56 A1 Fab sequence A1 bDS HC (SEQ ID NO: 26): 【Chem.】 A1 bDS LC (SEQ ID NO: 27): 【Chem.】 Anti-CD56 A2 Fab sequence A2 bDS HC (SEQ ID NO: 28): 【Chem.】 A2 bDS LC (SEQ ID NO: 29): 【Chem.】 Anti-CD56 A3 Fab sequence A3 bDS HC (SEQ ID NO: 30): 【Chem.】 A3 bDS LC (SEQ ID NO: 31): 【Chem.】 Anti-CD56 Rolovizumab Fab sequence Rolbocizumab bDS HC (SEQ ID NO: 32): 【Chem.】 Rolbocizumab bDS LC (SEQ ID NO: 33): 【Chem.】 Anti-CD2 RPA-2.10v1 Fab sequence RPA-2.10v1 bDS HC (SEQ ID NO: 34): 【Chem.】 RPA-2.10v1 bDS LC (SEQ ID NO: 35): 【Chem.】 Anti-CD137 4B4-1 Fab sequence 4B4-1 bDS HC (SEQ ID NO: 36): 【Chem.】 4B4-1 bDS LC (SEQ ID NO: 37): 【Chem.】 hSP34-hlam NoDS HC (SEQ ID NO: 38): 【Chem.】 hSP34-hlam NoDS LC (SEQ ID NO: 39): 【Chem.】 hSP34-hlam DS HC (SEQ ID NO: 40): 【Chem.】 hSP34-hlam DS LC (SEQ ID NO: 41): 【Chem.】 Anti-CD2 TS2 / 18.1 DS Fab TS2 / 18.1 DS HC (SEQ ID NO: 42): 【Chem.】 TS2 / 18.1 DS LC (SEQ ID NO: 43): 【Chem.】 Anti-CD2 9.6 DS Fab 9.6 DS HC (SEQ ID NO: 44): 【Chem.】 9.6 DS LC (SEQ ID NO: 45): 【Chem.】 hSP34-hlam bDS HC (SEQ ID NO: 46): 【Chem.】 hSP34-hlam bDS LC (SEQ ID NO: 47): 【Chem.】 Anti-CD3 TR66 bDS Fab Sequence TR66 bDS HC (SEQ ID NO: 48): 【Chem.】 TR66 bDS LC (SEQ ID NO: 49): 【Chem.】 Anti-CD3 TRX4 bDS Fab Sequence TRX4 bDS HC (SEQ ID NO: 50): 【Chem.】 TRX4 bDS LC (SEQ ID NO: 51): 【Chem.】 Anti-CD3 HzUCHT1 bDS Fab sequence HzUCHT1(Y59T) bDS HC (SEQ ID NO: 52): 【Chem.】 HzUCHT1 bDS LC (SEQ ID NO: 53): 【Chem.】 Anti-CD3 Teplizumab bDS Fab sequence Teplizumab bDS HC (SEQ ID NO: 54): 【Chem.】 Teplizumab bDS LC (SEQ ID NO: 55): 【Chem.】 Anti-CD8 TRX2 bDS Fab sequence TRX2 bDS HC (SEQ ID NO: 56): 【Chem.】 TRX2 bDS LC (SEQ ID NO: 57): 【Chem.】 Anti-CD2 Lo-CD2b bDS Fab sequence Lo-CD2b bDS HC (SEQ ID NO: 58): 【Chem.】 Lo-CD2b bDS LC (SEQ ID NO: 59): 【Chem.】 Anti-CD2 35.1 bDS Fab sequence 35.1 bDS HC (SEQ ID NO: 60): 【Chem.】 35.1 bDS LC (SEQ ID NO: 61): 【Chem.】 Anti-CD2 OKT11 bDS Fab sequence OKT11 bDS HC (SEQ ID NO: 62): 【Chem.】 OKT11 bDS LC (SEQ ID NO: 63): 【Chem.】 Anti-CD11a HzMHM24 bDS Fab sequence HzMHM24 bDS HC (SEQ ID NO: 64): 【Chem.】 HzMHM24 bDS LC (SEQ ID NO: 65): 【Chem.】 Anti-CD18 h1B4 bDS Fab sequence h1B4 bDS HC (SEQ ID NO: 66): 【Chem.】 h1B4 bDS LC (SEQ ID NO: 67): 【Chem.】 Anti-CD18 infliximab bDS Fab sequence Infliximab bDS HC (SEQ ID NO: 68): 【Chem.】 Infliximab bDS LC (SEQ ID NO: 69): 【Chem.】 Anti-CD4 / CD8 ibalizumab / TRX2 bDS Fab-ScFv sequence Ibalizumab / TRX2 bDS Fab-ScFv HC (SEQ ID NO: 70): 【Chemical formula】 Ibalizumab / TRX2 bDS Fab-ScFv LC (SEQ ID NO: 71): 【Chemical formula】 Anti-CD4 Ibalizumab NoDS Fab sequence Ibalizumab NoDS LC (SEQ ID NO: 72): 【Chemical formula】 Ibalizumab NoDS HC (SEQ ID NO: 73): 【Chemical formula】 Anti-CD4 OKT4 bDS Fab sequence OKT4 bDS LC (SEQ ID NO: 74): 【Chemical formula】 OKT4 bDS HC (SEQ ID NO: 75): 【Chemical formula】 Anti-CD4 T023200008 Nb sequence (SEQ ID NO: 76) CDR1, CDR2, and CDR3 underlined based on the IMGT representation: 【Chemical formula】 Anti-CD8 BDSn Nb sequence (SEQ ID NO: 77) CDR1, CDR2, and CDR3 underlined based on the IMGT representation: 【Chemical formula】 Anti-CD3 T0170117G03-A Nb sequence (SEQ ID NO: 78) 【Chemical formula】 Anti-CD3 T0170060E11 Nb sequence (SEQ ID NO: 79) 【Chemical formula】 Anti-CD7 V1 Nb sequence (SEQ ID NO: 80) 【Chemical formula】 Anti-TCR T017000700 Nb sequence (SEQ ID NO: 81) CDR1, CDR2, and CDR3 underlined based on IMGT representation: 【Chemical formula】 Anti-CD28 28CD065G01 Nb sequence (SEQ ID NO: 82) 【Chemical formula】 Anti-CD3 T0170061C09 Nb sequence (SEQ ID NO: 83) 【Chemical formula】 Anti-CD3 12D2 bDS Fab sequence 12D2 bDS HC (SEQ ID NO: 84): 【Chemical formula】 12D2 bDS LC (SEQ ID NO: 85): 【Chemical formula】 Anti-CD28 8G8A Fab sequence 8G8A bDS HC (SEQ ID NO: 86): 【Chemical formula】 8G8A bDS LC (SEQ ID NO: 87): 【Chemical formula】 Anti-CD28 2E12 Fab sequence 2E12 bDS HC (SEQ ID NO: 88): [Chemical formula] 2E12 bDS LC (SEQ ID NO: 89): [Chemical formula] Anti-CD28 CD28.9.3 Fab sequence CD28.9.3 bDS HC (SEQ ID NO: 90): [Chemical formula] CD28.9.3 bDS LC (SEQ ID NO: 91): [Chemical formula] Anti-CD28 HzTN228 Fab sequence HzTN228 bDS HC (SEQ ID NO: 92): [Chemical formula] HzTN228 bDS LC (SEQ ID NO: 93): [Chemical formula] Anti-CD28 TGN2122.C Fab sequence TGN2122.C bDS HC (SEQ ID NO: 94): [Chemical formula] TGN2122.C bDS LC (SEQ ID NO: 95): [Chemical formula] Anti-CD28 TGN2122.H Fab sequence TGN2122.H bDS HC (SEQ ID NO: 96): [Chemical formula] TGN2122.H bDS LC (SEQ ID NO: 97): [Chemical formula] Anti-CD8 TRX2 ScFv sequence (SEQ ID NO: 98): 【Chem.】 V1 VHH-CH1 bDS HC (SEQ ID NO: 99): 【Chem.】 【0283】 In some embodiments, the targeting moiety comprises the polypeptide sequences disclosed herein. In some embodiments, the targeting moiety comprises all six CDRs of the polypeptide sequences disclosed herein. In some embodiments, the targeting moiety comprises CDR1, CDR2, and CDR3 of an immunoglobulin single variable domain (ISVD) disclosed herein. In further embodiments, the targeting moiety binds to the same epitope on the targeting molecule to which the polypeptide sequences disclosed herein bind. In further embodiments, the targeting moiety competes with the polypeptide sequences disclosed herein and binds to the same epitope on the targeting molecule. 【0284】 In certain embodiments, the targeting group or immunocyte targeting group (e.g., a T cell targeting agent, a B cell targeting agent, or an NK cell targeting agent) may be covalently linked to a lipid via a polyethylene glycol (PEG)-containing linker. 【0285】 In other embodiments, the lipid used to make the conjugate is distearoyl-phosphatidylethanolamine (DSPE): 【Chem.】 Dipalmitoyl-phosphatidylethanolamine (DPPE): 【Chem.】 Dimyristoyl-phosphatidylethanolamine (DMPE): [Chemical formula] Distearoyl-glycero-phosphoglycerol (DSPG): [Chemical formula] Dimyristoyl glycerol (DMG): [Chemical formula] Distearoyl glycerol (DSG): [Chemical formula] and N-palmitoyl-sphingosine (C16-ceramide) [Chemical formula] may be selected from. 【0286】 The immune cell targeting group can be covalently linked to the lipid directly or via a linker, such as a polyethylene glycol (PEG)-containing linker. In certain embodiments, the PEG is PEG 1000, PEG 2000, PEG 3400, PEG 3000, PEG 3450, PEG 4000, or PEG 5000. In certain embodiments, the PEG is PEG 2000. 【0287】 In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.001 to 0.5 mole percent, 0.001 to 0.3 mole percent, 0.002 to 0.2 mole percent, 0.01 to 0.1 mole percent, 0.1 to 0.3 mole percent, or 0.1 to 0.2 mole percent. 【0288】 In certain embodiments, the lipid immunocyte targeting agent conjugate comprises DSPE, a PEG component, and a targeting antibody. In certain embodiments, the antibody is a T cell targeting agent, such as an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD7 antibody, an anti-CD8 antibody, or an anti-TCRβ antibody. 【0289】 Exemplary lipid-immunocyte targeting group conjugates include, for example, DSPE and PEG 2000, as described in Nellis et al. (2005) BIOTECHNOL. PROG. 21, 205-220. Exemplary conjugates include a structure of formula (III), wherein scFv represents an engineered antibody binding site that binds to a target of interest. In certain embodiments, the engineered antibody binding site binds to any of the above targets. In certain embodiments, the engineered antibody binding site can be, for example, an engineered anti-CD3 antibody or an engineered anti-CD8 antibody. In certain embodiments, the engineered antibody binding site can be, for example, an engineered anti-CD2 antibody or an engineered anti-CD7 antibody. 【0290】 Examples of the compounds of formula (III) are as follows: 【Chemical formula】 It is contemplated that the scFv in formula (III) can be replaced with an intact antibody or an antigen fragment thereof (e.g., Fab). 【0291】 Another example of the compounds of formula (IV) is as follows: 【Chemical formula】 as shown, and its production is described in Nellis et al. (2005) (supra) or U.S. Patent No. 7,022,336. It is contemplated that the Fab of formula (IV) can be replaced with an intact antibody or an antigen fragment thereof (e.g., (Fab’)2 fragment) or an engineered antibody binding site (e.g., scFv). 【0292】 Other lipid-immune cell target conjugate groups are described, for example, in U.S. Patent No. 7,022,336, and the targeting group can be replaced with a targeting group of interest, for example, a targeting group that binds to the T cell or NK cell surface antigen described above. 【0293】 In certain embodiments, the lipid component of an exemplary conjugate of formula (II) can be any of the lipids described herein. In some embodiments, the lipid component of the conjugate of formula (II) is based on an ionizable cationic lipid described herein, for example, an ionizable cationic lipid of formula (I), formula (Ia), formula (Ib), or a salt thereof. For example, exemplary ionizable cationic lipids can be selected from Table 1 or a salt thereof. 【0294】 In certain embodiments, the conjugate based on the lipids of the present disclosure is 【Chemical Formula】 It includes an scFv, which represents an engineered antibody binding site that binds to the above-mentioned targets, such as CD2, CD3, CD7, or CD8. In certain embodiments, the lipid blend may further include free PEG-lipids to reduce the amount of non-specific binding via the targeting group. The free PEG-lipids may be the same as or different from the PEG-lipids included in the conjugate. In certain embodiments, the free PEG-lipids are selected from the group consisting of PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DSG), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), DSPE-PEG-cysteine, or derivatives thereof, all having an average PEG length of 2000 - 5000, 2000, 3400, or 5000. The final composition may contain a mixture of two or more of these pegylated lipids. In certain embodiments, the LNP composition includes a mixture of PEG-lipids and myristoyl and stearic acid acyl chains. In certain embodiments, the LNP composition includes a mixture of PEG-lipids and palmitoyl and stearoyl acyl chains. 【0295】 In certain embodiments, the derivative of the PEG-lipid has a methoxy, hydroxyl, or carboxylic acid end group at the PEG terminus. 【0296】 The lipid-immunocyte targeting group conjugate can be incorporated, for example, into LNPs containing ionizable cationic lipids, sterols, neutral phospholipids, and PEG-lipids, as described below. In certain embodiments, the LNP containing the lipid immunocyte targeting group is the ionizable cationic lipid described herein, or, for example, the cationic lipids described in U.S. Patent No. 10,221,127, U.S. Patent No. 10,653,780, or U.S. Patent Application Publication No. 2018 / 0085474, U.S. Patent Application Publication No. 2016 / 0317676, International Publication No. 2009 / 086558 Pamphlet, or Miao et al. (2019) NATURE BIOTECH 37:1174-1185, or Jayaraman et al. (2012) ANGEW CHEM INT. 51:8529-8533. 【0297】 In some embodiments, the cationic lipid can be selected from the ionizable cationic lipids or salts thereof described in Table 1. 【0298】 [Table 5] 【0299】 [Table 6] 【0300】 [Table 7] 【0301】 [Table 8] 【0302】 [Table 9] 【0303】 【Table 10】 【0304】 【Table 11】 【0305】 【Table 12】 【0306】 【Table 13】 【0307】 【Table 14】 【0308】 【Table 15】 【0309】 【Table 16】 【0310】 【Table 17】 【0311】 The R provided in this specification 1 、R 2 、R 3 、R 1A 、R 2A 、R 3A 、R 1A1 、R 1A2 、R 1A3 、R 2A1 、R 2A2 、R 2A3 、R 3A1 、R 3A2 、R 3A3, R a1 , R a2 , R 3B , R 3B1 , R 3B2 , R 3B3 , R s1 , R s2 , R s3 , R s4 , R s5 , R s6 , R s7 , R s8 , R s9 , R s10 , R s11 , R s12 , R s13 , R s14 , or R s15 Any variation or embodiment of, as if each combination were individually and specifically recited, R 1 , R 2 , R 3 , R 1A , R 2A , R 3A , R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , R 3A3 , R a1 , R a2 , R 3B , R 3B1 , R 3B2 , R 3B3 , R s1 , R s2 , R s3 , R s4 , R s5 , R s6 , R s7 , R s8 , R s9 , R s10 , R s11 , R s12 , R s13 , R s14 , or R s15 can be combined with all other variations or embodiments of. 【0312】 The LNP can be formulated using the methods and other components described in the following sections. 【0313】 IV. Lipid Nanoparticle Composition The present invention provides a lipid nanoparticle (LNP) composition comprising a lipid blend containing an ionizable cationic lipid described herein and / or a lipid-immunocyte targeting agent conjugate described herein. In certain embodiments, the lipid blend may comprise an ionizable cationic lipid described herein and one or more of a sterol, a neutral phospholipid, a PEG-lipid, and a lipid-immunocyte targeting group conjugate. 【0314】 In certain embodiments, the ionizable cationic lipid described herein may be present in the lipid blend in the range of 30 to 70 mole percent, 30 to 60 mole percent, 30 to 50 mole percent, 40 to 70 mole percent, 40 to 60 mole percent, 40 to 50 mole percent, 50 to 70 mole percent, 50 to 60 mole percent, or at about 30 mole percent, about 35 mole percent, about 40 mole percent, about 45 mole percent, about 50 mole percent, about 55 mole percent, about 60 mole percent, about 65 mole percent or about 70 mole percent. 【0315】 Sterol In certain embodiments, the lipid blend of the lipid nanoparticles may comprise one or more sterols selected from the group consisting of sterol components such as cholesterol, fecosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol. In certain embodiments, the sterol is cholesterol. 【0316】 Sterols (e.g., cholesterol) can be present in the lipid blend in the range of 20 to 70 mole percent, 20 to 60 mole percent, 20 to 50 mole percent, 30 to 70 mole percent, 30 to 60 mole percent, 30 to 50 mole percent, 40 to 70 mole percent, 40 to 60 mole percent, 40 to 50 mole percent, 50 to 70 mole percent, 50 to 60 mole percent, or at about 20 mole percent, about 25 mole percent, about 30 mole percent, about 35 mole percent, about 40 mole percent, about 45 mole percent, about 50 mole percent, about 55 mole percent, about 60 mole percent or about 65 mole percent. 【0317】 Neutral phospholipid In certain embodiments, the lipid blend of the lipid nanoparticles can include one or more neutral phospholipids. The neutral phospholipids can be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM). 【0318】 Other neutral lipids may be selected from the group consisting of distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphocholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), dioleoyl-glycero-phosphoethanolamine (DOPE), dilinoleoyl-glycero-phosphocholine (DLPC), dimyristoyl-glycero-phosphocholine (DMPC), dioleoyl-glycero-phosphocholine (DOPC), dipalmitoyl-glycero-phosphocholine (DPPC), diundecanoyl-glycero-phosphocholine (DUPC), palmitoyl-oleoyl-glycero-phosphocholine (POPC), dioctadecenyl-glycero-phosphocholine, oleoyl-cholesteryl hemisuccinoyl-glycero-phosphocholine, hexadecyl-glycero-phosphocholine, dilinolenoyl-glycero-phosphocholine, diarachidonoyl-glycero-3-phosphocholine, didocosahexaenoyl-glycero-phosphocholine, or sphingomyelin. 【0319】 The neutral lipid may be present in the lipid blend in the range of 1 to 10 mole percent, 1 to 15 mole percent, 1 to 12 mole percent, 1 to 10 mole percent, 3 to 15 mole percent, 3 to 12 mole percent, 3 to 10 mole percent, 4 to 15 mole percent, 4 to 12 mole percent, 4 to 10 mole percent, 4 to 8 mole percent, 5 to 15 mole percent, 5 to 12 mole percent, 5 to 10 mole percent, 6 to 15 mole percent, 6 to 12 mole percent, 6 to 10 mole percent, or at about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, about 5 mole percent, about 6 mole percent, about 7 mole percent, about 8 mole percent, about 9 mole percent, about 10 mole percent, about 11 mole percent, about 12 mole percent, about 13 mole percent, about 14 mole percent or about 15 mole percent. 【0320】 PEG-lipid The lipid blend of the lipid nanoparticles can include one or more PEGs or PEG-modified lipids. Such species may also be referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. As described above, when a lipid immune cell targeting group is included in the lipid blend, free PEG-lipid can be included in the lipid blend to reduce or eliminate non-specific binding through the targeting group. 【0321】 The PEG lipid can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG lipid can be PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid. 【0322】 In certain embodiments, the blend may contain a free PEG-lipid selected from the group consisting of PEG-distearoyl glycerol (PEG-DSG), PEG-diacyl glycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE), and PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE). In some embodiments, the free PEG-lipid comprises a diacyl phosphatidylcholine containing a dipalmitoyl (C16) chain or a distearoyl (C18) chain. 【0323】 The PEG-lipid may be present in the lipid blend in an amount ranging from 1 to 10 mole percent, 1 to 8 mole percent, 1 to 7 mole percent, 1 to 6 mole percent, 1 to 5 mole percent, 1 to 4 mole percent, 1 to 3 mole percent, 2 to 8 mole percent, 2 to 7 mole percent, 2 to 6 mole percent, 2 to 5 mole percent, 2 to 4 mole percent, 2 to 3 mole percent, or at about 1 mole percent, about 2 mole percent, about 3 mole percent, about 4 mole percent, or about 5 mole percent. In some embodiments, the PEG-lipid is a free PEG-lipid. 【0324】 In some embodiments, the PEG-lipid may be present in the lipid blend in the range of 0.01 to 10 mole percent, 0.01 to 5 mole percent, 0.01 to 4 mole percent, 0.01 to 3 mole percent, 0.01 to 2 mole percent, 0.01 to 1 mole percent, 0.1 to 10 mole percent, 0.1 to 5 mole percent, 0.1 to 4 mole percent, 0.1 to 3 mole percent, 0.1 to 2 mole percent, 0.1 to 1 mole percent, 0.5 to 10 mole percent, 0.5 to 5 mole percent, 0.5 to 4 mole percent, 0.5 to 3 mole percent, 0.5 to 2 mole percent, 0.5 to 1 mole percent, 1 to 2 mole percent, 3 to 4 mole percent, 4 to 5 mole percent, 5 to 6 mole percent, or 1.25 to 1.75 mole percent. In some embodiments, the PET-lipid may be about 0.5 mole percent, about 1 mole percent, about 1.5 mole percent, about 2 mole percent, about 2.5 mole percent, about 3 mole percent, about 3.5 mole percent, about 4 mole percent, about 4.5 mole percent, about 5 mole percent, or about 5.5 mole percent of the lipid blend. In some embodiments, the PEG-lipid is a free PEG-lipid. 【0325】 In some embodiments, the lipid anchor length of the PEG-lipid is C14 (as in PEG-DMG). In some embodiments, the lipid anchor length of the PEG-lipid is C16 (as in the case of DPG). In some embodiments, the lipid anchor length of the PEG-lipid is C18 (as in PEG-DSG). In some embodiments, the backbone or head group of the PEG-lipid is diacylglycerol or phosphoethanolamine. In some embodiments, the PEG-lipid is a free PEG-lipid. 【0326】 The LNPs of the present disclosure may include one or more free PEG-lipids not conjugated to an immune cell targeting group and PEG-lipids conjugated to an immune cell targeting group. In some embodiments, the free PEG-lipid comprises a lipid that is the same as or different from the lipid in the lipid-immune cell targeting group conjugate. 【0327】 Immune cell-targeting base conjugate In certain embodiments, the lipid blend may also include a lipid-immune cell-targeting base conjugate. 【0328】 The lipid-immune cell-targeting base conjugate may be present in the lipid blend in the range of 0.001 to 0.5 mole percent, 0.001 to 0.1 mole percent, 0.01 to 0.5 mole percent, 0.05 to 0.5 mole percent, 0.1 to 0.5 mole percent, 0.1 to 0.3 mole percent, 0.1 to 0.2 mole percent, 0.2 to 0.3 mole percent, at about 0.01 mole percent, about 0.05 mole percent, about 0.1 mole percent, about 0.15 mole percent, about 0.2 mole percent, about 0.25 mole percent, about 0.3 mole percent, about 0.35 mole percent, about 0.4 mole percent, about 0.45 mole percent, or about 0.5 mole percent. 【0329】 In addition to the lipids present in the lipid blend, the LNP composition may further include a payload, such as the payload described below. In certain embodiments, the payload is a nucleic acid, such as DNA or RNA, such as mRNA, transfer RNA (tRNA), microRNA, or small interfering RNA (siRNA). 【0330】 In certain embodiments, the number of nucleotides in the nucleic acid is from about 400 to about 6000. 【0331】 Manufacture of lipid nanoparticles In some embodiments, the LNPs are generated by using either rapid mixing by an orbital vortex device or microfluidic mixing. Orbital vortex device mixing is achieved by rapidly adding a lipid solution in ethanol to an aqueous solution of the nucleic acid of interest, followed immediately by vortexing at 2,500 rpm. In some embodiments, the LNPs are generated using a microfluidic mixing step. In some embodiments, microfluidic mixing is achieved, for example, by mixing an aqueous stream and an organic stream at a controlled flow rate within a microfluidic channel using a NanoAssemblr device and a microfluidic chip featuring an optimized mixing chamber shape (Precision Nanosystems, Vancouver, BC). In some embodiments, the LNPs are generated using a microfluidic mixing step for rapidly mixing an ethanolic lipid solution and an aqueous nucleic acid solution, resulting in encapsulation of the nucleic acid into solid lipid nanoparticles. The nanoparticle suspension is then buffer-exchanged into an all-aqueous buffer using a membrane filtration device selected for ethanol removal and nanoparticle aging. 【0332】 In certain embodiments, the resulting LNP composition comprises, for example, a lipid blend containing from about 40 mole percent to about 60 mole percent of one or more ionizable cationic lipids described herein, from about 35 mole percent to about 50 mole percent of one or more sterols, from about 5 mole percent to about 15 mole percent of one or more neutral lipids, and from about 0.5 mole percent to about 5 mole percent of one or more PEG-lipids. 【0333】 Physical properties of lipid nanoparticles The characteristics of the LNP composition can depend on the components included in the lipid nanoparticle (LNP) composition, their absolute or relative amounts. The properties can also vary depending on the method of preparation and the preparation conditions of the LNP composition. 【0334】 LNP compositions can be characterized in various ways. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the LNP composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can be utilized to determine the particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can be used to measure multiple properties of the LNP composition, such as particle size, polydispersity index, and zeta potential. RNA encapsulation efficiency is determined by a combination of a method that depends on an RNA-binding dye (Ribogreen, SYBR Green for determining the dye-accessible RNA fraction) and LNP de-formulation, followed by HPLC analysis of the total RNA content. 【0335】 In some embodiments, the LNP can have an average diameter in the range of 1 to 250 nm, 1 to 200 nm, 1 to 150 nm, 1 to 100 nm, 50 to 250 nm, 50 to 200 nm, 50 to 150 nm, 50 to 100 nm, 75 to 250 nm, 75 to 200 nm, 75 to 150 nm, 75 to 100 nm, 100 to 250 nm, 100 to 200 nm, 100 to 150 nm. In certain embodiments, the LNP composition can have an average diameter of about 1 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. In some embodiments, the LNP has an average diameter of about 100 nm. 【0336】 In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit a change in average diameter of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40% after freeze-thaw. In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit a change in average diameter of less than 30% after freeze-thaw. In some embodiments, freeze-thaw and diameter measurements are performed using 10% sucrose in MES pH 6.5 buffer. 【0337】 In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit a change in average diameter of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40% upon insertion of the targeting antibody. In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit a change in average diameter of less than 15% upon insertion of the targeting antibody. In some embodiments, the change in diameter upon insertion of the targeting antibody is measured in pH 6.5 MES using incubation at 37 °C for 4 hours. 【0338】 In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have an average LNP diameter of less than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have an average LNP diameter of less than 100 nm. 【0339】 Alternatively or additionally, the LNP composition can have a polydispersity index in the range of 0.05 - 1, 0.05 - 0.75, 0.05 - 0.5, 0.05 - 0.4, 0.05 - 0.3, 0.05 - 0.2, 0.08 - 1, 0.08 - 0.75, 0.08 - 0.5, 0.08 - 0.4, 0.08 - 0.3, 0.08 - 0.2, 0.1 - 1, 0.1 - 0.75, 0.1 - 0.5, 0.1 - 0.4, 0.1 - 0.3, 0.1 - 0.2. In certain embodiments, the polydispersity index is in the range of 0.1 - 0.25, 0.1 - 0.2, 0.1 - 0.19, 0.1 - 0.18, 0.1 - 0.17, 0.1 - 0.16, or 0.1 - 0.15. 【0340】 In some embodiments, the LNP composition or LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a polydispersity of less than 0.4, 0.3, 0.25, 0.2, 0.15, 0.1, or 0.05. In some embodiments, the LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a polydispersity of less than 0.25. 【0341】 Alternatively or additionally, the LNP composition can have a zeta potential in the range of about -30 mV to about +30 mV. In certain embodiments, the LNP composition has a zeta potential in the range of about -10 mV to about +20 mV. The zeta potential can vary as a function of pH. As a result, in certain embodiments, the LNP composition can have a zeta potential of about 0 mV to about +30 mV, or about +10 mV to +30 mV, or about +20 mV to about +30 mV at pH 5.5 or pH 5, and / or a zeta potential of about -30 mV to about +5 mV or about -20 mV to about +15 mV at pH 7.4. 【0342】 In some embodiments, the LNP composition or LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a zeta potential greater than -10, -9, -8, -7, -6, -5.5, -5, -4.5, -4, -3.5, -3, -2.5, -2, -1.5, -1, or -0.5 mV at pH 7.4. In some embodiments, the LNP composition LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a zeta potential greater than -10 mV at pH 7.4. In some embodiments, the LNP composition LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a zeta potential greater than -1 mV at pH 7.4. In some embodiments, the LNP composition LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a zeta potential greater than -1, 0, 1, 2, 3, 4, 4.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 mV at pH 5.5. In some embodiments, the LNP composition LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a zeta potential greater than 5 mV at pH 5.5. In some embodiments, the LNP composition LNP comprising an ionizable cationic lipid described herein, prepared and characterized using the methods described herein, has a zeta potential greater than 15 mV at pH 5.5. 【0343】 V. Payload The LNP composition can include an agent, such as a nucleic acid molecule for delivery to a target cell (e.g., an immune cell) or tissue, such as a cell (e.g., an immune cell) or tissue. 【0344】 The LNP composition of the present invention may contain nucleic acids, such as DNA or RNA, such as mRNA, tRNA, microRNA, siRNA, gRNA (guide RNA), circRNA (circular RNA), ribozyme, decoy RNA or dicer substrate siRNA. The nucleic acid may contain naturally occurring components, such as naturally occurring bases, sugars or linking groups (e.g., phosphodiester linking groups), or it is contemplated that it may contain non-naturally occurring components or modifications (e.g., thioester linking groups). For example, the nucleic acid can be synthesized to contain base, sugar, linker modifications known to those skilled in the art. Further, the nucleic acid may be linear or circular, or may have any desired configuration. The LNP composition can contain multiple nucleic acid molecules, such as multiple RNA molecules, which may be the same or different. 【0345】 In certain embodiments, the payload is mRNA. In certain embodiments, a particular LNP composition can contain several mRNA molecules, which may be the same or different. In certain embodiments, one or more LNP compositions containing one or more different mRNAs can be combined with and / or contacted simultaneously with cells. The mRNA is thought to be able to contain one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure. The mRNA can encode a receptor such as a chimeric antigen receptor (CAR) for use, for example, in immunological disorders, inflammatory disorders or cancer. Further, the mRNA can encode an antigen for use in a therapeutic or prophylactic vaccine, for example, to treat or prevent infection by a pathogen, such as a microbial pathogen or a viral pathogen, or to reduce or ameliorate side effects directly or indirectly caused by such an infection. 【0346】 In certain embodiments, the LNP composition can contain one or more other components including, but not limited to, one or more pharmaceutically acceptable excipients, hydrophobic small molecules, therapeutic agents, carbohydrates, polymers, permeability enhancing molecules, and surface modifying agents. 【0347】 In some embodiments, the weight / weight ratio of the lipid component to the payload (e.g., mRNA) in the resulting LNP composition is from about 1:1 to about 50:1. In certain embodiments, the weight / weight ratio of the lipid component to the payload (e.g., mRNA) in the resulting composition is from about 5:1 to about 50:1. In certain embodiments, the wt / wt ratio is from about 5:1 to about 40:1. In certain embodiments, the wt / wt ratio is from about 10:1 to about 40:1. In certain embodiments, the wt / wt ratio is from about 15:1 to about 25:1. 【0348】 In certain embodiments, the encapsulation efficiency of the payload (e.g., mRNA) in the lipid nanoparticles is at least 50%. In certain embodiments, the encapsulation efficiency is at least 80%, at least 90%, or greater than 90%. 【0349】 In some embodiments, the LNPs containing the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit an encapsulation efficiency greater than 50, 55, 60, 65, 70, 75, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, or 99%. In some embodiments, the LNPs containing the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit an encapsulation efficiency greater than 87.5%. In some embodiments, the LNPs containing the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit less than 50, 45, 40, 35, 30, 25, 20, 17.5, 15, 12.5, 10, 7.5, 5, 2.5, or 1% dye-accessible RNA. In some embodiments, the LNPs containing the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit less than 12.5% dye-accessible RNA. 【0350】 In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit a total mRNA recovery rate of greater than 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%. In some embodiments, the LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit a total mRNA recovery rate of greater than 80%. 【0351】 RNA payload In certain embodiments, the RNA payload is an mRNA, tRNA, microRNA, or siRNA payload. 【0352】 In certain embodiments, the lipid nanoparticle composition is optimized for delivery of RNA, such as mRNA, to target cells for translation within the cell. The mRNA can be a naturally or non-naturally occurring mRNA. The mRNA can contain one or more modified nucleobases, nucleosides, or nucleotides. 【0353】 The nucleobase can be selected from the non-limiting group consisting of adenine, guanine, uracil, cytosine, 7-methylguanine, 5-methylcytosine, 5-hydroxymethylcytosine, thymine, pseudouracil, dihydrouracil, N1-methylpseudouracil, hypoxanthine, and xanthine. In some embodiments, the nucleobase is N1-methylpseudouracil. 【0354】 The nucleoside of the mRNA is a compound that includes a sugar molecule (e.g., a 5- or 6-carbon sugar such as pentose, ribose, arabinose, xylose, glucose, galactose, or their deoxy derivatives) in combination with a nucleobase. The nucleoside can be a canonical nucleoside (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, and thymidine) or an analog thereof, and can include one or more substitutions or modifications. 【0355】 The nucleotides of mRNA are compounds that include a nucleoside and a phosphate group or an alternative group (e.g., boranophosphate, thiophosphate, selenophosphate, phosphonate, alkyl group, amidate, and glycerol). The nucleotides can be canonical nucleotides (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine monophosphate) or analogs thereof, and can include one or more substitutions or modifications including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more condensations or ring openings; oxidations; and / or reductions of the nucleobase, sugar, and / or phosphate or alternative components. The nucleotides can include one or more phosphate groups or alternative groups. For example, the nucleotides can include a nucleoside and a triphosphate group. “Nucleoside triphosphates” (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) can refer to canonical nucleoside triphosphates or analogs or derivatives thereof, and can include one or more of the substitutions or modifications described herein. 【0356】 mRNA can include a 5' untranslated region, a 3' untranslated region, and / or a coding sequence or translated sequence. mRNA can include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of the nucleobases, nucleosides, or nucleotides can be analogs of canonical species, can be substituted, can be modified, or can not be naturally occurring. In certain embodiments, all of a particular nucleobase type can be modified. For example, all of the cytosines in the mRNA can be 5-methylcytosine. In certain embodiments, one or more or all of the uridine bases can be N1-methylpseudouridine. 【0357】 In certain embodiments, the mRNA can include a 5' cap structure, a chain-terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal. 【0358】 A cap structure or cap species is a compound comprising two nucleoside moieties linked by a linker, and can be selected from naturally occurring caps, non-naturally occurring caps or cap analogs. The cap species can include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7-position and linked by a triphosphate bond at their 5'-position, such as m7G(5')ppp(5')G, generally denoted as m7GpppG. The cap species can be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73’dGpppG, m7Gpppm7G, m73’dGpppG, and m 27 02’GppppG. 【0359】 Alternatively or additionally, the mRNA may contain a chain-terminating nucleoside. For example, the chain-terminating nucleosides can include nucleosides that are deoxygenated at the 2' and / or 3'-positions of their sugar moieties. Such species can include 3'-deoxyadenosine (cordycepin), 3'-deoxythymidine, 3'-deoxycytidine, 3'-deoxyguanosine, 3'-deoxythymidine, and 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymidine. 【0360】 Alternatively or additionally, the mRNA may contain a stem-loop, such as a histone stem-loop. The stem-loop can include 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, the stem-loop can include 4, 5, 6, 7 or 8 nucleotide base pairs. The stem-loop can be located in any region of the mRNA. For example, the stem-loop can be located in the untranslated region (5' untranslated region or 3' untranslated region), coding region, or in, before, or after the polyA sequence or tail. 【0361】 Alternatively or additionally, the mRNA may include a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or mostly of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail located adjacent to the 3' untranslated region of the mRNA. 【0362】 The mRNA may encode any polypeptide of interest, including any natural, non-natural, or other modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed intracellularly. In some embodiments, the mRNA may encode an antibody, an enzyme, a growth factor, a hormone, a cytokine, a viral protein (e.g., a viral capsid protein), an antigen, a vaccine, or a receptor. In some embodiments, the mRNA may encode an engineered receptor such as a CAR or an antigen for use in a therapeutic vaccine (e.g., a cancer vaccine) or a prophylactic vaccine (e.g., a vaccine for minimizing the risk or severity of infection by a microbial or viral pathogen). In some embodiments, the mRNA encodes a polypeptide capable of modulating the immune response in immune cells. In some embodiments, the mRNA encodes a polypeptide capable of reprogramming immune cells. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR). 【0363】 The lipid composition can be designed for one or more specific uses or targets. For example, the LNP composition can be designed to deliver mRNA to specific cells, tissues, organs, or systems in the body of a mammal, such as the renal system, or groups thereof. The physicochemical properties of the LNP composition can be altered to enhance selectivity for a specific target site within the subject. For example, the particle size can be adjusted based on the fenestration sizes of different organs. The mRNA included in the LNP composition can also depend on one or more desired delivery targets. For example, the mRNA can be selected for a specific indication, condition, disease, or disorder and / or for delivery to specific cells, tissues, organs, or systems, or groups thereof (e.g., local or specific delivery). 【0364】 The amount of mRNA in the lipid composition can depend on the size, sequence, and other characteristics of the mRNA. The amount of mRNA in the LNP can also depend on the size, composition, desired target, and other characteristics of the LNP composition. The relative amounts of mRNA and other elements (e.g., lipids) can also vary. The amount of mRNA in the LNP composition can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy). 【0365】 In some embodiments, one or more mRNAs, lipids, and polymers and their amounts can be selected to provide a specific N:P ratio (the ratio of positively charged lipid or polymer amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups). The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the mRNA. Generally, a lower N:P ratio is preferred. The N:P ratio can depend on the specific lipid and its pKa. In certain embodiments, the composition and / or relative amounts of the mRNA and LNP can be selected to provide an N:P ratio of about 1:1 to about 30:1, or about 1:1 to about 20:1. In certain embodiments, the N:P ratio can be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. In certain embodiments, the N:P ratio can be from about 2:1 to about 5:1. In certain embodiments, the N:P ratio can be about 4:1. In other embodiments, the N:P ratio is from about 4:1 to about 8:1. For example, the N:P ratio can be about 4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, about 5.0:1, about 5.1:1, about 5.2:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.6:1, about 5.7:1, about 6.0:1, about 6.5:1, or about 7.0:1. 【0366】 The amount of mRNA in the nanoparticle composition can depend on the size, sequence, and other characteristics of the mRNA. The amount of mRNA in the nanoparticle composition can also depend on the size, composition, desired target, and other properties of the nanoparticle composition. The relative amounts of mRNA and other elements (e.g., lipids) can also vary. In some embodiments, the weight / weight ratio of the lipid component to mRNA in the nanoparticle composition can be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the weight / weight ratio of the lipid component to mRNA can be from about 10:1 to about 40:1. The amount of mRNA in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy). 【0367】 The efficiency of mRNA encapsulation represents the amount of mRNA encapsulated after preparation or associated with the lipid composition compared to the initial amount provided. It is desirable for the encapsulation efficiency to be high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of mRNA in a solution containing the lipid composition before and after decomposing the LNP composition with one or more organic solvents or surfactants. Fluorescence can be used to measure the amount of free mRNA in the solution. For the LNP compositions of the present invention, the mRNA encapsulation efficiency can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain embodiments, the encapsulation efficiency can be at least 80%. 【0368】 VI. Formulations and Modes of Delivery The LNP compositions of the present invention can be formulated, in whole or in part, as pharmaceutical compositions. The pharmaceutical compositions can further comprise one or more pharmaceutically acceptable excipients or auxiliary components such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and drugs are available, for example, in Remington’s (2006) supra. Conventional excipients and auxiliary components can be used in any of the pharmaceutical compositions of the present invention, except insofar as any conventional excipient or auxiliary component may be incompatible with one or more components of the LNP compositions of the present invention. An excipient or auxiliary component can be incompatible with the components of the LNP composition if the combination with the components of the LNP composition can result in undesirable biological effects or other adverse effects. 【0369】 In some embodiments, one or more excipients or auxiliary components may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the LNP composition of the present invention. For example, one or more excipients or auxiliary components may constitute 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In certain embodiments, the excipients are approved for use in humans and veterinary use, for example, by the US Food and Drug Administration. In certain embodiments, the excipients are of pharmaceutical grade. In certain embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or International Pharmacopeia. 【0370】 The relative amounts of one or more lipids or LNPs, one or more pharmaceutically acceptable excipients, and / or any additional components in the pharmaceutical composition may vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route by which the composition is administered. 【0371】 A lipid composition and / or a pharmaceutical composition comprising one or more LNP compositions can be administered to any subject, including a human patient, who may benefit from the therapeutic effect provided by the delivery of a nucleic acid, such as RNA (e.g., mRNA, tRNA, or siRNA), to one or more specific cells, tissues, organs, or their systems or groups, such as the renal system. The description of the LNP compositions and pharmaceutical compositions comprising the LNP compositions provided herein relates primarily to compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications of compositions suitable for administration to humans to make them suitable for administration to various animals are understood. 【0372】 The pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a distinct quantity of a pharmaceutical composition that contains a predetermined amount of an active ingredient (e.g., payload). 【0373】 The pharmaceutical composition of the present invention can be prepared in various forms suitable for various administration routes and methods. For example, the pharmaceutical composition of the present invention can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injection dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms. 【0374】 Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixir formulations. In addition to the active ingredient, the liquid dosage form can include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, oral compositions can include adjuvants such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, and / or aromatic agents. 【0375】 Injectable preparations, for example, sterile aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing agents, wetting agents and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, United States Pharmacopeia, and isotonic sodium chloride solution. Sterile fixed oils have been conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used. Fatty acids such as oleic acid can be used in the preparation of injectables. 【0376】 Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use. 【0377】 Other ingredients Furthermore, it is contemplated that the pharmaceutical composition may contain one or more additional ingredients in addition to those described above. 【0378】 The pharmaceutical composition may also contain one or more permeation-enhancing molecules, carbohydrates, polymers, therapeutic agents, surface-modifying agents, or other ingredients. The permeation-enhancing molecules can be, for example, molecules described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs). 【0379】 The pharmaceutical composition may also contain surface modifiers, such as anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocysteine, epratrizone, mesna, ambroxol, sobrerol, domiodol, lectostin, streptonin, tiopronin, gelsolin, thymosin β4, dornase alpha, neretexin, and erdosteine), and DNase (e.g., rhDNase). The surface modifiers may be disposed inside and / or on the surface of the composition described herein. 【0380】 In addition to these components, the pharmaceutical composition containing the LNP composition of the present invention may contain any substance useful in pharmaceutical compositions. For example, the pharmaceutical composition may include, but is not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, flow promoters, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and one or more pharmaceutically acceptable excipients or auxiliary components such as other species. Excipients such as waxes, butters, colorants, coating agents, flavoring agents, and fragrances may also be included. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington’s (2006) supra). 【0381】 The dispersant can be selected from a non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resin, calcium carbonate, silicate, sodium carbonate, crosslinked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM (registered trademark)), sodium lauryl sulfate, quaternary ammonium compounds and / or combinations thereof. 【0382】 Surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, condurrox, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate,Sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate and / or combinations thereof may be mentioned. 【0383】 Examples of preservatives can include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants can include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents can include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antibacterial preservatives can include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives can include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives can include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives can include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, desulphoximine mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulphate (SLS), sodi...

Claims

[Claim 1] Equation (I): 【Chemistry 1】 A compound or salt thereof, wherein in the formula, R 1 , R 2 , and R 3 Each is independent, combined or C 1 ~ 3 It is alkylene, R 1A , R 2A , and R 3A are each independently a bond or C 1 to 10 alkylene, R 1A1 , R 1A2 , R 1A3 , R 2A1 , R 2A2 , R 2A3 , R 3A1 , R 3A2 , and R 3A3 These are each German Standing, H, C 1 ~ 20 Alkyl, C 1 ~ 20 Alkenyl, -(CH 2 ) 0 ~ 10 C(O)OR a1 , or - (CH 2 ) 0 ~ 10 OC(O)R a2 And, R a1 and R a2 Each of them is independent of C 1 ~ 20 Alkyl or C 1 ~ 20 It is alkenyl, R 3B teeth, 【Chemistry 2】 And, R 3B1 C 1 ~ 6 It is alkylene, R 3B2 and R 3B3 These are, independently, H or C 1 ~ 6 A compound or salt thereof that is alkyl. [Claim 2] R 3B1 The compound or salt thereof according to claim 1, wherein the compound is ethylene or propylene. [Claim 3] R 3B2 and R 3B3 The compound or salt thereof according to claim 1, wherein each is independently methyl or ethyl, and each is optionally substituted with one or more -OH groups. [Claim 4] R 3B2 and R 3B3 The compound or salt thereof according to claim 3, wherein each is an unsubstituted methyl group. 【Request Item 5】 【Chemistry 4】 but, 【Transformation 5】 The compound or salt thereof according to claim 1. [Claim 6] R 1 and R 2 However, each is methylene, and R 3 The compound or salt thereof according to claim 1, wherein the bond is a combination. [Claim 7] R 1 , R 2 , and R 3 The compound or salt thereof according to claim 1, wherein each of these is methylene. [Claim 8] R 1A , R 2A , and R 3A However, each is independent, bonded or -(CH 2 ) 1 ~ 10 - The compound or salt thereof according to claim 1. [Claim 9] R 1A and R 2A However, each is independent, bonded, -(CH 2 ) 2 -, - (CH 2 ) 4 -, - (CH 2 ) 6 -, - (CH 2 ) 7 - or - (CH 2 ) 8 - and R 3A However, the bond, -CH 2 -, - (CH 2 ) 2 - or - (CH 2 ) 7 - The compound or salt thereof according to claim 8. [Claim 10] (i) R 1A1 and R 2A1 are each independently, -CH=CH-(C 1 ~ 15 alkyl), -CH=CH-CH 2 -CH=CH-(C 1 ~ 10 alkyl), -(CH 2 ) 0 ~ 4 C(O)OCH(C 1-10 alkyl)(C 1 ~ 15 alkyl), or -(CH 2 ) 0 ~ 4 OC(O)CH(C 1 ~ 10 alkyl)(C 1 ~ 15 alkyl), and R 1A2 , R 1A3 , R 2A2 , and R 2A3 are each H; (ii) R 1A1 and R 2A1 However, each is C 1 ~ 15 It is alkyl, R 1A2 and R 2A2 However, each is C 1 ~ 15 It is alkyl, R 1A3 and R 2A3 However, each of them is H; (iii) R 1A1 and R 2A1 However, each of them is -(CH 2 ) 0 ~ 4 OC(O)CH 2 (C 1 ~ 15 It is alkyl, and R 1A2 and R 2A2 However, each of them is -(CH 2 ) 0 ~ 4 C(O)OCH 2 (C 1 ~ 15 It is alkyl, and R 1A3 and R 2A3 However, each is H; or (iv)R 1A1 and R 2A1 However, each is -C(O)OCH 2 (C 1 ~ 15 It is alkyl, and R 1A2 and R 2A2 However, each of them is -(CH 2 ) 0-4 C(O)OCH 2 (C 1 ~ 15 It is alkyl, and R 1A3 and R 2A3 However, each is H, The compound or salt thereof according to claim 1. [Claim 11] (i) R 1A1 and R 2A1 However, each of them, 【Transformation 7】 It is; (ii) R 1A1 and R 2A1 However, each of them, 【Transformation 8】 And, R 1A2 and R 2A2 However, each of them, 【Chemistry 9】 And R 1A3 and R 2A3 However, each of them is H; (iii) R 1A1 and R 2A1 However, each of them, 【Chemistry 10】 And, R 2A1 and R 2A2 However, each of them, 【Chemistry 11】 And R 1A3 and R 2A3 However, each of them is H; (iv)R 1A1 and R 2A1 However, each of them, 【Chemistry 12】 And, R 1A2 and R 2A2 However, each of them, 【Chemistry 13】 And R 1A3 and R 2A3 However, each is H; or, (v) R 1A1 and R 2A1 However, each of them, 【Chemistry 14】 And, R 2A1 and R 2A2 However, each of them, 【Chemistry 15】 And R 1A3 and R 2A3 However, each is H, The compound or salt thereof according to claim 10. [Claim 12] (i) R 3A1 and R 3A2 However, each is independent of C 1 ~ 15 It is alkyl, R 3A3 However, it is H; (ii) R 3A1 However, C 1 ~ 15 It is alkyl, R 3A2 and R 3A3 However, each of them is H; (iii) R 3A1 is -C(O)OCH(C 1 ~ 5 (Alkyl) (C 1 ~ 10 It is alkyl, and R 3A2 and R 3A3 However, each of them is H; (iv)R 3A1 However, - (CH 2 ) 0 ~ 4 OC(O)CH 2 (C 1 ~ 10 It is alkyl, and R 3A2 However, - (CH 2 ) 0 ~ 4 (O)OCH 2 (C 1 ~ 10 It is alkyl, and R 3A3 However, it is H; (v) R 3A1 However, - (CH 2 ) 0 ~ 4 C(O)OCH 2 (C 1 ~ 10 It is alkyl, and R 3A2 However, - (CH 2 ) 0 ~ 4 C(O)OCH 2 (C 1 ~ 10 It is alkyl, and R 3A3 is H; or (vi)R 3A1 , R 3A2 , and R 3A3 However, each is H, The compound or salt thereof according to claim 1. [Claim 13] (i) R 3A1 and R 3A2 However, each is independent of ethyl, 【Chemistry 16】 And R 3A3 However, it is H; (ii) R 3A1 but, 【Chemistry 17】 And R 3A2 and R 3A3 However, each of them is H; (iii) R 3A1 but, [Chemistry 18] And R 3A2 and R 3A3 However, each of them is H; (iv)R 3A1 but, 【Chemistry 19】 And, R 3A2 but, 【Chemistry 20】 And R 3A3 However, it is H; (v) R 3A1 but, 【Chemistry 21】 And, R 3A2 but, 【Chemistry 22】 And R 3A3 However, H is; or (vi)R 3A1 , R 3A2 , and R 3A3 However, each is H, The compound or salt thereof according to claim 12. [Claim 14] R a1 and R a2 However, each is independent of the other, - (CH 2 ) 0 ~ 15 CH 3 or -CH(C) 1 ~ 10 (Alkyl) (C 1 ~ 15 The compound or salt thereof according to claim 1, wherein it is alkyl. [Claim 15] R a1 and R a2 However, each is independent of the others. 【Chemistry 23】 The compound or salt thereof according to claim 14. [Claim 16] The compound or a salt thereof according to claim 1, wherein the compound is selected from Table 1. [Claim 17] The aforementioned compound, 【Chemistry 24】 The compound or salt thereof according to claim 1. [Claim 18] The aforementioned compound, 【Chemistry 25】 The compound or salt thereof according to claim 1. [Claim 19] The aforementioned compound, 【Chemistry 26】 The compound or salt thereof according to claim 1. [Claim 20] Lipid nanoparticles (LNPs) comprising a lipid blend for targeted delivery of nucleic acids to immune cells, wherein the lipid blend comprises (a) Formula (II): A lipid-immune cell targeting group conjugate containing a compound of [lipid]-[any linker]-[immune cell targeting group], (b) an ionizable cationic lipid comprising a compound or salt thereof as described in any one of claims 1 to 19, Lipid nanoparticles (LNPs) wherein the LNP further contains nucleic acids arranged therein. [Claim 21] The LNP according to claim 20, wherein the immune cell targeting group comprises an antibody that binds to a T cell antigen, and the T cell antigen is CD3, CD4, CD7, CD8, or a combination thereof (for example, both CD3 and CD8, both CD4 and CD8, or both CD7 and CD8). [Claim 22] The LNP according to claim 20, wherein the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen, and the NK cell antigen is CD7, CD8, CD56, or a combination thereof (for example, both CD7 and CD8). [Claim 23] (i) The immune cell targeting group is transmitted via a polyethylene glycol (PEG)-containing linker. The lipids in the aforementioned lipid blend are covalently linked to the lipids; (ii) The lipid covalently linked to the immune cell targeting group via a PEG-containing linker is distearoyl glycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl glycerol (DPG), or ceramide; (iii) The PEG is PEG2000 or PEG3400; (vi) The lipid-immune cell targeting group conjugate is present in the lipid blend in an amount of 0.001 to 0.5 mole percent (e.g., 0.002 to 0.2 mole percent); or (v) The ionizable cationic lipid is present in the lipid blend in an amount of 30 to 70 (e.g., 40 to 60) mole percent; or The LNP according to claim 20, any combination of (i) to (v). [Claim 24] The LNP according to claim 20, wherein the lipid blend further comprises one or more of structural lipids (e.g., sterols), neutral phospholipids, and free PEG-lipids. [Claim 25] (i) The sterol is present in the lipid blend in a range of 20 to 70 (e.g., 30 to 50) mole percent; (ii) The sterol is cholesterol; (iii) The neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and sphingomyelin; (vi) The neutral phospholipid is present in the lipid blend in an amount of 5 to 15 mole percent; (v) The free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. For example, the PEG lipid is PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol (PEG-DPG), PEG-dilinoleoylglycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoylphosphatidylethanolamine (PEG-DMPE) ), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, or PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipids; (vi) The free PEG-lipid comprises diacylphosphatidylethanolamine containing a dipalmitoyl (C16) chain or a distearoyl (C18) chain, and optionally the free PEG-lipid comprises PEG-DPG and PEG-DMG; (vii) The free PEG-lipid is present in the lipid blend in an amount of 1 to 4 mole percent; (viiii) The free PEG-lipid in the lipid-immune cell targeting group conjugate It contains the same or different lipids as the aforementioned lipids; or The LNP according to claim 24, any combination of (i) to (viiii). [Claim 26] (i) The LNP has an average diameter in the range of 50 to 200 nm; (ii) The LNP has a polydispersity index in the range of 0.05 to 1; or (iii) The LNP has a zeta potential of approximately +10 mV to approximately +30 mV at pH 5; or The LNP according to claim 20, any combination of (i) to (iii). [Claim 27] The nucleic acid is mRNA, and the mRNA is (i) Receptors, growth factors, hormones, cytokines, antibodies, antigens, enzymes, or vaccines; (ii) polypeptides capable of modulating the immune response in the immune cells; or (iii) polypeptides that can reprogram the immune cells; or Any combination of (i) through (iii) The LNP according to claim 20, which codes for the LNP. [Claim 28] The LNP according to claim 27, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR). [Claim 29] The LNP according to claim 20, wherein the immune cell targeting group comprises Fab, F(ab')2, Fab'-SH, Fv, or scFv fragment, or any combination thereof. [Claim 30] (i) The immunotherapy group comprises a Fab modified to knock out the native interchain disulfide bond at the C-terminus; (ii) The Fab, in Kabat numbering, includes a heavy chain fragment containing a C233S substitution and a light chain fragment containing a C214S substitution; (iii) The immune cell targeting group comprises a Fab having a non-natural interchain disulfide bond (e.g., an engineered embedded interchain disulfide bond); (vi) The Fab, in Kabat numbering, includes the F174C substitution in the heavy chain fragment and the S176C substitution in the light chain fragment; (v) The immune cell targeting group comprises a Fab containing cysteine ​​at the C-terminus of the heavy chain or light chain fragment; (vi) The Fab further comprises one or more amino acids between the heavy chain fragment of the Fab and the C-terminal cysteine; or (vii) The Fab comprises a heavy chain variable domain linked to the CH1 domain of the antibody and a light chain variable domain linked to the light chain constant domain of the antibody, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and the immune cell targeting group further comprises a single chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain by an amino acid linker, or The LNP according to claim 29, any combination of (i) to (vii). [Claim 31] The LNP according to claim 20, wherein the immune cell targeting group comprises an immunoglobulin monovariate (ISV) domain. [Claim 32] (i) The immunoglobulin monovariable domain contains cysteine ​​at its C-terminus; (ii) The immunoglobulin monovariable domain comprises a VHH domain, and further comprises a spacer comprising one or more amino acids between the VHH domain and the C-terminal cysteine; (iii) The immune cell targeting group comprises two or more V HH Including the domain; (vi) The two or more V HH The domains are linked by amino acid linkers; (v) The immune cell targeting group is linked to the antibody CH1 domain of a first V HH A second V linked to the domain and the constant domain of the antibody light chain HH The domain is included, and the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds; (vi) The immune cell targeting group is linked to the antibody CH1 domain. HH The antibody CH1 domain is linked to the antibody light chain constant domain by one or more disulfide bonds; (vii) The CH1 domain includes F174C and C233S substitutions in Kabat numbering, and the light chain steady domain includes S176C and C214S substitutions; or (viiii) The ISV domain is NanobodyR ISV; or The LNP according to claim 31, any combination of (i) to (viiii). [Claim 33] The aforementioned immune cell targeting group (a) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 1 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 2 or 3, or (b) Heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and light chain fragment containing the amino acid sequence of SEQ ID NO: 7 The LNP according to claim 20, comprising a Fab containing the [Claim 34] The aforementioned LNP is for delivering nucleic acids to immune cells. (i) The immune cells are NK cells, and the immune cell targeting group comprises an antibody that binds to CD56; (ii) The immune cell targeting group comprises an antibody that binds to CD7 or CD8, and the free PEG lipid is DMG-PEG or PEG-DPG; (iii) The LNP is bonded to CD3 and also to CD11a or CD18; (iv) The LNP binds to CD7 and CD8 on immune cells; (v) The LNP binds to a first antigen on the surface of a first type of immune cell and also to a second antigen on the surface of a second type of immune cell; (vi) The immune cell targeting group comprises a single antibody that binds to CD3 or CD7; (vii) The immune cell targeting group binds to CD7, CD8, or both CD7 and CD8; (viiii) The immune cell targeting group is bound to (a) both CD3 and CD56, (b) both CD8 and CD56, or (c) both CD7 and CD56; (ix) The immune cell targeting group includes a Fab lacking a natural interchain disulfide bond; or The LNP according to claim 20, any combination of (i) to (ix). [Claim 35] (i) The LNP comprises two conjugates, the first conjugate comprising an antibody that binds to CD3, and the second conjugate comprising an antibody that binds to CD11a or CD18; (ii) The LNP comprises a single conjugate, the conjugate comprising a bispecific antibody that binds to both CD3 and CD11a; (iii) The LNP comprises a single conjugate, the conjugate comprising a bispecific antibody that binds to both CD3 and CD18; (iv) The LNP comprises two conjugates, the first conjugate comprising an antibody bound to CD7 and the second conjugate bound to CD8; (v) The LNP comprises a conjugate, the conjugate comprising a bispecific antibody that binds to CD7 and CD8; (vi) The two different types of immune cells are CD4+ T cells and CD8+ T cells; (vii) The LNP comprises two conjugates, the first conjugate comprising a first antibody that binds to the first antigen of the first type of immune cell, and the second conjugate comprising a second antibody that binds to the second antigen of the second type of immune cell; (viiii) The LNP comprises a conjugate, the conjugate comprises a bispecific antibody, and the bispecific antibody binds to both the first antigen on the first type of immune cell and the second antigen on the second type of immune cell; (ix) The bispecific antibody is an immunoglobulin monovariable domain or Fab-ScFv; or (x) The Fab is manipulated to replace one or both of the cysteines on the natural steady light chain and the natural steady heavy chain that form the natural interchain disulfide with a non-cysteine ​​amino acid, thereby removing the natural interchain disulfide bond in the Fab; or The LNP according to claim 34, any combination of (i) to (x). [Claim 36] A method for targeting the delivery of nucleic acids to target immune cells, comprising contacting the immune cells with the LNP described in claim 20, wherein the LNP comprises the nucleic acid. [Claim 37] A method for expressing a target polypeptide in target immune cells, comprising contacting the immune cells with the LNP described in claim 20, wherein the LNP comprises a nucleic acid encoding the polypeptide. [Claim 38] A method for regulating the cellular function of target immune cells, comprising administering the LNP described in claim 20 to the subject, wherein the LNP comprises a nucleic acid that regulates the cellular function of the immune cells. [Claim 39] A method for treating, improving or preventing symptoms of a disorder or disease in a subject requiring treatment, improvement or prevention of symptoms of a disorder or disease, comprising administering an LNP to the subject for delivering nucleic acids to immune cells of the subject, wherein the LNP is the LNP described in claim 20, and the LNP comprises the nucleic acid. [Claim 40] (i) The disorder is an immune disorder, an inflammatory disorder, or cancer; and / or (ii) The method according to claim 39, wherein the nucleic acid encodes an antigen for use in a therapeutic or preventive vaccine for treating or preventing infection by a pathogen. [Claim 41] (i) The antibody is a human antibody or a humanized antibody; (ii) Less than 5% of non-immune cells are transfected by the LNP; (iii) The half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by the reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP; (iv) At least 10% of immune cells are transfected with the LNP; or (v) The expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by the reference LNP; or The method according to claim 39, any combination of (i) to (v).

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