Ionizable cationic lipids and lipid nanoparticles and methods of synthesis and use thereof

Abstract

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

Description

Technical field

[0001] Cross-reference of related applications This application is filed in U.S. Provisional Application No. 63 / 121,801, filed December 4, 2020; U.S. Provisional Application No. 63 / 166,205, filed March 25, 2021; 63 / 169,296; U.S. Provisional Application No. 63 / 169,395, filed April 1, 2021; and U.S. Provisional Application No. 63 / 172,024, filed April 7, 2021; The entire disclosures of , are incorporated herein by reference in their entirety.

[0002] Submit a sequence listing as an ASCII text file Contents of subsequent submission in ASCII text file: Sequence listing in computer readable format (CRF) (file name: 183952034040SEQLIST.TXT, date of record: December 3, 2021, size: 205,763 bytes), in its entirety by reference. Incorporated herein.

[0003] field of invention The present invention provides ionizable cationic lipids and lipid nanoparticles for the delivery of nucleic acids to immune cells, and methods of making and using such lipids and targeted lipid nanoparticles. [Background technology]

[0004] background Recently, several therapeutic modalities have been developed that involve the delivery of one or more nucleic acids to a subject. The treatment modality includes, for example, the introduction of the gene of interest in the form of deoxyribose nucleic acid (DNA) into cells, which is then expressed to treat disorders caused by or associated with the defect or absence of the gene product. including gene therapy, producing gene products, e.g. proteins, for In this approach, genes are transcribed into messenger ribonucleic acid (mRNA), which is then translated to produce the gene product. In another approach, mRNA rather than the gene of interest may be delivered to the cell. The resulting expression product can ameliorate the deficiency or absence of a particular protein in a subject (e.g., protein deficiencies that occur in certain types of cystic fibrosis or lysosomal storage disorders) or can, for example, be used in immunotherapy in a subject. Reprogram cells to initiate or otherwise modulate immune responses (e.g., as a therapeutic agent to treat cancer, or to prevent or minimize the risk or severity of microbial or viral infections) It can be used to modulate cell function (as a preventive vaccine).

[0005] However, the delivery of mRNA into cells for intracellular translation is affected by various factors, such as nuclease degradation of the mRNA before entry into the cell and subsequent, but before translation, after introduction into the cell. It was difficult.

[0006] RNA can be delivered to a subject using a variety of delivery vehicles, such as cationic polymer or lipid-based delivery vehicles that form nanoparticles with the RNA. Nanoparticles are intended to protect RNA from degradation, enable delivery of RNA to target sites, and facilitate cellular uptake and processing by target cells. For delivery effectiveness, in addition to 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 believed to reduce serum interactions, increase serum stability, and increase time in circulation, which may be beneficial for certain targeting approaches.

[0007] Compared to DNA delivery technologies used in certain gene therapies, mRNA-based gene treatments have several superior attributes, such as ease of manipulation, rapid and transient expression, and mutation-free It has no adaptive processing properties. [Summary of the invention] [Problem to be solved by the invention]

[0008] However, delivery of therapeutic RNA to cells is difficult due to the relative instability of RNA and low cell permeability. Accordingly, a need exists to develop methods and compositions that facilitate the delivery of RNA, eg, mRNA, to cells. [Means to solve the problem]

[0009] The present invention provides ionizable cationic lipids, lipid-immune cell targeting group conjugates, and such ionizable cationic lipid and / or lipid-immune cell (e.g., T cell) targeting group conjugates. lipid nanoparticle compositions comprising, medical kits containing such lipids and / or conjugates, and methods of making and using such lipids and conjugates.

[0010] The lipid nanoparticle compositions provided herein can further include a nucleic acid, such as RNA, such as messenger RNA or mRNA. Lipid nanoparticle compositions can be used for mRNA delivery to cells of interest (eg, immune cells such as T cells). Messenger RNA-based gene therapy requires efficient delivery of mRNA to circulating cells in the plasma (e.g. immune cells, e.g. T cells or NK cells) or to cells in a given tissue. . The main challenges associated with efficient mRNA delivery to achieve robust levels of protein expression include: (a) protecting the mRNA payload from predominant serum nucleases after administration to the subject; (b) the ability to specifically target mRNA delivery to target cell (e.g., T cell) populations, thereby maximizing protein expression there; and (c) the ability to target mRNA delivery to target cell (e.g., T cell) populations, thereby maximizing protein expression therein; Ability to maximally deliver mRNA payloads to the cytosolic compartment of cells (e.g., T cells) for translation.

[0011] The present invention provides lipid nanoparticle compositions for producing lipid nanoparticle compositions that facilitate the delivery of a payload (e.g., a nucleic acid, e.g., DNA or RNA, e.g., mRNA) placed therein to cells, e.g., mammalian cells, e.g., immune cells. ionizable cationic lipids. Lipids are designed to allow intracellular delivery of nucleic acids, such as mRNA, to the cytosolic compartment of target cell types and to rapidly degrade into non-toxic components. These complex functions are due to the interaction between the chemistry and geometry of the ionizable lipid headgroup in the ionizable cationic lipid, the hydrophobic "acyl-tail" group, and the linker connecting the headgroup and acyl-tail group. Achieved through interaction.

[0012] In one aspect, the invention provides compounds represented by formula I: [ka] or a salt thereof, where the variables are as defined herein.

[0013] In another aspect, the invention provides compounds represented by formula II: [ka] or a salt thereof, where the variables are as defined herein.

[0014] As used herein, a compound selected from the group consisting of: [ka] or a portion of its salt is provided.

[0015] In certain embodiments, the compound is of Formula III: [ka] or a salt thereof, where the variables are as defined herein.

[0016] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0017] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0018] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0019] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0020] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0021] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0022] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0023] As used herein, compounds of the following formula: [ka] Or its salt is also provided.

[0024] Herein, a lipid comprising a lipid blend comprising an ionizable cationic lipid and / or a lipid-immune cell targeting group conjugate (e.g., a lipid-T cell targeting group conjugate) provided herein Nanoparticles (LNPs) are also provided.

[0025] In another aspect, herein is provided a method of delivering a nucleic acid to an immune cell (e.g., a T cell), the method comprising: transferring the nucleic acid to an LNP described herein containing the nucleic acid; A method is provided that includes exposing an immune cell under enabling conditions.

[0026] In another aspect, herein is provided a method of delivering a nucleic acid to an immune cell (e.g., a T cell) of a subject in need thereof, the composition comprising a LNP as described herein containing a nucleic acid. A method is provided that includes administering a substance to a subject, thereby delivering the nucleic acid to an immune cell.

[0027] In another aspect, described herein is a method of targeting the delivery of a nucleic acid (e.g., mRNA) to an immune cell (e.g., a T cell) in a subject, the method comprising: facilitating targeted delivery of a nucleic acid to the immune cell. A method is provided that includes administering to a subject a LNP described herein containing a nucleic acid for the purpose of obtaining a nucleic acid.

[0028] In one aspect, provided herein are lipid nanoparticles (LNPs) comprising a lipid blend for targeted delivery of nucleic acids to immune cells. In some embodiments, the lipid blend comprises a lipid-immune cell targeting group conjugate comprising a compound of Formula IV: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, the lipid blend includes ionizable cationic lipids. In some embodiments, the ionizable cationic lipid is [ka] including. In some embodiments, the LNP includes a nucleic acid disposed therein.

[0029] In some embodiments, the immune cell targeting group comprises an antibody that binds a T cell antigen. In some embodiments, the T cell antigen is CD3, CD4, CD7 or CD8, or a combination thereof (eg, CD3 and CD8, CD4 and CD8, or CD7 and CD8). In some embodiments, the immune cell targeting group comprises an antibody that binds a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD7, CD8 or CD56, or a combination thereof (eg, CD7 and CD8). In some embodiments, the antibody is a human or humanized antibody.

[0030] In some embodiments, the immune cell targeting group is covalently attached to the lipid in the lipid blend through a polyethylene glycol (PEG)-containing linker. In some embodiments, the lipid that is covalently attached to the immune cell targeting group through a PEG-containing linker is distearoylglycerol (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.

[0031] In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.002-0.2 mole percent. In some embodiments, the lipid blend includes one or more of structured lipids (eg, sterols), neutral phospholipids, and free PEG-lipids. In some embodiments, the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent. In some embodiments, sterols are present in the lipid blend in a range of 30-50 mole percent. In some embodiments, sterols are present in the lipid blend in a range of 20-70 mole percent. In some embodiments, the sterol is cholesterol.

[0032] In some embodiments, the neutral phospholipid is 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. In some embodiments, neutral phospholipids are present in the lipid blend in a range of 1-10 mole percent.

[0033] In some embodiments, the free PEG-lipid is 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- selected from the group consisting of methylpolyoxyethylene (PEG-DSG), N-palmitoylsphingosine-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof. In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine containing dipalmitoyl (C16) 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, free PEG-lipid is present in the lipid blend in a 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 the same or a different lipid than the lipid in the lipid-immune cell targeting group conjugate.

[0034] In some embodiments, the LNPs have an average diameter in the range of 50-200 nm. In some embodiments, the LNPs have an average diameter of about 100 nm. In some embodiments, the LNPs have a polydispersity index ranging from 0.05 to 1. In some embodiments, the LNPs have a zeta potential of about -10 mV to about +30 mV at pH5.

[0035] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, siRNA 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 modulating an immune response in an immune cell. 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). In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:24. In some embodiments, the mRNA encoding the CAR comprises the polynucleotide sequence of SEQ ID NO: 25.

[0036] In some embodiments, the immune cell targeting group comprises an antibody, where the antibody is a Fab or an immunoglobulin single variable domain, eg, a Nanobody. In some embodiments, the immune cell targeting group comprises a Fab, F(ab')2, Fab'-SH, Fv or scFv fragment. In some embodiments, the immune cell targeting group comprises a Fab that is engineered to knock out one or more natural interchain disulfide bonds. For example, in some embodiments, according to Kabatt numbering, a Fab comprises a heavy chain fragment containing a C233S substitution and / or a light chain fragment containing a C214S substitution. In some embodiments, the immune cell targeting group comprises a Fab that is engineered to introduce one or more buried interchain disulfide bonds. For example, in some embodiments, according to Kabatt numbering, the Fab antibody comprises a heavy chain fragment comprising a F174C substitution and / or a light chain fragment comprising a S176C substitution according to Kabatt numbering. In some embodiments, the immune cell targeting group is configured to knock out one or more natural interchain disulfide bonds and to introduce one or more buried interchain disulfide bonds. Including the Fab that is being engineered. In some embodiments, the immune cell targeting group comprises a Fab that includes 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 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 one or Linked by further interchain disulfide bonds, 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. In some embodiments, the Fab antibody is a DS Fab (a Fab with wild-type (natural) interchain disulfide bonds), a NoDS Fab (a Fab in which the natural disulfide bonds have been knocked out, e.g., a heavy chain according to Kabat numbering). Fabs with C233S substitutions on the top and / or C214S substitutions on the light chain), bDS Fabs (Fabs with no natural disulfide bonds and with introduced non-natural interchain buried disulfide bonds, e.g. Kabat no. Fab with F174C and C233S on the heavy chain and / or S176C and C214S on the light chain by attachment), or bDS Fab-ScFv (with a linker such as (G4S)x, as illustrated in Figure 47). bDS Fab) linked to the ScFv through the bDS Fab).

[0037] In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain, eg, a nanobody. In some embodiments, the immunoglobulin single variable domain comprises a cysteine ​​at the C-terminus. In some embodiments, the nanobody is V HH It further includes a spacer comprising one or more amino acids between the domain and the C-terminal cysteine. In some embodiments, the immune cell targeting group has two or more V HH Contains domains. In some embodiments, two or more V HH The domains are connected by amino acid linkers. In some embodiments, the immune cell targeting group is the first V linked to the antibody CH1 domain. HH domain and a second V linked to the antibody light chain constant domain HH 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 is a V HH 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, and / or the light chain constant domain comprises S176C and C214S substitutions, according to Kabatt numbering. In some embodiments, as illustrated in Figure 47, the antibody is a ScFv, V HH (Nb), 2xV HH ,V HH -CH1 / empty Vκ, or V HH 1-CH1 / V HH -2-Nb bDS.

[0038] In some embodiments, the immune cell targeting group 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 immune cell targeting group 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.

[0039] 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; (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 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.

[0040] In some embodiments, the immune cell targeting group comprises a Fab, F(ab')2, Fab'-SH, Fv or scFv fragment. In some embodiments, the immune cell targeting group comprises a Fab that is engineered to knock out the natural interchain disulfide bond at the C-terminus. In some embodiments, according to Kabatt numbering, the Fab comprises a heavy chain fragment containing a C233S substitution and a light chain fragment containing a C214S substitution. In some embodiments, the immune cell targeting group comprises a Fab with a non-natural interchain disulfide bond (eg, an engineered, buried interchain disulfide bond). In some embodiments, the Fab includes a F174C substitution in the heavy chain fragment and a S176C substitution in the light chain fragment, according to Kabatt numbering. In some embodiments, the immune cell targeting group comprises a Fab that includes 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 fragment of the Fab and the C-terminal cysteine.

[0041] 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, two or more V HH The domains are connected by amino acid linkers. In some embodiments, the immune cell targeting group is the first V linked to the antibody CH1 domain. HH domain and a second V linked to the antibody light chain constant domain HH Contains domains. 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 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 includes the F174C and C233S substitutions and the light chain constant domain includes the S176C and C214S substitutions, according to Kabatt numbering.

[0042] In some embodiments, the immune cell targeting group 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; or (b) a light chain fragment comprising the amino acid sequence of SEQ ID NO: 6. Fab includes a heavy chain fragment comprising the amino acid sequence of SEQ ID NO:7 and a light chain fragment comprising the amino acid sequence of SEQ ID NO:7.

[0043] In another aspect, provided herein are methods of targeting the delivery of nucleic acids to immune cells in a subject. In some embodiments, the method includes contacting the immune cell with lipid nanoparticles (LNPs). In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising a compound of the following formula: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid.

[0044] In some embodiments, certain aspects of the present disclosure relate to LNPs disclosed herein or pharmaceutical compositions containing the same for use in methods of targeting the delivery of nucleic acids to immune cells of a subject. . Such methods may be for the treatment of diseases or disorders as hereinafter disclosed. In some embodiments, the methods disclosed herein can include contacting a subject's immune cells with lipid nanoparticles (LNPs) in vitro or ex vivo. In some embodiments, the LNP is a LNP described herein in this disclosure.

[0045] In some embodiments, LNP provides the following benefits: (i) increased specificity of targeted delivery to immune cells compared to reference LNPs; (ii) an increase in the half-life of the nucleic acid or polypeptide encoded by the nucleic acid in immune cells compared to a reference LNP; (iii) increased transfection rate compared to reference LNP; and (iv) providing at least one of: low levels (<15%) of dye-accessible mRNA and high RNA encapsulation efficiency; At least 80% mRNA was recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0046] In some embodiments, methods are provided for expressing a polypeptide of interest in targeted immune cells of a subject. In some embodiments, the method includes contacting the immune cell with lipid nanoparticles (LNPs). In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid encoding a polypeptide. In some embodiments, certain aspects of the present disclosure provide an LNP disclosed herein or a pharmaceutical composition containing the same for use in a method of expressing a polypeptide of interest in targeted immune cells of a subject. relating to things. Such methods may be for the treatment of diseases or disorders as hereinafter disclosed. In some embodiments, the methods disclosed herein can include contacting a subject's immune cells with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0047] In some embodiments, LNP provides the following benefits: (i) increased expression levels in immune cells compared to reference LNPs; (ii) increased specificity of expression in immune cells compared to reference LNPs; (iii) an increase in the half-life of the nucleic acid or polypeptide encoded by the nucleic acid in immune cells compared to a reference LNP; (iv) increased transfection rate compared to reference LNP; and (v) providing at least one of: low levels (<15%) of dye-accessible mRNA and high RNA encapsulation efficiency; At least 80% mRNA was recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0048] In some embodiments, methods of modulating cellular function of target immune cells in a subject are provided. In some embodiments, the method includes administering lipid nanoparticles (LNPs) to the subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid encoding a polypeptide for modulating cellular function of an immune cell. In some embodiments, certain aspects of the present disclosure provide LNPs disclosed herein or pharmaceutical compositions containing the same for use in a method of modulating cellular function of targeted immune cells in a subject. Regarding. Such methods may be for the treatment of diseases or disorders as hereinafter disclosed. In some embodiments, the methods disclosed herein can include contacting a subject's immune cells with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0049] In some embodiments, LNP provides the following benefits: (i) increased expression levels in immune cells compared to reference LNPs; (ii) increased specificity of expression in immune cells compared to reference LNPs; (iii) an increase in the half-life of the nucleic acid or polypeptide encoded by the nucleic acid in immune cells compared to a reference LNP; (iv) increased transfection rate compared to reference LNP; (v) the LNPs can be administered at lower doses compared to reference LNPs to reach the same biological effect on immune cells; and (vi) providing at least one of: low levels (<15%) of dye-accessible mRNA and high RNA encapsulation efficiency; At least 80% mRNA was recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0050] In some embodiments, modulating cellular function includes reprogramming immune cells to mount an immune response. In some embodiments, modulating cellular function includes modulating antigen specificity of immune cells.

[0051] In some embodiments, methods are provided for treating, ameliorating, or preventing symptoms of a disorder or disease in a subject in need thereof. In some embodiments, the method includes administering to the subject lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of the subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid.

[0052] In some embodiments, the nucleic acid modulates the immune response of immune cells, thereby treating or ameliorating the condition. In some embodiments, certain aspects of the present disclosure provide for the use of LNPs disclosed herein or the like for use in a method of treating, ameliorating, or preventing symptoms of a disorder or disease in a subject in need thereof. It relates to a pharmaceutical composition containing. The disease or disorder may be as disclosed hereinafter. In some embodiments, the methods disclosed herein can include contacting a subject's immune cells with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0053] In some embodiments, LNP provides the following benefits: (i) increased specificity of delivery of nucleic acids to immune cells compared to reference LNPs; (ii) an increase in the half-life of the nucleic acid or polypeptide encoded by the nucleic acid in immune cells compared to a reference LNP; (iii) increased transfection rate compared to reference LNP; (iv) LNPs can be administered at lower doses compared to reference LNPs to reach the same treatment effect; (v) increased level of gain of function by immune cells compared to reference LNPs; and (vi) providing at least one of: low levels (<15%) of dye-accessible mRNA and high RNA encapsulation efficiency; At least 80% mRNA was recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0054] In some embodiments, the disorder is an immune disorder, an inflammatory disorder or a cancer. In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for the treatment or prevention of infections caused by pathogens. In some embodiments, the Fab antibody comprises a heavy chain fragment comprising a F174C substitution according to Kabatt numbering and / or a light chain fragment comprising a S176C substitution according to Kabatt numbering.

[0055] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the non-immune cells are transfected by the LNPs. In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the unwanted immune cells that are not the intended destination of delivery The following are transfected by LNP. In some embodiments, the half-life of a nucleic acid delivered by a LNP or a polypeptide encoded by a nucleic acid delivered by a LNP to an immune cell is equal to at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100 of the half-life of the polypeptide encoded by the nucleic acid %, 1.5x, 2x, 3x, 4x, 5x, 10x, or longer.

[0056] 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% of the immune cells that are the intended destination of delivery; 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 by LNP transfected.

[0057] In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 5%, at least 10%, 1.5 times, 2 times, 3 times the expression level of the nucleic acid in the same immune cells delivered by a reference LNP. , 4x, 5x, 10x, 15x, 20x or more.

[0058] In one embodiment, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to NK cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other Structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. In some embodiments, the immune cell targeting group comprises an antibody that binds CD56.

[0059] In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other Structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. In some embodiments, the immune cell targeting group comprises an antibody that binds CD7 or CD8 and the free PEG lipid is DMG-PEG.

[0060] In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other (d) phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. In some embodiments, the immune cell targeting group comprises an antibody, where the antibody is a Fab or an immunoglobulin single variable domain. In some embodiments, the Fab is engineered to knock out the natural interchain disulfide at the C-terminus. In some embodiments, the Fab has buried interchain disulfides. In some embodiments, the antibody is an immunoglobulin single variable (ISV) domain, and the ISV domain is a Nanobody® ISV. In some embodiments, free PEG lipids include 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 comprises 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 PEG having a molecular weight of about 3000-3500 daltons.

[0061] In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other Structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. In some embodiments, the immune cell targeting group includes an antibody that binds to CD3 and an antibody that binds to CD11a or CD18.

[0062] In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other Structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. In some embodiments, the immune cell targeting group includes an antibody that binds CD7 and an antibody that binds CD8.

[0063] In one embodiment, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to two different types of immune cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other Structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids.

[0064] In some embodiments, the immune cell targeting group includes a bispecific targeting moiety. In some embodiments, a bispecific targeting moiety binds 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.

[0065] In one embodiment, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both CD4+ and CD8+ T cells of a subject. LNPs are composed of (a) an ionizable cationic lipid, (b) a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterol or other Structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. In some embodiments, the immune cell targeting group comprises a single antibody that binds CD3 or CD7.

[0066] Lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of interest are further provided, the LNPs comprising: (a) an ionizable cationic lipid; (b) the following structure: [lipid]-[optional (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids; and (f) nucleic acids. and immune cell targeting groups include Fabs that lack natural interchain disulfide bonds. In some embodiments, the Fab replaces one or both cysteines on the natural constant light chain and natural constant heavy chain that form a natural interchain disulfide with a non-cysteine ​​amino acid, thereby engineered to remove interchain disulfide bonds.

[0067] Immunoglobulin single variable domains (ISVDs) that bind human CD8 are also provided. In some embodiments, the ISVD comprises three complementarity determining domains CDR1, CDR2 and CDR3; (a) CDR1 contains GSTFSDYG (SEQ ID NO: 100), (b) CDR2 contains IDWNGEHT (SEQ ID NO: 101), (c) CDR3 contains AADALPYTVRKYNY (SEQ ID NO: 102).

[0068] In some embodiments, the ISVD is humanized.

[0069] In some embodiments, the ISVD comprises, consists of, or consists essentially of SEQ ID NO:77.

[0070] Also provided are polypeptides including GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101) and AADALPYTVRKYNY (SEQ ID NO: 102).

[0071] In some embodiments, the polypeptide comprises an ISVD described herein.

[0072] In some embodiments, the polypeptide further comprises a second binding moiety, the second binding moiety binding to CD8 or another different target. In some embodiments, the second binding moiety is also an ISVD.

[0073] In some embodiments, the polypeptide further comprises a detectable marker or therapeutic agent.

[0074] Compositions comprising the ISVDs or polypeptides described herein are also provided.

[0075] Further provided are pharmaceutical compositions comprising an ISVD or polypeptide described herein and a pharmaceutically acceptable carrier.

[0076] Further provided are methods of treating a CD8-related disease or disorder in a subject, the method comprising administering to the subject a pharmaceutical composition described herein.

[0077] In some embodiments, the disease is cancer. In some embodiments, the disorder is an immune disorder, an inflammatory disorder or a cancer.

[0078] In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for the treatment or prevention of infections caused by pathogens. In some embodiments, the ionizable cationic lipid is [ka] It is.

[0079] In some embodiments, the immune cell targeting group comprises an antibody that binds 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 a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD56. In some embodiments, the antibody is a human or humanized antibody.

[0080] In some embodiments, the immune cell targeting group is covalently attached to the lipid in the lipid blend through a polyethylene glycol (PEG)-containing linker. In some embodiments, the lipid that is covalently attached to the immune cell targeting group through a PEG-containing linker is distearoylglycerol (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.

[0081] In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.002-0.2 mole percent. In some embodiments, the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

[0082] In some embodiments, the sterol is cholesterol. In some embodiments, sterols are present in the lipid blend in a range of 30-50 mole percent. In some embodiments, the neutral phospholipid is 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.

[0083] In some embodiments, neutral phospholipids are present in the lipid blend in a range of 1-10 mole percent.

[0084] 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, PEG lipids include PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidylethanol Amine (PEG-DLPE), PEG-dimyristoylphosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoylphosphatidylethanolamine (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.

[0085] In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine containing dipalmitoyl (C16) or distearoyl (C18) chains. In some embodiments, free PEG-lipid is present in the lipid blend in a range of 2-4 mole percent. In some embodiments, the free PEG-lipid comprises the same or a different lipid than the lipid in the lipid-immune cell targeting group conjugate.

[0086] In some embodiments, the LNPs have an average diameter in the range of 50-200 nm. In some embodiments, the LNPs have an average diameter of about 100 nm. In some embodiments, the LNPs have a polydispersity index ranging from 0.05 to 1. In some embodiments, the LNPs have a zeta potential of about -10 mV to about +30 mV at pH5.

[0087] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA, tRNA, siRNA 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 modulating an immune response in an immune cell. 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).

[0088] In some embodiments, the immune cell targeting group comprises an antibody, where the antibody is a Fab or an immunoglobulin single variable domain. In some embodiments, the immune cell targeting group 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 group comprises a Fab that includes one or more interchain disulfide bonds. In some embodiments, according to Kabatt numbering, the Fab comprises a heavy chain fragment containing F174C and C233S substitutions and a light chain fragment containing S176C and C214S substitutions. In some embodiments, the immune cell targeting group comprises a Fab that includes a cysteine ​​at the C-terminus of the heavy chain or light chain fragment.

[0089] 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 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.

[0090] 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, two or more VHH domains are connected by an amino acid linker. In some embodiments, the immune cell targeting group 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 group 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 and the light chain constant domain comprises S176C and C214S substitutions according to Kabatt numbering.

[0091] In some embodiments, the immune cell targeting group 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: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 7; Including Fab.

[0092] In some embodiments, no more than 5% of non-immune cells are transfected with LNPs. 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 the half-life of the nucleic acid delivered by the reference LNP or the nucleic acid encoded by the nucleic acid delivered by the reference LNP. At least 10% longer than the half-life of the polypeptide. In some embodiments, at least 10% of the immune cells are transfected with LNPs. 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.

[0093] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cell targeting group comprises an antibody that binds CD56.

[0094] In some aspects, provided herein are lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds CD7 or CD8. In some embodiments, the free PEG lipid is DMG-PEG.

[0095] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. 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 immunoglobulin single variable domain.

[0096] In some embodiments, the Fab is engineered to knock out the natural interchain 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-natural interchain disulfide. In some embodiments, the Fab includes a F174C substitution in the heavy chain fragment and a S176C substitution in the light chain fragment. In some embodiments, the antibody is an immunoglobulin single variable (ISV) domain, and the ISV domain is a Nanobody® ISV. In some embodiments, free PEG lipids include 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 antibody is a Fab. In some embodiments, the Fab binds CD3 and the free PEG lipid in the LNP comprises 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 PEG having a molecular weight of about 3000-3500 daltons.

[0097] In some embodiments, the immune cell targeting group comprises two or more VHH domains. In some embodiments, two or more VHH domains are connected by an amino acid linker. In some embodiments, the immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain.

[0098] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid.

[0099] In some embodiments, the LNP binds CD3 and also binds CD11a or CD18. In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds CD3. In some embodiments, the second conjugate comprises an antibody that binds CD11a or CD18. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds CD3 and CD11a. In some embodiments, the conjugate includes a bispecific antibody that binds both CD3 and CD18. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

[0100] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the LNPs bind to CD7 and CD8 on immune cells.

[0101] In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate includes an antibody that binds CD7 and the second conjugate includes an antibody that binds CD8. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate includes a bispecific antibody that binds CD7 and CD8. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

[0102] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to two different types of immune cells in a subject. In some embodiments, the LNP comprises: an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the LNP binds a first antigen on the surface of a first type of immune cell and also binds 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 first antibody that binds to a first antigen of a second type of immune cell. It contains a second antibody that binds to two antigens. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate includes a bispecific antibody. In some embodiments, the bispecific antibody binds 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 Fab-ScFv.

[0103] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both CD4+ and CD8+ T cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group comprises a single antibody that binds CD3 or CD7.

[0104] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both T cells and NK cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group binds CD7, CD8, or both CD7 and CD8.

[0105] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to both T cells and NK cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid. In some embodiments, the immune cell targeting group binds (i) both CD3 and CD56; (ii) both CD8 and CD56; or (iii) both CD7 and CD56.

[0106] In some embodiments, the immune cell targeting group is covalently attached to the lipid in the lipid blend through a polyethylene glycol (PEG)-containing linker. In some embodiments, the lipid that is covalently attached to the immune cell targeting group through a PEG-containing linker is distearoylglycerol (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 lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.002-0.2 mole percent. In some embodiments, the lipid blend further comprises one or more of structured lipids (eg, sterols), neutral phospholipids, and free PEG-lipids.

[0107] In some embodiments, the ionizable cationic lipid is present in the lipid blend in a range of 40-60 mole percent.

[0108] In some embodiments, sterols are present in the lipid blend in a range of 30-50 mole percent. In some embodiments, the sterol is cholesterol.

[0109] In some embodiments, the neutral phospholipid is 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). In some embodiments, neutral phospholipids are present in the lipid blend in a range of 1-10 mole percent.

[0110] In some embodiments, the free PEG-lipid is 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-dimyristoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DMG) Palmitoyl-rac-glycero-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl-rac-glycerol, methoxypolyethylene glycol (DOG-PEG)1,2-distearoyl-rac-glycero-3-methyl selected from the group consisting of polyoxyethylene (PEG-DSG), N-palmitoylsphingosine-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), and DSPE-PEG-cysteine, or derivatives thereof. In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine containing dipalmitoyl (C16) or distearoyl (C18) chains. In some embodiments, free PEG-lipids are present in the lipid blend in the range of 1-2 mole percent. In some embodiments, the free PEG-lipid comprises the same or a different lipid than the lipid in the lipid immune cell targeting group conjugate.

[0111] In some embodiments, the LNPs have an average diameter in the range of 50-200 nm. In some embodiments, the LNPs have an average diameter of about 100 nm. In some embodiments, the LNPs have a polydispersity index ranging from 0.05 to 1. In some embodiments, the LNPs have a zeta potential of about -10 mV to about +30 mV at pH5.

[0112] 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 capable of modulating an immune response in an immune cell. 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).

[0113] In some embodiments, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNPs include ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising the following structure: [lipid]-[optional linker]-[immune cell targeting group]. In some embodiments, LNPs include sterols or other structured lipids. In some embodiments, the LNPs include neutral phospholipids. In some embodiments, the LNPs include free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises a nucleic acid.

[0114] In some embodiments, the immune cell targeting group comprises a Fab that lacks natural interchain disulfide bonds. In some embodiments, the Fab replaces one or both cysteines on the natural constant light chain and the natural constant heavy chain that form a natural interchain disulfide with a non-cysteine ​​amino acid, thereby engineered to remove interchain disulfide bonds.

[0115] In some embodiments, methods are provided for targeting the delivery of nucleic acids to immune cells in a subject. In some embodiments, the method includes contacting an immune cell with a lipid nanoparticle (LNP) provided herein. In some embodiments, the method is for targeting NK cells. In some embodiments, the immune cell targeting group binds CD56. In some embodiments, the method is for targeting T cells and NK cells simultaneously. In some embodiments, the immune cell targeting group binds CD7, CD8, or both CD7 and CD8. In some embodiments, the method is for targeting CD4+ and CD8+ T cells simultaneously. In some embodiments, the immune cell targeting group comprises a polypeptide that binds CD3 or CD7.

[0116] In some embodiments, methods are provided for expressing a polypeptide of interest in targeted immune cells of a subject. In some embodiments, the method includes contacting an immune cell with a lipid nanoparticle (LNP) provided herein.

[0117] In some embodiments, methods of modulating cellular function of target immune cells in a subject are provided. In some embodiments, the method includes administering to the subject a lipid nanoparticle (LNP) provided herein.

[0118] In some embodiments, methods are provided for treating, ameliorating, or preventing symptoms of a disorder or disease in a subject in need thereof. In some embodiments, the method includes administering to the subject a lipid nanoparticle (LNP) provided herein.

[0119] In some embodiments, an immunoglobulin single variable domain (ISVD) that binds 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 ADALPYTVRKYNY (SEQ ID NO: 102). In some embodiments, the ISVD is humanized. In some embodiments, the ISVD comprises SEQ ID NO:77.

[0120] In some embodiments, polypeptides are provided that include GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101), and ADALPYTVRKYNY (SEQ ID NO: 102). In another aspect, a polypeptide comprising an ISVD provided herein is provided. In some embodiments, the polypeptide includes a second binding moiety. In some embodiments, the second binding moiety binds CD8 or another different target. In some embodiments, the second binding moiety is also an ISVD. In some embodiments, the polypeptide includes a detectable marker. In some embodiments, the polypeptide includes a therapeutic agent.

[0121] In some embodiments, compositions are provided that include an ISVD provided herein or a polypeptide provided herein.

[0122] In some embodiments, a pharmaceutical composition is provided that includes an ISVD provided herein or a polypeptide provided herein and a pharmaceutically acceptable carrier.

[0123] In some embodiments, methods of treating a CD8-related disease or disorder in a subject are provided. In some embodiments, the method includes administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer.

[0124] Various aspects and embodiments of the invention are described in further detail below. [Brief explanation of the drawing]

[0125]

Figure 1

[0126] The present invention includes ionizable cationic lipids, lipid-immune cell targeting group conjugates, and such ionizable cationic lipid and / or lipid-immune cell (e.g., T cell) targeting group conjugates. Lipid nanoparticle compositions, medical kits containing such lipids and / or conjugates, and methods of making and using such lipids and conjugates are provided.

[0127] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, cell biology, and biochemistry. Such techniques are described in the literature, for example in "Comprehensive Organic Synthesis" (eds. B.M. Trost and I. Fleming, 1991-1992); "Current protocols in molecular biology" (eds. F.M. Ausubel et al., 1987 and periodic updates); Current protocols in immunology" (J.E. Coligan et al., eds., 1991), each of which is incorporated herein by reference in its entirety. Various embodiments of the invention are presented in the following sections; however, embodiments of the invention described in one particular section should not be limited to any particular section.

[0128] I. Definition To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used herein have their conventional meanings within the chemical and biological fields. Chemical structures and formulas shown herein are to be interpreted in accordance with standard rules of chemical valency known in the chemical art. In addition, for example, if the chemical group is a diradical, the chemical group can be bonded to their adjacent atoms of the rest of the structure in one or both directions, for example -OC(O)- It is understood that (O)O- is interchangeable or -OC(S)- is interchangeable with -C(S)O-.

[0130] The terms "a" and "an" as used herein mean "one or more" and include the plural unless the context indicates otherwise.

[0131] The term "alkyl," as used herein, refers to a saturated straight or branched hydrocarbon, such as referred to herein as C1-C12 alkyl, C1-C10 alkyl, and C1-C6 alkyl, respectively. , refers to a straight or branched group of 1 to 12, 1 to 10 or 1 to 6 carbon atoms. Exemplary alkyl groups include 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, Includes, but is not limited to, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, and the like.

[0132] The term "alkylene" refers to a diradical of an alkyl group. An exemplary alkylene group is -CH2CH2-.

[0133] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc.

[0134] The term "oxo" is art-recognized and refers to the "=O" substituent. For example, cyclopentane substituted with an oxo group is cyclopentanone.

[0135] The term "morpholinyl" means: [ka] Refers to a substituent having the structure.

[0136] The term "piperidinyl" means: [ka] Refers to a substituent having the structure.

[0137] Generally, the term "substituted" means that one or more hydrogens of the specified moiety are replaced by a suitable substituent, whether or not preceded by the term "optionally". It means there is. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and in any given structure more than one position is specified. When the substituents can be substituted by two or more substituents selected from the group, the substituents may be the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically viable compounds. In some embodiments, the optional substituents are C 1~6 Alkyl, cyano, halogen, -O-C 1~6 Alkyl, C 1~6 haloalkyl, C 3~7 can be selected from the group consisting of cycloalkyl, 3-7 membered heterocyclyl, 5-6 membered heteroaryl and phenyl, where R a is hydrogen or C 1~6 It is an alkyl. In some embodiments, the optional substituents are C 1~6 Alkyl, halogen, -O-C 1~6 Alkyl and -CH 2 N(R a ) 2 can be selected from the group consisting of, where R a is hydrogen or C 1~6 It is an alkyl.

[0138] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH 2 F, -CHF 2 ,-CF 3 ,-CH 2 C.F. 3 ,-CF 2 C.F. 3 Such.

[0139] The term "cycloalkyl" is used herein, for example, to refer to "C 4~8 a monovalent saturated cyclic, bicyclic, bridged cyclic (e.g. adamantyl) or spirocyclic hydrocarbon radical of 3 to 12, 3 to 8, 4 to 8 or 4 to 6 carbons, termed "cycloalkyl" refers to Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane. Unless otherwise specified, cycloalkyl groups are optionally in one or more ring positions, such as alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, Carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonate, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl or thio Substituted by carbonyl. In certain embodiments, a cycloalkyl group is unsubstituted, ie, it is unsubstituted.

[0140] The terms "heterocyclyl" and "heterocyclic group" are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures; contains 1 to 4 heteroatoms, such as nitrogen, oxygen and sulfur. The number of ring atoms in a heterocyclyl group is C x ~C x Nomenclature can be used to specify, where x is an integer specifying the number of ring atoms. For example, C 3 ~C 7 A 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. "C 3 ~C 7 '' designation indicates that the heterocyclic ring contains a total of 3 to 7 ring atoms, including any heteroatoms occupying ring atom positions. C 3One example of heterocyclyl is aziridinyl. A heterocycle may be, for example, a mono-, bi- or other polycyclic ring system (eg fused, spiro, bridged bicyclic). The heterocycle may be fused to one or more aryl, partially unsaturated or saturated rings. Heterocyclyl groups include, for example, 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-onyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, thiolanyl , thiomorpholinyl, thiopyranyl, xanthenyl, lactones, lactams such as azetidinone and pyrrolidinone, sultam, sultone, and the like. Unless otherwise specified, heterocyclic rings optionally include substituents in one or more positions, such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azide, Carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, oxo, phosphate, phosphonate, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl and thiocarbonyl. In certain embodiments, a heterocyclyl group is unsubstituted, ie, it is unsubstituted.

[0141] The term "aryl" is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term "aryl" refers to two or more carbocyclic rings in which two or more carbons are common to two adjacent rings (the rings are "fused" rings). including polycyclic ring systems, where at least one of the rings is aromatic, and other rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl and / or aryl. Unless otherwise specified, aromatic rings include, in one or more ring positions, e.g. halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, Carboxylic acid, -C(O)alkyl, CO 2 Alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF 3 , -CN, etc. In certain embodiments, aromatic rings are substituted at one or more ring positions with halogen, alkyl, hydroxyl or alkoxyl. In certain other embodiments, the aromatic ring is unsubstituted, ie, it is unsubstituted. In certain embodiments, the aryl group is a 6-10 membered ring structure.

[0142] The term "heteroaryl" is art-recognized and refers to an aromatic group containing at least one ring heteroatom. In certain cases, heteroaryl groups contain 1, 2, 3 or 4 ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, and the like. Unless otherwise specified, heteroaryl rings include, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, etc., in one or more ring positions. , carboxylic acid, C(O)alkyl, -CO 2 Alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF 3 , -CN, etc. The term "heteroaryl" also refers to two or more rings in which two or more carbons are common to two adjacent rings (the rings are "fused"). including polycyclic ring systems, where at least one of the rings is a heteroaromatic ring, for example, other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl and / or aryl . In certain embodiments, a heteroaryl ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl or alkoxyl. In certain other embodiments, the heteroaryl ring is unsubstituted, ie, it is unsubstituted. In certain embodiments, a heteroaryl group is a 5- to 10-membered ring structure, or a 5- to 6-membered ring structure containing 1, 2, 3, or 4 heteroatoms, such as nitrogen, oxygen, and Contains sulfur.

[0143] The terms "amine" and "amino" are art-recognized and include both unsubstituted and substituted amines, such as the general formula -N(R 10 )(R 11 ), where R 10 and R 11 are each independently hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, aralkyl or (CH 2 ) m -R 12 or R 10 and R 11 together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure; R 12 represents aryl, cycloalkyl, cycloalkenyl, heterocycle or polycycle; m is 0 or an integer within the range of 1 to 8; In certain embodiments, R 10 and R 11 are each independently hydrogen, alkyl, alkenyl or -(CH 2 ) m -R 12 represents.

[0144] The terms "alkoxyl" or "alkoxy" are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. An "ether" is two hydrocarbons covalently bonded by oxygen. Thus, substitutions on an alkyl that make it an ether are or resemble alkoxyl, such as -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH 2 ) m -R 12 where m and R 12 is explained above. The term "haloalkoxyl" refers to an alkoxyl group substituted with at least one halogen. For example, -O-CH 2 F, -O-CHF 2 ,-O-CF 3 Such. In certain embodiments, haloalkoxyl is an alkoxyl group substituted with at least one fluoro group. In certain embodiments, haloalkoxyl is an alkoxyl group substituted with 1-6, 1-5, 1-4, 2-4, or 3 fluoro groups.

[0145] symbol" [ka] ” indicates a joining point.

[0146] Compounds of the present disclosure may contain one or more chiral centers and / or double bonds and therefore exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term "stereoisomer" as used herein consists of all geometric isomers, enantiomers or diastereomers. These compounds can be designated by the symbols "R" or "S" depending on the configuration of substituents around the stereogenic carbon atom. The present invention encompasses the various stereoisomers of these compounds, and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Although mixtures of enantiomers or diastereomers may be designated in nomenclature by "(±)", those skilled in the art will recognize that the structure may implicitly indicate a chiral center. It is understood that graphical depictions of chemical structures, such as general chemical structures, include all stereoisomeric forms of a particular compound, unless otherwise indicated.

[0147] Individual stereoisomers of the compounds of the invention may be prepared synthetically from commercially available starting materials containing asymmetric or stereogenic centers or by preparation of racemic mixtures and subsequent resolution methods well known to those skilled in the art. can. These resolution methods include (1) coupling of the enantiomeric mixture to the asymmetric auxiliary, separation of the resulting diastereomeric mixture by recrystallization or chromatography, and separation of the optically pure product from the auxiliary; (2) salt formation using an optically active resolving agent, or (3) direct separation of optical enantiomeric mixtures in a chiral chromatography column. Stereoisomeric mixtures can also be obtained 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. , can be resolved into their component stereoisomers. Additionally, enantiomers can be separated using supercritical fluid chromatography (SFC) techniques described in the literature. Furthermore, stereoisomers can be obtained from stereomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0148] Also, geometric isomers can exist in the compounds of the invention. symbol" [ka] ” indicates a bond that may be a single, double or triple bond as described herein. The present invention encompasses various geometric isomers and mixtures thereof that result from the arrangement of substituents around carbon-carbon double bonds or from the arrangement of substituents around carbocyclic rings. Substituents around carbon-carbon double bonds are designated to be in the "Z" or "E" configuration, and the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both "E" and "Z" isomers.

[0149] Alternatively, substituents around a carbon-carbon double bond may be referred to as "cis" or "trans," where "cis" refers to substituents on the same side of the double bond, and "trans" refers to substituents on the same side of the double bond. Represents a substituent on the opposite side of a double bond. The arrangement of substituents around a carbocyclic ring is designated as "cis" or "trans." The term "cis" refers to substituents on the same side of the plane of the ring, and the term "trans" refers to substituents on opposite sides of the plane of the ring. Mixtures of compounds in which substituents are placed on both the same or opposite sides of the ring are designated as "cis / trans."

[0150] The present invention also provides that, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from that normally found in nature, Includes isotopically labeled compounds of the invention that are identical to the listed compounds. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as, respectively, 2 H, 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Contains Cl.

[0151] Certain isotopically labeled disclosed compounds (eg, those labeled with 3H and 14C) are useful in compound and / or substrate tissue distribution assays. Tritiated (ie, 3H) and carbon-14 (ie, 14C) isotopes are particularly preferred for their ease of preparation and detectability. Additionally, substitution with heavier isotopes, such as deuterium (i.e., 2H), may result in certain specific therapeutic benefits and therefore may be preferred in some situations. Isotopically labeled compounds of the invention are generally prepared by replacing isotopically labeled reagents with non-isotopically labeled reagents, e.g., by following procedures similar to those disclosed in the Examples herein. can be manufactured.

[0152] As used herein, the terms "subject" and "patient" refer to the organism to be treated by the methods of the invention. Such organisms are preferably mammals (eg mice, monkeys, horses, cows, pigs, dogs, cats, etc.) and more preferably humans.

[0153] As used herein, the term "pharmaceutical composition" refers to an active agent and an inactive or Refers to a combination with an active carrier.

[0154] As used herein, the term "pharmaceutically acceptable excipient" refers to standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions). and any of the various types of wetting agents. The composition may 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.

[0155] As known to those skilled in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include 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, and the like. Other acids, such as oxalic acid, which are not pharmaceutically acceptable on their own, are used in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. be able to.

[0156] Examples of bases include alkali metal hydroxides (e.g. sodium), alkaline earth metal hydroxides (e.g. magnesium), ammonia, and W 1~4 is alkyl, the formula NW 4 + These include, but are not limited to, compounds such as

[0157] Examples of salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclo Pentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, odor Hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate , pectate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, etc. Not limited to these. Other examples of salts include suitable cations such as Na + , N.H. 4 + and N.W. 4 + (In the formula, W is C 1~4 Included are anions of the compounds of the present invention combined with alkyl groups, such as alkyl groups.

[0158] Abbreviations used herein include: 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).

[0159] As used herein, the term "effective amount" refers to an amount of a compound (eg, a nucleic acid, eg, mRNA) sufficient to produce a beneficial or desired result. An effective amount may be administered in one or more administrations, applications or dosages and is not intended to be limited to any particular formulation or route of administration. The term effective amount can be considered to include therapeutically and / or prophylactically effective amounts of a compound.

[0160] The phrase "therapeutically effective amount," as used herein, refers to an amount that is administered in a mammal, e.g., a human or a subject (e.g., a human subject), at a reasonable benefit / risk ratio applicable to any medical treatment. Refers to 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 therapeutic effect in at least a subpopulation of cells. .

[0161] The phrase "prophylactically effective amount," as used herein, means that, at a reasonable benefit / risk ratio applicable to any medical treatment, reduces, minimizes or eliminates the risk of developing a condition; or is effective for producing some desired prophylactic effect in a mammal, e.g., a human or a subject (e.g., a human subject), at least in a subpopulation of cells, by reducing or minimizing the severity of a condition. Refers to an amount of a compound (eg, a nucleic acid, eg, mRNA), a material, or a composition comprising a compound (eg, a nucleic acid, eg, mRNA).

[0162] As used herein, the terms "treat," "treating," and "treatment" refer to any effect, e.g., alleviation, reduction, modulation, that results in amelioration of a condition, disease, disorder. including rate, amelioration or elimination, or amelioration of its symptoms.

[0163] The phrase "pharmaceutically acceptable" means that, within the scope of good medical judgment, the drug does not involve undue toxicity, irritation, allergic reactions or other problems or complications commensurate with the reasonable benefit / risk ratio. is used herein to refer to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissue.

[0164] In applications where an element or ingredient is said to be included in and / or selected from a list of enumerated elements or ingredients, the element or ingredient may be any of the enumerated elements or ingredients; It is to be understood that the or ingredients may be selected from the group consisting of two or more of the listed elements or ingredients.

[0165] Additionally, any elements and / or features of the compositions or methods described herein, whether express or implied herein, may be incorporated without departing from the spirit and scope of the invention. , it should be understood that various combinations are possible. For example, when reference is made to a particular compound, that compound is used in various embodiments of the compositions of the invention and / or in the methods of the invention, unless the context indicates otherwise. be able to. In other words, although the embodiments are described and depicted within this application in a manner that allows clear and concise application to be described and depicted, the embodiments may depart from the present teachings and inventions. It is intended and understood that they can be combined or separated in various ways without requiring any modification. For example, it is understood that all features described and depicted herein may be applicable to all aspects of the invention described and depicted herein.

[0166] The expression "at least one of", unless the context and usage dictate otherwise, means each of the enumerated objects that follows the expression, and each of the enumerated objects. It should be understood to include various combinations of two or more of the following. The expression "and / or" in relation to three or more listed objects should be understood to have the same meaning, unless the context indicates otherwise.

[0167] "include", "includes", "including", "have", "has", "having", The use of the terms "contain," "contains," or "containing" and their grammatical equivalents generally means that they are not specifically indicated otherwise. It should be understood as open and non-limiting, eg not excluding additional unlisted elements or steps, unless otherwise specified or understood from the context.

[0168] Where use of the term "about" precedes a quantitative value, the invention also includes the particular quantitative value itself, unless specifically indicated otherwise. As used herein, the term "about" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred.

[0169] As used herein, unless otherwise indicated, the term "antibody" refers to any antibody that includes at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. refers to an antigen-binding molecule or molecular complex. The term includes intact antibodies (e.g., intact monoclonal antibodies) or fragments thereof, such as Fc fragments of antibodies (e.g., Fc fragments of monoclonal antibodies) or antigen-binding fragments of antibodies (e.g., antigen-binding fragments of monoclonal antibodies). However, this is understood to include intact antibodies, antigen-binding fragments or Fc fragments that have been modified or engineered. Examples of antigen-binding fragments include Fab, Fab’, (Fab’) 2 , Fv, single chain antibodies (eg scFv), minibodies and diabodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (eg, bispecific antibodies). The term also refers to immunoglobulin single variable domains, e.g. nanobodies (e.g. V HH ).

[0170] As used herein, an "antibody that binds to X" (ie, where X is a specific antigen) or "anti-X antibody" is an antibody that specifically recognizes antigen X.

[0171] As used herein, "buried interchain disulfide bond" or "interchain buried disulfide bond" means that the disulfide bond is both a reducing agent and for conjugation to other hydrophilic PEGs. Refers to disulfide bonds on a polypeptide that are not readily accessible to water-soluble reducing agents, or that are effectively "buried" in hydrophobic regions of the polypeptide, such that they are not used for. Buried interchain disulfide bonds are further explained in WO2017096361A1, which publication is incorporated by reference in its entirety.

[0172] As used herein, specificity of targeted delivery by LNPs is defined as the % of the desired immune cell types that accept the delivered nucleic acid (e.g., on-target delivery) and the destination of delivery. % of undesired immune cell types that do not accept the delivered nucleic acid (eg, off-target delivery). For example, specificity is higher if more desired immune cells receive the delivered nucleic acid while fewer undesired immune cells receive the delivered nucleic acid. The specificity of targeted delivery by LNPs also determines the amount of nucleic acid delivered to desired immune cells (e.g., on-target delivery) versus the amount of nucleic acid delivered to undesired immune cells (e.g., off-target delivery). It can be defined as the ratio. Specificity of delivery can be determined using any suitable method. As a non-limiting example, the expression level of the nucleic acid in a desired immune cell type may be measured and compared to the expression level of the nucleic acid in a different immune cell type that is not considered the destination for delivery.

[0173] As used herein, in some embodiments, reference to an LNP is a LNP that does not have immune cell targeting groups, but is otherwise the same as the LNP being tested. In some other embodiments, references to LNPs are LNPs that have different ionizable cationic lipids, but are otherwise the same as the LNPs being tested. In some embodiments, reference to LNP refers to an ionizable cationic lipid that is different from, but otherwise the same as, the LNP that is tested. Contains D-Lin-MC3-DMA.

[0174] 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 to prevent or minimize an immune response in humans, e.g. modified to replace certain amino acids occurring at corresponding positions in the framework regions of the VH and VL domains in the sequence of antibodies of human origin, so as to increase their similarity to antibodies naturally produced in It is an antibody that exists. For example, using genetic engineering techniques, the variable domains of a non-human antibody of interest may be combined with the constant domains of a human antibody. The constant domains of humanized antibodies are most often human CH and CL domains.

[0175] As used herein, the term "structured lipid" refers to sterols and also lipids that contain sterol moieties.

[0176] It should be understood that the order of steps or order of performing certain tasks is not important so long as the invention remains operable. Furthermore, two or more steps or operations may be performed simultaneously.

[0177] At various places throughout this specification, substituents are disclosed in groups or ranges. The description is specifically intended to include each and every individual subcombination of the members of such groups and ranges. For example, “C 1~6 The term "alkyl" refers to C 1 ,C 2 ,C 3 ,C 4 ,C 5 ,C 6 ,C 1 ~C 6 ,C 1 ~C 5 ,C 1 ~C 4 ,C 1 ~C 3 ,C 1 ~C 2 ,C 2 ~C 6 ,C 2 C 5 ,C 2 C 4 ,C 2 C 3 ,C 3 C 6 ,C 3 C 5 ,C 3 C 4 ,C 4 C 6 ,C 4 C 5 and C 5 C 6 It is specifically intended that alkyl be disclosed individually. As another example, integers in the range 0-40 are 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 individually disclosed It is specifically intended that integers in the range 1 to 20 be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 and 20 are specifically intended to be disclosed individually.

[0178] The use of any and all example or exemplary language herein, such as "such" or "such as" or "including," merely better exemplifies the invention. No limitations on the scope of the invention are intended to be imposed unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0179] Compositions and kits are described as having, including, or comprising certain components, or processes and methods have or include certain steps. ) or comprising, in addition, that there are compositions and kits of the invention consisting essentially of or consisting of the recited ingredients; It is contemplated that there are processes and methods described herein that consist essentially of or consist of processing steps that include the following:

[0180] As a general matter, compositions specifying percentages are percentages by weight, unless otherwise specified. Additionally, if a variable is not accompanied by a definition, the variable's previous definition takes precedence.

[0181] immunoglobulin single variable domain In some embodiments, the immune cell targeting group of the LNP described herein comprises an immunoglobulin single variable domain, eg, a nanobody.

[0182] The term "immunoglobulin single variable domain" (ISV) is used interchangeably with "single variable domain" and means that the antigen-binding site is on a single immunoglobulin domain and that the antigen-binding site is on a single immunoglobulin domain. Define an immunoglobulin molecule, formed by This refers to immunoglobulin single variable domains as two immunoglobulin domains, especially 'conventional' immunoglobulins in which the two variable domains interact to form an antigen binding site (e.g. monoclonal antibodies) or fragments thereof (e.g. Fab, Fab', F(ab') 2 , scFv, di-scFv). Typically, in conventional immunoglobulins, the heavy chain variable domain (V H ) and light chain variable domain (V L ) interact to form an antigen binding site. In this case, V H and V L Both complementarity determining regions (CDRs) contribute to the antigen binding site, ie, a total of six CDRs are involved in antigen binding site formation. In terms of the above definition, Fab, F(ab ') 2 The antigen-binding domain of fragments, Fv fragments, such as disulfide-bonded Fv or scFv fragments, or diabodies (all known in the art), in which binding to each epitope of the antigen is usually one (single ) not by an immunoglobulin domain, but by a pair of immunoglobulin domains (associated immunoglobulin domains), e.g., a light chain and a heavy chain variable domain, i.e., immunoglobulin domains that bind jointly to an epitope of each antigen. V H -V L Because they can occur in pairs, they are not normally considered immunoglobulin single variable domains.

[0183] In contrast, an immunoglobulin single variable domain is capable of specifically binding an epitope of an antigen without pairing with additional immunoglobulin variable domains. The binding site for immunoglobulin single variable domains is a single V H , single V HH or a single V L formed by domains. Therefore, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0184] Therefore, a single variable domain consists of 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 array or V HH sequence) or a suitable fragment thereof; provided that it is a single antigen-binding unit (i.e., a single antigen-binding domain interacts with another variable domain to form a functional antigen-binding unit). It is possible to form a functional antigen-binding unit consisting essentially of a single variable domain, such that a functional antigen-binding unit is not required.

[0185] Immunoglobulin single variable domains (ISVs) are, for example, heavy chain ISVs, such as V H ,V HH These may be camelized V H or humanized V HH including. In one embodiment, it is V HH and this is camelization V H or humanized V HH including. Heavy chain ISVs can be derived from conventional four chain antibodies or from heavy chain antibodies.

[0186] For example, an immunoglobulin single variable domain is a domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or a dAb (or an amino acid sequence suitable for use as a dAb). Amino acid sequence), or Nanobody® ISV (as defined herein, V HH (including, but not limited to); other single variable domains, or any suitable fragment thereof.

[0187] In particular, the immunoglobulin single variable domain is a Nanobody® ISV (e.g. V HH ;humanized V HH Or camelization V H ) or a suitable fragment thereof. [Note: Nanobody® is a registered trademark of Ablynx N.V.].

[0188] "V HH "Domain" is V HH ,V HH Antibody fragments and V HH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"); Hamers-Casterman et al., 1993 (Nature 363:446 ~448). "V HH The term "domain" refers to these variable domains as the heavy chain variable domains (herein "V") present in conventional four-chain antibodies. H from the light chain variable domain present in conventional four-chain antibodies (referred to herein as the “V L ``domain''). V HH For further explanation, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74:277-302).

[0189] For the terms "dAb" and "domain antibody" see e.g. Ward et al. 1989 (Nature 341:544), Holt et al. 2003 (Trends Biotechnol. 21:484); and e.g. WO2004 / 068820, WO2006 / 030220, WO2006 Reference is made to Domantis Ltd. / 003388 and other published patent applications of Domantis Ltd. Furthermore, although a single variable domain derived from a particular type of shark is not of mammalian origin and is therefore less preferred in the context of the present invention, a single variable domain may be derived from a particular type of shark. It should be noted that (see, eg, so-called "IgNAR domains", eg WO2005 / 18629).

[0190] Typically, production of immunoglobulins involves immunization of laboratory animals, fusion of immunoglobulin-producing cells to create hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be produced by naive immunization or screening of synthetic libraries, eg, by phage display.

[0191] The production of immunoglobulin sequences, such as VHHs, has been extensively described in various publications, among them WO1994 / 04678, 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, camels are immunized with a target antigen to induce an immune response against said target antigen. The repertoire of VHHs obtained from said immunization is further screened for VHHs that bind the target antigen.

[0192] In these cases, antibody generation requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or in the process of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0193] Immunoglobulin sequences of various origins can be used herein, including mouse, rat, rabbit, donkey, human and camel immunoglobulin sequences. Also, fully human, humanized or chimeric sequences can be used in the methods described herein. For example, as described by Ward et al. 1989 (Nature 341:544), WO1994 / 04678 and Davis and Riechmann (1994, Febs Lett., 339:285-290; and 1996, Prot. Eng., 9:531-537). , camel immunoglobulin sequences and humanized camel immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs, can be used herein. Additionally, ISVs can be fused to form multivalent and / or multispecific constructs (one or more V HH For multivalent and multispecific polypeptides containing domains and their preparation, see also Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10.7346-7350), and for example WO1996 / 34103 and WO1999 / 23221. (reference is made to ).

[0194] “Humanized V HH ” is a naturally occurring V HH corresponds to the amino acid sequence of the domain, but is "humanized", i.e., the naturally occurring V HH one or more amino acid residues in the amino acid sequence (particularly within the framework sequences) of V H Includes amino acid sequences that have been "humanized" by replacing one or more of the amino acid residues that occur at corresponding positions in the domain. This can be done in a manner known per se, which is clear to the person skilled in the art, for example on the basis of the prior art (eg WO2008 / 020079). Again, such humanized V HH It is noted that the polypeptides can be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material. Should.

[0195] “Camelization V H ” is a naturally occurring V H corresponds to the amino acid sequence of the domain, but has been “camelized,” i.e., naturally occurring V from a conventional four-chain antibody. H V of a heavy chain antibody (camel heavy chain antibody) HH Includes amino acid sequences that have been "camelized" by replacing them with one or more of the amino acid residues that occur at corresponding positions in the domain. This is true, for example, in 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). This can be carried out in a manner known per se, which will be clear to the person skilled in the art on the basis of the description of . Such "camelization" substitutions are defined herein as V H -V L inserted at amino acid positions forming and / or present at the interface and / or at the so-called Camelidae hallmark residue (e.g. WO 1994 / 04678, and Davies and Riechmann (1994 and 1996, supra). ). In one embodiment, camelization V H V used as a starting material or starting point for producing or designing H The sequence is derived from mammalian V H sequence, e.g. human V H array, e.g. V H There are 3 arrays. However, such camelization V H can be obtained in any suitable manner known per se, thus naturally occurring V H It should be noted that the invention is not strictly limited to polypeptides obtained using polypeptides containing domains as starting material.

[0196] The structure of an immunoglobulin single variable domain sequence is referred to in the art and herein as "Framework Region 1" ("FR1"); "Framework Region 2" ("FR2"); "Framework Region 2" ("FR2"); can be thought of as consisting of four framework regions (“FR”), referred to as “Region 3” (“FR3”); and “Framework Region 4” (“FR4”); In the field and herein, "complementarity determining region 1" ("CDR1"); "complementarity determining region 2" ("CDR2"); and "complementarity determining region 3" ("CDR3"), respectively. It is interrupted by three complementarity determining regions (“CDRs”) called CDRs.

[0197] In such immunoglobulin sequences, the framework sequence may be any suitable framework sequence; examples of suitable framework sequences are found, for example, in standard handbooks and further disclosures and prior art cited herein. It is clear to a person skilled in the art based on the following.

[0198] The framework sequences are (suitable combinations of) immunoglobulin framework sequences or framework sequences derived (eg by humanization or camelization) from immunoglobulin framework sequences. For example, framework sequences may include light chain variable domains (e.g. V L sequence) and / or heavy chain variable domain (e.g. V H array or V HH It may be a framework sequence derived from a sequence). In one particular embodiment, the framework sequence is V HH A conventional V that is a framework sequence derived from a sequence (wherein said framework sequence is optionally partially or fully humanized) or that is camelized. H Sequence (as defined herein).

[0199] In particular, the framework sequences present in the ISV sequences described herein are such that the ISV sequence is a Nanobody® ISV, such as a humanized V HH or camelization V H V containing HH may contain one or more of the Characteristic Residues (as defined herein). Non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the further disclosure herein.

[0200] V H domain and V HH The total number of amino acid residues in the domain is usually in the range of 110-120, often 112-115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0201] However, the ISVs described herein may also be used with respect to the origin of the ISV sequence (or of the nucleotide sequence used to express it), from which the ISV sequence or nucleotide sequence is produced or obtained (or which is produced or obtained). It should be noted that there are no limitations on the method used). Thus, ISV sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In particular, but non-limiting embodiments, the ISV sequences are naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences, and are "humanized" (as defined herein). ) immunoglobulin sequences (e.g. partially or fully humanized mouse or rabbit immunoglobulin sequences, especially partially or fully humanized V HH sequence), “camelized” (as defined herein) immunoglobulin sequences (especially camelized V H sequence), and affinity maturation (e.g. starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, overlaying, combining combinatorial fragments from different immunoglobulin sequences, PCR using overlapping primers. or any suitable combination of any of the above; or any suitable combination of any of the foregoing.

[0202] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell). sequences, nucleotide sequences isolated from libraries (in particular expression libraries), introducing mutations into naturally occurring nucleotide sequences (using any suitable technique known per se, e.g. mismatch PCR). It may be a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using techniques for DNA synthesis known per se.

[0203] Generally, Nanobody® ISV (especially V HH sequence, (partially) humanized V HH Array and camelization V H Sequences) include one or more "characteristic residues" (also described herein) in one or more of the framework sequences (also described further herein). can be characterized by the presence of Therefore, in general, a Nanobody® ISV has a structure (general structure) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 can be defined as an immunoglobulin sequence having a characteristic One or more of the residues are as further defined herein.

[0204] In particular, Nanobody (registered trademark) ISV has a structure (general structure) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and may be an immunoglobulin sequence having a framework sequence. is as further defined herein.

[0205] More particularly, the Nanobody® ISV has a structure (general structure) FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, with the numbering according to Kabat. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 are selected from the characteristic residues listed in Table 2A below. Ru.

[0206] [Table 1-1] [Table 1-2]

[0207] In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the framework regions that are effective in preventing or reducing the binding of so-called "pre-existing antibodies" to the polypeptide. (i) the amino acid residue at position 112 is one of 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 one of K or Q; and in each of cases (iv)(i) to (iii), the amino acid residue at position 11 is preferably V. An ISV is described in WO2015 / 173325.

[0208] polypeptide An immunoglobulin single variable domain may form part of a protein or polypeptide, the protein or polypeptide comprising or consisting of one or more (at least one) immunoglobulin single variable domain. and may optionally further comprise one or more further amino acid sequences, all optionally linked via one or more suitable linkers. The term "immunoglobulin single variable domain" may also encompass such polypeptides. One or more immunoglobulin single variable domains are used as a binding unit in such a protein or polypeptide, which may optionally contain one or more additional amino acids that can act as a binding unit, Respectively, monovalent, multivalent or multispecific polypeptides of the invention can be provided (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also , Conrath et al. 2001 (J. Biol. Chem. 276:7346) and eg WO1996 / 34103, WO1999 / 23221 and WO2010 / 115998).

[0209] A polypeptide may comprise or consist essentially of one immunoglobulin single variable domain as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.

[0210] The term "multivalent" indicates the presence of multiple ISVs in a polypeptide. In one embodiment, the polypeptide is "bivalent," ie, comprises or consists of two ISVs. In one embodiment, the polypeptide is "trivalent," ie, comprises or consists of three ISVs. In another embodiment, the polypeptide is "tetravalent," ie, comprises or consists of four ISVs. Therefore, a polypeptide may be "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," "octavalent," "navivalent," etc. Often, ie, the polypeptide comprises or consists of 2, 3, 4, 5, 6, 7, 8, 9, etc. ISVs, respectively. 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.

[0211] In one embodiment, multivalent ISV polypeptides can also be multispecific. The term "multispecific" refers to binding to a large number of different target molecules (also called antigens). Thus, a multivalent ISV polypeptide may be "bispecific," "trispecific," "tetraspecific," etc., i.e., capable of targeting two, three, four, etc., different target molecules, respectively. can be combined with

[0212] For example, the polypeptide is a bispecific trivalent, e.g. polypeptide comprising or consisting of three ISVs, where two ISVs bind to a first target and one ISV binds to a first target. may be a polypeptide that binds to a different second target. In another example, the polypeptide is a polypeptide comprising or consisting of a trispecific tetravalent, e.g., four ISVs, where one ISV binds to the first target and two ISVs bind to the first target. It may be a polypeptide that binds to a second target that is different from the target, and one ISV binds to a third target that is different from the first and second targets. In yet another example, the polypeptide is a polypeptide comprising or consisting of trispecific pentavalents, e.g., five ISVs, wherein two ISVs bind to a first target and two ISVs bind to a first target. and one ISV binds a third target different from the first and second targets.

[0213] In one embodiment, a multivalent ISV polypeptide can also be multiparatopic. The term "multiparatopic" refers to binding to multiple different epitopes on the same target molecule (also called antigen). Thus, multivalent ISV polypeptides can be "biparatopic," "triparatopic," etc., ie, each can bind to two, three, etc. different epitopes on the same target molecule.

[0214] In another embodiment, a polypeptide of the invention comprising or consisting essentially of one or more immunoglobulin single variable domains (or a suitable fragment thereof) contains one or more other groups, It may further include residues, moieties or linking units. Such additional groups, residues, moieties, binding units or amino acid sequences may or may not provide additional functionality to the immunoglobulin single variable domain (and / or to the polypeptide in which it is present). The characteristics of the immunoglobulin single variable domain may or may not be altered.

[0215] For example, such additional groups, residues, moieties or binding units may include one or more additional It may also be an amino acid. In a preferred but non-limiting embodiment, said one or more other groups, residues, moieties or binding units are immunoglobulins. Even more preferably, said one or more other groups, residues, moieties or binding units are domain antibodies, amino acids suitable for use as domain antibodies, single domain antibodies, single domain antibodies. selected from the group consisting of amino acids suitable for use, "dAbs", amino acids suitable for use as dAbs, or nanobodies.

[0216] Alternatively, such groups, residues, moieties or linking units may be, for example, chemical groups, residues, moieties which may or may not themselves be biologically and / or pharmacologically active. It may be. For example, and without limitation, such a group can be linked to one or more immunoglobulin single variable domains to provide a "derivative" of an immunoglobulin single variable domain.

[0217] In another embodiment, the additional residues may be effective in preventing or reducing the binding of so-called "pre-existing antibodies" to the polypeptide. For this purpose, polypeptides and constructs may contain a C-terminal extension (X)n (SEQ ID NO: 150), where n is 1 to 10, preferably 1 to 5, such as 1, 2, 3 , 4 or 5 (preferably 1 or 2, such as 1); each X is independently selected and preferably independently alanine (A), glycine (G), valine (V), leucine ( L) or isoleucine (I), for which reference is made to WO2012 / 175741.Therefore, the polypeptide It may further include an extension (X)n (SEQ ID NO: 151), where n is 1 to 5, such as 1, 2, 3, 4 or 5, and X is a naturally occurring amino acid, preferably Not cysteine.

[0218] In the polypeptides described above, one or more immunoglobulin single variable domains and one or more groups, residues, moieties or linking units are directly connected to each other and / or one or They can be linked via any other suitable linker or spacer. For example, if one or more groups, residues, moieties or linking units are amino acids, the linker may also be an amino acid, such that the resulting polypeptide is a fusion protein or polypeptide.

[0219] As used herein, the term "linker" refers to a peptide that fuses two or more ISVs together into a single molecule. The use of linkers to connect two or more peptides (polypeptides) is well known in the art. Additional exemplary peptide linkers are shown in Table 2B. One commonly used class of peptide linkers is known as "Gly-Ser" or "GS" linkers. These consist essentially of glycine (G) and serine (S) residues and are usually peptide motifs, such as the GGGGS (SEQ ID NO: 154) motif, such as the formula (Gly-Gly-Gly-Gly-Ser)n( 152), where n may be 1, 2, 3, 4, 5, 6, 7 or more). Some commonly used examples of such GS linkers are the 9GS linker (GGGGGSGGGS, SEQ ID NO: 157), the 15GS linker (n=3) and the 35GS linker (n=7). Reference is made, for example, to 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).

[0220] [Table 2]

[0221] In one aspect, the present disclosure also provides amino acid sequences and / or Nanobodies capable of binding to and / or targeting CD8 and generally comprising CDR sequences as further defined herein. , suitable fragments thereof, and polypeptides comprising or consisting essentially of one or more of such Nanobodies and / or suitable fragments. In some embodiments, the present disclosure relates to a Nanobody having SEQ ID NO: 77. In particular, the present disclosure in some specific aspects: I) an amino acid sequence directed to CD8 and having at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with at least one of the amino acid sequences of SEQ ID NO: 77; II) an amino acid sequence that cross-blocks binding of the amino acid sequence of SEQ ID NO: 77 to CD8 and / or competes with at least the amino acid sequence of SEQ ID NO: 77 for binding to CD8; I will provide a.

[0222] Such amino acid sequences may be as further described herein (e.g., may be Nanobodies); polypeptides of the disclosure comprising one or more of such amino acid sequences may peptides (which may be as further described herein), and especially bispecific (or multispecific) polypeptides as described herein, and the amino acid sequences and polypeptides encoding such Nucleic acid sequences may also be as further described herein. Such amino acid sequences and polypeptides are free of any naturally occurring ligands.

[0223] In some embodiments, the CD8 is derived from a mammal, such as a human. In one specific but non-limiting aspect, the present disclosure provides: a) Amino acid sequence of SEQ ID NO: 77; b) an amino acid sequence having at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NO: 77; or c) an amino acid sequence having 3, 2 or 1 amino acid difference from at least one of the amino acid sequences of SEQ ID NO: 77; or any suitable combination thereof relates to an amino acid sequence directed to CD8, including.

[0224] In some embodiments, nanobodies to CD8 are disclosed that consist of four framework regions (FR1-FR4, respectively) and three complementarity-determining regions (CDR1-CDR3, respectively). In some embodiments, such Nanobodies: (I) CDR1 is 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 a substitution; and / or (2) said amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared to GSTFSDYG (SEQ ID NO: 100); and / or an amino acid sequence from the group consisting of amino acid sequences having two or only one 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 is an amino acid sequence that contains only amino acid substitutions and no amino acid deletions or insertions compared to GSTFSDYG (SEQ ID NO: 100). Containing or consisting essentially of.

[0225] (II) CDR2 is 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 a substitution; and / or (2) said amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared to IDWNGEHT (SEQ ID NO: 101); and / or an amino acid sequence from the group consisting of amino acid sequences having two or only one 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 is an amino acid sequence that contains only amino acid substitutions and no amino acid deletions or insertions compared to IDWNGEHT (SEQ ID NO: 101). Containing or consisting essentially of.

[0226] (III) CDR3 is the amino acid sequence of ADALPYTVRKYNY (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 a substitution; and / or (2) said amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions as compared to ADALPYTVRKYNY (SEQ ID NO: 102); and / or an amino acid sequence from the group consisting of amino acid sequences having two or only one 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 is an amino acid sequence that contains only amino acid substitutions and no amino acid deletions or insertions as compared to ADALPYTVRKYNY (SEQ ID NO: 102). Containing or consisting essentially of.

[0227] The CD8 Nanobodies disclosed herein may contain 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 nomenclature; CDR2: IDWNGEHT (SEQ ID NO: 101) according to IMGT nomenclature; and CDR3: AADALPYTVRKYNY (SEQ ID NO: 102) based on IMGT nomenclature including.

[0228] In the Nanobodies of the present disclosure comprising a combination of the CDRs mentioned above, each CDR has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably A CDR selected from the group consisting of amino acid sequences having at least 99% sequence identity: (1) Any amino acid substitutions are preferably conservative amino acid substitutions; and / or (2) Said amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions compared to said amino acid sequence. CDR; and / or a CDR selected from the group consisting of an amino acid sequence having 3, 2 or only 1 (indicated in the paragraph above) "amino acid differences" with one of the mentioned CDRs of the above amino acid sequence. hand: (1) Any amino acid substitutions are preferably conservative amino acid substitutions; and / or (2) Said amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions compared to said amino acid sequence. Can be replaced by CDR.

[0229] In one embodiment, the CD8 Nanobody is BDSn: Anti-CD8 BDSn Nb sequence (CDR1, CDR2, CDR3 are underlined based on IMGT nomenclature): [ka] It is.

[0230] In some embodiments, the CD8 Nanobodies of the present disclosure contain 10 -5 ~10 -12 mole / liter (M) or less, preferably 10 -7 ~10 -12 mole / liter (M) or less, more preferably 10 -8 ~10 -12 with a dissociation constant (KD) of moles / liter (M) and / or at least 10 7 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 binds to CD8 with an association constant (KA) of; in particular with a KD of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 μM. The KD and KA values ​​of the Nanobodies of the disclosure against 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.

[0231] It should be noted that, in general, the term nanobody, as used herein, in its broadest sense, is not limited to a particular biological source or to a particular method of preparation. For example, as explained in more detail below, Nanobodies can be created by: (1) isolating a naturally occurring VHH domain of a heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of a naturally occurring VHH domain (as described below) or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by any animal species, especially by “camelization” (as described below) of a naturally occurring VH domain from a mammalian species, e.g., from humans, or by expression of a nucleic acid encoding such a camelized VH domain; (5) (6) by "camelization" of "domain antibodies" or "Dabs" as described by Ward et al. (supra), or by expression of nucleic acids encoding such camelized VH domains; (6) proteins, polypeptides or other (7) preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis, and subsequently the nucleic acid so obtained; and / or (8) any combination of the above. Suitable methods and techniques for doing 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 herein below.

[0232] In some embodiments, the CD8 Nanobodies of the present disclosure are identical to the amino acid sequence of a naturally occurring VH domain, such as a naturally occurring VH domain from a mammal, particularly a human (i.e. (as a degree of 100% sequence identity with) the amino acid sequence.

[0233] One class of CD8 Nanobodies of the present disclosure correspond to the amino acid sequence of a naturally occurring VHH domain, but are "humanized," i.e., one or more of the amino acid sequences of said naturally occurring VHH sequence. "Humanized" by replacing the above amino acid residues with one or more of the amino acid residues that occur at the corresponding positions in the VH domain from a conventional four-chain antibody of human origin (as shown above). including Nanobodies having an amino acid sequence that is Such humanized CD8 Nanobodies of the present disclosure can be obtained in any suitable manner known per se (i.e. as indicated in points (1) to (8) above) and thus It should be noted that the invention is not strictly limited to polypeptides obtained using as starting material polypeptides containing VHH domains present in

[0234] Another class of CD8 Nanobodies of the present disclosure are those that are “camelized,” i.e., have one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain derived from a conventional four-chain antibody. having an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain that has been "camelized" by substitution by one or more of the amino acid residues that occur at the corresponding position in the VHH domain of the antibody. Contains nanobodies. This can be done in a manner known per se, which will be clear to the person skilled in the art, for example on the basis of the further description below. Reference is also made to WO94 / 04678. Such camelization may occur preferentially at amino acid positions present at the VH-VL interface and at so-called camel characteristic residues (see also e.g. WO94 / 04678), as also mentioned below. . In some embodiments, the VH domain or sequence used as a starting material or starting point for producing or designing a camelized Nanobody is a mammalian-derived VH sequence, such as a human VH sequence. Such camelized Nanobodies of the present disclosure may be obtained in any suitable manner known per se, and thus may be obtained using naturally occurring VH domain-containing polypeptides as starting material. It should be noted that it is not strictly limited to peptides.

[0235] For example, both "humanization" and "camelization" provide a nucleotide sequence encoding such a naturally occurring VHH domain or a VH domain, respectively, after which the new nucleotide sequence is modified, respectively, of the present disclosure. altering one or more codons in said nucleotide sequence in a manner known per se so as to code for a humanized or camelized Nanobody, and then the nucleotide sequence so obtained in a manner known per se; This can be done by expressing the desired Nanobody to provide the desired Nanobody. Alternatively, the amino acid sequences of the desired humanized or camelized Nanobodies of the present disclosure are designed based on the amino acid sequences of naturally occurring VHH domains or VH domains, respectively, and then for peptide synthesis known per se. It may also be synthesized de novo using the technique of It is also possible to design a nucleotide sequence encoding the desired humanized or camelized Nanobody based on the amino acid or nucleotide sequence of a naturally occurring VHH domain or VH domains, respectively, and subsequently for nucleic acid synthesis known per se. They may be synthesized de novo using techniques and the nucleotide sequences thus obtained may then be expressed in a manner known per se to provide the desired Nanobodies.

[0236] Starting from a naturally occurring VH domain or preferably a VHH domain (its amino acid sequence) and / or from a nucleotide sequence and / or nucleic acid encoding it, a Nanobody and / or a nucleotide sequence encoding it and / or Other suitable methods and techniques for obtaining nucleic acids will be apparent to those skilled in the art, for example, in a suitable manner naturally occurring VH domains (e.g. domains of one or more FRs and / or CDRs). one or more amino acid and / or nucleotide sequences derived from a naturally occurring VHH domain (e.g. one or more FR domains or CDR domains) In combination with an amino acid sequence and / or a nucleotide sequence to provide (a nucleotide sequence or nucleic acid encoding) a Nanobody. It also comprises or consists essentially of at least one such amino acid sequence and / or Nanobody of the present disclosure (or a suitable fragment thereof) and optionally one or more other groups, residues. Compounds and constructs, particularly proteins and polypeptides, further comprising , a moiety or a binding unit are provided. In some embodiments, such additional groups, residues, moieties, binding units or amino acid sequences provide additional functionality to the amino acid sequences and / or Nanobodies (and / or to the compounds or constructs in which they are present). may or may not be provided, and the amino acid sequence and / or properties of the Nanobody may or may not be modified.

[0237] The present disclosure also encompasses any polypeptide of the present disclosure that is typically glycosylated at one or more amino acid positions depending on the hot used to express the polypeptide. The polypeptide may comprise the amino acid sequence of a CD8 Nanobody of the present disclosure, which is fused at its amino terminus, at its carboxy terminus, or at both its amino terminus and its carboxy terminus, to at least one additional amino acid sequence. ing. Such further amino acid sequences may comprise at least one further Nanobody to provide a polypeptide comprising at least 2, such as 3, 4 or 5 Nanobodies, wherein said Nanobodies optionally include The linkage can be via one or more linker sequences (as defined herein). A polypeptide comprising a CD8 Nanobody and one or more other Nanobodies of the present disclosure is a multivalent polypeptide. In a multivalent polypeptide, the 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. against the same part or epitope of said antigen, or against two or more different parts or epitopes of said antigen; and / or: ·May be directed against different antigens; ·or a combination thereof It may be. Thus, a bivalent polypeptide may be, for example: ·May contain two identical nanobodies; a first Nanobody directed against a first part or epitope of an antigen, and a second Nanobody directed against the same part or epitope of said antigen or against another part or epitope of said antigen; may include nanobodies of; or may include a first Nanobody directed against a first antigen and a second Nanobody directed against a second antigen different from said first antigen; On the other hand, trivalent polypeptides of the invention may e.g. · may contain three identical or different Nanobodies directed against the same or different parts or epitopes of the same antigen; two identical or different Nanobodies directed against the same or different parts or epitopes on a first antigen, and a third directed against a second antigen different from said first antigen; may include nanobodies of; or a first Nanobody directed against a first antigen, a second Nanobody directed against a second antigen different from said first antigen, and said first and second antigens; may include a third Nanobody directed against a third antigen different from the third Nanobody.

[0238] The CD8 Nanobodies and polypeptides disclosed herein can also be used in one or more cells, tissues or organs of a multicellular organism, e.g. for prophylactic and / or therapeutic purposes (e.g. as gene therapy). may be introduced and expressed. To this end, the nucleotide sequences encoding the CD8 Nanobodies or polypeptides disclosed herein can be used in any suitable manner, e.g., as such (e.g., using liposomes) or in a suitable gene therapy. After insertion into a vector (eg, derived from a retrovirus, such as an adenovirus, or a parvovirus, such as an adeno-associated virus), it can be introduced into a cell or tissue. As will be clear to those skilled in the art, such gene therapy also involves administering a nucleic acid of the invention or a suitable gene therapy vector encoding it to a patient or to specific cells or specific tissues or organs of the patient. It may be carried out in vivo and / or in situ within the patient's body; alternatively, it may be carried out using suitable cells (often cells taken from the body of the patient to be treated, e.g. explanted lymph). cells, bone marrow aspirates or tissue biopsies) may be treated in vitro with the nucleotide sequences of the invention and then suitably introduced (re-introduced) into the body of the patient. All this is discussed in Culver, K. W., "Gene Therapy", 1994, p. 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;WO94 / 29469;WO97 / 00957, U.S. Patent No. 5,580,859 1 US Pat. No. 5,589,5466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, can be performed using gene therapy vectors, techniques and delivery systems well known to those skilled in the art. For example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and in situ expression of diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) Explained in the technical field.

[0239] Accordingly, also provided are nucleic acid sequences encoding the CD8 Nanobodies described herein, as well as expression constructs and host cells comprising the nucleic acid sequences.

[0240] Also disclosed are methods of using the CD8 Nanobodies and polypeptides of the present disclosure.

[0241] In some embodiments, polypeptides comprising CD8 Nanobodies can be used in lipid nanoparticles of the present disclosure to deliver nucleic acids to immune cells, as described herein. In some embodiments, the CD8 Nanobodies and polypeptides 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.

[0242] In some embodiments, polypeptides comprising CD8 Nanobodies can be used in contrast agents. In some embodiments, the contrast agent allows detection of human CD8, a specific biomarker found on the surface of a subset of T cells, for imaging of the immune system. CD8 imaging allows in vivo detection of T cell localization. Changes in T cell localization can reflect the progression of an immune response and can occur over time as a result of various therapeutic treatments or even as a disease state. In some embodiments, it is used to image T cell localization for immunotherapy.

[0243] In addition, CD8 plays a role in activating downstream signaling pathways that are important for the activation of cytolytic T cells that function to clear viral pathogens and provide immunity to tumors. . CD8-positive T cells can recognize short peptides presented within the MHCI protein of antigen-presenting cells. In some embodiments, a polypeptide comprising a CD8 Nanobody can enhance signaling through the T cell receptor and improve a subject's ability to clear viral pathogens and respond to tumor antigens. Thus, in some embodiments, the antigen binding constructs provided herein may be agonists and are capable of activating a CD8 target.

[0244] II. Ionizable cationic lipids To produce a lipid nanoparticle composition that facilitates the delivery of a payload (e.g. a nucleic acid, e.g. DNA or RNA, e.g. mRNA) placed within the lipid nanoparticle composition to a cell, e.g. a mammalian cell, e.g. an immune cell. Provided herein are ionizable cationic lipids that can be used for. Ionizable cationic lipids are designed to enable intracellular delivery of nucleic acids, such as mRNA, to the cytosolic compartment of target cell types and to rapidly degrade into non-toxic components. The complex functionality of ionizable cationic lipids is due to the chemical and geometric interaction of the ionizable lipid headgroup, the hydrophobic "acyl-tail" group, and the linker that connects the headgroup and acyl-tail group. Facilitated by action. Typically, the pK of the ionizable amine headgroup a is that it remains strongly cationic under acidic formulation conditions (e.g. pH 4~pH 5.5), is neutral at physiological pH (7.4), and is active in early and late endosomal compartments (e.g. pH 5.5~ It is designed to be cationic at a pH of 7) within the range of 6 to 8, for example between 6.2 and 7.4, or between 6.5 and 7.1. Acyl-tail groups play an important role in the fusion of lipid nanoparticles with endosomal membranes and in membrane destabilization through structural perturbation. The three-dimensional structure of the acyl-tail (determined by its length and degree and site of unsaturation), together with the relative sizes of the head and tail groups, promotes membrane fusion and, therefore, lipid nanoparticles. It is thought to play a role in particle endosomal evasion, an important prerequisite for delivery of nucleic acid payloads to the cytosol. The linker connecting the head group and the acyl tail group is designed to be degraded by physiologically prevalent enzymes (eg, esterases or proteases) or by acid-catalyzed hydrolysis.

[0245] In one embodiment, the present invention provides the formula I: [ka] or a salt thereof, in which: R 1 and R 2 is independently C 1~3 is alkyl or R 1 and R 2 together with the nitrogen atom forms optionally substituted piperidinyl or morpholinyl; Y is -O-, -OC(O)-, -OC(S)- and -CH 2 - selected from the group consisting of; X 1 ,X 2 ,X 3 and X 4 is hydrogen or X 1 and X 2 Or X 3 and X 4 independently and together form oxo; n is 0 or 3; o and p are independently integers selected from 2 to 6; The compound is [ka] or a salt thereof.

[0246] In certain embodiments, o and p may be 2. In certain embodiments, o and p may be 3. In other embodiments, o and p may be 4. In some embodiments, o and p may be 5. In other embodiments, o and p may be 6.

[0247] In certain embodiments, X 1 and X 2 may be taken together to form oxo, 3 and X 4 together form oxo. In other embodiments, X 1 ,X 2 ,X 3 and X 4 can be hydrogen.

[0248] In certain embodiments, Y is -O-, -OC(O)-, OC(S)- and -CH 2 - may be selected from the group consisting of. For example, in certain embodiments, Y can be -O-. In certain embodiments, Y can be -OC(O)-. In certain embodiments, Y is -CH 2 -Can be. In certain embodiments, Y can be -OC(S)-.

[0249] In certain embodiments, R 1 and R 2 is independently C 1~3 Can be alkyl. In other embodiments, R 1 and R 2 is -CH 3 It can be. In certain embodiments, R 1 and R 2 is -CH 2 CH 3 It is. In certain embodiments, R 1 and R 2 is C 3 It is an alkyl.

[0250] In certain embodiments, n can be 0. In other embodiments, n can be 3.

[0251] Also, in part, Formula II: [ka] Provided herein is a compound represented by or a salt thereof, where: R 1 and R 2 is independently C 1~3 is alkyl or R 1 and R 2 together with the nitrogen atom forms optionally substituted piperidinyl or morpholinyl; Y is -O-, -OC(O)-, -OC(S)- and -CH 2 - selected from the group consisting of; X 1 ,X 2 ,X 3 and X 4 is hydrogen or X 1 and X 2 Or X 3 and X 4 together form oxo; n is 0 to 4; o is 1 and r is an integer selected from 3 to 8; or o is 2 and r is an integer selected from 1 to 8; p is 1 and s is an integer selected from 3 to 8; or p is 2 and s is an integer selected from 1 to 8; here, If o and p are both 1, then r and s are independently 4, 5, 7 or 8; When o and p are both 2, r and s are independently 1, 2, 4 or 5.

[0252] In certain embodiments, X 1 and X 2 may be taken together to form oxo, 3 and X 4 may be taken together to form oxo. In other embodiments, X 1 ,X 2 ,X 3 and X 4 can be hydrogen.

[0253] In certain embodiments, Y is -O-, -OC(O)- and -CH 2 - may be selected from the group consisting of. For example, in certain embodiments, Y can be -O-. In certain embodiments, Y can be -OC(O)-. In certain embodiments, Y is -CH 2 -Can be. In certain embodiments, Y can be -OC(S)-.

[0254] In certain embodiments, R 1 and R 2 is independently C 1~3 Can be alkyl. In other embodiments, R 1 and R 2 is -CH 3 It can be. In certain embodiments, R 1 and R 2 is -CH 2 CH 3 It can be. In some embodiments, R 1 and R 2 is C 3 Can be alkyl. In certain embodiments, R 1 and R 2 together with the nitrogen atom form optionally substituted piperidinyl.

[0255] In certain embodiments, n can be 0. In other embodiments, n can be 3.

[0256] In part: [ka] Provided herein is a compound selected from the group consisting of: or a salt thereof.

[0257] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0258] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0259] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0260] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0261] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0262] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0263] In part, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0264] In certain embodiments, the compound has formula III: [ka] or a salt thereof, in the formula: R 1 and R 2 is independently C 1~3 is alkyl or R 1 and R 2 together with the nitrogen atom forms optionally substituted piperidinyl or morpholinyl; Y is -O-, -OC(O)-, -OC(S)- and -CH 2 - selected from the group consisting of; X 1 ,X 2 ,X 3 and X 4 is hydrogen or X 1 and X 2 Or X 3 and X 4 together form oxo; n is an integer selected from 0 to 4.

[0265] In certain embodiments, X 1 and X 2 may be taken together to form oxo, 3 and X 4 may be taken together to form oxo. In other embodiments, X 1 ,X 2 ,X 3 and X 4 can be hydrogen.

[0266] In certain embodiments, Y is -O-, -OC(O)- and -CH 2- may be selected from the group consisting of. For example, in certain embodiments, Y can be -O-. In certain embodiments, Y can be -OC(O)-. In certain embodiments, Y is -CH 2 -Can be. In certain embodiments, Y can be -OC(S)-.

[0267] In certain embodiments, R 1 and R 2 is independently C 1~3 Can be alkyl. In other embodiments, R 1 and R 2 is -CH 3 It can be. In certain embodiments, R 1 and R 2 is -CH 2 CH 3 It can be. In some embodiments, R 1 and R 2 is C 3 Can be alkyl. In certain embodiments, R 1 and R 2 together with the nitrogen atom form optionally substituted piperidinyl.

[0268] In certain embodiments, n can be 0. In other embodiments, n can be 3.

[0269] Also, the formula: [ka] Provided herein is a compound of or a salt thereof.

[0270] Scheme 1 - Synthetic scheme for producing lipids of formula (I) [ka]

[0271] Compounds of formula I can be prepared, for example, according to scheme 1. Hydroxy functional protected propane diol is converted to the corresponding dimethylamino group ether (Y=oxo) or ester (Y=O-C(O)). Ether bond formation results from the reaction of an alkyl halide with an alcohol in the presence of tert-butylammonium iodide / NaOH in THF at 80°C. Ester bond formation utilizes treatment of acid-functional dimethylamine with alcohol under carbodiimide activation (DCM, EDC, DIEPA, DMAP). Diol deprotection yields a vicinal diol intermediate, which is subsequently converted to the corresponding ether by treatment with TBAI / NaOH and bromo-acyl or by carbodiimide-mediated carboxylic acid activation for ester bond formation, respectively. bond, or converted into an ester-bonded diacyl lipid.

[0272] Scheme 2 - Synthetic scheme for producing lipid compositions of formula (II) [ka]

[0273] Compounds of formula II can be prepared, for example, according to scheme 2. The synthetic procedure is as outlined above for Scheme 1; however, in Scheme 2, either a bis-unsaturated acyl group or a mono-unsaturated acyl group is used to obtain a lipid of formula II. You can.

[0274] In some embodiments, the ionizable cationic lipids used in the LNPs of the present disclosure are selected from the lipids in Table 1 or combinations thereof. In some embodiments, the ionizable cationic lipid is: [ka] It is.

[0275] In some embodiments, the ionizable cationic lipid is not Dlin-MC3-DMA.

[0276] III. Lipid-immune cell targeting group conjugates As described herein, LNPs can be targeted to specific cell types, such as immune cells, such as T cells, B cells or natural killer (NK) cells. This can be accomplished using one or more of the lipids described herein. Additionally, targeting can be enhanced by including targeting groups on the solvent-accessible surface of the LNP particles. For example, targeting groups can include members of specific binding pairs, such as antibody-antigen pairs, ligand-receptor pairs, and the like. 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.

[0277] Optionally, the targeting moiety is an antibody fragment without an Fc component. Previous attempts to target circulating immune cells with LNPs used whole antibodies (WO2016 / 189532 Al). Liposomes or lipid-based particles conjugated with intact antibodies are cleared from the circulation more quickly due to Fc engagement and are less likely to reach the intended target cells (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 by antibody fragments are targeted to 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) as targeted liposomes, retaining their long circulation properties. In addition, very inefficient insertion when conjugating intact IgG (Ishida et al. (1999) FEBS Lett. 460, 129-133) or the need to complete conjugation directly to intact LNPs. (WO2016 / 189532 Al), lipid-based carriers can be prepared using a micellar insertion process that allows almost quantitative incorporation of antibody conjugates after separate production of antibody conjugates. (Nellis et al. (2005) Biotechnol Prog 21, 221-232). Alternatively, scFv, Fab or VHH fragments can be conjugated directly to activated PEG-lipids to produce insertable conjugates.

[0278] In certain embodiments, the targeting group may be a surface-bound antibody or surface-bound antigen-binding fragment thereof, which may allow for modulation of cell targeting specificity. This is particularly useful since highly specific antibodies can be generated against the epitope of interest for the desired targeting site. In one embodiment, a number of different antibodies can be incorporated and displayed on the surface of the LNP, where each antibody binds to a different epitope on the same antigen, or to a different epitope on a different antigen. Such approaches can increase avidity and specificity to target interactions to specific target cells.

[0279] The targeting group or combination of targeting groups can be selected based on the desired localization, function, or structural characteristics of a given target cell. one or more antibodies or antigen-binding fragments thereof or antigens that target T cells, e.g., via a T cell surface antigen, to target T cells, T cell populations or T cell subpopulations; Conjugated derivatives can 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) beta, TCR-α, TCR- including but not limited to α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, GL7, TLR2, TLR4, TLR5 and IL-15 receptors . To target NK cells or NK cell populations, one or more antibodies, antigen-binding fragments or antigen-binding derivatives thereof can be selected that target NK cells, e.g., via an NK cell surface antigen. can. Exemplary NK cell surface antigens include CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, Includes, but is not limited to, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, CD337 / NKP30. To target B cells or B cell populations, one or more antibodies, antigen-binding fragments or antigen-binding derivatives thereof can be selected that target B cells, for example via a B cell antigen. . Exemplary B cell antigens include CD19 for all B cells except plasma cells, CD19, CD25 and CD30 for activated B cells, CD27, CD38, CD78, CD138 and CD319 for plasma cells, and for memory cells. CD20, CD27, CD40, CD80 and PDL-2 for marginal zone B cells, Notch2, CD1, CD21 and CD27 for marginal zone B cells, CD21, CD22 and CD23 for follicular B cells, and CD1, CD5 for regulatory B cells, Includes, but is not limited to, CD21, CD24 and TLR4.

[0280] In certain embodiments, targeting can be performed, for example, by using the lipid-immune cell targeting group conjugates described herein. Exemplary lipid-immune cell targeting group conjugates are of formula IV: [lipid] - [optional linker] - [immune cell targeting group, e.g. T cell targeting molecule, anti-CD2 antibody, anti-CD3 antibody, anti-CD7 antibody or anti-CD8 antibody] (Formula IV) may contain compounds of

[0281] In some embodiments, the immune cell targeting group is a polypeptide and the lipid is conjugated to the N-terminus, C-terminus, or somewhere in the middle of the polypeptide.

[0282] In certain embodiments, the targeting group or targeting molecule is CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T cell receptor (TCR) beta, 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 A T cell targeting agent, such as an antibody, that binds to a T cell antigen that is expressed. In certain embodiments, the T cell antigen may be CD2 and the targeting group may be, for example, an anti-CD2 antibody. In certain embodiments, the T cell antigen may be CD3 and the targeting group may be, for example, an anti-CD3 antibody. In certain embodiments, the T cell antigen may be CD4 and the targeting group may be, for example, an anti-CD4 antibody. In certain embodiments, the T cell antigen may be CD5 and the targeting group may be, for example, an anti-CD5 antibody. In certain embodiments, the T cell antigen may be CD7 and the targeting group may be, for example, an anti-CD7 antibody. In certain embodiments, the T cell antigen may be CD8 and the targeting group may be, for example, an anti-CD8 antibody. In certain embodiments, the T cell antigen may be TCRβ and the targeting group may be, for example, an anti-TCRβ antibody. In some embodiments, the antibody is a human or humanized antibody.

[0283] Exemplary CD2 binding agents are 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 (US Patent No. 6849258B1), Cipiluzumab / MEDI -507 (US Patent No. 6849258B1), 35.1 (ATCC HB-222), OKT11 (ATCC CRL-8027), RPA-2.1 (PCT Publication WO2020023559A1), AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 (R&D Systems), MAB1856 (R&D Systems), PAB30359 (Abnova Corporation), 10299-1 (Abnova Corporation), and antigen-binding fragments thereof. In certain embodiments, the binder consists of AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 (R&D Systems), MAB1856 (R&D Systems), PAB30359 (Abnova Corporation) and 10299-1 (Abnova Corporation) The heavy chain variable domain (V H ) and light chain variable domain (V L )including. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art AF1856(R&D Systems), MAB18562(R&D Systems), MAB18561(R&D Systems), MAB1856(R&D Systems), PAB30359(Abnova Corporation) and 10299-1 as determined under any other CDR determination method known in the art. (Abnova Corporation) of an antibody selected from the group consisting of H and V L Sequence, heavy chain CDR 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0284] Exemplary CD2 binding agents are clone 9.6, 9-1, TS2 / 18.1.1, Lo-CD2b, Lo-CD2a, BTI-322, sipiluzumab, 35.1, OKT11, RPA-2.1, SQB-3.21, LT2, TS1 It is also possible to select from antibodies or antibody fragments using CDRs of / 8, UT329, 4F22, OX-34, UQ2 / 42, MU3, U7.4, NFN-76 or MOM-181-4-F(E).

[0285] Exemplary CD3 binding agents (CD3γ / δ / ε, CD3γ, CD3δ, CD3γ / ε, CD3δ / ε or CD3ε) include MEM-57 (CD3γ / δ / ε, EnzoLife 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- The antibody may be selected from the group consisting of AP (CD3δ, Invitrogen) and antigen-binding fragments thereof. In certain embodiments, the binding agent is MEM-57 (CD3γ / δ / ε, EnzoLife 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). H domain and V LContains domains. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art MEM-57 (CD3γ / δ / ε, EnzoLife Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Abnova Corporation), CD3 determined under any other CDR determination method known. -12 (CD3ε, Cell Signaling Technology), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, Novus Biologicals) and 16669-1-AP (CD3δ, Invitrogen). V of the antibody H and V L Sequence, heavy chain CDR 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0286] Exemplary CD3 binding agents include 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 (US Patent Application Publication No. 20200299409A1), REGN5458 (US 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) 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.

[0287] Exemplary CD4 binding agents are 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 binding agent is a V of an antibody selected from the group consisting of AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abcam). H domain and V L Contains domains. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art Selected from the group consisting of AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation) and CAL4 (Abcam), determined under any other CDR determination method known. V of the antibody to be H and V L Heavy chain CDR of sequence 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0288] Exemplary CD4 binding agents are 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 ilvalizumab.

[0289] Exemplary CD5 binding agents include He3, MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Abcam), CD5 / 54 / F6 (Abcam), The antibody may be selected from the group consisting of 65152 (Proteintech) and antigen-binding fragments thereof. In some embodiments, the binder is MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Abcam), CD5 / 54 / F6 (Abcam) and V of the antibody selected from the group consisting of 65152 (Proteintech). H domain and V L including. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Abcam), CD5 / determined under any other CDR determination method known in the art. V of an antibody selected from the group consisting of 54 / F6 (Abcam) and 65152 (Proteintech) H and V L Heavy chain CDR of sequence 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0290] Exemplary CD5 binding agents include 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 / It is also possible to select from antibodies or antibody fragments using the CDRs of the F6, Q-20, CC17, MOM-18539-S(P) or MOM-18885-S(P) clones.

[0291] Exemplary CD7 binding agents include MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), NBP2-38440 (Novus Biologicals) and their antigen binding properties. The antibody may be selected from the group consisting of fragments. In certain embodiments, the binding agent consists of MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals) and NBP2-38440 (Novus Biologicals) V of the antibody selected from the group H domain and V Lincluding. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals) and NBP2-38440 determined under any other CDR determination method known V of an antibody selected from the group consisting of (Novus Biologicals) H and V L Heavy chain CDR of sequence 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0292] Exemplary CD7 binding agents can also be selected from antibodies or antibody fragments that use the CDRs of clones TH-69, 3Afl1, T3-3A1, 124-1D1, 3A1f, CD7-6B7 or VHH6.

[0293] Exemplary CD8 (CD8α, CD8α / α, CD8α / β or CD8β) binding agents include 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 The antibody may be selected from the group consisting of antigen-binding fragments thereof. In certain embodiments, the binding agent is 2.43 (Invitrogen), 51.1 (ATCC HB-230), 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). H domain and V L Contains domains. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art 2.43 (Invitrogen), Du CD8-1 (CD8α, Invitrogen), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems) determined under any other CDR determination method known. ), ab4055 (CD8α, Abcam), C8 / 144B (CD8α, Novus Biologicals) and YTS105.18 (CD8α, Novus Biologicals). H and V L Heavy chain CDR of sequence 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0294] Exemplary CD8 binders include clones 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 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.

[0295] Exemplary CD137 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone 4B4-1, P566 or urelumab. Exemplary CD28 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone TAB08. Exemplary CD45 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clones BC8, 9.4, 4B2, Tu116 or GAP8.3. Exemplary CD18 binding agents 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 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone MHM24 or efalizumab. Exemplary IL-2 receptor binders include clone YTH 906.9HL, IL2R.1, BC96, B-B10, 216, MEM-181, ITYV, MEM-140, ICO-105, daclizumab, or IL2 or IL2 Antibodies or antibody fragments using CDRs from the group consisting of fragments of. Exemplary IL-15R binding agents can be selected from antibodies or antibody fragments that use CDRs from the group consisting of clones JM7A4 or OTI3D5, or from the group consisting of IL15 or fragments of IL15. Exemplary TLR2 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clones JM22-41, TL2.1, 11G7 or TLR2.45. Exemplary TLR4 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone HTA125 or 76B357-1. Exemplary TLR5 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone 85B152-5 or 9D759-2. Exemplary GL7 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone GL7.

[0296] Exemplary PD1 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clones MIH4, J116, J150, OTIB11, OTI17B10, OTI3A1 or OTI16D4. Additionally, exemplary anti-PD-1 antibodies include, for example, U.S. Pat. No. 697, No. 8,900,587, Described in Nos. 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) is included. Exemplary anti-PD-L1 antibodies are described, for example, in US Pat. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab and BMS 936559 (Bristol Myers Squibb Co.).

[0297] Exemplary CTLA-4 binders include clone ER4.7G.11 [7G11], OTI9G4, OTI9F3, OTI3A5, A3.4H2.H12, 14D3, OTI3A12, OTI1A11, OTI1E8, OTI3B11, OTI3D2, OTI10C8, OTI2E9, OTI6F1, It can be selected from antibodies or antibody fragments using the CDRs of OTI7D3, OTI85B, OTI12C6. Exemplary anti-CTLA-4 antibodies include U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; No. 8,263,073, 8,318,916, 8,017,114 WO 98 / 42752, WO 00 / 37504 and WO 01 / 14424, and EP 1212422. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0298] Exemplary TCRβ binding agents are 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. The antibody of choice may be the antibody of choice. In certain embodiments, the binding agent is selected from the group consisting of H57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα / β, Abcam) and E6Z3S (TRBC1 / TCRβ, Cell Signaling Technology) Antibody V H domain and V L including. In certain embodiments, the binding agent is Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (e.g., 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 in the art from H57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα / β, Abcam) and E6Z3S (TRBC1 / TCRβ, Cell Signaling Technology) determined under any other CDR determination method known. V of an antibody selected from the group H and V L Heavy chain CDR of sequence 1 ,CDR 2 and CDR 3 as well as light chain CDRs 1 ,CDR 2 and CDR 3 including.

[0299] Exemplary CD137 binding agents can be selected from antibodies or antibody fragments that use the CDRs of clone 4B4-1, P566 or urelumab.

[0300] In some embodiments, the immune cell targeting group 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 or humanized antibody. In some embodiments, the immune cell targeting group comprises a Fab or immunoglobulin single variable domain, such as a Nanobody. In some embodiments, the immune cell targeting group comprises a Fab that does not contain natural interchain disulfide bonds. For example, in some embodiments, according to Kabatt numbering, a Fab comprises a heavy chain fragment containing a C233S substitution and / or a light chain fragment containing a C214S substitution. In some embodiments, the immune cell targeting group comprises a Fab that includes one or more non-natural interchain disulfide bonds. In some embodiments, the interchain disulfide bond is between two non-natural cysteine ​​residues on the light chain fragment and the heavy chain fragment, respectively. For example, in some embodiments, according to Kabatt numbering, a Fab comprises a heavy chain fragment containing a F174C substitution and / or a light chain fragment containing a S176C substitution. In some embodiments, according to Kabatt numbering, the Fab comprises a heavy chain fragment containing F174C and C233S substitutions and / or a light chain fragment containing S176C and C214S substitutions. In some embodiments, the immune cell targeting group comprises a C-terminal cysteine ​​residue. In some embodiments, the immune cell targeting group comprises a Fab that includes 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 fragment of the Fab and the C-terminal cysteine. For example, in some embodiments, the Fab comprises two or more amino acids derived from an antibody hinge region (eg, a partial hinge sequence) between the C-terminus and the C-terminal cysteine ​​of the Fab. 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 one or more linked by interchain disulfide bonds, 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. In some embodiments, the Fab antibody is a DS Fab, NoDS Fab, bDS Fab, bDS Fab-ScFv, as illustrated in FIG. 47.

[0301] In some embodiments, the immune cell targeting group is an immunoglobulin single variable domain, e.g., a nanobody (e.g., V HH )including. In some embodiments, the Nanobody includes a cysteine ​​at the C-terminus. In some embodiments, the nanobody is V HH It further includes a spacer comprising one or more amino acids between the domain and the C-terminal cysteine. In some embodiments, the spacer includes one or more glycine residues, such as two glycine residues. In some embodiments, the immune cell targeting group has two or more V HH Contains domains. In some embodiments, two or more V HH The domains are connected by amino acid linkers. In some embodiments, the amino acid linker includes one or more glycine and / or serine residues (eg, one or more repeats of the sequence GGGGS). In some embodiments, the immune cell targeting group is the first V linked to the antibody CH1 domain. HH domain and a second V linked to the antibody light chain constant domain HH the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds (eg, an interchain disulfide bond). In some embodiments, the immune cell targeting group is a V HH 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 and the light chain constant domain comprises S176C and C214S substitutions according to Kabatt numbering. In some embodiments, as illustrated in Figure 47, the antibody is a ScFv, V HH , 2xV HH ,V HH -CH1 / empty Vκ, or V HH 1-CH1 / V HH -2-Nb bDS.

[0302] An exemplary targeting moiety may have the amino acid sequence shown below: Anti-CD3 hSP34-Fab sequence: hSP34 heavy chain (HC) sequence (SEQ ID NO: 1): EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSSDKTHTC hSP34-mlam light chain (LC) sequence (mouse lambda) (SEQ ID NO: 2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKS LSRADSS SP34-hlam LC (human lambda) (SEQ ID NO: 3): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPA ESS Anti-CD3 Hu291-Fab sequence: Hu291 HC (SEQ ID NO: 4): QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSSDKTHTC Hu 291 LC (SEQ ID NO: 5): MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD8 TRX2-Fab sequence: TRX2 HC (SEQ ID NO: 6): QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSSDKTHTC TRX2 LC (Sequence number 7): DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGES Anti-CD8 OKT8-Fab sequence: OKT8 HC (Sequence number 8): QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSSDKTHTC OKT8 LC (Sequence number 9): DIVMTQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGES Anti-CD4 Ibalizumab-Fab sequence: Ibalizumab HC (SEQ ID NO: 10): QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV NHKPSNTKVDKKVEPKSSDKTHTC Ibalizumab LC (SEQ ID NO: 11): DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGES Anti-CD5 He3-Fab sequence: He3 HC (SEQ ID NO: 12): EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSSDKTHTC He3 LC (SEQ ID NO: 13): DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGES Anti-CD7 TH-69-Fab sequence: TH-69 HC (SEQ ID NO: 14): EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTC TH-69 LC (Sequence number 15): DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Anti-CD2 TS2 / 18.1-Fab sequence: TS2 / 18.1 HC (SEQ ID NO: 16): EVQLVESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SSDKTHTC TS2 / 18.1 LC (SEQ ID NO: 17): DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHNFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGES Anti-CD2 9.6-Fab sequence: 9.6 HC (SEQ ID NO: 18): QVQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSSDKTHTC 9.6 LC (SEQ ID NO: 19): NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGES Anti-CD2 9-1-Fab sequence: 9-1 HC (SEQ ID NO: 20): QVQLQQPGTELVRPGSSVKLSCKASGYTFTSYWVNWVKQRPDQGLEWIGRIDPYDSETHYNQKFTDKAISTIDTSSNTAYMQLSTLTSDASAVYYCSRSPRDSSTNLADWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSSDKTHTC 9-1 LC (SEQ ID NO: 21): DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GES mutOKT8-Fab array: mutOKT8 HC (SEQ ID NO: 22): QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGAGAYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSSDKTHTC mutOKT8 LC (SEQ ID NO: 23): DIVMTQSPSSLSASVGDRVTITCRTSRSISAALAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGES

[0303] In certain embodiments, the targeting group or immune cell targeting group (e.g., T cell targeting agent, B cell targeting agent, or NK cell targeting agent) is linked via a polyethylene glycol (PEG) containing linker. It may also be covalently attached to lipids.

[0304] In other embodiments, the lipid used to create the conjugate is distearoyl-phosphatidylethanolamine (DSPE): [ka] , dipalmitoyl-phosphatidylethanolamine (DPPE): [ka] , dimyristoyl-phosphatidylethanolamine (DMPE): [ka] , distearoyl-glycero-phosphoglycerol (DSPG): [ka] , dimyristoyl-glycerol (DMG): [ka] , distearoylglycerol (DSG): [ka] and N-palmitoyl-sphingosine (C16-ceramide) [ka] You can choose from.

[0305] Immune cell targeting groups can be covalently attached to the lipid directly or through a linker, such as a polyethylene glycol (PEG) containing linker. In certain embodiments, the PEG is PEG1000, PEG2000, PEG3400, PEG3000, PEG3450, PEG4000 or PEG5000. In certain embodiments, the PEG is PEG2000.

[0306] In some embodiments, the lipid-immune cell targeting group conjugate is 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 Present in the lipid formulation in the range of 0.2 mole percent.

[0307] In certain embodiments, the lipid-immune cell targeting agent conjugate includes DSPE, a PEG moiety, and a targeting antibody. In certain embodiments, the antibody is a T cell targeting agent, such as an anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody or anti-TCR beta antibody.

[0308] Exemplary lipid immune cell targeting group conjugates include DSPE and PEG 2000, as described, for example, in Nellis et al. (2005) BIOTECHNOL. PROG. 21, 205-220. Exemplary conjugates include the structure of Formula V, where scFv represents an engineered antibody binding site that binds to the target of interest. In certain embodiments, the engineered antibody binding site binds to any of the targets described herein above. 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.

[0309] Examples of compounds of formula (V) are as shown below: [ka] . It is contemplated that the scFv in Formula V can be replaced with an intact antibody or antigenic fragment thereof (eg, Fab).

[0310] Another example of a compound of formula (VI) is as shown below: [ka] , the production of which is described in Nellis et al. (2005) (supra) or US Pat. No. 7,022,336. Fab in formula VI is an intact antibody or an antigenic fragment thereof (e.g. (Fab') 2 It is contemplated that fragments) or engineered antibody binding sites (eg, scFv) can be substituted.

[0311] Other lipid immune cell targeting group conjugates are described, for example, in U.S. Pat. No. 7,022,336, where the targeting group is a targeting group of interest, e.g., T Targeting groups that bind to cell or NK cell surface antigens can be substituted.

[0312] In certain embodiments, the lipid component of the exemplary conjugate of Formula IV is an ionizable cationic lipid described herein, such as an ionizable cationic lipid of Formula I, Formula II, or Formula III. may be based on. For example, exemplary ionizable cationic lipids are: [ka] or salts thereof.

[0313] In certain embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0314] In some embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0315] In other embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0316] In certain embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0317] In some embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0318] In other embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0319] In certain embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0320] In some embodiments, exemplary ionizable cationic lipids have the formula: [ka] or a salt thereof.

[0321] In certain embodiments, the lipid-based conjugate of Formula III is: [ka] , where scFv represents an engineered antibody binding site that binds to a target as described herein above, such as CD2, CD3, CD7 or CD8.

[0322] In certain embodiments, the lipid blend can further include free PEG-lipid to reduce the amount of non-specific binding through the targeting group. The free PEG-lipid may be the same or different from the PEG-lipid included in the conjugate. In certain embodiments, the free PEG-lipid is 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- selected from the group consisting of methylpolyoxyethylene (PEG-DSG), N-palmitoylsphingosine-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), DSPE-PEG-cysteine, or derivatives thereof, all of which are Between 2000 and 5000, with an average PEG length of 2000, 3400 or 5000. The final composition can contain a mixture of two or more of these pegylated lipids. In certain embodiments, the LNP composition comprises a mixture of PEG-lipids and myristoyl and stearacyl chains.

[0323] In certain embodiments, the PEG-lipid derivative has a hydroxyl or carboxylic acid end group at the PEG terminus.

[0324] Lipid-immune cell targeting group conjugates can be incorporated into LNPs, e.g., containing ionizable cationic lipids, sterols, neutral phospholipids, and PEG-lipids, as described below. can. In certain embodiments, the LNPs containing lipid-immune cell targeting groups are the ionizable cationic lipids described herein or, e.g., U.S. Pat. Publication No. 2018 / 0085474, US Patent Application Publication No. 2016 / 0317676, International Publication WO2009 / 086558, or Miao et al. (2019) NATURE BIOTECH 37:1174-1185 or Jayaraman et al. (2012) ANGEW CHEM INT.51:8529 It is thought that it can contain the cationic lipids described on pages ~8533. In other embodiments, the cationic lipid can be selected from the ionizable cationic lipids shown in Table 1.

[0325] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0326] LNPs can be formulated using the methods and other ingredients described below in the following sections.

[0327] IV. Lipid nanoparticle composition The present invention provides lipid nanoparticle (LNP) compositions comprising lipid blends containing ionizable cationic lipids as described herein and / or lipid-immune cell targeting agent conjugates as described herein. provide something. In certain embodiments, the lipid blend comprises an ionizable cationic lipid described herein and one or more of sterols, neutral phospholipids, PEG-lipids and lipid-immune cell targeting group conjugates. It can contain more than that.

[0328] In certain embodiments, the ionizable cationic lipids described herein are 30-70 mole percent, 30-60 mole percent, 30-50 mole percent, 40-70 mole percent, 40-60 mole percent. percent, in the range of 40-50 mole percent, 50-70 mole percent, 50-60 mole percent, or about 30 mole percent, about 35 mole percent, about 40 mole percent, about 45 mole percent, about 50 mole percent, about It can be present in the lipid blend at 55 mole percent, about 60 mole percent, about 65 mole percent or about 70 mole percent.

[0329] sterol In certain embodiments, the lipid blend of lipid nanoparticles comprises a sterol component, e.g., from the group consisting of cholesterol, fecosterol, beta-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, brassicasterol. One or more selected sterols may be included. In certain embodiments, the sterol is cholesterol.

[0330] Sterol (e.g. cholesterol) is 20-70 mole percent, 20-60 mole percent, 20-50 mole percent, 30-70 mole percent, 30-60 mole percent, 30-50 mole percent, 40-70 mole percent, in the range of 40-60 mole percent, 40-50 mole percent, 50-70 mole percent, 50-60 mole percent, or about 20 mole percent, about 25 mole percent, about 30 mole percent, about 35 mole percent, about 40 It can be present in the lipid blend at a mole percent of about 45 mole percent, about 50 mole percent, about 55 mole percent, about 60 mole percent, or about 65 mole percent.

[0331] neutral phospholipid In certain embodiments, the lipid blend of lipid nanoparticles can contain one or more neutral phospholipids. Neutral phospholipids include phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- It can be selected from the group consisting of dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and sphingomyelin (SM).

[0332] Other neutral phospholipids are distearoyl-phosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-glycero-phosphocholine (DSPC), 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-cholesterylhemisuccinoyl-glycero-phosphocholine, hexadecyl-glycero-phosphocholine, dilinolenoyl-glycero-phosphocholine, dialachidonoyl-glycero-3-phosphocholine, didocosahexaenoyl - Glycero-phosphocholine or sphingomyelin.

[0333] Neutral phospholipids are 1-10 mole percent, 1-15 mole percent, 1-12 mole percent, 1-10 mole percent, 3-15 mole percent, 3-12 mole percent, 3-10 mole percent, 4- 15 mole percent, 4-12 mole percent, 4-10 mole percent, 4-8 mole percent, 5-15 mole percent, 5-12 mole percent, 5-10 mole percent, 6-15 mole percent, 6-12 mole percent, in the range of 6 to 10 mole percent, or 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 %, 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.

[0334] PEG-lipid The lipid blend of lipid nanoparticles can include one or more PEG or PEG-modified lipids. Such species can alternatively be referred to as pegylated lipids. PEG lipids are lipids modified with polyethylene glycol. As mentioned above, free PEG-lipids may be included in the lipid blend to reduce or eliminate non-specific binding through the targeting group when the lipid-immune cell targeting group is included in the lipid blend. .

[0335] 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, PEG lipids include PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidylethanol amine (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.

[0336] In certain embodiments, the blend includes PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, e.g., PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl- Contains free PEG-lipids that can be selected from the group consisting of glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) and PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE). be able to. In some embodiments, the free PEG-lipid comprises diacylphosphatidylcholine containing dipalmitoyl (C16) or distearoyl (C18) chains.

[0337] PEG-lipids are 1-10 mole percent, 1-8 mole percent, 1-7 mole percent, 1-6 mole percent, 1-5 mole percent, 1-4 mole percent, 1-3 mole percent, 2-8 mole percent, in the range of 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 about 1 mole percent, about 2 mole percent, about 3 mole %, about 4 mole percent or about 5 mole percent. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0338] In some embodiments, the PEG-lipid is 0.01-10 mole percent, 0.01-5 mole percent, 0.01-4 mole percent, 0.01-3 mole percent, 0.01-2 mole percent, 0.01-1 mole percent, 0.1- 10 mole percent, 0.1-5 mole percent, 0.1-4 mole percent, 0.1-3 mole percent, 0.1-2 mole percent, 0.1-1 mole percent, 0.5-10 mole percent, 0.5-5 mole percent, 0.5-4 mole percent, 0.5-3 mole percent, 0.5-2 mole percent, 0.5-1 mole percent, 1-2 mole percent, 3-4 mole percent, 4-5 mole percent, 5-6 mole percent or 1.25-1.75 mole percent can be present in the lipid blend in a range. In some embodiments, the PET-lipid is 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 of the lipid blend, It may be about 4 mole percent, about 4.5 mole percent, about 5 mole percent, or about 5.5 mole percent. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0339] In some embodiments, the lipid anchor length of the PEG-lipid is C14 (as in the case of 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 the case of PEG-DSG). In some embodiments, the backbone or headgroup of the PEG-lipid is diacylglycerol or phosphoethanolamine. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0340] LNPs of the present disclosure can include one or more free PEG-lipids not conjugated to immune cell targeting groups and PEG-lipids conjugated to immune cell targeting groups. In some embodiments, the free PEG-lipid comprises the same or a different lipid than the lipid in the lipid immune cell targeting group conjugate.

[0341] Immune cell targeting group conjugates In certain embodiments, the lipid blend can also include a lipid-immune cell targeting group conjugate as described in Section III above.

[0342] The lipid-immune cell targeting group conjugate is 0.001-0.5 mole percent, 0.001-0.1 mole percent, 0.01-0.5 mole percent, 0.05-0.5 mole percent, 0.1-0.5 mole percent, 0.1-0.3 mole percent, 0.1-0.2 mole percent, in the range of 0.2-0.3 mole percent, 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 in the lipid blend.

[0343] In addition to the lipids present in the lipid blend, the LNP composition can further include payloads, such as those described below. In certain embodiments, the payload is a nucleic acid, eg, DNA or RNA, eg, mRNA, transfer RNA (tRNA), microRNA, or small interfering RNA (siRNA).

[0344] In certain embodiments, the number of nucleotides in the nucleic acid is about 400 to about 6000.

[0345] Production of lipid nanoparticles Generally, LNPs are produced using rapid mixing with an orbital vortexer or microfluidic mixing. Orbital vortexer mixing is accomplished by rapid addition of a lipid solution in ethanol to an aqueous solution of the nucleic acid of interest followed immediately by vortexing at 2,500 rpm. Microfluidic mixing can be performed, for example, by using a NanoAssemblr device consisting of an optimized mixing vessel geometry (Precision Nanosystems, Vancouver, BC) and a microfluidic chip to mix aqueous fluids at controlled flow rates in microfluidic channels. and organic streams.

[0346] In certain embodiments, the resulting LNP composition contains, for example, about 40 mole percent to about 60 mole percent of one or more ionizable cationic lipids described herein, about 35 mole percent ~about 50 mole percent of one or more sterols, about 5 mole percent to about 15 mole percent of one or more neutral lipids, and about 0.5 mole percent to about 5 mole percent of one or more of PEG-lipids.

[0347] Physical properties of lipid nanoparticles Characteristics of LNP compositions may depend on the components, their absolute or relative amounts, contained in the lipid nanoparticle (LNP) composition. Characteristics may also vary depending on the method and conditions of manufacture of the LNP composition.

[0348] LNP compositions can be characterized by various methods. For example, microscopy (eg, 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 potentiometric measurements (eg, potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of LNP compositions such as particle size, polydispersity index and zeta potential. RNA encapsulation efficiency is determined by a combination of methods that rely on RNA-binding dyes (ribogreen, cybergreen to determine dye-accessible RNA fractions) and LNP deformulation, followed by HPLC analysis of total RNA content.

[0349] In some embodiments, the LNPs are 1-250nm, 1-200nm, 1-150nm, 1-100nm, 50-250nm, 50-200nm, 50-150nm, 50-100nm, 75-250nm, 75-200nm, It can have an average diameter in the range of 75-150nm, 75-100nm, 100-250nm, 100-200nm, 100-150nm. In certain embodiments, the LNP composition has 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 LNPs have an average diameter of about 100 nm.

[0350] Alternatively, or in addition, the LNP composition is 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, It can have a polydispersity index in the range of 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 ranges from 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.

[0351] Alternatively, or in addition, the LNP composition can have a zeta potential of about -30 mV to about +30 mV. In certain embodiments, the LNP composition has a zeta potential of about -10 mV to about +20 mV. Zeta potential can vary as a function of pH. As a result, in certain embodiments, the LNP composition has a zeta potential of about -10 mV to about +30 mV, or about 0 mV to +30 mV, or about +5 mV to about +30 mV at pH 5.5 or pH 5, and or can have a zeta potential of about -30 mV to about +5 mV or about -20 mV to about +15 mV at pH 7.4.

[0352] V. Payload LNP compositions can include agents, eg, nucleic acid molecules, for delivery to cells (eg, immune cells) or tissues, eg, cells (eg, immune cells) or tissues of a subject.

[0353] LNP compositions of the invention can include nucleic acids, such as DNA or RNA, such as mRNA, tRNA, microRNA, siRNA or Dicer substrate siRNA. Nucleic acids can contain naturally occurring components, such as naturally occurring bases, sugars or linking groups (e.g., phosphodiester linking groups) or non-naturally occurring components or modifications (e.g., thioester linking groups). ). For example, nucleic acids can be synthesized to contain base, sugar, and linker modifications known to those skilled in the art. Additionally, nucleic acids may be linear or circular or have any desired configuration. A LNP composition can include multiple nucleic acid molecules, such as multiple RNA molecules, which may be the same or different.

[0354] 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 and / or contacted simultaneously with cells. It is contemplated that the mRNA may include 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 in, for example, immune disorders, inflammatory disorders or cancer. Furthermore, the mRNA may be used in therapeutic or prophylactic vaccines, for example, for the treatment or prevention of infections by pathogens, such as microbial or viral pathogens, or for use directly or indirectly by such infections. Antigens can be encoded to reduce or ameliorate side effects caused by.

[0355] In certain embodiments, LNP compositions include one or more pharmaceutically acceptable excipients, small hydrophobic molecules, therapeutic agents, carbohydrates, polymers, permeability-enhancing molecules, and surface-altering agents. One or more other ingredients may be included, including without limitation.

[0356] In some embodiments, the wt / wt ratio of lipid component to payload (eg, mRNA) in the resulting LNP composition is from about 1:1 to about 50:1. In certain embodiments, the wt / wt ratio of lipid component to payload (eg, 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.

[0357] In certain embodiments, the encapsulation efficiency of the payload (eg, mRNA) in the lipid nanoparticle is at least 50%. In certain embodiments, the encapsulation efficiency is at least 80%, at least 90% or greater than 90%.

[0358] RNA payload In certain embodiments, the RNA payload is an mRNA, tRNA, microRNA or siRNA payload.

[0359] In certain embodiments, the lipid nanoparticle composition is optimized for delivery of RNA, eg, mRNA, to target cells for translation in the cell. The mRNA may be naturally occurring or non-naturally occurring mRNA. mRNA can include one or more modified nucleobases, nucleosides or nucleotides.

[0360] 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, hypoxanthine and xanthine. .

[0361] A nucleoside of mRNA is a compound that includes a sugar molecule (eg, a 5- or 6-carbon sugar such as pentose, ribose, arabinose, xylose, glucose, galactose or its deoxy derivative) together with a nucleobase. The nucleoside may be a regular nucleoside (e.g., adenosine, guanosine, cytidine, uridine, ribothymidine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine) or an analog thereof, with one or more substitutions or modifications. can include.

[0362] Nucleotides of mRNA are compounds containing nucleosides and phosphate groups or alternative groups such as boranophosphate, thiophosphate, selenophosphate, phosphonate, alkyl groups, amidates and glycerol. The nucleotides may be regular nucleotides (e.g. adenosine, guanosine, cytidine, uridine, ribothymidine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine and thymidine monophosphate) or analogs thereof, including alkyl, aryl, halo, oxo, hydroxyl, alkyloxy and / or thio substitution; one or more fusions or ring openings; oxidation; and / or reduction of nucleobases, sugars and / or phosphates or alternative moieties; One or more substitutions or modifications may be included. A nucleotide can include one or more phosphate or alternative groups. For example, a nucleotide can include a nucleoside and a triphosphate group. "Nucleoside triphosphate" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate and uridine triphosphate) may refer to regular nucleoside triphosphates or analogs or derivatives thereof; It may include one or more substitutions or modifications as described in the specification.

[0363] The mRNA can include a 5' untranslated region, a 3' untranslated region and / or a coding or translated sequence. mRNA can contain 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 may be analogs of the original species, substituted, modified, or otherwise non-naturally occurring. Good too. In certain embodiments, all of a particular nucleobase type may be modified. For example, all cytosines in the mRNA may be 5-methylcytosines.

[0364] 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.

[0365] A cap structure or cap species is a compound comprising two nucleoside moieties connected by a linker and can be selected from naturally occurring caps, non-naturally occurring caps or cap analogs. A cap species can include one or more modified nucleosides and / or linker moieties. For example, the natural mRNA cap consists of guanine nucleotides and guanine (G) nucleotides methylated at the 7 position, linked by a triphosphate linkage at their 5' positions, e.g., m7G, commonly written as m7GpppG. (5')ppp(5')G can be included. The cap species may be an anti-reverse cap analog. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m7Gpppm7G, m73'dGpppG and m27 02'GppppG.

[0366] Alternatively, or in addition, the mRNA can include chain-terminating nucleosides. For example, chain-terminating nucleosides can include nucleosides that are deoxygenated at the 2' and / or 3' positions of their sugar groups. Such species include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine and 2',3-dideoxynucleosides such as 2' ,3-dideoxyadenosine, 2',3-dideoxyuridine, 2',3-dideoxycytosine, 2',3-dideoxyguanosine and 2',3-dideoxythymine.

[0367] Alternatively, or in addition, the mRNA can include a stem-loop, such as a histone stem-loop. A stem-loop can contain 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, a stem-loop can contain 4, 5, 6, 7 or 8 nucleotide base pairs. Stem loops can be located in any region of the mRNA. For example, a stem-loop can be located in, before, or after an untranslated region (5' untranslated region or 3' untranslated region), a coding region, or a polyA sequence or tail.

[0368] Alternatively, or in addition, the mRNA can include a polyA sequence and / or a polyadenylation signal. The polyA sequence may be composed entirely or in large part 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.

[0369] The mRNA can encode any polypeptide of interest, including any naturally occurring or non-naturally occurring or otherwise modified polypeptide. Polypeptides encoded by mRNA can be of any size and have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA can have a therapeutic effect when expressed in a cell. In some embodiments, the mRNA can encode antibodies, enzymes, growth factors, hormones, cytokines, viral proteins (eg, viral capsid proteins), antigens, vaccines, or receptors. In some embodiments, the mRNA is for use in therapeutic vaccines (e.g., cancer vaccines) or prophylactic vaccines (e.g., vaccines to minimize the risk or severity of infections caused by microbial or viral pathogens). It can encode an engineered receptor, such as a CAR, or an antigen. In some embodiments, the mRNA encodes a polypeptide capable of modulating an immune response in an immune cell. 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).

[0370] Lipid compositions can be designed for one or more specific applications or targets. For example, LNP compositions may be designed to deliver mRNA to a particular cell, tissue, organ or system or group thereof within a mammal's body, such as the renal system. The physiochemical properties of the LNP composition may be altered to increase selectivity for specific target sites within a subject. For example, particle size may be adjusted based on fenestration sizes of various organs. The mRNA included in the LNP composition may also depend on the desired delivery target(s). For example, the mRNA is selected for a particular indication, condition, disease or disorder and / or for delivery to a particular cell, tissue, organ or system or group thereof (e.g. localized or specific delivery). You can.

[0371] The amount of mRNA in a lipid composition may depend on the size, sequence and other characteristics of the mRNA. Additionally, the amount of mRNA in LNPs may depend on the size, composition, desired target, and other characteristics of the LNP composition. Also, the relative amounts of mRNA and other elements (eg, lipids) may vary. The amount of mRNA in a LNP composition can be measured using, for example, absorption spectroscopy (eg, ultraviolet-visible spectroscopy).

[0372] In some embodiments, one or more of the mRNAs, lipids, and polymers and their amounts are determined at a specific N:P ratio (of positively chargeable lipid or polymer amine (N=nitrogen) groups). may be selected to provide a charged nucleic acid phosphate (P) group). The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the mRNA. Generally, lower N:P ratios are preferred. The N:P ratio may depend on the particular lipid and its pKa. In certain embodiments, the mRNA and LNP compositions and / or their relative amounts provide an N:P ratio of about 1:1 to about 30:1, or about 1:1 to about 20:1. You may choose as follows. 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 is 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.

[0373] The amount of mRNA in a nanoparticle composition can depend on the size, sequence and other characteristics of the mRNA. The amount of mRNA in a nanoparticle composition may also depend on the size, composition, desired target, and other characteristics of the nanoparticle composition. Also, the relative amounts of mRNA and other elements (eg, lipids) may vary. In some embodiments, the wt / wt ratio of lipid components to mRNA in the nanoparticle composition is 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, Can be 25:1, 30:1, 35:1, 40:1, 45:1 and 50:1. For example, the wt / wt ratio of lipid component to mRNA can be from about 10:1 to about 40:1. The amount of mRNA in a nanoparticle composition can be measured using, for example, absorption spectroscopy (eg, ultraviolet-visible spectroscopy).

[0374] Efficiency of mRNA encapsulation describes the amount of mRNA encapsulated in or otherwise associated with the lipid composition after preparation compared to the initial amount provided. It is desirable that the encapsulation efficiency be high (for example, close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of mRNA in a solution containing the lipid composition before and after separation of the LNP composition by one or more organic solvents or surfactants. . Fluorescence may be used to measure the amount of free mRNA in solution. For LNP compositions of the invention, the encapsulation efficiency of mRNA is at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, It can be 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain embodiments, the encapsulation efficiency can be at least 80%.

[0375] VI. Mode of formulation and delivery The LNP compositions of the invention can be formulated in whole or in part as pharmaceutical compositions. The pharmaceutical composition may further include one or more pharmaceutically acceptable excipients or adjunct ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's (2006) (supra). Conventional excipients and auxiliary ingredients are included in the present invention, except to the extent that any conventional excipients or auxiliary ingredients may be incompatible with one or more components of the LNP compositions of the present invention. Can be used in any pharmaceutical composition. An excipient or auxiliary ingredient may be incompatible with the components of the LNP composition if its combination with the component may result in any undesirable biological effect or other adverse effect.

[0376] In some embodiments, one or more excipients or auxiliary ingredients may constitute more than 50% of the total weight or volume of a pharmaceutical composition comprising an LNP composition of the invention. For example, one or more excipients or auxiliary ingredients may constitute 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In certain embodiments, the excipient is approved for human use and for veterinary use, eg, by the US Food and Drug Administration. In certain embodiments, the excipients are pharmaceutical grade. In certain embodiments, the excipients meet the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0377] The relative amounts of one or more lipids or LNPs, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition may be the same in the subject being treated. This will vary depending on the sex, size and / or condition, and also on the route by which the composition is administered.

[0378] A pharmaceutical composition comprising a lipid composition and / or one or more LNP compositions may contain a nucleic acid, e.g. or to any subject, including human patients, who can benefit from the therapeutic effect provided by delivery to the renal system or group thereof, such as the renal system. Although the description provided herein of LNP compositions and pharmaceutical compositions comprising LNP compositions is primarily directed to compositions that are suitable for administration to humans, such compositions may include any other Those skilled in the art will appreciate that they are generally suitable for administration to mammals. Modifications of compositions suitable for human administration to make them suitable for administration to a variety of animals are understood.

[0379] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient (eg, payload).

[0380] Pharmaceutical compositions of the invention can be prepared in a variety of forms suitable for various routes and methods of administration. For example, the pharmaceutical compositions of the invention can be used in liquid dosage forms (e.g. emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable 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 It can be prepared with

[0381] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and / or elixirs. In addition to the active ingredient, the liquid dosage form contains inert diluents commonly used in the art, such as water or other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Solubilizers and emulsifiers such as propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene Glycols and fatty acid esters of sorbitan, and mixtures thereof may be included. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents.

[0382] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing, wetting agents, and / or suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension and / or emulsion in a non-toxic parenterally acceptable diluent and / or solvent, for example as a solution in 1,3-butanediol. You can. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0383] Injectable preparations may contain 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, for example, by filtration through a bacteria-retaining filter and / or Can be sterilized by incorporation.

[0384] other ingredients Additionally, it is contemplated that the pharmaceutical compositions may include one or more ingredients in addition to those described above by the inventors.

[0385] Pharmaceutical compositions may also include one or more permeability enhancer molecules, carbohydrates, polymers, therapeutic agents, surface modifiers or other ingredients. The permeability enhancer molecule can be, for example, a molecule described in US Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (eg, glucose) and polysaccharides (eg, glycogen and its derivatives and analogs).

[0386] Pharmaceutical compositions may also contain, for example, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclo dextrins), polymers (e.g. heparin, polyethylene glycols and poloxamers), mucolytics (e.g. acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol) , domiodol, retostein, stepronin, tiopronin, gelsolin, thymosin β4, dornase alpha, neltenexin and erdostein) and DNases (eg, rhDNase). Surface modifiers can be placed within and / or on the surface of the compositions described herein.

[0387] In addition to these ingredients, pharmaceutical compositions containing LNP compositions of the present invention may include any materials useful in pharmaceutical compositions. For example, the pharmaceutical composition may include, but is not limited to, one or more solvents, dispersion media, diluents, dispersing aids, suspending aids, granulation aids, disintegrants, fillers, glidants. one or more pharmaceutically acceptable agents, such as liquid vehicles, binders, surfactants, tonicity agents, thickening or emulsifying agents, buffers, lubricants, oils, preservatives and other species. may contain excipients or auxiliary ingredients. Excipients such as waxes, butters, colorants, coatings, flavors and perfumes may also be included. Pharmaceutically acceptable excipients are well known in the art (see, eg, Remington's (2006) supra).

[0388] Dispersants include 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, carbonic acid. Sodium, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline from the non-limiting list consisting of pure starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0389] As surfactants and / or emulsifiers, natural emulsifiers such as, but not limited to, gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, waxes and lecithin), colloidal clays (e.g. bentonite [aluminium 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 polymers and carboxyvinyl polymers) ), carrageenan, cellulose derivatives (e.g. sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN® 20 ], polyoxyethylene sorbitan [TWEEN(R) 60], polyoxyethylene sorbitan monooleate [TWEEN(R) 80], sorbitan monopalmitate [SPAN(R) 40], sorbitan monostearate [SPAN (registered trademark) 60], sorbitan tristearate [SPAN (registered trademark) 65], glyceryl monooleate, sorbitan monooleate [SPAN (registered trademark) 80]), polyoxyethylene esters (e.g., polyoxyethylene monooleate Stearates [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR (registered trademark)), polyoxyethylene ether, (e.g., polyoxyethylene lauryl ether [BRIJ (registered trademark) 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, docusate sodium and / or combinations thereof.

[0390] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcoholic preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, gallic acid. Propyl acid, sodium ascorbate, sodium bisulfite, sodium metabisulfite and / or sodium sulfite may be mentioned. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid and / or or edetate trisodium. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, Mention may be made of hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and / or Examples include sorbic acid. Examples of alcoholic preservatives 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 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, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), lauryl ether Examples include sodium sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, and potassium metabisulfite.

[0391] Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluvionate, calcium gluceptate, calcium gluconate. , d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, gluconic acid Potassium, potassium mixture, potassium phosphate dibasic, potassium phosphate monobasic, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium phosphate dibasic, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or a combination thereof.

[0392] In certain embodiments, lipid nanoparticle compositions and formulations thereof are suitable for administration intravenously, intramuscularly, intradermally, subcutaneously, intraarterially, intratumorally, or by inhalation. . In certain embodiments, a dose of about 0.001 mg / kg to about 10 mg / kg is administered to the subject. Compositions according to the present disclosure can be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.

[0393] The appropriate dose level (e.g., for imaging) that is particularly therapeutically, prophylactically, or otherwise effective for any particular patient will depend on the severity and identification, if any, of the disorder being treated; or more of the mRNA utilized; the particular composition utilized; the patient's age, weight, general health, sex and diet; time of administration, route of administration and excretion of the particular pharmaceutical composition utilized. It will vary depending on a variety of factors, including the rate; the duration of treatment; the drugs used in combination with or concomitantly with the particular pharmaceutical composition utilized; and similar factors well known in the medical arts.

[0394] VII. Method The present disclosure provides methods of delivering a payload to target cells or tissues, eg, target cells or tissues in a subject, and LNPs or pharmaceutical compositions containing LNPs for use in such methods.

[0395] In certain embodiments, the invention provides methods for producing polypeptides of interest (e.g., proteins of interest) in mammalian cells and LNPs or pharmaceutical compositions containing LNPs for use in such methods. provide. A method of producing a polypeptide in such a cell includes contacting the cell with an LNP composition comprising an RNA of interest (eg, an mRNA encoding a polypeptide of interest (eg, protein of interest)). When cells are contacted with the LNP composition, the mRNA is taken up by the cells and translated to produce the polypeptide of interest.

[0396] Generally, contacting mammalian cells with a LNP composition comprising mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, or in vitro. The amount of LNP composition contacted with cells and / or the amount of mRNA therein will depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics of the LNP composition and the mRNA therein (e.g., size , charge and chemical composition) and other factors. Generally, an effective amount of the LNP composition will enable efficient polypeptide production in the cell. Efficiency metrics can include polypeptide translation (as indicated by polypeptide expression), levels of mRNA degradation, and immune response indicators.

[0397] Contacting the LNP composition containing mRNA with the cell may include or cause transfection in which the LNP composition can fuse with the membrane of the cell, allowing delivery of the mRNA into the cell. Once introduced into the cell's cytoplasm, the mRNA is then translated into proteins or peptides via the protein synthesis machinery within the cell's cytoplasm.

[0398] In certain embodiments, the LNP compositions described herein are used to deliver therapeutic or prophylactic agents to a subject. For example, the mRNA included in the LNP composition may encode a polypeptide upon contact with and / or entry into a cell (eg, transfection) to produce a therapeutic or prophylactic polypeptide. In certain embodiments, the mRNA included in the LNP compositions of the invention may encode a polypeptide that can improve or increase immunity in a subject.

[0399] In certain embodiments, contacting a cell with a LNP composition that includes mRNA can reduce the cell's innate immune response to exogenous nucleic acids. The cell can be contacted with a first LNP composition comprising a first exogenous mRNA comprising a first amount of a translatable region, and the level of the cell's innate immune response to the first exogenous mRNA can be determined. . Thereafter, the cell can be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount comprising a lesser amount of the first exogenous mRNA as compared to the first amount. Exogenous mRNA. Alternatively, the second composition can include a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The step of contacting the cells with the first and second compositions can be repeated one or more times.

[0400] Additionally, the efficiency of polypeptide production in the cells can optionally be determined, and the cells can be repeatedly recontacted with the first and / or second compositions until the target protein production efficiency is achieved.

[0401] The present disclosure provides methods of delivering nucleic acids (eg, mRNA) to mammalian cells or tissues, eg, mammalian cells or tissues in a subject. Delivery of mRNA to such cells or tissues includes, for example, administering a LNP composition comprising mRNA to a subject via injection, eg, intramuscular injection or intravascular delivery to the subject. After administration, the LNPs can target and / or contact cells, eg, immune cells such as T cells. Upon contacting the cell with the LNP composition, the translatable mRNA is translated in the cell to produce the polypeptide of interest.

[0402] In certain embodiments, the LNP compositions of the invention can be targeted to specific types or classes of cells. This targeting is facilitated using the lipids described herein to form LNPs that also contain targeting groups to target cells of interest. In certain embodiments, specific delivery involves targeting a targeted destination (e.g., cells that do not express or express the receptor of interest only at low levels) as compared to another destination (e.g., cells that do not express or express the receptor of interest only at low levels). Increase the amount of mRNA by more than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold into cells that express or express high levels of the receptor of interest that binds to the immune cell targeting group of LNP. can bring about

[0403] LNP compositions of the invention may be useful for treating diseases, disorders or conditions characterized by lost or abnormal protein or polypeptide activity. Upon delivery of mRNA encoding a missing or abnormal polypeptide into a cell, translation of the mRNA produces the polypeptide, thereby eliminating the problems caused by the lack of activity or abnormal activity caused by the polypeptide. can be reduced or eliminated. Because translation can occur rapidly, the methods and compositions of the invention can be useful in the treatment of acute diseases, disorders, or conditions such as sepsis, stroke, and myocardial infarction. The mRNA included in the LNP compositions of the invention can also alter the rate of transcription in a given species, thereby influencing gene expression.

[0404] Diseases, disorders and / or conditions characterized by dysfunctional or aberrant protein or polypeptide activity to which the compositions of the invention are administered include, but are not limited to, cancer and proliferative diseases, genetic diseases ( Examples include cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardio- and renal-vascular diseases and metabolic diseases. Multiple diseases, disorders and / or conditions may be characterized by protein activity that is lost (or substantially reduced, such that proper protein function does not occur). Such proteins may be absent and they may be essentially non-functional. A specific example of a dysfunctional protein is a missense mutant variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein and causes cystic fibrosis. . The present disclosure provides methods for treating such diseases, disorders and / or conditions in a subject by administering LNP compositions comprising mRNA and lipid components including KL10, phospholipids (optionally unsaturated), PEG lipids and structured lipids. provides a method of treating a subject, wherein the mRNA encodes a polypeptide that antagonizes or otherwise overcomes aberrant protein activity present in a subject's cells.

[0405] The therapeutic and / or prophylactic compositions described herein are effective for preventing, treating, diagnosing or imaging diseases, disorders and / or conditions, and / or for any other purpose. It can be administered to a subject in any reasonable amount and using any route of administration. The particular amount administered to a given subject may vary depending on the species, age and general condition of the subject, purpose of administration, particular composition, mode of administration, and the like. Compositions according to the present disclosure can be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.

[0406] LNP compositions containing one or more mRNAs can be administered by a variety of routes, such as orally, intravenously, intramuscularly, intraarterially, intrathecally, intrathecally, subcutaneously, It can be administered intraventricularly, percutaneously or intradermally, intradermally, rectally, intravaginally, intraperitoneally, topically, mucosally, nasally, intratumorally. In certain embodiments, LNP compositions can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, or subcutaneously. However, the present disclosure encompasses delivery of the LNP compositions of the invention by any suitable route given the potential for advances in the science of drug delivery. In general, the most appropriate route of administration will depend on the nature of the LNP composition containing one or more mRNAs (e.g., its stability in various body environments such as the bloodstream and gastrointestinal tract), the patient's condition (e.g., It depends on a variety of factors, including whether the patient can tolerate a particular route of administration.

[0407] In certain embodiments, compositions according to the present disclosure provide from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg in a given dose. / kg, about 0.01mg / kg to about 10mg / kg, about 0.05mg / kg to about 10mg / kg, about 0.1mg / kg to about 10mg / kg, about 1mg / kg to about 10mg / kg, about 2mg / kg ~10mg / kg, 5mg / kg~10mg / kg, 0.0001mg / kg~5mg / kg, 0.001mg / kg~5mg / kg, 0.005mg / kg~5mg / kg, approx. 0.01mg / kg to about 5mg / kg, about 0.05mg / kg to about 5mg / kg, about 0.1mg / kg to about 5mg / kg, about 1mg / kg to about 5mg / kg, about 2mg / kg to about 5mg / kg kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, Approximately 0.05mg / kg to approximately 2.5mg / kg, approximately 0.1mg / kg to approximately 2.5mg / kg, approximately 1mg / kg to approximately 2.5mg / kg, approximately 2mg / kg to approximately 2.5mg / kg, approximately 0.0001mg / kg~about 1mg / kg, about 0.001mg / kg~about 1mg / kg, about 0.005mg / kg~about 1mg / kg, about 0.01mg / kg~about 1mg / kg, about 0.05mg / kg~about 1mg / kg , about 0.1mg / kg to about 1mg / kg, about 0.0001mg / kg to about 0.25mg / kg, about 0.001mg / kg to about 0.25mg / kg, about 0.005mg / kg to about 0.25mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg; A dose of 1 mg / kg provides 1 mg of the composition per kg of subject body weight.

[0408] In certain embodiments, doses of the LNP compositions of the invention from about 0.001 mg / kg to about 10 mg / kg can be administered. In other embodiments, a dose of the LNP composition from about 0.005 mg / kg to about 2.5 mg / kg can be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg can be administered. In other embodiments, doses of about 0.05 mg / kg to about 0.25 mg / kg can be administered. Doses can be administered in the same or different amounts one or more times per day to obtain the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic or imaging effect. The desired dosage may be administered, for example, three times a day, twice a day, once a day, every other day, once every third day, every week, every two weeks, every three weeks, or every four weeks. Can be delivered. In certain embodiments, the desired dosage is divided into multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 1, 13, 14 or more doses). In some embodiments, a single dose can be administered, for example, before or after a surgical procedure, or in the case of an acute disease, disorder or condition.

[0409] LNP compositions containing one or more mRNAs can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" does not mean that the agents must be administered simultaneously and / or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. It is. For example, one or more LNP compositions containing one or more different mRNAs can be administered in combination. The composition can be administered simultaneously with, before, or after one or more other desired therapeutic agents or medical procedures. Generally, each drug is administered at a dose and / or on a time schedule determined for that drug. In some embodiments, the present disclosure is combined with agents that improve its bioavailability, reduce and / or modify its metabolism, inhibit its excretion, and / or modify its distribution within the body. delivery of the compositions of the invention or their imaging, diagnostic or prophylactic compositions.

[0410] It will be further understood that therapeutically, prophylactically, diagnostically active or imaging active agents utilized in combination can be administered together in a single composition or separately in different compositions. Generally, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually.

[0411] The particular combination of therapies (therapeutic agents or procedures) for utilization in a combination regimen takes into account the suitability of the desired therapeutic agents and / or procedures and the desired therapeutic effect to be achieved. The therapies utilized can achieve a desired effect for the same disorder (e.g., a composition useful for treating cancer can be administered simultaneously with a chemotherapeutic drug), or they can achieve different effects ( For example, it will be understood that the control of any adverse effects).

[0412] In some embodiments, no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, 9 %, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% are transfected by LNP. In some embodiments, the cells that are not targeted for delivery are non-immune cells of interest. In some embodiments, cells that are not targeted for delivery are cells that are not targeted by the method. In some embodiments, cells that are not targeted for delivery are cells of interest that are not targeted by the method.

[0413] In some embodiments, the half-life of a nucleic acid delivered by a LNP described herein or a polypeptide encoded by a nucleic acid delivered by a LNP and expressed in an immune cell is at least 1%, at least 5%, at least 10%, at least 15%, at least 20% less 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 and expressed in the immune cell. %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least twice, at least three times, At least 4 times or at least 5 times longer.

[0414] In some embodiments, the composition of the LNP includes the type of ionizable cationic lipid, the relative amount of ionizable cationic lipid, the length of the lipid anchor in the PEG lipid, the backbone or headgroup of the PEG lipid, It differs from the composition of the reference LNP in the relative amount of PEG lipids, or the type of immune cell targeting group, or any combination thereof. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only by the type of ionizable cationic lipid. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only by the amount of PEG lipid. In some embodiments, the reference LNP comprises the cationic lipid DLin-MC3-DMA or lipid 7, but is otherwise the same as the tested LNP. In some embodiments, the PEG lipid is a free PEG lipid.

[0415] In some embodiments, at least 1%, 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% of the immune cells, 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%, or at least 95% are transfected by the LNP. In some embodiments, the immune cell is a subject's immune cell. In some embodiments, the immune cell is the target of the method. In some embodiments, the immune cell is the immune cell of the subject that is the target of the method.

[0416] In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 1%, at least 5%, at least 10%, at least 15%, at least 20% compared to the expression level of the nucleic acid delivered by the reference LNP. %, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least twice, at least three times, At least 4 times, at least 5 times, at least 6 t...

Claims

1. 1. A lipid nanoparticle (LNP) for targeted delivery of nucleic acids to immune cells, comprising a lipid blend, the lipid blend comprising: (a) a lipid-immune cell targeting group conjugate comprising a compound of formula IV: [lipid]-[optional linker]-[immune cell targeting group]; and (b) [Case 1] An ionizable cationic lipid comprising wherein the LNP further comprises a nucleic acid disposed therein.

2. The LNP of claim 1 , wherein the immune cell targeting group comprises an antibody that binds to a T cell antigen.

3. The LNP of claim 2 , wherein the T cell antigen is CD3, CD4, CD7, CD8 or a combination thereof (e.g., CD3 and CD8, CD4 and CD8, or CD7 and CD8).

4. The LNP of claim 2 , wherein the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen.

5. The LNP of claim 4 , wherein the NK cell antigen is CD7, CD8, CD56 or a combination thereof (e.g., CD7 and CD8).

6. The LNP of any one of claims 1 to 5, wherein the immune cell targeting group is covalently attached to a lipid in the lipid blend through a polyethylene glycol (PEG)-containing linker.

7. 7. The LNP of claim 6, wherein the lipid covalently attached to the immune cell targeting group through a PEG-containing linker is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG) or ceramide.

8. The LNP of claim 6 or 7, wherein the PEG is PEG 2000 or PEG 3400.

9. 9. The LNP of any one of claims 1 to 8, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in a range of 0.001 to 0.5 mole percent (e.g., 0.002 to 0.2 mole percent).

10. The LNP of any one of claims 1 to 9, wherein the lipid blend further comprises one or more of a structured lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid.

11. The LNP of any one of claims 1 to 10, wherein the ionizable cationic lipid is present in the lipid blend in a range of 30 to 70 (e.g., 40 to 60) molar percent.

12. The LNP of claim 10, wherein the sterol is present in the lipid blend in a range of 20-70 (e.g., 30-50) mole percent.

13. The LNP of claim 10 or 12, wherein the sterol is cholesterol.

14. The LNP according to any one of claims 10 to 13, wherein 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.

15. The LNP of any one of claims 10 to 14, wherein the neutral phospholipid is present in the lipid blend in the range of 1 to 10 mole percent.

16. The LNP of any one of claims 10 to 15, wherein 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 selected from the group consisting of PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DG), PEG-diisopropyl ... G-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoylphosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, For example, it may be a 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.

17. The LNP of any one of claims 10 to 15, wherein the free PEG-lipid comprises a diacylphosphatidylethanolamine containing a dipalmitoyl (C16) chain or a distearoyl (C18) chain.

18. The LNP of any one of claims 10 to 17, wherein the free PEG-lipid is present in the lipid blend in the range of 1 to 4 mole percent.

19. The LNP of any one of claims 10 to 18, wherein 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.

20. The LNP of any one of claims 1 to 19, having an average diameter in the range of 50 to 200 nm.

21. The LNP of claim 20 having an average diameter of about 100 nm.

22. The LNP of any one of claims 1 to 21, having a polydispersity index in the range of 0.05 to 1.

23. The LNP of any one of claims 1 to 22, having a zeta potential of about -10 mV to about +30 mV at pH 5.

24. The LNP of any one of claims 1 to 23, wherein the nucleic acid is DNA or RNA.

25. The LNP of claim 24, wherein the RNA is mRNA.

26. The LNP of claim 25, wherein the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme or a vaccine.

27. The LNP of claim 25, wherein the mRNA encodes a polypeptide capable of regulating an immune response in the immune cell.

28. The LNP of claim 25, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

29. The LNP of claim 27, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR).

30. The LNP of any one of claims 1 to 29, wherein the immune cell targeting group comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain (e.g., a nanobody).

31. The LNP of any one of claims 1 to 29, wherein the immune cell targeting group comprises a Fab, F(ab')2, Fab'-SH, Fv or scFv fragment.

32. The LNP of claim 30 or claim 31 , wherein the immune cell targeting group comprises a Fab that has been engineered to knock out the native interchain disulfide bond at the C-terminus.

33. 33. The LNP of claim 32, wherein the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution, according to Kabat numbering.

34. The LNP of any one of claims 31 to 33, wherein the immune cell targeting group comprises a Fab having a non-native interchain disulfide bond (e.g., an engineered, buried interchain disulfide bond).

35. 35. The LNP of claim 34, wherein said Fab comprises an F174C substitution in said heavy chain fragment and an S176C substitution in said light chain fragment, according to Kabat numbering.

36. The LNP of any one of claims 31 to 35, wherein the immune cell targeting group comprises a Fab that contains a cysteine ​​at the C-terminus of the heavy or light chain fragment.

37. The LNP of claim 36, wherein the Fab further comprises one or more amino acids between the heavy chain fragment and the C-terminal cysteine ​​of the Fab.

38. The LNP of claim 30, wherein the immune cell targeting group comprises an immunoglobulin single variable domain.

39. The LNP of claim 30 or claim 38, wherein the immunoglobulin single variable domain comprises a cysteine ​​at the C-terminus.

40. The LNP of claim 39, wherein 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.

41. The LNP of any one of claims 31 and 38-40, wherein the immune cell targeting group comprises two or more VHH domains.

42. The two or more V ＨＨ The LNP of claim 41 , wherein the domains are linked by an amino acid linker.

43. The immune cell targeting group comprises a first V ＨＨ a second V domain linked to an antibody light chain constant domain; ＨＨ 42. The LNP of claim 41, comprising an antibody CH1 domain and an antibody light chain constant domain, wherein the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds.

44. The LNP of any one of claims 30 and 38-40, wherein the immune cell targeting group comprises a VHH domain linked to an antibody CH1 domain, and the antibody CH1 domain is linked to an antibody light chain constant domain by one or more disulfide bonds. .

45. The LNP of claim 43 or 44, wherein the CH1 domain comprises a F174C and a C233S substitution, and the light chain constant domain comprises a S176C and a C214S substitution, according to the Kabat numbering system.

46. The immune cell targeting group comprises: (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 The LNP of any one of claims 1 to 27, comprising a Fab comprising:

47. 1. A method for targeting delivery of a nucleic acid to an immune cell of a subject, comprising: contacting the immune cells with a lipid nanoparticle (LNP), the LNP comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising a compound of the following formula: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids, (e) free polyethylene glycol (PEG) lipids, and (f) Nucleic acid and wherein the LNP has the following benefits: (i) increased specificity of targeted delivery to said immune cells compared to a reference LNP; (ii) an increase in the half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell compared to a reference LNP; (iii) an increase in transfection rate compared to a reference LNP; and (iv) low levels of dye-accessible mRNA (<15%) and high RNA encapsulation efficiency (at least 80% of mRNA is recovered in the final formulation relative to the total RNA used in the LNP batch preparation); The method of claim 1, further comprising:

48. 1. A method for expressing a polypeptide of interest in targeted immune cells of a subject, comprising: contacting the immune cells with a lipid nanoparticle (LNP), the LNP comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids, (e) free polyethylene glycol (PEG) lipids, and (f) a nucleic acid encoding the polypeptide. A method comprising:

49. The LNP provides the following benefits: (i) an increased expression level in said immune cells compared to a reference LNP; (ii) increased specificity of expression in said immune cells compared to a reference LNP; (iii) an increase in the half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell compared to a reference LNP; (iv) an increase in transfection rate compared to a reference LNP; and (v) Low levels of dye-accessible mRNA (<15%) and high RNA encapsulation 3. mRNA purification efficiency (at least 80% mRNA recovery in the final formulation relative to the total RNA used in the LNP batch preparation) 49. The method of claim 48, further comprising providing at least one of:

50. 1. A pharmaceutical composition comprising a lipid nanoparticle (LNP) for use in a method for modulating a cellular function of a target immune cell in a subject, comprising: The method includes administering to the subject a lipid nanoparticle (LNP), the LNP comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) a nucleic acid encoding a polypeptide for modulating said cellular function of said immune cell.

13. A pharmaceutical composition comprising:

51. The LNP provides the following benefits: (i) an increased expression level in said immune cells compared to a reference LNP; (ii) increased specificity of expression in said immune cells compared to a reference LNP; (iii) an increase in the half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell compared to a reference LNP; (iv) an increase in transfection rate compared to a reference LNP; and (v) the LNPs can be administered at lower doses compared to a reference LNP to achieve the same biological effect on the immune cells; and (vi) low levels of dye-accessible mRNA (<15%) and high RNA encapsulation efficiency (at least 80% of mRNA is recovered in the final formulation relative to the total RNA used in the LNP batch preparation); 51. The pharmaceutical composition of claim 50, providing at least one of:

52. 52. The pharmaceutical composition of claim 50 or 51, wherein the modulation of cell function comprises reprogramming the immune cell to mount an immune response.

53. 52. The pharmaceutical composition of claim 50 or 51, wherein said modulation of cellular function comprises modulating antigen specificity of said immune cell.

54. 1. A pharmaceutical composition comprising a lipid nanoparticle (LNP) for use in a method for treating, ameliorating or preventing a symptom of a disorder or disease in a subject in need thereof, comprising: The method includes administering to the subject a lipid nanoparticle (LNP) for delivery of a nucleic acid to an immune cell of the subject, the LNP comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein said nucleic acid modulates said immune response of said immune cell, thus treating or ameliorating said condition.

55. The LNP provides the following benefits: (i) increased specificity of delivery of said nucleic acid to said immune cells compared to a reference LNP; (ii) an increase in the half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell compared to a reference LNP; (iii) increased transfection rate compared to reference LNPs; (iv) the LNPs can be administered at lower doses compared to a reference LNP to reach the same therapeutic effect; and (v) low levels of dye-accessible mRNA (<15%) and high RNA encapsulation efficiency (at least 80% of mRNA is recovered in the final formulation relative to the total RNA used in the LNP batch preparation); 55. The pharmaceutical composition of claim 54, providing at least one of:

56. 56. The pharmaceutical composition of claim 54 or 55, wherein the disorder is an immune disorder, an inflammatory disorder or cancer.

57. 56. The pharmaceutical composition of claim 54 or 55, wherein the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine for the treatment or prevention of infection by a pathogen.

58. The ionizable cationic lipid [Case 2] The method according to any one of claims 47 to 57, wherein

59. 59. The method of any one of claims 47 to 58, wherein the immune cell targeting group comprises an antibody that binds to a T cell antigen.

60. 60. The method of claim 59, wherein the T cell antigen is CD3, CD8, or both CD3 and CD8.

61. 59. The method of any one of claims 47 to 58, wherein the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen.

62. 62. The method of claim 61, wherein the NK cell antigen is CD7, CD8 or CD56.

63. The method of any one of claims 58 to 62, wherein the antibody is a human or humanized antibody.

64. The immune cell targeting group is attached via a polyethylene glycol (PEG)-containing linker.

64. The method of any one of claims 47 to 63, wherein the lipid blend is covalently bound to a lipid.

65. 65. The method of claim 64, wherein the lipid covalently attached to the immune cell targeting group through a PEG-containing linker is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG) or ceramide.

66. 66. The method of claim 64 or 65, wherein the PEG is PEG 2000.

67. 67. The method of any one of claims 47-66, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002 to 0.2 mole percent.

68. 68. The method of any one of claims 47 to 67, wherein the ionizable cationic lipid is present in the lipid blend in the range of 40 to 60 mole percent.

69. 69. The method of any one of claims 47 to 68, wherein the sterol is cholesterol.

70. 70. The method of any one of claims 47 to 69, wherein the sterol is present in the lipid blend in the range of 30 to 50 mole percent.

71. The method according to any one of claims 47 to 70, wherein 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 (SM).

72. 72. The method of any one of claims 47 to 71, wherein the neutral phospholipid is present in the lipid blend in the range of 1 to 10 mole percent.

73. 73. The method of any one of claims 47 to 72, wherein 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 may be selected from the group consisting of PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoylphosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG- The lipid may be a PEG-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.

74. 74. The method of any one of claims 47 to 73, wherein the free PEG-lipid comprises a diacylphosphatidylethanolamine comprising a dipalmitoyl (C16) or distearoyl (C18) chain.

75. 75. The method of any one of claims 47 to 74, wherein the free PEG-lipid is present in the lipid blend in the range of 2 to 4 mole percent.

76. 76. The method of any one of claims 66-75, wherein 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.

77. The method of any one of claims 47 to 74, wherein the LNPs have an average diameter in the range of 50 to 200 nm.

78. 76. The method of claim 75, wherein the LNPs have an average diameter of about 100 nm.

79. 79. The method of any one of claims 47 to 78, wherein the LNPs have a polydispersity index in the range of 0.05 to 1.

80. The method of any one of claims 47 to 79, wherein the LNPs have a zeta potential of about -10 mV to about +30 mV at pH 5.

81. The method of any one of claims 47 to 80, wherein the nucleic acid is DNA or RNA.

82. 82. The method of claim 81, wherein the RNA is mRNA, tRNA, siRNA or microRNA.

83. 83. The method of claim 82, wherein the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme, or a vaccine.

84. 83. The method of claim 82, wherein the mRNA encodes a polypeptide capable of modulating an immune response in the immune cell.

85. 83. The method of claim 82, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

86. 83. The method of claim 82, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR).

87. 87. The method of any one of claims 47 to 86, wherein the immune cell targeting group comprises an antibody, the antibody being a Fab or an immunoglobulin single variable domain.

88. 87. The method of any one of claims 47 to 86, wherein the immune cell targeting group comprises an antibody fragment selected from the group consisting of Fab, F(ab')2, Fab'-SH, Fv and scFv fragments.

89. 89. The method of claim 87 or 88, wherein the immune cell targeting group comprises a Fab comprising one or more interchain disulfide bonds.

90. 90. The method of claim 89, wherein said Fab comprises a heavy chain fragment comprising F174C and C233S substitutions and a light chain fragment comprising S176C and C214S substitutions according to Kabat numbering.

91. 91. The method of any one of claims 87-90, wherein the immune cell targeting group comprises a Fab that comprises a cysteine ​​at the C-terminus of the heavy or light chain fragment.

92. 88. The method of claim 87, wherein the Fab further comprises one or more amino acids between the heavy chain fragment and the C-terminal cysteine ​​of the Fab.

93. 93. The method of any one of claims 88-92, wherein 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, the CH1 domain and the light chain constant domain being 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.

94. 88. The method of claim 87, wherein the immune cell targeting group comprises an immunoglobulin single variable domain.

95. 95. The method of claim 87 or 94, wherein the immunoglobulin single variable domain comprises a cysteine ​​at the C-terminus.

96. 96. The method of claim 95, wherein 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.

97. 97. The method of any one of claims 87 and 94-96, wherein the immune cell targeting group comprises two or more VHH domains.

98. The method of claim 97, wherein the two or more VHH domains are linked by an amino acid linker.

99. 98. The method of claim 97, wherein said immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain, wherein said antibody CH1 domain and said antibody light chain constant domain are linked by one or more disulfide bonds.

100. 97. The method of any one of claims 87 and 94-96, wherein said immune cell targeting group comprises a VHH domain linked to an antibody CH1 domain, said antibody CH1 domain being linked to an antibody light chain constant domain by one or more disulfide bonds.

101. 99. The method of claim 97 or 98, wherein the CH1 domain comprises a F174C and a C233S substitution and the light chain constant domain comprises a S176C and a C214S substitution according to Kabat numbering.

102. The immune cell targeting group comprises: (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:6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO:7 The method of any one of claims 47 to 86, comprising a Fab comprising:

103. The method of any one of claims 47 to 102, wherein no more than 5% of non-immune cells are transfected by said LNPs.

104. The method of any one of claims 47 to 103, wherein 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 a reference LNP or the polypeptide encoded by the nucleic acid delivered by a reference LNP.

105. The method of any one of claims 47 to 104, wherein at least 10% of immune cells are transfected by said LNPs.

106. The method of any one of claims 47 to 105, wherein 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 a reference LNP.

107. A lipid nanoparticle (LNP) 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) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell is a NK cell and the immune cell targeting group comprises an antibody that binds to CD56.

108. A lipid nanoparticle (LNP) 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) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell targeting group comprises an antibody that binds to CD7 or CD8, and the free PEG lipid is DMG-PEG.

109. A lipid nanoparticle (LNP) 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) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell targeting group comprises an antibody, the antibody being a Fab or an immunoglobulin monoclonal antibody. A single variable domain, lipid nanoparticle (LNP).

110. The LNP of claim 109, wherein the Fab has been engineered to knock out the native interchain disulfide at the C-terminus.

111. The LNP of claim 110, wherein the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution.

112. The LNP of claim 111, wherein the Fab comprises a non-natural interchain disulfide.

113. The LNP of claim 111, wherein the Fab comprises an F174C substitution in the heavy chain fragment and an S176C substitution in the light chain fragment.

114. The LNP of claim 109, wherein the antibody is an immunoglobulin single variable (ISV) domain, and the ISV domain is a Nanobody® ISV.

115. The LNP of claim 114, wherein the free PEG lipid comprises a PEG having a molecular weight of at least 2000 daltons.

116. The LNP of claim 115, wherein the PEG has a molecular weight of about 3,000 to 5,000 daltons.

117. The LNP of claim 109, wherein the antibody is a Fab.

118. The LNP of claim 117, wherein the Fab binds to CD3 and the free PEG lipid in the LNP comprises a PEG having a molecular weight of about 2000 daltons.

119. The LNP of claim 117, wherein the Fab is an anti-CD4 antibody and the free PEG lipid in the LNP comprises a PEG having a molecular weight of about 3000-3500 daltons.

120. The LNP of claim 109, wherein the immune cell targeting group comprises two or more VHH domains.

121. The LNP of claim 120, wherein the two or more VHH domains are linked by an amino acid linker.

122. The LNP of claim 121, wherein the immune cell targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain.

123. A lipid nanoparticle (LNP) 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) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; and a lipid nanoparticle (LNP) that binds to CD3 and also binds to CD11a or CD18.

124. The LNP of claim 123, comprising 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.

125. The LNP of claim 123, comprising one conjugate, said conjugate comprising a bispecific antibody that binds to CD3 and CD11a.

126. The LNP of claim 123, comprising one conjugate, said conjugate comprising a bispecific antibody that binds to CD3 and CD18.

127. The LNP of claim 125 or 126, wherein the bispecific antibody is an immunoglobulin single variable domain or a Fab-ScFv.

128. A lipid nanoparticle (LNP) 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) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; and a lipid nanoparticle (LNP) that binds to CD7 and CD8 of said immune cells.

129. The LNP of claim 128, comprising two conjugates, a first conjugate comprising an antibody that binds to CD7 and a second conjugate comprising an antibody that binds to CD8.

130. The LNP of claim 128, comprising one conjugate, said conjugate comprising a bispecific antibody that binds to CD7 and CD8.

131. The LNP of claim 130, wherein the bispecific antibody is an immunoglobulin single variable domain or a Fab-ScFv.

132. A lipid nanoparticle (LNP) for delivery of nucleic acids to two different types of immune cells in a subject: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein 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.

133. The LNP of claim 132, wherein the two different types of immune cells are CD4+ T cells and CD8+ T cells.

134. The method comprises the steps of: (a) administering to said patient a first type of immune cell; The LNP of claim 132, wherein the second conjugate comprises a first antibody that binds to the first antigen of the second type of immune cell, and the second conjugate comprises a second antibody that binds to the second antigen of the second type of immune cell.

135. The LNP of claim 132, comprising a conjugate, said conjugate comprising a bispecific antibody, said bispecific antibody binding to both said first antigen on said first type of immune cell and said second antigen on said second type of immune cell.

136. The LNP of claim 135, wherein the bispecific antibody is an immunoglobulin single variable domain or a Fab-ScFv.

137. A lipid nanoparticle (LNP) for delivering a nucleic acid to CD4+ and CD8+ T cells in a subject, comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell targeting group comprises a single antibody that binds to CD3 or CD7.

138. A lipid nanoparticle (LNP) for delivering nucleic acids to both T cells and NK cells of a subject, comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell targeting group binds to CD7, CD8, or both CD7 and CD8.

139. A lipid nanoparticle (LNP) for delivering nucleic acids to both T cells and NK cells of a subject, comprising: (a) an ionizable cationic lipid; (b) a conjugate comprising the structure: [lipid]-[optional linker]-[immune cell targeting group]; (c) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell targeting group comprises: (i) both CD3 and CD56; (ii) both CD8 and CD56; or (iii) both CD7 and CD56 Binding to lipid nanoparticles (LNPs).

140. The LNP of any one of claims 107 to 139, wherein the immune cell targeting group is covalently attached to a lipid in the lipid blend through a polyethylene glycol (PEG)-containing linker.

141. The LNP of claim 140, wherein the lipid covalently attached to the immune cell targeting group through a PEG-containing linker is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG) or ceramide.

142. The LNP of any one of claims 107-131, wherein the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 mole percent.

143. The LNP of any one of claims 107 to 142, wherein the lipid blend further comprises one or more of a structured lipid (e.g., a sterol), a neutral phospholipid, and a free PEG-lipid.

144. The LNP of any one of claims 107 to 143, wherein the ionizable cationic lipid is present in the lipid blend in the range of 40 to 60 mole percent.

145. The LNP of claim 143, wherein the sterol is present in the lipid blend in the range of 30-50 mole percent.

146. The LNP of claim 143 or 145, wherein the sterol is cholesterol.

147. The LNP of any one of claims 139 to 146, wherein 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), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

148. The LNP of any one of claims 107 to 147, wherein the neutral phospholipid is present in the lipid blend in the range of 1 to 10 mole percent.

149. The free PEG-lipid may be 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-methyl 148. The LNP of any one of claims 107 to 147, wherein the LNP is selected from the group consisting of 1,2-dioleoyl-rac-glycerol, 1,2-distearoyl-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-palmitoylsphingosine-1-{succinyl[methoxy(polyethylene glycol)](PEG-ceramide), and DSPE-PEG-cysteine, or a derivative thereof.

150. The LNP of any one of claims 107 to 149, wherein the free PEG-lipid comprises a diacylphosphatidylethanolamine comprising a dipalmitoyl (C16) chain or a distearoyl (C18) chain.

151. The LNP of any one of claims 107 to 150, wherein the free PEG-lipid is present in the lipid blend in the range of 1 to 2 mole percent.

152. The LNP of any one of claims 107 to 151, wherein 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.

153. The LNP of any one of claims 107 to 152, having an average diameter in the range of 50 to 200 nm.

154. The LNP of claim 153, having an average diameter of about 100 nm.

155. The LNP of any one of claims 107 to 154, having a polydispersity index in the range of 0.05 to 1.

156. The LNP of any one of claims 107 to 155, having a zeta potential of about -10 mV to about +30 mV at pH 5.

157. The LNP of any one of claims 107 to 156, wherein the nucleic acid is DNA or RNA.

158. The LNP of claim 157, wherein the RNA is mRNA.

159. The LNP of claim 158, wherein the mRNA encodes a receptor, a growth factor, a hormone, a cytokine, an antibody, an antigen, an enzyme or a vaccine.

160. The LNP of claim 158, wherein the mRNA encodes a polypeptide capable of regulating an immune response in the immune cell.

161. The LNP of claim 160, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

162. The LNP of claim 161, wherein the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR).

163. A lipid nanoparticle (LNP) 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) sterols or other structured lipids; (d) neutral phospholipids; (e) free polyethylene glycol (PEG) lipids, and (f) nucleic acid; wherein the immune cell targeting group comprises a Fab that lacks native interchain disulfide bonds.

164. The LNP of claim 163, wherein the Fab has been engineered to replace one or both cysteines on the native constant light chain and the native constant heavy chain that form native interchain disulfides with non-cysteine ​​amino acids, thus eliminating the native interchain disulfide bonds of the Fab.

165. A method for targeting immune cells of a subject for delivery of a nucleic acid, comprising contacting said immune cells with a lipid nanoparticle (LNP) described in any one of claims 107 to 164.

166. 166. The method of claim 165, for targeting NK cells, wherein said immune cell targeting group binds to CD56.

167. 166. The method of claim 165, for simultaneously targeting both T cells and NK cells, wherein the immune cell targeting group binds to CD7, CD8, or both CD7 and CD8.

168. 166. The method of claim 165, for simultaneously targeting both CD4+ and CD8+ T cells, wherein the immune cell targeting group comprises a polypeptide that binds to CD3 or CD7.

169. A method for expressing a polypeptide of interest in targeted immune cells of a subject, comprising contacting said immune cells with a lipid nanoparticle (LNP) described in any one of claims 107 to 164.

170. A pharmaceutical composition comprising a lipid nanoparticle (LNP) according to any one of claims 107 to 160 for use in a method for modulating a cellular function of a target immune cell in a subject, said method comprising administering to said subject a lipid nanoparticle (LNP) according to any one of claims 107 to 160.

171. A pharmaceutical composition comprising a lipid nanoparticle (LNP) according to any one of claims 107 to 164 for use in a method for treating, ameliorating or preventing a symptom of a disorder or disease in a subject in need thereof, said method comprising administering to said subject a lipid nanoparticle (LNP) according to any one of claims 107 to 164.

172. comprising 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); An immunoglobulin single variable domain (ISVD) that binds to human CD8.

173. The ISVD of claim 172, which is humanized.

174. The ISVD of claim 173 comprising sequence number 77.

175. A polypeptide comprising GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101) and AADALPYTVRKYNY (SEQ ID NO: 102).

176. A polypeptide comprising the ISVD described in claim 172.

177. 177. The polypeptide of claim 176, further comprising a second binding moiety, said second binding moiety binding to CD8 or another different target.

178. The polypeptide of claim 177, wherein the second binding moiety is also an ISVD.

179. 177. The polypeptide of claim 176, further comprising a detectable marker.

180. The polypeptide of claim 176, further comprising a therapeutic agent.

181. 172-174, or a composition comprising a polypeptide according to any one of claims 175-180.

182. A pharmaceutical composition comprising an ISVD according to any one of claims 172-174, or a polypeptide according to any one of claims 175-180, and a pharma- ceutically acceptable carrier.

183. 183. The pharmaceutical composition of claim 182 for use in a method of treating a CD8-associated disease or disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 182.

184. The pharmaceutical composition of claim 183, wherein the disease or disorder is cancer.

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