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

Abstract

Ionizable cationic lipids and lipid nanoparticles for delivering nucleic acids to cells (e.g., immune cells) are provided, as well as methods for preparing and using such lipids and targeted lipid nanoparticles.

Description

Technical field

[0001] The present invention provides ionizable cationic lipids and lipid nanoparticles for the delivery of nucleic acids to immune cells, as well as methods of making and using such lipids and targeting lipid nanoparticles.

Prior Technology

[0002] In recent years, a number of therapeutic modalities involving the delivery of one or more nucleic acids to a subject have been developed. Treatment modalities include, for example, gene therapy, in which a gene of interest in the form of deoxyribonucleic acid (DNA) is introduced into cells and then expressed to produce a gene product (eg, protein) for the treatment of A disorder caused by or related to a defect or deficiency. In this method, a gene is transcribed into messenger ribonucleic acid (mRNA), which is then translated to produce the gene product. In another approach, mRNA can be delivered to cells instead of the gene of interest. The resulting expressed product may ameliorate a specific protein deficiency or deficiency in a subject (for example, protein deficiency that occurs in certain forms of cystic fibrosis or lysosomal storage disorders), or may be used to modulate cellular function, For example, reprogramming immune cells to initiate or otherwise modulate an immune response in a subject (e.g., as a therapeutic agent for treating cancer or as a therapeutic agent for preventing or rendering a microbial or viral infection risky or severe) preventive vaccines to a minimum).

[0003] However, delivery of mRNA to cells for translation within the cell is challenging due to factors such as nuclease degradation of the mRNA prior to entry into the cell and after introduction into the cell but prior to translation.

[0004] Different delivery vehicles can be used to deliver RNA to a subject, for example based on cationic polymers or lipids that form nanoparticles with the RNA. Nanoparticles are designed to protect RNA from degradation, enable delivery of RNA to target sites, and facilitate cellular uptake and processing by target cells. For delivery efficacy, besides the molecular composition, parameters like finesse, charge or grafting with molecular moieties such as polyethylene glycol (PEG) or ligands also play a role. Grafting with PEG is thought to reduce serum interactions, increase serum stability, and increase circulation time, which may be helpful for certain targeting approaches.

[0005] Compared with DNA delivery technologies used in some gene therapies, mRNA-based gene therapy has many superior characteristics, such as ease of manipulation, rapid and transient performance, and self-regulating switchability without mutagenesis.

[0006] However, delivery of therapeutic RNA to cells is difficult given the relative instability and low cell permeability of RNA. Accordingly, there is a need to develop methods and compositions for facilitating the delivery of RNA, such as mRNA, to cells.

Content of invention

[0007] The present invention provides ionizable cationic lipids, lipid-immune cell targeting group conjugates, and lipid-immune cell (e.g., T cell) targeting compounds comprising such ionizable cationic lipids and / or lipid-immune cell (e.g., T cell) targeting Lipid nanoparticle compositions of radical conjugates, medical kits containing such lipids and / or conjugates, and methods of making and using such lipids and conjugates.

[0008] The lipid nanoparticle compositions provided herein can further comprise a nucleic acid, such as RNA, eg, messenger RNA or mRNA. The lipid nanoparticle compositions can be used to deliver mRNA to cells (eg, immune cells, such as T cells) of a subject. Messenger RNA-based gene therapy requires efficient delivery of mRNA to circulating cells in plasma (for example, immune cells such as T cells or NK cells) or to cells in a given tissue. Major challenges associated with efficient mRNA delivery to achieve robust protein expression levels include: (a) the ability to protect the mRNA payload from ubiquitous serum nucleases when administered to a subject; The ability of mRNA delivery to be specifically targeted to a population of target cells (e.g., T cells) thereby maximizing expression of the protein therein; and (c) maximal delivery of mRNA payloads to cells (e.g., T cells) The ability of the plasmic compartment to be translated into proteins within the cytoplasm.

[0009] The present invention provides ionizable cationic lipids for use in producing lipid nanoparticle compositions that facilitate the placement of payloads (e.g., nucleic acids, such as DNA or RNA) into them. , such as mRNA) delivered to cells, such as mammalian cells, such as immune cells. The lipids are designed to enable intracellular delivery of nucleic acids (eg, mRNA) to the cytoplasmic compartment of target cell types and rapid degradation into nontoxic components. These complex functions are via the interplay between the chemistry and geometry of the ionizable lipid headgroup, the hydrophobic "acyl tail" group, and the linker linking the headgroup and acyl tail group in ionizable cationic lipids. function is achieved.

[0010] In one aspect, the invention provides a compound represented by Formula I: (Formula I), or a salt thereof, wherein the variables are as defined herein.

[0011] In another aspect, the present invention provides a compound represented by Formula II: (Formula II), or a salt thereof, wherein the variables are as defined herein.

[0012] Provided in part herein is a compound selected from the group consisting of: ,,,,,, and, or a salt thereof.

[0013] In certain embodiments, the compound is a compound of Formula III: (Formula III), or a salt thereof, wherein the variables are as defined herein.

[0014] This paper also provides a compound of the following formula: , or a salt thereof.

[0015] This paper also provides a compound of the following formula: , or a salt thereof.

[0016] This paper also provides a compound of the following formula: , or a salt thereof.

[0017] This paper also provides a compound of the following formula: , or a salt thereof.

[0018] This paper also provides a compound of the following formula: , or a salt thereof.

[0019] This paper also provides a compound of the following formula: , or a salt thereof.

[0020] This paper also provides a compound of the following formula: , or a salt thereof.

[0021] This paper also provides a compound of the following formula: , or a salt thereof.

[0022] Also provided herein is a lipid nanoparticle (LNP) comprising a lipid blend comprising an ionizable cationic lipid and / or a lipid-immune cell target as provided herein. Targeting moiety conjugates (eg, lipid-T cell targeting moiety conjugates).

[0023] In another aspect, provided herein is a method of delivering a nucleic acid to an immune cell (e.g., a T cell), the method comprising causing the immune cell to Exposure to the LNP described herein containing the nucleic acid.

[0024] In another aspect, provided herein is a method of delivering a nucleic acid to immune cells (e.g., T cells) of a subject in need thereof, the method comprising administering to the subject a method comprising The composition of the described nucleic acid-containing LNP, thereby delivering the nucleic acid to the immune cell.

[0025] In another aspect, provided herein is a method of targeting the delivery of a nucleic acid (eg, mRNA) to immune cells (eg, T cells) in a subject, the method comprising administering to the subject The LNP containing the nucleic acid described herein is administered to facilitate targeted delivery of the nucleic acid to the immune cells.

[0026] 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 moiety conjugate comprising a compound of formula IV: [lipid] - [optional linker] - [immune cell target to the group]. In some embodiments, the lipid blend comprises ionizable cationic lipids. In some embodiments, the ionizable cationic lipid comprises or . In some embodiments, the LNP comprises a nucleic acid disposed therein.

[0027] In some embodiments, the immune cell targeting moiety 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, both CD3 and CD8, both CD4 and CD8, or both CD7 and CD8). In some embodiments, the immune cell targeting moiety comprises an antibody that binds a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD7, CD8 or CD56 or a combination thereof (eg, both CD7 and CD8). In some embodiments, the antibodies are human or humanized antibodies.

[0028] In some embodiments, the immune cell targeting moiety is covalently coupled to the lipid in the lipid blend via a polyethylene glycol (PEG) containing linker. In some embodiments, the lipid covalently coupled to the immune cell targeting moiety via a PEG-containing linker is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE ), dimyristyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglycerol (DSPG), dimyristyl-glycerol (DMG), dipalmityl-phosphatidylethanolamine (DPPE), dipalmitoyl Acyl-glycerol (DPG) or ceramide. In some embodiments, the PEG is PEG 2000.

[0029] In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 molar percent. In some embodiments, the lipid blend comprises 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 the range of 40-60 molar percent. In some embodiments, the sterol is present in the lipid blend in the range of 30-50 molar percent. In some embodiments, the sterol is present in the lipid blend in the range of 20-70 molar percent. In some embodiments, the sterol is cholesterol.

[0030] In some embodiments, the neutral phospholipid is selected from phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1,2-distearyl -sn-glycero-3-phosphocholine (DSPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycero-3-phosphocholine alkali (DOPC), sphingomyelin (SM). In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1-10 molar percent.

[0031] In some embodiments, the free PEG-lipid is selected from the group consisting of PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1,2-dimyristyl- rac-glycerol-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl- rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DSG), N-palmityl-sphingamine Alcohol-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide) and DSPE-PEG-cysteine ​​or its derivatives. In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine comprising dipalmitoyl (C16) chains or distearoyl (C18) chains. In some embodiments, the free PEG-lipid is a mixture of two or more unique free PEG-lipids. In some embodiments, the free PEG-lipid is present in the range of 1-4 molar percent (such as about 1-2 molar percent, or about 2-4 molar percent, or about 1.5 molar percent) in the in the lipid blend. 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.

[0032] 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 LNP has a polydispersity index ranging from 0.05 to 1. In some embodiments, the LNP has a zeta potential at pH 5 of from about -10 mV to about +30 mV.

[0033] 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, said mRNA encodes a polypeptide capable of modulating an immune response in said immune cell. In some embodiments, said mRNA encodes a polypeptide capable of reprogramming said immune cell. 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.

[0034] In some embodiments, the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain, such as a Nanobody. In some embodiments, the immune cell targeting moiety comprises a Fab, F(ab')2, Fab'-SH, Fv or scFv fragment. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to delete one or more native interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution, numbered according to Kabat; and / or a light chain fragment comprising a C214S substitution, numbered according to Kabat. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to introduce one or more buried interchain disulfide bonds. For example, in some embodiments, the Fab antibody comprises a heavy chain fragment comprising a F174C substitution, numbered according to Kabat; and / or a light chain fragment comprising a S176C substitution, numbered according to Kabat. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to delete one or more native interchain disulfide bonds and introduce one or more buried interchain disulfide bonds. In some embodiments, said immune cell targeting moiety comprises a Fab comprising a cysteine ​​at the C-terminus of said heavy or light chain fragment. In some embodiments, said Fab further comprises one or more amino acids between the heavy chain fragment of said Fab and said 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 The chain constant domains are linked via one or more interchain disulfide bonds, and wherein the immune cell targeting moiety further comprises a single chain that is linked to the C-terminus of the light chain constant domain via an amino acid linker. variable fragment (scFv). In some embodiments, the Fab antibody is a DS Fab (Fab with wild-type (native) interchain disulfide bonds), NoDS Fab (Fab with natural disulfide bonds removed, such as a C233S substitution on the heavy chain and / or Fab with C214S substitution on the light chain, according to Kabat numbering), bDS Fab (Fab without natural disulfide bonds and introduced non-native interchain buried disulfide bonds, such as F174C and C233S on the heavy chain and / or Fab with S176C and C214S substitutions on the light chain, according to Kabat numbering) or bDS Fab-ScFv (bDS Fab linked to ScFv via a linker such as (G4S)x), as shown in Figure 47.

[0035] In some embodiments, the immune cell targeting group comprises an immunoglobulin single variable domain, such as a nanobody (Nanobody). In some embodiments, said immunoglobulin single variable domain comprises a cysteine ​​at the C-terminus. In some embodiments, said Nanobody further comprises a spacer comprising one or more amino acids between said VHH domain and said C-terminal cysteine. In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked via an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain, and wherein the antibody CH1 structure domain and said antibody light chain constant domain are linked via one or more disulfide bonds. In some embodiments, the immune cell targeting moiety comprises a VHH domain linked to an antibody CH1 domain, and wherein the antibody CH1 domain is linked to an antibody light chain constant domain via one or more disulfide bonds . In some embodiments, said CH1 domain comprises F174C and C233S substitutions, and / or said light chain constant domain comprises S176C and C214S substitutions, according to Kabat numbering. In some embodiments, the antibody is ScFv, VHH(Nb), 2xVHH, VHH-CH1 / empty Vk, or VHH1-CH1 / VHH-2-Nb bDS, as shown in FIG. 47 .

[0036] In some embodiments, the immune cell targeting moiety comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and an amino acid comprising SEQ ID NO: 2 or 3 sequence of light chain fragments. In some embodiments, the immune cell targeting moiety comprises a Fab comprising a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 6 and a light chain fragment comprising the amino acid sequence of SEQ ID NO: 7 . In some embodiments, the antibody is an antibody described in the Examples.

[0037] 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 comprising SEQ ID NO: 2 or 3 The light chain fragment of the amino acid sequence; (b) the heavy chain fragment containing the amino acid sequence of SEQ ID NO: 4 and the light chain fragment containing the amino acid sequence of SEQ ID NO: 5; (c) containing the amino acid sequence of SEQ ID NO: ID NO: the heavy chain fragment of the amino acid sequence of 6 and the light chain fragment containing the amino acid sequence of SEQ ID NO: 7; (d) the heavy chain fragment containing the amino acid sequence of SEQ ID NO: 8 and containing A light chain fragment of the amino acid sequence of SEQ ID NO: 9; (e) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 10 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 11; (f) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 12 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 13; (g) containing the amino acid sequence of SEQ ID NO: 14 A heavy chain fragment and a light chain fragment containing the amino acid sequence of SEQ ID NO: 15; (h) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence containing SEQ ID NO: 17 (i) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 18 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 19; (j) containing the amino acid sequence of SEQ ID NO: 20 A heavy chain fragment of an amino acid sequence and a light chain fragment containing the amino acid sequence of SEQ ID NO: 21; or (k) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 22 and a light chain fragment containing the amino acid sequence of SEQ ID NO: The light chain fragment of the amino acid sequence of 23.

[0038] In some embodiments, the immune cell targeting moiety comprises a Fab, F(ab')2, Fab'-SH, Fv or scFv fragment. In some embodiments, the immune cell targeting moiety comprises a Fab engineered to delete the natural interchain disulfide bond at the C-terminus. In some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution, numbered according to Kabat. In some embodiments, the immune cell targeting moiety comprises a Fab with non-native interchain disulfide bonds (eg, engineered embedded interchain disulfide bonds). In some embodiments, said Fab comprises a F174C substitution in said heavy chain fragment and a S176C substitution in said light chain fragment, numbering according to Kabat. In some embodiments, said immune cell targeting moiety comprises a Fab comprising a cysteine ​​at the C-terminus of said heavy or light chain fragment. In some embodiments, said Fab further comprises one or more amino acids between the heavy chain fragment of said Fab and said C-terminal cysteine.

[0039] In some embodiments, the immune cell targeting moiety comprises an immunoglobulin single variable domain. In some embodiments, said immunoglobulin single variable domain comprises a cysteine ​​at the C-terminus. In some embodiments, said immunoglobulin single variable domain comprises a VHH domain, and further comprises a spacer comprising one or more spacers between said VHH domain and said C-terminal cysteine an amino acid. In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked via an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain. In some embodiments, the antibody CH1 domain and the antibody light chain constant domain are linked via one or more disulfide bonds. In some embodiments, the immune cell targeting moiety comprises a VHH domain linked to an antibody CH1 domain. In some embodiments, the antibody CH1 domain is linked to the antibody light chain constant domain via one or more disulfide bonds. In some embodiments, said CH1 domain comprises F174C and C233S substitutions and said light chain constant domain comprises S176C and C214S substitutions, numbering according to Kabat.

[0040] 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 comprising SEQ ID NO: 2 or 3 or (b) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 7.

[0041] In another aspect, provided herein are methods of targeting delivery of nucleic acids to immune cells of a subject. In some embodiments, the methods comprise contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNP comprises ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising a compound of the formula: [lipid] - [optional linker] - [immune cell targeting group]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid.

[0042] In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition containing the same, for use in a method of targeting the delivery of a nucleic acid to an immune cell of a subject . Such methods can be used to treat diseases or disorders as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo. In some embodiments, the LNP is a LNP as described herein in this disclosure.

[0043] In some embodiments, the LNP provides at least one of the following benefits: (i) increased specificity of targeted delivery to the immune cells compared to the reference LNP; (ii) compared to the reference LNP (iii) increased transfection efficiency compared to reference LNP; and (iv) low levels of dye-accessible mRNA (< 15 %) and high RNA encapsulation efficiency in which at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0044] In some aspects, methods of expressing a polypeptide of interest in targeted immune cells of a subject are provided. In some embodiments, the methods comprise contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises a nucleic acid encoding said polypeptide. In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition comprising the same, for use in a method of expressing a polypeptide of interest in targeted immune cells of a subject. Such methods can be used to treat diseases or disorders as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo.

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

[0046] In some aspects, methods of modulating cellular function of target immune cells in a subject are provided. In some embodiments, the method comprises administering lipid nanoparticles (LNPs) to the subject. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises a nucleic acid encoding a polypeptide for modulating a cellular function of said immune cell. In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition containing the same, for use in a method of modulating a cellular function targeting an immune cell in a subject. Such methods can be used to treat diseases or disorders as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0047] In some embodiments, the LNP provides at least one of the following benefits: (i) increased expression levels in the immune cells compared to a reference LNP; (ii) compared to a reference LNP, an increase in specificity exhibited in said immune cells; (iii) an increase in the half-life of said nucleic acid or a polypeptide encoded by said nucleic acid in said immune cells compared to a reference LNP; (iv) compared to a reference LNP , increased transfection efficiency; (v) the LNP can be administered at a lower dose than a reference LNP to achieve the same biological effect in the immune cells; and (vi) low levels of dye accessibility mRNA (<15%) and high RNA encapsulation efficiency where at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0048] In some embodiments, said modulation of cellular function comprises reprogramming said immune cells to initiate an immune response. In some embodiments, said modulation of cellular function comprises modulating antigen specificity of said immune cells.

[0049] In some aspects, methods of treating, ameliorating or preventing symptoms of a disorder or disease in a subject in need thereof are provided. In some embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP) to deliver a nucleic acid to immune cells of the subject. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid.

[0050] In some embodiments, the nucleic acid modulates the immune response of the immune cells, thereby treating or ameliorating the symptom. In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition containing the same, for use in a method of treating, ameliorating or preventing symptoms of a disorder or disease in a subject in need thereof use. A disease or disorder may be as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0051] In some embodiments, the LNP provides at least one of the following benefits: (i) increased specificity of delivery of the nucleic acid to the immune cells compared to a reference LNP; (ii) compared to a reference LNP; The half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell is increased compared to LNP; (iii) the transfection rate is increased compared to the reference LNP; (iv) the LNP can be compared with the reference LNP (v) increased levels of gain-of-function in immune cells compared to reference LNP; and (vi) low levels of dye-accessible mRNA (<15%) and High RNA encapsulation efficiency, where at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0052] In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer. In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine to treat or prevent infection by a pathogen. In some embodiments, the Fab antibody comprises a heavy chain fragment comprising a F174C substitution, numbered according to Kabat; and / or a light chain fragment comprising a S176C substitution, numbered according to Kabat.

[0053] 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 LNP . In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the undesired Immune cells were transfected with the LNPs. In some embodiments, the nucleic acid delivered to the immune cell by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP has a shorter half-life than the nucleic acid delivered to the immune cell by a reference LNP or the polypeptide encoded by the nucleic acid delivered by the LNP. The half-life of the polypeptide encoded by said nucleic acid delivered with reference to the LNP is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% longer , 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or longer.

[0054] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the immune cells intended to be targeted for said delivery are The LNP transfection.

[0055] In some embodiments, the level of expression of the nucleic acid delivered by the LNP is at least 5%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, 1.5 times, 2x, 3x, 4x, 5x, 10x, 15x, 20x or more.

[0056] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to NK cells of a subject are provided. The LNP comprises (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 as described. In some embodiments, the immune cell targeting moiety comprises an antibody that binds CD56.

[0057] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (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 as described. In some embodiments, the immune cell targeting moiety comprises an antibody that binds CD7 or CD8, and the free PEG lipid is DMG-PEG.

[0058] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (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 as described. In some embodiments, the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain. In some embodiments, the Fab is engineered to delete the native interchain disulfide bond at the C-terminus. In some embodiments, the Fab has buried interchain disulfide bonds. In some embodiments, the antibody is an immunoglobulin single variable (ISV) domain, and the ISV domain is a Nanobody® ISV. In some embodiments, the free PEG lipid comprises PEG having a molecular weight of at least 2000 Daltons. In some embodiments, the PEG has a molecular weight of about 3000 to 5000 Daltons. In some embodiments, said Fab is an anti-CD3 antibody, and said free PEG lipid in said LNP comprises PEG having a molecular weight of about 2000 Daltons. In some embodiments, said Fab is an anti-CD4 antibody, and said free PEG lipid in said LNP comprises PEG having a molecular weight of about 3000 to 3500 Daltons.

[0059] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (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 as described. In some embodiments, the immune cell targeting moiety comprises an antibody that binds CD3 and an antibody that binds CD11a or CD18.

[0060] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. The LNP comprises (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 as described. In some embodiments, the immune cell targeting moiety comprises an antibody that binds CD7 and an antibody that binds CD8.

[0061] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to two different types of immune cells of a subject are provided. The LNP comprises (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 as described.

[0062] In some embodiments, the immune cell targeting moiety comprises a bispecific targeting moiety. In some embodiments, the bispecific targeting moiety binds to the 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.

[0063] In one aspect, lipid nanoparticles (LNPs) for delivering nucleic acids to both CD4+ and CD8+ T cells of a subject are provided. The LNP comprises (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 as described. In some embodiments, the immune cell targeting moiety comprises a single antibody that binds to CD3 or CD7.

[0064] There is further provided a lipid nanoparticle (LNP) for nucleic acid delivery to immune cells of a subject, wherein the LNP comprises: (a) ionizable cationic lipids; (b) containing the following Structural conjugates: [lipid] - [optional linker] - [immune cell targeting group]; (c) sterol or other structural lipid; (d) neutral phospholipid; (e) free polymer ethylene glycol (PEG ) lipids; and (f) the nucleic acid, wherein the immune cell targeting moiety comprises a Fab lacking native interchain disulfide bonds. In some embodiments, the Fab is engineered to replace one or both of the native interchain disulfide bond-forming cysteines on the native constant light chain and the native constant heavy chain with a non-cysteine ​​amino acid , thereby removing the native interchain disulfide bonds in the Fab.

[0065] Also provided is an immunoglobulin single variable domain (ISVD) that binds to human CD8. In some embodiments, the ISVD comprises three complementarity determining domains CDR1, CDR2 and CDR3, wherein (a) the CDR1 comprises GSTFSDYG (SEQ ID NO: 100), (b) the CDR2 comprises IDWNGEHT (SEQ ID NO: 101), and (c) the CDR3 comprises AADALPYTVRKYNY (SEQ ID NO: 102).

[0066] In some embodiments, the ISVD is humanized.

[0067] In some embodiments, the ISVD comprises, consists of, or consists essentially of SEQ ID NO: 77.

[0068] Also provided is a polypeptide comprising GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101) and AADALPYTVRKYNY (SEQ ID NO: 102).

[0069] In some embodiments, the polypeptide comprises an ISVD as described herein.

[0070] In some embodiments, the polypeptide further comprises a second binding moiety, wherein the second binding moiety binds CD8 or another different target. In some embodiments, the second binding moiety is also an ISVD.

[0071] In some embodiments, the polypeptide further comprises a detectable label or therapeutic agent.

[0072] Also provided is a composition comprising an ISVD or polypeptide as described herein.

[0073] There is further provided a pharmaceutical composition comprising the ISVD or polypeptide as described herein and a pharmaceutically acceptable carrier.

[0074] Further provided is a method of treating a CD8-related disease or disorder in a subject, the method comprising administering to the subject a pharmaceutical composition as described herein.

[0075] In some embodiments, the disease is cancer. In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer.

[0076] In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine to treat or prevent infection by a pathogen. In some embodiments, the ionizable cationic lipid is or .

[0077] In some embodiments, the immune cell targeting moiety comprises an antibody that binds a T cell antigen. In some embodiments, the T cell antigen is CD3, CD8, or both CD3 and CD8. 60. In some embodiments, the immune cell targeting moiety comprises an antibody that binds a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD56. In some embodiments, the antibodies are human or humanized antibodies.

[0078] In some embodiments, the immune cell targeting moiety is covalently coupled to the lipid in the lipid blend via a polyethylene glycol (PEG) containing linker. In some embodiments, the lipid covalently coupled to the immune cell targeting moiety via a PEG-containing linker is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE ), dimyristyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglycerol (DSPG), dimyristyl-glycerol (DMG), dipalmityl-phosphatidylethanolamine (DPPE), dipalmitoyl Acyl-glycerol (DPG) or ceramide. In some embodiments, the PEG is PEG 2000.

[0079] In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 molar percent. In some embodiments, the ionizable cationic lipid is present in the lipid blend in the range of 40-60 molar percent.

[0080] In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is present in the lipid blend in the range of 30-50 molar percent. In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-di Stearoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycero-3 - Phosphocholine (DOPC), sphingomyelin (SM).

[0081] In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1-10 molar percent.

[0082] 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 Dialkylamines, PEG-modified diacylglycerols and PEG-modified dialkylglycerols. For example, the PEG lipids can be PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-diglycol Oleoyl-Glyceryl-Phosphatidylethanolamine (PEG-DLPE), PEG-Dimyristyl-Phosphatidylethanolamine (PEG-DMPE), PEG-Dipalmitoyl-Phosphatidylethanolamine (PEG-DPPE), PEG-Distearyl Acylglycerol (PEG-DSG), PEG-Diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-Ceramide, PEG-Distearyl-Glycerol-Phosphoglycerol (PEG-DSPG), PEG-dioleyl-glycerol-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or PEG- Distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

[0083] In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine comprising dipalmitoyl (C16) chains or distearoyl (C18) chains. In some embodiments, the free PEG-lipid is present in the lipid blend in the range of 2-4 molar 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.

[0084] 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 LNP has a polydispersity index ranging from 0.05 to 1. In some embodiments, the LNP has a zeta potential at pH 5 of from about -10 mV to about +30 mV.

[0085] 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, said mRNA encodes a polypeptide capable of modulating an immune response in said immune cell. In some embodiments, said mRNA encodes a polypeptide capable of reprogramming said immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR).

[0086] In some embodiments, the immune cell targeting moiety comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain. In some embodiments, the immune cell targeting moiety comprises an antibody fragment selected from the group consisting of Fab, F(ab')2, Fab'-SH, Fv and scFv fragments. In some embodiments, the immune cell targeting moiety comprises a Fab comprising one or more interchain disulfide bonds. In some embodiments, the Fab comprises a heavy chain fragment comprising the F174C and C233S substitutions and a light chain fragment comprising the S176C and C214S substitutions, numbered according to Kabat. In some embodiments, said immune cell targeting moiety comprises a Fab comprising a cysteine ​​at the C-terminus of said heavy or light chain fragment.

[0087] 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, said CH1 domain and said light chain constant domain are linked via one or more interchain disulfide bonds. In some embodiments, the immune cell targeting moiety further comprises a single chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker.

[0088] In some embodiments, the immune cell targeting moiety comprises an immunoglobulin single variable domain. In some embodiments, said immunoglobulin single variable domain comprises a cysteine ​​at the C-terminus. In some embodiments, said immunoglobulin single variable domain comprises a VHH domain, and further comprises a spacer comprising one or more spacers between said VHH domain and said C-terminal cysteine an amino acid. In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked via an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain. In some embodiments, the antibody CH1 domain and the antibody light chain constant domain are linked via one or more disulfide bonds. In some embodiments, the immune cell targeting moiety comprises a VHH domain linked to an antibody CH1 domain. In some embodiments, the antibody CH1 domain is linked to the antibody light chain constant domain via one or more disulfide bonds. In some embodiments, said CH1 domain comprises F174C and C233S substitutions and said light chain constant domain comprises S176C and C214S substitutions, numbering according to Kabat.

[0089] 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 comprising SEQ ID NO: 2 or 3 (b) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 7.

[0090] In some embodiments, no more than 5% of the non-immune cells are transfected with the LNP. In some embodiments, the half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is greater than that of the nucleic acid delivered by a reference LNP or the nucleic acid delivered by the reference LNP The half-life of the encoded polypeptide is at least 10% longer. In some embodiments, at least 10% of the immune cells are transfected with the LNP. In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by a reference LNP.

[0091] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cell targeting moiety comprises an antibody that binds CD56.

[0092] In some aspects, provided herein are lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the immune cell targeting moiety comprises an antibody that binds CD7 or CD8. In some embodiments, the free PEG lipid is DMG-PEG.

[0093] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the immune cell targeting moiety comprises an antibody. In some embodiments, the antibody is a Fab or immunoglobulin single variable domain.

[0094] In some embodiments, the Fab is engineered to delete the native interchain disulfide bond at the C-terminus. In some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and a light chain fragment comprising a C214S substitution. In some embodiments, the Fab comprises non-native interchain disulfide bonds. In some embodiments, said Fab comprises a F174C substitution in said heavy chain fragment and a S176C substitution in said 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, the free PEG lipid comprises PEG having a molecular weight of at least 2000 Daltons. In some embodiments, the PEG has a molecular weight of about 3000 to 5000 Daltons. In some embodiments, the antibody is a Fab. In some embodiments, said Fab binds CD3 and said free PEG lipid in said LNP comprises PEG having a molecular weight of about 2000 Daltons. In some embodiments, said Fab is an anti-CD4 antibody, and said free PEG lipid in said LNP comprises PEG having a molecular weight of about 3000 to 3500 Daltons.

[0095] In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked via an amino acid linker. In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain.

[0096] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid.

[0097] 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 a conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD3 and CD11a. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD3 and CD18. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

[0098] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the LNP binds CD7 and CD8 of the immune cells.

[0099] In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds CD7 and the second conjugate comprises an antibody that binds CD8. In some embodiments, the LNP comprises a conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds CD7 and CD8. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

[0100] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to two different types of immune cells of a subject are provided. 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the LNP binds to a first antigen on the surface of a first type of immune cell and also binds to a second antigen on the surface of a second type of immune cell. In some embodiments, the two different types of immune cells are CD4+ T cells and CD8+ T cells. In some embodiments, the LNP comprises two conjugates. In some embodiments, a first conjugate comprises a first antibody that binds to said first antigen of said first type of immune cell, and a second conjugate comprises said first antibody that binds to said second type of immune cell. Secondary Antigen Binding Secondary Antibody. In some embodiments, the LNP comprises a conjugate. In some embodiments, the conjugate comprises a bispecific antibody. In some embodiments, said bispecific antibody binds to both said first antigen on said first type of immune cell and said second antigen on said second type of immune cell. In some embodiments, the bispecific antibody is an immunoglobulin single variable domain or Fab-ScFv.

[0101] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to both CD4+ and CD8+ T cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the immune cell targeting moiety comprises a single antibody that binds to CD3 or CD7.

[0102] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to both T cells and NK cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD8.

[0103] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to both T cells and NK cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid. In some embodiments, the immune cell targeting moiety binds to (i) both CD3 and CD56; (ii) both CD8 and CD56; or (iii) both CD7 and CD56.

[0104] In some embodiments, the immune cell targeting moiety is covalently coupled to the lipid in the lipid blend via a polyethylene glycol (PEG) containing linker. In some embodiments, the lipid covalently coupled to the immune cell targeting moiety via a PEG-containing linker is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE ), dimyristyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglycerol (DSPG), dimyristyl-glycerol (DMG), dipalmityl-phosphatidylethanolamine (DPPE), dipalmitoyl Acyl-glycerol (DPG) or ceramide. In some embodiments, the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 molar percent. In some embodiments, the lipid blend further comprises one or more of structured lipids (eg, sterols), neutral phospholipids, and free PEG-lipids.

[0105] In some embodiments, the ionizable cationic lipid is present in the lipid blend in the range of 40-60 molar percent.

[0106] In some embodiments, the sterol is present in the lipid blend in the range of 30-50 molar percent. In some embodiments, the sterol is cholesterol.

[0107] In some embodiments, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1, 2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn- Glycero-3-phosphocholine (DOPC). In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1-10 molar percent.

[0108] In some embodiments, the free PEG-lipid is selected from the group consisting of PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1,2-dimyristyl- rac-glycerol-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl- rac-glycerol, methoxypolyethylene glycol (DOG-PEG), 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DSG), N-palmityl-sphingamine Alcohol-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide) and DSPE-PEG-cysteine ​​or its derivatives. In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine comprising dipalmitoyl (C16) chains or distearoyl (C18) chains. In some embodiments, the free PEG-lipid is present in the lipid blend in the range of 1-2 molar 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.

[0109] 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 LNP has a polydispersity index ranging from 0.05 to 1. In some embodiments, the LNP has a zeta potential at pH 5 of from about -10 mV to about +30 mV.

[0110] 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, said mRNA encodes a polypeptide capable of modulating an immune response in said immune cell. In some embodiments, said mRNA encodes a polypeptide capable of reprogramming said immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR).

[0111] In some aspects, lipid nanoparticles (LNPs) for delivering nucleic acids to immune cells of a subject are provided. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid.

[0112] In some embodiments, the immune cell targeting moiety comprises a Fab that lacks native interchain disulfide bonds. In some embodiments, the Fab is engineered to replace one or both of the native interchain disulfide bond-forming cysteines on the native constant light chain and the native constant heavy chain with a non-cysteine ​​amino acid , thereby removing the native interchain disulfide bonds in the Fab.

[0113] In some aspects, methods of targeting delivery of nucleic acids to immune cells of a subject are provided. In some embodiments, the methods comprise contacting the immune cells with a lipid nanoparticle (LNP) provided herein. In some embodiments, the methods are used to target NK cells. In some embodiments, the immune cell targeting moiety binds to CD56. In some embodiments, the methods are used to target both T cells and NK cells simultaneously. In some embodiments, the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD8. In some embodiments, the methods are used to target both CD4+ and CD8+ T cells simultaneously. In some embodiments, the immune cell targeting moiety comprises a polypeptide that binds CD3 or CD7.

[0114] In some aspects, methods of expressing a polypeptide of interest in targeted immune cells of a subject are provided. In some embodiments, the methods comprise contacting the immune cells with a lipid nanoparticle (LNP) provided herein.

[0115] In some aspects, methods of modulating cellular function of target immune cells in a subject are provided. In some embodiments, the methods comprise administering to the subject a lipid nanoparticle (LNP) provided herein.

[0116] In some aspects, methods of treating, ameliorating or preventing symptoms of a disorder or disease in a subject in need thereof are provided. In some embodiments, the methods comprise administering to the subject a lipid nanoparticle (LNP) provided herein.

[0117] In some aspects, an immunoglobulin single variable domain (ISVD) that binds to human CD8 is provided. In some embodiments, the ISVD comprises three complementarity determining domains, CDR1, CDR2 and CDR3. In some embodiments, the CDR1 comprises GSTFSDYG (SEQ ID NO: 100). In some embodiments, the CDR2 comprises IDWNGEHT (SEQ ID NO: 101). In some embodiments, the CDR3 comprises AADALPYTVRKYNY (SEQ ID NO: 102). In some embodiments, the ISVD is humanized. In some embodiments, the ISVD comprises SEQ ID NO:77.

[0118] In some aspects, polypeptides comprising GSTFSDYG (SEQ ID NO: 100), IDWNGEHT (SEQ ID NO: 101 ), and AADALPYTVRKYNY (SEQ ID NO: 102) are provided. In another aspect, a polypeptide comprising an ISVD provided herein is provided. In some embodiments, the polypeptide comprises 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 comprises a detectable label. In some embodiments, the polypeptide comprises a therapeutic agent.

[0119] In some aspects, compositions are provided comprising an ISVD provided herein or a polypeptide provided herein.

[0120] In some aspects, pharmaceutical compositions are provided comprising an ISVD provided herein or a polypeptide provided herein and a pharmaceutically acceptable carrier.

[0121] In some aspects, methods of treating a CD8-related disease or disorder in a subject are provided. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer.

[0122] Various aspects and embodiments of the invention are described in further detail below.

Implementation

[0208] CROSS-REFERENCE TO RELATED APPLICATIONS

[0209] This application claims priority and benefit to the following provisional applications: U.S. Provisional Application No. 63 / 121,801, filed December 4, 2020; U.S. Provisional Application No. 63 / 166,205, filed March 25, 2021; 2021 U.S. Provisional Application No. 63 / 169,296, filed April 1, 2021; 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, which The entire disclosure is hereby incorporated by reference in its entirety. Submission of ASCII Text File Sequence Listing

[0210] The contents of the following submitted ASCII text file are hereby incorporated by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (file name: 183952034042SEQLIST.TXT, date of record: December 3, 2021, size : 205,763 bytes).

[0211] The present invention provides ionizable cationic lipids, lipid-immune cell targeting moiety conjugates, and lipid-immune cell (e.g., T cell) targeting compounds comprising such ionizable cationic lipids and / or lipid-immune cell (e.g., T cell) targeting Lipid nanoparticle compositions of radical conjugates, medical kits containing such lipids and / or conjugates, and methods of making and using such lipids and conjugates.

[0212] The practice of the present invention employs, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, cell biology and biochemistry. Such techniques are explained in the literature, eg, 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 regularly updated) and "Current protocols in immunology" (J.E. Coligan et al. eds., 1991), each of which is incorporated herein by reference in its entirety. Various aspects of the invention are set forth in the following sections; however, aspects of the invention described in a particular section are not limited to any particular section. I. Definition

[0213] In order to facilitate the understanding of the present invention, a number of terms and phrases are defined below.

[0214] 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 fields of chemistry and biology. Chemical structures and formulas set forth herein are to be interpreted in accordance with standard rules of chemical valence known in the chemical art. Also, for example, when the chemical group is a diradical, it is understood that the chemical group can bond to its adjacent atoms in the rest of the structure in one or both directions, e.g. -OC(O)- can bond with -C(O)O- is interchangeable, or -OC(S)- is interchangeable with -C(S)O-.

[0215] Unless the context is inappropriate, the terms "a / a (a)" and "one / a (an)" as used herein mean "one or more )" and includes the plural.

[0216] The term "alkyl" as used herein refers to a saturated straight chain or branched chain hydrocarbon, such as a straight chain or branched chain group of 1-12, 1-10 or 1-6 carbon atoms, respectively Known as C1-C12 alkyl, C1-C10 alkyl and C1-C6 alkyl. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl , 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1 -Pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl Base-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tertiary butyl, pentyl, isopentyl, neo Pentyl, hexyl, heptyl, octyl, etc.

[0217] The term "alkylene" refers to a diradical of an alkyl group. An exemplary alkylene group is -CH2CH2-.

[0218] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3 and the like.

[0219] The term "side oxy" is art recognized and refers to an "=O" substituent. For example, a cyclopentane substituted with a pendant oxy group is cyclopentanone.

[0220] The term "morpholinyl" refers to a substituent having the structure: .

[0221] The term "piperidinyl" refers to a substituent having the structure: .

[0222] In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be selected from the specified When a group of substituents is substituted, the substituents at each position may be the same or different. Combinations of substituents envisioned under the present invention are preferably those that result in the formation of stable or chemically feasible compounds. In some embodiments, optional substituents may be selected from: C1-6 alkyl, cyano, halogen, -O-C1-6 alkyl, C1-6 haloalkyl, C3-7 cycloalkyl, 3- 7-membered heterocyclyl, 5-6 membered heteroaryl and phenyl, wherein Ra is hydrogen or C1-6 alkyl. In some embodiments, optional substituents may be selected from: C1-6 alkyl, halogen, -O-C1-6 alkyl, and -CH2N(Ra)2, where Ra is hydrogen or C1-6 alkyl.

[0223] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3 and the like.

[0224] The term "cycloalkyl" means a monovalent saturated cyclic, bicyclic, bridged ring of 3-12, 3-8, 4-8, or 4-6 carbons derived from a cycloalkane (e.g., adamantyl ) or spirocyclic hydrocarbon group, referred to herein as, for example, "C4-8 cycloalkyl". Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane. Unless otherwise stated, cycloalkyl is optionally replaced at one or more ring positions by, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amine , aryl, arylalkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic substituted by hydroxy, imino, ketone, nitro, phosphate, phosphonic acid, phosphinic acid, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, a cycloalkyl group is unsubstituted, ie, it is unsubstituted.

[0225] The terms "heterocyclyl" and "heterocyclic group" are art recognized and refer to saturated, partially unsaturated or aromatic 3 to 10 membered ring structures, alternatively 3 to 7 membered rings, which Ring structures include from one to four heteroatoms, such as nitrogen, oxygen and sulfur. The number of ring atoms in a heterocyclyl group can be designated using the Cx-Cx nomenclature, where x is an integer designating the number of ring atoms. For example, C3-C7 heterocyclyl refers to a saturated or partially unsaturated 3 to 7 membered ring structure containing one to four heteroatoms such as nitrogen, oxygen and sulfur. The designation "C3-C7" indicates that the heterocycle contains a total of from 3 to 7 ring atoms, including any heteroatoms occupying ring atom positions. An example of a C3 heterocyclyl is aziridyl. Heterocycles can be, for example, monocyclic, bicyclic, or other polycyclic ring systems (eg, fused, spiro, bridged bicyclic). A heterocycle can be fused to one or more aryl, partially unsaturated or saturated rings. Heterocyclic groups include, for example, biotinyl, chromenyl, dihydrofuryl, indolinyl, dihydropyranyl, dihydrothienyl, bithiazolyl, homopiperidinyl, imidazolidinyl, isoquinyl Linyl, isothiazolyl, isoxazolyl, morpholinyl, oxolyl, oxazolyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyryl Azolidinyl, pyrazolinyl, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl Linyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, thiopyryl, lactone, lactam (such as azetidinone and pyrrolidone), sulphonyl , sultone, etc. Unless otherwise stated, heterocyclic rings are optionally replaced at one or more positions by groups such as alkacyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amine, aryl, aryl Alkyl, azido, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, Substituents such as amine, ketone, nitro, pendant oxygen, phosphate, phosphonic acid, phosphinic acid, sulfate, sulfide, sulfonamide, sulfonyl and thiocarbonyl. In certain embodiments, a heterocyclyl group is unsubstituted, ie it is unsubstituted.

[0226] 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" includes polycyclic ring systems having two or more carbon rings, wherein two or more carbons are common to two adjacent rings (the rings are "fused rings"), wherein at least One ring is aromatic and, for example, the other ring or rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. Unless otherwise stated, aromatic rings can be replaced at one or more ring positions by, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amine, Nitro, mercapto, imino, amide, carboxylic acid, -C(O) alkyl, CO2 alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone , Aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF3, -CN etc. substitution. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halo, alkyl, hydroxy, or alkoxy. In certain other embodiments, the aromatic ring is unsubstituted, ie it is unsubstituted. In certain embodiments, aryl is a 6-10 membered ring structure.

[0227] The term "heteroaryl" is art recognized and refers to an aromatic group that includes at least one ring heteroatom. In certain instances, heteroaryl groups contain 1, 2, 3 or 4 ring heteroatoms. Representative examples of heteroaryl include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridyl, pyrimidinyl, and the like . Unless otherwise stated, a heteroaryl ring can be replaced at one or more ring positions by, for example, halo, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amine group, nitro group, mercapto group, imino group, amido group, carboxylic acid, C(O) alkyl group, -CO2 alkyl group, carbonyl group, carboxyl group, alkylthio group, sulfonyl group, sulfonamide group, sulfonamide group , ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moiety, -CF3, -CN and other substitutions. The term "heteroaryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings (the rings are "fused rings"), wherein At least one ring is heteroaromatic, for example, the 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 halo, alkyl, hydroxy, or alkoxy. In certain other embodiments, the heteroaryl ring is unsubstituted, ie, it is unsubstituted. In certain embodiments, heteroaryl is a 5 to 10 membered ring structure, alternatively a 5 to 6 membered ring structure, which ring structure includes 1, 2, 3 or 4 heteroatoms, such as nitrogen, oxygen and sulfur.

[0228] The terms "amine" and "amino" are art recognized and refer to both unsubstituted and substituted amines, such as moieties represented by the general formula -N(R10)(R11), wherein R10 and R11 are each independently represents hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, aralkyl or (CH2)m-R12; or R10 and R11 together with the N atom to which they are attached form a ring structure a heterocyclic ring having from 4 to 8 atoms; R12 represents aryl, cycloalkyl, cycloalkenyl, heterocyclic or polycyclic; and m is zero or an integer ranging from 1 to 8. In certain embodiments, R10 and R11 each independently represent hydrogen, alkyl, alkenyl or -(CH2)m-R12.

[0229] The term "alkoxyl" or "alkoxy" is art recognized and refers to an alkyl group as defined above having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. An "ether" is two hydrocarbons covalently linked via oxygen. Thus, the substituent of an alkyl group that renders the alkyl group an ether is or resembles an alkoxy group, as can be represented by -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH2)m- One of R12 represents, wherein m and R12 are as described above. The term "haloalkoxy" refers to an alkoxy group substituted with at least one halogen. For example, -O-CH2F, -O-CHF2, -O-CF3 and the like. In certain embodiments, haloalkoxy is alkoxy substituted with at least one fluoro group. In certain embodiments, haloalkoxy is alkoxy substituted with from 1-6, 1-5, 1-4, 2-4, or 3 fluoro groups.

[0230] The symbol "" indicates an attachment point.

[0231] The compounds of this disclosure may contain one or more chiral centers and / or double bonds, and thus be classified as stereoisomers (such as geometric isomers, enantiomers or diastereomers) exist. As used herein, the term "stereoisomers" consists of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols "R" or "S", depending on the configuration of substituents around the stereocarbon atoms. The present invention encompasses the various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated by "(±)" in nomenclature, but those skilled in the art will recognize that structures may implicitly represent chiral centers. It is to be understood that unless otherwise indicated, graphical depictions of chemical structures (eg, generic chemical structures) encompass all stereoisomeric forms of a given compound.

[0232] Individual stereoisomers of the compounds of the present invention may be prepared synthetically from commercially available starting materials containing asymmetric or stereocenters, or by preparation of racemic mixtures followed by resolution procedures well known to those of ordinary skill in the art to prepare. These resolution methods are exemplified by the following: (1) Attachment of enantiomeric mixtures to chiral auxiliaries, separation of the resulting diastereomeric mixtures via recrystallization or chromatography, and separation of enantiomeric auxiliaries from the auxiliaries (2) salt formation using an optically active resolving agent; or (3) direct separation of a mixture of optical enantiomers on a chiral column. Stereoisomeric mixtures can also be resolved into their constituent stereoisomers via well-known methods, such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallization of compounds as chiral phase salt complexes or crystallize compounds in chiral solvents. Further, enantiomers can be separated using supercritical fluid chromatography (SFC) techniques described in the literature. Still further, stereoisomers may be obtained from stereoisomerically pure intermediates, reagents and catalysts via well known asymmetric synthetic methods.

[0233] Geometric isomers may also exist in the compounds of the present invention. The symbol "" denotes a bond which may be a single, double or triple bond as described herein. The present invention encompasses various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated in the "Z" or "E" configuration, where the terms "Z" and "E" are used according to IUPAC standards. Unless otherwise stated, structures depicting double bonds encompass both "E" and "Z" isomers.

[0234] Substituents around a carbon-carbon double bond may alternatively be referred to as "cis" or "trans", where "cis" means that the substituents are on the same side of the double bond, and "trans" means The substituents are on opposite sides of the double bond. The arrangement of substituents around a carbocycle is designated as "cis" or "trans". The term "cis" means that the substituents are on the same side of the plane of the ring, and the term "trans" means that the substituents are on the opposite side of the plane of the ring. Compound mixtures in which substituents are placed on the same and opposite sides of the ring plane are designated "cis / trans".

[0235] The invention also includes isotopically labeled compounds of the invention which are identical to the compounds described herein except that one or more atoms are assigned an atomic mass or mass number different from the atomic mass or mass normally found in nature. Atomic substitution of numbers. Examples of isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 31P, 32P, respectively , 35S, 18F and 36Cl.

[0236] Certain isotopically labeled disclosed compounds (eg, compounds labeled with3H and14C) 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. Further, substitution with heavier isotopes such as deuterium (2H) may confer certain therapeutic advantages due to greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements), so in some cases The following may be preferred. Isotopically labeled compounds of the invention can generally be prepared via procedures analogous to those disclosed, for example, in the Examples herein, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0237] As used herein, the terms "subject" and "patient" refer to an organism to be treated via the methods of the invention. Such organisms are preferably mammals (eg, murines, simians, equines, bovines, porcines, canines, felines, etc.), and more preferably humans.

[0238] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier such that the composition is particularly suitable for diagnostic or therapeutic use, in vivo or ex vivo.

[0239] As used herein, the term "pharmaceutically acceptable excipient" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (such as, for example, oil / water or water / oil emulsions), and 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 ed., A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.

[0240] "Salts" of the compounds of the present invention may be derived from inorganic or organic acids and inorganic or organic bases, as known to those skilled in the art. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, Acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids, such as oxalic acid, while not themselves pharmaceutically acceptable, can be used to prepare salts which are useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

[0241] Examples of bases include, but are not limited to, alkali metal (eg, sodium) hydroxides, alkaline earth metal (eg, magnesium) hydroxides, ammonia, and compounds of formula NW4+ (wherein W is C1-4 alkyl) and the like.

[0242] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citric acid Salt, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, lauryl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerin Phosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate , 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate , tartrate, thiocyanate, tosylate, undecanoate, etc. Other examples of salts include anions of compounds of the invention complexed with suitable cations such as Na+, NH4+ and NW4+ (wherein W is C1-4 alkyl) and the like.

[0243] Abbreviations as used herein include diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); tetrabutylammonium iodide (TBAI); 1-ethyl-3-(3 -Dimethylaminopropyl)carbodiimide (EDC); benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate (PyBOP); 9-fluorenylmethoxycarbonyl (Fmoc ); Tetrabutyldimethylsilyl chloride (TBDMSCl); Hydrogen fluoride (HF); Phenyl (Ph); Bis(trimethylsilyl)amine (HMDS); Dimethylformamide (DMF) ; Dichloromethane (DCM); Tetrahydrofuran (THF); High Performance Liquid Chromatography (HPLC); Mass Spectrometry (MS); Evaporative Light Scattering Detector (ELSD); Electrospray (ES); Nuclear Magnetic Resonance Spectroscopy (NMR ).

[0244] As used herein, the term "effective amount" refers to an amount of a compound (eg, a nucleic acid such as mRNA) sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations, applications or doses and is not intended to be limited to a particular formulation or route of administration. The term effective amount may be considered to include a therapeutically and / or prophylactically effective amount of a compound.

[0245] As used herein, the phrase "therapeutically effective amount" means that amount of a compound (e.g., a nucleic acid, such as mRNA), a material, or a composition comprising a compound (e.g., a nucleic acid, such as mRNA) that is suitable for use in A reasonable benefit / risk ratio for any medical treatment effective to produce some desired therapeutic effect in at least a subpopulation of cells in a mammal (eg, a human) or a subject (eg, a human subject).

[0246] As used herein, the phrase "prophylactically effective amount" means that amount of a compound (e.g., nucleic acid, such as mRNA), material, or composition comprising a compound (e.g., nucleic acid, such as mRNA) that is suitable for use in A reasonable benefit / risk ratio for any medical treatment that is effective in a mammal (e.g., a human) or subject (e.g., a human subject) by reducing, minimizing, or eliminating the risk of developing, or reducing or minimizing the severity of some desired prophylactic effect in at least one subpopulation of cells in the subject).

[0247] As used herein, the terms "treat", "treating" and "treatment" include any effect that results in amelioration or amelioration of the symptoms of a condition, disease, disorder, etc., such as reduction, alleviation , adjust, improve or eliminate.

[0248] The phrase "pharmaceutically acceptable" is used herein to refer to substances suitable for use in contact with tissues of humans and animals without undue toxicity, irritation, allergic reaction or other problems or complications within the scope of sound medical judgment. , those compounds, materials, compositions and / or dosage forms commensurate with a reasonable benefit / risk ratio.

[0249] In the present application, where an element or component is said to be included in and / or selected from a list of enumerated elements or components, it should be understood that said element or component may be an enumerated element or any one of the components, or the elements or components may be selected from two or more of the listed elements or components.

[0250] Further, it should be understood that the elements and / or features of the compositions or methods described herein may be combined in various ways without departing from the spirit and scope of the invention, no matter what is expressly stated herein. Still implied. For example, where a particular compound is referred to, that compound may be used in various embodiments of the compositions of the invention and / or the methods of the invention, unless otherwise understood from the context. In other words, within this application, the embodiments have been described and depicted in a manner that enables a clear and concise application to be written and drawn, but it is intended and understood that the embodiments may be made without departing from the teachings and one or more of the present inventions. Cases of multiple inventions are combined or separated in various ways. For example, it should be understood that all features described and depicted herein can be applied to all aspects of one or more inventions described and depicted herein.

[0251] It should be understood that unless otherwise understood from context and use, the expression "at least one of" includes each and every one of the items enumerated after said expression / Each and two or more / various combinations of two or more of the listed items. The expression "and / or" combined with three or more / three or more listed items should be understood as having the same meaning unless otherwise understood from the context.

[0252] Unless expressly stated otherwise or otherwise understood from the context, the terms "include", "includes", "including", "have", "has" , "having", "contains", "contains" or "containing" (including their grammatical equivalents) should generally be construed as open-ended and non-limiting , such as does not exclude additional unlisted elements or steps.

[0253] Where the term "about" is used before a numerical value, unless expressly stated otherwise, the invention also includes the specific numerical value itself. As used herein, unless otherwise indicated or inferred, the term "about" refers to a variation of ±10% from the nominal value.

[0254] As used herein, unless otherwise indicated, the term "antibody" means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen. It will be understood that the term encompasses whole antibodies (e.g., intact monoclonal antibodies) or fragments thereof such as the Fc fragment of an antibody (e.g., the Fc fragment of a monoclonal antibody) or an antigen-binding fragment of an antibody (e.g., an antigen-binding antibody of a monoclonal antibody). Fragments), including whole 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 antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (eg, bispecific antibodies). The term also encompasses immunoglobulin single variable domains, such as Nanobodies (eg, VHH).

[0255] As used herein, an "antibody that binds to X" (ie, X is a specific antigen) or an "anti-X antibody" is an antibody that specifically recognizes antigen X.

[0256] As used herein, "embedded interchain disulfide bond" or "interchain embedded disulfide bond" refers to a disulfide bond on a polypeptide that is not easily accessible or effectively removed by water-soluble reducing agents. "Buried" in the hydrophobic region of the polypeptide, making it unavailable to either reducing agents or conjugation with other hydrophilic PEGs. Buried interchain disulfide bonds are further described in WO 2017096361 A1, which is incorporated by reference in its entirety.

[0257] As used herein, the specificity of targeted delivery of LNPs is determined by the percentage of desired immune cell types that receive the delivered nucleic acid (e.g., on-target delivery) versus those that are not intended to be the target of said delivery. However, the ratio between the % of undesired immune cell types that receive the delivered nucleic acid (eg, off-target delivery) is defined. For example, specificity is higher when more desired immune cells receive the delivered nucleic acid while fewer undesired immune cells receive the delivered nucleic acid. The specificity of targeted delivery of LNPs can also be defined as the ratio of the amount of nucleic acid delivered to desired immune cells (eg, on-target delivery) to the amount of nucleic acid delivered to undesired immune cells (eg, off-target delivery). The specificity of delivery can be determined using any suitable method. As a non-limiting example, the level of expression of the nucleic acid in a desired immune cell type can be measured and compared to the level of expression of the nucleic acid in a different immune cell type not intended to be the target of the delivery.

[0258] As used herein, in some embodiments, a reference LNP is a LNP that does not have an immune cell targeting group but is otherwise identical to the LNP being tested. In some other embodiments, the reference LNP is an LNP that has a different ionizable cationic lipid but is otherwise identical to the LNP being tested. In some embodiments, the reference LNP comprises D-Lin-MC3-DMA as an ionizable cationic lipid that is different from the ionizable cationic lipid in the tested LNP, but is otherwise identical to the tested LNP.

[0259] As used herein, a humanized antibody is an antibody that is of wholly or partially non-human origin and whose protein sequence has been modified to replace certain amino acids, such as those found in the sequence of an antibody from a human. Amino acids at one or more corresponding positions in the framework regions of the VH and VL domains to increase their similarity to antibodies naturally produced in humans in order to avoid or minimize human immune responses. For example, using genetic engineering techniques, the variable domains of a non-human antibody of interest can be combined with the constant domains of a human antibody. The constant domains of humanized antibodies are often human CH and CL domains.

[0260] As used herein, the term "structured lipid" refers to sterols, and also refers to lipids that contain sterol moieties.

[0261] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

[0262] At various places in this specification, substituents are disclosed in groups or in ranges. Specifically, it is intended that the description includes each individual subcombination of members of such groups and ranges. For example, the term "C1-6 alkyl" is specifically intended to disclose individually C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6 , C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5 and C5-C6 alkyl. By way of other examples, integers in the range 0 to 40 are specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 and 40, and integers in the range of 1 to 20 are specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0263] The use of any and all examples, or exemplary language (eg, "such as" or "including") herein, is intended merely to better illuminate the invention and does not impose any imposition on the scope of the invention unless claimed. limit. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0264] Throughout the specification, where compositions and kits are described as having, comprising, or comprising specific components, or where processes and methods are described as having, comprising, or comprising specific steps, it is contemplated that There are additionally compositions and kits of the invention that consist essentially of or consist of the recited components, and that consist essentially of or consist of the recited process steps. Processes and Methods of the Invention.

[0265] In general, unless otherwise indicated, percentages of compositions given are by weight. Further, if a variable has no accompanying definition, the previous definition of said variable prevails. immunoglobulin single variable domain

[0266] In some embodiments, the immune cell targeting moiety of a LNP as described herein comprises an immunoglobulin single variable domain, such as a Nanobody.

[0267] The term "immunoglobulin single variable domain" (ISV), used interchangeably with "single variable domain", defines a domain in which the antigen binding site is present on and formed by a single immunoglobulin domain of immunoglobulin molecules. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g. monoclonal antibodies) or fragments thereof (e.g. Fab, Fab', F(ab')2, scFv, di-scFv) , wherein two immunoglobulin domains, particularly two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) interact to form the antigen-binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will participate in the formation of the antigen binding site. In view of the above definitions, conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD or IgE molecules; known in the art) or Fab, F(ab')2 fragments, Fv fragments derived from such conventional 4-chain antibodies (such as bis Sulfur-bonded Fv or scFv fragments) or diabodies (all known in the art) will generally not be considered as immunoglobulin single variable domains, because in these cases, the antigen-binding domain associated with the corresponding epitope of the antigen Binding usually does not occur via one (single) immunoglobulin domain, but rather via a pair of (associated) immunoglobulin domains that together bind to an epitope of the corresponding antigen (e.g. light chain and heavy chain variable structure domain), that is, via the VH-VL pair of an immunoglobulin domain.

[0268] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of an antigen without pairing with another immunoglobulin variable domain. The binding site for an immunoglobulin single variable domain is formed by a single VH, single VHH or single VL domain. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0269] Thus, the single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0270] An immunoglobulin single variable domain may for example be a heavy chain ISV, such as a VH, VHH, including a camelid VH or a humanized VHH. In one embodiment it is a VHH, including camelid VH or humanized VHH. Heavy chain ISVs can be derived from conventional four-chain antibodies or heavy chain antibodies.

[0271] For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or a dAb (or suitable for use as a dAb amino acid sequence) or Nanobody® ISV (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment of either.

[0272] In particular, the immunoglobulin single variable domain may be a Nanobody® ISV (such as VHH, including humanized VHH or camelized VH) or a suitable fragment thereof. [Note: Nanobody® is a registered trademark of Ablynx N.V.].

[0273] "VHH domains" (also known as VHH, VHH antibody fragments, and VHH antibodies) have originally been described for "heavy chain antibodies" (i.e., "antibodies devoid of light chains"; Hamers-Casterman et al. 1993 Nature 363: 446-448) Antigen-binding immunoglobulin variable domains. The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains present in conventional 4 chain antibodies (which are referred to herein as "VH domains") and those present in conventional 4 chain antibodies. Light chain variable domains (which are referred to herein as "VL domains") in V-chain antibodies are distinguished. For a further description of VHH, refer to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74: 277-302).

[0274] Regarding the terms "dAb" and "domain antibody", reference is made to eg Ward et al. 1989 (Nature 341: 544), Holt et al. 2003 (Trends Biotechnol. 21: 484); and eg WO 2004 / 068820, WO 2006 / 030220, WO 2006 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention, since they are not of mammalian origin, single variable domains may be derived from certain shark species (e.g. the so-called "IgNAR domains", see eg WO 2005 / 18629).

[0275] In general, immunoglobulin production involves immunizing experimental animals, fusing immunoglobulin-producing cells to generate fusionomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced via screening of naive, immune or synthetic libraries, for example via phage display.

[0276] The generation of immunoglobulin sequences such as VHH has been extensively described in various publications, including WO 1994 / 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, a camelid is immunized with a target antigen in order to induce an immune response against the target antigen. The VHH library obtained from the immunization is further screened for VHH binding to the target antigen.

[0277] In these cases, antibody production requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production. Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0278] Immunoglobulin sequences of various origins can be used herein, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. In addition, fully human, humanized or chimeric sequences can be used in the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelid domain antibodies, can be used herein, e.g. as in Ward et al. 1989 (Nature 341: 544), WO 1994 / 04678 and camelized dAbs described by Davis and Riechmann (1994, Febs Lett., 339:285-290; and 1996, Prot. Eng., 9:531-537). Furthermore, ISVs are fused to form multivalent and / or multispecific constructs (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350) and eg WO 1996 / 34103 and WO 1999 / 23221).

[0279] A "humanized VHH" comprises an amino acid sequence corresponding to a naturally occurring VHH domain but has been "humanized" (i.e. via use in a VH structure derived from a conventional 4-chain antibody from humans (such as indicated above) One or more amino acid residues occurring at one or more corresponding positions in the domain replace one or more amine groups in the amino acid sequence of said naturally occurring VHH sequence, especially in the framework sequence acid residues) amino acid sequence. This can be done in a manner known per se, which should be clear to a person skilled in the art, eg based on the prior art (eg WO 2008 / 020079). Also, it should be noted that such humanized VHHs may be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides which have been obtained using polypeptides comprising naturally occurring VHH domains as starting material.

[0280] A "camelized VH" comprises an amino acid sequence corresponding to a naturally occurring VH domain but has been "camelized" (i.e. via the VHH domain of a (camelid) heavy chain antibody One or more amino acid residues occurring at one or more corresponding positions replace one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain of a conventional 4-chain antibody) amino acid sequence. This can be carried out in a manner known per se, which should be clear to the person skilled in the art, e.g. based on the descriptions of the prior art (e.g. Davies and Riechman 1994, FEBS 339: 285; 1995, Biotechnol. 13: 475 ; 1996, Prot. Eng. 9: 531; and Riechman 1999, J. Immunol. Methods 231: 25). As defined herein, such "camelizing" substitutions are inserted at amino acid positions forming and / or present at the VH-VL interface and / or at so-called camelid marker residues (see e.g. WO 1994 / 04678 and Davies and Riechmann (1994 and 1996, supra)). In one embodiment, the VH sequence used as the starting material or starting point for generating or designing a camelized VH is a VH sequence from a mammal, such as a human VH sequence, such as a VH3 sequence. However, it should be noted that such camelized VHs may be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides which have been obtained using polypeptides comprising naturally occurring VH domains as starting material.

[0281] The structure of an immunoglobulin single variable domain sequence can be considered to be composed of four framework regions ("FR"), which are referred to in the art and herein as "Framework Region 1" ("FR1"); "Architectural Region 2" ("FR2"); "Architectural Region 3" ("FR3"); and "Architectural Region 4" ("FR4"); said architectural region is interrupted by three complementarity determining regions ("CDRs") , the three complementarity-determining regions are referred to in the art and herein as "complementarity-determining region 1" ("CDR1"); "complementarity-determining region 2" ("CDR2"); and "complementarity-determining region 3" (" CDR3").

[0282] In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, e.g. based on standard manuals and herein Further disclosures and prior art mentioned.

[0283] A framework sequence is an immunoglobulin framework sequence or a framework sequence (a suitable combination of) that has been derived (eg, via humanization or camelization) from an immunoglobulin framework sequence. For example, the framework sequence may be a framework sequence derived from a light chain variable domain (eg VL sequence) and / or a heavy chain variable domain (eg VH sequence or VHH sequence). In a particular aspect, the framework sequences are framework sequences that have been derived from VHH sequences (where said framework sequences may optionally have been partially or fully humanized) or conventional VH sequences (as defined herein) that have been camelized .

[0284] In particular, the framework sequences present in the ISV sequences described herein may contain one or more marker residues (as defined herein), such that the ISV sequences are Nanobody® ISVs, e.g. like VHHs, including humanized VHHs Or camelized VH. Non-limiting examples of (suitable combinations of) such architectural sequences will become apparent from further disclosure herein.

[0285] The total number of amino acid residues in the VH and VHH domains will generally range from 110 to 120, usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0286] However, it should be noted that, with respect to the origin of the ISV sequence (or the nucleotide sequence used to express it), and with respect to the manner in which the ISV sequence or nucleotide sequence was produced or obtained (or has been produced or obtained), The ISVs described herein are not limited. Thus, ISV sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In one specific but non-limiting aspect, ISV sequences are naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences, including but not limited to "humanized" (as defined herein) immunoglobulins sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, especially partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences (especially camelized VH sequences), and ISVs that have been obtained through techniques such as: affinity maturation (e.g. starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combinations derived from different immunoglobulins Fragments of sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art; or any suitable combination of any of the foregoing.

[0287] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be a sequence isolated, for example via PCR, from a suitable naturally occurring template (eg isolated from a cell). DNA or RNA), nucleotide sequences that have been isolated from libraries (especially expression libraries), nucleotide sequences that have been prepared by introducing mutations into naturally occurring nucleotide sequences (using any suitable technique known per se, such as mismatch PCR), nucleotide sequences that have been prepared via PCR using overlapping primers, or nucleotide sequences that have been prepared using DNA synthesis techniques known per se.

[0288] In general, Nanobody® ISVs (particularly VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) may be characterized by the presence in one or more of the framework sequences (also as further described herein) One or more "marker residues" (as described herein). Thus, in general, a Nanobody® ISV can be defined as an immunoglobulin sequence with the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0289] wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more marker residues are as further defined herein.

[0290] In particular, the Nanobody® ISV may be an immunoglobulin sequence having the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3 respectively, and wherein the framework sequence is as further defined herein.

[0291] More particularly, the Nanobody® ISV may be an immunoglobulin sequence having the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein: one or more amine groups at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering The acid residues were selected from the labeled residues mentioned in Table 2A below. Table 2A: Labeled residues in Nanobody® ISV Location Person V H 3 labeled residue 11 L, V; mainly L L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L 37 V, I, F; usually V f (1) , Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F (1) or Y 44 (8) G E. (3) , Q (3) , G (2) , D, A, K, R, L, P, S, V, H, T, N, W, M, I; Preferred G (2) ,E (3) or Q (3) ; most preferably G (2) or Q (3) 。 45 (8) L L (2) , R (3) , P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L (2) or R (3) 47 (8) W, Y f (1) , L (1) or W (2) , G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W (2) , L (1) or F (1) 83 R or K; usually R R, K (5) , T, E (5) , Q, N, S, I, V, G, M, L, A, D, Y, H; preferably K or R; most preferably K 84 A, T, D; mostly A P (5) , S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; preferably P 103 W W (4) , R (6) , G, S, K, A, M, Y, L, F, T, N, V, Q, P (6) , E, C; preferred W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108 L, M, or T; mostly L Q, L (7) , R, P, E, K, S, T, M, A, H; preferably Q or L (7) Notice: In particular, but not exclusively, in combination with KERE (SEQ ID NO: 103) or KQRE (SEQ ID NO: 104) at positions 43-46. Usually at position 44-47 as GLEW (SEQ ID NO: 105). Usually at positions 43-46 as KERE (SEQ ID NO: 103) or KQRE (SEQ ID NO: 104), for example at positions 43-47 as KEREL (SEQ ID NO: 106), KEREF (SEQ ID NO: 107 ), KQREL (SEQ ID NO: 108), KQREF (SEQ ID NO: 109), KEREG (SEQ ID NO: 110), KQREW (SEQ ID NO: 111) or KQREG (SEQ ID NO: 112). Alternatively, such as TERE (SEQ ID NO: 113) (for example TEREL (SEQ ID NO: 114)), TQRE (SEQ ID NO: 115) (for example TQREL (SEQ ID NO: 116)), KECE (SEQ ID NO: 116)), KECE (SEQ ID NO: : 117) (such as KECEL (SEQ ID NO: 118) or KECER (SEQ ID NO: 119)), KQCE (SEQ ID NO: 120) (such as KQCEL (SEQ ID NO: 121)), RERE (SEQ ID NO: 122) (eg REREG (SEQ ID NO: 123)), RQRE (SEQ ID NO: 124) (eg RQREL (SEQ ID NO: 125), RQREF (SEQ ID NO: 126) or RQREW (SEQ ID NO: 127) ), QERE (SEQ ID NO: 128) (eg QEREG (SEQ ID NO: 129)), QQRE (SEQ ID NO: 130), (eg QQREW (SEQ ID NO: 131), QQREL (SEQ ID NO: 132) or QQREF (SEQ ID NO: 133)), KGRE (SEQ ID NO: 134) (eg KGREG (SEQ ID NO: 135)), KDRE (SEQ ID NO: 136) (eg KDREV (SEQ ID NO: 137)) Equal sequences are also possible. Some other possible but less preferred sequences include eg DECKL (SEQ ID NO: 138) and NVCEL (SEQ ID NO: 139). Has GLEW (SEQ ID NO: 105) at positions 44-47 and KERE (SEQ ID NO: 103) or KQRE (SEQ ID NO: 104) at positions 43-46. Usually found in naturally occurring V HH Positions 83-84 of the domain serve as KP or EP. In particular, but not exclusively, in combination with GLEW (SEQ ID NO: 105) at positions 44-47. provided that when positions 44-47 are GLEW (SEQ ID NO: 105), (dehumanized) V HH Position 108 is always a Q in the sequence that also contains a W at 103 . The GLEW group also contains GLEW-like sequences at positions 44-47, such as GVEW (SEQ ID NO: 140), EPEW (SEQ ID NO: 141), GLER (SEQ ID NO: 142), DQEW (SEQ ID NO: 143), DLEW (SEQ ID NO: 144), GIEW (SEQ ID NO: 145), ELEW (SEQ ID NO: 146), GPEW (SEQ ID NO: 147), EWLP (SEQ ID NO: 148), GPER ( SEQ ID NO: 149), GLER (SEQ ID NO: 142) and ELEW (SEQ ID NO: 146).

[0292] In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the framework regions that are effective to prevent or reduce the binding of so-called "pre-existing antibodies" to the polypeptide . Such ISVs have been described in WO2015 / 173325, wherein (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 (iv) in (i) to (iii) In each case, the amino acid at position 11 is preferably V. polypeptide The immunoglobulin single variable domain may form part of a protein or polypeptide which may comprise or consist essentially of one or more (at least one) immunoglobulin single variable domain , and may optionally further comprise one or more other amino acid sequences (all optionally linked via one or more suitable linkers). The term "immunoglobulin single variable domain" may also encompass such polypeptides. The one or more immunoglobulin single variable domains may be used as binding units in such proteins or polypeptides which may optionally contain one or more other amine groups useful as binding units acid, in order to provide monovalent, multivalent or multispecific polypeptides of the invention, respectively (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 WO 1996 / 34103, WO 1999 / 23221 and WO 2010 / 115998).

[0294] The polypeptide may comprise or consist essentially of an immunoglobulin single variable domain, as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.

[0295] 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 ISVDs. Thus, the polypeptide may be "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", "heptavalent", "octavalent", " "nine-valent" and so on, that is, the polypeptide comprises or consists of two, three, four, five, six, seven, eight, nine and other ISVs respectively. In one embodiment, said multivalent ISV polypeptide is trivalent. In another embodiment, said multivalent ISV polypeptide is tetravalent. In yet another embodiment, said multivalent ISV polypeptide is pentavalent.

[0296] In one embodiment, the multivalent ISV polypeptide may also be multispecific. The term "multispecific" refers to binding to multiple different target molecules (also known as antigens). Therefore, the multivalent ISV polypeptide can be "bispecific", "trispecific", "tetraspecific", etc., that is, it can bind to two, three, four, etc. different target molecules, respectively. .

[0297] For example, the polypeptide may be bispecific-trivalent, such as a polypeptide comprising or consisting of three ISVs, wherein two ISVs bind to a first target and one ISV binds to a target different from the first target. Secondary target binding of the target. In another example, the polypeptide may be trispecific-tetravalent, such as a polypeptide comprising or consisting of four ISVs, wherein one ISV binds to a first target and two ISVs bind to a target different from the first target. A second target of the target binds, and an ISV binds a third target different from the first and second targets. In yet another example, the polypeptide can be trispecific-pentavalent, such as a polypeptide comprising or consisting of five ISVs, wherein two ISVs bind to the first target and two ISVs are different from the first target. A second target of one target binds, and an ISV binds a third target different from the first and second targets.

[0298] In one embodiment, the multivalent ISV polypeptide may also be multiparatopic. The term "multiparatopic" refers to binding to multiple different epitopes on the same target molecule (also known as an antigen). Thus, the multivalent ISV polypeptide may be "biparatopic", "triparatopic", etc., ie, may bind to two, three, etc. different epitopes on the same target molecule, respectively.

[0299] In another aspect, the invention comprises or consists essentially of one or more immunoglobulin single variable domains (or suitable fragments thereof) A polypeptide consisting of suitable fragments) may further comprise one or more other groups, residues, moieties or binding units. Such other groups, residues, moieties, binding units or amino acid sequences may or may not provide additional functions to the immunoglobulin single variable domain (and / or the polypeptide in which it is present), and may alter Or the properties of the immunoglobulin single variable domain may not be altered.

[0300] For example, such further groups, residues, moieties or binding units may be one or more additional amino acids such that the compound, construct or polypeptide is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, 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 selected from domain antibodies, amino acids suitable for use as domain antibodies, single domain antibodies, suitable for use as single domain Amino acids of antibodies, "dAbs", amino acids suitable for use as dAbs or Nanobodies.

[0301] Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not themselves have biological and / or pharmacological activity. For example, without limitation, such groups may be linked to said one or more immunoglobulin single variable domains so as to provide "derivatives" of said immunoglobulin single variable domains.

[0302] In another embodiment, the other residues are effective to prevent or reduce the binding of so-called "pre-existing antibodies" to the polypeptide. For this purpose, the polypeptides and constructs may contain a C-terminal extension (X)n (SEQ ID NO: 150) (wherein n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 ( Preferably 1 or 2, such as 1); and each X is independently selected from, preferably independently selected from alanine (A), glycine (G), extractine (V), leucine (L) or the (preferably naturally occurring) amino acid residues of isoleucine (I), for which reference is made to WO 2012 / 175741). Accordingly, the polypeptide may further comprise a C-terminal extension (X)n (SEQ ID NO: 151), wherein n is 1 to 5, such as 1, 2, 3, 4 or 5, and wherein X is a naturally occurring amine group Acid, preferably other than cysteine. In the above polypeptides, said one or more immunoglobulin single variable domains and said one or more groups, residues, moieties or binding units may be linked to each other directly and / or via one or more a suitable linker or spacer. For example, when the one or more groups, residues, moieties or binding units are amino acids, the linker may also be an amino acid such that the resulting polypeptide is a fusion protein or fusion polypeptide.

[0304] As used herein, the term "linker" refers to a peptide that fuses two or more ISVs together to form a single molecule. The use of linkers to link two or more (poly)peptides is well known in the art. Other exemplary peptide linkers are shown in Table 2B. One class of commonly used peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, and usually contain one or more repeats of a peptide motif such as GGGGS (SEQ ID NO: 154) motif (e.g., of formula (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 152), wherein n can be 1, 2, 3, 4, 5, 6, 7 or greater) . Some commonly used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 157), the 15GS linker (n=3) and the 35GS linker (n=7). See for example Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27 (10): 325-330). Table 2B: Linker sequences ("ID" refers to SEQ ID NO as used herein) name ID amino acid sequence 3A linker 153 AAA 5GS linker 154 GGGGS 7GS linker 155 SGGSGGS 8GS linker 156 GGGGSGGS 9GS linker 157 GGGGSGGGS 10GS linker 158 GGGGSGGGGS 15GS linker 159 GGGGSGGGGSGGGGS 18GS linker 160 GGGGSGGGGSGGGGSGGS 20GS linker 161 GGGGSGGGGSGGGGSGGGGS 25GS linker 162 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS linker 163 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS linker 164 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS linker 165 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 166 EPKSCDKTHTCPPCP 9GS-G1 hinge 167 GGGGSGGGSEPKSCDKTHTCPPCP Llama upper long hinge region 168 EPKTPKPQPAAA G3 hinge 169 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTTPPPPCPRCPEPKSCDTPPPCPRCP

[0305] In one aspect, the present disclosure also relates to such amino acid sequences and / or Nanobodies which can bind to and / or be directed against CD8 and which comprise CDR sequences generally as further defined herein; suitable fragments thereof; As well as polypeptides comprising or consisting essentially of one or more such Nanobodies and / or suitable fragments. In some aspects, the disclosure relates to a Nanobody having SEQ ID NO: 77. In particular, in certain aspects, this disclosure provides:

[0306] 1) an amino acid sequence directed against CD8 and having at least 80%, preferably at least 85% (such as 90% or 95% or higher) sequence identity with at least one amino acid sequence of SEQ ID NO: 77;

[0307] II) cross-blocking of the amino acid sequence of SEQ ID NO: 77 binding to CD8 and / or at least competing with the amino acid sequence of SEQ ID NO: 77 in combination with the amino acid sequence of CD8;

[0308] Such amino acid sequences may be as further described herein (and may for example be Nanobodies); and references of the disclosure comprising one or more such amino acid sequences (which may be as further herein described) Polypeptides, especially bispecific (or multispecific) polypeptides as described herein, and nucleic acid sequences encoding such amino acid sequences and polypeptides. Such amino acid sequences and polypeptides do not include any naturally occurring ligands.

[0309] In some embodiments, CD8 is derived from a mammal, such as a human. In one specific but non-limiting aspect, the disclosure relates to an amino acid sequence directed against CD8 comprising:

[0310] a) the amino acid sequence of SEQ ID NO: 77;

[0311] b) an amino acid sequence having at least 80% amino acid identity to at least one amino acid sequence of SEQ ID NO: 77, or

[0312] c) an amino acid sequence having 3, 2 or 1 amino acid difference with at least one amino acid sequence of SEQ ID NO: 77;

[0313] or any suitable combination thereof.

[0314] In some embodiments, Nanobodies directed against CD8 are disclosed that consist of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively). In some embodiments, in such Nanobodies:

[0315] (I) CDR1 comprises or consists essentially of the amino acid sequence of GSTFSDYG (SEQ ID NO: 100),

[0316] Or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity to GSTFSDYG (SEQ ID NO: 100), wherein (1) any amino acid substitution All are conservative amino acid substitutions; and / or (2) Compared with GSTFSDYG (SEQ ID NO: 100), the amino acid sequence only contains amino acid substitutions, and does not contain amino acid deletions or insertions; and / or selected from an amino acid sequence having 2 or only 1 amino acid difference with GSTFSDYG (SEQ ID NO: 100), wherein

[0318] Any amino acid substitution is a conservative amino acid substitution; and / or

[0319] Compared to GSTFSDYG (SEQ ID NO: 100), the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions.

[0320] (II) CDR2 comprises or consists essentially of the following amino acid sequence: the amino acid sequence of IDWNGEHT (SEQ ID NO: 101),

[0321] Or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or higher sequence identity to IDWNGEHT (SEQ ID NO: 101), wherein (1) any amino acid substitution All are conservative amino acid substitutions; and / or (2) Compared with IDWNGEHT (SEQ ID NO: 101), the amino acid sequence only contains amino acid substitutions, and does not contain amino acid deletions or insertions; and / or selected from an amino acid sequence having 2 or only 1 amino acid difference with IDWNGEHT (SEQ ID NO: 101), wherein

[0323] Any amino acid substitution is a conservative amino acid substitution; and / or

[0324] Compared to IDWNGEHT (SEQ ID NO: 101), the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions.

[0325] (III) CDR3 comprises or consists essentially of the amino acid sequence of AADALPYTVRKYNY (SEQ ID NO: 102), or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99% or greater sequence identity to AADALPYTVRKYNY (SEQ ID NO: 102), wherein (1) any amino acid substitution All are conservative amino acid substitutions; and / or (2) Compared with AADALPYTVRKYNY (SEQ ID NO: 102), the amino acid sequence only contains amino acid substitutions and does not contain amino acid deletions or insertions; and / or selected from an amino acid sequence having 2 or only 1 amino acid difference from AADALPYTVRKYNY (SEQ ID NO: 102), wherein

[0328] Any amino acid substitution is a conservative amino acid substitution; and / or

[0329] Compared to AADALPYTVRKYNY (SEQ ID NO: 102), the amino acid sequence contains only amino acid substitutions and no amino acid deletions or insertions.

[0330] A CD8 Nanobody as disclosed herein may comprise one, two or all three of the CDRs explicitly listed above. In some embodiments, the CD8 Nanobody comprises:

[0331] CDR1: GSTFSDYG (SEQ ID NO: 100), based on the IMGT name;

[0332] CDR2: IDWNGEHT (SEQ ID NO: 101), based on the IMGT name; and

[0333] CDR3: AADALPYTVRKYNY (SEQ ID NO: 102), based on the IMGT name.

[0334] In the Nanobodies of the disclosure comprising combinations of the above-mentioned CDRs, each CDR may be selected from the group having at least 80%, preferably at least 90%, more preferably at least 95% of the mentioned CDRs. , even more preferably CDR substitutions of amino acid sequences of at least 99% sequence identity; wherein (1) Any amino acid substitutions are preferably conservative amino acid substitutions; and / or

[0336] (2) Compared with the above one or more amino acid sequences, the amino acid sequence preferably only contains amino acid substitutions, and does not contain amino acid deletions or insertions; and / or selected from those having 3, 2 or only 1 (as indicated in the preceding paragraph) "amino acid differences" from one of the amino acid sequences above the mentioned one or more CDRs Amino acid sequence, where:

[0338] (1) Any amino acid substitutions are preferably conservative amino acid substitutions; and / or

[0339] (2) Compared with the above one or more amino acid sequences, the amino acid sequence preferably only contains amino acid substitutions, and does not contain amino acid deletions or insertions.

[0340] In one embodiment, the CD8 Nanobody is BDSn: Anti-CD8 BDSn Nb Sequence (CDR1, CDR2, CDR3 underlined, based on IMGT name):

[0341] EVQLVESGGGLVQAGGSLRLSCAASGSTFSDYGVGWFRQAPGKGREFVADIDWNGEHTSYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYCAADALPYTVRKYNYWGQGTQVTVSSGGCGGHHHHHH (SEQ ID NO: 77)

[0342] In some embodiments, the CD8 Nanobody of the disclosure is dosed at 10-5 to 10-12 moles / liter (M) or less, preferably 10-7 to 10-12 moles / liter (M). ) or less, more preferably from 10-8 to 10-12 moles / liter (M), and / or with a dissociation constant (KD) of at least 107M-1, preferably at least 108M-1, more preferably at least 109M-1 (e.g. at least 1012 M-1), especially binds to CD8 with a KD of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM (e.g., 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.

[0343] In general, it should be noted that 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 will be discussed in more detail below, Nanobodies can be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a protein encoding a naturally occurring VHH domain. (3) via "humanization" (described below) of a naturally occurring VHH domain or via expression of a nucleic acid encoding such a humanized VHH domain; (4) via "camelization" (described below) (described above) from a naturally occurring VH domain from any animal species, particularly a mammalian species (such as from a human), or via expression of a nucleic acid encoding such a camelized VH domain; (5) via, for example, Ward et al. ) "camelized" "domain antibodies" or "Dab" as described, or via expression of nucleic acids encoding such camelized VH domains; (6) using synthetic methods to prepare proteins, polypeptides, or other amino acid sequences (7) via the use of nucleic acid synthesis techniques to prepare nucleic acids encoding Nanobodies and then express the nucleic acids thus obtained; and / or (8) via any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to those skilled in the art based on the disclosure herein, and include, for example, those described in more detail below.

[0344] In some embodiments, the CD8 Nanobodies of the present disclosure do not have an amino acid sequence identical to that of a naturally occurring VH domain (such as an amino acid sequence from a naturally occurring VH domain from a mammal, particularly from a human). An amino acid sequence that is completely identical (ie, has a degree of 100% sequence identity) to it.

[0345] A class of CD8 Nanobodies of the present disclosure comprises an amino acid sequence corresponding to a naturally occurring VHH domain but has been "humanized" (i.e. via use in conventional 4-chain antibodies from humans (such as above One or more amino acid residues occurring at one or more corresponding positions in the VH domain as indicated) replace one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence base) amino acid sequence of nanobodies. It should be noted that such humanized CD8 Nanobodies of the present disclosure may be obtained in any suitable manner known per se (i.e. as indicated under points (1)-(8) above), and therefore are not It is not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring VHH domains as starting material.

[0346] Another class of CD8 Nanobodies of the present disclosure comprises a CD8 Nanobody having an amino acid sequence corresponding to a naturally occurring VH domain that has been "camelized" (i.e. via use in the VHH domain of a heavy chain antibody). Replacement of one or more amino acid residues in the amino acid sequence from a naturally occurring VH domain of a conventional 4-chain antibody by one or more amino acid residues occurring at one or more corresponding positions of Amino acid sequence nanobodies. This can be done in a manner known per se, which should be clear to a person skilled in the art, eg on the basis of the further description below. Reference is also made to WO 94 / 04678. This camelization may preferentially occur at amino acid positions present at the VH-VL interface and at so-called camelid marker residues (see also eg WO 94 / 04678), also mentioned below. In some embodiments, the VH domain or sequence used as the starting material or starting point for generating or designing a camelized Nanobody is a VH sequence from a mammal, such as a human VH sequence. It should be noted that such camelized Nanobodies of the present disclosure may be obtained in any suitable manner known per se and are therefore not strictly limited to having used polypeptides comprising naturally occurring VH domains as starting material obtained peptides.

[0347] For example, both "humanization" and "camelization" can be carried out by providing a nucleotide sequence encoding such a naturally occurring VHH domain or VH domain, respectively, and then using the per se known One or more codons in the nucleotide sequence are changed in such a way that the new nucleotide sequence encodes the humanized or camelized Nanobodies of the disclosure, respectively, and then behaves thereby in a manner known per se Nucleotide sequences obtained in order to provide the desired Nanobodies. Alternatively, the desired amino acid sequences of humanized or camelized Nanobodies of the disclosure can be designed based on the amino acid sequences of naturally occurring VHH domains or VH domains, respectively, and then used per se De novo synthesis by known peptide synthesis techniques. Furthermore, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, one can design a nucleotide sequence encoding a desired humanized or camelized Nanobody, and then use the known nucleic acid synthesis techniques, the nucleotide sequence thus obtained can then be expressed in a manner known per se in order to provide the desired Nanobody. Other suitable means and techniques for obtaining Nanobodies and / or nucleotide sequences and / or nucleic acids encoding them (from (amino acid sequences of) naturally occurring VH domains or preferably VHH domains and / or starting from the nucleotide sequence and / or nucleic acid sequence encoding it) will be clear to those skilled in the art, and may, for example, include in a suitable manner from a naturally occurring VH domain (such as a one or more amino acid sequences and / or nucleotide sequences from a naturally occurring VHH domain (such as one or more FRs or CDRs) acid sequence and / or nucleotide sequence in order to provide a Nanobody (nucleotide sequence or nucleic acid encoding it). Also provided are compounds and constructs, particularly proteins and polypeptides, which comprise or consist essentially of at least one such amino acid sequence and / or Nanobody (or a suitable fragment thereof) of the disclosure, and any Optionally further comprising one or more other groups, residues, moieties or binding units. In some embodiments, such other groups, residues, moieties, binding units or amino acid sequences may or may not be directed towards said amino acid sequence and / or Nanobody (and / or the compound in which it is present). or constructs) provide other functions and may or may not change the amino acid sequence and / or properties of the Nanobody.

[0349] The disclosure also encompasses any polypeptide of the disclosure that has been glycosylated at one or more amino acid positions, generally depending on the host used to express the polypeptide. The polypeptide may comprise the amino acid sequence of a CD8 Nanobody of the disclosure, at its amino terminus, at its carboxy terminus, or at both its amino terminus and at its carboxyl terminus, combined with at least one Other amino acid sequence fusions. Such other amino acid sequences may comprise at least one other Nanobody, so as to provide a polypeptide comprising at least two (such as three, four or five) Nanobodies, wherein said Nanobodies may optionally be obtained via One or more linker sequences (as defined herein) are linked. A polypeptide comprising a CD8 Nanobody of the disclosure and one or more additional Nanobodies is a multivalent polypeptide. In a multivalent polypeptide, the two or more Nanobodies may be the same or different. For example, said 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 and / or ˙ may be directed against different antigens; ˙ or combinations thereof. Thus, a bivalent polypeptide, for example: ˙ may comprise two identical Nanobodies; ˙ may comprise a first Nanobody directed against a first part or epitope of an antigen and either directed against the same part or epitope of said antigen or directed against all a second Nanobody to another part or epitope of said antigen; or may comprise a first Nanobody directed against a first antigen and a second Nanobody directed against a second antigen different from said first antigen; and Trivalent polypeptides of the invention, for example: ˙ may comprise three identical or different Nanobodies against the same or different parts or epitopes of the same antigen; two identical or different Nanobodies and a third Nanobody directed against a second antigen different from said first antigen; or ˙ may comprise a first Nanobody directed against a first antigen, directed against said second antigen A second Nanobody to a second antigen of an antigen and a third Nanobody to a third antigen different from said first and second antigens.

[0350] CD8 Nanobodies and polypeptides as disclosed herein may also be introduced and expressed 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). For this purpose, the nucleotide sequence encoding a CD8 Nanobody or polypeptide as disclosed herein may be introduced into said cells or tissues in any suitable manner, for example as such (e.g. using liposomes) or after the They are inserted behind a suitable gene therapy vector (eg derived from a retrovirus such as an adenovirus or a parvovirus such as an adeno-associated virus). As those skilled in the art should also be clear, the nucleic acid of the present invention or a suitable gene therapy vector encoding the nucleic acid can be administered to the patient or the patient's specific cells or specific tissues or organs. and / or carry out this gene therapy in situ; or suitable cells (usually obtained from the body of the patient to be treated, such as transplanted lymphocytes, bone marrow aspirate or tissue can be treated in vitro) with the nucleotide sequence of the present invention biopsy) and then appropriately (re)introduced into the patient's body. All of these can be performed using gene therapy vectors, techniques and delivery systems well known to those skilled in the art, Culver, K. W., "Gene Therapy", 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.; Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994) , 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, Gene Ther. 5 ( 1998), 692-699; Nabel, Ann. N.Y. Acad. Sci.: 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996) , 714-716; WO 94 / 29469; WO 97 / 00957; US Patent No. 5,580,8591; US ​​Patent No. 5,589,5466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) have been described in the art. bit performance.

[0351] Accordingly, there is also provided a nucleic acid sequence encoding a CD8 Nanobody as described herein, as well as expression constructs and host cells comprising said nucleic acid sequence.

[0352] Also disclosed are methods of using the CD8 Nanobodies and polypeptides of the disclosure.

[0353] In some embodiments, polypeptides comprising CD8 Nanobodies can be used in lipid nanoparticles of the 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 disorder or disease in a subject in need thereof. In some embodiments, such conditions or diseases include, but are not limited to, cancer, infection, immune disorder, autoimmune disease.

[0354] In some embodiments, a polypeptide comprising a CD8 Nanobody can be used in an imaging agent. In some embodiments, the imaging agent allows for the detection of human CD8, a specific biomarker found on the surface of T cell subsets for diagnostic imaging of the immune system. Imaging of CD8 allows detection of T cell localization in vivo. Changes in T cell localization can reflect the progression of the immune response and may occur over time as a result of various therapeutic treatments or even disease states. In some embodiments, it is used to image T cell localization for immunotherapy.

[0355] In addition, CD8 plays a role in initiating downstream signaling pathways important for initiating cytolytic T cells that function to clear viral pathogens and provide immunity against 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 via a T cell receptor and enhance a subject's ability to clear viral pathogens and respond to tumor antigens. Thus, in some embodiments, the antigen binding constructs provided herein can be agonists and can activate CD8 targeting. II. Ionizable Cationic Lipids

[0356] Provided herein are ionizable cationic lipids that can be used to generate lipid nanoparticle compositions to facilitate delivery of payloads (e.g., nucleic acids, such as DNA or RNA, such as mRNA) placed therein to cells, For example mammalian cells, such as immune cells. The ionizable cationic lipids have been engineered to enable intracellular delivery of nucleic acids (eg, mRNA) to the cytoplasmic compartment of target cell types and rapid degradation into nontoxic components. The complex functionality of the ionizable cationic lipids is via a relationship between the chemistry and geometry of the ionizable lipid headgroup, the hydrophobic "acyl tail" group, and the linker linking the headgroup and the acyl tail group. to facilitate the interaction between them. Typically, the pKa of the ionizable amine headgroup is designed to be in the range of 6-8, such as between 6.2-7.4 or between 6.5-7.1, so that it is stable under acidic formulation conditions (e.g., pH 4 - pH 5.5 ), remain strongly cationic at physiological pH (7.4), and remain cationic in early and late endosomal compartments (eg, pH 5.5 - pH 7). The acyl tail group plays a key role in the fusion of the lipid nanoparticles to the endosomal membrane and membrane destabilization via structural perturbation. The three-dimensional structure of the acyl tail (determined by its length, degree of unsaturation, and location) and the relative sizes of the head and tail groups are thought to play a role in promoting membrane fusion and thus within lipid nanoparticles. function in body escape, a key requirement for cytoplasmic delivery of nucleic acid payloads. The linker linking the head group and the acyl tail group is designed to degrade via physiologically ubiquitous enzymes (eg, esterase or protease) or via acid-catalyzed hydrolysis.

[0357] In one aspect, the present invention provides a compound represented by Formula I: (Formula I), or a salt thereof, wherein: R1 and R2 are independently C1-3 alkyl, or R1 and R2 form together with a nitrogen atom Optionally substituted piperidinyl or morpholinyl; Y is selected from -O-, -OC(O)-, -OC(S)- and -CH2-; X1, X2, X3 and X4 are hydrogen, or X1 and X2 or X3 and X4 independently form a side oxygen group together; n is 0 or 3; o and p are independently an integer selected from 2-6; wherein the compound is not a compound selected from the following: ,,,,, and , or its salts.

[0358] In certain embodiments, o and p may be two. In some embodiments, o and p can be 3. In other embodiments, o and p can be four. In some embodiments, o and p may be 5. In other embodiments, o and p may be 6.

[0359] In certain embodiments, X1 and X2 may together form a pendant oxy group, and X3 and X4 together form a pendant oxy group. In other embodiments, X1, X2, X3, and X4 can be hydrogen.

[0360] In certain embodiments, Y may be selected from -O-, -OC(O)-, OC(S)-, and -CH2-. For example, in certain embodiments, Y can be -O-. In certain embodiments, Y can be -OC(O)-. In certain embodiments, Y can be -CH2-. In certain embodiments, Y can be -OC(S)-.

[0361] In certain embodiments, R1 and R2 can independently be C1-3 alkyl. In other embodiments, R1 and R2 may be -CH3. In certain embodiments, R1 and R2 are -CH2CH3. In certain embodiments, R1 and R2 are C3 alkyl.

[0362] In some embodiments, n may be zero. In other embodiments, n may be three.

[0363] Also provided herein is a compound represented in part by Formula II: (Formula II), or a salt thereof, wherein: R1 and R2 are independently C1-3 alkyl, or R1 and R2 together with a nitrogen atom form an optional Substituted piperidinyl or morpholinyl; Y is selected from -O-, -OC(O)-, -OC(S)- and -CH2-; X1, X2, X3 and X4 are hydrogen, or X1 and X2 or X3 and X4 together form a side oxygen group; n is 0-4; o is 1 and r is an integer selected from 3-8, or o is 2 and r is an integer selected from 1-8, p is 1 and s is An integer selected from 3-8, or p is 2 and s is an integer selected from 1-8, wherein, when both o and p are 1, r and s are independently 4, 5, 7 or 8, And when o and p are both 2, r and s are independently 1, 2, 4 or 5.

[0364] In certain embodiments, X1 and X2 can together form a pendant oxy group, and X3 and X4 can together form a pendant oxy group. In other embodiments, X1, X2, X3, and X4 can be hydrogen.

[0365] In certain embodiments, Y may be selected from -O-, -OC(O)-, and -CH2-. For example, in certain embodiments, Y can be -O-. In certain embodiments, Y can be -OC(O)-. In certain embodiments, Y can be -CH2-. In certain embodiments, Y can be -OC(S)-.

[0366] In certain embodiments, R1 and R2 can be independently C1-3 alkyl. In other embodiments, R1 and R2 may be -CH3. In certain embodiments, R1 and R2 may be -CH2CH3. In some embodiments, R1 and R2 can be C3 alkyl. In certain embodiments, R1 and R2 are taken together with a nitrogen atom to form optionally substituted piperidinyl.

[0367] In some embodiments, n may be zero. In other embodiments, n may be three.

[0368] Provided herein, in part, is a compound selected from the group consisting of: ,,,,,, and, or a salt thereof.

[0369] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0370] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0371] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0372] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0373] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0374] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0375] Provided herein, in part, are compounds of the formula: , or a salt thereof.

[0376] In certain embodiments, the compound is a compound of Formula III: (Formula III), or a salt thereof, wherein: R1 and R2 are independently C1-3 alkyl, or R1 and R2 are together with a nitrogen atom Form optionally substituted piperidinyl or morpholinyl; Y is selected from -O-, -OC(O)-, -OC(S)- and -CH2-; X1, X2, X3 and X4 are hydrogen, or X1 together with X2 or X3 and X4 to form a pendant oxy group; and n is an integer selected from 0-4.

[0377] In certain embodiments, X1 and X2 can together form a pendant oxy group, and X3 and X4 can together form a pendant oxy group. In other embodiments, X1, X2, X3, and X4 can be hydrogen.

[0378] In certain embodiments, Y may be selected from -O-, -OC(O)-, and -CH2-. For example, in certain embodiments, Y can be -O-. In certain embodiments, Y can be -OC(O)-. In certain embodiments, Y can be -CH2-. In certain embodiments, Y can be -OC(S)-.

[0379] In certain embodiments, R1 and R2 can independently be C1-3 alkyl. In other embodiments, R1 and R2 may be -CH3. In certain embodiments, R1 and R2 may be -CH2CH3. In some embodiments, R1 and R2 can be C3 alkyl. In certain embodiments, R1 and R2 are taken together with a nitrogen atom to form optionally substituted piperidinyl.

[0380] In some embodiments, n may be zero. In other embodiments, n may be three.

[0381] Also provided herein is a compound of the formula: , or a salt thereof. Scheme 1 - Synthetic scheme for the preparation of lipids of formula (I)

[0382] Compounds of Formula I can be prepared, for example, according to Scheme 1. Conversion of hydroxy-functional protected propylene glycols to the corresponding dimethylamino-functional ethers (Y = pendant oxy) or esters (Y = O-C(O)). The formation of ether linkages is due to the reaction of alkyl halides with alcohols in the presence of tertiary butylammonium iodide / NaOH in THF at 80 ºC. Formation of ester bonds utilizes treatment of acid-functional dimethylamine with alcohol under carbodiimide activation (DCM, EDC, DIEPA, DMAP). Diol deprotection yields vicinal diol intermediates, which are subsequently converted to the corresponding ether linkages or ester linkages via treatment with TBAI / NaOH and bromide or via carbodiimide-mediated carboxylic acid activation to form ester linkages, respectively of diacyl lipids. Scheme 2 - Synthetic Scheme for the Preparation of Lipid Compositions of Formula (II)

[0383] Compounds of formula II can be prepared, for example, according to Scheme 2. The synthetic procedure is outlined above for Scheme 1; however, in Scheme 2, diunsaturated acyl or monounsaturated acyl groups can be used to obtain lipids of formula II.

[0384] 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: , or in some embodiments, the ionizable cationic lipid is not Dlin-MC3-DMA. III. Lipid-Immune Cell Targeting Molecule Conjugates

[0385] As discussed herein, the 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 achieved through the use of one or more lipids described herein. Furthermore, targeting can be enhanced via the inclusion of targeting groups on the solvent-accessible surface of LNP particles. For example, targeting groups can include members of specific binding pairs (eg, antibody-antigen pairs, ligand-receptor pairs, etc.). In certain embodiments, the targeting moiety is an antibody. Targeting can be performed, for example, via the use of the lipid-immune cell targeting moiety conjugates described herein.

[0386] Optionally, the targeting moiety is an antibody fragment without an Fc component. Previous attempts to target circulating immune cells with LNPs have employed whole antibodies (WO 2016 / 189532 Al). Liposomes or lipid-based particles conjugated with whole antibodies are cleared from circulation faster due to Fc engagement, reducing their likelihood of reaching target cells of interest (Harding et al. (1997) Biochim Biophys. Acta 1327, 181-192; Sapra et al. (2004) Clin Cancer Res 10, 1100-1111; Aragnol et al., (1986) Proc Natl Acad Sci USA 83, 2699-2703). Liposomes targeted with antibody fragments retain their long-circulating properties, such as targeting to EGFR (Mamot et al., (2005) Cancer Res 65, 11631-11638), ErbB2 (Park et al. (2002) Clin Cancer Res 8, 1172-1181) or liposomes of EphA2 (Kamoun et al., 2019 Nat. Biomed. Eng 3, 264-280). Alternatively, lipid-based vectors can be prepared using micellar insertion methods that allow insertions that are inefficient when conjugating whole IgG (Ishida et al. (1999) FEBS Lett. 460, 129-133) or require direct insertion of intact LNPs. Antibody conjugates are almost quantitatively incorporated after their manufacture alone (Nellis et al. (2005) Biotechnol Prog 21, 221-232) compared to the conjugation done on (WO 2016 / 189532 Al). scFv, Fab or VHH fragments can also be directly conjugated to activated PEG-lipids to form insertable conjugates.

[0387] In certain embodiments, the targeting moiety may be a surface-bound antibody or surface-bound antigen-binding fragment thereof, which may allow modulation of cell targeting specificity. This is particularly useful as highly specific antibodies can be raised against the epitope of interest at the desired targeting site. In one example, multiple different antibodies can be incorporated and presented on the surface of the LNP, where each antibody binds to a different epitope on the same antigen or a different epitope on a different antigen. Such methods can increase the avidity and specificity of targeted interactions with specific target cells.

[0388] A targeting group or combination of targeting groups can be selected based on the desired localization, function or structural characteristics of a given target cell. For example, to target T cells, T cell populations, or T cell subpopulations, one or more antibodies or antigen-binding fragments or antigen-binding derivatives thereof that target T cells (eg, via T cell surface antigens) can be selected. Exemplary T cell surface antigens include, but are not limited to, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD39, CD69, CD103, CD137, CD45, T cell receptor (TCR) beta, TCR-alpha, TCR- α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, GL7, TLR2, TLR4, TLR5, and IL-15 receptor. To target NK cells or populations of NK cells, one or more antibodies, antigen-binding fragments or antigen-binding derivatives thereof that target NK cells (eg, via NK cell surface antigens) can be selected. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, CD337 / NKP30. To target a B cell or population of B cells, one or more antibodies, antigen-binding fragments or antigen-binding derivatives thereof that target B cells (eg, via a B cell antigen) can be selected. Exemplary B cell antigens include, but are not limited to, CD19 (for all B cells except plasma cells), CD19, CD25, and CD30 (for primed B cells), CD27, CD38, CD78, CD138, and CD319 (for plasma cells), CD20, CD27, CD40, CD80, and PDL-2 (for memory cells), Notch2, CD1, CD21, and CD27 (for marginal zone B cells), CD21, CD22, and CD23 (for follicular B cells), and CD1, CD5 , CD21, CD24, and TLR4 (for regulatory B cells).

[0389] In certain embodiments, targeting can be performed, for example, via the use of lipid-immune cell targeting moiety conjugates described herein. Exemplary lipid-immune cell targeting moiety conjugates may include compounds of formula IV, [lipid] - [optional linker] - [immune cell targeting moiety, such as a T cell targeting molecule, e.g., Anti-CD2 antibody, anti-CD3 antibody, anti-CD7 antibody or anti-CD8 antibody] (Formula IV).

[0390] In some embodiments, the immune cell targeting moiety is a polypeptide, and the lipid is attached to the N-terminus, C-terminus, or anywhere in the middle of the polypeptide.

[0391] In certain embodiments, the targeting moiety or targeting molecule is a T cell targeting agent (e.g., an antibody) that binds a T cell antigen selected from: CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor , CD11a, TLR2, TLR4, TLR5, IL-7 receptor or IL-15 receptor. In certain embodiments, the T cell antigen can be CD2, and the targeting moiety can be, for example, an anti-CD2 antibody. In certain embodiments, the T cell antigen can be CD3, and the targeting moiety can be, for example, an anti-CD3 antibody. In certain embodiments, the T cell antigen can be CD4, and the targeting moiety can be, for example, an anti-CD4 antibody. In certain embodiments, the T cell antigen can be CD5, and the targeting moiety can be, for example, an anti-CD5 antibody. In certain embodiments, the T cell antigen can be CD7, and the targeting moiety can be, for example, an anti-CD7 antibody. In certain embodiments, the T cell antigen can be CD8, and the targeting moiety can be, for example, an anti-CD8 antibody. In certain embodiments, the T cell antigen can be TCR β, and the targeting moiety can be, for example, an anti-TCR β antibody. In some embodiments, the antibodies are human or humanized antibodies.

[0392] An exemplary CD2 binding agent may be an antibody selected from: 9.6 (https: / / academic.oup.com / intimm / article / 10 / 12 / 1863 / 744536), 9-1 (https: / / academic.oup.com / intimm / article / 10 / 12 / 1863 / 744536), .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 6849258B1), Sipilzumab / MEDI-507 (US Patent 6849258B1 / en), 35.1 (ATCC HB-222), OKT11 (ATCC CRL-8027), RPA-2.1 (PCT Publication WO 2020023559A1), 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 binding agent comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody selected from the group consisting 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). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 ( R&D Systems), MAB1856 (R&D Systems), PAB30359 (Abnova Corporation), and 10299-1 (Abnova Corporation).

[0393] Exemplary CD2 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following strains: 9.6, 9-1, TS2 / 18.1.1, Lo-CD2b, Lo-CD2a, BTI-322, Hep Zizumab, 35.1, OKT11, RPA-2.1, SQB-3.21, LT2, TS1 / 8, UT329, 4F22, OX-34, UQ2 / 42, MU3, U7.4, NFN-76, or MOM-181-4- F(E).

[0394] An exemplary CD3 binding agent (CD3γ / δ / ε, CD3γ, CD3δ, CD3γ / ε, CD3δ / ε, or CD3ε) can be an antibody selected from the group consisting of: 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-AP (CD3δ, Invitrogen) and antigen-binding fragments thereof. In certain embodiments, the binding agent comprises a VH domain and a VL domain of an antibody selected from the group consisting of: 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). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: 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).

[0395] Exemplary CD3 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following strains: hsp34, OKT-3, UCHT1, 38.1, HIT3a, RFT8, SK7, BC3, SP34-2, HU291, TRX4, Catumaxomab, 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 20200299409A1), REGN5458 (US Patent Application 20200024356A1), blinatumomab (https: / / go.drugbank.com / drugs / DB09052 / polypeptide_sequences.fasta). In some embodiments, the conjugate comprises a Fab, wherein the Fab comprises (a) a heavy chain fragment comprising the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence comprising SEQ ID NO: 2 or 3 light chain fragments.

[0396] An exemplary CD4 binding agent may be an antibody selected from the group consisting of Ibalizumab (https: / / www.genome.jp / dbget-bin / www_bget?D09575), AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Abnova Corporation), CAL4 (Abcam) and antigen-binding fragments thereof. In certain embodiments, the binding agent comprises a VH domain and a VL domain 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). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 ( Affinity Biosciences), PAB31115 (Abnova Corporation), and CAL4 (Abcam).

[0397] Exemplary CD4 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following strains: 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 ilbalizumab.

[0398] An exemplary CD5 binding agent may be an antibody selected from the group consisting of He3, MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Abeam), CD5 / 54 / F6 (Abcam), 65152 (Proteintech) and antigen-binding fragments thereof. In some embodiments, the binding agent comprises a VH domain and a VL domain of an antibody selected from the group consisting of: MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473 + CD5 / 54 / F6 (Abcam), CD5 / 54 / F6 (Abcam), and 65152 (Proteintech). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 ( R&D Systems), C5 / 473 + CD5 / 54 / F6 (Abcam), CD5 / 54 / F6 (Abcam), and 65152 (Proteintech).

[0399] Exemplary CD5 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following strains: zolimomab, 5D7, L17F12, and UCHT2, 1D8, 3I21, 4H10, 8J23, 504, 4H2 , 5G2, 8G8, 6M4, 2E3, 4E24, 4F10, 7J9, 7P9, 8E24, 6L18, 7H7, 1E7, 8J21, 7I11, 8M9, 1P21, 2H11, 3M22, 5M6, 5H8, 7I19, 1A2, 8E15, 8C10, 3P16 , 4F3, 5M24, 5O24, 7B16, 1E8, 2H16, BLa1, 1804, DK23, Cris1, MEM-32, H65, 4C7, OX-19, Leu-1, 53-7.3, 4H8E6, T101, EP2952, D-9 , H-3, HK231, N-20, Y2 / 178, H-300, CD5 / 54 / F6, Q-20, CC17, MOM-18539-S(P) or MOM-18885-S(P).

[0400] An exemplary CD7 binding agent may be an antibody selected from the group consisting of MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), NBP2-38440 ( Novus Biologicals) and antigen-binding fragments thereof. In certain embodiments, the binding agent comprises a VH domain and a VL domain of an antibody selected from MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abcam), 1G10D8 (Proteintech), NBP2- 32097 (Novus Biologicals) and NBP2-38440 (Novus Biologicals). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 ( Abcam), 1G10D8 (Proteintech), NBP2-32097 (Novus Biologicals), and NBP2-38440 (Novus Biologicals).

[0401] Exemplary CD7-binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following strains: TH-69, 3Afl1, T3-3A1, 124-1D1, 3A1f, CD7-6B7, or VHH6.

[0402] Exemplary CD8 (CD8α, CD8α / α, CD8α / β, or CD8β) binding agents may be antibodies selected from the group consisting of 2.43 (Invitrogen), Du CD8-1 (CD8α, Invitrogen), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abcam), C8 / 144B (CD8α, Novus Biologicals), YTS105.18 (CD8α, Novus Biologicals), TRX2 (https: / / patents.justia .com / patent / 20170198045) and antigen-binding fragments thereof. In certain embodiments, the binding agent comprises a VH domain and a VL domain of an antibody selected from the group consisting of: 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). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: 2.43 (Invitrogen), Du CD8-1 (CD8α, Invitrogen) , 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abcam), C8 / 144B (CD8α, Novus Biologicals) and YTS105.18 (CD8α, Novus Biologicals).

[0403] Exemplary CD8 binding agents may also be selected from antibodies or antibody fragments employing the CDRs of the following strains: 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, wherein the Fab comprises 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.

[0404] Exemplary CD137 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: 4B4-1, P566, or Urelumab. Exemplary CD28-binding agents can be selected from antibodies or antibody fragments employing the CDRs of the clone TAB08. Exemplary CD45-binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: BC8, 9.4, 4B2, Tu116, or GAP8.3. Exemplary CD18 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: 1B4, TS1 / 18, MEM-48, YFC118-3, TA-4, MEM-148 or R3-3, 24. Exemplary CD11a binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: MHM24 or Efalizumab. Exemplary IL-2 receptor binding agents can be selected from antibodies or antibody fragments employing the CDRs of the following strains: YTH 906.9HL, IL2R.1, BC96, B-B10, 216, MEM-181, ITYV, MEM-140, ICO-105, Daclizumab, or IL2 or IL2 fragments. Exemplary IL-15R-binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: JM7A4 or OTI3D5, or from IL15 or IL15 fragments. Exemplary TLR2-binding agents can be selected from antibodies or antibody fragments employing the CDRs of the following strains: JM22-41, TL2.1, 11G7, or TLR2.45. Exemplary TLR4-binding agents can be selected from antibodies or antibody fragments employing the CDRs of the following strains: HTA125 or 76B357-1. Exemplary TLR5-binding agents can be selected from antibodies or antibody fragments employing the CDRs of the following strains: 85B152-5 or 9D759-2. Exemplary GL7-binding agents can be selected from antibodies or antibody fragments that employ the CDRs of the clone GL7.

[0405] Exemplary PD1-binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: MIH4, J116, J150, OTIB11, OTI17B10, OTI3A1, or OTI16D4. Additionally, exemplary anti-PD-1 antibodies are described in, e.g., U.S. Pat. 87, 8,735,553 and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals) and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, eg, in US Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 ( Bristol Myers Squibb Co.).

[0406] Exemplary CTLA-4 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: ER4.7G.11 [7G11], OTI9G4, OTI9F3, OTI3A5, A3.4H2.H12, 14D3, OTI3A12, OTI1A11, OTI1E8, OTI3B11, OTI3D2, OTI10C8, OTI2E9, OTI6F1, OTI7D3, OTI85B, OTI12C6. Exemplary anti-CTLA-4 antibodies are described in U.S. Pat. 3, 8,318,916, 8,017,114, 8,784,815, and 8,883,984; International (PCT) Publication No. WO 98 / 42752, WO 00 / 37504 and WO 01 / 14424; and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0407] An exemplary TCR β binding agent may be an antibody selected from the group consisting of: H57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα / β, Abcam), E6Z3S (TRBC1 / TCRβ, Cell Signaling Technology) and its antigen-binding fragment. In certain embodiments, the binding agent comprises a VH domain and a VL domain of an antibody selected from H57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα / β, Abcam), and E6Z3S ( TRBC1 / TCRβ, Cell Signaling Technology). In certain embodiments, the binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of the VH and VL sequences of an antibody selected from Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C and Lesk A M, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (See, MacCallum R M et al., (1996) J. MOL. BIOL. 262: 732-745) or any other CDR determination method known in the art: H57-597 (Invitrogen), 8A3 (Novus Biologicals), R73 (TCRα / β, Abcam) and E6Z3S (TRBC1 / TCRβ, Cell Signaling Technology).

[0408] Exemplary CD137 binding agents may be selected from antibodies or antibody fragments employing the CDRs of the following strains: 4B4-1, P566, or Urelumab.

[0409] In some embodiments, the immune cell targeting moiety comprises an antibody selected from the group consisting of Fab, F(ab')2, Fab'-SH, Fv and scFv fragments. In some embodiments, the antibodies are human or humanized antibodies. In some embodiments, the immune cell targeting moiety comprises a Fab or immunoglobulin single variable domain, such as a Nanobody. In some embodiments, the immune cell targeting moiety comprises a Fab that does not contain native interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment comprising a C233S substitution and / or a light chain fragment comprising a C214S substitution, numbered according to Kabat. In some embodiments, the immune cell targeting moiety comprises a Fab comprising one or more non-native interchain disulfide bonds. In some embodiments, the interchain disulfide bond is located between two non-native cysteine ​​residues on the light chain segment and the heavy chain segment, respectively. For example, in some embodiments, the Fab comprises a heavy chain fragment comprising a F174C substitution and / or a light chain fragment comprising a S176C substitution, numbered according to Kabat. In some embodiments, the Fab comprises a heavy chain fragment comprising F174C and C233S substitutions and / or a light chain fragment comprising S176C and C214S substitutions, according to Kabat numbering. In some embodiments, the immune cell targeting moiety comprises a C-terminal cysteine ​​residue. In some embodiments, said immune cell targeting moiety comprises a Fab comprising a cysteine ​​at the C-terminus of said heavy or light chain fragment. In some embodiments, said Fab further comprises one or more amino acids between the heavy chain of said Fab and said 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 of the Fab and the C-terminal cysteine. amino acids. 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 The chain constant domains are linked via one or more interchain disulfide bonds, and wherein the immune cell targeting moiety further comprises a single chain that is linked to the C-terminus of the light chain constant domain via an amino acid linker. variable fragment (scFv). In some embodiments, the Fab antibody is DS Fab, NoDS Fab, bDS Fab, bDS Fab-ScFv, as shown in FIG. 47 .

[0410] In some embodiments, the immune cell targeting moiety comprises an immunoglobulin single variable domain, such as a Nanobody (eg, VHH). In some embodiments, the Nanobody comprises a cysteine ​​at the C-terminus. In some embodiments, said Nanobody further comprises a spacer comprising one or more amino acids between said VHH domain and said C-terminal cysteine. In some embodiments, the spacer comprises one or more glycine residues, such as two glycine residues. In some embodiments, the immune cell targeting moiety comprises two or more VHH domains. In some embodiments, the two or more VHH domains are linked via an amino acid linker. In some embodiments, the amino acid linker comprises one or more glycine and / or serine residues (eg, one or more repeats of the sequence GGGGS). In some embodiments, the immune cell targeting moiety comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain, and wherein the antibody CH1 structure domain and the antibody light chain constant domain are linked via one or more disulfide bonds (eg, interchain disulfide bonds). In some embodiments, the immune cell targeting moiety comprises a VHH domain linked to an antibody CH1 domain, and wherein the antibody CH1 domain is linked to an antibody light chain constant domain via one or more disulfide bonds . In some embodiments, said CH1 domain comprises F174C and C233S substitutions and said light chain constant domain comprises S176C and C214S substitutions, numbering according to Kabat. In some embodiments, the antibody is a ScFv, VHH, 2xVHH, VHH-CH1 / empty Vk, or VHH1-CH1 / VHH-2-Nb bDS, as shown in FIG. 47 .

[0411] An exemplary targeting moiety may have an amine sequence as set forth below: Anti-CD3 hSP34-Fab Sequence: hSP34 Heavy Chain (HC) Sequence (SEQ ID NO: 1 ): EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYY CVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC hSP34-mlam light chain (LC) sequence (mouse lambda) (SEQ ID NO: 2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVT QGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADSS SP34-hlam LC (human lambda) (SEQ ID NO: 3): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSN RWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS Anti-CD3 Hu291-Fab sequence: Hu291 HC (SEQ ID NO: 4): QVQLVQSGAEVKKPGAS VKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSLGTQTYICN VNHKPSNTKVDKKVEPKSSDKTHTC Hu291 LC (SEQ ID NO: 5): MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD8 TRX2-Fab Sequence: TRX2 HC (SEQ ID NO: 6): QVQLVESGGGVVQPGRSLRLSCAASGTFFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC TRX2 LC (SEQ ID NO: 7 ): DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD8 OKT8-Fab sequence: OKT8 HC (SEQ ID NO: 8): QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC OKT8 LC (SEQ ID NO: 9): DIVMTQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFLTISSLQPEDFATYYCQQHNENPLTFGQGT KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD4 ibalizumab-Fab sequence: Ibalizumab HC (SEQ ID NO: 10): QVQLQQSGPEVVKPGASVK MSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSLGTQTYICNVNHKPS NTKVDKKVEPKSSDKTHTC Ibalizumab LC (SEQ ID NO: 11): DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD5 He3-Fab Sequence: He3 HC (SEQ ID NO: 12): EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQIN SLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSSDKTHTC He3 LC (SEQ ID NO: 13): DIQMTQSPSSLSASVGDRVTIT CRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISLQYEDFGIYYCQQYDESPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGES anti-CD7 TH-69-Fab sequence: TH-69 HC (SEQ ID NO: 14): EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC TH-69 LC (SEQ ID NO: 15): DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-CD2 TS2 / 18.1-Fab sequence: TS2 / 18.1 HC (SEQ ID NO: 16): EVQLVESGGGLVM PGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSLGTQTYICNVNHK PSNTKVDKKVEPKSSDKTHTC TS2 / 18.1 LC ( SEQ ID NO: 17): DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHNFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD2 9.6-Fab sequence: 9.6 HC (SEQ ID NO: 18): QVQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC 9.6 LC (SEQ ID NO: 19): NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFLTISSVQPEDLAVYYCHQ YLSSHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD2 9-1-Fab Sequence: 9-1 HC (SEQ ID NO: 20): QVQLQQPGTELVRP GSSVKLSCKASGYTFTSYWVNWVKQRPDQGLEWIGRIDPYDSETHYNQKFTDKAISTIDTSSNTAYMQLSTLTSDASAVYYCSRSPRDSSTNLADWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSLGTQTYICNVN HKPSNTKVDKKVEPKSSDKTHTC 9-1 LC (SEQ ID NO: 21): DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES mutOKT8-Fab sequence: mutOKT8 HC (SEQ ID NO: 22): QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSSNTAYMELSSLRSEDTAVYYCGR GAGAYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSSDKTHTC mutOKT8 LC (SEQ ID NO: 23): DIVMTQSPSSLSASVGDRVTITCRTRSISAALAWY QEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES. .

[0412] In certain embodiments, the targeting moiety or immune cell targeting moiety (e.g., T cell targeting agent, B cell targeting agent or NK cell targeting agent) can be The alcohol (PEG) linker is covalently coupled to the lipid.

[0413] In other embodiments, the lipid used to generate the conjugate may be selected from the group consisting of distearoyl-phosphatidylethanolamine (DSPE): , dipalmitoyl-phosphatidylethanolamine (DPPE): , dimyristyl Dimethyl-phosphatidylethanolamine (DMPE): , Distearyl-glyceroyl-phosphoglycerol (DSPG): , Dimyristyl-glycerol (DMG): , Distearyl-glycerol (DSG): , and N-palmitoyl - Sphingosine (C16-ceramide).

[0414] The immune cell targeting moiety can be covalently coupled to the lipid either directly or via a linker (eg, a polyethylene glycol (PEG) containing linker). In certain embodiments, the PEG is PEG 1000, PEG 2000, PEG 3400, PEG 3000, PEG 3450, PEG 4000 or PEG 5000. In certain embodiments, the PEG is PEG 2000.

[0415] In some embodiments, the lipid-immune cell targeting moiety conjugate is used at 0.001-0.5 molar percentage, 0.001-0.3 molar percentage, 0.002-0.2 molar percentage, 0.01-0.1 molar percentage , 0.1-0.3 molar percent, or a range of 0.1-0.2 molar percent present in the lipid blend.

[0416] In certain embodiments, the lipid-immune cell targeting agent conjugate comprises DSPE, a PEG component and a targeting antibody. In certain embodiments, the antibody is a T cell targeting agent, such as an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD7 antibody, an anti-CD8 antibody, or an anti-TCR beta antibody.

[0417] Exemplary lipid-immune cell targeting group conjugates comprise DSPE and PEG 2000, eg as described in Nellis et al. (2005) BIOTECHNOL. PROG. 21, 205-220. Exemplary conjugates comprise the structure of Formula V, wherein the scFv represents an engineered antibody binding site that binds to a target of interest. In certain embodiments, the engineered antibody binding site binds to any of the targets described 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.

[0418] Examples of compounds of formula (V) are shown below: (Formula V). It is contemplated that the scFv in Formula V may be replaced by a whole antibody or an antigenic fragment thereof (eg, Fab). Another example of a compound of formula (VI) is shown below: (Formula VI), the production of which is described in Nellis et al. (2005) supra or in US Patent No. 7,022,336. It is contemplated that the Fab in Formula VI may be replaced by an intact antibody or an antigenic fragment thereof (eg, (Fab')2 fragment) or an engineered antibody binding site (eg, scFv).

[0419] Other lipid-immune cell targeting group conjugates are described, e.g., in U.S. Pat. Targeting group for NK cell surface antigen) replacement.

[0420] In certain embodiments, the lipid component of an exemplary conjugate of Formula IV can be based on an ionizable cationic lipid described herein, eg, an ionizable cationic lipid of Formula I, Formula II, or Formula III. For example, exemplary ionizable cationic lipids may be selected from: ,,,,,, and, or salts thereof.

[0421] In certain embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0422] In some embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0423] In other embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0424] In certain embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0425] In some embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0426] In other embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0427] In certain embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0428] In some embodiments, an exemplary ionizable cationic lipid can be a compound of the formula: , or a salt thereof.

[0429] In certain embodiments, the lipid-based conjugate of Formula III may comprise: wherein the scFv represents an engineered antibody binding to a target described above (e.g., CD2, CD3, CD7, or CD8) location.

[0430] In certain embodiments, the lipid blend may further comprise free PEG-lipid in order to reduce the amount of non-specific binding via 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 selected from PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristyl-phosphatidylethanolamine (PEG-DMPE), N- (Methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), 1,2-dimyristyl-rac-glycero-3-methyl Polyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleyl-rac-glycerol, methoxypoly Ethylene glycol (DOG-PEG), 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DSG), N-palmitoyl-sphingosine-1-{succinyl[ Methoxy(polyethylene glycol)] (PEG-ceramide), DSPE-PEG-cysteine ​​or derivatives thereof, all having an average PEG length of 2000, 3400 or 5000 between 2000-5000 . The final composition may contain a mixture of two or more of these pegylated lipids. In certain embodiments, the LNP composition comprises a mixture of PEG-lipids having myristyl and stearyl chains.

[0431] In certain embodiments, the PEG-lipid derivative has a hydroxyl or carboxylic acid end group at the PEG terminus.

[0432] The lipid-immune cell targeting group conjugates may be incorporated into LNPs as described below, e.g. LNP. It is contemplated that in certain embodiments, LNPs containing such lipid-immune cell targeting groups may contain ionizable cationic lipids as described herein, or as described, for example, in U.S. Patent Nos. 10,221,127, 10,653,780 or U.S. Published Application No. Cationic lipids in US2018 / 0085474, US2016 / 0317676, International Publication No. WO2009 / 086558, or Miao et al. (2019) NATURE BIOTECH 37:1174-1185 or Jayaraman et al. (2012) ANGEW CHEM INT. 51: 8529-8533 . In other embodiments, the cationic lipid can be selected from the ionizable cationic lipids set forth in Table 1. Table 1. (Lipid 1) (Lipid 2) (Lipid 3) (Lipid 6) (Lipid 4) (Lipid 5) (Lipid 7) (Lipid 8) (Lipid 14) Lipid 15 (Lipid 9) (Lipid 10) (Lipid 11) (Lipid 12) (Lipid 13)

[0433] The LNP can be formulated using the methods and other components described in the following sections below. IV. Lipid Nanoparticle Compositions

[0434] The present invention provides lipid nanoparticle (LNP) compositions comprising a lipid blend comprising an ionizable cationic lipid as described herein and / or a lipid as described herein. Plasma-immune cell targeting agent conjugates. In certain embodiments, the lipid blend may comprise ionizable cationic lipids described herein in combination with sterols, neutral phospholipids, PEG-lipids, and lipid-immune cell targeting group conjugates. one or more of .

[0435] In certain embodiments, the ionizable cationic lipids described herein can be present at 30-70 molar percent, 30-60 molar percent, 30-50 molar percent, 40-70 molar percent, 40 - a range of 60 molar percent, 40-50 molar percent, 50-70 molar percent, 50-60 molar percent, or about 30 molar percent, about 35 molar percent, about 40 molar percent, about 45 A molar percent, about 50 molar percent, about 55 molar percent, about 60 molar percent, about 65 molar percent, or about 70 molar percent is present in the lipid blend. Sterols

[0436] In certain embodiments, the lipid blend of lipid nanoparticles may comprise a sterol component, such as one or more sterols selected from the group consisting of cholesterol, fucosterol, Beta-sitosterol, ergosterol, campesterol, stigmasterol, stigmasterol, brassicasterol. In certain embodiments, the sterol is cholesterol.

[0437] The sterol (for example, cholesterol) can be expressed in 20-70 molar percent, 20-60 molar percent, 20-50 molar percent, 30-70 molar percent, 30-60 molar percent, 30 -50 molar percent, 40-70 molar percent, 40-60 molar percent, 40-50 molar percent, 50-70 molar percent, 50-60 molar percent, or about 20 molar percent, about 25 molar percent, about 30 molar percent, about 35 molar percent, about 40 molar percent, about 45 molar percent, about 50 molar percent, about 55 molar percent, about 60 molar percent, or about 65 Molar percentages are present in the lipid blend. neutral phospholipids

[0438] In certain embodiments, the lipid blend of lipid nanoparticles can comprise one or more neutral phospholipids. The neutral phospholipid may be selected from phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphate Choline (DSPC), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPC), sphingomyelin ( SM).

[0439] Other neutral phospholipids may be selected from distearoyl-phosphatidylethanolamine (DSPE), dimyristyl-phosphatidylethanolamine (DMPE), distearoyl-glycerol-phosphocholine (DSPC), dioleoyl Dioleoyl-glycero-phosphoethanolamine (DOPE), Dilinoleoyl-glycero-phosphocholine (DLPC), Dimyristyl-glycero-phosphocholine (DMPC), Dioleoyl-glycero-phosphocholine (DOPC) , dipalmitoyl-glyceroyl-phosphorylcholine (DPPC), eicosyl-glyceroyl-phosphorylcholine (DUPC), palmitoyl-oleyl-glycero-phosphorylcholine (POPC), octadecenyl- Glycero-Phosphocholine, Oleoyl-Cholesterol Hemisuccinyl-Glycero-Phosphocholine, Hexadecyl-Glycero-Phosphocholine, Dilinolenoyl-Glycero-Phosphocholine, Diarachidonoyl-Glycero-3 - phosphorylcholine, docosahexaenoyl-glycero-phosphocholine or sphingomyelin.

[0440] The neutral phospholipid can be 1-10 mole percentage, 1-15 mole percentage, 1-12 mole percentage, 1-10 mole percentage, 3-15 mole percentage, 3-12 mole percentage percent, 3-10 molar percent, 4-15 molar percent, 4-12 molar percent, 4-10 molar percent, 4-8 molar percent, 5-15 molar percent, 5-12 molar percent , 5-10 molar percent, 6-15 molar percent, 6-12 molar percent, 6-10 molar percent range, or about 1 molar percent, about 2 molar percent, about 3 molar percent, about 4 molar percent, about 5 molar percent, about 6 molar percent, about 7 molar percent, about 8 molar percent, about 9 molar percent, about 10 molar percent, about 11 molar percent, about 12 A molar percent, about 13 molar percent, about 14 molar percent, or about 15 molar percent is present in the lipid blend. PEG-lipid

[0441] The lipid blend of lipid nanoparticles can include one or more PEG or PEG-modified lipids. Such species may alternatively be referred to as pegylated lipids. PEG lipids are lipids modified with polyethylene glycol. As noted above, when lipid-immune cell targeting groups are included in the lipid blend, free PEG-lipids can be included in the lipid blend to reduce or Non-specific binding via targeting groups is eliminated.

[0442] PEG lipids may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified - Modified diacylglycerols and PEG-modified dialkylglycerols. For example, the PEG lipids can be PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-diglycol Oleoyl-Glyceryl-Phosphatidylethanolamine (PEG-DLPE), PEG-Dimyristyl-Phosphatidylethanolamine (PEG-DMPE), PEG-Dipalmitoyl-Phosphatidylethanolamine (PEG-DPPE), PEG-Distearyl Acylglycerol (PEG-DSG), PEG-Diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-Ceramide, PEG-Distearyl-Glycerol-Phosphoglycerol (PEG-DSPG), PEG-dioleyl-glycerol-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or PEG- Distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

[0443] In certain embodiments, the blend may contain free PEG-lipid, which may be selected from PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG , such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristyl-glycerol (PEG-DMG), PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE), and PEG-dimyristyl Acyl-phosphatidylethanolamine (PEG-DMPE). In some embodiments, the free PEG-lipid comprises diacylphosphatidylcholine comprising dipalmitoyl (C16) chains or distearoyl (C18) chains.

[0444] The PEG-lipid can be 1-10 molar percentage, 1-8 molar percentage, 1-7 molar percentage, 1-6 molar percentage, 1-5 molar percentage, 1-4 molar percentage Ear percentage, 1-3 molar percentage, 2-8 molar percentage, 2-7 molar percentage, 2-6 molar percentage, 2-5 molar percentage, 2-4 molar percentage, 2-3 molar percentage A range of percentages, or about 1 molar percent, about 2 molar percent, about 3 molar percent, about 4 molar percent, or about 5 molar percent is present in the lipid blend. In some embodiments, the PEG-lipid is free PEG-lipid.

[0445] In some embodiments, the PEG-lipid can be expressed in 0.01-10 molar percentage, 0.01-5 molar percentage, 0.01-4 molar percentage, 0.01-3 molar percentage, 0.01-2 molar percentage Percentage, 0.01-1 molar percentage, 0.1-10 molar percentage, 0.1-5 molar percentage, 0.1-4 molar percentage, 0.1-3 molar percentage, 0.1-2 molar percentage, 0.1-1 molar percentage , 0.5-10 mole percentage, 0.5-5 mole percentage, 0.5-4 mole percentage, 0.5-3 mole percentage, 0.5-2 mole percentage, 0.5-1 mole percentage, 1-2 mole percentage, A range of 3-4 molar percent, 4-5 molar percent, 5-6 molar percent, or 1.25-1.75 molar percent is present in the lipid blend. In some embodiments, the PET-lipid can be about 0.5 molar percent, about 1 molar percent, about 1.5 molar percent, about 2 molar percent, about 2.5 molar percent of the lipid blend percent, about 3 molar percent, about 3.5 molar percent, about 4 molar percent, about 4.5 molar percent, about 5 molar percent, or about 5.5 molar percent. In some embodiments, the PEG-lipid is free PEG-lipid.

[0446] In some embodiments, the lipid anchor hook length of the PEG-lipid is C14 (as in PEG-DMG). In some embodiments, the lipid anchor length of the PEG-lipid is C16 (as in DPG). In some embodiments, the lipid anchor length of the PEG-lipid is C18 (as in PEG-DSG). In some embodiments, the backbone or head group of the PEG-lipid is diacylglycerol or phosphoethanolamine. In some embodiments, the PEG-lipid is free PEG-lipid.

[0447] The LNPs of the present disclosure may comprise one or more free PEG-lipids not conjugated to an immune cell targeting group and PEG-lipids conjugated to an immune cell targeting group. In some embodiments, the free PEG-lipid comprises the same or a different lipid than the lipid in the lipid-immune cell targeting group conjugate. Immune Cell Targeting Group Conjugates

[0448] In certain embodiments, the lipid blend can also include a lipid-immune cell targeting group conjugate as described above in Section III.

[0449] The lipid-immune cell targeting group conjugate can be expressed in 0.001-0.5 mole percentage, 0.001-0.1 mole percentage, 0.01-0.5 mole percentage, 0.05-0.5 mole percentage, 0.1-0.5 mole percentage Molar percent, 0.1-0.3 molar percent, 0.1-0.2 molar percent, 0.2-0.3 molar percent range, about 0.01 molar percent, about 0.05 molar percent, about 0.1 molar percent, about 0.15 molar percent, About 0.2 molar percent, about 0.25 molar percent, about 0.3 molar percent, about 0.35 molar percent, about 0.4 molar percent, about 0.45 molar percent, or about 0.5 molar percent present in the lipid blend middle.

[0450] In addition to the lipids present in the lipid blend, the LNP composition may further comprise a payload, such as the payload described below. In certain embodiments, the payload is a nucleic acid, such as DNA or RNA, such as mRNA, transfer RNA (tRNA), microRNA or small interfering RNA (siRNA).

[0451] In certain embodiments, the number of nucleotides in the nucleic acid is from about 400 to about 6000. Production of lipid nanoparticles

[0452] Typically, the LNPs are produced through the use of rapid mixing via an orbital vortex or via microfluidic mixing. Orbital vortex mixing was accomplished via rapid addition of the ethanol solution of the lipids to the aqueous solution of the nucleic acid of interest followed by immediate vortexing at 2,500 rpm. Microfluidic mixing is achieved via mixing aqueous and organic flows at controlled flow rates in microfluidic channels using, for example, NanoAssemblr devices featuring optimized mixing chamber geometries and microfluidic chips (Precision Nanosystems, Vancouver, BC). .

[0453] In certain embodiments, the resulting LNP composition comprises a lipid blend containing, for example, from about 40 molar percent to about 60 molar percent of one or more ionizable cations described herein Lipid, one or more sterols from about 35 molar percent to about 50 molar percent, one or more neutral lipids from about 5 molar percent to about 15 molar percent, and from about 0.5 A molar percent to about 5 molar percent of one or more PEG-lipids. Physical properties of lipid nanoparticles

[0454] The characteristics of the LNP composition may depend on the components contained in the lipid nanoparticle (LNP) composition, their absolute or relative amounts. Characteristics may also vary depending on the preparation method and conditions of the LNP composition.

[0455] LNP compositions can be characterized via a variety of methods. For example, microscopy (eg, transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of LNP compositions. Dynamic light scattering or potentiometry (eg, potentiometric titration) can be used to measure the zeta potential. Dynamic Light Scattering can also be used to determine subtleties. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Urcestershire, UK) can also be used to measure various characteristics of LNP compositions such as fineness, polydispersity index and zeta potential. RNA encapsulation efficiency was determined via a combination of methods relying on RNA binding dyes (ribogreen, cybergreen for determining the fraction of dye-accessible RNA) and LNP de-formulation followed by HPLC analysis of total RNA content.

[0456] In some embodiments, the LNP has a 1-250 nm, 1-200 nm, 1-150 nm, 1-100 nm, 50-250 nm, 50-200 nm, 50-150 nm, 50 - Average diameter in the range of 100 nm, 75-250 nm, 75-200 nm, 75-150 nm, 75-100 nm, 100-250 nm, 100-200 nm, 100-150 nm. In certain embodiments, the LNP composition may have a particle size of about 1 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, Average diameter of 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.

[0457] Alternatively or additionally, the LNP composition may have an Polydispersity index in the range of 0.08-0.5, 0.08-0.4, 0.08-0.3, 0.08-0.2, 0.1-1, 0.1-0.75, 0.1-0.5, 0.1-0.4, 0.1-0.3, 0.1-0.2. In certain embodiments, the polydispersity index is in the range of 0.1-0.25, 0.1-0.2, 0.1-0.19, 0.1-0.18, 0.1-0.17, 0.1-0.16, or 0.1-0.15.

[0458] Alternatively or additionally, the LNP composition may 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. The zeta potential can vary with changes in pH. Thus, in certain embodiments, the LNP composition may have a pH 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 may have a zeta potential of about -30 mV to about +5 mV or about -20 mV to about +15 mV at pH 7.4. V. Payload

[0459] The LNP composition can comprise an agent, such as a nucleic acid molecule, for delivery to a cell (eg, immune cell) or tissue (eg, a cell (eg, immune cell) or tissue of a subject).

[0460] The LNP compositions of the present invention may comprise nucleic acids, eg, DNA or RNA, such as mRNA, tRNA, microRNA, siRNA or dicer substrate siRNA. It is contemplated that nucleic acids may contain naturally occurring components, such as naturally occurring bases, sugars, or linking groups (e.g., phosphodiester linking groups); or may contain non-naturally occurring components or modifications (e.g., thioester connecting group). For example, the nucleic acid can be synthesized to contain base, sugar, linker modifications known to those skilled in the art. Furthermore, the nucleic acid can be linear or circular, or have any desired configuration. The LNP composition can include multiple nucleic acid molecules (eg, multiple RNA molecules), which can be the same or different.

[0461] In certain embodiments, the payload is mRNA. In certain embodiments, a particular LNP composition may contain many mRNA molecules, which may be the same or different. In certain embodiments, one or more LNP compositions comprising 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 may encode a receptor, such as a chimeric antigen receptor (CAR), for use in, for example, immune disorders, inflammatory disorders or cancer. In addition, the mRNA may encode an antigen for use in a therapeutic or prophylactic vaccine, for example, for treating or preventing infection by a pathogen (e.g., a microbial or viral pathogen), or for reducing or ameliorating an infection directly or indirectly caused by such an infection. indirect side effects.

[0462] In certain embodiments, the LNP composition may include one or more other components including, but not limited to, one or more pharmaceutically acceptable excipients, hydrophobic small molecules, therapeutic agents, carbohydrates, polymers, permeability-enhancing molecules, and surface-altering agents.

[0463] In some embodiments, the wt / wt ratio of the lipid component to the 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 the lipid component to the 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.

[0464] In certain embodiments, the encapsulation efficiency of the payload (eg, mRNA) in the lipid nanoparticles is at least 50%. In certain embodiments, the encapsulation efficiency is at least 80%, at least 90%, or greater than 90%. RNA payload

[0465] In certain embodiments, the RNA payload is an mRNA, tRNA, microRNA or siRNA payload.

[0466] In certain embodiments, the lipid nanoparticle composition is optimized for delivery of RNA (eg, mRNA) to target cells for translation within the cells. The mRNA can be naturally or non-naturally occurring mRNA. An mRNA may include one or more modified nucleobases, nucleosides or nucleotides.

[0467] The nucleobase can be selected from the non-limiting group consisting of: adenine, guanine, uracil, cytosine, 7-methylguanine, 5-methylcytosine, 5-hydroxymethyl Cytosine, thymine, pseudouracil, dihydrouracil, hypoxanthine, and xanthine.

[0468] Nucleosides of mRNA are combinations of sugar molecules (e.g., 5- or 6-carbon sugars, such as pentose, ribose, arabinose, xylose, glucose, galactose, or deoxygenated derivatives thereof) and nucleobases compound of. Nucleosides can be classical nucleosides (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine glycosides) or analogs thereof, and may include one or more substitutions or modifications.

[0469] Nucleotides of mRNA are those containing nucleosides and phosphate groups or alternative groups (e.g., boronic acid phosphate, phosphorothioate, phosphoroselenoate, phosphonate, alkyl, amidate, and glycerol) compound of. Nucleotides can be classical nucleotides (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine and thymidine monophosphate) or analogs thereof, and may include one or more substitutions or modifications including, but not limited to, alkyl, aryl, halo, pendant oxy, hydroxy, alkoxy and / or thio substitution ; one or more fused or ring-opened rings; oxidation of nucleobases, sugars and / or phosphates or replacement components; and / or reduction. Nucleotides may include one or more phosphate or substitution groups. For example, a nucleotide can include a nucleoside and a triphosphate group. "Nucleoside triphosphates" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) may refer to classical nucleoside triphosphates or analogs or derivatives thereof, and may include The one or more substitutions or modifications.

[0470] The RNA may include 5' untranslated regions, 3' untranslated regions and / or coding or translation sequences. An mRNA can comprise any number of base pairs, including tens, hundreds or thousands of base pairs. Any number (eg, all, some or none) of the nucleobases, nucleosides or nucleotides may be substituted, modified or otherwise non-naturally occurring analogs of the classical species. In certain embodiments, all of a particular nucleobase type can be modified. For example, all cytosines in the mRNA can be 5-methylcytosine.

[0471] In certain embodiments, an mRNA may include a 5' cap structure, a chain-terminating nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal.

[0472] A cap structure or cap species is a compound comprising two nucleoside moieties connected via a linker, and may be selected from naturally occurring caps, non-naturally occurring caps or cap analogs. Cap species may include one or more modified nucleoside and / or linker moieties. For example, a natural mRNA cap can include a guanine nucleotide at its 5' position via a triphosphate bond and a guanine (G) nucleotide methylated at the 7 position, such as m7G(5')ppp( 5')G, usually written as m7GpppG. Cap species can also be anti-reverse cap analogs. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m7Gpppm7G, m73'dGpppG, and m2702'GppppG.

[0473] Alternatively or additionally, the mRNA may include chain terminating nucleosides. For example, chain-terminating nucleosides may include those nucleosides that are deoxygenated at the 2' and / or 3' positions of their sugar groups. Such species may 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'-Dideoxythymidine.

[0474] Alternatively or additionally, the mRNA may comprise a stem-loop, such as a histone stem-loop. A stem-loop may comprise 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, a stem loop can comprise 4, 5, 6, 7 or 8 nucleotide base pairs. The stem-loop can be located in any region of the mRNA. For example, the stem-loop may be located within, before or after an untranslated region (5' or 3' untranslated region), a coding region, or a polyA sequence or tail.

[0475] Alternatively or additionally, the mRNA may comprise a polyA sequence and / or a polyadenylation signal. The polyA sequence may consist entirely or predominantly of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail located near the 3' untranslated region of the mRNA.

[0476] The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The polypeptide encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell. In some embodiments, the mRNA can encode an antibody, enzyme, growth factor, hormone, cytokine, viral protein (eg, viral capsid protein), antigen, vaccine, or receptor. In some embodiments, the mRNA can encode an engineered receptor (such as a CAR) or be used in a therapeutic vaccine (for example, a cancer vaccine) or a prophylactic vaccine (for example, to reduce the risk of microbial or viral pathogen infection or Antigens used in vaccines that minimize severity). In some embodiments, said mRNA encodes a polypeptide capable of modulating an immune response in said immune cell. In some embodiments, said mRNA encodes a polypeptide capable of reprogramming said immune cell. In some embodiments, the mRNA encodes a synthetic T cell receptor (synTCR) or a chimeric antigen receptor (CAR).

[0477] Lipid compositions can be tailored for one or more specific applications or targets. For example, LNP compositions can be designed to deliver mRNA to a particular cell, tissue, organ or system or group thereof (eg, the renal system) of the mammalian body. Physicochemical properties of LNP compositions can be altered in order to increase selectivity for specific target sites within a subject. For example, subtlety can be adjusted based on the fenestration size of different organs. The mRNA included in the LNP composition can also depend on one or more desired delivery targets. For example, mRNA can be selected for a particular indication, condition, disease or disorder, and / or for delivery to a particular cell, tissue, organ or system or group thereof (eg, local or specific delivery).

[0478] The amount of mRNA in the lipid composition can depend on the size, sequence and other characteristics of the mRNA. The amount of mRNA in the LNP can also depend on the size, composition, desired target, and other characteristics of the LNP composition. The relative amounts of mRNA and other elements (eg, lipids) can also vary. The amount of mRNA in the LNP composition can be measured, for example, using absorption spectroscopy (eg, UV-Vis spectroscopy).

[0479] In some embodiments, the one or more mRNAs, lipids and polymers and their amounts can be selected to provide a specific N:P ratio (possibly positively charged lipid or polymer amine ( N = ratio of nitrogen) groups to negatively charged nucleic acid phosphate (P) groups). The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in the one or more lipids to the number of phosphate groups in the mRNA. In general, lower N:P ratios are preferred. The N:P ratio can depend on the particular lipid and its pKa. In certain embodiments, the mRNA and the LNP composition and / or relative amounts thereof can be selected to provide a N of from about 1:1 to about 30:1 or from about 1:1 to about 20:1 : P ratio. 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 may be from about 2:1 to about 5:1. In certain embodiments, the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is from about 4:1 to about 8:1. For example, the N:P ratio can be about 4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, about 5.0:1, about 5.1:1, about 5.2 :1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.6:1, about 5.7:1, about 6.0:1, about 6.5:1 or about 7.0:1.

[0480] The amount of mRNA in the nanoparticle composition can depend on the size, sequence and other characteristics of the mRNA. The amount of mRNA in a nanoparticle composition can also depend on the size, composition, desired target, and other characteristics of the nanoparticle composition. The relative amounts of mRNA and other elements (eg, lipids) can also vary. In some embodiments, the wt / wt ratio of the lipid component to mRNA in the nanoparticle composition may be from about 5:1 to about 50:1, such as 5:1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 and 50:1. For example, the wt / wt ratio of the 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, for example, using absorption spectroscopy (eg, UV-Vis spectroscopy).

[0481] The encapsulation efficiency of mRNA describes the amount of mRNA that is encapsulated or otherwise associated with a lipid composition after preparation relative to the initial amount provided. Encapsulation efficiency is desirably high (eg, 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 decomposition of the LNP composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free mRNA in solution. For the LNP composition of the invention, the encapsulation efficiency of mRNA may be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In certain embodiments, the encapsulation efficiency can be at least 80%. VI. Method of Formulation and Delivery

[0482] The LNP compositions of the present invention may be formulated in whole or in part as pharmaceutical compositions. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for formulating and manufacturing pharmaceutical compositions and medicaments are available, eg, in Remington's (2006) supra. Conventional excipients and auxiliary ingredients may be used in any pharmaceutical composition of the invention, unless any conventional excipient or auxiliary ingredient may be incompatible with one or more components of the LNP composition of the invention. An excipient or auxiliary ingredient may be incompatible with a component of an LNP composition if its combination is likely to result in any undesired biological or otherwise deleterious effect.

[0483] In some embodiments, one or more excipients or auxiliary ingredients may constitute greater than 50% of the total mass or volume of a pharmaceutical composition comprising an LNP composition of the invention. For example, the 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 use in humans and for veterinary use, eg, by the US Food and Drug Administration. In certain embodiments, the excipients are pharmaceutical grade. In certain embodiments, the excipient complies with the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia and / or International Pharmacopoeia.

[0484] The relative amounts of the one or more lipids or LNPs, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in the pharmaceutical composition will depend on the amount of the subject being treated. Identity, body size and / or condition and further vary according to the route of administration of the composition.

[0485] Lipid compositions and / or pharmaceutical compositions comprising one or more LNP compositions can be administered to any subject, including those that can benefit from the administration of nucleic acids such as RNA (e.g., mRNA, tRNA or siRNA) Delivery to a human patient of a therapeutic effect provided by one or more specific cells, tissues, organs or systems or groups thereof (eg, the renal system). Although the descriptions of LNP compositions and pharmaceutical compositions including LNP compositions provided herein are primarily directed to compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to any other mammal. It is understood that compositions suitable for administration to humans are modified in order to render the compositions suitable for administration to various animals.

[0486] Pharmaceutical compositions according to the present disclosure may be prepared, packaged and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete quantity of pharmaceutical composition that contains a predetermined quantity of active ingredient (eg, payload).

[0487] The pharmaceutical compositions of the present invention can be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, the pharmaceutical compositions of the present invention can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules , lozenges, 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.

[0488] 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, liquid dosage forms can also contain inert diluents commonly used in the industry, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoic acid Benzyl esters, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, Fatty acid esters of polyethylene glycol and sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and / or perfuming agents.

[0489] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing agents, 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. Acceptable vehicles and solvents that may be employed include 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 find use in the preparation of injectables.

[0490] Injectable formulations can be sterilized, for example, by filtration through bacteria-retaining filters, and / or incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water prior to use. sterile water or other sterile injectable media. other components

[0491] Additionally, it is contemplated that the pharmaceutical composition may include one or more components other than those described above.

[0492] The pharmaceutical composition may also include one or more permeability enhancer molecules, carbohydrates, polymers, therapeutic agents, surface altering agents, or other components. The permeability enhancer molecule can be a molecule described, for example, in US Patent Application Publication No. 2005 / 0222064. Carbohydrates can include monosaccharides (eg, glucose) and polysaccharides (eg, glycogen and its derivatives and analogs).

[0493] The pharmaceutical composition may also contain surface-altering agents including, for example, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyloctadecyl-bromide ammonium chloride), sugars or sugar derivatives (e.g., cyclodextrins), polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytics (e.g., acetylcysteine, mugwort ( mugwort), bromelain, papain, clerodendrum, bromhexine, carbocisteine, iprazone, mesna, ambroxol, sobrirol, domiorol, letoxol Gelsolin, stilonine, tiopronin, gelsolin, thymosin beta 4, dornase alfa, neticosine, and erdosteine) and DNase (eg, rhDNase). Surface-altering agents can be placed within and / or on the surface of the compositions described herein.

[0494] In addition to these components, pharmaceutical compositions containing the LNP compositions of the present invention may include any material useful in pharmaceutical compositions. For example, the pharmaceutical composition may include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as but not limited to one or more solvents, dispersion media, diluents, dispersion aids, suspension aids , granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives and other kinds. Excipients such as waxes, butters, coloring agents, coatings, flavoring and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see eg, Remington's (2006) supra).

[0495] Dispersants may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pomace, agar, bentonite, cellulose, and wood products, natural sponge, cation exchange resin, calcium carbonate, silicate, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxy Methylcellulose, Croscarmellose Sodium (Crosscarmellose), Methylcellulose, Pregelatinized Starch (Starch 1500), Microcrystalline Starch, Water Insoluble Starch, Carmellose Calcium , magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0496] Surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (such as bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols ( such as stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carboxyl Boomers (such as carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulosic organisms (such as sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (such as polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene dehydrated Sorbitan [TWEEN® 60], Polyoxyethylene sorbitan monooleate [TWEEN® 80], Sorbitan monopalmitate [SPAN® 40], Sorbitan monostearate [SPAN® 60] , sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [ MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), poly Oxyethylene ethers (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, oil Potassium Oleate, Ethyl Oleate, Oleic Acid, Ethyl Laurate, Sodium Lauryl Sulfate, PLURONIC® F 68, POLOXAMER® 188, Cetrimonium Bromide, Cetylpyridinium Chloride, Benzalkonium Chloride, Docusate Sodium and / or combinations thereof.

[0497] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, Sodium bisulfite, sodium metabisulfite and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, Tartaric acid and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chlorine Xylenol, cresol, ethanol, glycerol, hexetidine, mirenide, phenol, phenoxyethanol, phenethyl 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 sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethyl alcohol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenols, chlorobutanol, parabens, 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, tocopheryl acetate, deferoxamine mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, lauryl sulfate Sodium (SLS), Sodium Lauryl Ether Sulfate (SLES), Sodium Bisulfite, Sodium Metabisulfite, Potassium Sulphite, Potassium Metabisulfite.

[0498] Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, glucose Calcium heptonate, calcium gluconate, d-gluconate, calcium glycerophosphate, calcium lactate, calcium lacturonate, propionic acid, calcium levylpropionate, valeric acid, dibasic calcium phosphate, phosphoric acid, tribasic Calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, lemon Sodium phosphate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, sulfamate buffer (eg HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water , isotonic saline, Ringer's solution, ethanol, and / or combinations thereof.

[0499] In certain embodiments, the lipid nanoparticle compositions and formulations thereof are suitable for intravenous, intramuscular, intradermal, subcutaneous, intraarterial, intratumoral or via inhalation administration. 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. However, it should be understood that the total daily usage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0500] The specific therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for any particular patient will depend on a variety of factors, including the severity of the disorder being treated, if any and identity; the mRNA or mRNAs employed; the particular composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion; duration of treatment; drugs used in combination or concomitantly with the particular pharmaceutical composition employed; and similar factors well known in the medical art. VII. Method

[0501] This disclosure provides methods of delivering a payload to a target cell or tissue (e.g., a target cell or tissue of a subject) as well as LNPs for use in such methods or pharmaceutical combinations containing said LNPs thing. In certain embodiments, the present invention provides methods for producing a polypeptide of interest (e.g., a protein of interest) in mammalian cells and LNPs for use in such methods or pharmaceutical compositions containing said LNPs . Methods of producing a polypeptide in such cells involve contacting the cell with an LNP composition comprising RNA of interest (eg, mRNA encoding a polypeptide of interest (eg, protein of interest)). After contacting the cell with the LNP composition, the mRNA can be taken up and translated in the cell to produce the polypeptide of interest.

[0503] In general, the step of contacting a mammalian cell with an LNP composition comprising mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, or in vitro. The amount of the LNP composition and / or the amount of mRNA therein contacted with cells may depend on the type of cell or tissue contacted, the mode of administration, the physicochemical 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 allow efficient production of the polypeptide in the cell. Efficiency metrics can include polypeptide translation (indicated by polypeptide expression), levels of mRNA degradation, and indicators of immune response.

[0504] The step of contacting an LNP composition comprising mRNA with a cell may involve or result in transfection, wherein the LNP composition may fuse with a cell membrane to allow delivery of the mRNA into the cell. After introduction into the cytoplasm of the cell, the mRNA is then translated into proteins or peptides via the protein synthesis machinery within the cell's cytoplasm.

[0505] In certain embodiments, the LNP compositions described herein can be used to deliver a therapeutic or prophylactic agent to a subject. For example, mRNA included in an LNP composition can encode a polypeptide and upon contact with and / or enter (eg, transfect) a cell, produce a therapeutic or prophylactic polypeptide. In certain embodiments, the mRNA included in the LNP composition of the present invention may encode a polypeptide that improves or increases immunity in a subject.

[0506] In certain embodiments, contacting a cell with an LNP composition comprising mRNA reduces the innate immune response of the cell to the exogenous nucleic acid. A cell can be contacted with a first LNP composition comprising a first amount of a first exogenous mRNA, the first exogenous mRNA comprising a translatable region, and the cell's innate sensitivity to the first exogenous mRNA can be determined level of immune response. Subsequently, the cells may be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount being a lesser amount of the first exogenous mRNA than the first amount exogenous mRNA. Alternatively, the second composition may include a first amount of a second exogenous mRNA different from the first exogenous mRNA. The step of contacting the cells with the first composition and the second composition may be repeated one or more times.

[0507] Additionally, the efficiency of polypeptide production in the cells can optionally be determined, and the cells can be repeatedly recontacted with the first composition and / or the second composition until the target protein is reached Generate efficiency.

[0508] The present disclosure provides methods of delivering nucleic acids (eg, mRNA) to mammalian cells or tissues (eg, mammalian cells or tissues of a subject). Delivery of mRNA to such cells or tissues involves administering to a subject a LNP composition comprising the mRNA, eg, via injection (eg, via intramuscular injection) or intravascular delivery to the subject. Following administration, the LNP may target and / or contact cells, eg immune cells, such as T cells. After contacting the cell with the LNP composition, the translatable mRNA can be translated in the cell to produce a polypeptide of interest.

[0509] In certain embodiments, LNP compositions of the invention can be targeted to specific types or classes of cells. Such targeting can be facilitated using the lipids described herein to form LNPs, which can also include targeting groups for targeting cells of interest. In certain embodiments, specific delivery can result in reaching a targeted target (e.g., expressing or expressing at high levels the receptor of interest) compared to other targets (e.g., cells that do not express or express only low levels of the receptor of interest). The amount of mRNA of cells to which the immune cell targeting group of the LNP binds) is greater than 2-fold, 5-fold, 10-fold, 15-fold or 20-fold.

[0510] The LNP compositions of the invention are useful in the treatment of diseases, disorders or conditions characterized by a lack or abnormality of protein or polypeptide activity. After delivery of mRNA encoding a deleted or abnormal polypeptide to a cell, translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the deletion of the polypeptide or abnormal activity caused by the polypeptide. Because translation can occur rapidly, the methods and compositions of the invention are 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 of a given species, thereby affecting gene expression. Diseases, disorders, and / or conditions characterized by aberrant or abnormal protein or polypeptide activity that can be administered to compositions of the invention include, but are not limited to, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis ), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases. Various diseases, disorders, and / or conditions can be characterized by a loss of protein activity (or a substantial reduction such that proper protein function does not occur). Such proteins may not be present, or they may be essentially non-functional. A specific example of a dysfunctional protein is a missense mutation variant of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein, which causes cystic fibrosis. The present disclosure provides methods for treating such diseases, disorders and / or conditions in a subject via administration of an LNP composition comprising mRNA and a lipid component comprising KL10, phospholipids ( Optionally unsaturated), PEG lipids and structured lipids, wherein the mRNA encodes a polypeptide that antagonizes or otherwise overcomes the abnormal protein activity present in the cells of the subject. Therapeutic and / or prophylactic compositions described herein may use any reasonable amount and any route of administration effective for preventing, treating, diagnosing or imaging a disease, disorder and / or condition and / or any other purpose administered to the subjects. The specific amount administered to a given subject can vary depending on the species, age and general condition of the subject, the purpose of the administration, the particular composition, the 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. However, it should be understood that the total daily usage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0513] LNP compositions comprising one or more mRNAs may be administered via a variety of routes, such as orally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, subcutaneously, intraventricularly, transdermally or intradermally, Intradermal, rectal, intravaginal, intraperitoneal, topical, transmucosal, nasal, intratumoral administration. In certain embodiments, LNP compositions can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, or subcutaneously. However, the present disclosure contemplates delivery of the LNP compositions of the invention via any suitable route (taking into account possible advances in the science of drug delivery). In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the LNP composition comprising one or more mRNAs (e.g., its stability in various bodily environments such as the bloodstream and gastrointestinal tract). ), the condition of the patient (eg, whether the patient can tolerate a particular route of administration), etc.

[0514] In certain embodiments, it may be sufficient to deliver 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 / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 2 mg / kg to about 5 mg / kg, from about 0.0001 mg / kg to about 2.5 mg / kg, from about 0.001 mg / kg to about 2.5 mg / kg, from about 0.005 mg / kg to about 2.5 mg / kg, from about 0.01 mg / kg to about 2.5 mg / kg, from about 0.05 mg / kg to about 2.5 mg / kg, From about 0.1 mg / kg to about 2.5 mg / kg, from about 1 mg / kg to about 2.5 mg / kg, from about 2 mg / kg to about 2.5 mg / kg, from about 0.0001 mg / kg to about 1 mg / kg kg, from about 0.001 mg / kg to about 1 mg / kg, from about 0.005 mg / kg to about 1 mg / kg, from about 0.01 mg / kg to about 1 mg / kg, from about 0.05 mg / kg to about 1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 0.0001 mg / kg to about 0.25 mg / kg, from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to Combinations of given doses of about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg A dose level of the composition delivers a composition according to the present disclosure, wherein a dose of 1 mg / kg provides 1 mg of the composition per 1 kg of body weight of a subject.

[0515] In particular embodiments, a dose of an LNP composition of the invention may be administered from about 0.001 mg / kg to about 10 mg / kg. In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of the LNP composition may be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg may be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg may be administered. One or more doses per day may be administered in the same or different amounts to achieve the desired level of mRNA expression and / or therapeutic, diagnostic, prophylactic or imaging effect. The desired dose may be delivered, for example, three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, Thirteen, fourteen or more administrations) to deliver the desired dose. In some embodiments, a single dose may be administered, eg, before or after surgery or in the context of an acute disease, disorder or condition.

[0516] LNP compositions comprising one or more mRNAs may be used in combination with one or more other therapeutic, prophylactic, diagnostic or imaging agents. "In combination with" is not intended to imply that the agents must be administered and / or formulated for delivery together, but such methods of delivery are within the scope of this disclosure. For example, one or more LNP compositions comprising one or more different mRNAs can be administered in combination. Compositions can be administered simultaneously with, prior to, or subsequent to one or more other desired therapeutic agents or medical procedures. Typically, each agent will be administered at a dose and / or schedule established for that agent. In some embodiments, the present disclosure contemplates delivery of compositions of the invention, or imaging, diagnostic or prophylactic compositions thereof, in combination with agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their Excretion and / or modify its distribution in the body.

[0517] It is further understood that therapeutic, prophylactic, diagnostic or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels no greater than when they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually.

[0518] The particular combination of therapies (therapeutics or procedures) to be employed in a combination regimen will take into account the compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It is also understood that the therapies employed may achieve the desired effect for the same disorder (e.g., a composition useful in the treatment of cancer may be administered concurrently with a chemotherapeutic agent), or that they may achieve different effects (e.g., control any harmful effects).

[0519] 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%, no more More than 9%, not more than 10%, not more than 15%, not more than 20%, not more than 25%, not more than 30%, not more than 35%, not more than 40%, not more than 45% or not more than 50% The cells targeted for the delivery are transfected with the LNP. In some embodiments, the cells not intended to be targeted by said delivery are non-immune cells of the subject. In some embodiments, cells that are not intended to be targeted by the delivery are cells that are not targeted by the methods. In some embodiments, cells not intended to be targeted by said delivery are cells of a subject not targeted by said method.

[0520] In some embodiments, the nucleic acid delivered to the immune cell by the LNP described herein or the polypeptide encoded by the nucleic acid delivered by the LNP and expressed in the immune cell has a half-life ratio determined by reference The half-life of the nucleic acid delivered by the LNP to said immune cell or the polypeptide encoded by said nucleic acid delivered by said reference LNP and expressed in said immune cell is at least 1%, at least 5%, at least 10%, at least 15% longer , at least 20%, 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 2 times, at least 3 times, at least 4 times or at least 5 times.

[0521] In some embodiments, the composition of the LNP differs from the composition of the reference LNP in the following aspects: type of ionizable cationic lipid, relative amount of ionizable cationic lipid, lipid in PEG lipid The length of the anchor hook, the backbone or headgroup of the PEG lipid, the relative amount of the PEG lipid, or the type of immune cell targeting moiety, or any combination thereof. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only in the type of ionizable cationic lipid. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only in the amount of PEG lipid. In some embodiments, the reference LNP comprises the cationic lipid DLin-MC3-DMA or Lipid 7, but is otherwise identical to the LNP tested. In some embodiments, the PEG lipids are free PEG lipids.

[0522] 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%, 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% of said immune cells are transfected by said LNP. In some embodiments, the immune cells are immune cells of a subject. In some embodiments, the immune cell is an immune cell targeted by the method. In some embodiments, the immune cells are immune cells of the subject targeted by the method. 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% higher than 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 2 times, at least 3 times, At least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In some embodiments, performance levels are measured and compared using the methods described herein. In some embodiments, expression levels are measured via the ratio of cells expressing the encoded polypeptide. In some embodiments, performance levels are measured using FACS. In some embodiments, expression levels are measured via the average amount of encoded polypeptide expressed in cells. In some embodiments, performance levels are measured as mean fluorescence intensity. In some embodiments, the level of expression is measured via the amount of encoded polypeptide or other material secreted by the cell.

[0524] In another aspect, provided herein are methods of targeting delivery of nucleic acids to immune cells of a subject. In some embodiments, the methods comprise contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNP comprises ionizable cationic lipids. In some embodiments, the LNP comprises a conjugate comprising a compound of the formula: [lipid] - [optional linker] - [immune cell targeting group]. In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid.

[0525] In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition containing the same, for use in a method of targeting the delivery of a nucleic acid to an immune cell of a subject . Such methods can be used to treat diseases or disorders as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo. In some embodiments, the LNP is a LNP as described herein in this disclosure.

[0526] In some embodiments, the LNP provides at least one of the following benefits: (i) increased specificity of targeted delivery to the immune cells compared to a reference LNP; (ii) compared to a reference LNP (iii) increased transfection efficiency compared to reference LNP; and (iv) low levels of dye-accessible mRNA (< 15 %) and high RNA encapsulation efficiency in which at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0527] In some aspects, methods of expressing a polypeptide of interest in targeted immune cells of a subject are provided. In some embodiments, the methods comprise contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises a nucleic acid encoding said polypeptide. In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition comprising the same, for use in a method of expressing a polypeptide of interest in targeted immune cells of a subject. Such methods can be used to treat diseases or disorders as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0528] In some embodiments, the LNP provides at least one of the following benefits: (i) increased expression levels in the immune cells compared to a reference LNP; (ii) compared to a reference LNP, an increase in specificity exhibited in said immune cells; (iii) an increase in the half-life of said nucleic acid or a polypeptide encoded by said nucleic acid in said immune cells compared to a reference LNP; (iv) compared to a reference LNP , increased transfection efficiency; and (v) low levels of dye-accessible mRNA (<15%) and high RNA encapsulation efficiency, wherein at least 80% of the total RNA was recovered in the final formulation relative to the total RNA used in the LNP bulk preparation. % mRNA.

[0529] In some aspects, methods of modulating cellular function of target immune cells in a subject are provided. In some embodiments, the method comprises administering lipid nanoparticles (LNPs) to the subject. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises a nucleic acid encoding a polypeptide for modulating a cellular function of said immune cell. In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition containing the same, for use in a method of modulating a cellular function targeting an immune cell in a subject. Such methods can be used to treat diseases or disorders as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0530] In some embodiments, the LNP provides at least one of the following benefits: (i) increased expression levels in the immune cells compared to a reference LNP; (ii) compared to a reference LNP, an increase in specificity exhibited in said immune cells; (iii) an increase in the half-life of said nucleic acid or a polypeptide encoded by said nucleic acid in said immune cells compared to a reference LNP; (iv) compared to a reference LNP , increased transfection efficiency; (v) the LNP can be administered at a lower dose than a reference LNP to achieve the same biological effect in the immune cells; and (vi) low levels of dye accessibility mRNA (<15%) and high RNA encapsulation efficiency where at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0531] In some embodiments, said modulation of cellular function comprises reprogramming said immune cells to initiate an immune response. In some embodiments, said modulation of cellular function comprises modulating antigen specificity of said immune cells.

[0532] In some aspects, methods of treating, ameliorating or preventing symptoms of a disorder or disease in a subject in need thereof are provided. In some embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP) to deliver a nucleic acid to immune cells of the subject. In some embodiments, the LNP comprises 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, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, said LNP comprises said nucleic acid.

[0533] In some embodiments, the nucleic acid modulates the immune response of the immune cells, thereby treating or ameliorating the condition. In some embodiments, an aspect of the present disclosure relates to a LNP as disclosed herein, or a pharmaceutical composition containing the same, for use in a method of treating, ameliorating or preventing symptoms of a disorder or disease in a subject in need thereof use. A disease or disorder may be as disclosed below. In some embodiments, a method as disclosed herein may comprise contacting said immune cells of a subject with lipid nanoparticles (LNPs) in vitro or ex vivo.

[0534] In some embodiments, the LNP provides at least one of the following benefits: (i) increased specificity of delivery of the nucleic acid to the immune cells compared to a reference LNP; (ii) compared to a reference LNP; The half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell is increased compared to LNP; (iii) the transfection rate is increased compared to the reference LNP; (iv) the LNP can be compared with the reference LNP (v) increased levels of gain-of-function in immune cells compared to reference LNP; and (vi) low levels of dye-accessible mRNA (<15%) and High RNA encapsulation efficiency, where at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in the LNP bulk preparation.

[0535] In some embodiments, the disorder is an immune disorder, an inflammatory disorder, or cancer. In some embodiments, the nucleic acid encodes an antigen for use in a therapeutic or prophylactic vaccine to treat or prevent infection by a pathogen.

[0536] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of non-immune cells are transfected with said LNP . In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the undesired Immune cells were transfected with the LNPs. In some embodiments, the nucleic acid delivered to the immune cell by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP has a shorter half-life than the nucleic acid delivered to the immune cell by a reference LNP or the polypeptide encoded by the nucleic acid delivered by the LNP. The half-life of the polypeptide encoded by said nucleic acid delivered with reference to the LNP is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% longer , 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times or longer.

[0537] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the immune cells intended to be targeted for said delivery are The LNP transfection. In some embodiments, the level of expression of said nucleic acid delivered by said LNP is at least 5%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, at least 10%, 1.5 times, 2x, 3x, 4x, 5x, 10x, 15x, 20x or more.

[0539] In some aspects, methods of targeting delivery of nucleic acids to immune cells of a subject are provided. In some embodiments, the methods comprise contacting the immune cells with a lipid nanoparticle (LNP) provided herein. In some embodiments, the methods are used to target NK cells. In some embodiments, the immune cell targeting moiety binds to CD56. In some embodiments, the methods are used to target both T cells and NK cells simultaneously. In some embodiments, the immune cell targeting moiety binds to CD7, CD8, or both CD7 and CD8. In some embodiments, the methods are used to target both CD4+ and CD8+ T cells simultaneously. In some embodiments, the immune cell targeting moiety comprises a polypeptide that binds CD3 or CD7.

[0540] In some aspects, methods of expressing a polypeptide of interest in targeted immune cells of a subject are provided. In some embodiments, the methods comprise contacting the immune cells with a lipid nanoparticle (LNP) provided herein.

[0541] In some aspects, methods of modulating cellular function of target immune cells in a subject are provided. In some embodiments, the methods comprise administering to the subject a lipid nanoparticle (LNP) provided herein.

[0542] In some aspects, methods of treating, ameliorating or preventing symptoms of a disorder or disease in a subject in need thereof are provided. In some embodiments, the methods comprise administering to the subject a lipid nanoparticle (LNP) provided herein.

[0543] In some aspects, methods of treating a CD8-related disease or disorder in a subject are provided. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition described herein. In some embodiments, the disease or disorder is cancer.

[0544] The LNPs disclosed and claimed in this disclosure are suitable for use in the methods described above. VIII. Kits for use in medical applications

[0545] Another aspect of the invention provides a kit for treating a disorder. The kit comprises: ionizable cationic lipids, lipid-immune cell targeting group conjugates, lipid nanoparticles comprising ionizable cationic lipids and / or lipid-immune cell targeting group conjugates Compositions (with or without encapsulated payload (eg, mRNA)) and instructions for use in the treatment of medical disorders such as cancer or microbial or viral infections. example

[0546] The present invention now being generally described, it will be more readily understood by reference to the following examples, which are included for the purpose of illustrating certain aspects and embodiments of the invention only and are not intended to are limiting the invention. Example 1 - Preparation of ionizable cationic lipids This example describes the synthesis of various cationic lipids. cationic lipid 1

[0547] Synthesis of Cationic Lipid 1 (shown in the formula below) was prepared as described in Scheme 1 below. (plan 1)

[0548] Ether intermediate 1-3 was prepared via the following method: the hydroxyl functional protected 1,2-diol starting material (1-1) (0.151 mol, 24 g) and dimethylaminopropane The base chloride compound 1-2 (0.051 mol, 24 g, 1 equiv.) and TBAI (0.0015 mol, 554 mg, 0.01 equiv.) were reacted overnight at 80oC in the presence of NaOH (32%) / THF to afford the ether Intermediate compound 1-3 (20.1 g, 0.1 mol). Thereafter, compound 1-3 (2.2 g, 0.01 mol) was deprotected (THF, 6 M HCl, 4 h) to obtain the vicinal diol intermediate compound 1-4 (1.6 g, 0.009 mol) in quantitative yield. Using EDC (3.8 g, 0.019 mol, 3.2 equiv.), using fatty acid 1-5 in DCM (5.9 mL, 0.018 mol, 3 equiv.) (using 10 equiv. EQ DIPEA) diacylated compound 1-4 ( 1.12 g, 0.006 mol), to provide lipid compound 1 (238 mg, 0.0034 mol). Lipid Compound 1 was purified via preparative HPLC (CombiFlash Nextgen 300+ Teledyne ISCO) and the final product was 98% pure (RP-HPLC-ELSD using Durashell-C18, 4.6 x 50 mm, 3 uM, cat. no. DC930505-0 ).

[0549] The purified lipid 1 free base (C44H79NO5, molecular weight 702.12 g / mol) was characterized in CDCl3 via proton NMR spectroscopy (400 MHz), as shown in Figure 1; and via LC-MS to confirm the structure ( NMR, m / z) and purity (NMR, LC), as shown in Figure 2A and Figure 2B. cationic lipid 2

[0550] Synthesis of Cationic Lipid 2 (shown in the formula below) was prepared as described in Scheme 2 below. (Scenario 2)

[0551] Ester intermediate 2-3 was prepared via: EDCl (0.03 mol, 5.73 g, 1 eq.), DIPEA (0.12 mol, 12.9 g, 4.0 eq.) in DCM (300 mL) solution and DMAP (0.006 mol, 733 mg, 0.2 eq.) to acylate the protected 1,2-diol (1, 2-O-isopropylidene-D-glycerol) starting material (2-1), to obtain 5.44 g (0.02 mol) of 2-3. Intermediate 2-3 (670 mg, 0.0027 mol, 1.0 eq.) was deprotected in a mixture of 10 mL TFA / water (50:50 v / v) and 10 mL 6 M HCl at room temperature to afford 560 mg (0.0027 mol) of compounds 2-4. Using EDC (0.009 mol, 1.72 g, 1 eq.), DIPEA (0.001 mol, 122 mg, 0.33 eq.), with fatty acids 1-5 (0.009 mol, 2.52 g, 3 eq.) in 50 mL of DCM Compounds 2-4 (0.003 mol, 641 mg, 1 eq.) were esterified to provide cationic lipid 2 (260 mg, 0.0003 mol).

[0552] Lipid Compound 2 was purified via preparative HPLC. The resulting product was greater than 99% pure (reversed phase HPLC-ELSD using Durashell-C18, 4.6 x 50 mm, 3 uM, cat. no. DC930505-0).

[0553] The purified lipid 2 free base (C45H79NO6, molecular weight 730.10 g / mol) was characterized in CDCl3 via proton NMR spectroscopy (400 MHz), as shown in Figure 3; and via LC-MS to confirm the structure ( NMR, m / z) and purity (NMR, LC), as shown in Figure 4A and Figure 4B.

[0554] Lipid 6 was synthesized using a method similar to that of Lipid 2, except that diethylaminobutyric acid was used to generate a tertiary amine head group instead of the dimethylamine group used for Lipid 2 butyric acid.

[0555] Purified lipid 6 was characterized via proton NMR spectroscopy, as shown in Figure 41; and via mass spectrometry and reverse phase HPLC, as shown in Figure 42A and Figure 42B. cationic lipid 3

[0556] Synthesis of Cationic Lipid 3 (shown in the formula below) was prepared as described in Scheme 3 below. (Option 3)

[0557] Fatty acids 3-10 were prepared using the following Scheme 3a: (Scheme 3a)

[0558] The fatty acid 3-10(9Z,12Z)-hexadexadec-9,12-dienoic acid was synthesized using the Wittig reaction method as shown in Scheme 3a. To generate compound 3-7, 9-bromononanoic acid (0.0148 mol, 3.50 g) was combined with PPh3 (0.0148 mol, 3.87 g, 1 eq.) in 5 mL of toluene and refluxed for 48 hours to provide 7.23 g of bromine Phosphonium Chlorides 3-7. To produce compound 3-9, compound 3-8 (0.0076 mol, 0.87 g) was oxidized using Dess Martin oxidant (0.0082 mol, 3.48 g, 1.1 eq.) in 20 mL of DCM to afford aldehyde 0.85 g of 3- 9. Compound 3-9 (0.0076 mol, 085 g) was reacted with compound 3-7 (0.0076 mol, 3.78 g, 1 eq.) in 7.6 mL of 40% NAHMDS in 60 mL THF to provide 400 mg of fatty acid 3 -10.

[0559] As shown in Scheme 3, intermediate 3-3 was produced via the following method: using tosyl chloride (3-2) (0.034 mol, 6.5 g, 1 eq. ) in 250 mL DCM at room temperature and triethylamine (0.034 mol, 4.9 mL, 1 eq.), DMAP (0.011 mol, 200 mg, 0.3 eq.) tosylate the protected 1,2,4-butanetriol starting material (3 - 1) Free hydroxyl groups on (0.0342 mol, 5.0 g, 1 eq.). Nucleophilic displacement of compound 3-3 (0.0032 mol, 1.0 g, 1 eq.) with dimethylamine (0.03 mol, 1.5 g, 10 eq.) in 16.6 mL of THF afforded 400 mg of the tertiary amine 3- 4. Compound 3-4 was deprotected with 2 M HCl in MeOH to provide 410 mg of compound 3-5. Using EDC (0.0018 mol, 306 mg, 3 eq.), DIPEA (0.0024 mol, 8.8 mL, 4.5 eq.) in 5.4 mL of DCM with fatty acid 3-10 (0.0016 mol, 400 mg, 3 eq.) Compounds 3-5 (90 mg) were esterified for 2 hours to provide 12 mg of ionizable lipid 3. Lipid compound 3 was purified via preparative HPLC.

[0560] Lipid Compound 3 was purified via preparative HPLC (CombiFlash Nextgen 300+ Teledyne ISCO). Product purity was determined to be 99% via reverse phase HPLC-ELSD (using Durashell-C18, 4.6 x 50 mm, 3 uM, cat# DC930505-0). cationic lipid 4

[0561] Synthesis of Cationic Lipid 4 (shown in the formula below) was prepared as described in Scheme 4 below. (Option 4)

[0562] Using the 9-bromononanoic acid heptanal starting material, fatty acid 9-1 was synthesized using a protocol similar to that used above for the synthesis of fatty acids 4-6.

[0563] Fatty acid 9-1 (0.01 mol, 2.14 g, 3 eq.) Diacylation of 1–4 (0.003 mol, 600 mg) provided 203 mg of ionizable lipid 4 (mass, yield). Lipid compound 4 was purified via preparative HPLC (CombiFlash Nextgen 300+ Teledyne ISCO) and yielded a 99% pure product (HPLC-ELSD using Durashell-C18, 4.6 x 50 mm, 3 uM, Cat# DC930505-0) .

[0564] Purified Lipid 4 was characterized via proton NMR spectroscopy, as shown in Figure 6; and via mass spectrometry and reverse phase HPLC, as shown in Figures 7A and 7B. cationic lipid 5

[0565] Synthesis of Cationic Lipid 5 (shown in the formula below) was prepared as described in Scheme 5 below. (Option 5)

[0566] As shown in Scheme 5, intermediate 5-3 was produced via the following method: using tosyl chloride (5-2) (0.039 mol, 7.5 g, 1 eq. ) in 250 mL DCM at room temperature and triethylamine (0.039 mol, 5.6 mL, 1 eq.), DMAP (0.013 mol, 230 mg, 0.3 eq.) tosylate the protected 1,2,4-butanetriol starting material (5 - 1) Free hydroxyl groups on (0.0342 mol, 5.0 g, 1 eq.). Nucleophilic displacement of compound 5-3 (0.0032 mol, 1.0 g, 1 eq.) with dimethylamine (0.03 mol, 1.5 g, 10 eq.) in 16.6 mL of THF overnight provided 1.1 g of tertiary amine 5 -4. Compound 5-4 (712 mg) was deprotected in 6 M HCl in water (5 mL) to provide 551 mg of compound 5-5. Use EDC.HCl (35.5 mol, 6.7 g, 3 eq.), DIPEA (47 mmol, 6.7 mL, 4 eq.) in 55 mL of DCM with fatty acids 1-5 (35.5 mmol, 8.88 g, 3 eq. ) compound 5-5 (2 g) was esterified for 2 h to provide 1.09 g of ionizable lipid 5. Lipid compound 5 was purified via preparative HPLC.

[0567] Lipid compound 5 was purified via preparative HPLC (CombiFlash Nextgen 300+ Teledyne ISCO). Product purity was determined to be 99% via reverse phase HPLC-ELSD (using Durashell-C18, 4.6 x 50 mm, 3 uM, cat# DC930505-0).

[0568] The purified lipid 5 free base (C42H74NO4, molecular weight 657.57 g / mol) was characterized in CDCl3 via proton NMR spectroscopy (400 MHz), as shown in Figure 39; and via mass spectrometry to confirm the structure (NMR , m / z) and purity (LC-ELSD), as shown in Figure 40A and Figure 40B.

[0569] Lipid 7 was synthesized using a method similar to that of Lipid 5, except that diethylamine was used instead of dimethylamine to incorporate a tertiary amine headgroup.

[0570] Purified lipid 7 was characterized via proton NMR spectroscopy, as shown in Figure 43; and via mass spectrometry and reverse phase HPLC, as shown in Figure 44A and Figure 44B.

[0571] Scheme 6 below depicts the synthesis of Lipid 9, Lipid 10 and Lipid 11: (Scheme 6) Cationic Lipid 9

[0572] The ester intermediate D-2 was prepared via: dimethylalanine compound D-1 (33.3 mmol, 6.06 g, 1.71 eq), EDCI (33.2 4 g (30.2 mmol) of protected 1,2-diol were acylated with DIPEA (121.0 mmol, 21.08 mL, 4.0 eq) and DMAP (6 mmol, 740 mg, 0.2 eq) in mmol, 6.28 g, 1.1 eq) (1,2-O-isopropylidene-D-glycerol) starting material (1), 2.6 g (0.02 mol) of D-2 were obtained in 37% yield. Intermediate D-2 (2.5 g, 10.68 mmol, 1.0 eq.) was deprotected in a 1:3 (v / v) mixture of 1 M aqueous HCl and THF (total volume 20 mL) at room temperature to afford 2.4 g (12.6 mmol) of crude compound D-3. With fatty acid 2 (8.9 g, 2.5 eq, 31.8 mmol), EDCI (6.1 g, 2.5 eq, 31.8 mmol), DIPEA (5.53 mL, 2.5 eq, 31.8 mmol), DMAP (285 mg, 0.2 eq, 2.6 mmol), Crude compound D-3 (12.6 mmol, 2.4 g, 1 eq.) was esterified with DCM (100 mL) to provide 7 g of crude ionizable lipid 9. The crude product (3 g) was purified via preparative HPLC to obtain 100 mg of pure lipid (measured via reverse phase HPLC-ELSD (using Durashell-C18, 4.6 x 50 mm, 3 uM, cat# DC930505-0)> 99% pure).

[0573] Purified Lipid 9 free base (C44H77NO6, molecular weight 716.10 g / mol) was characterized via proton NMR spectroscopy (400 MHz) (in CDCl3) and mass spectrometry to confirm the structure (Figure 48A and Figure 48B), and Characterized via LC-ELSD to determine purity (Figure 48C). cationic lipid 10

[0574] The ester intermediate E-2 was prepared via: dimethylalanine compound E-1 (4.76 g, 1.1 eq, 33 mmol), EDCI (6.38 g, 1.1 eq, 33 mmol), DIPEA (21.08 mL, 4.0 eq, 120 mmol) and DMAP (680 mg, 0.2 eq, 6 mmol) acylated 4 g (30.2 mmol) of the protected 1,2-diol (1,2-O-isopropylidene-D-glycerol) starting material (1), 1.9 g (7.38 mmol) of E-2 were obtained in 25% yield. Intermediate E-2 (1.9 g, 7.38 mmol, 1 eq) was deprotected in a 1:3 (v / v) mixture of 1 M aqueous HCl and THF (total volume 80 mL) at room temperature to afford 1.58 g (7.27 mmol) of crude compound E-3. With fatty acid 2 (18.2 mmol, 5.1 g, 2.5 eq), EDCI (18.2 mmol, 3.48 g, 2.5 eq), DIPEA (18.2 mmol, 3.16 mL, 2.5 eq) and DMAP (1.4 mmol, 160 mg, 0.2 eq) of crude compound E-3 (7.27 mmol, 1.58 g, 1 eq.) was esterified to provide about 7.5 g of crude ionizable lipid 10. The crude product (3 g) was purified via preparative HPLC to obtain 105 mg of pure lipid (measured via reverse phase HPLC-ELSD (using Durashell-C18, 4.6 x 50 mm, 3 uM, cat# DC930505-0)> 99% pure).

[0575] Purified Lipid 10 free base (C46H79NO6, molecular weight 742.14 g / mol) was characterized via proton NMR spectroscopy (400 MHz) (in CDCl3) and mass spectrometry to confirm the structure (Figure 49A and Figure 49B), and Characterized via LC-ELSD to determine purity (Figure 49C). cationic lipid 11

[0576] The ester intermediate F-2 was prepared via: dimethylalanine compound F-1 (6.06 g, 1.38 eq, 41.7 mmol), EDCI (6.38 g, 1.1 eq, 33.3 mmol), DIPEA (21.08 mL, 4.0 eq, 121.0 mmol) and DMAP (740 mg, 0.2 eq, 6 mmol) acylated 4 g (30.2 mmol) of the protected 1,2-diol (1,2-O-isopropylidene-D-glycerol) starting material (1), 3.3 g (12.7 mmol) of F-2 were obtained in 41.7% yield. Intermediate F-2 (3.2 g, 12.3 mmol, 1 eq) was deprotected in a 1:3 (v / v) mixture of 1 M aqueous HCl and THF (total volume 80 mL) at room temperature to afford 3.1 g (14.1 mmol) of crude compound F-3. With fatty acid 2 (35.3 mmol, 9.9 g, 2.5 eq), EDCI (6.8 g, 2.5 eq, 35.3 mmol), DIPEA (6.2 mL, 2.5 eq, 35.3 mmol) and DMAP (316 mg, 0.2 eq, 2.8 mmol) to esterify crude compound F-3 (14.13 mmol, 3.1 g, 1 eq.) to provide approximately 9 g of crude ionizable lipid 11. The crude product (3 g) was purified via preparative HPLC to obtain 55 mg of pure lipid 11 (measured via reverse phase HPLC-ELSD (using Durashell-C18, 4.6 x 50 mm, 3 uM, cat# DC930505-0) > 99% pure).

[0577] Purified Lipid 11 free base (C46H81NO6, molecular weight 744.16 g / mol) was characterized via proton NMR spectroscopy (400 MHz) (in CDCl3) and mass spectrometry to confirm the structure (Figure 50A and Figure 50B), and Characterized via LC-ELSD to determine purity (Figure 50C).

[0578] Scheme 7 and Scheme 8 below depict the synthesis of Lipid 12 and Lipid 13: (Scheme 7) (Scheme 8) Cationic Lipid 12

[0579] From (R)-(2,2-dimethyl-1,3-dioxolane-4- base) Methanol (1) 2.0 g (1.0 eq, 15.2 mmol) yielded the Fmoc-protected intermediate 3A, providing 3.8 g of 3A in 71% yield (Step 1, Scheme 7). 3A 2.3 g (1.0 eq, 6.5 mmol) was selectively deprotected in 1 M HCl:THF (1:3, 20 mL) and 0.5 mL methanol to provide approximately 2 g of 4A (step 2, scheme 7). Linoleic acid 1-5 (9.2 mmol, 2.9 mL, 2.2 eq) O-acyl Fe4A 1.3 g (1.0 eq, 4.2 mmol). The combined products from the two batches provided a total of 1.6 g (21%) of pure intermediate H-8' (step 3, scheme 7). Removal of Fmoc (step 4, scheme 7) from H-8' 2.05 g (1.0 eq, 2.44 mmol) using 1% piperidine in THF (25 mL) (0 ºC, 4 h) yielded in 45% yield 680 mg of purified intermediate H-9 were obtained. The key intermediate H-9 was used in subsequent steps to generate lipid 12 (via O-acylation at G-4') as well as to generate lipid 13 (via O-acylation at H-5', as relevant below section).

[0580] Preparation of protected compound G-4' 3-(( 2-((tertiary butyldimethylsilyl)oxy)ethyl)(methyl)amino)propanoic acid. Make methyl acrylate (H-2') 1.6 ml (1 eq, 17.8 mmol) with G-1' (2 g, 1.5 eq, 26.6 mmol) and alumina (904 mg, 0.5 eq, 8.9 mmol) at room temperature Reaction was carried out for 3 h under solvent-free conditions to provide 2.58 g (91%) of G-2′ (step 7, scheme 8). G-2' 1.33 g (1 eq, 8.26 mmol) was converted to the tertiary butyldimethylsilyl-protected intermediate G-3' (step 8, scheme 8) overnight at room temperature, resulting in the recovery of approximately 1.13 g (50%) of purified G-3 '. G-3' 1.14 g (1 eq, 4 mmol) was subsequently selectively deprotected in THF / MeOH / 1 M HCl (3 / 2 / 1 (v / v); total volume 6 ml) overnight at room temperature, 1.13 g of G-4' were produced (Step 9, Scheme 8). G-4' and H-9 were combined to yield intermediate G-6 (Step 5A, Scheme 8). G-4' (0.98 mmol , 268 mg, 1.5 eq) acylated H-9 400 mg (1.0 eq, 0.65 mmol) to provide 308 mg (55%) of crude G-6. Deprotection of crude G-6 (308 mg) in 6.0 mL of HF.pyridine (9.0 mmol, 641 µL, 25 eq) in THF (Step 6A, Protocol 7) yielded 308 mg of crude lipid 12. The crude product was purified twice using preparative HPLC to isolate 213 mg (79%) of purified lipid 12. (>99% pure via reversed-phase HPLC-ELSD using Durashell-C18, 4.6 x 50 mm, 3 uM, Cat# DC930505-0).

[0581] Purified Lipid 12 free base (C45H79NO7, molecular weight 746.13 g / mol) was characterized via proton NMR spectroscopy (400 MHz) (in CDCl3) and mass spectrometry to confirm the structure (Figure 51A and Figure 51B), and Characterized via LC-ELSD to determine purity (Figure 51C). cationic lipid 13

[0582] Preparation of protected compound 3-(bis (2-((tertiary butyldimethylsilyl)oxy)ethyl)amino)propanoic acid (H5'). Make methyl acrylate (H-2') 1.65 g (1 eq, 19.2 mmol) with H-1' (28.5 mmol, 3.0 g, 1.5 eq), alumina (960 mg, 0.5 eq, 9.6 mmol) at room temperature Reaction was carried out for 3 hours without solvent to provide 3.53 g (97%) of H-3'. H-3' 830 mg (1 eq, 4.3 mmol) was converted to the tertiary butyldimethylsilyl-protected intermediate H-4′ overnight at room temperature, resulting in the recovery of approximately 1.51 g (83%) of purified H-4′. Subsequent selective deprotection of H-4' 600 mg (1 eq, 1.4 mmol) in THF (3 ml) / MeOH (2 ml) / 1 M LiOH (1 ml) overnight at room temperature yielded ~500 mg H-5'. Intermediate H- 10. H-5' and H-9 were combined to give intermediate H-10 (step 5, scheme 7). Use EDCI (0.36 mmol, 74 mg, 1.5 eq), DIPEA (0.36 mmol, 62 µL, 1.5 eq), DMAP (0.048 mmol, 6 mg, 0.2 eq) in 1.0 mL DCM with H-5' (0.36 mmol , 150 mg, 1.5 eq) acylated H-9 150 mg (1.0 eq, 0.24 mmol) to provide 108 mg (44%) of crude H-10. Deprotection of 108 mg (1.0 eq, 0.11 mmol) of crude H-10 in HF.pyridine (2.75 mmol, 200 µL, 25 eq) in 2.0 mL THF yielded 41 mg (48%) of crude lipid 13 . The crude products from the two batches were combined and purified twice using preparative HPLC to isolate 71 mg of purified lipid 13. (>99% pure via reverse phase HPLC-ELSD using Durashell-C18, 4.6 x 50 mm, 3 uM, cat. no. DC930505-0).

[0583] Purified Lipid 13 free base (C46H81NO8, molecular weight 776.15 g / mol) was characterized via proton NMR spectroscopy (400 MHz) (in CDCl3) and mass spectrometry to confirm the structure (Figure 52A and Figure 52B), and Characterized via LC-ELSD to determine purity (Figure 52C). Example 2 - Preparation of LNP via Vortex Mixing Using Exemplary Ionizable Lipids

[0584] Using cationic lipid 2 and cationic lipid 5 synthesized as in Example 1 and commercially available cationic lipid 8 and cationic lipid DLin-MC3-DMA (MedChemExpress, NJ, USA; Cat. No. HY-112758) to generate examples sex LNP.

[0585] LNPs with encapsulated mRNA payload and lipid blend were generated via vortex mixing of aqueous mRNA solution and ethanolic lipid solution. The mRNA (9:1 w / w mixture of eGFP-encoding mRNA and Cy5-labeled eGFP mRNA, TriLink Biotechnologies, CA, USA) was mixed with pH 4 acetate buffer to provide a solution containing 133 µg / mL mRNA and 21.7 Final aqueous mRNA solution in mM acetate buffer. The lipid components were dissolved in absolute ethanol in the relative ratios set forth in Table 3 below. table 3 Lipid source Lipids and mRNA The ratio (nmol lipid / 100 µg mRNA) in lipid solution Concentration in (mM) Theoretical LNP lipid composition (mol%) ionizable cation Lipid - 1,500 6 48.8 cholesterol Dishman, Netherlands 1,200 4.8 39.0 DSPC Avanti Polar Lipids, Alabama, USA 300 1.2 9.8 DMG-PEG (2000) Avanti Polar Lipids, Alabama, USA 75 0.3 2.4

[0586] Briefly, the mRNA solution (375 µL) was transferred to a conical bottom centrifuge tube, and the lipid solution (125 µL) was quickly added to the tube containing the mRNA solution (v of mRNA solution to lipid solution / v ratio is 3:1). Tubes containing the mixture were immediately capped and vortexed at 2,500 rpm for 15 s, followed by incubation at room temperature for no less than 5 min before ethanol removal and buffer exchange.

[0587] After mixing, the resulting LNP suspension was subjected to ethanol removal and buffer exchange via gravity flow using a Sephadex G-25 resin packed SEC column (PD MiniTrap G-25, Cytiva, Massachusetts, USA). Briefly, the SEC column was washed five times with 2.5 mL of exchange buffer (25 mM pH 7.4 HEPES buffer with 150 mM NaCl) and then loaded with 425 µL of LNP suspension. Once the LNP suspension was completely transferred into the resin bed, 75 µL stacking volumes of exchange buffer were applied to the column according to the manufacturer's instructions to achieve the column's specified target loading volume and maximize recovery. After the stacking volume had been completely transferred into the resin bed, the SEC column was transferred to a new centrifuge tube, and the LNP suspension was eluted by adding 1.0 mL of exchange buffer to the column. The LNP-containing eluate in exchange buffer was recovered and stored at 4 ºC until further use. Example 3 - Characterization of LNP

[0588] This example describes the characterization of the LNP produced in Example 2.

[0589] The LNP samples produced in Example 2 were characterized to determine mean hydrodynamic diameter, zeta potential and mRNA content (total and dye accessible). Hydrodynamic diameters were determined via dynamic light scattering (DLS) using a Zetasizer model ZEN3600 (Malvern Pananalytical, UK). Zeta potential was measured via laser Doppler electrophoresis using a Zetasizer in 5 mM pH 5.5 MES buffer and 5 mM pH 7.4 HEPES buffer.

[0590] The RNA content of the nanoparticles was measured using the Thermo Fisher Quant-iT RiboGreen RNA Assay Kit. Dye-accessible RNA (which includes both unincorporated RNA and RNA near the nanoparticle surface) was measured by diluting the nanoparticles to approximately 1 µg / mL mRNA using HEPES-buffered saline, followed by Quant- iT reagent was added to the mixture. Total RNA content was measured by diluting the particles to 1 µg / mL mRNA using HEPES-buffered saline, disrupting the nanoparticles by heating them to 60ºC for 30 minutes in HEPES-buffered saline containing 0.5% Triton, and then adding Quant-It reagents. RNA was quantified via measurement of fluorescence at 485 / 535 nm, and concentrations were determined relative to a concurrently run RNA standard curve. The results are set forth in Table 5. table 5 Preparation serial number ionizable lipid DLS Z The average diameter (nm) DLS PDI at pH 5.5 zeta potential (mV) at pH 7.4 zeta potential (mV) Dye accessible mRNA (%) 1 cationic lipid 2 120 0.19 twenty one 5.4 16 2 cationic lipid 5 109 0.14 twenty three 0.3 13 3 cationic lipid 8 91 0.12 20 0.9 6.3 4 Cationic lipid DLn-MC3-DMA 96 0.14 16 -0.4 9.4 Example 4 - Preparation of Fab conjugates for T cell targeting

[0591] This example describes the generation of exemplary lipid-immune cell targeting group conjugates.

[0592] The anti-CD3 Fab (hSP34 with mouse lambda and human lambda) was made via covalent coupling between the maleimide group and the C-terminal cysteine ​​in the heavy chain (HC), see below amino acid sequence) conjugated with DSPE-PEG(2k)-maleimide. Anti-CD3 Fab strain Hu291, anti-CD8 Fab strain TRX2, anti-CD8 Fab strain OKT8, non-functional mutant OKT8 (mutOKT8), anti-CD4 from the ibalizumab sequence were also ligated using similar methods described herein. Fab, anti-CD5 Fab strain He3, anti-CD7 Fab strain TH-69, anti-CD2 Fab strain TS2 / 18.1, anti-CD2 Fab strain 9.6, anti-CD2 Fab strain 9-1 with human kappa. After buffer exchange into anaerobic pH 7 phosphate buffer, proteins (3–4 mg / mL) were reduced in 2 mM TCEP in anaerobic pH 7 phosphate buffer for 1 hr at room temperature. Reduced proteins were separated using a 7 kDa SEC column to remove TCEP and buffer exchanged into fresh anaerobic pH 7 phosphate buffer.

[0593] DSPE-PEG-maleimide (Avanti Polar Lipids, Alabama, USA) and 30 mg / mL DSPE were added in anaerobic pH 5.7 citrate buffer (1 mM citrate). - 10 mg / mL micellar suspension of PEG-OCH3 (vanti Polar Lipids, Alabama, USA) (use a weight ratio of 1:1 to 1:3 depending on the protein) to initiate the conjugation reaction. Concentrate the protein solution to 3-4 mg / mL using a 10 kDa regenerated cellulose membrane, followed by buffer exchange into anaerobic pH 7 phosphate buffer using a 40 kDa size exclusion column. Conjugation reactions were performed using 2-4 mg / mL protein and a 3.5 molar excess of maleimide at 37ºC for 2 hours, followed by an additional 12-16 hours at room temperature.

[0594] Production of the resulting conjugate was monitored via HPLC, and the reaction was quenched in 2 mM cysteine. The resulting conjugate (DSPE-PEG(2k)-anti-hSP34 Fab) was isolated by filtration using a pH 7.4 HEPES-buffered saline (25 mM HEPES, 150 mM NaCl) buffer using a 100 kDa Millipore regenerated cellulose membrane and stored in Store at 4ºC. After quenching, the final micellar composition consisted of a mixture of DSPE-PEG-Fab, DSPE-PEG-maleimide (terminated with cysteine), and DSPE-PEG-OCH3. The ratio of these three components is DSPE-PEG-Fab : DSPE-PEG-maleimide (terminated with cysteine) : DSPE-PEG-OCH3 = 1 : 2.45 : 3.45-10.35 (by molar ).

[0595] The resulting conjugates showed binding to recombinant rhesus monkey CD3ε comparable to unconjugated anti-CD3 Fab as measured by ELISA assay. Anti-CD3 hSP34-Fab sequence: hSP34 HC (SEQ ID NO: 1): EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC hSP34-mlam LC (mouse lambda) (SEQ ID NO: 2): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFL APGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADSS SP34-hlam LC (human lambda) (SE Q ID NO: 3 ): QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSY LSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS Anti-CD3 Hu291-Fab Sequence: Hu291 HC (SEQ ID NO: 4): QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSA YYDYDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC Hu291 LC (SEQ ID NO: 5): MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSSSL SASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGES Anti-CD8 TRX2-Fab Sequence: TRX2 HC (SEQ ID NO: 6): QVQLVESGGGVVQPGRSLRLSCAASGTFFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC TRX2 LC (SEQ ID NO: 7): DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD8 OKT8-Fab sequence: OKT8 HC (SEQ ID NO: 8): QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAY MELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSSDKTHTC OKT8 LC (SEQ ID NO: 9): DIVMTQSPSSLSASVG DRVTITCRTRSQYLAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGES anti-CD4 ibalizumab-Fab sequence : Ibalizumab HC (SEQ ID NO: 10): QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC Ibalizumab LC (SEQ ID NO: 11): DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFLTISSVQA EDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD5 He3-Fab sequence: He3 HC (SEQ ID NO: 12): EIQLV QSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC He3 LC (SEQ ID NO: 13): DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES Anti-CD7 T...

Claims

1. A lipid nanoparticle (LNP) for targeted delivery of nucleic acids to immune cells, comprising a 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) an ionizable cationic lipid, wherein the LNP further comprises a nucleic acid disposed therein; and wherein the cationic lipid comprises a compound of formula I: (Formula I), or a salt thereof, wherein: R1 and R2 are independently C1-3 alkyl groups, or R1 and R2 together with a nitrogen atom form an optionally substituted piperidinyl or morpholinyl group; Y is selected from -O-, -OC(O)-, -OC(S)-, and -CH2-; X1, X2, X3, and X4 are hydrogens, or X1 and X2 or X3 and X4 independently form a side oxygen group; n is 0 or 3; o and p are independently integers selected from 2 to 6.

2. The LNP as claimed in claim 1, wherein the cationic lipid comprises (lipid 5), or a salt thereof.

3. The LNP as claimed in claim 1, wherein the cationic lipid comprises (lipid 2), or a salt thereof.

4. The LNP as claimed in claim 1, wherein the cationic lipid comprises (lipid 6), or a salt thereof.

5. The LNP of any one of claims 1 to 4, wherein the immune cell targeting group comprises an antibody that binds to a T cell antigen, wherein the T cell antigen is CD3, CD4, CD7, CD8 or a combination thereof.

6. The LNP as claimed in any one of claims 1 to 4, wherein the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen, wherein the NK cell antigen is CD7, CD8, CD56, or a combination thereof.

7. The LNP as claimed in any one of claims 1 to 4, wherein (i) the immune cell targeting group is covalently coupled to the lipids in the lipid blend via a linker containing polyethylene glycol (PEG); (ii) the lipid covalently coupled to the immune cell targeting group via a linker containing PEG is distearate (DSG), distearate-phosphatidylethanolamine (DSPE), distearate-phosphatidylethanolamine (DMPE), distearate-glycerol-phosphoglyceride (DSPG), distearate-glycerol (DMG), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide; (iii) the PEG is PEG 2000 or PEG 3400; (iv) the lipid-immune cell targeting group conjugate is present in the lipid blend in the range of 0.001-0.5 mole percentage; or (v) The ionizable cationic lipids are present in the lipid blend in the range of 30-70 moles; or any combination of (i) to (v).

8. The LNP as claimed in any one of claims 1 to 4, wherein the lipid blend further comprises one or more of structured lipids, neutral phospholipids, and free PEG-lipids.

9. The LNP as claimed in claim 8, wherein the structural lipid is a sterol, and wherein (i) the sterol is present in the lipid blend in the range of 20-70 moles; and (ii) the sterol is cholesterol; (iii) The neutral phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipryl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipryl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin; (iv) The neutral phospholipid is present in the lipid blend in the range of 1-10 moles. (v) The free PEG-lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; (vi) The free PEG-lipid comprises diacylphosphatidylethanolamine, which comprises a dipalmitoyl (C16) chain or a distearate (C18) chain; (vii) The free PEG-lipid is present in the lipid blend in the range of 1-4 moles; or (viii) The free PEG-lipid comprises lipids that are the same as or different from the lipids in the lipid-immune cell targeting group conjugate; or any combination of (i) to (viii).

10. The LNP as claimed in claim 9, wherein the PEG lipid is PEG-dioleoglycerol (PEG-DOG), PEG-dimyristicoglycerol (PEG-DMG), PEG-dispalmitoglycerol (PEG-DPG), PEG-dilinoleoglycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dispalmitoglycerol-phosphatidylethanolamine (PEG-DMPE), or PEG-dispalmitoglycerol-phosphatidylethanolamine (PEG-D). PPE), PEG-distearylglycerol (PEG-DSG), PEG-diacetylglycerol (PEG-DAG), PEG-ceramide, PEG-distearyl-glycerol-phosphate glycerol (PEG-DSPG), PEG-diacetyl-glycerol-phosphate ethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-bistetradecyl acetamide or PEG-distearyl-phosphatidylethanolamine (PEG-DSPE) lipids.

11. The LNP as claimed in any one of claims 1 to 4, wherein the LNP has (i) an average diameter in the range of 50-200 nm; (ii) a polydispersity index in the range of 0.05 to 1; or (iii) the LNP has a zeta potential in the range of about -10 mV to about +30 mV at pH 5; or any combination of (i) to (iii).

12. The LNP as claimed in any one of claims 1 to 4, wherein the nucleic acid is mRNA, and the mRNA (i) encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine; (ii) a polypeptide capable of regulating an immune response in the immune cell; or (iii) a polypeptide capable of reprogramming the immune cell; or any combination of (i) to (iii).

13. The LNP as claimed in claim 12, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

14. The LNP as claimed in any one of claims 1 to 4, wherein the immune cell targeting group comprises a Fab, F(ab')2, Fab'-SH, Fv, or scFv fragment.

15. The LNP as claimed in claim 14, wherein (i) the immune cell targeting group comprises a Fab engineered to remove the natural interchain disulfide bond at the C-terminus; (ii) the Fab comprises a heavy chain segment containing a C233S substitution and a light chain segment containing a C214S substitution, according to Kabat numbering; (iii) the immune cell targeting group comprises a Fab having a non-natural interchain disulfide bond; (iv) the Fab contains an F174C substitution in the heavy chain segment and an S176C substitution in the light chain segment, according to Kabat numbering; (v) the immune cell targeting group comprises a Fab containing a cysteine ​​at the C-terminus of the heavy chain or light chain segment; (vi) the Fab further comprises one or more amino acids between the heavy chain segment of the Fab and the C-terminal cysteine; (vii) The Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked via one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker; or any combination of (i) to (vii).

16. The LNP of any one of claims 1 to 4, wherein the immune cell targeting group comprises an immunoglobulin monovariable domain.

17. The LNP as claimed in claim 16, wherein (i) the immunoglobulin monovariable domain comprises a cysteine ​​at its C-terminus; (ii) the immunoglobulin monovariable domain comprises a VHH domain and further comprises a spacer containing one or more amino acids between the VHH domain and the C-terminal cysteine; (iii) the immune cell targeting group comprises two or more VHH domains; (iv) the two or more VHH domains are linked by amino acid linkers; and (v) 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 CH1 domain. (vi) The antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds; (vii) The immune cell targeting group comprises a VHH domain linked to the antibody CH1 domain, wherein the antibody CH1 domain is linked to the antibody light chain constant domain by one or more disulfide bonds; (vii) The CH1 domain comprises F174C and C233S substitutions, and the light chain constant domain comprises S176C and C214S substitutions, according to Kabat numbering; or any combination of (i) to (vii).

18. The LNP of any one of claims 1 to 4, wherein the immune cell targeting group comprises Fab, which comprises: (a) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 1 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 2 or 3; or (b) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO:

7.

19. The LNP as claimed in claim 8, wherein (i) the immune cell is an NK cell and the immune cell targeting group comprises an antibody binding CD56; (ii) the immune cell targeting group comprises an antibody binding CD7 or CD8, and the free PEG lipid is DMG-PEG; (iii) the LNP binds CD3 and also binds CD11a or CD18; (iv) the LNP binds CD7 and CD8 of the immune cell; (v) 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; (vi) the immune cell targeting group comprises a single antibody binding to CD3 or CD7; (vii) the immune cell targeting group binds to CD7, CD8, or both CD7 and CD8; (viii) the immune cell targeting group binds to: (a) both CD3 and CD56; (b) both CD8 and CD56; or (c) Both CD7 and CD56; or (ix) the immune cell targeting group comprising Fab lacking natural interchain disulfide bonds; or any combination of (i) to (ix).

20. Use of a lipid nanoparticle (LNP) as claimed in any one of claims 1 to 19 for the preparation of a medicament for the purpose of delivering nucleic acids to immune cells of a subject, expressing a target peptide in the target immune cells of a subject, or modulating the cellular function of the target immune cells of a subject.

21. Use of any of claims 1 to 19 of the lipid nanoparticles (LNPs) for the preparation of a medicament for treating, improving or preventing symptoms of a disorder or disease in a subject of need, wherein a nucleic acid is delivered to the subject's immune cells and the nucleic acid modulates the immune response of the immune cells, thereby treating or improving the symptoms.

22. The use as claimed in claim 21, wherein (i) the barrier is an immune disorder, an inflammatory disorder, or cancer; (ii) the nucleic acid encodes an antigen for use in a therapeutic or preventive vaccine for treating or preventing infection by a pathogen; or (iii) the antibody is a human or humanized antibody; or any combination of (i) to (iii).

23. A compound of formula I: (Formula I), or a salt thereof, wherein: R1 and R2 are independently C1-3 alkyl groups, or R1 and R2 together with a nitrogen atom form an optionally substituted piperidinyl or morpholinyl group; Y is selected from the group consisting of -O-, -OC(O)-, -OC(S)-, and -CH2-; X1, X2, X3, and X4 are hydrogens, or X1 and X2 or X3 and X4 independently form a side oxygen group; n is 0 or 3; o and p are independently integers selected from 2 to 6; wherein the compound or its salt is not a compound selected from the group consisting of , , , , and , or its salt.

Images