Chimeric antigen receptor (CAR) comprising an Anti-CD19 VHH as targeting domain

Abstract

The present disclosure relates generally to compositions and methods for in vivo gene therapy, and more specifically to delivering mRNA encoding anti-CD19 chimeric antigen receptors (CARs) to immune cells (e.g., T cells, such as CD8+ T cells) in vivo.

Description

Attorney Reference: 15979-20208.40IN VIVO T CELL GENE EDITINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 753,894, filed February 4, 2025, and U.S. Provisional Application No. 63 / 796,975, filed April 29, 2025, the contents of each which are incorporated herein by reference in their entireties.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (159792020840seqlist.xml; Size: 786,216 bytes; and Date of Creation: February 3, 2026) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates generally to compositions and methods for in vivo gene therapy, and more specifically to delivering mRNA-based therapeutics to immune cells in vivo.BACKGROUND

[0004] Genetic modification of T cells with chimeric antigen receptors (CARs) to target specific diseases has shown impressive clinical responses in patients with hematologic malignancies. However, several barriers remain before this therapy is available to a broader patient population. Currently, CAR-T cell therapy production is carried out ex vivo, including genetic modification of the patient’ s T cells in culture before infusing the cells back into the patient. The ex vivo methods required to generate sufficient numbers of tumor- specific T cells are complex thereby hindering widespread application to treat cancer patients. Finally, CAR T cell therapy results in acute and chronic toxicides that limit its overall therapeutic index in cancer patients [5]. The onset of immune activation, known as cytokine release syndrome (CRS), is the most prevalent adverse effect following CAR T cell infusion. In order to overcome these significant challenges and limitations, more innovative strategies are required to program T cells to express CARs.

[0005] In-vitro transcribed (IVT) mRNA has, in recent years, proven an effective technology to express therapeutic proteins and antigens in cells, making it a promising1MF-366657479Attorney Reference: 15979-20208.40alternative to DNA-based products. By utilizing IVT mRNA, researchers allow for transient protein expression without causing integration into the genome, and do not require nuclear import or slow cytoplasmic diffusion. However, to function in vivo, effective and stable delivery platforms, protecting the mRNA from degradation and allowing for specific target cell uptake, are required. One such delivery system that has entered the clinic and proven successful is lipid nanoparticles (LNPs), employing an ionizable cationic lipid to condense nucleic acids into a relatively uniform solid lipid nanoparticle approximating 100 nm in diameter. Remarkably, the recent authorization of two coronavirus (COVID- 19) vaccines utilizing LNPs to deliver mRNA intramuscularly stands out as noticeable examples. However, to date, the use and design of LNPs for systemic delivery have primarily allowed for cellular uptake by hepatocytes and Kupffer cells of the liver. Delivery of a nuclei acid payload toward a cell type of interest may be achieved with the addition of targeting ligands.

[0006] Thus, what is needed is a delivery platform that overcomes some of the barriers in existing T cell therapies. More particularly, what is needed is a delivery platform that overcomes some of the barriers in existing CAR T cell therapies for B cell hematologic malignancies.BRIEF SUMMARY

[0007] The present disclosure in one aspect provides a polypeptide comprising an antiCD 19 chimeric antigen receptor (anti-CD19 CAR), wherein the anti-CD19 CAR comprises: (a) an anti-CD19 single domain antibody (sdAb or VHH) as disclosed herein; (b) a hinge domain, (c) a transmembrane domain, (d) an intracellular signaling domain, and (e) optionally a co- stimulatory domain. The present disclosure in another aspect provides anti-CD19 single domain antibodies (sdAbs or VHH antibodies) as disclosed herein. The sdABs of the disclosure can be used independently (e.g., as an immunotherapy), as a component of a bispecific a multispecific antibody, for diagnostic applications, or can be used to engineer CAR-T cells.

[0008] The present disclosure in one embodiment provides a polypeptide comprising an anti-CD19 chimeric antigen receptor (anti-CD19 CAR), wherein the anti-CD19 CAR comprises: (a) an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an antiCD 19 VHH comprising: i) an H-CDR1 comprising the amino acid sequence (Kabat numbering) of SEQ ID NO:427, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 428 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID 2MF-366657479Attorney Reference: 15979-20208.40NO:429 (Kabat numbering), or (ii) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 475 (Kabat numbering), an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 476 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 477 (Kabat numbering); (b) a hinge domain, (c) a transmembrane domain, (d) an intracellular signaling domain, and (e) optionally a co-stimulatory domain.

[0009] In some embodiments according to any of the polypeptides described above, the anti-CD19 sdAb comprises a VHH comprising the amino acid sequence of SEQ ID NO: 433 or 481 , or an amino acid sequence comprising at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 433 or 481, or an amino acid sequence comprising at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 433 or SEQ ID NO: 481, or an amino acid sequence comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 433 or SEQ ID NO: 481, or an amino acid sequence comprising at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 433 or SEQ ID NO: 481, or an amino acid sequence comprising at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 433 or SEQ ID NO: 481, or an amino acid sequence comprising at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 433 or SEQ ID NO: 481.

[0010] The present disclosure in one embodiment provides a polypeptide comprising an anti-CD19 chimeric antigen receptor (anti-CD19 CAR), wherein the anti-CD19 CAR comprises: (a) an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an antiCD 19 VHH comprising: i) an H-CDR1 comprising the amino acid sequence (Kabat numbering) of SEQ ID NO: 740, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 741 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 743 (Kabat numbering), or (ii) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 740 (Kabat numbering), an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 741 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 744 (Kabat numbering); (b) a hinge domain, (c) a transmembrane domain, (d) an intracellular signaling domain, and (e) optionally a co-stimulatory domain.

[0011] In some embodiments according to any of the polypeptides described above, the anti-CD19 sdAb comprises a VHH comprising the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 85% sequence identity 3MF-366657479Attorney Reference: 15979-20208.40to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721.

[0012] The present disclosure in another embodiment provides a polypeptide comprising an anti-CD19 chimeric antigen receptor (anti-CD19 CAR), wherein the anti-CD19 CAR comprises: (a) an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an antiCD 19 VHH comprising: an H-CDR1 comprising the amino acid sequence (Kabat numbering) of SEQ ID NO: 850, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 851 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 852 (Kabat numbering); (b) a hinge domain, (c) a transmembrane domain, (d) an intracellular signaling domain, and (e) optionally a co- stimulatory domain.

[0013] In some embodiments according to any of the polypeptides described above, the anti-CD19 sdAb comprises a VHH comprising the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712, or an amino acid sequence comprising at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712, or an amino acid sequence comprising at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712, or an amino acid sequence comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712, or an amino acid sequence comprising at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712, or an amino acid sequence comprising at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712, or an amino acid sequence comprising at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 711 or SEQ ID NO: 712.

[0014] In some embodiments according to any of the polypeptides described above, the hinge domain is derived from a protein selected from the group consisting of CD28, CD5, CD9, CD2, CD27, CD28, IgG, IgK, and CD8a. In some embodiments, the hinge domain is derived from CD28 or CD8a.4MF-366657479Attorney Reference: 15979-20208.40

[0015] In some embodiments according to any of the polypeptides described above, the transmembrane domain is derived from a protein selected from the group consisting of CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD147, 4-1BB (CD137), CD152, PD-1, CD2, CD27, CDllb, CDlld, CDlla, CDllc, CD84, and CD86. In some embodiments, the transmembrane domain is derived from CD28 or CD8a.

[0016] In some embodiments according to any of the polypeptides described above, the hinge domain and the transmembrane domain are derived from the same protein. In some embodiments, the hinge domain and the transmembrane domain are derived from CD28 and comprises the amino acid sequence of SEQ ID NO: 431 or 434.

[0017] In some embodiments according to any of the polypeptides described above, the intracellular signaling domain is derived from a protein selected from the group consisting of CD3^, FceRip, FceRIy, FcRp, CD3y, CD36, CD3e, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain is derived from CD3(^. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 544.

[0018] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR further comprises a co-stimulatory domain derived from a protein selected from the group consisting of CD27, CD28, CD137 (4-1BB), 0X40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, EIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40EG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, EGALS9, CD83, ligands of CD83, and any combination thereof. In some embodiments, the co-stimulatory domain is derived from CD137 (4-1BB) or CD28. In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO: 432 or 543.

[0019] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR further comprises a signal peptide sequence that comprises a signal peptide, wherein the lead peptide sequence is connected to the N-terminus of the anti-CD19 CAR. In some embodiments, the signal peptide sequence is derived from a CD8a propeptide. In some embodiments, the signal peptide sequence comprises the amino acid sequence of SEQ ID NO: 515.

[0020] In some embodiments according to any of the polypeptides described above, the C-terminus of the anti-CD19 sdAb and the N-terminus of the hinge domain are connected by a peptide linker. In some embodiments, the peptide linker is AS or AAA.5MF-366657479Attorney Reference: 15979-20208.40

[0021] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of any one of SEQ ID NOs: 49-51 and 435-437.

[0022] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 49.

[0023] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 50.

[0024] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 51.

[0025] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 435.

[0026] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 436.

[0027] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 437.

[0028] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of any one of SEQ ID NOs: 576-623. In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of any one of SEQ ID NOs: 624-681.

[0029] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 584.

[0030] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 632.

[0031] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 585.

[0032] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 633.

[0033] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 621.

[0034] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 675.

[0035] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 591.6MF-366657479Attorney Reference: 15979-20208.40

[0036] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 601.

[0037] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 602.

[0038] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 639.

[0039] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 649.

[0040] In some embodiments according to any of the polypeptides described above, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 650.

[0041] In another aspect, there is provided an isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:46-48, 114, 145, 441-458, 482-511, and 570-575.

[0042] Also provided herein are isolated nucleic acids encoding any of the polypeptides described above, vectors comprising any of the isolated nucleic acids described herein, and cells comprising any of the vectors or isolated nucleic acids described herein or any of the polypeptides described above. In some embodiments, the isolated nucleic acid comprises or is RNA. In some embodiments, the RNA comprises or is mRNA. In some embodiments, the RNA comprises or is any one of SEQ ID NOs:570-575. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the cell is a human cell.

[0043] In another aspect, there is provided a lipid nanoparticle (LNP) comprising: (a) a lipid-immune cell targeting group conjugate comprising the compound of Formula (II): [Lipid] - [optional linker] - [binding domain], wherein the binding domain specifically binds to human CD8a, (b) an ionizable cationic lipid, and (c) a nucleic acid encoding any of the polypeptides described above, wherein the nucleic acid is encapsulated in the LNP.

[0044] In another aspect, there is provided a lipid nanoparticle (LNP) comprising: (a) a lipid-immune cell targeting group conjugate comprising the compound of Formula (II): [Lipid] - [optional linker] - [binding domain], wherein the binding domain specifically binds to human CD8a, (b) an ionizable cationic lipid, and (c) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:46-48, 114, 145, 441-458, 482-511, and 570-575, wherein the nucleic acid is encapsulated in the LNP.

[0045] In some embodiments according to any of the LNPs described above, the binding domain is an immunoglobulin single variable domain (ISVD) that specifically binds to human 7MF-366657479Attorney Reference: 15979-20208.40CD8a, wherein the ISVD comprises a complementarity-determining region 1 (CDR1), a CDR2, and a CDR3 of an ISVD having the sequence selected from the group consisting of SEQ ID NOs: 160-179; or the binding domain is an immunoglobulin single variable domain (ISVD) that specifically binds to human CD8a, wherein the ISVD specifically binding to CD8a comprises a CDR1, a CDR2, and a CDR3 according to the Abm CDR definition, wherein CDR1 is chosen from the group consisting of: (i) SEQ ID NO: 244; and (ii) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO: 244; wherein CDR2 is chosen from the group consisting of: (i) SEQ ID NO: 246; and (ii) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO: 246; and wherein CDR3 is chosen from the group consisting of: (i) SEQ ID NO: 248; and (ii) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO: 248.

[0046] In some embodiments according to any of the LNPs described above, the binding domain specifically binding to human CD8a is covalently coupled to the Lipid in Formula (II) via a linker comprising polyethylene glycol (PEG). In some embodiments, the Lipid in Formula (II) covalently coupled to the binding domain is distearoylglycerol (DSG), distearoylphosphatidylethanolamine (DSPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), or ceramide. In some embodiments, the Lipid in Formula (II) covalently coupled to the binding domain is DSPE. In some embodiments, the PEG is PEG 3400 (PEG 3.4K).

[0047] In some embodiments according to any of the LNPs described above, the immunoglobulin single variable domain comprises SEQ ID NO: 9, SEQ ID NO: 169, or SEQ ID NO: 44.

[0048] In some embodiments according to any of the LNPs described above, the LNP further comprises a structural lipid, a neutral phospholipid, or a free PEG-lipid, or any combination thereof. In some embodiments, the structural lipid comprises or is sterol. In some embodiments, the sterol comprises or is cholesterol.

[0049] In some embodiments according to any of the LNPs described above, 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), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and sphingomyelin. In some embodiments, the neutral phospholipid comprises or is DSPC.8MF-366657479Attorney Reference: 15979-20208.40

[0050] In some embodiments according to any of the LNPs described above, the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. In some embodiments, the free PEG lipid is PEG- dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid. In some embodiments, the PEG-DAG comprises PEG-DMG, PEG-DPG, or PEG-DSG, or any combination thereof. In some embodiments, the free PEG-lipid comprises PEG-DPG. In some embodiments, the PEG-DPG comprises or is PEG 2000-DPG (DPG-PEG 2000).

[0051] In some embodiments according to any of the LNPs described above, the nucleic acid comprises or is RNA. In some embodiments, the RNA comprises or is mRNA. In some embodiments, the RNA comprises or is any one of SEQ ID NOs:570-575. In some embodiments, the mRNA comprises a 5' Cap, a 5' untranslated region (UTR), a sequence encoding a polypeptide, a 3' UTR, and optionally a polyA tail. In some embodiments, the nucleic acid comprises: (1) optionally, a 5' cap; (2) optionally, a 5' UTR region; (3) nucleotides encoding any of the polypeptides described above; (4) optionally, a 3' UTR region; and (5) optionally, a polyA tail. In some embodiments, the polypeptide encoded by the nucleic acid comprises the following formula, arranged from N-terminus to C-terminus: [Signal peptide sequence (optional)] - [anti-CD19 sdAb] - [Peptide Linker (optional)] - [Hinge domain] -[Transmembrane domain] - [Co- stimulatory domain] - [Intracellular signaling domain]. In some embodiments, the nucleic acid comprises pseudouridine. In some embodiments, the pseudouridine is Nl-methyl-pseudouridine.

[0052] In some embodiments according to any of the LNPs described above, the ionizable cationic lipid comprises a compound of Formula (I):9MF-366657479Attomey Reference: 15979-20208.40or a salt thereof, or both, wherein: R1, R2, and R3are each independently a bond or C1-3 alkylene; R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene; R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)o-ioC(0)ORal, or -(CH2)o-ioOC(0)Ra2; Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl; R3BisR3B1is C1-6 alkylene; and R3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.

[0053] In some embodiments according to any of the LNPs described above, R1, R2, and R3are each independently a bond or methylene; R1Aand R2Aare each C1-10 alkylene; R3Ais C1-5 alkylene; R1A1, R1A2, R2A1, R2A2, R3A1, and R3A2are each H; R1A3and R2A3are each C1-20 alkenyl; R3A3is -C(0)0(Ci-2o alkyl); R3Bisp3B2 R3B^p3B3R3B1is C2-4 alkylene; and R3B2and R3B3are each methyl. In some embodiments, R3B1is -(CH2)s-

[0054] In some embodiments according to any of the LNPs described above, the ionizable cationic lipid comprises10MF-366657479Attorney Reference: 15979-20208.40or a salt thereof, or both.

[0055] In some embodiments according to any of the LNPs described above, the cationic lipid has a concentration between about 10 mol% and about 60 mol% of the LNP. In some embodiments, the LNP comprises cholesterol at a concentration between about 25 mol% and about 45 mol% of the LNP. In some embodiments, the LNP comprises DSPC at a concentration between about 5 mol% and about 25% mol% of the LNP. In some embodiments, the LNP comprises DPG-PEG2K at a concentration between about 0.5 mol% and about 2.5 mol% of the LNP. In some embodiments, the LNP comprises cationic lipid at a concentration between about 49 mol% and about 50 mol% of the LNP, such as about 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, or 49.9 mol%. In some embodiments, the LNP comprises cholesterol at a concentration between about 25 mol% and about 30 mol% of the LNP, such as about 25, 26, 27, 28, 29, or 30 mol%. In some embodiments, the LNP comprises DSPC at a concentration between about 9 mol% and about 21 mol% of the LNP. In some embodiments, the LNP comprises DPG-PEG2K at a concentration between about 1.4 mol% and about 1.6 mol% of the LNP.

[0056] In some embodiments according to any of the LNPs described above, the LNP comprises cationic lipid at a concentration between about 10 and about 20 g per gram of mRNA in the LNP. In some embodiments, the LNP comprises cholesterol at a concentration between about 3.0 and about 5.0 g per gram of mRNA in the LNP. In some embodiments, the LNP comprises DSPC at a concentration between about 2.0 and about 5.0 g per gram of mRNA in the LNP. In some embodiments, the LNP comprises DPG-PEG2K at a concentration between about 1.0 and about 1.5 g per gram of mRNA in the LNP. In some embodiments, the LNP comprises a DSPE-PEG3.4-antibody conjugate at a concentration between about 0.05 to 0.1 g per gram of mRNA in the LNP.

[0057] In some embodiments according to any of the LNPs described above, (i) the cationic lipid has a concentration about 49.2 mol% of the LNP; (ii) the cholesterol has a concentration about 39.4 mol% of the LNP; (iii) the DSPC has a concentration about 9.8 mol% of the LNP; and (iv) the DPG-PEG2K has a concentration about 1.5% of the LNP.11MF-366657479Attorney Reference: 15979-20208.40

[0058] In some embodiments according to any of the LNPs described above, (i) the cationic lipid has a concentration about 49.2 mol% of the LNP; (ii) the cholesterol has a concentration about 29.3 mol% of the LNP; (iii) the DSPC has a concentration about 20.0 mol% of the LNP; and (iv) the DPG-PEG2K has a concentration about 1.5% of the LNP.

[0059] In some embodiments according to any of the LNPs described above, (i) the cationic lipid has a concentration about 14.2 g / g mRNA in the LNP; (ii) the cholesterol has a concentration about 4.64 g / g mRNA in the LNP; (iii) the DSPC has a concentration about 2.37 g / g mRNA in the LNP; (iv) the DPG-PEG2K has a concentration about 1.15 g / g mRNA in the LNP; and (v) the DSPE-PEG3.4K-anti-CD8 antibody conjugate has a concentration about 0.084 g / g to 0.15 g / g mRNA in the LNP.

[0060] Also provided herein is an in vitro transcribed mRNA derived from an isolated nucleic acid encoding any of the polypeptides described above, an immune cell comprising said in vitro transcribed mRNA, and an immune cell expressing any of the polypeptides described herein. Also provided herein is a pharmaceutical composition comprising any of the polypeptides described herein, any of the isolated nucleic acids described herein, any of the vectors described herein, any of the cells of described herein, and / or any of the LNPs described herein.

[0061] In another aspect is provided a method of preparing an LNP, comprising combining any of the isolated nucleic acids described above with a mixture of lipids.

[0062] In another aspect is provided a method of delivering a nucleic acid sequence into a human cell, comprising administering any of the pharmaceutical compositions described herein. In some embodiments, the human cell is a human immune cell. In some embodiments, the human immune cell is a human T cell (such as a human CD8+ T cell). Also provided herein is a use of any of the LNPs or pharmaceutical compositions described above for the manufacture of a medicament for delivering a nucleic acid to a target cell. In some embodiments, the target cell is an immune cell (e.g., a T cell, such as a CD8+ T cell).

[0063] In another aspect is provided a method of modulating immune response in a human subject, comprising administering any of the LNPs described above, any of the isolated nucleic acids described above, any of the vectors described above, and / or any of the cells described above to the human subject, and / or expressing any of the polypeptides described above in the human subject.

[0064] In another aspect is provided a method of treating a B cell malignancy in a human subject comprising administering any of the LNPs described above, any of the isolated nucleic acids described above, any of the vectors described above, and / or any of the cells described 12MF-366657479Attorney Reference: 15979-20208.40above to the human subject, and / or expressing any of the polypeptides described above in the human subject. Also provided herein is a use of any of the polypeptides described above, any of the isolated nucleic acids described above, any of the vectors described above, any of the cells described above, any of the LNPs described above, and / or any of the pharmaceutical compositions described above for the manufacture of a medicament for the treatment of a B cell malignancy. In some embodiments, the B cell malignancy is a hematological cancer or an autoimmune disorder. In some embodiments, the B cell malignancy is a hematological cancer, wherein the cancer is hematological cancer is a B cell lymphoma. In some embodiments, the B cell lymphoma is diffuse large B cell lymphoma (DLBCL). In some embodiments, the B cell malignancy is an autoimmune disorder selected from the group consisting of rheumatoid arthritis (RA), multiple sclerosis, type 1 diabetes mellitus (T1D), primary Sjogren’s syndrome (pSS), and systemic lupus erythematosus (SLE).

[0065] In another aspect, the disclosure provides anti-CD19 single domain antibodies (sdAbs or VHH antibodies). In some embodiments, the sdAb has an amino acid sequence selected from the group consisting of any one of SEQ ID Nos: 684-739, or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence selected from the group consisting of any one of SEQ ID Nos: 684-739.

[0066] In one embodiment, the sdAb comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (H-CDR1 to H-CDR3, respectively), wherein:H-CDR1 (AbM definition) consists of an amino acid sequence selected from: a) the amino acid sequence of GFTFSSYAMA (SEQ ID NO: 747);b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of GFTFSSYAMA (SEQ ID NO: 747); andc) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of GFTFSSYAMA (SEQ ID NO: 747);andH-CDR2 (AbM definition) consists of an amino acid sequence selected from: d) the amino acid sequence of VISESGGSVD (SEQ ID NO: 748);e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of VISLSGGSVD (SEQ ID NO: 748); andf) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of VISLSGGSVD (SEQ ID NO: 748);13MF-366657479Attorney Reference: 15979-20208.40andH-CDR3 (AbM definition) consists of an amino acid sequence selected from: g) the amino acid sequence of RPVYXiKWSERDFDY (SEQ ID NO: 838), wherein the amino acid residue Xi is selected from M, I, L and V; h) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of RPVYXiKWSERDFDY (SEQ ID NO: 838), wherein the amino acid residue Xi is selected from M, I, L and V; andi) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of RPVYXiKWSERDFDY (SEQ ID NO: 838). wherein the amino acid residue Xi is selected from M, I, L and V.

[0067] In some embodiments, the disclosure provides an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an anti-CD19 VHH comprising: i) an H-CDR1 comprising the amino acid sequence (Abm numbering) of SEQ ID NO: 747, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 748 (Abm numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 749 (Abm numbering), or (ii) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 747 (Abm numbering), an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 748 (Abm numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 750 (Abm numbering).

[0068] In another aspect, the disclosure provides anti-CD19 single domain antibodies (sdAbs or VHH antibodies). In one embodiment, the sdAb comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (H-CDR1 to H-CDR3, respectively), wherein:H-CDR1 (Kabat definition) consists of an amino acid sequence selected from: a) the amino acid sequence of SYAMA (SEQ ID NO: 740);b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SYAMA (SEQ ID NO: 740); andc) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SYAMA (SEQ ID NO: 740);andH-CDR2 (Kabat definition) consists of an amino acid sequence selected from: d) the amino acid sequence of VISLSGGSVDYX2DSVKG (SEQ ID NO: 839), wherein X2 is R or A;14MF-366657479Attorney Reference: 15979-20208.40e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of VISLSGGSVDYX2DSVKG (SEQ ID NO: 839), wherein X2is R or A; andf) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of VISLSGGSVDYX2DSVKG (SEQ ID NO: 839), wherein X2is R or A;andH-CDR3 (Kabat definition) consists of an amino acid sequence selected from: g) the amino acid sequence of RPVYXiKWSERDFDY (SEQ ID NO: 815), wherein the amino acid residue Xi is selected from M, I, L and V; h) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of RPVYXiKWSERDFDY (SEQ ID NO: 815), wherein the amino acid residue Xi is selected from M, I, L and V; andi) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of RPVYXiKWSERDFDY (SEQ ID NO: 815). wherein the amino acid residue Xi is selected from M, I, L and V.

[0069] In some embodiments, the disclosure provides an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an anti-CD19 VHH comprising: i) an H-CDR1 comprising the amino acid sequence (Kabat numbering) of SEQ ID NO: 740, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 741 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 743 (Kabat numbering), or (ii) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 740 (Kabat numbering), an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 741 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 744 (Kabat numbering).

[0070] In some embodiments according to any of the polypeptides described above, the anti-CD19 sdAb comprises a VHH comprising the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:15MF-366657479Attorney Reference: 15979-20208.40699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721, or an amino acid sequence comprising at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 699, SEQ ID NO: 739 or SEQ ID NO: 721.

[0071] In some embodiments, the disclosure provides an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an anti-CD19 VHH comprising: i) an H-CDR1 comprising the amino acid sequence (Kabat numbering) of SEQ ID NO: 850, an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 851 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 852 (Kabat numbering), or (ii) an H-CDR1 comprising the amino acid sequence of SEQ ID NO: 857 (Abm numbering), an H-CDR2 comprising the amino acid sequence of SEQ ID NO: 858 (Kabat numbering), and an H-CDR3 comprising the amino acid sequence of SEQ ID NO: 859 (Kabat numbering).DESCRIPTION OF THE FIGURES

[0072] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0073] The present application can be understood by reference to the following description taken in conjunction with the accompanying figures.

[0074] FIG. 1 depicts proton NMR spectrum of intermediate 13-11.

[0075] FIGS. 2A-2C depict proton NMR spectra of intermediates. FIG.2A depicts proton NMR spectrum of intermediate 13-1 la; FIG.2B depicts proton NMR spectrum of intermediate 13-1 lb; and FIG. 2C depicts LC-ELSD of intermediate 13-1 lb.

[0076] FIGS. 3A-3B depict the proton NMR spectrum and LC-CAD chromatogram of intermediate 13-10. FIG. 3A depicts proton NMR spectrum of intermediate 13-10; FIG. 3B depicts LC-CAD chromatogram of intermediate 13-10.

[0077] FIGS. 4A-1 and 4A-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 1. FIG. 4A-1 depicts proton NMR spectrum for Lipid 1; FIG. 4A-2 depicts the LC-CAD chromatogram of Lipid 1.16MF-366657479Attorney Reference: 15979-20208.40

[0078] FIGS. 4B-1 and 4B-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 3. FIG. 4B-1 depicts proton NMR spectrum of Lipid 3; FIG. 4B-2 depicts the LC-CAD chromatogram of Lipid 3.

[0079] FIGS. 4C-1 and 4C-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 4. FIG. 4C-1 depicts proton NMR spectrum of Lipid 4; FIG. 4C-2 depicts the LC-CAD chromatogram L of Lipid 4.

[0080] FIGS. 4D-1 and 4D-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 5A. FIG. 4D-1 depicts proton NMR spectrum of Lipid 5A; FIG. 4D-2 depicts the LC-CAD chromatogram of Lipid 5A.

[0081] FIGS. 4E-1 and 4E-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 6. FIG. 4E-1 depicts proton NMR spectrum of Lipid 6; FIG. 4E-2 depicts the LC-CAD chromatogram of Lipid 6.

[0082] FIGS. 4F-1 and 4F-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 7. FIG. 4F-1 depicts proton NMR spectrum of Lipid 7; FIG. 4F-2 depicts the LC-CAD chromatogram of Lipid 7.

[0083] FIGS. 4G-1 and 4G-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 2. FIG. 4G-1 depicts proton NMR spectrum of Lipid 2; FIG. 4G-2 depicts the LC-CAD chromatogram of Lipid 2.

[0084] FIGS. 4H-1 and 4H-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 8. FIG. 4H-1 depicts proton NMR spectrum of Lipid 8; FIG. 4H-2 depicts the LC-CAD chromatogram of Lipid 8.

[0085] FIGS. 41-1 and 41-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 9. FIG.41-1 depicts proton NMR spectrum of Lipid 9; FIG.41-2 depicts the LC-CAD chromatogram of Lipid 9.

[0086] FIGS. 4J-1 and 4J-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 10A. FIG. 4J-1 depicts proton NMR spectrum of Lipid 10A; FIG. 4J-2 depicts the LC-CAD chromatogram of Lipid 10 A.

[0087] FIGS. 4K-1 and 4K-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 11 A. FIG. 4K-1 depicts proton NMR spectrum of Lipid 11 A; FIG.4K-2 depicts the LC-CAD chromatogram of Lipid 11 A.

[0088] FIGS. 4L-1 and 4L-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 12. FIG. 4L-1 depicts proton NMR spectrum of Lipid 12; FIG. 4L-2 depicts the LC-CAD chromatogram of Lipid 12.17MF-366657479Attorney Reference: 15979-20208.40

[0089] FIGS. 4M-1 and 4M-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 13. FIG. 4M-1 depicts proton NMR spectrum of Lipid 13; FIG. 4M-2 depicts the LC-CAD chromatogram of Lipid 13.

[0090] FIGS. 4N-1 and 4N-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 15. FIG. 4N-1 depicts proton NMR spectrum of Lipid 15; FIG. 4N-2 depicts the LC-CAD chromatogram of Lipid 15.

[0091] FIGS. 40-1 and 40-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 16. FIG. 40-1 depicts proton NMR spectrum of Lipid 16; FIG. 40-2 depicts the LC-CAD of Lipid 16.

[0092] FIGS. 4P-1 and 4P-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 19. FIG. 4P-1 depicts proton NMR spectrum of Lipid 19; FIG. 4P-2 depicts the LC-ELSD chromatogram of Lipid 19.

[0093] FIGS. 4Q-1 and 4Q-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 20. FIG. 4Q-1 depicts proton NMR spectrum of Lipid 20; FIG. 4Q-2 depicts the LC-ELSD chromatogram of Lipid 20.

[0094] FIGS. 4R-1 and 4R-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 31. FIG. 4R-1 depicts proton NMR spectrum of Lipid 31; FIG. 4R-2 depicts the LC-CAD chromatogram of Lipid 31.

[0095] FIGS. 4S-1 and 4S-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 32. FIG. 4S-1 depicts proton NMR spectrum of Lipid 32; FIG. 4S-2 depicts the LC-CAD chromatogram of Lipid 32.

[0096] FIGS. 4T-1 and 4T-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 33. FIG. 4T-1 depicts proton NMR spectrum of Lipid 33; FIG. 4T-2 depicts the LC-CAD chromatogram of Lipid 33.

[0097] FIGS. 4U-1 and 4U-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 34. FIG. 4U-1 depicts proton NMR spectrum of Lipid 34; FIG. 4U-2 depicts the LC-CAD chromatogram of Lipid 34.

[0098] FIGS. 4V-1 and 4V-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 14A. FIG. 4V-1 depicts proton NMR spectrum of Lipid 14A; FIG.4V-2 depicts the LC-CAD chromatogram of Lipid 14A.

[0099] FIGS. 4W-1 and 4W-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 17A. FIG. 4W-1 depicts proton NMR spectrum of Lipid 17A; FIG.4W-2 depicts the LC-CAD chromatogram of Lipid 17A.18MF-366657479Attorney Reference: 15979-20208.40

[0100] FIGS. 4X-1 and 4X-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 18A. FIG. 4X-1 depicts proton NMR spectrum of Lipid 18A; FIG.4X-2 depicts the LC-CAD chromatogram of Lipid 18 A.

[0101] FIGS. 4Y-1 and 4Y-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 21A. FIG. 4Y-1 depicts proton NMR spectrum of Lipid 21A; FIG.4Y-2 depicts the LC-CAD chromatogram of Lipid 21 A.

[0102] FIGS. 4Z-1 and 4Z-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 22. FIG. 4Z-1 depicts proton NMR spectrum of Lipid 22; FIG. 4Z-2 depicts the LC-CAD chromatogram of Lipid 22.

[0103] FIGS. 4AA-1 and 4AA-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 23A. FIG. 4AA-1 depicts proton NMR spectrum of Lipid 23A; FIG.4AA-2 depicts the LC-CAD chromatogram of Lipid 23A.

[0104] FIGS. 4AC-1 and 4AC-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 25A. FIG. 4AC-1 depicts proton NMR spectrum of Lipid 25A; FIG.4AC-2 depicts the LC-CAD chromatogram of Lipid 25A.

[0105] FIGS. 4AE-1 and 4AE-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 27. FIG.4AE-1 depicts proton NMR spectrum of Lipid 27; FIG.4AE-2 depicts the LC-CAD chromatogram of Lipid 27.

[0106] FIGS. 4AF-1 and 4AF-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 28. FIG.4AF-1 depicts proton NMR spectrum of Lipid 28; FIG.4AF-2 depicts the LC-CAD chromatogram of Lipid 28.

[0107] FIGS. 4AG-1 and 4AG-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 29. FIG.4AG-1 depicts proton NMR spectrum of Lipid 29; FIG.4AG-2 depicts the LC-CAD chromatogram of Lipid 29.

[0108] FIGS. 4AH-1 and 4AH-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 37A. FIG. 4AH-1 depicts proton NMR spectrum of Lipid 37A; FIG.4AH-2 depicts the LC-CAD chromatogram of Lipid 37A.

[0109] FIGS. 4AI-1 and 4AI-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 19A. FIG. 4AI-1 depicts proton NMR spectrum of Lipid 19A; FIG.4AI-2 depicts the LC-CAD chromatogram of Lipid 19A.

[0110] FIGS. 4AJ-1 and 4AJ-2 depict the proton NMR spectrum and LC-CAD chromatogram of Lipid 20A. FIG. 4AJ-1 depicts proton NMR spectrum of Lipid 20A; FIG.4AJ-2 depicts the LC-CAD chromatogram of Lipid 20A.19MF-366657479Attorney Reference: 15979-20208.40

[0111] FIG. 5A depicts diameter (DLS, nm) of LNPs based on Lipid 1 to Lipid 8 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0112] FIG. 5B depicts polydispersity (DLS) of LNPs based on Lipid 1 to Lipid 8 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0113] FIG. 5C depicts charge (Zeta potential, DLS) of LNPs based on Lipid 1 to Lipid 8 in pH 5.5 MBS, pH 7.4 HBS.

[0114] FIG. 5D depicts % RNA recovery and dye accessible RNA in LNPs based on Lipid 1 to Lipid 8.

[0115] FIG. 6A depicts diameter (DLS, nm) of LNPs based on Lipids 9, 10, 11, and 15 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0116] FIG. 6B depicts polydispersity (DLS) of LNPs based on Lipids 9, 10, 11, and 15 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0117] FIG. 6C depicts charge (Zeta potential, DLS) of LNPs based on Lipids 9, 10, 11, and 15 in pH 5.5 MBS, pH7.4 HBS.

[0118] FIG. 6D depicts % RNA recovery and dye accessible RNA in LNPs based on Lipids 9, 10, 11, and 15.

[0119] FIG. 7A depicts diameter (DLS, nm) of LNPs based on Lipid 31 to Lipid 34 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0120] FIG. 7B depicts polydispersity (DLS) of LNPs based on Lipid 31 to Lipid 34 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0121] FIG. 7C depicts charge (Zeta potential, DLS) of LNPs based on Lipid 31 to Lipid 34 in pH 5.5 MBS, pH 7.4 HBS.

[0122] FIG. 7D depicts % RNA recovery and dye accessible RNA in LNPs based on Lipid 31 to Lipid 34.

[0123] FIG. 8A depicts diameter (DLS, nm) of LNPs based on Lipids 1, 3, 4, 5, 9, and 15 in pH 7.4 HBS, pH 6.5 MBS, Post inserted with aCD8 antibody conjugates TRX-2 and T8.

[0124] FIG. 8B depicts polydispersity (DLS) of LNPs based on Lipids 1, 3, 4, 5, 9, and 15 in pH 7.4 HBS, pH 6.5 MBS, Post inserted with aCD8 antibody conjugates TRX-2 and T8.

[0125] FIG. 9A depicts diameter (DLS, nm) of LNPs based on Lipids 1, 8, 9, 10, 11, and 15 in pH 7.4 HBS, pH 6.5 MBS, Post inserted with aCD8 antibody conjugates TRX-2 and T8.

[0126] FIG. 9B depicts polydispersity (DLS) of LNPs based on Lipids 1, 8, 9, 10, 11, and 15 in pH 7.4 HBS, pH 6.5 MBS, Post inserted with aCD8 antibody conjugates TRX-2 and T8.20MF-366657479Attorney Reference: 15979-20208.40

[0127] FIG. 10A depicts diameter (DLS, nm) of LNPs based on Lipid 3, 4, 33, and 34 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0128] FIG. 10B depicts polydispersity (DLS) of LNPs based on Lipid 3, 4, 33, and 34 in pH 7.4 HBS, pH 6.5 MBS, Post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0129] FIG. 10C depicts charge (Zeta potential, DLS) of LNPs based on Lipid 3, 4, 33, and 34 in pH 5.5 MBS, pH7.4 HBS, pH6.5 MBS, post antibody (aCD3, hSP34) insertion and post freeze-thaw (-80°C).

[0130] FIG. 10D depicts % RNA recovery and dye accessible RNA in LNPs based on Lipid 3, 4, 33, and 34.

[0131] FIG. 11A depicts GFP expression in primary human T cells; transfected by aCD8 (hsp34) targeted LNPs based on ALC-0315, DLin-MC3-DMA, Lipid 3, Lipid 6, and Lipid 7, stored at 4°C; % GFP+ T cells at 24 hours.

[0132] FIG. 11B depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on ALC-0315, DLin-MC3-DMA, Lipid 3, Lipid 6, and Lipid 7, after 1 freeze-thaw cycle (-80°C storage); % GFP+ T cells at 24 hours.

[0133] FIG. 11C depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on ALC-0315, DLin-MC3-DMA, Lipid 3, Lipid 6, and Lipid 7, stored at 4°C; GFP MFI in live T cells at 24 hours.

[0134] FIG. 11D depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on ALC-0315, DLin-MC3-DMA, Lipid 3, Lipid 6, and Lipid 7, after 1 freeze-thaw cycle (-80°C storage); GFP MFI in live T cells at 24 hours.

[0135] FIG. HE depicts % live T cells transfected by aCD3 (hsp34) targeted LNPs based on ALC-0315, DLin-MC3-DMA, Lipid 3, Lipid 6, and Lipid 7, after 1 freeze-thaw cycle (-80°C storage); % live T cells at 24 hours.

[0136] FIG. 12A depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, Lipid 4 stored at 4°C; % GFP+ T cells at 24 hours.

[0137] FIG. 12B depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, Lipid 4, after 1 freezethaw cycle (-80°C storage); % GFP+ T cells at 24 hours.21MF-366657479Attorney Reference: 15979-20208.40

[0138] FIG. 12C depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, Lipid 4, stored at 4°C; GFP MFI in live T cells at 24 hours.

[0139] FIG. 12D depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, Lipid 4, after 1 freezethaw cycle (-80°C storage); GFP MFI in live T cells at 24 hours.

[0140] FIG. 12E depicts % live T cells transfected with by aCD3 (hsp34) targeted LNPs based on SM-102, DLin-KC2-DMA, Lipid 3, Lipid 4, after 1 freeze-thaw cycle (-80°C storage); % live T cells at 24 hours.

[0141] FIG. 13A depicts GFP expression in primary human T cells; transfected by targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 1 (4°C stored), Lipid 3 (4°C stored), and Lipid 5 (4°C stored); % GFP+ T cells.

[0142] FIG. 13B depicts GFP expression in primary human T cells; transfected by targeted LNPs based on DLin-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycle (-80°C storage); % GFP+ T cells.

[0143] FIG. 13C depicts GFP expression in primary human T cells; transfected by targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 1 (4°C stored), Lipid 3 (4°C stored), and Lipid 5 (4°C stored), GFP MFI in live T cells.

[0144] FIG. 13D depicts GFP expression in primary human T cells; transfected by a targeted LNPs based on DLin-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycle (-80°C storage); GFP MFI in live T cells.

[0145] FIG. 13E depicts % live T cells transfected with targeted LNPs based on DLin-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 stored at -80°C.

[0146] FIG. 14A depicts GFP expression in primary human T cells; transfected by aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA, Lipid 1 (4°C stored), Lipid 8 (4°C stored), and Lipid 8 (-80°C stored); % GFP+ T cells.

[0147] FIG. 14B depicts GFP expression in primary human T cells; transfected by aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA, Lipid 1 (4°C stored), Lipid 8 (4°C stored), and Lipid 8 (-80°C stored); GFP MFI in live T cells.

[0148] FIG. 14C depicts % living cells with targeted LNPs based on DLin-KC2-DMA, Lipid 1 (4°C stored), Lipid 8 (4°C stored), and Lipid 8 (-80°C stored).

[0149] FIG. 15A depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 8 (4°C stored), Lipid 9 (4°C stored), and Lipid 10 (4°C stored); % GFP+ T cells.22MF-366657479Attorney Reference: 15979-20208.40

[0150] FIG. 15B depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 8 (-80°C stored) and Lipid 10 (-80°C stored); % GFP+ T cells.

[0151] FIG. 15C depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 8 (4°C stored), Lipid 9 (4°C stored), and Lipid 10 (4°C stored); GFP MFI in live T cells.

[0152] FIG. 15D depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 8 (-80°C stored) and Lipid 10 (-80°C stored); GFP MFI in live T cells.

[0153] FIG. 15E depicts % live T cells transfected with aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 8 (4°C stored), Lipid 9 (4°C stored), and Lipid 10 (4°C. stored); % live T cells.

[0154] FIG. 15F depicts % live T cells transfected with aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 8 (-80°C stored) and Lipid 10 (-80°C stored); % live T cells.

[0155] FIG. 16A depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), Lipid 15 (4°C stored); % GFP+ T cells.

[0156] FIG. 16B depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 15 (-80°C stored); % GFP+ T cells.

[0157] FIG. 16C depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), Lipid 15 (4°C stored), GFP MFI in live T cells.

[0158] FIG. 16D depicts GFP expression in primary human T cells; transfected by aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 15 (-80°C stored); GFP MFI in live T cells.

[0159] FIG. 16E depicts % live T cells transfected with aCD3 (hsp34) targeted LNPs based on DLin-KC2-DMA (-80° C. stored), Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), and Lipid 15 (-80°C stored).

[0160] FIG. 17A depicts GFP expression in primary human T cells; transfected by aCD8 (TRX2) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs; % GFP+ T cells.23MF-366657479Attorney Reference: 15979-20208.40

[0161] FIG. 17B depicts GFP expression in primary human T cells; transfected by aCD8 (TRX2) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs; GFP MFI in live T cells.

[0162] FIG. 17C depicts % +Dil T cell with aCD8 (TRX2) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs.

[0163] FIG. 17D depicts Dil MFI in live T cells with aCD8 (TRX2) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs.

[0164] FIG. 17E depicts % live T cells transfected with aCD8 (TRX2) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs.

[0165] FIG. 18A depicts GFP expression in primary human T cells; transfected by aCD8 (T8) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs; % GFP+ T cells.

[0166] FIG. 18B depicts GFP expression in primary human T cells; transfected by aCD8 (T8) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs; GFP MFI in live T cells.

[0167] FIG. 18C depicts % -i-Dil T cell with aCD8 (T8) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs.

[0168] FIG. 18D depicts Dil MFI in live T cells with aCD8 (T8) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs.

[0169] FIG. 18E depicts % live T cells transfected with aCD8 (T8) targeted LNPs based on Lipid 3, Lipid 4, Lipid 9, Lipid 15, and compared with the corresponding non-targeted parent LNPs.

[0170] FIG. 19A depicts GFP expression in primary human T cells; transfected by aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA, Lipid 2, Lipid 3, Lipid 31, and Lipid 32; stored at 4°C; % GFP+ T cells.

[0171] FIG. 19B depicts GFP expression in primary human T cells; transfected by aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA, Lipid 2, Lipid 3, Lipid 31, and Lipid 32; stored at 4°C; GFP MFI in live T cells.

[0172] FIG. 19C depicts % living T cells with aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA, Lipid 2, Lipid 3, Lipid 31, and Lipid 32; stored at 4°C.24MF-366657479Attorney Reference: 15979-20208.40

[0173] FIG. 20A depicts GFP expression in primary human T cells; transfected with aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (4°C stored), Lipid 33 (4°C stored), Lipid 34 (4°C stored), or transfected with aCD8 (muOKT8) targeted LNPs based on Lipid 33 (4°C stored) Lipid 34 (4°C stored); % GFP+ T cells.

[0174] FIG. 20B depicts GFP expression in primary human T cells; transfected with aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 33 (-80°C stored), Lipid 34 (-80°C stored); % GFP+ T cells.

[0175] FIG. 20C depicts GFP expression in primary human T cells; transfected with aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 33 (4°C stored), Lipid 34 (4°C stored), or transfected with aCD8 (muOKT8) targeted LNPs based on Lipid 33 (4°C stored) Lipid 34 (4°C stored); GFP MFI in live T cells.

[0176] FIG. 20D depicts GFP expression in primary human T cells; transfected with aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 33 (-80°C stored), Lipid 34 (-80°C stored); GFP MFI in live T cells.

[0177] FIG. 20E depicts % live T cells transfected with aCD3 (hSP34) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 33 (-80°C stored), Lipid 34 (-80°C stored), or transfected with aCD8 (muOKT8) targeted LNPs based on Lipid 33 (4°C stored) and Lipid 34 (4°C stored).

[0178] FIG. 21A depicts % aCD20 CAR+ T cells transfected by aCD3 (hSP34) targeted LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), and Lipid 33 (4°C stored); as illustrated by % Ml value.

[0179] FIG. 21B depicts % aCD20 CAR+ T cells transfected by aCD3 (hSP34) targeted LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored); as illustrated by % Ml value.

[0180] FIG. 21C depicts % aCD20 CAR MFI in T cells transfected by aCD3 (hSP34) targeted LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), and Lipid 33 (4°C stored).

[0181] FIG. 21D depicts % aCD20 CAR MFI in T cells transfected by aCD3 (hSP34) targeted LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored).

[0182] FIG. 21E depicts % live T cells transfected with aCD3 (hSP34) targeted aCD20 CAR LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), and Lipid 33 (4°C stored).25MF-366657479Attorney Reference: 15979-20208.40

[0183] FIG. 21F depicts % live T cells transfected with aCD3 (hSP34) targeted aCD20 CAR LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored).

[0184] FIG. 22A depicts % aCD20 CAR+ T cells (CD8 population) with aCD8 (T8) targeted LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), Lipid 33 (4°C stored), as illustrated by CD4- % Ml value.

[0185] FIG. 22B depicts aCD20 CAR MFI in T cells (CD8 population) with aCD8 (T8) targeted LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), Lipid 33 (4°C stored), as illustrated by CD4- Ml MFI value.

[0186] FIG. 22C depicts aCD20 CAR level in CD4+ T cells transfected with aCD8 (T8) targeted LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), Lipid 33 (4°C stored); as illustrated by the Ml % value.

[0187] FIG. 22D depicts aCD20 CAR level in CD4+ T cells transfected with aCD8 (T8) targeted LNPs based on DLin-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 33 (-80°C stored), Lipid 34 (-80°C stored); as illustrated by the Ml MFI value.

[0188] FIG. 22E depicts % live T cells (CD4 / CD8 populations) transfected with aCD8 (T8) targeted aCD20 CAR LNPs based on Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), Lipid 33 (4°C stored).

[0189] FIG. 23A depicts % aCD20 CAR+ T cells (CD8 population) transfected with aCD8 (T8) targeted LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored) after one Freeze-Thaw cycle, as illustrated by CD4-% Ml value.

[0190] FIG. 23B depicts aCD20 CAR MFI in T cells (CD8 population) transfected with aCD8 (T8) targeted LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored) after one Freeze-Thaw cycle, as illustrated by CD4-M1 MFI value.

[0191] FIG. 23C depicts aCD20 CAR level in CD4+ T cells transfected with aCD8 (T8) targeted LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored); as illustrated by CD4+% Ml value.

[0192] FIG. 23D depicts aCD20 CAR level in CD4+ T cells transfected with aCD8 (T8) targeted LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored); as illustrated by CD4+M1 MFI value.26MF-366657479Attorney Reference: 15979-20208.40

[0193] FIG. 23E depicts % live T cells transfected with aCD8 (T8) targeted aCD20 CAR LNPs based on Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), Lipid 33 (-80°C stored).

[0194] FIG. 24A depicts GFP expression in CD8+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by % GFP+ T cells.

[0195] FIG. 24B depicts GFP expression in CD8+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by GFP MFI.

[0196] FIG. 24C depicts GFP expression in CD4+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by % GFP+ T cells.

[0197] FIG. 24D depicts GFP expression in CD4+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by GFP MFI.

[0198] FIG. 24E depicts % Dil+CD8+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by % Dil+ T cells.

[0199] FIG. 24F depicts Dil MFI in CD8+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by Dil MFI.

[0200] FIG. 24G depicts % Dil+CD4+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by % Dil+ T cells.

[0201] FIG. 24H depicts Dil MFI in CD4+ T cells transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to vector control (mutOKT8) and un-transfected; as illustrated by Dil MFI.

[0202] FIG. 25A depicts GFP expression in NK cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by % GFP+NK cells.

[0203] FIG. 25B depicts GFP expression in NK cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or27MF-366657479Attorney Reference: 15979-20208.40DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by GFP MFI.

[0204] FIG. 25C depicts GFP expression in granulocytes in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and untransfected; as illustrated by % GFP+ granulocytes.

[0205] FIG. 25D depicts GFP expression in granulocytes in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and untransfected; as illustrated by GFP MFI.

[0206] FIG. 25E depicts GFP expression in B cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by % GFP+ B cells.

[0207] FIG. 25F depicts GFP expression in B cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by GFP MFI.

[0208] FIG. 26A depicts LNP binding to NK cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by % Dil+NK cells.

[0209] FIG. 26B depicts LNP binding to NK cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by Dil MFI.

[0210] FIG. 26C depicts LNP binding to granulocytes in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by % Dil+ granulocytes.

[0211] FIG. 26D depicts LNP binding to granulocytes in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by Dil MFI.28MF-366657479Attorney Reference: 15979-20208.40

[0212] FIG. 26E depicts LNP binding to B cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by % Dil+ B cells.

[0213] FIG. 26F depicts LNP binding to B cells in whole blood samples transfected with aCD3 (hSP34) targeted or aCD8 (TRX2) targeted LNPs based on Lipid 9, Lipid 15, or DLin-KC3-DMA compared to non-binding control (mutOKT8) and un-transfected; as illustrated by Dil MFI.

[0214] FIG. 27 depicts structures of various Fab, VHH (Nb), ScFv, Fab-ScFv and Fab-VHH hybrids.

[0215] FIG. 28A depicts GFP expression in T cells transfected with aCD3 targeted LNPs based on Lipid 9, Lipid 10, Lipid 13, Lipid 15, or DLin-KC2-DMA, stored at either 4°C or post freeze-thaw (-80°C stored), as illustrated by % GFP+ T cells.

[0216] FIG. 28B depicts GFP expression in T cells transfected with aCD3 targeted LNPs based on Lipid 9, Lipid 10, Lipid 13, Lipid 15, or DLin-KC2-DMA, stored at either 4°C or post freeze-thaw (-80°C stored), as illustrated by GFP MFI.

[0217] FIG. 28C depicts % Dil+ T cells transfected with aCD3 targeted LNPs based on Lipid 9, Lipid 10, Lipid 13, Lipid 15, or DLin-KC2-DMA, stored at either 4°C or post freezethaw (-80°C stored), as illustrated by % Dil+ T cells.

[0218] FIG. 28D depicts Dil MFI in live T cells transfected with aCD3 targeted LNPs based on Lipid 9, Lipid 10, Lipid 13, Lipid 15, or DLin-KC2-DMA, stored at either 4°C or post freeze-thaw (-80°C stored), as illustrated by Dil % MFI.

[0219] FIG. 28E depicts % live T cells transfected with aCD3 targeted LNPs based on Lipid 9, Lipid 10, Lipid 13, Lipid 15, or DLin-KC2-DMA, stored at either 4°C or post freezethaw (-80°C stored).

[0220] FIGS. 29A-29C depict % GFP+ T cells (CD4 and CD8 populations) in Blood (FIG.29A), Spleen (FIG.29B), and Liver (FIG.29C) samples (analyzed for additional cell types of interest per legend) at 24 hours post injection of GFP RNA using Lipid 15, DLin-KC3-DMA, and Lipid 9 LNP formulations and a-CD8 targeting with TRX-2 antibody.

[0221] FIGS. 30A-30C depict % Dil+ T cells (CD4 and CD8 populations) in Blood (FIG.30A), Spleen (FIG.30B), and Liver (FIG.30C) samples (analyzed for additional cell types of interest per legend) at 24 hours post injection of GFP RNA using Lipid 15 (DiLdye labelled), DLin-KC3-DMA (No DiLdye label used), and Lipid 9 LNP (DiLdye labelled) formulations and a-CD8 targeting with TRX-2 antibody.29MF-366657479Attorney Reference: 15979-20208.40

[0222] FIGS. 31A-31B provide a comparison of the CD69 signal in Jurkat T cells (FIG.31 A) and primary T cells (FIG. 31B) resulting from anti-CD19 CAR mRNA transfection and co-culture with CD19-expressing Raji cells. Untreated Jurkat T cells or primary T cells were used as a negative control (i.e., signal / noise ratio of 1). Anti-CD19 CARs were designed using any one of aCD19#l-16 VHH clones and a CD28 hinge of 40 amino acids (“AAs”), a CD8a hinge of 26 amino acids (i.e., “short”), a CD8a hinge of 26 amino acids (i.e., “intermediate”), or CD8a hinge of 66 amino acids (i.e., “long”).

[0223] FIG. 32 shows the anti-CD19 CAR protein expression levels after 24 hours in primary human CD8+ T cells resulting from CAR mRNA transfection. FMC63 was used as a benchmark control.

[0224] FIG. 33A depicts %DiI+ CD8+ T cells transfected with aCD8 targeted LNPs based on Lipids 10, 15, 16, 24, and 26 and ALC-0315 as a comparator, as illustrated by %DiI+ T cells.

[0225] FIG. 33B depicts Dil MFI of CD8+ T cells transfected with aCD8 targeted LNPs based on Lipids 10, 15, 16, 24, and 26 and ALC-0315 as a comparator, as illustrated by Dil MFI.

[0226] FIG. 33C depicts %GFP+ CD8+ T cells transfected with aCD8 targeted LNPs based on Lipids 10, 15, 16, 24, and 26 and ALC-0315 as a comparator, as illustrated by %GFP+ T cells.

[0227] FIG. 33D depicts GFP MFI of CD8+ T cells transfected with aCD8 targeted LNPs based on Lipids 10, 15, 16, 24, and 26 and ALC-0315 as a comparator, as illustrated by GFP MFI.

[0228] FIG. 34-1A depicts the viability of CD8+ T cells transfected with aCD8 targeted LNPs based on DLn-KC2-DMA, as illustrated by % Live T cells.

[0229] FIG. 34-1B depicts %DiI+ CD8+ T cells transfected with aCD8 targeted LNPs based on DLn-KC2-DMA, as illustrated by %DiI+ T cells.

[0230] FIG. 34-1C depicts Dil MFI in CD8+ T cells transfected with aCD8 targeted LNPs based on DLn-KC2-DMA, as illustrated by Dil MFI.

[0231] FIG. 34-1D depicts GFP expression in CD8+ T cells transfected with aCD8 targeted LNPs based on DLn-KC2-DMA, as illustrated by %GFP+ T cells.

[0232] FIG. 34-1E depicts GFP MFI in CD8+ T cells transfected with aCD8 targeted LNPs based on DLn-KC2-DMA, as illustrated by GFP MFI.30MF-366657479Attorney Reference: 15979-20208.40

[0233] FIG. 35A depicts Dil expression in CD8+ T cells transfected with aCD3- and aCD8-targeted LNPs based on Lipid 15, at mRNA doses of 3, 1, 0.33, 0.11, and 0.037 ug / mL, as illustrated by %DiI+ T cells.

[0234] FIG. 35B depicts Dil MFI in CD8+ T cells transfected with aCD3- and aCD8-targeted LNPs based on Lipid 15, at mRNA doses of 3, 1, 0.33, 0.11, and 0.037 ug / mL, as illustrated by Dil MFI.

[0235] FIG. 35C depicts GFP expression in CD8+ T cells transfected with aCD3- and aCD8-targeted LNPs based on Lipid 15, at mRNA doses of 3, 1, 0.33, 0.11, and 0.037 ug / mL, as illustrated by %GFP+ T cells.

[0236] FIG. 35D depicts GFP MFI in CD8+ T cells transfected with aCD3- and aCD8-targeted LNPs based on Lipid 15, at mRNA doses of 3, 1, 0.33, 0.11, and 0.037 ug / mL, as illustrated by GFP MFI.

[0237] FIG. 36A depicts GFP expression in CD8+ T cells transfected with aCD3- and aCD8-targeted LNPs based on Lipid 15, at various time points, as illustrated by Green Integrated Intensity.

[0238] FIG. 36B depicts level of LNP association (Dil signal) in CD8+ T cells transfected with aCD3- and aCD8-targeted LNPs based on Lipid 15, at various time points, as illustrated by NIR Integrated Intensity.

[0239] FIG. 37A depicts level of LNP association (Dil signal) in CD8+ T cells transfected with DLin-KC3-DMA (KC3) LNPs containing different densities of aCD8 (TRX2 and 15C01), at various dose levels, as illustrated by %DiI+ CD8+ T cells.

[0240] FIG. 37B depicts level of LNP association (Dil signal) in CD8+ T cells transfected with KC3 LNPs containing different densities of aCD8 (TRX2 and 15C01), at various dose levels, as illustrated by Dil MFI of CD8+ T cells.

[0241] FIG. 37C depicts level of GFP expression in CD8+ T cells transfected with KC3 LNPs containing different densities of aCD8 (TRX2 and 15C01), at various dose levels, as illustrated by %GFP+ CD8+ T cells.

[0242] FIG. 37D depicts level of GFP expression in CD8+ T cells transfected with KC3 LNPs containing different densities of aCD8 (TRX2 and 15C01), at various dose levels, as illustrated by GFP MFI of CD8+ T cells.

[0243] FIG. 37E depicts level of GFP expression in CD4+ T cells transfected with KC3 LNPs containing different densities of aCD8 (TRX2 and 15C01), at various dose levels, as illustrated by %GFP+ CD4+ T cells.31MF-366657479Attorney Reference: 15979-20208.40

[0244] FIG. 37F depicts level of GFP expression in CD4+ T cells transfected with KC3 LNPs containing different densities of aCD8 (TRX2 and 15C01), at various dose levels, as illustrated by GFP MFI of CD4+ T cells.

[0245] FIG. 38A depicts viability of CD8+ T cells transfected with Lipid 15 LNPs containing different variants of the 15C01 aCD8 targeting moiety, at various densities and dose levels, as illustrated by %Live T cells.

[0246] FIG. 38B depicts LNP association levels (Dil signal) of CD8+ T cells transfected with Lipid 15 LNPs containing different variants of the 15C01 aCD8 targeting moiety, at various densities and dose levels, as illustrated by %DiI+ T cells.

[0247] FIG. 38C depicts LNP association levels (Dil signal) of CD8+ T cells transfected with Lipid 15 LNPs containing different variants of the 15C01 aCD8 targeting moiety, at various densities and dose levels, as illustrated by Dil MFI of T cells.

[0248] FIG. 38D depicts GFP expression levels of CD8+ T cells transfected with Lipid 15 LNPs containing different variants of the 15C01 aCD8 targeting moiety, at various densities and dose levels, as illustrated by %GFP+ T cells.

[0249] FIG. 38E depicts GFP expression levels of CD8+ T cells transfected with Lipid 15 LNPs containing different variants of the 15C01 aCD8 targeting moiety, at various densities and dose levels, as illustrated by GFP MFI of T cells.

[0250] FIG. 39A depicts CD69 expression levels of CD8+ T cells transfected with Lipid 15 LNPs containing aCD3 (SP34) or aCD8 (15C01v8 or TRX2) targeting moieties, at various dose levels, as illustrated by CD69 MFI of T cells.

[0251] FIG. 39B illustrates a histogram of CD69 expression levels of CD8+ T cells transfected with Lipid 15 LNPs containing aCD3 (SP34) or aCD8 (15C01v8 or TRX2) targeting moieties, at a dose of 1 ug / mL mRNA.

[0252] FIG. 40 depicts mCherry expression levels of primary NHP CD8+ T cells transfected with Lipid 15 LNPs containing different aCD8 targeting moieties (15C01 and TRX2) as illustrated by mCherry MFI of T cells.

[0253] FIG. 41 illustrates the general CAR design (top panel) consisting of an anti-CD19 VHH domain connected by a linker, an extracellular hinge domain, a transmembrane domain, and an intracellular co- stimulatory domain and signaling domain. The bottom panel illustrates three different CAR cassette designs with varying intracellular co-stimulatory and / or antiCD 19 VHH domains.

[0254] FIG. 42 shows the comparison of IFN-y secretion in primary human CD8+ T cells electroporated with exemplary anti-CD19 CAR constructs and assessed by IFN-y ELISA.32MF-366657479Attorney Reference: 15979-20208.40

[0255] FIGS. 43A-43F show the levels of anti-CD19 CAR-mediated cytotoxicity of CD8+ T cells transfected with CAR-comprising LNPs against target cells at effector-to-target cell ratios (E:Ts) of 8: 1 or 1: 1. FIG.43A shows the levels of anti-CD19 CAR- mediated cytotoxicity against wild-type Nalm6 cells at effector-to-target cell ratios (E:Ts) of 8:1. FIG. 43B shows the levels of anti-CD19 CAR-mediated cytotoxicity against wild- type Nalm6 cells at effector-to-target cell ratios (E:Ts) of 1:1. FIG. 43C shows the levels of anti-CD19 CAR- mediated cytotoxicity against wild- type Raji cells at effector-to-target cell ratios (E:Ts) of 8: 1. FIG.43D shows the levels of anti-CD19 CAR- mediated cytotoxicity against wild- type Raji cells at effector-to-target cell ratios (E:Ts) of 1:1. FIG. 43E shows the levels of anti-CD19 CAR-mediated cytotoxicity against K562 cells at effector-to-target cell ratios (E:Ts) of 8:1. FIG.43F shows the levels of anti-CD19 CAR-mediated cytotoxicity against K562 cells at effector-to-target cell ratios (E:Ts) of 1:1.

[0256] FIG. 44 shows the level of anti-CD19 CAR-mediated cytotoxicity of Raji cells resulting from anti-CD19 CAR mRNA transfection by electroporation and subsequent coculture with CD19-expressing Raji cells. FMC63 was used as a benchmark control.

[0257] FIG. 45 shows the level of B cell depletion in non-human primate (NHP; i.e., rhesus macaques and cynomolgus monkeys) and human primary PBMCs. The level of B cell depletion was assessed by gating on the CD20+ cells and determining the % of dead cells via live / dead stain.

[0258] FIG. 46 shows the repeated CAR-mediated cytotoxicity of Nalm6 target cells resulting from co-culture with CD8+ T cells after being transfected with Lipid 15 LNPs comprising an anti-CD8-targeting moiety as assessed by the live cell imaging SX5 Incucyte system. Repeated addition of Nalm6 target cells to the culture was performed every 48 or 72 hours as indicated by the cell schematic beneath the x-axis.

[0259] FIG. 47 depicts the Anion Exchange Chromatography profile of VHH 15C01 -GGC after capture and subsequent polish. The product was loaded on Capto Q ImpRes (Cytiva) in a 25 mM Tirs pH7.5 buffer and eluted with the same buffer with a salt gradient from 0 to 2000 mM of NaCl.

[0260] FIG. 48 depicts the Anion Exchange Chromatography profile of VHH 15C01 -GGC after capture, a reduction step in TCEP and subsequent polish. The product was loaded on Capto Q ImpRes (Cytiva) in a 25 mM Tirs pH7.5 buffer and eluted with the same buffer with a salt gradient from 0 to 2000 mM of NaCl.

[0261] FIG. 49 depicts the Flow Cytometry gating strategy for ex vivo whole blood.

[0262] FIG. 50 depicts concept and compositions of an exemplary targeted LNP.33MF-366657479Attorney Reference: 15979-20208.40

[0263] FIG. 51 depicts exemplary method for making phospholipid-PEG-ISVD conjugate.

[0264] FIG. 52 depicts an example of using click chemistry to make phospholipid-PEG-ISVD conjugate.

[0265] FIG. 53 depicts an exemplary method for making targeted LNP through antibody conjugated lipid-PEG micelles and parent LNPs.

[0266] FIGS. 54A-54G depict Lipid 15-based LNPs show highly efficient mRNA delivery to primary human T cells in vitro and 1.5% DPG-PEG shows superior transfection in vivo.FIG. 54A shows an illustration of the LNP formulation workflow. The left panel depicts how an aqueous solution containing the mRNA and an organic solution containing the various lipids are mixed by microfluidic mixing using a NanoAssemblr Ignite. The right panel depicts how a protein-based targeting moiety conjugate is post-inserted into the non-targeted LNP to form a targeted LNP (created with BioRender.com). FIG. 54B shows the fraction of DiLpositive primary human T cells, demonstrating the level of association and binding to T cells 24 hours after incubation with 1 pg / mL CD8-targeted LNPs formulated with different ionizable lipids and encapsulating mRNA encoding GFP. FIGS. 54C-54D depict the fraction of GFP-positive T cells (FIG. 54C) and the GFP MFI (FIG. 54D) evaluating the level of protein expression in T cells 24 hours after incubation with 1 pg / mL and 0.25 pg / mL CD8-targeted LNPs formulated with different ionizable lipids and encapsulating mRNA encoding GFP. Data are mean of values in n = 2 replicates. The displayed p-values are from a two-way ANOVA. FIG. 54E depicts histograms of Dil and GFP values in one representative donor illustrating the differences between CD8-targeted LNPs based on Lipid 15 and ALC-0315. FIG. 54F shows an in vivo comparison of 1 mg / kg CD3-targeted (SP34) LNPs based on DLin-KC2-DMA (KC2) and with 2.5% DMG-PEG, DPG-PEG, and DSG-PEG, respectively, demonstrating the level of GFP-positive cells of CD4+ and CD8+ T cells, 24 hours after intravenous injection into humanized NSG mice. Data are from biologically independent mice (n = 4 in LNP-treated groups, n = 3 in vehicle group). The displayed p-values are from a two-way ANOVA. FIG.54G shows an in vivo comparison of 0.3 mg / kg CD3-targeted (SP34) LNPs based on KC2 and with different percentages of DMG-PEG, DPG-PEG, and DPPE-PEG, demonstrating the level of GFP-positive cells of CD8+ T cells, 24 hours after intravenous injection into humanized NSG mice. Data are from biologically independent mice (n = 4 in LNP groups, n = 2 in vehicle group). The displayed p-values are from an ordinary one-way ANOVA.

[0267] FIGS. 55A-55E depict aCD8 ISVD-targeted LNPs show dose-dependent and specific delivery to CD8+ cells without inducing cytokine release in vitro. FIGS. 55A-55B depicts the fraction of Dil- (FIG. 55A) and GFP-positive (FIG. 55B) primary human T cells 34MF-366657479Attorney Reference: 15979-20208.4024 hours after incubation with 0.33 |ig / mL mRNA encapsulated in targeted LNPs inserted with aCD8 (anti-CD8 Fab (TRX2) or Nb8), aCD3 (anti-CD3 Fab (SP34)), or non-binding (Fab (mutOKT8)) targeting moieties in three independent donors. Each bar represents the mean of technical replicates (n = 3). The displayed p-values are from a two-way ANOVA. FIG. 55C depicts the level of GFP expression over time in T cells evaluated with the Incucyte SX5 and illustrated as the Green Integrated Intensity (GCU). Data are from biologically independent replicates (n = 3). FIG. 55D shows the levels of IFN-y detected in the supernatants 24 hours after incubation with 0.33 pg / mL mRNA encapsulated in targeted LNPs. Each bar represents the mean of technical replicates (n = 3). FIG. 55E provides an ex vivo whole blood experiment enabling investigation of expression in Granulocytes, B cells, T cells, and NK cells by flow cytometry (left panel). Fraction of GFP-positive cells in various cell subsets 24 hours after incubation with mRNA-encapsulating LNPs inserted with the humanized aCD8 VHH (Nb8-H3) or non-binding (Fab (mutOKT8)) targeting moieties (right panel). Data are from biologically independent replicates (n = 8) (created with BioRender.com).

[0268] FIGS. 56A-56F depicts Lipid chemistry of various lipids. Chemical structures of DLin-KC2-DMA (KC2-DMA) (FIG. 56A), KC3-DMA (FIG. 56B), ALC-0315 (FIG. 56C), SM-102 (FIG. 56D), Dialkyl lipid (FIG. 56E), and phospholipid degradation (FIG. 56F).

[0269] FIGS. 57A-57C depict chemical structures of novel ionizable lipids and PEG lipids. FIG. 57A provides chemical structures of proprietary branched lipids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 24A, and 26. FIG. 57B provides chemical structures of DMG-PEG, DPG-PEG, and DSG-PEG. FIG. 57C provides chemical structures of DMPE-PEG, DPPE-PEG, and DSPE-PEG.

[0270] FIGS. 58A-58B depict size and PDI of LNPs formulated with novel ionizable lipids 1-8. FIG. 58A provides diameter (DLS, nm) of LNPs based on Lipid 1 to Lipid 8 in pH 7.4 HBS, pH 6.5 MBS, post- antibody (aCD3, SP34) insertion and post- freeze-thaw (-80°C). FIG.58B provides polydispersity (DLS) of LNPs based on Lipid 1 to Lipid 8 in pH 7.4 HBS, pH 6.5 MBS, post- antibody (aCD3, SP34) insertion and post- freeze-thaw (-80°C). Data are mean of values in n = 2 replicates

[0271] FIGS. 59A-59B depict size and PDI of LNPs formulated with novel ionizable lipids 9, 10, 11, and 15. FIG. 59A provides diameter (DLS, nm) of LNPs based on Lipids 9, 10, 11, and 15 in pH 7.4 HBS, pH 6.5 MBS, post- antibody (aCD3, SP34) insertion and post- freezethaw (-80°C). FIG.59B provides polydispersity (DLS) of LNPs based on Lipids 9, 10, 11, and 15 in pH 7.4 HBS, pH 6.5 MBS, post- antibody (aCD3, SP34) insertion and post- freeze-thaw (-80°C). Data are mean of values in n = 2 replicates.35MF-366657479Attorney Reference: 15979-20208.40

[0272] FIGS. 60A-60D depict LNP characterization of LNPs formulated with ionizable lipids 10, 15, 16, 24A, and 26. FIG. 60A provides diameter (DLS, nm) of targeted LNPs (aCD8, Nb8 VHH) based on Lipids 10, 15, 16, 24A, 26, and ALC-0315 pre- and post- freezethaw. FIG. 60B provides polydispersity of targeted LNPs (aCD8, Nb8 VHH) based on Lipids 10, 15, 16, 24A, 26, and ALC-0315 pre- and post- freeze-thaw. FIG. 60C provides zeta Potential (mV) of targeted LNPs (aCD8, Nb8 VHH) based on Lipids 10, 15, 16, 24A, 26, and ALC-0315 pre- and post- freeze-thaw in pH 5.5 MES and pH 7.4 HBS. FIG. 60D provides total mRNA recovery (ug / mL) and % dye-accessible mRNA of targeted LNPs (aCD8, Nb8 VHH) based on Lipids 10, 15, 16, 24A, 26, and ALC-0315 pre- and post- freeze-thaw.

[0273] FIGS. 61A-61E depicts transfection of human T cells with CD3-targeted LNPs based on Lipids 1, 3, and 5 pre- and post- freeze-thaw. FIG. 61 A provides %GFP+ primary human T cells after transfection with CD3-targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 1 (4°C stored), Lipid 3 (4°C stored), and Lipid 5 (4°C stored). FIG.61B provides %GFP+ primary human T cells after transfection with CD3-targeted LNPs based on DLn-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycle (-80°C storage). FIG.61C provides GFP MFI in primary human T cells after transfection with CD3-targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 1 (4°C stored), Lipid 3 (4°C stored), and Lipid 5 (4°C stored).FIG. 61D provides GFP MFI in primary human T cells after transfection with CD3-targeted LNPs based on DLn-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycle (-80°C storage). FIG. 61E provides %Live primary human T cells after transfection with CD3-targeted LNPs based on DLn-KC2-DMA, Lipid 1, Lipid 3, and Lipid 5 after freeze-thaw cycle (-80°C storage).

[0274] FIGS. 62A-62C depict transfection of human T cells with CD3-targeted LNPs based on Lipids 1 and 8 pre- and post- freeze-thaw. FIG.62A provides %GFP+ primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA, Lipid 1 (4°C stored), Lipid 8 (4°C stored), and Lipid 8 (-80°C stored). FIG.62B provides GFP MFI in primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA, Lipid 1 (4°C stored), Lipid 8 (4°C stored), and Lipid 8 (-80°C stored). FIG. 62C provides %Live primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA, Lipid 1 (4°C stored), Lipid 8 (4°C stored), and Lipid 8 (-80°C stored).

[0275] FIGS. 63A-63D depict transfection of human T cells with CD3-targeted LNPs based on Lipids 8, 9, and 10 pre- and post- freeze-thaw. FIG. 63A provides %GFP+ primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA 36MF-366657479Attorney Reference: 15979-20208.40(-80°C stored), Lipid 8 (4°C stored), Lipid 9 (4°C stored), and Lipid 10 (4°C stored). FIG.63B provides %GFP+ primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 8 (-80°C stored), and Lipid 10 (-80°C stored).FIG. 63C provides GFP MFI in primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 8 (4°C stored), Lipid 9 (4°C stored), and Lipid 10 (4°C stored). FIG.63D provides GFP MFI in primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 8 (-80°C stored), and Lipid 10 (-80°C stored).

[0276] FIGS. 64A-64E depict transfection of human T cells with CD3 -targeted LNPs based on Lipids 3, 4, 9, and 15 pre- and post- freeze-thaw. FIG.64A provides %GFP+ primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), and Lipid 15 (4°C stored). FIG. 64B provides %GFP+ primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), and Lipid 15 (-80°C stored). FIG. 64C provides GFP MFI in primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 3 (4°C stored), Lipid 4 (4°C stored), Lipid 9 (4°C stored), and Lipid 15 (4°C stored). FIG.64D provides GFP MFI in primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), and Lipid 15 (-80°C stored).FIG. 64E provides %Live primary human T cells after transfection with aCD3 (SP34) targeted LNPs based on DLn-KC2-DMA (-80°C stored), Lipid 3 (-80°C stored), Lipid 4 (-80°C stored), Lipid 9 (-80°C stored), and Lipid 15 (-80°C stored).

[0277] FIGS. 65A-65E depict transfection of human T cells with CD8-targeted vs nontargeted LNPs based on Lipids 3, 4, 9, and 15. FIG. 65A provides %GFP+ primary human T cells after transfection with aCD8 (Nb8) targeted or non-targeted LNPs based on Lipid 3, Lipid, 4, Lipid 9, and Lipid 15. FIG. 65B provides GFP MFI in primary human T cells after transfection with aCD8 (Nb8) targeted or non-targeted LNPs based on Lipid 3, Lipid, 4, Lipid 9, and Lipid 15. FIG.65C provides %DiI+ primary human T cells after transfection with aCD8 (Nb8) targeted or non-targeted LNPs based on Lipid 3, Lipid, 4, Lipid 9, and Lipid 15. FIG.65D provides Dil MFI in primary human T cells after transfection with aCD8 (Nb8) targeted or non-targeted LNPs based on Lipid 3, Lipid, 4, Lipid 9, and Lipid 15. FIG. 65E provides %Live primary human T cells after transfection with aCD8 (Nb8) targeted or non-targeted LNPs based on Lipid 3, Lipid, 4, Lipid 9, and Lipid 15.37MF-366657479Attorney Reference: 15979-20208.40

[0278] FIGS. 66A-66E depict transfection of human T cells with CD8-targeted LNPs based on Lipids 10, 15, 16, 24A, and 26 pre- and post- freeze-thaw. FIG.66A provides %GFP+ primary human T cells after transfection with aCD8 (Nb8) targeted LNPs based on Lipids 10, 15, 16, 24A, and 26 and comparator lipid ALC-0315 pre- and post- freeze-thaw. FIG. 66B provides GFP MFI in primary human T cells after transfection with aCD8 (Nb8) targeted LNPs based on Lipids 10, 15, 16, 24A, and 26 and comparator lipid ALC-0315 pre- and post- freezethaw. FIG. 66C provides %DiI+ primary human T cells after transfection with aCD8 (Nb8) targeted LNPs based on Lipids 10, 15, 16, 24A, and 26 and comparator lipid ALC-0315 pre-and post- freeze-thaw. FIG.66D provides Dil MFI in primary human T cells after transfection with aCD8 (Nb8) targeted LNPs based on Lipids 10, 15, 16, 24A, and 26 and comparator lipid ALC-0315 pre- and post- freeze-thaw. FIG. 66E provides %Live primary human T cells after transfection with aCD8 (Nb8) targeted LNPs based on Lipids 10, 15, 16, 24A, and 26 and comparator lipid ALC-0315 pre- and post- freeze-thaw.

[0279] FIGS. 67A-67F depict aCD3-targeted LNPs induce cytokine secretion and upregulation of CD69 in human T cells in vitro. FIG.67A provides the Dil MFI and FIG.67B provides GFP MFI of human T cells 24 hours after incubation with varying doses of mRNA-encapsulating LNPs inserted with aCD8 (Nb8 or CD8 Fab), aCD3 (CD3 Fab), or non-binding (Fab control) targeting moieties in 3 independent human donors. Data are from technical replicates (n = 3). FIG. 67C provides CD69 MFI of human T cells 24 hours after incubation with varying doses of mRNA-encapsulating LNPs inserted with aCD8 (Nb8 or CD8 Fab), aCD3 (CD3 Fab), or non-binding (Fab control) targeting moieties in 3 independent human donors. Data are from technical replicates (n = 3). FIG. 67D provides histograms of CD69 values in one representative donor (Donor 1) illustrating the differences between cells treated with aCD3-targeted LNPs and aCD8-targeted LNPs. FIG. 67E provides the level of DiLLNP association over time with T cells evaluated with the Incucyte SX5 and illustrated as the Near InfraRed (NIR) Integrated Intensity (NIRCU). Data are from biologically independent replicates (n = 3). FIG. 67F provides levels of TNF-a detected in the supernatants 24 hours after incubation with varying doses of mRNA-encapsulating LNPs in 3 independent human donors. Data are from technical replicates (n = 3). The displayed p-values are from a two-way ANOVA.

[0280] FIGS. 68A-68E depict comparison of LNPs inserted with humanized variants of Nb8. FIG. 68A provides the fraction of live T cells 24 hours after incubation with 1, 0.3, and 0.06 pg / mL mRNA encapsulated in targeted LNPs inserted with humanized variants of Nb8 at different densities. Each bar represents the mean of technical duplicates (n = 2). FIG. 68B 38MF-366657479Attorney Reference: 15979-20208.40provides the Dil-positive fraction and FIG. 68C provides Dil MFI of primary human T cells 24 hours after incubation with 1, 0.3, and 0.06 pg / mL mRNA encapsulated in targeted LNPs inserted with humanized variants of Nb8 at different densities. Data are mean of values in n = 2 replicates. FIG. 68D provides the GFP-positive fraction and FIG. 68 provides GFP MFI of primary human T cells 24 hours after incubation with 1, 0.3, and 0.06 g / mL mRNA encapsulated in targeted LNPs inserted with humanized variants of Nb8 at different densities. Data are mean of values in n = 2 replicates.

[0281] FIGS. 69A-69B depict the comparison of CAR expression level on NK cells and CD8+ T cells (FIG. 69A) and B cell aplasia (FIG. 69B) in the tissues (e.g., spleen) of rhesus macaques dosed with CD8-targeted LNP comprising mRNA encoding anti-CD19 CAR#1.FIG. 69A shows the percent (%) of anti-CD19 CAR#1 expression on CD8+ T cells (top panel) and NK cells (bottom panel). FIG. 69B shows the B cell count (top panel) and percent (%) of live B cells in the spleens of rhesus macaques dosed with CD8-targeted LNP comprising mRNA encoding anti-CD19 CAR#1.

[0282] FIGS. 70A-70B depict the degree of B cell depletion in spleen and axillary lymph nodes of rhesus macaques dosed with CD8-targeted LNP comprising mRNA encoding antiCD 19 CAR#1 or anti-CD19 CAR #3. FIG. 70A shows the results of immunohistochemical (IHC) staining of the spleens rhesus macaques dosed with CD8-targeted LNP comprising mRNA encoding anti-CD19 CAR#1 or anti-CD19 CAR #3 for CD19-positive cells. FIG. 70B shows shows the results of immunohistochemical (IHC) staining of the spleens of rhesus macaques dosed with CD8-targeted LNP comprising mRNA encoding anti-CD19 CAR#1 or anti-CD19 CAR #3 for CD20-positive cells.DETAILED DESCRIPTION - LIPID NANOPARTICLES

[0283] The following description sets forth exemplary methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.I. Definitions

[0284] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0285] 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 this39MF-366657479Attorney Reference: 15979-20208.40invention belongs. The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein should be construed according to the standard rules of chemical valency known in the chemical arts. In addition, when a chemical group is a diradical, for example, it is understood a that the chemical groups can be bonded to their adjacent atoms in the remainder of the structure in one or both orientations, for example, -OC(O)- is interchangeable with -C(O)O- or -OC(S)- is interchangeable with -C(S)O-.

[0286] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. In some embodiments, “one or more” is 1 or 2. In some embodiments, “one or more” is 1, 2, or 3. In some embodiments, “one or more” is 1, 2, 3, or 4. In some embodiments, “one or more” is 1, 2, 3, 4, or 5. In some embodiments, “one or more” is 1, 2, 3, 4, 5, or more.

[0287] The term “alkyl” as used herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as Ci-Ci2alkyl, Ci-Cioalkyl, or Ci-Cealkyl, respectively. In some embodiments, alkyl is optionally substituted. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3-methyl-l -butyl, 2-methyl-3-butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3-methyl-l -pentyl, 4-methyl-l-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-1 -butyl, 2-ethyl-l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc.

[0288] The term “alkylene” refers to a diradical of an alkyl group. In some embodiments, alkylene is optionally substituted. An exemplary alkylene group is -CH2CH2-.

[0289] The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0290] “Alkenyl” refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8, or 2 to 6 carbon atoms) and at least one carbon-carbon double bond. The group may be in either the cis or trans configuration (Z or E configuration) about the double bond(s). Alkenyl groups include, but are not limited to, ethenyl, propenyl (e.g., prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl), and butenyl (e.g., but-l-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2-en-1-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl).

[0291] “Alkynyl” refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one 40MF-366657479Attorney Reference: 15979-20208.40carbon-carbon triple bond. Alkynyl groups include, but are not limited to, ethynyl, propynyl (e.g., prop-l-yn-l-yl, prop-2-yn-l-yl) and butynyl (e.g., but-l-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl).

[0292] The term “oxo” is art-recognized and refers to a “=O” substituent. For example, a cyclopentane substituted with an oxo group is cyclopentanone.

[0293] The term “morpholinyl” refers to a substituent having the structure of:, which is optionally substituted.The term “piperidinyl” refers to a substituent having a structure of:>, which is optionally substituted.

[0295] 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 with 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 substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this invention are preferably those that result in the formation of stable or chemically feasible compounds. In some embodiments, “optionally substituted” is equivalent to “unsubstituted or substituted.” In some embodiments, “optionally substituted” indicates that the designated atom or group is optionally substituted with one or more substituents independently selected from optional substituents provided herein. In some embodiments, optional substituent may be selected from the group consisting of: Ci-ealkyl, cyano, halogen, -O-Ci-ealkyl, Ci-ehaloalkyl, Cs vcycloalkyl, 3- to 7-membered heterocyclyl, 5-to 6-membered heteroaryl, and phenyl. In some embodiments, optional substituent is alkyl, cyano, halogen, halo, azide, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, -CChalkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl, or heteroaryl. In some embodiments, optional substituent is -ORsl, -NRs2Rs3, -C(O)Rs4, -C(O)ORs5, C(O)NRS6RS7, -OC(O)RS8, -OC(O)ORS9, -OC(O)NRsl0Rn, -NRsl2C(O)Rs13, or -NRS14C(O)ORS15, wherein Rsl, Rs2, Rs3, Rs4, Rs5, Rs6, Rs7, Rs8, Rs9, Rsl°, Rsl1, Rsl2, Rsl3, Rsl4, and Rsl5are each independently H, Ci-6 alkyl, C3-10 cycloalkyl, Ce-14 aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl, each of which is optionally substituted.41MF-366657479Attorney Reference: 15979-20208.40

[0296] The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0297] The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, bridged cyclic (e.g., adamantyl), or spirocyclic hydrocarbon group of 3-12, 3-10, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8cycloalkyl,” derived from a cycloalkane. In some embodiments, cycloalkyl is optionally substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0298] 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, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. In some embodiments, heterocyclyl is optionally substituted. The number of ring atoms in the heterocyclyl group can be specified using Cx-Cxnomenclature where x is an integer specifying the number of ring atoms. For example, a Ci-Cvheterocyclyl group 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 heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. One example of a Csheterocyclyl is aziridinyl. Heterocycles may be, for example, mono-, bi-, or other multi-cyclic ring systems (e.g., fused, spiro, bridged bicyclic). A heterocycle may be fused to one or more aryl, partially unsaturated, or saturated rings. Heterocyclyl groups include, for example, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl, isothiazolidinyl, isooxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Unless specified otherwise, the heterocyclic ring is optionally substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido,42MF-366657479Attorney Reference: 15979-20208.40amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, oxo, phosphate, phosphonato, phosphinate, sulfate, sulfide, sulfonamide, sulfonyl and thiocarbonyl. In certain embodiments, the heterocyclyl group is not substituted, i.e., it is unsubstituted.

[0299] The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. In some embodiments, aryl is optionally substituted. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, CChalkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, or the like. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the aryl group is a 6- to 10-membered ring structure. In some embodiments, the aryl group is a C6-C14 aryl.

[0300] The term “heteroaryl” is art-recognized and refers to aromatic groups that include at least one ring heteroatom. In some embodiments, heteroaryl is optionally substituted. In certain instances, a heteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Unless specified otherwise, the heteroaryl ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, C(O)alkyl, -CChalkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, or the like. The term “heteroaryl” also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. In certain embodiments, the heteroaryl ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the 43MF-366657479Attorney Reference: 15979-20208.40heteroaryl ring is not substituted, i.e., it is unsubstituted. In certain embodiments, the heteroaryl group is a 5- to 10-membered ring structure, alternatively a 5- to 6-membered ring structure, whose ring structure includes 1, 2, 3, or 4 heteroatoms, such as nitrogen, oxygen, and sulfur.

[0301] The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety represented by the general formula -N(R10)(Rn), wherein R10and R11each independently represent hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, aryl, aralkyl, or (CH2)m-R12; or R10and R11, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R12represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, R10and R11each independently represent hydrogen, alkyl, alkenyl, or -(CH2)m-R12.

[0302] The terms “alkoxy!” or “alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto. In some embodiments, alkoxyl is optionally substituted. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH2)m-R12, where m and R12are described above. The term “haloalkoxyl” refers to an alkoxyl group that is substituted with at least one halogen. For example, -O-CH2F, -O-CHF2, -O-CF3, and the like. In certain embodiments, the haloalkoxyl is an alkoxyl group that is substituted with at least one fluoro group. In certain embodiments, the haloalkoxyl is an alkoxyl group that is substituted with from 1-6, 1-5, 1-4, 2-4, or 3 fluoro groups.

[0303] The symbol “ ~~ ” indicates a point of attachment.

[0304] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist 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 stereogenic carbon atom. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “(+)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise.44MF-366657479Attorney Reference: 15979-20208.40

[0305] Individual stereoisomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Further, enantiomers can be separated using supercritical fluid chromatographic (SFC) techniques described in the literature. Still further, stereoisomers can be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0306] Geometric isomers can also exist in the compounds of the present invention. The symbol > ” denotes a bond that may be a single, double or triple bond as described herein. The present invention encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z’ or “E” configuration wherein the terms “Z’ and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers.

[0307] Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis / trans.”

[0308] The present disclosure also embraces isotopically labeled compounds of the present disclosure which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or 45MF-366657479Attorney Reference: 15979-20208.40mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as2H,3H,13C,14C,15N,180,170,31P,32P,35S,18F, and36C1, respectively.

[0309] Certain isotopically-labeled disclosed compounds (e.g., those labeled with3H and14C) are useful in compound and / or substrate tissue distribution assays. Tritiated (i.e.,3H) and carbon- 14 (i.e.,14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labeled compounds of the present disclosure can generally be prepared by following procedures analogous to those disclosed in, e.g., the Examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0310] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0311] By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific 46MF-366657479Attorney Reference: 15979-20208.40binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0312] As used herein, the terms “subject” and “patient” refer to organisms to be treated by the methods of the present invention. Such organisms are preferably mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably humans.

[0313] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0314] As used herein, the term “pharmaceutically acceptable excipient” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.

[0315] As is known to those of skill in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the present disclosure and their pharmaceutically acceptable acid addition salts.

[0316] Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NWT, wherein W is Ci-4 alkyl, and the like.

[0317] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,47MF-366657479Attorney Reference: 15979-20208.40cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NW4+(wherein W is a C1-4 alkyl group), and the like.

[0318] Abbreviations as used herein include diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); tetrabutylammonium iodide (TBAI); l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) ; benzotriazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 9-Fluorenylmethoxycarbonyl (Fmoc), tetrabutyldimethylsilyl chloride (TBDMSC1), hydrogen fluoride (HF), phenyl (Ph), bis(trimethylsilyl)amine (HMDS), dimethylformamide (DMF); methylene chloride (DCM); tetrahydrofuran (THF); high-performance liquid chromatography (HPEC); mass spectrometry (MS), evaporative light scattering detector (EESD), electrospray (ES)); nuclear magnetic resonance spectroscopy (NMR).

[0319] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a nucleic acid, e.g., an mRNA) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. The term effective amount can be considered to include therapeutically and / or prophylactically effective amounts of a compound.

[0320] The phrase “therapeutically effective amount” as used herein means that amount of a compound (e.g., a nucleic acid, e.g., an mRNA), material, or composition comprising a compound (e.g., a nucleic acid, e.g., an mRNA) which is effective for producing some desired therapeutic effect in at least a sub-population of cells in a mammal, for example, a human, or a subject (e.g., a human subject) at a reasonable benefit / risk ratio applicable to any medical treatment.

[0321] The phrase “prophylactically effective amount” as used herein means that amount of a compound (e.g., a nucleic acid, e.g., an mRNA), material, or composition comprising a compound (e.g., a nucleic acid, e.g., an mRNA) which is effective for producing some desired prophylactic effect in at least a sub-population of cells in a mammal, for example, a human, or a subject (e.g., a human subject) by reducing, minimizing or eliminating the risk of developing 48MF-366657479Attorney Reference: 15979-20208.40a condition or the reducing or minimizing severity of a condition at a reasonable benefit / risk ratio applicable to any medical treatment.

[0322] As used herein, the terms “treat,” “treating,” and “treatment” include any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.

[0323] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0324] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0325] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0326] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.49MF-366657479Attorney Reference: 15979-20208.40

[0327] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0328] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0329] As used herein, unless otherwise indicated, the term “antibody” means any antigenbinding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen. It is understood the term encompasses an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody), including an intact antibody, antigen-binding fragment, or Fc fragment that has been modified or engineered. Examples of antigen-binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term also encompasses an immunoglobulin single variable domain, such as a VHH (including a humanized VHH), a VH (including a camelized VH, a human VH, a camelized human VH, and a dAb) or a VL.

[0330] As used here, an “antibody that binds to X” (i.e., X being a particular antigen), or “an anti-X antibody”, is an antibody that specifically recognizes the antigen X.

[0331] As used herein, a “buried interchain disulfide bond” or an “interchain buried disulfide bond” refers to a disulfide bond on a polypeptide which is not readily accessible to water soluble reducing agents, or is effectively “buried” in the hydrophobic regions of the polypeptide, such that it is unavailable to both reducing agents and for conjugation to other hydrophilic PEGs. Buried interchain disulfide bonds are further described in WO2017096361A1, which is incorporated by reference in its entirety.

[0332] As used herein, specificity of the targeted delivery by an LNP is defined by the ratio between % of a desired immune cell type that receives the delivered nucleic acid (e.g., on-target delivery), and % of an undesired immune cell type that is not meant to be the destination of the delivery, but receives the delivered nucleic acid (e.g., off-target delivery). For example,50MF-366657479Attorney Reference: 15979-20208.40the specificity is higher when more desired immune cells receive the delivered nucleic acid, while less undesired immune cells receive the delivered nucleic acid. Specificity of the targeted delivery by an LNP can also be defined by the ratio of amount of nucleic acid being delivered to the desired immune cells (e.g., on-target delivery) and amount of nucleic acid being delivered to the undesired immune cells (e.g., off-target delivery). Specificity of the delivery can be determined using any suitable method. As a non-limiting example, expression level of the nucleic acid in the desired immune cell type can be measured and compared to that of a different immune cell type that is not meant to be the destination of the delivery.

[0333] As used herein, in some embodiments, a reference LNP is an LNP that does not have the immune cell targeting group but is otherwise the same as the tested LNP. In some other embodiments, a reference LNP is an LNP that has a different ionizable cationic lipid but is otherwise the same as the tested LNP. In some embodiments, a reference LNP comprises D-Lin-MC3-DMA as the ionizable cationic lipid which is different from the ionizable cationic lipid in a tested LNP, but is otherwise the same as the tested LNP.

[0334] As used herein, a humanized antibody is an antibody which is wholly or partially of non-human origin and whose protein sequence has been modified to replace certain amino acids, for instance that occur at the corresponding position(s) in the framework regions of the VH and VL domains in a sequence of antibody from a human being, to increase its similarity to antibodies produced naturally in humans, in order to avoid or minimize an immune response in humans. For example, using techniques of genetic engineering, the variable domains of a non-human antibodies of interest may be combined with the constant domains of human antibodies. The constant domains of a humanized antibody are most of the time human CH and CL domains.

[0335] As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties.

[0336] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0337] At various places in the present specification, substituents are disclosed in groups or in ranges. It is specifically intended that the description includes each and every individual subcombination of the members of such groups and ranges. For example, the term “Ci-6 alkyl” is specifically intended to individually disclose Ci, C2, C3, C4, C5, Ce, Ci-Ce, C1-C5, C1-C4, Ci-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, an integer in the range of 0 to 40 is specifically intended to individually 51MF-366657479Attorney Reference: 15979-20208.40disclose 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 an integer in the range of 1 to 20 is 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.

[0338] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the present disclosure unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0339] As used herein, the term “pseudouridine” refers to the natural product which is a C-glycosyl pyrimidine that consists of uracil having a beta-D-ribofuranosyl residue attached at position 5 (i.e., 5-(beta-D-Ribofuranosyl)uracil). In some embodiments, the term refers to m'acphi / (1- methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the term refers to mlvP (1-methylpseudouridine). In another embodiment, the term refers to *| / m (2'-O- methylpseudouridine. In another embodiment, the term refers to m5D (5-methyldihydrouridine). In another embodiment, the term refers to m3\| / (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0340] As used herein, the terms “lipid-PEG” and “PEG-lipid” are interchangeable, referring to PEG derivatives in which PEG is attached with a lipid moiety. PEG-lipid can be used to improve circulation times for liposome encapsulated (LNP) drugs and reduce nonspecific uptakes. If the lipid is a phospholipid, the molecule can be referred as “phospholipid-PEG” or “PEG-phospholipid”. Any suitable chemistry may be used to conjugate a polypeptide to the PEG of the PEG-lipid, see Parhiz et al., Journal of Controlled Release 291:106-115, 2018; Kolb et al., Angewandte Chemie International Edition 40(ll):2004-2021, 2001; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007. For example, lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, PEG-dibenzocyclooctyne (DBCO), lipid-PEG-bromo maleimide, lipid-PEG-alkylnoic amide, PEG-alkynoic imide, and lipid-PEG-azide can be used to produce a Lipd-PEG-polypeptide conjugate.

[0341] Throughout the description, where compositions and kits are described as having, including, or comprising specific components, or where processes and methods are described 52MF-366657479Attorney Reference: 15979-20208.40as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions and kits of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0342] In some embodiments, when a domain, antibody, or sequence is derived from another domain, antibody, or sequence, then the domain, antibody, or sequence is the same as the other domain, antibody, or sequence. In some embodiments, when a domain, antibody, or sequence is derived from another domain, antibody, or sequence, the domain, antibody, or sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the other domain, antibody, or sequence. In some embodiments, when a domain, antibody, or sequence is derived from another domain, antibody, or sequence, the domain, antibody, or sequence has 3, 2, or 1 amino acid difference.II. Antibody Moieties (Immunoglobulin single variable domain)

[0343] The present disclosure provides immune cell targeting LNPs comprising an immune cell targeting group. In some embodiments, the immune cell targeting group of the LNPs as described herein comprises an antibody moiety, wherein the antibody moiety is selected from the group consisting of: a full-length antibody, an scFv, a VH, a VL, an scFv-scFv, an Fv, a Fab, a Fab’, a (Fab’)2, a minibody, a diabody, a domain antibody variant (dAb), a single domain antibody (sdAb or VHH), a camelid VHH antibody, a Nanobody® ISVD, a fibronectin 3 domain variant, an ankyrin repeat variant, and other antigen-specific binding domains derived from other protein scaffolds. In some embodiments, the immune cell targeting group of the LNPs as described herein comprises an immunoglobulin single variable domain (ISVD), such as a VHH (including a humanized VHH), a VH (including a camelized VH, a human VH, a camelized human VH, and a dAb) or a VL.

[0344] The term “immunoglobulin single variable domain” (ISVD), interchangeably used with “single variable domain,” defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g., monoclonal antibodies) or their fragments (such as Fab, Fab’, F(ab’)z, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the53MF-366657479Attorney Reference: 15979-20208.40complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab, a F(ab')2 fragment, an Fv fragment such as a disulfide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

[0345] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0346] As such, the single variable domain may be a light chain variable domain sequence (e.g., a V -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a Vu-sequence or 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 that essentially consists of the 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).

[0347] An immunoglobulin single variable domain (ISVD) can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH. In one embodiment, it is a VHH, including a camelized VH or humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.

[0348] For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody® ISVD (as defined herein and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof.

[0349] In particular, the immunoglobulin single variable domain may be a Nanobody® ISVD (such as a VHH, including a humanized VHH or camelized VH) or a suitable fragment thereof. Nanobody® and Nanobodies® is a registered trademark of Ablynx N.V.54MF-366657479Attorney Reference: 15979-20208.40

[0350] ‘VHH domains”, also known as VHHS, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin variable domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al.1993 (Nature 363: 446-448). The term “VHH domain” has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”). For a further description of VHH’S, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74: 277-302).

[0351] For the term “dAb’s” and “domain antibody”, reference is for example made to Ward et al. 1989 (Nature 341: 544), to Holt et al. 2003 (Trends Biotechnol. 21: 484); as well as to for example 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 because they are not of mammalian origin, single variable domains can be derived from certain species of shark (for example, the so-called “IgNAR domains”, see for example WO 2005 / 18629).

[0352] Typically, the generation of immunoglobulins involves the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities. Alternatively, immunoglobulins can be generated by screening of naive, immune or synthetic libraries, e.g., by phage display.

[0353] The generation of immunoglobulin sequences, such as VHHs, has been described extensively in various publications, among which 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, camelids are immunized with the target antigen in order to induce an immune response against said target antigen. The repertoire of VHHs obtained from said immunization is further screened for VHHs that bind the target antigen.

[0354] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0355] Immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences can be used herein. Also, fully human, humanized or chimeric sequences can be used in the method described herein. For example,55MF-366657479Attorney Reference: 15979-20208.40camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g., camelized dAb as described by Ward et al. 1989 (Nature 341: 544), WO 1994 / 04678, and Davis and Riechmann (1994, Febs Lett., 339:285-290; and 1996, Prot. Eng., 9:531-537) can be used herein. Moreover, the ISVDs are fused forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350) as well as to for example WO 1996 / 34103 and WO 1999 / 23221).

[0356] A “humanized VHH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g., indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the prior art (e.g., WO 2008 / 020079). Again, it should be noted that such humanized VHHS can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0357] A “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a (camelid) heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the description in the prior art (e.g., Davies and Riechman 1994, FEBS 339: 285; 1995, Biotechnol. 13: 475; 1996, Prot. Eng. 9: 531; and Riechman 1999, J. Immunol. Methods 231: 25). Such “camelizing” substitutions are inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 1994 / 04678 and Davies and Riechmann (1994 and 1996, supra). In one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, such as the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner known per 56MF-366657479Attorney Reference: 15979-20208.40se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0358] The structure of an immunoglobulin single variable domain sequence can be considered to be comprised of four framework regions (“FRs”), which are referred to in the art and herein as “Framework region 1” (“FR1”); as “Framework region 2” (“FR2”); as “Framework region 3” (“FR3”); and as “Framework region 4” (“FR4”), respectively; which framework regions are interrupted by three complementary determining regions (“CDRs”), which are referred to in the art and herein as “Complementarity Determining Region 1” (“CDR1”); as “Complementarity Determining Region 2” (“CDR2”); and as “Complementarity Determining Region 3” (“CDR3”), respectively.

[0359] The amino acid residues of an ISVD can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example Figure 2 of this publication). It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. That is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0360] In the present application, unless indicated otherwise, CDR sequences were determined according to the AbM numbering as described in Kontermann and Diibel (Eds.2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp.33-51). According to this method, FR1 of an ISVD comprises the amino acid residues at positions 1-25, CDR1 of an ISVD comprises the amino acid residues at positions 26-35, FR2 of an ISVD comprises the amino acids at positions 36-49, CDR2 of an ISVD comprises the amino acid residues at positions 50-58, FR3 of an ISVD comprises the amino acid residues at positions 59-94, CDR3 of an ISVD comprises the amino acid residues at positions 95-102, and FR4 of an ISVD comprises the amino acid residues at positions 103-113.57MF-366657479Attorney Reference: 15979-20208.40

[0361] Determination of CDR regions may also be done according to different methods. In the CDR determination according to Kabat, FR1 of an ISVD comprises the amino acid residues at positions 1-30, CDR1 of an ISVD comprises the amino acid residues at positions 31-35, FR2 of an ISVD comprises the amino acids at positions 36-49, CDR2 of an ISVD comprises the amino acid residues at positions 50-65, FR3 of an ISVD comprises the amino acid residues at positions 66-94, CDR3 of an ISVD comprises the amino acid residues at positions 95-102, and FR4 of an ISVD comprises the amino acid residues at positions 103-113.

[0362] In such an immunoglobulin sequence, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

[0363] The framework sequences are (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g., a V -sequence) and / or from a heavy chain variable domain (e.g., a Vn-sequence or VHH sequence). In one particular aspect, the framework sequences are either framework sequences that have been derived from a Vnu-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).

[0364] In particular, the framework sequences present in the ISVD sequence described herein may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a Nanobody® ISVD, such as, e.g., a VHH, including a humanized VHH or camelized VH. Non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0365] The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0366] However, it should be noted that the ISVDs described herein is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi- 58MF-366657479Attorney Reference: 15979-20208.40synthetic sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized VH sequences), as well as ISVDs that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.

[0367] Similarly, nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi- synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

[0368] Generally, Nanobody® ISVDs (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences (again as further described herein). Thus, generally, a Nanobody® ISVD can be defined as an immunoglobulin sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

[0369] In particular, a Nanobody® ISVD can be an immunoglobulin sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

[0370] More in particular, a Nanobody® ISVD can be an immunoglobulin sequence with the (general) structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4, in which FR1 to FR459MF-366657479Attorney Reference: 15979-20208.40refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A below.Table A: Hallmark Residues in Nanobody® ISVDs60MF-366657479Attorney Reference: 15979-20208.40

[0371] In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the framework regions effective in preventing or reducing binding by so-called “pre-existing antibodies” to the polypeptides. To this end, in one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD. In one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD. Accordingly, part of the present disclosure are also ISVDs and polypeptides as described above that have been sequence optimized with a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD, such as in all ISVDs. Examples of ISVDs and polypeptides that comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD are depicted in Table C-2 (SEQ ID NOs: 161-169, 171-179 and 28-36 and 44).

[0372] In one embodiment, the ISVD or polypeptide has a C-terminal end of the sequence VTVSS(X)n (SEQ ID NO: 353), in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5, and in which each X is an amino acid residue that is independently chosen. In one embodiment, the polypeptide comprises such an ISVD at its C-terminal end. In one embodiment, n is 1 or 2, such as 1. In one embodiment, X is a naturally occurring amino acid. In one embodiment, X is chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I).61MF-366657479Attorney Reference: 15979-20208.40

[0373] In another embodiment the polypeptide comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVD comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 354), VQVSS (SEQ ID NO: 355), VTVKS (SEQ ID NO: 356), VTVQS (SEQ ID NO: 357), VKVKS (SEQ ID NO: 358), VKVQS (SEQ ID NO: 359), VQVKS (SEQ ID NO: 360), or VQVQS (SEQ ID NO: 361) such that after addition of a single alanine the C-terminus of the polypeptide for example comprises the sequence VTVSSA (SEQ ID NO: 362), VKVSSA (SEQ ID NO: 363), VQVSSA (SEQ ID NO: 364), VTVKSA (SEQ ID NO: 365), VTVQSA (SEQ ID NO: 366), VKVKSA (SEQ ID NO: 367), VKVQSA (SEQ ID NO: 368), VQVKSA (SEQ ID NO: 369), or VQVQSA (SEQ ID NO: 370). In one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and comprises an extension of 1 to 5 (naturally occurring) amino acids (as defined above), such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD, such that the C-terminus of the polypeptide for example comprises the sequence VTVSSA (SEQ ID NO: 362), VKVSSA (SEQ ID NO: 363) or VQVSSA (SEQ ID NO: 364). See e.g. WO2012 / 175741 and WO2015 / 173325 for further information in this regard.

[0374] The immunoglobulin single variable domains may form part of a protein or polypeptide, which may comprise or essentially consist of one or more (at least one) immunoglobulin single variable domains and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). The term “immunoglobulin single variable domain” may also encompass such polypeptides. The one or more immunoglobulin single variable domains may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acids that can serve as a binding unit, so as to provide a monovalent, multivalent or multispecific polypeptide of the present disclosure, respectively (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. 2001 (J. Biol. Chem. 276: 7346), as well as to for example WO 1996 / 34103, WO 1999 / 23221 and WO 2010 / 115998).

[0375] The polypeptides may comprise or essentially consist of one immunoglobulin single variable domain, as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.62MF-366657479Attorney Reference: 15979-20208.40

[0376] The term “multivalent” indicates the presence of multiple ISVDs in a polypeptide. In one embodiment, the polypeptide is “bivalent”, i.e., comprises or consists of two ISVDs. In one embodiment, the polypeptide is “trivalent”, i.e., comprises or consists of three ISVDs. In another embodiment, the polypeptide is “tetravalent”, i.e. comprises or consists of four ISVDs. The polypeptide can thus be “bivalent”, “trivalent”, “tetravalent”, “pentavalent”, “hexavalent”, “heptavalent”, “octavalent”, “nonavalent”, etc., i.e., the polypeptide comprises or consists of two, three, four, five, six, seven, eight, nine, etc., ISVDs, respectively. In one embodiment the multivalent ISVD polypeptide is trivalent. In another embodiment the multivalent ISVD polypeptide is tetravalent. In still another embodiment, the multivalent ISVD polypeptide is pentavalent.

[0377] In one embodiment, the multivalent ISVD polypeptide can also be multispecific. The term “multispecific” refers to binding to multiple different target molecules (also referred to as antigens). The multivalent ISVD polypeptide can thus be “bispecific”, “trispecific”, “tetraspecific”, etc., i.e., can bind to two, three, four, etc., different target molecules, respectively.

[0378] For example, the polypeptide may be bispecific-trivalent, such as a polypeptide comprising or consisting of three ISVDs, wherein two ISVDs bind to a first target and one ISVD binds to a second target different from the first target. In another example, the polypeptide may be trispecific-tetravalent, such as a polypeptide comprising or consisting of four ISVDs, wherein one ISVD binds to a first target, two ISVDs bind to a second target different from the first target and one ISVD binds to a third target different from the first and the second target. In still another example, the polypeptide may be trispecific-pentavalent, such as a polypeptide comprising or consisting of five ISVDs, wherein two ISVDs bind to a first target, two ISVDs bind to a second target different from the first target and one ISVD binds to a third target different from the first and the second target.

[0379] In one embodiment, the multivalent ISVD polypeptide can also be multiparatopic. The term “multiparatopic” refers to binding to multiple different epitopes on the same target molecules (also referred to as antigens). The multivalent ISVD polypeptide can thus be “biparatopic”, “triparatopic”, etc., i.e., can bind to two, three, etc., different epitopes on the same target molecules, respectively.

[0380] In another aspect, the polypeptide of the present disclosure that comprises or essentially consists of one or more immunoglobulin single variable domains (or suitable fragments thereof), may further comprise one or more other groups, residues, moieties or binding units. Such further groups, residues, moieties, binding units or amino acid sequences 63MF-366657479Attorney Reference: 15979-20208.40may or may not provide further functionality to the immunoglobulin single variable domain (and / or to the polypeptide in which it is present) and may or may not modify the properties of the immunoglobulin single variable domain.

[0381] 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 chosen from the group consisting of domain antibodies, amino acids that are suitable for use as a domain antibody, single domain antibodies, amino acids that are suitable for use as a single domain antibody, “dAb”s, amino acids that are suitable for use as a dAb, VHHs (including humanized VHHs), VHs (including human VHs, camelized VHs and camelized human VHs), and VLs.

[0382] Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and / or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more immunoglobulin single variable domain so as to provide a “derivative” of the immunoglobulin single variable domain.

[0383] In another embodiment, said further residues may be effective in preventing or reducing binding by so-called “pre-existing antibodies” to the polypeptides. For this purpose, the polypeptides and constructs may contain a C-terminal extension (X)n (in which n is 1 to 10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (E) or isoleucine (I), for which reference is made to WO 2012 / 175741. Accordingly, the polypeptide may further comprise a C-terminal extension (X)n, in which n is 1 to 5, such as 1, 2, 3, 4 or 5, and in which X is a naturally occurring amino acid, preferably no cysteine.

[0384] In one embodiment, the polypeptide may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased (in vivo) half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units. In vivo half-life extension means, for example, that the polypeptide has an increased half-life in a mammal,64MF-366657479Attorney Reference: 15979-20208.40such as a human subject, after administration. Half-life can be expressed for example as tl / 2beta.

[0385] The type of groups, residues, moieties or binding units is not generally restricted and may for example be chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0386] More specifically, said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life can be chosen from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is a binding unit that can bind to human serum albumin. In one embodiment, the binding unit is an ISVD.

[0387] For example, WO 2004 / 041865 and WO 2006 / 122787 describes ISVDs binding to serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other ISVDs binding to a desired target) in order to increase the half-life of said protein. These ISVDs include the ISVDs called Alb-1 (SEQ ID NO: 52 in WO 2006 / 122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 2006 / 122787). Again, these can be used to extend the half-life of therapeutic proteins and polypeptide and other therapeutic entities or moieties. Moreover, WO 2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0388] In one embodiment, the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 (WO 2006 / 122787) and Alb-23. In one embodiment, the serum albumin binding moiety is Alb-8 or Alb-23 or its variants, as shown on pages 7-9 of WO 2012 / 175400. In one embodiment, the serum albumin binding moiety is selected from the albumin binders described in WO 2012 / 175741, WO2015 / 173325, W02017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, and WO2018 / 134234. Some serum albumin binders are also shown in Table B. In one embodiment, the serum albumin binder is AlbXOOOOl (SEQ ID NO: 334). In one embodiment the serum albumin binder is Alb23002 (SEQ ID NO: 335).

[0389] In the polypeptides described above, the one or more immunoglobulin single variable domains and the one or more groups, residues, moieties or binding units may be linked directly to each other and / or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acids, the linkers may also be an amino acid, so that the resulting polypeptide is a fusion protein or fusion polypeptide.65MF-366657479Attorney Reference: 15979-20208.40

[0390] As used herein, the term “linker” denotes a peptide that fuses together two or more ISVDs into a single molecule. The use of linkers to connect two or more (poly)peptides is well known in the art.

[0391] In some embodiments, the further exemplary peptide linkers are shown in Table B.One often used class of peptide linker are known as the “Gly-Ser” or “GS” linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 337) motif (for example, having the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more). Some often-used examples of such GS linkers are 9GS linkers (GGGGSGGGS, SEQ ID NO: 340), 15GS linkers (n=3) and 35GS linkers (n=7). Reference is for example made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sei. 27 (10): 325-330).Table B: Serum albumin binding ISVD sequences, Linker sequences and some proposed ISVD C-terminal ends (amino acids 109-112 or 109-113, according to Kabat numbering) (“ID” refers to the SEQ ID NO as used herein).66MF-366657479Attorney Reference: 15979-20208.4067MF-366657479Attorney Reference: 15979-20208.40369 VQVKSA

[0392] In one aspect, the disclosure relates to CD8 binders that can bind to and / or are directed against CD8. CD8 is a disulfide-linked dimer of a and P chain, two a chains, or two P chains (see, Weber and Cantor, Encyclopedia of Immunology, p475-478, 1998; Li et al., Front Immunol. 4:206, 2013). The most common form of CD8 is composed of a CD8-a and CD8-P chain. In mammals, the CD8 receptor acts as a co-receptor with the TCR complex. The CD8a and CD8P chains display high homology to immunoglobulin variable domains. The CD8aP and CD8aa dimers bind to MHC class I molecules and help stabilize ligand binding during antigen presentation and T cell receptor activation. In some embodiments, the CD8 binder specifically recognizes a human CD8. In some embodiments, the CD8 binder specifically recognizes a cynomolgus monkey and / or rhesus macaque CD8. In some embodiments, the CD8 binder specifically recognizes both human and cynomolgus monkey and / or rhesus macaque CD8. CD8 as used herein may be from various animal species, including human, primate, mouse, rat, rabbit, or other mammals. CD8 as used herein can be wild-type or mutant (e.g., has insertion, deletion, and / or amino acid substitution compared to wild-type). In some embodiments, the CD8 binder specifically binds to the extracellular domain of one or both of CD8a and CD8p. In some embodiments, the CD8 binder specifically binds to the extracellular domain of CD8a. In some embodiments, the CD8 binder specifically binds to the extracellular domain of CD8p.

[0393] In some embodiments, the CD8 binder comprises an anti-CD8 antibody moiety (e.g., full-length, sdAb, scFv, or Fab) that specifically binds to both human and non-human primate (such as cynomolgus monkey and / or rhesus macaque) CD8. Exemplary anti-human CD8 antibodies and antigen binding fragments thereof with cross reactivity to human and monkey CD8 include, but are not limited to, CM-T807 mouse-human chimeric monoclonal antibody (see, for example, Schmitz, J.E. et al., Am J Pathol. 154(6): 1923- 1932, 1999), REGN5054 human monoclonal antibody (see, for example, Fredriksson, F. et al., J Nucl Med. 61(1): abstract 158), 3B5 mouse monoclonal antibody (see, for example, Woolridge, L. et al., J. Immunol. 171(12):6650-6660, 2003), LT8 mouse monoclonal antibody (see, for example, Sestak, K. et al., Vet Immunol Immunopathol. 119(l-2):21-26, 2007), FK18 mouse monoclonal antibody (see, for example, Stevens, H.P.J.D. et al., J Med Primatol. 20(8):386-393, 1991), or RPA-T8 monoclonal mouse antibody (see, for example, Bozkus, C.C. et al., STAR Protoc. 2(3): 100758, 2021). Use of such anti-CD8 antibody moieties with cross- 68MF-366657479Attorney Reference: 15979-20208.40reactivity to monkey CD8 may facilitate toxicity studies in non-human primates, which can provide more relevant safety assessments for human clinical trial candidates, without having to perform toxicity studies in chimpanzees or using surrogate molecules.

[0394] In some embodiments, the anti-CD8 antibody moiety (e.g., full-length, sdAb, scFv, or Fab) is derived from an anti-CD8 antibody or antigen binding fragment thereof that does not have cross -reactivity to non-human primates. Such exemplary anti-CD8 antibody moieties include, but are not limited to, the QA18A37 mouse monoclonal antibody (see, for example, Paulikat, A.D. et al. J Innate Immun.l4(5):569-580, 2022), MEM-31 mouse monoclonal antibody (see, for example, Horejsi, V. et al., Folia Biol. 34(l):23-24, 1988), UCHT-4 mouse monoclonal antibody (see, for example, Willemsen, R.A. et al. J Immunol.177(2):991-998, 2006), and OKT8 mouse monoclonal antibody (see, for example, Shiratsuchi, H. and Tsuyuguchi, I., Clin Exp Immunol. 57(2):271-278, 1984). Further CD8 binding molecules contemplated herein include CD8 binding molecules described in US11,535,869; WO2021 / 046159; WO2019 / 023148; WO2019 / 033043; WO1993 / 008817; WO2014 / 164553; the contents of each of which are incorporated herein by reference in their entirety.

[0395] In one aspect, the disclosure also relates to ISVDs as described herein that can bind to and / or are directed against CD8a (CD8alpha) and that comprise CDR sequences that are generally as further defined herein, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such ISVDs and / or suitable fragments. In some aspect, the disclosure relates to an ISVDs comprising a sequence selected from the group consisting of SEQ ID NOs: 160 to 179. In particular, the disclosure in some specific aspects provides:(I) ISVDs that are directed against CD8a and that have at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with an ISVD comprising a sequence selected from the group consisting of SEQ ID NOs: 160 to 179;(II) ISVDs that cross-block the binding of the amino acid sequence selected from the group consisting of SEQ ID NOs: 160 to 179 to CD8a and / or that compete with at least the ISVD selected from the group consisting of SEQ ID NOs: 160 to 179 for binding to CD8a.

[0396] Such ISVDs may be as further described herein (and may for example be VHHs, including humanized VHHs, VHs, including human VHs, camelized VHs and camelized human VHs); as well as polypeptides of the disclosure that comprise one or more of such amino acid sequences (which may be as further described herein), and particularly bispecific (or69MF-366657479Attorney Reference: 15979-20208.40multispecific) polypeptides as described herein, and nucleic acid sequences that encode ISVDs and polypeptides. Such ISVDs and polypeptides do not include any naturally occurring ligands.

[0397] In some embodiments, the CD8a is derived from a mammalian animal, such as a human being. In one specific, but non-limiting aspect, the disclosure relates to an ISVD directed against CD8a, that comprises:a) the amino acid sequence selected from the group consisting of SEQ ID NOs: 160-179;b) amino acid sequences that have at least 80% amino acid identity with a sequence selected from the group consisting of SEQ ID NOs: 160-179, orc) amino acid sequences that have 3, 2, or 1 amino acid difference with a sequence selected from the group consisting of SEQ ID NOs: 160-179;or any suitable combination thereof.

[0398] In some embodiments, disclosed is an ISVD against CD8a, which consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively). In some embodiments, in such an ISVD:(I) CDR1 comprises or essentially consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering; or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering, in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering; and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering, in which any amino acid substitution is a conservative amino acid substitution; and / or said amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 181,70MF-366657479Attorney Reference: 15979-20208.40188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering.(II) CDR2 comprises or essentially consists of an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering, or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering; and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering in which any amino acid substitution is a conservative amino acid substitution; and / or said amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering.(III) CDR3 comprises or essentially consists of an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering, and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering, or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering, in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a 71MF-366657479Attorney Reference: 15979-20208.40sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering; and / or from the group consisting of amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering, in which any amino acid substitution is a conservative amino acid substitution; and / orsaid amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 4255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering.

[0399] In one embodiment, the ISVD comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 244, a CDR2 that is the amino acid sequence of SEQ ID NO: 246 and a CDR3 that is the amino acid sequence of SEQ ID NO: 248 (Abm numbering) or that comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 314, a CDR2 that is the amino acid sequence of SEQ ID NO: 316 and a CDR3 that is the amino acid sequence of SEQ ID NO: 318 (Kabat numbering).

[0400] CD8 binding ISVDs as disclosed herein may comprise one, two or all three of the CDRs explicitly listed above. In some embodiments, the anti-CD8a ISVD is selected from the ISVDs described in the tables below. In some embodiments, the anti-CD8aISVD is selected from the ISVDs that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ISVDs described Table C-l, Table C-2, and Table C-3. In some embodiments, the anti-CD8 ISVD is selected from the ISVDs that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ISVDs described in Table C-4.72MF-366657479Attorney Reference: 15979-20208.40Table C-l. Anti-CD8a ISVDs.Table C-2. Anti-CD8a ISVD full sequences73MF-366657479Attorney Reference: 15979-20208.4074MF-366657479Attorney Reference: 15979-20208.4075MF-366657479Attorney Reference: 15979-20208.40Table C-3. anti-CD8a ISVD CDRs.76MF-366657479Attorney Reference: 15979-20208.4077MF-366657479Attorney Reference No. 15979-20208.40

[0401] In some embodiments, the ISVDs as described herein may further comprise additional amino acids at either the N-terminus and / or the C-terminus. In some embodiments, the additional amino acids are at the C-terminus. In some embodiments, the additional amino acids form a tag peptide that can facilitate purification. In some embodiments, the tag peptide is a poly-histidine (such as 6 X His). In some embodiments, the additional amino acids can form a tag for downstream modification of the ISVD, such as a tag for conjugation reaction. In some embodiments, the tag for conjugation reaction comprises at least one cysteine, which can help with a downstream cysteine-based click chemistry reaction. In some embodiments, the tag for click chemistry reaction comprises GGC. In some embodiments, the additional amino acid can form both a tag for click chemistry, and a tag that can facilitate purification. In some embodiments, the tag for click chemistry reaction is GGC. For example, GGC can be added to the C-terminus of an ISVD (e.g. an anti-CD8a ISVD) as described herein, such as any ISVD of SEQ ID Nos: 160 to 179, to form new ISVD of SEQ ID Nos: 28-36 and 44. In some embodiments, the phospholipid-PEG-anti-CD8a conjugate is DSPE-PEG3.4K-A044300805_v8_GGC (a.k.a., T0347015C01v8, SEQ ID NO: 44).

[0402] In some embodiments, the ISVDs as described herein comprise at least one internal disulfide bridge. In some embodiments, the ISVDs as described herein comprise two internal disulfide bridge, such as a canonical disulfide bridge and a VHH-specific bridge (e.g., a VHH1 specific disulfide bridge). In some embodiments, the ISVD is a VHHl-type ISVD. In some embodiments, the VHHl-type ISVD has two internal disulfide bridges (e.g., 1 x canonical + 1 x exposed VHH1 -specific), and one free cysteine for conjugation at the C-terminus (e.g., a GGC linker).

[0403] In the ISVDs of the disclosure that comprise the combinations of CDRs mentioned above, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the mentioned CDRs; in which:(1) any amino acid substitution is preferably a conservative amino acid substitution; and / or(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s); and / or chosen from the group consisting of amino acid sequences that have 3, 2 or only 1 (as indicated in the preceding paragraph) “amino acid difference(s)” with the mentioned CDR(s) of one of the above amino acid sequences, in which:78MF-366657479Attorney Reference No. 15979-20208.40(1) any amino acid substitution is preferably a conservative amino acid substitution; and / or(2) said amino acid sequence preferably only contains amino acid substitutions, and no amino acid deletions or insertions, compared to the above amino acid sequence(s).

[0404] In one embodiment, the anti-CDa 8 ISVD is BDSn:Anti-CD8 BDSn Nb sequence (CDR1, CDR2, CDR3 underlined based on IMGT designation):EVOLVESGGGLVQAGGSLRLSCAASGSTFSDYGVGWFROAPGKGREFVADIDWNG EHTSYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYCAADALPYTVRKYNY WGQGTQVTVSSGGCGGHHHHHH (SEQ ID NO: 419).

[0405] In some embodiments, the anti-CD8a ISVD in a phospholipid-PEG-antibody conjugate is derived from SEQ ID NO: 419, such as listed in the Table C-4 below.Table C-4. Anti-CD8 BDSn modified ISVDs.79MF-366657479Attorney Reference No. 15979-20208.40

[0406] In some embodiments, the anti-CD8a ISVD in a phospholipid-PEG-antibody conjugate is T0347015C01, A044300805, A044300805_vl, A044300805_v2, A044300805_v3, A044300805_v4, A044300805_v5, A044300805_v6, A044300805_v7, or A044300805_v8 (e.g., SEQ ID NOs: 160 to 169), or a modified version wherein a polypeptide comprising cysteine (e.g., GGC) is added to the C-terminus of each (e.g., SEQ ID NOs: 28-36 and 44). In some embodiments, the anti-CD8a ISVD in the phospholipid-PEG-antibody conjugate is SEQ ID NO: 44.

[0407] In some embodiments, an anti-CD8a ISVD of the present disclosure binds to CD8 with an dissociation constant (KD) of 10“5to 10“12moles / liter (M) or less, and preferably 10“7to 10-12 moles / liter (M) or less and more preferably 10“8to 10“12moles / liter (M), and / or with an association constant (KA) of at least 107 M-1, preferably at least 108M-1, more preferably at least 109M-1, such as at least 1012M-1; and in particular with a KD less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The KD and KA values of the ISVD of the disclosure against CD8 can be determined in a manner known per se, for example using the assay described herein. More generally, the ISVDs described herein preferably have a dissociation constant with respect to CD8 that is as described in this paragraph.

[0408] Generally, it should be noted that the term ISVD as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the ISVD can be obtained (1) by isolating the VHH80MF-366657479Attorney Reference No. 15979-20208.40domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” (as described below) of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by “camelization” (as described below) of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelisation” of a “domain antibody” or “Dab” as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a VHH domain, VH domain and / or dAb using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail hereinbelow.

[0409] In some embodiments, the anti-CD8a ISVDs of the present disclosure do not have an amino acid sequence that is exactly the same as (i.e. as a degree of sequence identity of 100% with) the amino acid sequence of a naturally occurring VH domain, such as the amino acid sequence of a naturally occurring VH domain from a mammal, and in particular from a human being.

[0410] One class of anti-CD8a ISVDs of the disclosure comprises ISVDs with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g., indicated above). It should be noted that such humanized anti-CD8a ISVDs of the present disclosure can be obtained in any suitable manner known per se (i.e. as indicated under points (l)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0411] Another class of anti-CD8a ISVDs of the present disclosure comprises Nanobody® ISVD with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding 81MF-366657479Attorney Reference No. 15979-20208.40position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description below. Reference is also made to WO 94 / 04678. Such camelization may preferentially occur at amino acid positions which are present at the VH-VL interface and at the so-called Camelidae hallmark residues (see for example also WO 94 / 04678), as also mentioned below. In some embodiments, the VH domain or sequence that is used as a starting material or starting point for generating or designing the camelized ISVD is a VH sequence from a mammal, e.g.,VH sequence of a human being. It should be noted that such camelized ISVD of the present disclosure can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0412] For example, both “humanization” and “camelization” can be performed by providing a nucleotide sequence that encodes such a naturally occurring VHH domain or VH domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence such that the new nucleotide sequence encodes a humanized or camelized ISVD of the present disclosure, respectively, and then expressing the nucleotide sequence thus obtained in a manner known per se so as to provide the desired ISVD. Alternatively, based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, the amino acid sequence of the desired humanized or camelized ISVD of the present disclosure, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized ISVD can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleotide sequence thus obtained can be expressed in a manner known per se so as to provide the desired ISVD.

[0413] Other suitable ways and techniques for obtaining ISVDs and / or nucleotide sequences and / or nucleic acids encoding the same, starting from (the amino acid sequence of) naturally occurring VH domains or preferably VHH domains and / or from nucleotide sequences and / or nucleic acid sequences encoding the same will be clear from the skilled person, and may for example comprising combining one or more amino acid sequences and / or nucleotide sequences from naturally occurring VH domains (such as one or more FR's and / or CDR's) with one or more one or more amino acid sequences and / or nucleotide sequences from naturally occurring VHH domains (such an one or more FR's or CDR's), in a suitable manner so as to 82MF-366657479Attorney Reference No. 15979-20208.40provide (a nucleotide sequence or nucleic acid encoding) an ISVD. Also provided are compounds and constructs, and in particular proteins and polypeptides that comprise or essentially consists of at least one such amino acid sequence and / or ISVD of the disclosure (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. In some embodiments, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence and / or ISVD (and / or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence and / or ISVD.

[0414] The disclosure also encompasses any polypeptide of the present disclosure that has been glycosylated at one or more amino acid positions, usually depending on the host used to express the polypeptide. A polypeptide can comprise an amino acid sequence of an anti-CD8a ISVD of the present disclosure, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end with at least one further amino acid sequence. Such further amino acid sequence may comprise at least one further ISVD, so as to provide a polypeptide that comprises at least two, such as three, four or five, ISVDs, in which said ISVDs may optionally be linked via one or more linker sequences (as defined herein). Polypeptides of comprising the anti-CD8a ISVD of the present disclosure and one or more other ISVDs are multivalent polypeptides. In a multivalent polypeptide, the two or more ISVDs may be the same or different. For example, the two or more ISVDs in a multivalent polypeptide:• may be directed against the same antigen, i.e. against the same parts or epitopes of said antigen or against two or more different parts or epitopes of said antigen; and / or:• may be directed against the different antigens;• or a combination thereof.Thus, a bivalent polypeptide, for example:• may comprise two identical ISVDs;• may comprise a first ISVD directed against a first part or epitope of an antigen and a second ISVD directed against the same part or epitope of said antigen or against another part or epitope of said antigen;or may comprise a first ISVD directed against a first antigen and a second ISVD directed against a second antigen different from said first antigen;whereas a trivalent polypeptide of the present disclosure for example:83MF-366657479Attorney Reference No. 15979-20208.40• may comprise three identical or different ISVDs directed against the same or different parts or epitopes of the same antigen;• may comprise two identical or different ISVDs directed against the same or different parts or epitopes on a first antigen and a third ISVD directed against a second antigen different from said first antigen; or• may comprise a first ISVD directed against a first antigen, a second ISVD directed against a second antigen different from said first antigen, and a third ISVD directed against a third antigen different from said first and second antigen.

[0415] The anti-CD8a ISVDs and polypeptides as disclosed herein can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and / or therapeutic purposes (e.g., as a gene therapy). For this purpose, the nucleotide sequences encoding the anti-CD8a ISVDs or polypeptides as disclosed herein can be introduced into the cells or tissues in any suitable way, for example as such (e.g., using liposomes) or after they have been inserted into a suitable gene therapy vector (for example derived from retroviruses such as adenovirus, or parvoviruses such as adeno-associated virus). As will also be clear to the skilled person, such gene therapy may be performed in vivo and / or in situ in the body of a patient by administering a nucleic acid of the present disclosure or a suitable gene therapy vector encoding the same to the patient or to specific cells or a specific tissue or organ of the patient; or suitable cells (often taken from the body of the patient to be treated, such as explanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated in vitro with a nucleotide sequence of the present disclosure and then be suitably (reintroduced into the body of the patient. All this can be performed using gene therapy vectors, techniques and delivery systems which are well known to the skilled person, for 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, U.S. Pat. No. 5,580,859; 1 U.S. Pat. No. 5,589,5466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been described in the art.84MF-366657479Attorney Reference No. 15979-20208.40

[0416] The disclosure also encompasses compositions comprising at least one ISVD or at least one polypeptide or construct of the present technology, at least one nucleic acid molecule encoding a polypeptide of the present technology or at least one vector comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and / or compounds.

[0417] Accordingly, nucleic acid sequences encoding the anti-CD8a ISVDs as described herein, and expression construct and host cells comprising the nucleic acid sequence are also provided. Suitable host cells or host organisms are clear to the skilled person, and are for example any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. In one embodiment, the host is Pichia pastoris.

[0418] The present technology also provides a method for producing the ISVD and / or polypeptide of the present technology. The method may comprise transforming / transfecting a (non-human) host cell or host organism with a nucleic acid encoding the ISVD and / or polypeptide, expressing the ISVD and / or polypeptide in the host, optionally followed by one or more isolation and / or purification steps. In an embodiment, the method may comprise: a) expressing, in a suitable (non-human) host cell or host organism or in another suitable expression system, a nucleic acid sequence or genetic construct encoding the ISVD and / or polypeptide; optionally followed by:b) isolating and / or purifying the ISVD and / or polypeptide.

[0419] In another embodiment, the method may comprise:a) cultivating and / or maintaining a (non-human) host or host cell that is capable of expressing the ISVD and / or polypeptide of the present technology and / or that comprises a nucleic acid or a genetic construct encoding the ISVD and / or polypeptide of the present technology, under suitable circumstances that are such that said (non-human) host or host cell is expresses the ISVD and / or polypeptide of the present technology;optionally followed by:b) isolating and / or purifying the ISVD and / or polypeptide produced.

[0420] Suitable (non-human) host cells or host organisms for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific85MF-366657479Attorney Reference No. 15979-20208.40examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. In one embodiment, the host is Pichia pastoris.

[0421] Also disclosed are methods of using anti-CD8a ISVDs and polypeptides of the present disclosure.

[0422] In some embodiments, a polypeptide comprising an anti-CD8a ISVD can be used in the lipid nanoparticles of the present disclosure for delivering a nucleic acid into an immune cell, as described herein. In some embodiments, anti-CD8a ISVDs and polypeptides of the present disclosure can be used to treat a condition or a disease in a subject in need thereof. In some embodiments, such conditions or diseases include, but are not limited to, cancer, infections, immune disorders, autoimmune diseases.

[0423] Also provided is the ISVD of the present disclosure, the polypeptide or construct comprising the ISVD, the nucleic acid molecule or vector as described, or the composition comprising the ISVD, polypeptide, construct, nucleic acid molecule or vector of the present disclosure, for use in the diagnosis, prevention and / or treatment of at least one disease and / or disorder that is associated with CD8a, with its biological or pharmacological activity, and / or with the biological pathways or signaling in which CD8a is involved.

[0424] Also provided is the ISVD of the present disclosure, the polypeptide or construct comprising the ISVD, the nucleic acid molecule or vector as described, or the composition comprising the ISVD, polypeptide, construct, nucleic acid molecule or vector of the present disclosure for use in the diagnosis, prevention and / or treatment of a proliferative disease (such as cancer), an inflammatory disease, and / or an infectious disease. Therefore, also provided is the ISVD of the present disclosure, the polypeptide or construct of the present disclosure, the nucleic acid molecule or vector as described, or the composition comprising the ISVD, polypeptide, construct, nucleic acid molecule or vector of the present disclosure for use in the diagnosis, prevention and / or treatment of a proliferative disease (such as cancer), an inflammatory disease, and / or an infectious disease.

[0425] In some embodiments, a polypeptide comprising an anti-CD8a ISVD can be used in an imaging agent. In some embodiments, the imaging agent allows for the detection of human CD8, which is a specific biomarker found on the surface of a subset of T cells for diagnostic imaging of the immune system. Imaging of CD8 allows for the in vivo detection of T cell localization. Changes in T cell localization can reflect the progression of an immune response and can occur over time as a result of various therapeutic treatments or even disease states. In some embodiments, it is used for imaging T cell localization for immunotherapy.86MF-366657479Attorney Reference No. 15979-20208.40

[0426] In addition, CD8 plays a role in activating downstream signaling pathways that are important for the activation of cytolytic T cells that function to clear viral pathogens and provide immunity to tumors. CD8 positive T cells can recognize short peptides presented within the MHCI protein of antigen presenting cells. In some embodiments, a polypeptide comprising an anti-CD8a ISVD can potentiate signaling through the T cell receptor and enhance the ability of a subject 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 the CD8 target.

[0427] General methods for purifying ISVD polypeptides (such as VHs and VHHs) can be used to produce ISVD that is used to make phospholipid-PEG-ISVD (e.g., VHH) conjugate, such as those described in WO 2010 / 125187 and WO 2012 / 056000.III. Lipid Nanoparticles (LNPs) and compositions

[0428] In one aspect, provided is a lipid nanoparticle (LNP) or LNP composition. In some embodiments, the LNP or LNP composition comprises (i) a lipid-immune cell targeting group conjugate.

[0429] In some embodiments, the LNPs also comprise (ii) an ionizable cationic lipid.

[0430] In some embodiments, the LNPs further comprise (iii) a structural lipid.

[0431] In some embodiments, the LNP further comprise (iv) a neutral phospholipid.

[0432] In some embodiments, the LNPs further comprise (v) a free PEG-lipid.

[0433] In some embodiments, the LNPs further comprise (vi) a payload.

[0434] In some embodiments, the LNPs comprises (i) to (vi) and any combination thereof.

[0435] In some embodiments, the LNPs comprise a formulation as illustrated in FIG. 50.

[0436] In some embodiments, the LNP or LNP composition comprises DSPE-PEG 3.4K-anti-CD8a ISVD conjugate (lipid-immune cell targeting group conjugate), Lipid 15 (ionizable cationic lipid), mRNA (payload), cholesterol (structural lipid), DSPC (neutral phospholipid), and PEG 2000-DPG (same as DPG-PEG 2K; free PEG-lipid). In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR) comprises an anti-CD19 antibody, such as an anti-CD19 single domain antibody (sdAb or VHH). Non-limiting examples of each element are described herein.

[0437] In some embodiments, the LNP or LNP composition further comprises one or more additional components. For example, it may further comprise one or more additional87MF-366657479Attorney Reference No. 15979-20208.40components that are included in the LNP due to a manufacturing process that is used to produce the LNP. For example, as described herein, to produce a phospholipid-PEG-antibody conjugate, a phospholipid-PEG that does not have a bioconjugation linker may be included with the LNP or the LNP composition. In some embodiments, such phospholipid-PEG not having a bioconjugation linker is DSPE-PEG. In some embodiments, the DSPE-PEG has a PEG with a molecular weight smaller than the PEG in the phospholipid-PEG-antibody conjugate. In some embodiments, the DSPE-PEG has a PEG with a molecular weight of about 2.0 kDa, and the PEG in the phospholipid-PEG-antibody conjugate has a molecular weight of about 3.4 kDa.

[0438] In some embodiments, the DSPE-PEG2.0k has a concentration of less than about 0.1 mol%, 0.09 mol%, 0.08 mol%, 0.07 mol%, 0.06 mol%, 0.05 mol%, 0.04 mol%, 0.03 mol%, 0.02 mol%, 0.01 mol%, 0.009 mol%, 0.008 mol%, 0.007 mol%, 0.006 mol%, 0.005 mol%, 0.004 mol%, 0.003 mol%, 0.002 mol%, or 0.001 mol% in the LNP, or a composition comprising the LNP, excluding solvent.

[0439] In some embodiments, the DSPE-PEG2.0k has a concentration of less than about 0.1 mol%, 0.09 mol%, 0.08 mol%, 0.07 mol%, 0.06 mol%, 0.05 mol%, 0.04 mol%, 0.03 mol%, 0.02 mol%, 0.01 mol%, 0.009 mol%, 0.008 mol%, 0.007 mol%, 0.006 mol%, 0.005 mol%, 0.004 mol%, 0.003 mol%, 0.002 mol%, or 0.001 mol% in the LNP, or a composition comprising the LNP, excluding solvent. In some embodiments, the DSPE-PEG2.0k has a concentration of between about 0.01 mol% and about 0.02 mol% or between about 0.04 mol% and about 0.08 mol%, in the LNP, or a composition comprising the LNP, excluding solvent.A. Lipid-immune cell targeting group conjugates

[0440] In some embodiments, the lipid-immune cell targeting group conjugate comprises the compound of Formula (II): [Lipid] - [optional linker] - [antibody]. In some embodiments, the Lipid of Formula (II) is a phospholipid. In some embodiments, the optional linker of Formula (II) is PEG. In some embodiments, the lipid-immune cell targeting group conjugate comprises DSPE-PEG 3.4K-anti CD8 antibody conjugate. In some embodiments, the conjugate is produced from conjugating an anti-CD8a immunoglobulin single variable domain (ISVD, such as a VHH) such as those described in the “II. Immunoglobulin single variable domain” section above, and DSPE-PEG having a bioconjugation linker, such as maleimide. In some embodiments, the DSPE-PEG maleimide is DSPE-PEG3.4K-maleimide. In some embodiments, the anti-CD8a ISVD in the conjugate comprises any one of SEQ ID NOs: 16088MF-366657479Attorney Reference No. 15979-20208.40to 169 or any one of SEQ ID NOs 28-36 and 44, such as SEQ ID NO: 44, or any one of SEQ ID NOs: 10 to 27.

[0441] In some embodiments, the antibody of Formula (II) comprises an ISVD that can bind to and / or are directed against CD8a (CD8alpha) and that comprise CDR sequences that are generally as further defined herein, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such ISVDs and / or suitable fragments. In some embodiments, the antibody of Formula (II) comprises an ISVD comprising a sequence selected from the group consisting of SEQ ID NOs: 160 to 179. In some embodiments, the antibody of Formula (II) comprisesI) ISVD that is directed against CD8a and that has at least 80%, preferably at least 85%, such as 90% or 95% or more sequence identity with an ISVD comprising a sequence selected from the group consisting of SEQ ID NOs: 160 to 179; and / orII) ISVD that cross-blocks the binding of the amino acid sequence selected from the group consisting of SEQ ID NOs: 160 to 179 to CD8a and / or that compete with at least the ISVD selected from the group consisting of SEQ ID NOs: 160 to 179 for binding to CD8a.

[0442] Such ISVDs, as part of Formula (II), may be as further described herein (and may for example be VHHs, including humanized VHHs, VHs, including human VHs, camelized VHs and camelized human VHs); as well as polypeptides of the disclosure that comprise one or more of such amino acid sequences (which may be as further described herein), and particularly bispecific (or multispecific) polypeptides as described herein, and nucleic acid sequences that encode ISVDs and polypeptides. Such ISVDs and polypeptides do not include any naturally occurring ligands.

[0443] In some embodiments, the CD8a is derived from a mammalian animal, such as a human being. In some embodiments, the antibody of Formula (II) comprises an ISVD directed against CD8a, that comprises:a) the amino acid sequence selected from the group consisting of SEQ ID NOs: 160 to 179; b) amino acid sequences that have at least 80% amino acid identity with a sequence selected from the group consisting of SEQ ID NOs: 160 to 179, orc) amino acid sequences that have 3, 2, or 1 amino acid difference with a sequence selected from the group consisting of SEQ ID NOs: 160 to 179;or any suitable combination thereof.

[0444] In some embodiments, the antibody of Formula (II) comprises an ISVD against CD8a, which consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively). In some embodiments, in such an ISVD:89MF-366657479Attorney Reference No. 15979-20208.40(I) CDR1 comprises or essentially consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering;or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering, in which (I) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering; and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering, in whichany amino acid substitution is a conservative amino acid substitution; and / orsaid amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 181, 188, 195, 202, 209, 216, 223, 230, 237, and 244 according to Abm numbering, and SEQ ID NOs: 251, 258, 265, 272, 279, 286, 293, 300, 307, and 314 according to Kabat numbering. (II) CDR2 comprises or essentially consists of an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering,or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 183,90MF-366657479Attorney Reference No. 15979-20208.40190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering; and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering, and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering in whichany amino acid substitution is a conservative amino acid substitution; and / orsaid amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 183, 190, 197, 204, 211, 218, 225, 232, 239 and 246 according to Abm numbering and SEQ ID NOs: 253, 260, 267, 274, 281, 288, 295, 302, 309 and 316 according to Kabat numbering. (Ill) CDR3 comprises or essentially consists of an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering, and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering,or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering, in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering; and / or from the group consisting of amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering, in which any amino acid substitution is a conservative amino acid substitution; and / orsaid amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199, 206, 213, 220, 227, 234, 241 and 248 according to Abm numbering and SEQ ID NOs: 4255, 262, 269, 276, 283, 290, 297, 304, 311 and 318 according to Kabat numbering.91MF-366657479Attorney Reference No. 15979-20208.40

[0445] In one embodiment, the antibody of Formula (II) comprises ISVD that comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 244, a CDR2 that is the amino acid sequence of SEQ ID NO: 246 and a CDR3 that is the amino acid sequence of SEQ ID NO: 248 (Abm numbering) or that comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 314, a CDR2 that is the amino acid sequence of SEQ ID NO: 316 and a CDR3 that is the amino acid sequence of SEQ ID NO: 318 (Kabat numbering).

[0446] CD8 binding ISVDs as disclosed herein may comprise one, two or all three of the CDRs explicitly listed above. In some embodiments, the anti-CD8aISVD is selected from the ISVDs described in Table C-l, Table C-2, and Table C-3 above. In some embodiments, the anti-CD8aISVD is selected from the ISVDs that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ISVDs described in Table C-l, Table C-2, and Table C-3 above. In some embodiments, the anti-CD8ISVD is selected from the ISVDs that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ISVDs described in Table C-4 above.

[0447] In some embodiments, the lipid-immune cell targeting group conjugate is present in the LNP in a range of 0.001-0.5 mol percent, 0.001-0.1 mol%, 0.01-0.5 mol%, 0.05-0.5 mol%, 0.1-0.5 mol%, 0.1-0.3 mol%, 0.1-0.2 mol%, 0.2-0.3 mol%, of about 0.01 mol%, about 0.05 mol%, about 0.1 mol%, about 0.15 mol%, about 0.2 mol%, about 0.25 mol%, about 0.3 mol%, about 0.35 mol%, about 0.4 mol%, about 0.45 mol%, or about 0.5 mol%. In some embodiments, the lipid-immune cell targeting group conjugate is present in the LNP in a range of 0.01 to 0.03 mol percent. In some embodiment, the lipid- immune cell targeting group conjugate is present in the LNP in about 0.015 to about 0.016 mol percent.

[0448] In some embodiments, the lipid-immune cell targeting group conjugate is presented in the LNP at a density from about 0.4 to 11.4, about 1.9 to 9.5, about 3.8 to 7.6, about 4.6 to 6.5, about 5.3 to 6.1, about 3.8, or about 5.7 micromoles of conjugate per gram of mRNA in the LNP. In some embodiments, the lipid-immune cell targeting group conjugate comprises SEQ ID NO: 9, SEQ ID NO: 169, or SEQ ID NO: 44.

[0449] In one aspect, the present disclosure provides methods for producing the lipid-immune cell targeting group conjugate. In some embodiments, the lipid-immune cell targeting group conjugate comprises lipid-linker-antibody, such as a phospholipid-PEG-antibody. In some embodiments, the antibody is an ISVD. In some embodiments, the methods comprise (i) purifying the ISVD; and (ii) conjugating the ISVD to a phospholipid-PEG, such as a DSPE-PEG (e.g., DSPE-PEG3.4k-maileimide).92MF-366657479Attorney Reference No. 15979-20208.40

[0450] In some embodiments, non-limiting examples for the method of producing the conjugate is as illustrated in FIG. 51 and FIG. 52.

[0451] In some embodiments, after the production / expression of the polypeptide of an ISVD, the host cell proteins and other impurities can be removed from the culture medium by routine means. For example, the host cell proteins and other impurities can be removed by centrifugation or filtration. The solution obtained by removal of the host cell proteins and other impurities from the culture medium is also referred to as culture supernatant or clarified culture supernatant.

[0452] The ISVD mixture product can be purified from culture supernatant by methods described herein. In some embodiments, the methods include, but are not limited to chromatographic methods, including size exclusion chromatography (SEC), ion exchange chromatography (IEX), affinity chromatography (AC), hydrophobic interaction chromatography (HIC), mixed-mode chromatography (MMC). These methods can be performed alone or in combination with other purification methods, e.g., precipitation. Suitable combinations of purification methods for ISVDs and ISVD containing polypeptides can be chosen as needed.

[0453] Any or all chromatographic steps can be carried out by any suitable mechanical means. Chromatography may be carried out, for example, in a column. The column may be run with or without pressure and from top to bottom or bottom to top. The direction of the flow of fluid in the column may be reversed during the chromatography process. Chromatography may also be carried out using a batch process in which the solid media is separated from the liquid used to load, wash, and elute the sample by any suitable means, including gravity, centrifugation, or filtration.

[0454] Chromatography may also be carried out by contacting the sample with a filter that absorbs or retains some molecules in the sample more strongly than others. In the following description, the various embodiments are mostly described in the context of chromatography carried out in a column. It is understood, however, that use of a column is merely one of several chromatographic modalities that may be used, and the illustration using a column does not limit the application to column chromatography, as those skilled in the art may readily apply the teachings to other modalities as well, such as those using a batch process or filter.

[0455] Suitable supports may be any currently available or later developed materials having the characteristics necessary to practice the claimed method, and may be based on any synthetic, organic, or natural polymer. For example, commonly used support substances include organic materials such as cellulose, polystyrene, agarose, sepharose, polyacrylamide,93MF-366657479Attorney Reference No. 15979-20208.40polymethacrylate, dextran and starch, and inorganic materials, such as charcoal, silica (glass beads or sand) and ceramic materials. Suitable solid supports are disclosed, for example, in Zaborsky “Immobilized Enzymes” CRC Press, 1973, Table IV on pages 28-46.

[0456] General method conditions, solutions and / or buffers, as well as their concentration ranges for use in the different chromatographic processes can be determined based on standard handbooks on chromatography (see e.g. Gunter Jagschies, Eva Lindskog (ed.) Biopharmaceutical Processing, Development, Design, and Implementation of Manufacturing Processes, 1st Ed. 2017, Elsevier).

[0457] The first step of an ISVD polypeptide purification process is often referred to as “the capture step”. The purpose of the capture step is to have a first reduction of process-related impurities (for example, but not limited to, host cell proteins (HCPs), color and DNA) and to capture the ISVD polypeptide product while maintaining a high recovery. In one embodiment, the capture step refers to the first purification step on protein A chromatography in bind and elute mode.

[0458] In one embodiment, the ISVD polypeptide containing preparations may be purified by Protein A chromatography. Staphylococcal Protein A (SpA) is a 42 kDa protein composed of five nearly homologous domains named as E, D, A, B and C in order from the N-terminus (Sjodhal Eur. J. Biochem. 78: 471-490 (1977); Uhlen et al. J. Biol. Chem. 259: 1695-1702 (1984)). These domains contain approximately 58 residues, each sharing about 65%-90% amino acid sequence identity. Binding studies between Protein A and antibodies have shown that while all five domains of SpA (E, D, A, B and C) bind to an IgG via its Fc region, domains D and E exhibit significant Fab binding (Ljungberg et al. Mol. Immunol. 30(14): 1279-1285 (1993); Roben et al. J. Immunol. 154: 6437-6445 (1995); Starovasnik et al. Protein Sei. 8: 1423-1431 (1999). The Z- domain, a functional analogue and energy-minimized version of the B domain (Nilsson et al. Protein Eng. 1: 107-113 (1987)), was shown to have negligible binding to the antibody variable domain region (Cedergren et al. Protein Eng. 6(4): 441-448 (1993); Ljungberg et al. (1993) supra; Starovasnik et al. (1999) supra).

[0459] Until recently, commercially available Protein A stationary phases employed SpA (isolated from Staphylococcus aureus or expressed recombinantly) as their immobilized ligand. Using these columns, it has not been possible to use alkaline conditions for column regeneration and sanitation as is typically done with other modes of chromatography using non-proteinaceous ligands (Ghose et al. Biotechnology and Bioengineering Yol. 92 (6): 665-73 (2005)). A new resin (MabSELECT™ SuRe) has been developed to withstand stronger alkaline conditions (Ghose et al. (2005) supra). Using protein engineering techniques, a number 94MF-366657479Attorney Reference No. 15979-20208.40of asparagine residues were replaced in the Z-domain of protein A and a new ligand was created as a tetramer of four identically modified Z-domains (Ghose et al. (2005) supra).

[0460] Accordingly, purification methods can be carried out using commercially available Protein A columns according to manufacturers’ specification. For instance, MabSELECT™ columns or MabSELECT™ SuRe columns (GE Healthcare Products) can be used. MabSELECT™ is a commercially available resin containing recombinant SpA as its immobilized ligand. Other commercially available sources of Protein A column including, but not limited to, PROSEP-ATM (Millipore, U.K.), which consists of Protein A covalently coupled to controlled pore glass, can be usefully employed. Other useful Protein A formulations include Protein A Sepharose FAST FLOW™ (Amersham Biosciences, Piscataway, NJ), AmsphereTM A3 (JSR Life Sciences), and TOYOPEARL™ 650M Protein A (TosoHaas Co., Philadelphia, PA).

[0461] Protein purification by Protein A-based chromatography may be performed in a column containing an immobilized Protein A ligand (typically a column packed with modified support of methacrylate copolymer or agarose beads to which is affixed an adsorbent consisting of Protein A or functional derivatives thereof). The column is typically equilibrated with a buffer and a sample containing a mixture of proteins (the target protein, plus contaminating proteins) is loaded onto the column. As the mixture passes through the column, the target protein binds to the adsorbent (Protein A or derivative thereof) within the column, while some unbound impurities and contaminants flow through. Bound protein is then eluted from the column. In this process the target protein is bound to the column while impurities and contaminants flow through. Target protein is subsequently recovered from the eluate.

[0462] After host cell proteins are removed, a sample containing ISVD can be subjected to a process often referred to as “the polish step” which aims at purity improvement. For instance, a chromatography step (e.g., ion exchange chromatography) in bind and elute mode can be used to remove / reduce product related variants (e.g., but not limited to, High-molecular Weight (HMW) species, Low-Molecular Weight (LMW) species, and other charged variants) as well as some process related impurities (e.g., but not limited to, HCP, residual Protein A, DNA) still present after the capture step.Method for purifying ISVD monomers

[0463] Due to the free cysteine linker at the C-terminus of the ISVD molecules, ISVD harvested from host cells normally present as a mixture of both monomers and dimers (e.g., two ISVD molecules are bound together through S-S). For example, a typical fermentation95MF-366657479Attorney Reference No. 15979-20208.40process may lead to a mixture having over 60% ISVD dimers and less than 40% monomers. Accordingly, there is a need to isolate only ISVD monomers from the mixture before they are conjugated to the phospholipid-PEG.

[0464] Therefore, in one aspect, the present disclosure provides a method for preparing a composition comprising monomers of an ISVD with a cysteine containing linker at its C-terminal end from a mixture of monomers of the ISVD and dimers of the ISVD. In some embodiments, the method comprises (a) reducing ISVD dimers in the mixture to ISVD monomers with a first reducing agent. In some embodiments, the first reducing agent comprises TCEP (tris (2-carboxyethyl)phosphine). In some embodiments, the step (a) is conducted around 15 to 25 °C, optionally around 20-22 °C. In some embodiments, the firstreducing agent comprises 20X TCEP. In some embodiments, the step (a) takes about 16 to 20 hours. Optionally, the mixture of monomers of the ISVD and dimers of the ISVD is subjected to a step of removing host cell proteins and DNA before being subjected to step (a). In some embodiments, Protein A chromatography is used to remove host cell proteins and DNA.

[0465] In some embodiments, the method further comprises (b) purifying the ISVD monomers obtained in step (a) to get a purified composition comprising the ISVD monomers. In some embodiments, the step (b) comprises using a chromatograph, such as an ion exchange chromatography (IEX).

[0466] In some embodiments, the method further comprises (c) reducing the purified composition obtained in step (b) with a second reducing agent. In some embodiments, the second reducing agent is as the same or different from the first reducing agent used in step (a). In some embodiments, the reducing agent also comprises TCEP. In some embodiments, the first reducing agent comprises 10X TCEP. In some embodiments, the step (c) is conducted around 15°C to 25°C, optionally around 20-22°C. In some embodiments, the step (c) takes about 16 to 20 hours.

[0467] In some embodiments, the method further comprises (d) purifying the reduced composition obtained in step (c) to obtain a composition comprising monomers of the ISVD. In some embodiments, the step (d) comprises Ultrafiltration / Diafiltration (UF / DF). In some embodiments, the UF / DF membrane has a molecular weight cut-off of 10 kDa.

[0468] In some embodiments, 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 ISVD in the composition obtained from step (d) are in monomeric form.Method for conjugating ISVD to lipid or LNPs containing lipid96MF-366657479Attorney Reference No. 15979-20208.40

[0469] In one aspect, also provided is a method for preparation of a composition comprising a lipid-PEG-antibody conjugate (e.g., a lipid-PEG-ISVD conjugate, such as a phospholipid-PEG-ISVD conjugate). In some embodiments, the lipid-PEG-ISVD is a phospholipid- PEG-ISVD. In some embodiments, the method comprises the following steps: (a) mixing a first composition comprising monomers of an ISVD comprising a linker (e.g., a linker for click chemistry reaction, such as a cysteine linker (e.g., GGC)), with a second composition comprising a first phospholipid-PEG comprising a bioconjugation linker under conditions that the phospholipid-PEG and the ISVD monomer can form a conjugate through click chemistry; (b) adding a quenching agent to the mixture obtained in step (a) under conditions that the conjugation reaction is quenched, wherein a composition comprising the phospholipid-PEG ISVD conjugate is obtained. As used herein, the term “quenching agent” refers to a compound that is able to compete with at least one of the substrates of the conjugation reaction there to slow down or stop the reaction. For example, the quenching agent can be cysteine when the conjugation click chemistry is based on cysteine-assisted click chemistry. In some embodiments, the composition obtained from the method comprises micelles wherein the micelles comprise the phospholipid-PEG-ISVD.

[0470] In some embodiments, the click chemistry reaction takes place in the mixture in step (a) under 15°C to 25 °C, such as about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. In some embodiments, the click chemistry reaction takes place under 20-22°C.

[0471] The click chemistry reaction in step (a) can take as long as needed to ensure a complete reaction. In some embodiments, the reaction takes about 1 to about 3 hours. In some embodiments, the reaction takes about 2 hours.

[0472] Step (b) as described in the method is a quenching step in which excessive quenching agent (e.g., cysteine for a cysteine-based click chemistry reaction) is added into the reaction so that the conjugation reaction is slowed down or stopped. In some embodiments, molar ratio between the added quenching agent and the phospholipid-PEG-maleimide is about 5:1 to about 1:1, such as about 5.0:1, 4.9:1, 4.8:1, 4.7:1, 4.6:1, 4.5:1, 4.4:1, 4.3:1, 4.2:1, 4.1:1, 4.0:1, 3.9:1, 3.8:1, 3.7:1, 3.6:1, 3.5:1, 3.4:1, 3.3:1, 3.2:1, 3.1:1, 3.0:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, or 1.0:1. In some embodiments, the quenching step take about 5 minutes to 60 minutes, such as about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes or 60 minutes. In some embodiments, the quenching step takes place in the mixture in step (a) under 15°C to 25°C, such as about 15°C,97MF-366657479Attorney Reference No. 15979-20208.4016°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. In some embodiments, the click chemistry reaction takes place under 20-22°C.

[0473] In some embodiments, 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%, at least 99% or more of the ISVDs in the first composition are in monomeric form. In some embodiments, the phospholipid in the first phospholipid-PEG is a derivative of phosphatidylethanolamine. In some embodiments, the phospholipid in the first phospholipid-PEG comprises stearic acid acyl chains. In some embodiments, phospholipid in the first phospholipid-PEG is 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). The PEG in the first phospholipid-PEG can have a weight from about 1 kDa to about 10 kDa, such as about 1 kDa, 1.5 kDa, 2.0 kDa, 2.5 kDa, 3.0 kDa, 3.5 kDa, 4.0 kDa, 4.5 kDa, 5.0 kDa, 5.5 kDa, 6.0 kDa, 6.5 kDa, 7.0 kDa, 7.5 kDa, 8.0 kDa, 8.5 kDa, 9.0 kDa, 9.5 kDa, or 10 kDa. In some embodiments, the PEG in the first phospholipid-PEG has a weight of about 3.4kDa, and the conjugate is a DSPE-PEG 3.4K-ISVD conjugate. In some embodiments, the ISVD in the DSPE-PEG 3.4K-ISVD conjugate is an anti-CD8a ISVD, such as those described herein.

[0474] In some embodiments, the phospholipid-PEG has a bioconjugation linker, so that under proper conditions the first phospholipid-PEG molecules and the ISVD monomers can form a conjugate through click chemistry reaction. In some embodiments, the bioconjugation linker in the phospholipid-PEG has a maleimide group (e.g., phospholipid-PEG-maleimide, such as DSPE-PEG 3.4K-maleimide).

[0475] In some embodiments, a mixture obtained from the method described herein that contains micelles comprising the phospholipid-PEG-ISVD is contacted with a composition comprising LNPs. In some embodiments, such contact leads to that molecules in the micelles diffuse from the micelles and insert into the LNPs. For example, the phospholipid-PEG-ISVD in the micelles can diffuse from the micelles and insert into the LNPs to form new LNPs that comprise the phospholipid-PEG-ISVD. Depending on the binding specificity of the ISVD, the resulted LNPs can target to a specific cell type or tissue that the ISVD can specifically bind to. In some embodiments, the LNPs before step (a) comprise elements selected from the group of (i) an ionizable cationic lipid, (ii) a structural lipid, (iii) a neutral phospholipid, (iv) a free PEG-lipid, (v) a payload, such as mRNA, and (vi) any combination thereof. In some embodiments, the resulted LNPs obtained from the method comprise (i) the ionizable cationic lipid, (ii) the structural lipid, (iii) the neutral phospholipid, (iv) the free PEG-lipid, (v) the payload, such as mRNA, and (vi) the phospholipid-PEG-ISVD conjugate. In some embodiments, the LNPs resulted from the method comprise Lipid 15 (ionizable cationic lipid), cholesterol (structural 98MF-366657479Attorney Reference No. 15979-20208.40lipid), DSPC (neutral phospholipid), PEG 2000-DPG (same as DPG-PEG 2K; free PEG-lipid), mRNA encodes a chimeric antigen receptor (CAR) comprises an anti-CD19 antibody, such as an anti-CD19 single domain antibody (sdAb or VHH), and DSPE-PEG 3.4K-anti-CD8a ISVD conjugate (lipid-immune cell targeting group conjugate).

[0476] Optionally, the composition comprising the phospholipid-PEG that has a bioconjugation linker for the conjugation reaction (e.g., a click chemistry reaction), further contains a second phospholipid-PEG that does not react with the ISVD. For example, the second phospholipid-PEG does not have the bioconjugation linker (e.g., a phospholipid-PEG that is not reactive in the bioconjugation reaction). In some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker stabilizes the composition. For example, in some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker stabilizes the micelle structure of the composition formed by the first phospholipid-PEG having a bioconjugation linker. In some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker is commercially available. In some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker to be used has at least a GMP grade. In some embodiments, the micelles formed through the bioconjugation reaction comprises a conjugate formed by the process as described herein. In some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker stabilizes the micelle structure of the composition formed by the first phospholipid-PEG having a bioconjugation linker and the ISVD, and the second phospholipid-PEG that does not have the bioconjugation linker. In some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker can be the same phospholipid in the first phospholipid-PEG, and the PEG in the second phospholipid-PEG has a different or the same molecular weight as the first phospholipid-PEG that has the bioconjugation linker. In some other embodiments, the second phospholipid-PEG that does not have the bioconjugation linker has a different phospholipid as in the first phospholipid-PEG, and the PEG in the second phospholipid-PEG has a different or the same molecular weight as the first phospholipid-PEG that has the bioconjugation linker. In some embodiments, the PEG in the second phospholipid-PEG has a molecular weight from about 0.5kDa to about 10 kDa, such as about 0.5 kDa, 1.0 kDa, 1.5 kDa, 2.0 kDa, 2.5 kDa, 3.0 kDa, 3.5 kDa, 4.0 kDa, 4.5 kDa, 5.0 kDa, 5.5 kDa, 6.0 kDa, 6.5 kDa, 7.0 kDa, 7.5 kDa, 8.0 kDa, 8.5 kDa, 9.0 kDa, 9.5 kDa or 10.0 Kda. In some embodiments, the second phospholipid-PEG that does not have the bioconjugation linker can be the same phospholipid in the first phospholipid-PEG, and the PEG in the second phospholipid-PEG has a smaller molecular weight compared to the first phospholipid-PEG that has the bioconjugation 99MF-366657479Attorney Reference No. 15979-20208.40linker. In some embodiments, the PEG in the first phospholipid-PEG with a bioconjugation linker has a molecular weight of about 3.4kDa, and the PEG in the second phospholipid-PEG without a bioconjugation linker has a molecular weight of about 2.0kDa. In some embodiments, the second phospholipid-PEG is also DSPE-PEG but with a smaller PEG size (e.g., DSPE-PEG2.0k) compared to the first DSPE-PEG having a bioconjugation linker (e.g., DSPE-PEG3.4k-maleimide). In some embodiments, the first DSPE-PEG is DSPE-PEG3.4k-maleimide and the second phospholipid-PEG is DSPE-PEG2.0k-OMeH.

[0477] In some embodiments, the first phospholipid-PEG molecules comprising the bioconjugation linker and the second phospholipid-PEG that does not have the bioconjugation linker are mixed first to form micelles before being conjugated to the antibody (e.g., the ISVD). In some embodiments, the molar ratio of the first phospholipid-PEG molecules comprising the bioconjugation linker to the second phospholipid-PEG that does not have the bioconjugation linker before they are mixed to form the micelles, or in the formed micelles is about 1:3 to about 1:1, such as about 1:3, about 2:3, about 1:1. In some embodiments, the molar ratio of the phospholipid-PEG molecules comprising the bioconjugation linker to the second phospholipid-PEG that does not have the bioconjugation linker before they are mixed to form micelles, or the ratio of the two components in formed micelles is about 2:3.

[0478] In some embodiments, the formed micelles are conjugated to one or more ISVD monomers that have a linker through a clicking reaction, such as a thiol-maleimide clicking reaction (e.g., through the reaction between a cysteine tag on the ISVD and a maleimide tail on the phospholipid-PEG). In some embodiments, the molar ratio of the ISVD monomers, the phospholipid-PEG molecules comprising the bioconjugation linker, and the second phospholipid-PEG that does not have the bioconjugation linker before the clicking reaction or in the formed conjugate is about 1:1:4 or 1:2:3. In some embodiments, the micelles having the phospholipid-PEG-antibody conjugated to them are mixed with a composition comprising LNPs to form targeted LNPs having the phospholipid-PEG-antibody conjugate.

[0479] As a result of the method described herein, micelles comprising the phospholipid-PEG-antibody (e.g., ISVD) conjugate are obtained. In some embodiments, the micelles also comprise a second phospholipid-PEG. In some embodiments, the micelles comprise DSPE-PEG3.4K-antibodody (e.g., ISVD) conjugate. In some embodiments, the micelles comprise DSPE-PEG3.4K-ISVD conjugate and DSPE-PEG-3.4-maleimide not conjugated to the antibody (e.g., ISVD). In some embodiments, the micelles comprise DSPE-PEG3.4K-ISVD conjugate, DSPE-PEG-3.4-maleimide not conjugated to the antibody (e.g., ISVD), and / or a second phospholipid-PEG that does not have a bioconjugate linker, such as DSPE-PEG2k. In 100MF-366657479Attorney Reference No. 15979-20208.40some embodiments, the DSPE-PEG3.4K-ISVD conjugate has a concentration about 0.084 g / g to 0.15 g / g mRNA in the LNP. In some embodiments, the DSPE-PEG3.4K-ISVD conjugate has a concentration of about 0.084 g / g mRNA in the LNP. In some embodiments, the DSPE-PEG3.4K-ISVD conjugate has a concentration of about 0.11 g / g mRNA in the LNP. In some embodiments, the DSPE-PEG3.4K-ISVD conjugate has a concentration about 0.05 g / g to 0.15 g / g mRNA in the LNP. In some embodiments, the DSPE-PEG3.4K-ISVD conjugate is DSPE-PEG3.4K-anti-CD8 antibody conjugate.

[0480] The obtained micelles comprising the phospholipid-PEG-antibody (e.g., ISVD) can be further used to produce targeted LNPs described in the present disclosure.

[0481] In another aspect, also provided is a method for conjugating an antibody (e.g., an ISVD) to a LNP comprising at least one lipid-PEG (e.g., a phospholipid-PEG). In some embodiments, the lipid-PEG-ISVD is a phospholipid-PEG-ISVD. In some embodiments, the method comprises the following steps: (a) mixing a first composition comprising monomers of an ISVD comprising a cysteine containing linker, with a second composition comprising a LNP that contains a first phospholipid-PEG, wherein the first phospholipid-PEG comprises a bioconjugation linker under conditions that the phospholipid-PEG and the ISVD monomer can form a conjugate through click chemistry; (b) adding a quenching agent to the mixture obtained in step (a) under conditions that the conjugation reaction is quenched, wherein a composition comprising LNPs comprising the phospholipid-PEG ISVD conjugate is obtained. In some embodiments, the LNPs that contain first phospholipid-PEG molecules also comprise other elements selected from the group of (i) an ionizable cationic lipid, (ii) a structural lipid, (iii) a neutral phospholipid, (iv) a free PEG-lipid, (v) a payload, such as mRNA, and (vi) any combination thereof. In some embodiments, the LNPs comprise Lipid 15 (ionizable cationic lipid), mRNA (payload), cholesterol (structural lipid), DSPC (neutral phospholipid), and PEG 2000-DPG (same as DPG-PEG 2K; free PEG-lipid). In some embodiments, the mRNA encodes a chimeric antigen receptor (CAR) comprises an anti-CD19 antibody, such as an antiCD 19 single domain antibody (sdAb or VHH). In some embodiments, the LNPs produced from a method as described herein further comprises DSPE-PEG 3.4K-anti-CD8a ISVD conjugate (lipid-immune cell targeting group conjugate).

[0482] In some embodiments, the antibody in the phospholipid-PEG-antibody is an immunoglobulin single variable domain. In some embodiments, the antibody in the phospholipid-PEG-antibody is selected from a VHH or sdAb (including humanized VHH or sdAb), a VH (including camelized VH, human VH, camelized human VH, dAb) and a VL. In101MF-366657479Attorney Reference No. 15979-20208.40some embodiments, the antibody in the phospholipid-PEG-antibody is an immunoglobulin single variable domain that comprises at least two disulfide bridges.

[0483] It is known that all VHHs contain at least one disulfide bridge, between the cysteine residue at position 22 and the cysteine residue at position 92 (numbering according to Kabat; Muyldermans and Lauwereys 1999, J. Mol. Recognit. 12: 131). Although most VHHs contain only this single disulfide bridge, it is also known that some VHHs contain a total of two (or in exceptional cases three) disulfide bridges. For example, a class of VHHs referred to as “VHH-1 type”, “VHH-1 class”, or the like (as further defined herein) commonly has a second disulfide bridge between the cysteine residue in CDR2 at position 50 and a cysteine residue present in CDR3 (or in exceptional cases in CDR1 or CDR2). Also, some VHHs derived from camels or dromedaries often have a disulfide bridge between a cysteine residue present in CDR1 (or at position 45 in FR2) and a cysteine residue present in CDR3 (Vu et al. 1997, Mol. Immunol.34: 1121; Muyldermans and Eauwereys 1999). Some VHHs derived from llamas sometimes have a disulfide bridge between cysteine residues present in CDR1 (such as at position 33) and a cysteine residue present in CDR3 (Vu et al., 1997).

[0484] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in one of the framework regions and a cysteine residue in one of the CDR regions.

[0485] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in one of the framework regions and a cysteine residue in CDR3.

[0486] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in framework two (FR2) and a cysteine residue in CDR3.

[0487] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue at position 45 in framework two (FR2) and a cysteine residue in CDR3 (as in some VHHs derived from camels and dromedaries).102MF-366657479Attorney Reference No. 15979-20208.40

[0488] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in one CDR and a cysteine residue in another CDR.

[0489] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR3 and a cysteine residue in another CDR (in particular in CDR1, as in some VHHs derived from camels, dromedaries and llamas).

[0490] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR1 and a cysteine residue in another CDR.

[0491] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR1 and a cysteine residue in CDR1.

[0492] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR1 and a cysteine residue in CDR3 (as in some VHHs derived from camels, dromedaries and llamas).

[0493] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine at position 33 and a cysteine residue in CDR3 (as in some VHHs derived from camels, dromedaries and llamas).

[0494] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR2 and a cysteine residue in another CDR.

[0495] In some embodiments, the immunoglobulin single variable domain comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position103MF-366657479Attorney Reference No. 15979-20208.4092, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR2 and a cysteine residue in CDR2.

[0496] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue in CDR2 and a cysteine residue in CDR3 (as in some VHHs derived from llamas).

[0497] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue at position 50 and a cysteine residue in another CDR, such as CDR1, CDR2 or CDR3 (as in VHHs of the VHH1 type).

[0498] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody comprises a disulfide bridge between the cysteine residue at position 22 and the cysteine residue at position 92, and further comprises a disulfide bridge that is formed between a cysteine residue at position 50 and a cysteine residue in CDR3 (as in VHHs of the VHH1 type).

[0499] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody may be a “VHH1 type immunoglobulin single variable domain”. An amino acid such as e.g., an immunoglobulin single variable domain or polypeptide is said to be a “VHH1 type immunoglobulin single variable domain” or “VHH type 1 sequence”, if said VHH1 type immunoglobulin single variable domain or VHH type 1 sequence has 85% identity (using the blastp algorithm with standard setting, i.e. blosom62 scoring matrix) to the VHH1 consensus sequence (SEQ ID NO: 569): QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGS TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) and mandatorily has a cysteine in position 50, i.e. Cys50 (using Kabat numbering). These VHH1 type immunoglobulin single variable domains generally have (or are capable of forming) a disulfide bridge between Cys50 and a cysteine residue in CDR3 (or in exceptional cases CDR1 or CDR2).

[0500] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody is another immunoglobulin single variable domains that comprise two or more disulfide bridges (such as (single) domain antibodies, dAbs, IgNAR domains from sharks, etc.). In some embodiments, the immunoglobulin single variable domain in the 104MF-366657479Attorney Reference No. 15979-20208.40phospholipid-PEG-antibody comprises at least two disulfide bridges. In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody belongs to the group of VHH1 type immunoglobulin single variable domains.

[0501] In some embodiments, the immunoglobulin single variable domain in the phospholipid-PEG-antibody does not belong to the group of VHH1 type immunoglobulin single variable domains, but belongs to another group of immunoglobulin single variable domains, such as the VHH2, VHH3 or any other type of immunoglobulin single variable domains.

[0502] As such, in some embodiments, the lipid-immune cell targeting group conjugate comprises DSPE-PEG 3.4K-ISVD conjugate. In some embodiments, the lipid-immune cell targeting group conjugate comprises DSPE-PEG 3.4K-VHH conjugate. In some embodiments, the lipid-immune cell targeting group conjugate comprises DSPE-PEG 3.4K-anti-CD8a VHH conjugate. In some embodiments, the lipid-immune cell targeting group conjugate comprises DSPE-PEG 3.4K-VHH1 conjugate. In some embodiments, the lipid-immune cell targeting group conjugate comprises DSPE-PEG 3.4K-anti-CD8a VHH1 conjugate.

[0503] In some embodiments, the immunoglobulin single variable domain comprised in the phospholipid-PEG-antibody is an ISVD directed against CD8a, that comprises:a) the amino acid sequence selected from the group consisting of SEQ ID NOs: 160 to 179; b) amino acid sequences that have at least 80% amino acid identity with a sequence selected from the group consisting of SEQ ID NOs: 160 to 179, orc) amino acid sequences that have 3, 2, or 1 amino acid difference with a sequence selected from the group consisting of SEQ ID NOs: 160 to 179;or any suitable combination thereof.

[0504] In some embodiments, the immunoglobulin single variable domain comprised in the phospholipid-PEG-antibody an ISVD against CD8a, which consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively) in which:(I) CDR1 comprises or essentially consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 181 and 188, according to Abm numbering, and SEQ ID NOs: 251, according to Kabat numbering;or of amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 181 and 188, according to Abm numbering, and SEQ ID NOs: 251, according to Kabat numbering, in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2)105MF-366657479Attorney Reference No. 15979-20208.40said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 181 and 188, according to Abm numbering, and SEQ ID NOs: 251, according to Kabat numbering; and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 181 and 188, according to Abm numbering, and SEQ ID NO: 251, according to Kabat numbering, in which any amino acid substitution is a conservative amino acid substitution; and / or said amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 181 and 188, according to Abm numbering, and SEQ ID NO: 251, according to Kabat numbering.(II) CDR2 comprises or essentially consists of an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 183 and 190, according to Abm numbering, and SEQ ID NOs: 253 and 260, according to Kabat numbering,or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 183 and 190, according to Abm numbering, and SEQ ID NOs: 253 and 260, according to Kabat numbering in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 183 and 190, according to Abm numbering, and SEQ ID NOs: 253 and 260, according to Kabat numbering;and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 183 and 190, according to Abm numbering, and SEQ ID NOs: 253 and 260, according to Kabat numbering, in which any amino acid substitution is a conservative amino acid substitution; and / or said amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 183 and 190, according to Abm numbering and SEQ ID NOs: 253 and 260, according to Kabat numbering.(III) CDR3 comprises or essentially consists of an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199 and 206, according to Abm numbering, and SEQ ID NOsl85, 192, 199 and 206, according to Kabat numbering, or amino acid sequences that have at least 80%, at least 90%, at least 95%, at least 99% or more sequence identity with a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199 and 206, according to Abm numbering and SEQ ID NOs: 185, 192, 199 and 206, according 106MF-366657479Attorney Reference No. 15979-20208.40to Kabat numbering, in which (1) any amino acid substitution is a conservative amino acid substitution; and / or (2) said amino acid sequence only contains amino acids substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199 and 206, according to Abm numbering and SEQ ID NOs: 185, 192, 199 and 206, according to Kabat numbering;and / or amino acids sequences that have 2 or only 1 amino acid difference(s) with a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199 and 206, according to Abm numbering and SEQ ID NOs: 185, 192, 199 and 206, according to Kabat numbering, in which any amino acid substitution is a conservative amino acid substitution; and / or said amino acid sequence only contains amino acid substitutions, and no amino acid deletions or insertions, compared to a sequence selected from the group consisting of SEQ ID NO: 185, 192, 199 and 206, according to Abm numbering and SEQ ID NOs: 185, 192, 199 and 206, according to Kabat numbering.

[0505] In one embodiment, the immunoglobulin single variable domain comprised in the phospholipid-PEG-antibody comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 244, a CDR2 that is the amino acid sequence of SEQ ID NO: 246 and a CDR3 that is the amino acid sequence of SEQ ID NO: 248 (Abm numbering) or comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 314, a CDR2 that is the amino acid sequence of SEQ ID NO: 316 and a CDR3 that is the amino acid sequence of SEQ ID NO: 318 (Kabat numbering).

[0506] CD8 binding ISVDs in the phospholipid-PEG-antibody may comprise one, two or all three of the CDRs explicitly listed above. In some embodiments, the anti-CD8aISVD in the phospholipid-PEG-antibody is selected from the ISVDs described in Table C-l, Table C-2, and Table C-3. In some embodiments, the anti-CD8aISVD in the phospholipid-PEG-antibody is selected from the ISVDs that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ISVDs described in Table C-l, Table C-2, and Table C-3. In some embodiments, the anti-CD8ISVD in the phospholipid-PEG-antibody is selected from the ISVDs that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the ISVDs described in Table C-4.

[0507] In some embodiments, the anti-CD8a ISVD comprises or is an ISVD described herein, such as: a) the amino acid sequence selected from the group consisting of SEQ ID NOs: 160 to 179; b) amino acid sequences that have at least 80% amino acid identity with a sequence selected from the group consisting of SEQ ID NOs: 160 to 179; or c) amino acid sequences that have 3, 2, or 1 amino acid difference with a sequence selected from the group consisting 107MF-366657479Attorney Reference No. 15979-20208.40of SEQ ID NOs: 160 to 179. In some embodiments, the anti-CD8aISVD comprises SEQ ID NP: 9, SEQ ID NO: 169, or SEQ ID NO: 179.

[0508] In some embodiments, the phospholipid in the phospholipid-PEG-antibody is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), ceramide. In some embodiments, the phospholipid is DSPE.

[0509] In some embodiments, the antibody is covalently coupled to the phospholipid in Formula (II) ([Lipid] - [optional linker] - [antibody]), phospholipid- linker- antibody, or phospholipid-PEG-antibody, wherein the linker comprises polyethylene glycol (PEG). In some embodiments, PEG has a molecular weight of about 1.0 kDa to about 6 kDa. In some embodiments, the PEG has a molecular weight about 3400 Da (e.g., PEG 3400 (PEG 3.4K)). In some embodiments, phospholipid-PEG molecule is conjugated to the antibody through a click chemistry reaction. In some embodiments, the antibody is an ISVD. The phospholipid-PEG can be conjugated to the ISVD according to methods described herein.

[0510] The lipid-PEG-antibody conjugate of the present disclosure can be inserted into an LNP to form a targeted LNP. In some embodiment, the lipid-PEG-antibody conjugate can help the LNP to target a specific cell, a specific tissue, or a specific organ. In some embodiments, the lipid-PEG-antibody conjugate comprises an immune cell targeting group. For example, the antibody in the conjugate can be an antibody that specifically binds to immune cells. In some embodiments, the lipid-immune cell targeting group conjugate in the LNP of the present disclosure comprises DSPE-PEG-anti-CD8a antibody. In some embodiments, the conjugate comprises DSPE-PEG3.4k-anti-CD8a antibody. In some embodiments, the conjugate comprises DSPE-PEG3.4k-A044300805_v8 (SEQ ID NO: 9, SEQ ID NO: 169 or SEQ ID NO: 179).

[0511] In some embodiments, the conjugate of the present disclosure (e.g., DSPE-PEG3.4k-A044300805_v8) can be present in the LNP in a range of 0.001-0.5 mol percent, 0.001-0.1 mol%, 0.01-0.5 mol%, 0.05-0.5 mol%, 0.1-0.5 mol%, 0.1-0.3 mol%, 0.1-0.2 mol%, 0.2-0.3 mol%, of about 0.01 mol%, about 0.05 mol%, about 0.1 mol%, about 0.15 mol%, about 0.2 mol%, about 0.25 mol%, about 0.3 mol%, about 0.35 mol%, about 0.4 mol%, about 0.45 mol%, or about 0.5 mol%. In some embodiments, the conjugate comprises DSPE-PEG3.4k-A044300805_v8, and is present in the LNP in a range of 0.01 to 0.02 mol percent. In some embodiment, the conjugate comprising DSPE-PEG3.4k-A044300805_v8 is present in the LNP is about 0.015 to about 0.016 mol percent.108MF-366657479Attorney Reference No. 15979-20208.40

[0512] An exemplary method for making a targeted LNP that comprises any of the ISVD described herein is shown in FIG. 53.B. Ionizable Cationic Lipids

[0513] Provided herein are ionizable cationic lipids that can be used to produce lipid nanoparticle compositions to facilitate the delivery of a payload (e.g., a nucleic acid, such as a DNA or RNA, such as an mRNA, such as any of SEQ ID NOs:570-575) disposed therein to cells, e.g., mammalian cells, e.g., immune cells. In some embodiments, an ionizable cationic lipid of the present disclosure is used to produce LNPs as described herein. The ionizable cationic lipids have been designed to enable intracellular delivery of a nucleic acid, e.g., mRNA, to the cytosolic compartment of a target cell type and rapidly degrade into non-toxic components. The complex functionalities of the ionizable cationic lipids are facilitated by the interplay between the chemistry and geometry of the ionizable lipid head group, the hydrophobic “acyl-tail” groups and the linkers connecting the head group and the acyl tail groups. Typically, the substituents of the ionizable amine head group are chosen to tune the apparent pKa of amine head group in the LNP formulation to fall in the range of 6-8, such as between 6.2-7.4, or between 6.7-7.2. As such, the amine head groups remains strongly cationic under acidic formulation conditions (e.g., pH 4 - pH 5.5), neutral or slightly anionic or slightly cationic (typical zeta potential of 0 ± 5 mV) in physiological pH (7.4), but strongly cationic in the early and late endosomal compartments (e.g., pH 5.5 - pH 7). The acyl-tail groups play a key role in fusion of the lipid nanoparticle with endosomal membranes and membrane destabilization through structural perturbation. The three-dimensional structure of the acyl-tail (determined by its length, and degree and site of unsaturation) along with the relative sizes of the head group and tail group are thought to play a role in promoting membrane fusion, and hence lipid nanoparticle endosomal escape (a key requirement for cytosolic delivery of a nucleic acid payload). The linker connecting the head group and acyl tail groups is designed to degrade by physiologically prevalent enzymes (e.g., esterases, or proteases) or by acid catalyzed hydrolysis.109MF-366657479Attorney Reference No. 15979-20208.40

[0514] In one aspect, the present invention provides a compound represented by Formulaor a salt thereof, wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1.20 alkenyl, -(CH2)o-ioC(0)ORal, or -(CH2)o-ioOC(0)Ra2;Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;R3B1is C1-6 alkylene; andR3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.

[0515] In some embodiments, provided is a compound of Formula (1-1):110MF-366657479Attorney Reference No. 15979-20208.40or a salt thereof, wherein:R1, R2, and R3are each independently a bond or methylene;R1Aand R2Aare each Ci-io alkylene;R3Ais Ci-5 alkylene;R1A1, R1A2, R2A1, R2A2, R3A1, and R3A2are each H;R1A3and R2A3are each C1-20 alkenyl;R3A3is -C(O)O(Ci-20 alkyl);R3B1is C2-4 alkylene; andR3B2and R3B3are each methyl.

[0516] In some embodiments, R3B1is -(CFb -.

[0517] In some embodiments, the ionizable cationic lipid is

[0518] In one aspect, the present invention provides a compound of Formula (I-A):111MF-366657479Attorney Reference No. 15979-20208.40or a salt thereof, wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;RIAR2Aan(j R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1.20 alkenyl, -(CH2)o-ioC(0)ORal, or -(CH2)o-ioOC(0)Ra2;Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;R3B1is C1-6 alkylene; andR3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with one or more substituents each independently selected from the group consisting of -OH and -O-(Ci-6alkyl).

[0519] Any of the variables or substituents provided herein is unsubstituted or substituted with one or more substituents. In some embodiments, any of the variables or substituents provided herein is optionally substituted. In some embodiments, any of the variables or substituents provided herein is optionally substituted with one or more substituents independently selected from the group consisting of -ORsl, -NRs2Rs3, -C(O)Rs4, -C(O)ORs5, C(O)NRS6RS7, -OC(O)RS8, -OC(O)ORS9, -OC(O)NRsl0Rn, -NRsl2C(O)Rs13, and -NRS14C(O)ORS15, wherein Rsl, Rs2, Rs3, Rs4, Rs5, Rs6, Rs7, Rs8, Rs9, Rsl°, Rsl1, Rsl2, Rsl3, Rsl4, and Rsl5are each independently H, C1-6 alkyl, C3-10 cycloalkyl, Ce-14 aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl, each of which is optionally substituted.112MF-366657479Attorney Reference No. 15979-20208.40

[0520] In some embodiments, R1, R2, and R3are each independently a bond or C1-3 alkylene. In some embodiments, R1, R2, and R3are each independently a bond or methylene. In some embodiments, R1and R2are each methylene and R3is a bond. In some embodiments, R1, R2, and R3are each methylene. In some embodiments, R1, R2, and R3are each independently unsubstituted or substituted. In some embodiments, R1, R2, and R3are unsubstituted.

[0521] In some embodiments, R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene. In some embodiments, R1A, R2A, and R3Aare each independently a bond or -(CH2)I-10-. In some embodiments, R1Aand R2Aare each independently a bond, -CH2-, -(CEh -, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CEh)?-, or -(CH2)8-. In some embodiments, R1Aand R2Aare each a bond, each -CH2-, each -(CH2)2-, each -(CH2)3-, each -(CH2)4-, each -(CH2)s-, each -(CH2)6-, each -(Clfc)?-, or each -(CH2)s-. In some embodiments, R1Aand R2Aare each independently a bond, -(CH2)2-, -(CH2)4-, -(CH2)e-, -(Clfc)?-, or -(CH2)s-. In some embodiments, R1Aand R2Aare each a bond, each -(CH2)2-, each -(CH2)4-, each -(CH2)e-, each -(CEb)?-, or each -(CH2)s-. In some embodiments, R3Ais a bond, -CH2-, -(CEhh-, or -(CEh)?-. In some embodiments, R1A, R2A, and R3Aare each independently unsubstituted or substituted. In some embodiments, R1A, R2A, and R3Aare unsubstituted.

[0522] In some embodiments, R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)o-ioC(0)ORal, or -(CH2)o-ioOC(0)Ra2. In some embodiments, R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-15 alkyl, -CH=CH-(Ci-is alkyl), -CH=CH-CH2-CH=CH-(Ci-io alkyl), -(CH2)O-4C(0)OCH(CI-IO alkyl)(Ci-i5 alkyl), -(CH2)O-4OC(0)CH(CI-IO alkyl)(Ci-i5 alkyl), -(CH2)o-4C(0)OCH2(Ci-i5 alkyl), or -(CH2)o-40C(0)CH2(Ci-i5 alkyl). In some embodiments, R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, R3A3, R1, R2, R3, R1A, R2A, and R3Aare each independently unsubstituted or substituted. In some embodiments, R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, R3A3, R1, R2, R3, R1A, R2A, and R3Aare each unsubstituted. In some embodiments, R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently unsubstituted or substituted. In some embodiments, R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each unsubstituted. In some embodiments, R1, R2, R3, R1A, R2A, and R3Aare each independently unsubstituted or substituted. In some embodiments, R1, R2, R3, R1A, R2A, and R3Aare each unsubstituted. In some embodiments, R1, R2, and R3are each unsubstituted.

[0523] In some embodiments, R3B1is unsubstituted. In some embodiments, R3B1is not substituted with oxo.113MF-366657479Attorney Reference No. 15979-20208.40

[0524] In some embodiments, R1A1and R2A1are each independently -CH=CH-(Ci-is alkyl), -CH=CH-CH2-CH=CH-(CI IO alkyl), -(CH2)O-4C(0)OCH(CI-IO alkyl)(Ci-i5 alkyl), or -(CH2)o-4OC(0)CH(CI-IO alkyl)(Ci-i5 alkyl); and R1A2, R1A3, R2A2, and R2A3are each H. In some embodiments, R1A1and R2A1are each -CH=CH-(Ci-is alkyl), -CH=CH-CH2-CH=CH-(CI-IO alkyl), -(CH2)O-4C(0)OCH(CI-IO alkyl)(Ci-i5 alkyl), or -(CH2)0.4OC(0)CH(CI-IO alkyl)(Ci-i5 alkyl); and R1A2, R1A3, R2A2, and R2A3are each H. In some embodiments, R1A1and R2A1areembodiments, R1A1and R2A1are eachsome embodiments, R1A2, R1A3, R2A2, and R2A3are each H.

[0525] In some embodiments, R1A1and R2A1are each C1-15 alkyl; R1A2and R2A2are each Ci-15 alkyl; and R1A3and R2A3are each H. In some embodiments, R1A1and R2A1are each. , . embodiments, R1Aand R2Aare each a bond.

[0526] In some embodiments, R1A1and R2A1are each -(CH2)O-4OC(0)CH2(CI-IS alkyl); R2A1and R2A2are each -(CH2)O-4C(0)OCH2(CI-IS alkyl); and R1A3and R2A3are each H. Insome embodiments, R1A1and R2A1are each114MF-366657479Attorney Reference No. 15979-20208.40R2A2are eachsome embodiments, R1A3and R2A3are each H. In some embodiments, R1Aand R2Aare each a bond.

[0527] In some embodiments, R1A1and R2A1are each -C(O)OCH2(CI-IS alkyl); R1A2and R2A2are each -(CH2)o-4C(0)OCH2(Ci-i5 alkyl); and R1A3and R2A3are each H. In someembodiments, R1A1and R2A1are eachR2A2are each. In someembodiments, R1A1and R2A1are eachR2A1and R2A2are each. In some embodiments, R1A3and R2A3are each H. In some embodiments, R1Aand R2Aare each a bond.

[0528] In some embodiments, R3A1, R3A2, and R3A3are each independently H, C1-15 alkyl, -(CH2)O-4C(0)OCH(CI-5alkyl)(Ci-io alkyl), -(CH2)O-40C(0)CH(CI-5alkyl)(Ci-io alkyl), - (CH2)O-4C(0)OCH2(CI-IO alkyl), or -(CH2)O-4OC(0)CH2(CI-IO alkyl).

[0529] In some embodiments, R3A1and R3A2are each independently C1-15 alkyl; and R3A3is H. In some embodiments, R3A1and R3A2are each independently ethyl, propyl, butyl, pentyl, hexyl, or heptyl. In some embodiments, R3A1and R3A2are each independently ethyl,or. In some embodiments, R3A3is H. In some embodiments, R3Ais a bond.115MF-366657479Attorney Reference No. 15979-20208.40

[0530] In some embodiments, R3A1is C1-15 alkyl; and R3A2and R3A3are each H. In someembodiments, R3A1is. In some embodiments, R3A2and R3A3are each H. In some embodiments, R3Ais a bond.

[0531] In some embodiments, R3A1is -C(O)OCH(CI-5 alkyl)(Ci-io alkyl); and R3A2and116MF-366657479Attorney Reference No. 15979-20208.40

[0534] In some embodiments, R3A1, R3A2, and R3A3are each H.

[0535] Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl. In some embodiments, Raland R;'2are each independently -(CH2)o-i5CH3 or -CH(Ci-io alkyl)(Ci-i5alkyl). In some embodiments, Raland Ra2are each independentlywhich is optionally substituted. In some embodiments, Raland Ra2are each independently unsubstituted or substituted. In some embodiments, Raland Ra2are unsubstituted.

[0536] In some embodiments,some embodiments, R3Bis H. In some embodiments, R3Bis unsubstituted or substituted. In some embodiments, R3Bis unsubstituted.

[0537] In some embodiments, R3B1is C1-6 alkylene. In some embodiments, R3B1is ethylene or propylene. In some embodiments, R3B1is unsubstituted or substituted. In some embodiments, R3B1is optionally substituted.

[0538] In some embodiments, R3B2and R3B3are each independently and optionally substituted. In some embodiments, R3B2and R3B3are each independently H or C1-6 alkyl optionally substituted with one or more substituents each independently selected from the group consisting of -OH and -O-(Ci-6 alkyl). In some embodiments, R3B2and R3B3are each independently H or C1-6 alkyl optionally substituted with one or more substituents independently selected from the group consisting of -ORsl, -NRs2Rs3, -C(O)Rs4, -C(O)ORs5, C(O)NRS6RS7, -OC(O)RS8, -OC(O)ORS9, -OC(O)NRsl0Rn, -NRsl2C(O)Rs13, and -117MF-366657479Attorney Reference No. 15979-20208.40NRS14C(O)ORS15, wherein Rsl, Rs2, Rs3, Rs4, Rs5, Rs6, Rs7, Rs8, Rs9, Rsl°, Rsl1, Rsl2, Rsl3, Rsl4, and Rsl5are each independently H, Ci-6 alkyl, C3-10 cycloalkyl, Ce-14 aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl, each of which is optionally substituted. In some embodiments, R3B2and R3B3are each independently H, methyl, ethyl, propyl, butyl, or pentyl, each of which is optionally substituted with one or more substituents each independently selected from the group consisting of -OH and -O-(Ci-6 alkyl). In some embodiments, R3B2and R3B3are each independently methyl or ethyl, each optionally substituted with one or more -OH. In some embodiments, R3B2and R3B3are each methyl or each ethyl, each optionally substituted with one or more -OH. In some embodiments, R3B2and R3B3are each unsubstituted methyl.or a salR3A3, R3B1, R3B2, and R3B3are as defined for Formula (I) or any variation or embodiment thereof.118MF-366657479Attorney Reference No. 15979-20208.40

[0541] In one aspect, the present invention provides a compound represented by Formula (lb):or a saland R3A3are as defined for Formula (I) or any variation or embodiment thereof.

[0542] In some embodiments, the cationic lipid in a targeted LNP of the present disclosure has a concentration between about 10 mol% to about 60 mol% of the LNP, such as about 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, 16 mol%, 17 mol%, 18 mol%, 19 mol%, 20 mol%, 21 mol%, 22 mol%, 23 mol%, 24 mol%, 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, or 60 mol%. In some embodiments, the cationic lipid in a targeted LNP of the present disclosure has a concentration between about 48.0 mol% to about 50.0 mol%, such as 48.0 mol%, 48.1 mol%, 48.2 mol%, 48.3 mol%, 48.4 mol%, 48.5 mol%, 48.6 mol%, 48.7 mol%, 48.8 mol%, 48.9 mol%, 49.0 mol%, 49.1 mol%, 49.2 mol%, 49.3 mol%, 49.4 mol%, 49.5 mol%, 49.6 mol%, 49.7 mol%, 49.8 mol%, 49.9 mol%, or 50.0 mol%.

[0543] In some embodiments, the cationic lipid in the targeted LNP comprises Lipid 15.119MF-366657479Attorney Reference No. 15979-20208.40

[0544] In some aspects, the ionizable lipid is DLin-KC2-DMA:some embodiments, theionizable lipid(KC3). In some embodiments, the ionizable lipid is DLin-MC3-DMA:

[0545] In some embodiments, the cationic lipid, or a salt thereof, is a compound selected from the compounds listed in Table 1, or a slat thereof.120MF-366657479Attorney Reference No. 15979-20208.40121MF-366657479Attorney Reference No. 15979-20208.40122MF-366657479Attorney Reference No. 15979-20208.40123MF-366657479Attorney Reference No. 15979-20208.40124MF-366657479Attorney Reference No. 15979-20208.40125MF-366657479Attorney Reference No. 15979-20208.40126MF-366657479Attorney Reference No. 15979-20208.40127MF-366657479Attorney Reference No. 15979-20208.40128MF-366657479Attorney Reference No. 15979-20208.40129MF-366657479Attorney Reference No. 15979-20208.40130MF-366657479Attorney Reference No. 15979-20208.40

[0546] In some aspects, the ionizable lipid comprises one or more lipids as described in International PCT Publication Nos. W02009028824A2, WO2011043913 A2 WO2012091523A2, W02015074085A1, W02016081029A1, W02017117530A1 W02018118102A1, WO2018119163A1, WO2019045897A1, W02020069445A1 W02020106903A1, WO2020219876A1, WO2021055892A1, WO2021163339A1 WO2021188389A2, WO2021202694A1, W02022016070A1, WO2022119883A2 WO2022120388A2, WO2022159475A1, WO2022207938A1, WO2022218957A1 WO2022246555A1, W02023023410A2, W02023091490A1, WO2023091787A1 WO2023114937A2, WO2023114944A1, WO2023115221A1, WO2023121970A1 WO2023121975A1, WO2023141624A1, WO2023144798A1, WO2023183616A1 WO2023196444A1, WO2023196527 A2, W02023207101A1, WO2023235589A1 WO2023240156A1, W02024008967A1, W02024010330A1, W02024019770A1, and W02024035710A2.C. Payload131MF-366657479Attorney Reference No. 15979-20208.40

[0547] In some embodiments, the payload is a drug substance. In some embodiments, the payload is a nucleic acid. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, mRNA encodes a synthetic T cell receptor (synTCR) or a Chimeric Antigen Receptor (CAR). In some embodiments, mRNA comprises a 5’ Cap, a 5’ untranslated region (UTR), a payload nucleic acid sequence encoding a payload polypeptide, a 3’ UTR, and a polyA tail. In some embodiments, the payload polypeptide comprises an antibody specifically binding to a B cell, a hinge and transmembrane domain, a co- stimulatory domain, and a signaling domain.

[0548] In some embodiments, the payload is an mRNA that encodes a payload polypeptide comprising: an optional Lead peptide sequence (e.g., a signal peptide), an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), a hinge domain, a transmembrane domain, a Co-stimulatory domain, and a Signaling domain. In some embodiments, each of the domains described herein is connected to another directly without any linker. In some embodiments, one or more linkers are used to connect any two of the domains described herein. In some embodiments, the payload is an mRNA that encodes a payload polypeptide having the following formula, arranged from N-terminus to C-terminus:[Lead peptide sequence (optional)] - [antibody heavy chain variable region (VHH)] - [Linker A (optional)] - [Hinge domain] - [Transmembrane domain] - [Co- stimulatory domain] -[Signaling domain]. In some embodiments, exemplary payload polypeptides are demonstrated in FIG. 41.

[0549] In some embodiments, the payload is an mRNA that comprises a 5’ Cap (optional), a 5’ UTR (optional), nucleotides encoding a Lead polypeptide sequence, nucleotides encoding a VHH domain, nucleotides encoding a Linker A, nucleotides encoding a Hinge, nucleotides encoding a Transmembrane domain, nucleotides encoding Co-stimulatory domain, nucleotides encoding a Signaling domain, a 3’UTR (optional), and polyA tail (optional). In some embodiments, the payload has the following formula, arranged from 5’ to 3’:5’ Cap (optional) - 5’ UTR (optional) - nucleotides encoding Lead peptide sequence (optional) - nucleotides encoding a VHH - nucleotides encoding a Linker A (optional)- nucleotides encoding a Hinge - nucleotides encoding a Transmembrane domain - nucleotides encoding Co- stimulatory domain - nucleotides encoding Signaling domain - 3’UTR (optional) - polyA tail (optional).

[0550] In some embodiments, the 5’ UTR comprises SEQ ID NO: 516 or SEQ ID NO: 518. In some embodiments, the 3’ UTR comprises SEQ ID NO: 517 or SEQ ID NO: 519. In132MF-366657479Attorney Reference No. 15979-20208.40some embodiments, the 5’ UTR comprises SEQ ID NO: 516 and the 3’ UTR comprises SEQ ID NO: 517. In some embodiments, the 5’ UTR comprises SEQ ID NO: 518 and the 3’ UTR comprises SEQ ID NO: 519.

[0551] In some embodiments, an mRNA of the present disclosure comprises a 5' cap structure. In some embodiments, the 5’ cap structure is IRES, CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, CleanCapTM, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225. In one embodiment, the mRNA comprises a 5'-cap and wherein at least one of the uridines in the molecule is a modified uridine, preferably Nl-methyl-pseudouridine (Im T). In some embodiments, the mRNA comprises a 5' cap having the sequence NpppNU, wherein the U in the 5' cap is an unmodified uridine. In one embodiment, the 5' cap has the sequence NpppAU with A representing a modified or unmodified adenosine nucleotide. For example, a modified nucleotide N or A 3' to the triphosphate linkage may have a modified ribose structure such as a 2'-O- methylated ribose (Nm or Am) resulting in a so-called “Cap 1”. In contrast, a cap comprising a nucleotide N or A 3' to the triphosphate linkage having an unmethylated ribose is usually referred to as “Cap 0”.

[0552] In some embodiments, the payload is an mRNA that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises an antibody binding domain that specifically binds to B cells. In some embodiments, the antibody binding domain specifically binding to B cells is an antibody binding domain that specifically binds to human CD19 (i.e., “antiCD 19 CAR”). Exemplary anti-CD19 CAR constructs are described herein.(i) Anti-CD19 antibody binding domain

[0553] In some embodiments, the anti-CD19 antigen binding domain specifically binds to a target epitope of a CD 19 molecule. CD 19 antigens may be from various animal species, including human, non-human primate, mouse, rat, rabbit, or other non-human mammals. In some embodiments, the CD19 molecule is human CD19. In some embodiments, the CD19 molecule is rhesus macaque CD19. In some embodiments, the CD19 molecule is cynomolgus monkey CD 19. In some embodiments, the CD 19 molecule is wild- type. In some embodiments, the CD19 molecule is a mutant (e.g., comprises one or more insertions, deletions, and / or amino acid substitutions compared to a wild-type CD19 sequence from the same species). In some embodiments, the anti-CD19 antigen binding domain cross-reacts with a human CD19 and a133MF-366657479Attorney Reference No. 15979-20208.40cynomolgus monkey CD 19 and / or a rhesus macaque CD 19. In some embodiments, the antiCD 19 antigen moiety binds to wild-type human CD 19.

[0554] In some embodiments, the anti-CD19 antigen binding domain (e.g., full-length, sdAb, scFv, or Fab) binds to a CD19 antigen with a Kd < 1 pM, such as <100 nM, preferably <10 nM, more preferably <1 nM. For example, the Kd value of the anti-CD19 antigen binding domain is between about 1 nM and about 1 pM. In some embodiments, the anti-CD19 antigen binding domain binds to a human CD19 and / or a monkey (e.g., cynomolgus, rhesus macaque) CD 19 with a Kd of from about IxlO11M to about IxlO-7M (e.g., from about IxlO11M to about IxlO10M, from about IxlO11M to about IxlO-9M, from about IxlO11M to about IxlO-8M, from about IxlO10M to about IxlO-9M, from about IxlO10M to about IxlO-8M, from about IxlO10M to about IxlO-7M, from about IxlO-9M to about IxlO-7M, from about IxlO-9M to about IxlO-8M, or from about IxlO-8M to about IxlO-7M).

[0555] The anti-CD19 antigen binding domains described herein can be of any format and derived from any suitable anti-CD19 antibodies or antigen-binding fragments thereof. For example, the anti-CD19 antigen binding domain can be selected from a full-length antibody, an scFv, a VH, a VL, an scFv-scFv, an Fv, a Fab, a Fab’, a (Fab’)2, a minibody, a diabody, a domain antibody variant (dAb), a single domain antibody (sdAb or VHH), a camelid VHH antibody, a Nanobody® ISVD, a fibronectin 3 domain variant, an ankyrin repeat variant, and other antigen-specific binding domains derived from other protein scaffolds. In some embodiments, the anti-CD19 antigen binding domain is an sdAb, a VHH, or a Nanobody® ISVD. In some embodiments, the anti-CD19 antigen binding domain is a Fab. In some embodiments, the anti-CD19 antigen binding domain is an scFv. In some embodiments, the anti-CD19 antigen binding domain is humanized. In some embodiments, the anti-CD19 antigen binding domain is chimeric. In some embodiments, the anti-CD19 antigen binding domain is derived from a monoclonal antibody of mouse, rat, monkey, or rabbit. In some embodiments, the anti-CD19 antigen binding domain is derived from any anti-CD19 monoclonal antibodies known in the art. In some embodiments, the anti-CD19 antigen binding domain is derived from a fully human antibody, for example, developed using phage-display, yeast-display, or transgenic mice bearing human Ig genes. In some embodiments, the anti-CD19 antigen binding domain is a monospecific antibody, a multispecific antibody, a monovalent antibody, or a multivalent (e.g., bivalent) antibody.

[0556] In some embodiments, the anti-CD19 antigen binding domain specifically binds to both human and non-human primates (such as cynomolgus monkey and / or rhesus macaque) CD19. In some embodiments, the anti-CD19 antigen binding domain only binds to human 134MF-366657479Attorney Reference No. 15979-20208.40CD 19. Exemplary anti-CD19 antibodies and antigen binding fragments thereof include, but are not limited to, tafasitamab, loncastuximab tesirine, blinatumomab, MOR00208, DI-B4, FMC63, and those disclosed in: US 7,968,687; US 11,091,532; US 11,639,386; US 11,253,547; US 10,533,055; US 11,975,026; US 11,427,633; US 10,301,388; US 11,364,289; W02018161017; and WO2017055328, the content of each of which is specifically incorporated herein by reference. Chimeric antigen receptor constructs employing such antiCD 19 antigen binding domains with cross -reactivity to monkey CD 19 may facilitate toxicity studies in non-human primates, which can provide more relevant safety assessments for human clinical trial candidates, without having to perform toxicity studies in chimpanzees or using surrogate molecules.Anti-CD19 single domain antibody (sdAb; VHH; Nanobody® ISVD)

[0557] In some embodiments, the antigen binding domain specifically binding to B cells comprises a single domain antibody (sdAb) comprising a VHH. In some embodiments, the anti-CD19 antigen binding domain (e.g., scFv or Fab) specifically recognizes CD19.

[0558] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 433. In some embodiments, the antiCD 19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 481. In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising one, two, or three CDRs selected from either one of SEQ ID NO: 433 or 481.

[0559] In some embodiments, the anti-CD19 antigen binding domain comprises: (i) a VHH amino acid sequence as set forth in SEQ ID NO: 433, or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 433; or (ii) a VHH sequence as set forth in SEQ ID NO: 481, or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 481. In some embodiments, the anti-CD19 antigen binding domain comprises: (i) a VHH sequence as set forth in SEQ ID NO: 433; or (ii) a VHH sequence as set forth in SEQ ID NO: 481. In some embodiments, the anti-CD19 antigen binding domain comprises SEQ ID NO: 433 or SEQ ID NO: 481.

[0560] In some embodiments, the anti-CD19 antigen binding domain binds to essentially the same epitope as an antibody comprising: (i) a VHH amino acid sequence as set forth in SEQ135MF-366657479Attorney Reference No. 15979-20208.40ID NO: 433 or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 433; or (ii) a VHH amino acid sequence as set forth in SEQ ID NO: 481 or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 481.

[0561] In some embodiments, the anti-CD19 antigen binding domain competes for binding to a CD 19 epitope with an antibody comprising: (i) a VHH amino acid sequence as set forth in SEQ ID NO: 433 or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 433; or (ii) a VHH amino acid sequence as set forth in SEQ ID NO: 481 or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 481.

[0562] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH of any one of SEQ ID NOs: 684-739.

[0563] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 699.

[0564] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 739.

[0565] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 721.

[0566] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 687.

[0567] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 701.

[0568] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 706.

[0569] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 707.

[0570] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 714.136MF-366657479Attorney Reference No. 15979-20208.40

[0571] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 715.

[0572] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 718.

[0573] In some embodiments, the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 711.

[0574] the anti-CD19 antigen binding domain comprises a VHH comprising an amino acid sequence of SEQ ID NO: 712.

[0575] In some embodiments, the anti-CD19 antigen binding domain comprises: (i) a VHH amino acid sequence as set forth in SEQ ID NO: 699, or an amino sequence comprising at least about 80% (such as at least about any of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 699; (ii) a VHH sequence as set forth ...

Claims

Attorney Reference No. 15979-20208.40CLAIMSWhat is claimed is:

1. A polypeptide comprising an anti-CD19 chimeric antigen receptor (anti-CD19 CAR), wherein the anti-CD19 CAR comprises:(a) an anti-CD19 antigen-binding domain, wherein the anti-CD19 antigen-binding domain is an anti-CD19 single domain antibody (anti-CD19 sdAb) domain comprising an anti-CD19 VHH comprising:(i) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:427, an H- CDR2 comprising the amino acid sequence of SEQ ID NO:428, and an H- CDR3 comprising the amino acid sequence of SEQ ID NO:429, or (ii) an H-CDR1 comprising the amino acid sequence of SEQ ID NO:475, an H-CDR2 comprising the amino acid sequence of SEQ ID NO:476, and an H-CDR3 comprising the amino acid sequence of SEQ ID NO:477;(b) a hinge domain,(c) a transmembrane domain,(d) an intracellular signaling domain, and(e) optionally a co-stimulatory domain.

2. The polypeptide of claim 1, wherein the anti-CD19 sdAb comprises a VHH comprising the amino acid sequence of SEQ ID NO:433 or 481, or an amino acid sequence comprising at least about 80% sequence identity to the amino acid sequence of SEQ ID NO:433 or 481.

3. The polypeptide of claim 1 or 2, wherein the hinge domain is derived from a protein selected from the group consisting of CD28, CD5, CD9, CD2, CD27, CD28, IgG, IgK, and CD8a.

4. The polypeptide of claim 3, wherein in the hinge domain is derived from CD28 or CD8a.

5. The polypeptide of any one of claims 1-4, wherein the transmembrane domain is derived from a protein selected from the group consisting of CD28, CD3e, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD147, 4-1BB (CD137), CD152, PD-1, CD2, CD27, CD1 lb, CD1 Id, CD1 la, CD11c, CD84, and CD86.

6. The polypeptide of claim 5, wherein in the transmembrane domain is derived from CD28 or CD8a.407MF-366657479Attorney Reference No. 15979-20208.

407. The polypeptide of any one of claims 1-6, wherein the hinge domain and the transmembrane domain are derived from the same protein.

8. The polypeptide of claim 7, wherein the hinge domain and the transmembrane domain are derived from CD28 and comprises the amino acid sequence of SEQ ID NO:431 or 434.

9. The polypeptide of any one of claims 1-8, wherein the intracellular signaling domain is derived from a protein selected from the group consisting of CD3(^, FceRip, FceRIy, FcRp, CD3y, CD36, CD3e, CD5, CD22, CD79a, CD79b, and CD66d.

10. The polypeptide of claim 9, wherein the intracellular signaling domain is derived from CD3 .

11. The polypeptide of claim 9 or 10, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:544.

12. The polypeptide of any one of claims 1-11, wherein the anti-CD19 CAR comprises a co- stimulatory domain derived from a protein selected from the group consisting of CD27, CD28, CD137 (4-1BB), 0X40, CD40, PD-1, EFA-1, ICOS, CD2, CD7, EIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40EG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, EGALS9, CD83, ligands of CD83, and any combination thereof.

13. The polypeptide of claim 12, wherein the co-stimulatory domain is derived from CD137 (4-1BB) or CD28.

14. The polypeptide of claim 12 or 13, wherein the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:432 or 543.

15. The polypeptide of any one of claims 1-14, further comprising a signal peptide sequence that comprises a signal peptide, wherein the lead peptide sequence is connected to the N-terminus of the anti-CD19 CAR.

16. The polypeptide of claim 15, wherein the signal peptide sequence is derived from a CD8a propeptide.

17. The polypeptide of claim 15 or 16, wherein the signal peptide sequence comprises the amino acid sequence of SEQ ID NO:515.

18. The polypeptide of any one of claims 1-17, wherein the C-terminus of the anti-CD19 sdAb and the N-terminus of the hinge domain are connected by a peptide linker, optionally wherein the peptide linker is AS or AAA.408MF-366657479Attorney Reference No. 15979-20208.4019. The polypeptide of any one of claims 1-18, wherein the anti-CD19 CAR comprises the amino acid sequence of any one of SEQ ID NOs:49-51 and 435-437.

20. An isolated nucleic acid encoding the polypeptide of any one of claims 1-19.

21. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:46-48, 114, 145, 441-458, 482-511, and 570-575.

22. The isolated nucleic acid of claim 20 or 21, wherein the nucleic acid is RNA.

23. The isolated nucleic acid of claim 22, wherein the RNA comprises a nucleic acid sequence of any one of SEQ ID NOs:570-575.

24. A vector comprising the isolated nucleic acid of any one of claims 20-23.

25. A cell comprising the polypeptide of any one of claims 1-19, the isolated nucleic acid of any one of claims 20-23, or the vector of claim 24.

26. The cell of claim 25, wherein the cell is an immune cell.

27. The cell of claim 26, wherein the immune cell is a T cell.

28. The cell of any one of claims 25-27, wherein the cell is a human cell.

29. A lipid nanoparticle (LNP) comprising:(a) a lipid- immune cell targeting group conjugate comprising the compound of Formula (II): [Lipid] - [optional linker] - [binding domain], wherein the binding domain specifically binds to human CD8a,(b) an ionizable cationic lipid, and(c) a nucleic acid encoding the polypeptide of any one of claims 1-19, wherein the nucleic acid is encapsulated in the LNP.

30. A lipid nanoparticle (LNP) comprising:(a) a lipid- immune cell targeting group conjugate comprising the compound of Formula (II): [Lipid] - [optional linker] - [binding domain], wherein the binding domain specifically binds to human CD8a,(b) an ionizable cationic lipid, and(c) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:46-48, 114, 145, 441-458, 482-511, and 570-575, wherein the nucleic acid is encapsulated in the LNP.409MF-366657479Attorney Reference No. 15979-20208.4031. The LNP of claim 29 or 30, wherein:a. the binding domain is an immunoglobulin single variable domain (ISVD) that specifically binds to human CD8a, wherein the ISVD comprises a complementarity-determining region 1 (CDR1), a CDR2, and a CDR3 of an ISVD having the sequence selected from the group consisting of SEQ ID NOs: 160-179; orb. the binding domain is an immunoglobulin single variable domain (ISVD) that specifically binds to human CD8a, wherein the ISVD specifically binding to CD8a comprises a CDR1, a CDR2, and a CDR3 according to the Abm CDR definition,wherein CDR1 is chosen from the group consisting of:(i) SEQ ID NO: 244; and(ii) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO: 244; wherein CDR2 is chosen from the group consisting of:(iii) SEQ ID NO: 246; and(iv) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO: 246; and wherein CDR3 is chosen from the group consisting of:(v) SEQ ID NO: 248; andamino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NO: 248.

32. The LNP of any one of claims 29-31, wherein the binding domain specifically binding to human CD8a is covalently coupled to the Lipid in Formula (II) via a linker comprising polyethylene glycol (PEG).

33. The LNP of claim 32, wherein the Lipid in Formula (II) covalently coupled to the binding domain is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyrstoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.

34. The LNP of claim 33, wherein the Lipid in Formula (II) covalently coupled to the binding domain is DSPE.410MF-366657479Attorney Reference No. 15979-20208.4035. The LNP of any one of claims 32-34, wherein the PEG is PEG 3400 (PEG 3.4K).

36. The LNP of any one of claims 32-35, wherein the immunoglobulin single variable domain comprises SEQ ID NO: 9, SEQ ID NO: 169, or SEQ ID NO: 44.

37. The LNP of any one of claims 29-36, wherein the LNP further comprises a structural lipid, a neutral phospholipid, or a free PEG-lipid, or any combination thereof.

38. The LNP of claim 37, wherein the structural lipid comprises or is sterol.

39. The LNP of claim 37 or 38, wherein the sterol comprises or is cholesterol.

40. The LNP of any one of claims 37-39, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and sphingomyelin.

41. The LNP of any one of claims 37-40, wherein the neutral phospholipid comprises or is DSPC.

42. The LNP of any one of claims 37-41, wherein the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

43. The LNP of any one of claims 37-42, wherein the free PEG lipid is PEG-dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyrstoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (Cl 6) chain or Distearoyl (Cl 8) chain, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

44. The LNP of claim 43, wherein the PEG-DAG comprises PEG-DMG, PEG-DPG, or PEG-DSG, or any combination thereof.411MF-366657479Attorney Reference No. 15979-20208.4045. The LNP of claim 44, wherein the free PEG-lipid comprises PEG-DPG.

46. The LNP of claim 45, wherein the PEG-DPG comprises or is PEG 2000-DPG (DPG- PEG 2000).

47. The LNP of any one of claims 29-46, wherein the nucleic acid comprises RNA.

48. The LNP of claim 47, wherein the RNA comprises mRNA.

49. The LNP of claim 48, wherein the mRNA comprises a 5' Cap, a 5' untranslated region (UTR), a sequence encoding a polypeptide, a 3' UTR, and optionally a polyA tail.

50. The LNP of claim 49, wherein the mRNA comprises SEQ ID NO: 570.

51. The LNP of claim 49, wherein the mRNA comprises SEQ ID NO: 571.

52. The LNP of claim 49, wherein the mRNA comprises SEQ ID NO: 572.

53. The LNP of claim 49, wherein the mRNA comprises SEQ ID NO: 573.

54. The LNP of claim 49, wherein the mRNA comprises SEQ ID NO: 574.

55. The LNP of claim 49, wherein the mRNA comprises SEQ ID NO:575.

56. The LNP of claim 55, wherein the mRNA comprises a 5' Cap, a 5' untranslated region (UTR), a sequence encoding a polypeptide, a 3' UTR, and optionally a polyA tail.

57. The LNP of any one of claims 29-56, wherein the nucleic acid comprises:(1) optionally, a 5' cap;(2) optionally, a 5' UTR region;(3) nucleotides encoding the polypeptide of any one of claims 1-19;(4) optionally, a 3' UTR region; and(5) optionally, a polyA tail.

58. The LNP of claim 29 or 30, wherein the polypeptide encoded by the nucleic acid comprises the following formula, arranged from N-terminus to C-terminus:[Signal peptide sequence (optional)] - [anti-CD19 sdAb] - [Peptide Linker (optional)] [Hinge domain] - [Transmembrane domain] - [Co-stimulatory domain] - [Intracellular signaling domain],59. The LNP of any one of claims 29-58, wherein the nucleic acid comprises pseudouridine.

60. The LNP of claim 59, wherein the pseudouridine is Nl-methyl-pseudouridine.412MF-366657479Attorney Reference No. 15979-20208.4061. The LNP of any one of claims 29-60, wherein the ionizable cationic lipid comprises a compound of Formula (I):or a salt thereof, or both, wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)o-ioC(0)ORal, or - (CH2)o-ioOC(0)Ra2;Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;p3B2 R3B^R3Bis R3B3;R3B1is C1-6 alkylene; andR3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.

62. The LNP of claim 54, wherein:R1, R2, and R3are each independently a bond or methylene;R1Aand R2Aare each Ci-io alkylene;R3Ais Ci-5 alkylene;R1A1, R1A2, R2A1, R2A2, R3A1, and R3A2are each H;R1A3and R2A3are each C1-20 alkenyl;R3A3is -C(O)O(Ci-20 alkyl);413MF-366657479Attorney Reference No. 15979-20208.40R3B1is C2-4 alkylene; andR3B2and R3B3are each methyl.

63. The LNP of claim 61 or 62, wherein R3B1is -(CEL)?-.

64. The LNP of any one of claims 29-63, wherein the ionizable cationic lipid comprisessalt thereof, or both.

65. The LNP of any one of claims 29-64, wherein the cationic lipid has a concentration between about 10 mol% and about 60 mol% of the LNP.

66. The LNP of any one of claims 29-65, wherein the LNP comprises cholesterol at a concentration between about 25 mol% and about 45 mol% of the LNP.

67. The LNP of any one of claims 29-66, wherein the LNP comprises DSPC at a concentration between about 5 mol% and about 25% mol% of the LNP.

68. The LNP of any one of claims 29-67, wherein the LNP comprises DPG-PEG2K at a concentration between about 0.5 mol% and about 2.5 mol% of the LNP.

69. The LNP of any one of claims 29-68, wherein the LNP comprises cationic lipid at a concentration between about 49 mol% and about 50 mol% of the LNP, such as about 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, or 49.9 mol%.

70. The LNP of any one of claims 29-69, wherein the LNP comprises cholesterol at a concentration between about 25 mol% and about 30 mol% of the LNP, such as about 25, 26, 27, 28, 29, or 30 mol%.

71. The LNP of any one of claims 29-70, wherein the LNP comprises DSPC at a concentration between about 9 mol% and about 21 mol% of the LNP.

72. The LNP of any one of claims 29-71, wherein the LNP comprises DPG-PEG2K at a concentration between about 1.4 mol% and about 1.6 mol% of the LNP.414MF-366657479Attorney Reference No. 15979-20208.4073. The LNP of any one of claims 29-64, wherein the LNP comprises cationic lipid at a concentration between about 10 and about 20 g per gram of mRNA in the LNP.

74. The LNP of any one of claims 29-73, wherein the LNP comprises cholesterol at a concentration between about 3.0 and about 5.0 g per gram of mRNA in the LNP.

75. The LNP of any one of claims 29-74, wherein the LNP comprises DSPC at a concentration between about 2.0 and about 5.0 g per gram of mRNA in the LNP.

76. The LNP of any one of claims 29-75, wherein the LNP comprises DPG-PEG2K at a concentration between about 1.0 and about 1.5 g per gram of mRNA in the LNP.

77. The LNP of any one of claims 29-76, wherein the LNP comprises DSPE-PEG3.4-antibody conjugate at a concentration between about 0.05 to 0.1 g per gram of mRNA in the LNP.

78. The LNP of any one of claims 29-72, wherein(i) the cationic lipid has a concentration about 49.2 mol% of the LNP;(ii) the cholesterol has a concentration about 39.4 mol% of the LNP;(iii) the DSPC has a concentration about 9.8 mol% of the LNP; and(iv) the DPG-PEG2K has a concentration about 1.5% of the LNP.

79. The LNP of any one of claims 29-72, wherein(i) the cationic lipid has a concentration about 49.2 mol% of the LNP;(ii) the cholesterol has a concentration about 29.3 mol% of the LNP;(iii) the DSPC has a concentration about 20.0 mol% of the LNP; and(iv) the DPG-PEG2K has a concentration about 1.5% of the LNP.

80. The LNP of any one of claims 29-77, wherein(i) the cationic lipid has a concentration about 14.2 g / g mRNA in the LNP;(ii) the cholesterol has a concentration about 4.64 g / g mRNA in the LNP;(iii) the DSPC has a concentration about 2.37 g / g mRNA in the LNP;(iv) the DPG-PEG2K has a concentration about 1.15 g / g mRNA in the LNP; and (v) the DSPE-PEG3.4K-anti-CD8 antibody conjugate has a concentration about 0.084 g / g to 0.15 g / g mRNA in the LNP.

81. An in vitro transcribed mRNA derived from the isolated nucleic acid of any one of claims 20-23.415MF-366657479Attorney Reference No. 15979-20208.4082. An immune cell comprising the in vitro transcribed mRNA of claim 81.

83. An immune cell expressing the polypeptide of any one of claims 1-19.

84. A method of preparing an LNP, comprising combining the isolated nucleic acid of any one of claims 20-23 with a mixture of lipids.

85. A pharmaceutical composition, comprising the polypeptide of any one of claims 1-19, the isolated nucleic acid of any one of claims 20-23, the vector of claim 24, the cell of any one of claims 25-28, and / or the LNP of any one of claims 29-80.

86. A method of delivering a nucleic acid sequence into a human cell, comprising administering the pharmaceutical composition of claim 85.

87. A method of modulating immune response in a human subject, comprising administering the LNP of any one of claims 29-80, the isolated nucleic acid of any one of claims 20-23, the vector of claim 24, and / or the cell of any one of claims 25-28 to the human subject, and / or expressing the polypeptide of any one of claims 1-19 in the human subject.

88. A method of treating a B cell malignancy in a human subject comprising administering the LNP of any one of claims 29-80, the isolated nucleic acid of any one of claims 20-23, the vector of claim 24, and / or the cell of any one of claims 25-28 to the human subject, and / or expressing the polypeptide of any one of claims 1-19 in the human subject.

89. The method of claim 88, wherein the B cell malignancy is hematological cancer or an autoimmune disorder.

90. The method of claim 89, wherein the B cell malignancy is a hematological cancer.

91. The method of claim 89 or 90, wherein the hematological cancer is a B cell lymphoma.

92. The method of claim 91 , wherein the B cell lymphoma is diffuse large B cell lymphoma (DLBCL).

93. The method of claim 92, wherein the B cell malignancy is an autoimmune disorder.

94. The method of claim 89 or 93, wherein the autoimmune disorder is selected from the group consisting of rheumatoid arthritis (RA), multiple sclerosis, type 1 diabetes mellitus (T1D), primary Sjogren’s syndrome (pSS), and systemic lupus erythematosus (SLE).

95. Use of the polypeptide of any one of claims 1-19, the isolated nucleic acid of any one of claims 20-23, the vector of claim 24, the cell of any one of claims 25-28, LNP of any one of416MF-366657479Attorney Reference No. 15979-20208.40claims 29-80, and / or the pharmaceutical composition of claim 85 for the manufacture of a medicament for the treatment of a B cell malignancy.

96. The use of claim 88, wherein the B cell malignancy is a hematological cancer or an autoimmune disorder.

97. The use of claim 96, wherein the B cell malignancy is a hematological cancer.

98. The use of claim 96 or 97, wherein the hematological cancer is a B cell lymphoma.

99. The use of claim 98, wherein the B cell lymphoma is diffuse large B cell lymphoma (DLBCL).

100. The use of claim 96, wherein the B cell malignancy is an autoimmune disorder selected from the group consisting of rheumatoid arthritis (RA), multiple sclerosis, type 1 diabetes mellitus (T1D), primary Sjogren’s syndrome (pSS), and systemic lupus erythematosus (SLE).

101. Use of the LNP of any one of claims 29-80 or the pharmaceutical composition of claim 78 for the manufacture of a medicament for delivering a nucleic acid to a target cell.

102. The use of claim 101, wherein the target cell is an immune cell.417MF-366657479

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