Delivery composition

Abstract

The present disclosure provides novel lipids, e.g., suitable for use in lipid nanoparticles, for therapeutic delivery of nucleic acids. Methods of making and using these lipids are also provided.

Description

Related applications

[0001] This application claims priority to U.S. Serial No. 63 / 552,586, filed February 12, 2024; U.S. Serial No. 63 / 666,081, filed June 28, 2024; and U.S. Serial No. 63 / 725,955, filed November 27, 2024, the entire contents of each of which are incorporated herein by reference. sequence list

[0002] This application contains a sequence list that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on February 11, 2025, named A2002-7000TW_SL.xml, and is 129,622 bytes in size. Technical Field

[0003] This invention provides novel ionizable lipids and lipid nanoparticles for the in vitro and in vivo delivery of therapeutic nucleic acids (e.g., siRNA, mRNA, gRNA). Background Technology

[0004] Due to the relative instability and low cell permeability of nucleic acids, effective targeted delivery of these types to cells is challenging. Therefore, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents (such as nucleic acids) to cells. Preferably, these lipid-nucleic acid particles should be well-tolerated and provide a sufficient therapeutic index so that patient treatment with an effective dose of nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present invention provides these and related advantages. Summary of the Invention

[0005] This invention recognizes the need for compositions, formulations, nanoparticles and / or nanomaterials, and methods of using them. Among other things, this disclosure recognizes that the structural characteristics of compositions, formulations, nanoparticles and / or nanomaterials influence functional responses in vivo, in vitro, and ex vivo. For example, this disclosure describes, among other things, that the selection and combination of one or more components described herein affects the functional activity of lipid nanoparticles. In some embodiments, functional activity may, for example, refer to desired tropism, stability, and / or drug delivery efficacy. In some embodiments, among other things, this disclosure describes that different ratios of one or more components affect one or more functional activities of the compositions, formulations, nanoparticles and / or nanomaterials described herein.

[0006] The lipid nanoparticle compositions provided herein may further comprise nucleic acids, such as RNA, for example messenger RNA (mRNA). These lipid nanoparticle compositions can be used to deliver mRNA to the cells of a subject. Messenger RNA-based gene therapies require the efficient delivery of mRNA to circulating cells in plasma (e.g., immune cells) or to cells in a given tissue. Key challenges associated with efficient mRNA delivery to achieve robust protein expression levels include: (a) the ability to protect the mRNA payload from ubiquitous serum nucleases when administered to a subject; (b) the ability to specifically target mRNA delivery to a target cell population to maximize protein expression therein; and (c) the ability to deliver the mRNA payload maximally to the cytoplasmic compartments of the cell for intracellular translation into protein.

[0007] This invention provides ionizable cationic lipids for producing lipid nanoparticle compositions that facilitate the delivery of payloads (e.g., nucleic acids) placed therein to cells, such as mammalian cells, like immune cells. These lipids are engineered to deliver nucleic acids (e.g., mRNA) intracellularly to cytoplasmic compartments of target cell types and rapidly degrade into non-toxic components. Without wishing to be bound by theory, in some embodiments, these complex functions are achieved through the chemical and geometric interactions between the ionizable lipid head group, the hydrophobic "acyl tail" group, and the linker connecting the head group and the acyl tail group in the ionizable cationic lipid.

[0008] In one aspect, the present invention provides a compound represented by formula (A1) or formula (A-II):

[0009] or

[0010] (AI)(A-II)

[0011] Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein

[0012] L is an optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0013] L1 is an optionally substituted C1-6 alkylene or C2-6 heteroalkylene;

[0014] Each L2 is independently a C1-6 alkylene group;

[0015] L3 is an optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0016] L4 is a non-existent, optionally substituted C1- 10 Alkylene or optionally substituted C2- 10 Heteroalkyl;

[0017] L5 is an optionally substituted C1- 10 Alkyl, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Alkyne group or optionally substituted C2- 10 Heteroalkyl;

[0018] X is non-existent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-;

[0019] Each X2 is non-existent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-;

[0020] Each of R is independently hydrogen, OR6, or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;

[0021] R1 is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered aliphatic ring, optionally substituted 3- to 7-membered heterocyclic group containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -CN, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, - NR2C(O)N(R2)2, -NR2C(S)N(R2)2, -NR2C(NR2)N(R2)2, -NR2C(CHR2)N(R2)2, -N(OR 2)C(O)R2, -N(OR2)S(O)2R2, -N(OR2)C(O)OR2, -N(OR2)C(O)N(R2)2, -N(OR2)C(S)N( R2)2, -N(OR2)C(NR2)N(R2)2, -N(OR2)C(CHR2)N(R2)2, -C(NR2)N(R2)2, -C(NR2)R2, -C(O)N(R2)OR2, -C(R2)N(R2)2C(O)OR2, -CR2(R3)2, -OP(O)(OR2)2 or -P(O)(OR2)2; or

[0022] R1 is selected from the following rings: 3- to 7-membered aliphatic rings and 3- to 7-membered heterocyclic groups (containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur), wherein the aliphatic or heterocyclic ring is optionally substituted by 1 to 4 R2 or R3 groups;

[0023] Each R2 group is independently hydrogen, oxo, -CN, -NO2, -OR4, -S(O)2R4, -S(O)2N(R4)2, or -(CH2). n-R4 or optionally substituted groups selected from: C1-6 aliphatic, phenyl, 3- to 7-membered aliphatic, 5- to 6-membered monocyclic heteroaryl (containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), and 3- to 7-membered heterocyclic groups (containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur); or two R2s together with one or more atoms attached to them form an optionally substituted 4- to 7-membered heterocyclic group, which contains 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R3 is independently -(CH2)n-R4; or two R3s together with one or more atoms attached to them form an optionally substituted 4- to 7-membered heterocyclic group, which contains 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R3 is independently -(CH2)n-R4; or two R3s together with one or more atoms attached to them form an optional substituted 4- to 7-membered heterocyclic group. One or more atoms together form an optionally substituted 5- to 6-membered heterocyclic group, the heterocyclic group comprising 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R4 is independently hydrogen, -OR5, -N(R5)2, -OC(O)R5, -OC(O)OR5, -CN, -C(O)N(R5)2, -NR5C(O)R5, -OC(O)N(R5)2, -N(R5)C(O)OR5, -NR5S(O)2R5, -NR5C(O)N(R5)2, -NR5C(S)N(R5)2, or -NR5C(NR5)N(R5)2; and

[0024] Each R5 is independently hydrogen or optionally substituted C. 1-6 Aliphatic; or the two R5s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups, which contain 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R6 is independently C 4-12 Aliphatic; and each n is independently 0 to 4.

[0025] In some embodiments, R1 is -N(R2)2.

[0026] In some embodiments, L1-R1 is selected from the group consisting of:

[0027] .

[0028] In some embodiments, Choose from the following groups: ,

[0029] In some embodiments, yes .

[0030] In some embodiments, yes , where R 1000 It is an optional substituted C1- 10 Alkyl, optionally substituted C2- 10alkenyl, optionally substituted C2- 10 Alkyne group, optionally substituted C3- 10 cycloalkyl or optionally substituted C5- 10 Heteroalkyl groups.

[0031] In some embodiments, yes .

[0032] In some embodiments, yes .

[0033] In some embodiments, x is -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-.

[0034] In some embodiments, x is -OC(O)-.

[0035] In some embodiments, when at least one no At that time, yes , where R 1000 It is an optional substituted C1- 10 Alkyl, optionally substituted C2- 10 alkenyl, optionally substituted C2- 10 Alkyne group, optionally substituted C3- 10 cycloalkyl or optionally substituted C5- 10 Heteroalkyl groups.

[0036] In some embodiments, when no At that time, yes , where R 1000 It is an optional substituted C1- 10 Alkyl, optionally substituted C2- 10 alkenyl, optionally substituted C2- 10 Alkyne group, optionally substituted C3- 10 cycloalkyl or optionally substituted C5- 10 Heteroalkyl groups.

[0037] In some embodiments, compounds having formula (A-III)

[0038] (A-III); where L1-L5, X2, R1, R” and R 1000 As defined above.

[0039] In some embodiments, X2 is OC(O) or C(O)O; and "-L3-R" is independently selected from:

[0040]

[0041] In some embodiments, compounds having formula (A-IV)

[0042] (A-IV): where each X 100 Independently, it is OC(O), C(O)O, OC(O)O, OC(O)NH, or NHC(O)O; L 100 It is a C1-C6 alkylene group; and L1-L5, X2, R1, R” and R 1000 As defined above.

[0043] In some embodiments, L 100 It is CH2 and X 100 It is OC(O).

[0044] In some embodiments, L4 is absent, and L 100 It is CH2 and X 100 It is OC(O).

[0045] In some embodiments, L2 is CH2, X2 is OC(O), and L3 is CH2.

[0046] In some embodiments, L2 is CH2, X2 is OC(O), L3 is CH2, L4 is non-existent, and L... 100 It is CH2 and X 100 It is OC(O).

[0047] In some embodiments, L1 is a C2-C6 alkylene group.

[0048] In some embodiments, -L1-R1 is selected from:

[0049]

[0050] In some embodiments, compounds having formula (BI):

[0051] (BI)

[0052] Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein

[0053] Each R' is independently a non-existent, optionally substituted C1-6 alkyl or optionally substituted C1-6 heteroalkyl;

[0054] Each of L is independently a C1- that has been optionally substituted. 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0055] Each of X is independently O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O;

[0056] Each Z is independent , or The condition is that at least one or two of Z are or ;

[0057] Each of m is independently 1-9;

[0058] t is 1, 2, or 3;

[0059] X 10 It is non-existent, O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O;

[0060] "R" is H, , or ;

[0061] L 10 It is an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0062] R 11 Each of them is independently C2- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, C2-C substituted bicyclic compounds with 7- to 12-membered bridging substitutions containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 10 Aliphatic, 1-adamantyl, 2-adamantyl, sterol or phenyl; and

[0063] R10 Each of these is independently hydrogen, OR6, or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl.

[0064] In some embodiments, Z is .

[0065] In some embodiments, Z is .

[0066] In some embodiments, R' is H or CH3.

[0067] In some embodiments, the compound represented by formula (B-II):

[0068] (B-II)

[0069] Z and R are each independent of each other. , or The condition is that at least two or three of Z and R are or .

[0070] In some embodiments, Selected from the following group, which consists of the following:

[0071]

[0072] In some embodiments, Selected from the following group, which consists of the following:

[0073] , , ,and .

[0074] In some embodiments, compounds having formula (CI):

[0075] (CI)

[0076] Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein

[0077] X 20 Each of these is independent of the following: O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O;

[0078] L 20 Each of them is independently and optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0079] Z 20 Each of them is independently , or The condition is Z. 20 At least one of them is or ;

[0080] Each of m is independently 1-9;

[0081] t is 1, 2, or 3;

[0082] L 10 It is an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0083] R 11 Each of them is independently C2- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, C2-C substituted bicyclic compounds with 7- to 12-membered bridging substitutions containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 10 Aliphatic, 1-adamantyl, 2-adamantyl, sterol or phenyl; and

[0084] R 10 Each of these is independently hydrogen, OR6, or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl;

[0085] q can be 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0086] It is a 4- to 8-membered ring or a heterocyclic ring;

[0087] L1 is absent, a C1-6 alkylene group, or a C2-6 heteroalkylene group;

[0088] R1 is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered aliphatic ring, optionally substituted 3- to 7-membered heterocyclic group containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -CN, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, - NR2C(O)N(R2)2, -NR2C(S)N(R2)2, -NR2C(NR2)N(R2)2, -NR2C(CHR2)N(R2)2, -N(OR 2)C(O)R2, -N(OR2)S(O)2R2, -N(OR2)C(O)OR2, -N(OR2)C(O)N(R2)2, -N(OR2)C(S)N( R2)2, -N(OR2)C(NR2)N(R2)2, -N(OR2)C(CHR2)N(R2)2, -C(NR2)N(R2)2, -C(NR2)R2, -C(O)N(R2)OR2, -C(R2)N(R2)2C(O)OR2, -CR2(R3)2, -OP(O)(OR2)2 or -P(O)(OR2)2; or

[0089] R1 is selected from the following rings: 3- to 7-membered aliphatic rings and 3- to 7-membered heterocyclic groups (containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur), wherein the aliphatic or heterocyclic ring is optionally substituted by 1 to 4 R2 or R3 groups;

[0090] Each R2 group is independently hydrogen, oxo, -CN, -NO2, -OR4, -S(O)2R4, -S(O)2N(R4)2, or -(CH2). n -R4 or optionally substituted groups selected from: C1-6 aliphatic, phenyl, 3- to 7-membered aliphatic, 5- to 6-membered monocyclic heteroaryl (containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur), and 3- to 7-membered heterocyclic groups (containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur); or R2 appearing twice together with one or more atoms to which they are attached to form optionally substituted 4- to 7-membered heterocyclic groups, which contain 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R3 is independently -(CH2). n-R4; or two R3s together with one or more atoms to which they are attached form optionally substituted 5- to 6-membered heterocyclic groups, the heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R4 is independently hydrogen, -OR5, -N(R5)2, -OC(O)R5, -OC(O)OR5, -CN, -C(O)N(R5)2, -NR5C(O)R5, -OC(O)N(R5)2, -N(R5)C(O)OR5, -NR5S(O)2R5, -NR5C(O)N(R5)2, -NR5C(S)N(R5)2, or -NR5C(NR5)N(R5)2; and

[0091] Each R5 is independently hydrogen or optionally substituted C. 1-6 Aliphatic; or the two R5s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups, which contain 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R6 is independently C 4-12 Aliphatic; and each n is independently 0 to 4.

[0092] In some embodiments, Choose from the following groups:

[0093]

[0094] In some embodiments, Choose from the following groups:

[0095]

[0096] In some embodiments, yes or .

[0097] In some embodiments, L1 is absent, and R1 is -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2 or -NR2C(O)N(R2)2; and R2 is as defined above.

[0098] In some embodiments, R1 is -OR2 or -C(O)OR2.

[0099] In some embodiments, L1-R1 is selected from the group consisting of:

[0100] In some embodiments, -L1-R1 is selected from:

[0101]

[0102] In some embodiments, the compound represented by formula (C-II):

[0103] (C-II)

[0104] Where L1 is non-existent, R1 is OR2, q is 2-9, and X is... 20 It is OC(O) or C(O)O, R2, L 20 and Z 20 As defined above.

[0105] In some embodiments, the compound represented by formula (C-III):

[0106] (C-III)

[0107] Where L1 is non-existent, R1 is OR2, q is 2-9, and X is... 20 It is OC(O) or C(O)O, R2, L 20 and Z 20 As defined above.

[0108] In some embodiments, -L 20 -Z 20 Choose from the following groups:

[0109]

[0110] In some embodiments, the compound represented by formula (C-IV):

[0111] (C-IV)

[0112] Where L1 is non-existent, R1 is OR2, q is 2-9, and X is... 20 It is OC(O) or C(O)O, R2, L 20 and Z 20 As defined above.

[0113] In some embodiments, Selected from the following group, which consists of the following:

[0114]

[0115] In some embodiments, Selected from the following group, which consists of the following:

[0116] , , ,and .

[0117] In some embodiments, -L1-R1 is selected from:

[0118]

[0119] In some embodiments, compounds having formula (DI):

[0120]

[0121] Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein

[0122] L 101 and L 102 Each is independently a C1-6 alkylene group;

[0123] L 103 and L 104 Each is independently an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0124] X 101 and X 102 Each of these can be independently nonexistent: -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-.

[0125] L 105、 L 106 L 107 and L 108 Each is independently an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0126] R 100 R 101 R 102 and R 103 Each is independently hydrogen, OC 1-3 Aliphatic, OR6, or optionally substituted groups selected from the following: C6-20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl;

[0127] L 109 It is a C1-6 alkylene group;

[0128] X 103 It does not exist, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -OC(O)O- or -NR 105 -;

[0129] R 105 It is hydrogen, C1- 10 Alkanes, C2- 10 Alkenes or C 3-12 Cycloalkanes;

[0130] L 110 It is a C1-6 alkylene group;

[0131] X 104 Is it non-existent or -NR 106 -;

[0132] L 111 It is a C1-6 alkylene group;

[0133] R 106 It is hydrogen, C1- 10 Alkanes, C2- 10 Alkenes or C 3-12 Cycloalkanes;

[0134] R 104 It is hydrogen, OR 107 C1- 10 Alkanes, C2- 10 Olefins, C 3-12 Cycloalkanes or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterol, and phenyl; and

[0135] R 107 Is it H or C? 1-12 Aliphatic.

[0136] In some embodiments, compounds having formula (D-II):

[0137] ,

[0138] Where R 100R 101 R 102 and R 103 Each is independently hydrogen, OC 1-3 Aliphatic, OR6, or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl;

[0139] L 105、 L 106 L 107 and L 108 Each is independently an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0140] X 103 It does not exist, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -OC(O)O- or -NR 105 -;

[0141] R 105 It is hydrogen, C1- 10 Alkanes, C2- 10 Alkenes or C 3-12 Cycloalkanes;

[0142] L 110 It is a C1-6 alkylene group;

[0143] X 104 Is it non-existent or -NR 106 -;

[0144] L 111 It is a C1-6 alkylene group;

[0145] R 106 It is hydrogen, C1- 10 Alkanes, C2- 10 Alkenes or C 3-12 Cycloalkanes; and

[0146] R 104 It is hydrogen, OR 107 C1- 10 Alkanes, C2- 10 Olefins, C 3-12Cycloalkanes or optionally substituted groups selected from the following: C6-20 aliphatic, 3- to 12-membered cycloaliphatic, 7- to 12-membered bridged bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 1-adamantyl, 2-adamantyl, sterol, or phenyl; and

[0147] R 107 Is it H or C? 1-12 Aliphatic.

[0148] In some embodiments, and Each person independently chooses from the following groups:

[0149] In some embodiments, and Each person independently chooses from the following groups:

[0150] , , , and .

[0151] In some embodiments, Selected from the following group, which consists of the following:

[0152] , , ,and .

[0153] In some embodiments, -L1-R1 is selected from:

[0154]

[0155] In some embodiments, compounds having formula (EI) are:

[0156] (EI)

[0157] Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein

[0158] X 30 Each of these is independent of the following: O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O;

[0159] L 30 Each of them is independently and optionally substituted C1- 10Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0160] X 31 Each of them is independently NR”; where R” is H, , or The condition is Z. 30 At least one of them is or ;

[0161] Z 30 Each of them is independently , or The condition is Z. 30 At least one of them is or ;

[0162] Each of m is independently 1 to 9;

[0163] t is 1, 2, or 3;

[0164] L 10 It is an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl;

[0165] R 11 Each of them is independently C2- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, C2-C substituted bicyclic compounds with 7- to 12-membered bridging substitutions containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 10 Aliphatic, 1-adamantyl, 2-adamantyl, sterol or phenyl; and

[0166] R 10 Each of them is optionally substituted with a group selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl.

[0167] In some embodiments, the compound represented by formula (E-II):

[0168] (E-II)

[0169] Where X 30 L 30 X 31 and Z 30 As defined above.

[0170] In some embodiments, X 30 It is C(O)O, OC(O), C(O)NH, NHC(O) or O.

[0171] In some embodiments, Selected from the following group, which consists of the following:

[0172]

[0173] In some embodiments, Selected from the following group, which consists of the following:

[0174] , , ,and .

[0175] In some embodiments, X 30 It is C(O)NH.

[0176] In some embodiments, X 30 It is C(O)O.

[0177] In some embodiments, X 31 It's NR.

[0178] In some embodiments, Z 30 Choose from the following groups:

[0179]

[0180] In some embodiments, -L1-R1 is selected from:

[0181]

[0182] In some embodiments, the pKa of the protonated form of the compound is about 4.5 to about 8.0.

[0183] In some embodiments, the cationic lipid may be selected from the ionizable cationic lipids listed in the table below, or their isomers or salts.

[0184] Table A

[0185]

[0186] Table B

[0187]

[0188] Table C

[0189]

[0190] Table D

[0191]

[0192] This article also provides lipid nanoparticles (LNPs) comprising lipid blends containing ionizable cationic lipids and / or lipid-immune cell targeting conjugates (e.g., lipid-T cell targeting conjugates) provided herein.

[0193] In some embodiments, the lipid blend comprises one or more of structured lipids (e.g., sterols), neutral phospholipids, and free PEG-lipids.

[0194] In some embodiments, the LNP comprises the ionizable lipid of the present invention, distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), optionally in a molar ratio of about 48.5 : 10 : 40 : 1.5.

[0195] In some embodiments, the LNP comprises the ionizable lipid of the present invention, distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), optionally in a molar ratio of about 48.5 : 10 : 40 : 1.5.

[0196] In some embodiments, the LNP comprises the ionizable lipid of the present invention, distearate phosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), optionally in a molar ratio of about 48.5 : 10 : 39 : 2.5.

[0197] The amounts and ratios of LNP components can vary in any quantity depending on the desired form, structure, function, cargo, target, or any combination thereof. The amount of each component can be expressed in various embodiments as a percentage (mol%) of the total molar mass of all lipids or lipid conjugates of the specified component. The amount of each component can be expressed in various embodiments as a relative ratio (molar ratio) of the components based on molar mass. The amount of each component can be expressed in various embodiments as the weight (mg or equivalent) of each component used to formulate the LNP prior to manufacturing. The amount of each component can be expressed in various embodiments by any other method known in the art. Any formulation given in one form of component amount (“unit”) is expressly intended to cover any formulation expressed in different units of component amount, wherein these expressions are effectively equivalent when converted to the same unit. In some embodiments, “effectively equivalent” means that two or more values ​​differ from each other by less than about 10%.

[0198] In some aspects, methods are provided for expressing a target peptide in targeted cells (e.g., immune cells) of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs) provided herein.

[0199] In some aspects, methods for modulating the cellular function of target cells (e.g., target immune cells) of a subject are provided. In some embodiments, the method includes administering lipid nanoparticles (LNPs) provided herein to the subject.

[0200] In some respects, methods are provided for treating, improving, or preventing symptoms of a disorder or disease in a subject in need. In some embodiments, the method includes administering lipid nanoparticles (LNPs) provided herein to the subject.

[0201] The various aspects and embodiments of the present invention are described in further detail below. Detailed Implementation

[0202] This invention provides ionizable cationic lipids and lipid nanoparticle compositions comprising such ionizable cationic lipids, medical kits containing such lipids, and methods for preparing and using such lipids and conjugates. The compositions may further comprise lipid-immune cell-targeting group conjugates.

[0203] Unless otherwise indicated, the practice of this invention employs conventional techniques of organic chemistry, pharmacology, cell biology, and biochemistry. These techniques are elucidated in the following literature, such as “Comprehensive Organic Synthesis” (BMTrost and I. Fleming, eds., 1991–1992); “Current protocols in molecular biology” (FM Ausubel et al., eds., 1987, and periodically updated); and “Current protocols in immunology” (JEColigan et al., eds., 1991), each of which is incorporated herein by reference in its entirety. Various aspects of the invention are set forth in the following sections; however, the aspects of the invention described in one particular section are not limited to any particular section.

[0204] I. Definition

[0205] To facilitate understanding of this invention, many terms and phrases are defined below.

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations used herein have their conventional meanings in the chemical and biological fields. Chemical structures and formulas described herein should be interpreted according to the standard rules of chemical valence known in the field of chemistry. Furthermore, for example, when a chemical group is a diradical, it should be understood that the chemical group can bond to its adjacent atoms in one or both directions in the remainder of the structure; for example, -OC(O)- can be interchanged with -C(O)O-, or -OC(S)- can be interchanged with -C(S)O-.

[0207] The terms “cationic lipid” or “ionizable cationic lipid” are used interchangeably or together herein and refer to lipids that are protonated (e.g., >50% protonated) at low pH (e.g., pH 4), which makes them positively charged, but they remain neutral at physiological pH (e.g., pH 7.4).

[0208] Unless the context is inappropriate, as used herein, the term "a / species (a and an)" means "one / species or more / species" and includes the plural. In some embodiments, "one / species or more / species" is 1 or 2. In some embodiments, "one / species or more / species" is 1, 2, or 3. In some embodiments, "one / species or more / species" is 1, 2, 3, or 4. In some embodiments, "one / species or more / species" is 1, 2, 3, 4, or 5. In some embodiments, "one / species or more / species" is 1, 2, 3, 4, 5, or greater.

[0209] As used herein, the term "alkyl" refers to a saturated straight-chain or branched hydrocarbon, such as a straight-chain or branched group with 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C1, respectively. 12 Alkyl, C1-C 10 Alkyl or C1-C6 alkyl. In some embodiments, the alkyl group is optionally substituted. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc.

[0210] The term "alkyleneyl" or "alkylene" refers to a diradical of an alkyl group. In some embodiments, the alkylene group is optionally substituted. An exemplary alkylene group is -CH2CH2-.

[0211] The term "alkenylenyl" or "alkenylene" refers to a diradical of an alkenyl group. In some embodiments, the alkenyl group is optionally substituted. An exemplary alkenyl group is -CH=CH-.

[0212] The term "akynylenyl" or "alkynylene" refers to a diradical of an alkenyl group. In some embodiments, the akynylenyl group is optionally substituted. An exemplary akynylenyl group is... .

[0213] The term "halogenated alkyl" refers to an alkyl group that has been substituted with at least one halogen. Examples include -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc.

[0214] As used herein, the terms “aliphatic” or “aliphatic group” mean a fully saturated or branched, substituted or unsubstituted linear (i.e., unbranched) or branched hydrocarbon chain containing one or more unsaturated units, or a fully saturated or bicyclic hydrocarbon (also referred to herein as “carbocyclic,” “carbocyclic,” “aliphatic,” or “cycloalkyl”) containing one or more unsaturated units, said unsaturated units having a single attachment point to the remainder of the molecule. Unless otherwise specified, an aliphatic group contains 1 to 6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 5 carbon atoms. In some embodiments, an aliphatic group contains 1 to 4 carbon atoms. In some embodiments, an aliphatic group contains 1 to 3 carbon atoms, and in some embodiments, an aliphatic group contains 1 to 2 carbon atoms. In some embodiments, “carbocyclic” (or “cycloaliphatic” or “carbocycle” or “cycloalkyl”) means a optionally substituted monocyclic C3-C8 hydrocarbon, or optionally substituted C6-C8 hydrocarbon. 12 Bicyclic hydrocarbons are hydrocarbons that are fully saturated or contain one or more unsaturated units, but are not aromatic, and have a single attachment point to the remainder of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0215] The term "bridged bicyclic" refers to any bicyclic ring system having at least one bridge, i.e., a carbocyclic or heterocyclic ring, saturated or partially unsaturated. As defined by IUPAC, a "bridge" is a non-branched or valence bond connecting multiple atoms or a single atom to two bridgeheads, wherein a "bridgehead" is any skeletal atom of the ring system bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those groups listed below, wherein each group is attached to the remainder of the molecule at any substituted carbon or nitrogen atom. Unless otherwise specified, the bridged bicyclic group is optionally substituted with one or more substituents as listed for aliphatic groups. Additionally or alternatively, any substituted nitrogen atom in the bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include, but are not limited to:

[0216]

[0217] The terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably herein and refer to a stable 3- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (e.g., one to four) heteroatoms in addition to a carbon atom, as defined above. When referring to the ring atom of a heterocyclic compound, the term “nitrogen” includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or NR. + (e.g., in N-substituted pyrrolidinyl groups). Heterocycles can be attached to their side groups at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, aza-butyl, oxa-butyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolyl, piperazine, tetrahydropyranyl, dioxyl, dioxopentyl, diaza-butyl, oxazolyl, thioazolyl, morpholinyl, thiomorpholinyl, and... The heterocyclic group can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, and more preferably monocyclic or bicyclic. The term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group, wherein the alkyl and heterocyclic moieties are optionally substituted. Bicyclic heterocycles also include groups in which the heterocycle is fused to one or more aryl, heteroaryl, or cycloaliphatic rings. Exemplary bicyclic heterocyclic groups include dihydroindolyl, isodihydroindolyl, benzodioxanepentenyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocycles can also be spirocyclic systems (e.g., 7- to 11-membered spirocyclic fused heterocycles having one or more heteroatoms as defined above (e.g., one, two, three, or four heteroatoms) in addition to a carbon atom). Bicyclic heterocycles can also be bridging ring systems (e.g., 7- to 11-membered bridging heterocycles having one, two, or three bridging atoms).

[0218] "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 a cis or trans configuration (Z or E configuration) with respect to one or more double bonds. Alkenyl groups include, but are not limited to, vinyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl) and butenyl (e.g., buten-1-en-1-yl, buten-1-en-2-yl, 2-methyl-propen-1-en-1-yl, buten-2-en-1-yl, buten-2-en-2-yl, buten-1,3-dien-1-yl, buten-1,3-dien-2-yl).

[0219] "Alynyl" refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one carbon-carbon triple bond. Alynyl groups include, but are not limited to, ethynyl, propynyl (e.g., prop-1-yn-1-yl, prop-2-yn-1-yl), and butynyl (e.g., but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl).

[0220] The term "oxo" is recognized in the art and refers to an "=O" substituent. For example, cyclopentane substituted with an oxo group is cyclopentanone.

[0221] The term "morpholino" refers to a substituent having the following structure:

[0222]

[0223] It is arbitrarily replaced.

[0224] The term "piperidinyl" refers to a substituent having the following structure:

[0225] It is arbitrarily replaced.

[0226] Generally, the term "substituted," regardless of whether the term "optionally" precedes it, indicates that one or more hydrogens of the specified moiety are replaced by suitable substituents. Unless otherwise indicated, a "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure can be substituted by more than one substituent selected from the specified group. Substituent combinations contemplated under this invention are preferably combinations that result in the formation of stable or chemically viable compounds. In some embodiments, "optionally substituted" is equivalent to "unsubstituted or substituted." In some embodiments, "optionally substituted" means that the specified atom or group is optionally substituted by one or more substituents independently selected from the optional substituents provided herein. In some embodiments, the optional substituents may be selected from the group consisting of: C 1-6 Alkyl, cyano, halogen, -OC 1-6 Alkyl, C 1-6 Haloalkyl, C 3-7 Cycloalkyl, 3- to 7-membered heterocyclic groups, 5- to 6-membered heteroaryl groups, and phenyl groups. In some embodiments, optional substituents are alkyl, cyano, halogen, halogenated, azide, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amide, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclic, aryl, or heteroaryl. In some embodiments, optional substituents are -OR s1 -NR s2 R s3 -C(O)R s4 -C(O)OR s5 C(O)NR s6 R s7 -OC(O)R s8 -OC(O)OR s9 -OC(O)NR s10 R s11 -NR s12 C(O)R s13 , or -NR s14 C(O)OR s15 , where R s1 R s2 R s3 R s4 R s5 R s6 R s7 R s8 Rs9 R s10 R s11 R s12 R s13 R s14 and R s15 H and C are independent of each other. 1-6 Alkyl, C 3-10 cycloalkyl, C 6-14 The aryl group, 5- to 10-membered heteroaryl group, or 3- to 10-membered heterocyclic group are each optionally substituted.

[0227] The term "halogenated alkyl" refers to an alkyl group that has been substituted with at least one halogen. Examples include -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc.

[0228] The term "cycloalkyl" refers to a monovalent saturated cyclic, bicyclic, bridged (e.g., adamantyl) or spirocyclic hydrocarbon group derived from cycloalkanes of 3-12, 3-10, 3-8, 4-8, or 4-6 carbons, and is referred to herein as, for example, "C". 4-8 "Cycloalkyl". In some embodiments, the cycloalkyl group is optionally substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclohexane, cyclopentane, cyclobutane, and cyclopropane. Unless otherwise stated, the cycloalkyl group is optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amide, amidine, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic, hydroxyl, imino, ketone, nitro, phosphate, phosphonic acid, hypophosphonic acid, sulfate, sulfide, sulfonamide, sulfonyl, or thiocarbonyl. In some embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0229] The terms "heterocyclic group" and "heterocyclic group" are recognized in the art and refer to a saturated, partially unsaturated, or aromatic 3- to 10-membered ring structure, alternatively a 3- to 7-membered ring, whose ring structure includes one to four heteroatoms, such as nitrogen, oxygen, and sulfur. In some embodiments, the heterocyclic group is optionally substituted. The number of ring atoms in the heterocyclic group can be determined using C1. x -C xThe nomenclature specifies that x is an integer representing the number of ring atoms. For example, C3-C7 heterocyclic groups refer to saturated or partially unsaturated 3- to 7-membered ring structures containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The name "C3-C7" indicates that the heterocycle contains a total of 3 to 7 ring atoms, including any heteroatoms occupying ring positions. An example of a C3 heterocyclic group is an azirropropyl group. The heterocycle can be, for example, a monocyclic, bicyclic, or other polycyclic ring system (e.g., fused, spirocyclic, bridged bicyclic). The heterocycle can be fused with one or more aryl groups, or partially unsaturated or saturated rings. Heterocyclic groups include, for example, biotinyl, chromanyl, dihydrofuranyl, dihydroindolyl, dihydropyranyl, dihydrothiophenyl, dithiazolyl, homopiperidinyl, imidazolyl, isoquinolinyl, isothiazolinyl, isoxazolinyl, morpholinyl, oxazolinyl, phenoxanthenyl, piperazine, piperidinyl, pyranyl, pyrazolyl, pyrazolinyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolidin-2-one, pyrrololinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl, tetrahydroquinolinyl, thiazolyl, thioheteropentyl, thiomorpholinyl, thiaranyl, xanthenyl, lactone, lactam (such as aziridine ketone and pyrrolidone), sulfonamide, sulfonyl lactone, etc. Unless otherwise stated, the heterocycle may optionally be substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amide, amidyl, amino, aryl, arylalkyl, azide, carbamate, carbonate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic, hydroxyl, imino, ketone, nitro, oxo, phosphate, phosphonate, hypophosphonate, sulfate, sulfide, sulfonamide, sulfonyl, and thiocarbonyl. In some embodiments, the heterocyclic group is not substituted, i.e., it is unsubstituted.

[0230] The term "aryl" is recognized in the art and refers to a carbocyclic aromatic group. In some embodiments, the aryl group is optionally substituted. Representative aryl groups include phenyl, naphthyl, anthracene, etc. The term "aryl" includes polycyclic systems having two or more carbon rings, wherein the two or more carbons are common to two adjacent rings (the ring is a "fused ring"), wherein at least one ring is aromatic, and for example, the other one or more rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. Unless otherwise stated, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amide, carboxylic acid, -C(O)alkyl, CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclic, aryl or heteroaryl moiety, -CF3, -CN, etc. In some embodiments, the aromatic ring is substituted with a halogen, alkyl, hydroxyl, or alkoxy group at one or more ring positions. In some other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In some embodiments, the aryl group is a 6- to 10-membered ring structure. In some embodiments, the aryl group is C6-C. 14 Aryl.

[0231] The term "heteroaryl" is recognized in the art and refers to an aromatic group comprising at least one cyclic heteroatom. In some embodiments, the heteroaryl group is optionally substituted. In some cases, the heteroaryl group contains 1, 2, 3, or 4 cyclic heteroatoms. Representative examples of heteroaryl groups include pyrrole, furanyl, phenylthio, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl, and pyrimidinyl. Unless otherwise stated, the heteroaryl ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amide, carboxylic acid, C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamide, sulfonamide, ketone, aldehyde, ester, heterocyclic group, aryl or heteroaryl moiety, -CF3, -CN, etc. The term "heteroaryl" also includes polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings (the rings are "fused rings"), and wherein at least one ring is heteroaromatic; for example, other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, and / or aryl. In some embodiments, the heteroaryl ring is substituted with a halogen, alkyl, hydroxyl, or alkoxy group at one or more ring positions. In some other embodiments, the heteroaryl ring is not substituted, i.e., it is unsubstituted. In some 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.

[0232] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines, for example, those of the general formula -N(R 10 (R) 11 The part represented by ) where R 10 and R 11 Each can independently represent hydrogen, alkyl, cycloalkyl, heterocyclic, alkenyl, aryl, aralkyl, or (CH2). m -R 12 Or R 10 and R 11 Together with the N atoms to which they are attached, they form heterocycles with 4 to 8 atoms in the ring structure; R 12 Represents aryl, cycloalkyl, cycloalkenyl, heterocyclic, or polycyclic; and m is zero or an integer in the range of 1 to 8. In some embodiments, R 10 and R 11 Each can independently represent hydrogen, alkyl, alkenyl, or -(CH2). m -R 12 .

[0233] The term "alkoxyl" or "alkoxy" is recognized in the art and refers to an alkyl group having an oxygen radical attached thereto as defined above. In some embodiments, the alkoxy group is optionally substituted. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. An "ether" is two hydrocarbons covalently linked by oxygen. Therefore, the substituent of the alkyl group that makes it an ether is or similar to an alkoxy group, such as -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH2) m -R 12 One of the representations, where m and R 12 As described above, the term "haloalkoxy" refers to an alkoxy group substituted with at least one halogen. Examples include -O-CH2F, -O-CHF2, -O-CF3, etc. In some embodiments, the haloalkoxy group is an alkoxy group substituted with at least one fluorine group. In some embodiments, the haloalkoxy group is an alkoxy group substituted with 1-6, 1-5, 1-4, 2-4, or 3 fluorine groups.

[0234] symbol" "Instruction attachment point."

[0235] The compounds disclosed herein may contain one or more chiral centers and / or double bonds, and thus exist as stereoisomers (such as geometric isomers, enantiomers, or diastereomers). When used herein, the term "stereoisomer" encompasses all geometric isomers, enantiomers, or diastereomers. These compounds may be designated by the symbol "R" or "S," depending on the configuration of the substituents surrounding the stereocarbon atom. This invention covers various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated in nomenclature by "(±)", but those skilled in the art will recognize that the structure may implicitly represent the chiral center. It should be understood that, unless otherwise indicated, graphical depictions of the chemical structure (e.g., a general chemical structure) cover all stereoisomeric forms of the specified compound.

[0236] Individual stereoisomers of the compounds of the present invention can be prepared by synthesis from commercially available starting materials containing asymmetric or stereocenters, or by preparing racemic mixtures followed by resolution methods well known to those skilled in the art. These resolution methods are exemplified by: (1) attaching an enantiomer mixture to a chiral auxiliary agent, separating the resulting diastereomer mixture by recrystallization or chromatography, and releasing an optically pure product from the auxiliary agent; (2) forming a salt using an optically active resolving agent; or (3) directly separating the optically enantiomer mixture on a chiral chromatographic column. The stereoisomer mixture can also be resolved into its constituent stereoisomers by well-known methods such as chiral phase gas chromatography, chiral phase high-performance liquid chromatography, crystallizing the compound into a chiral salt complex, or crystallizing the compound in a chiral solvent. Furthermore, enantiomers can be separated using supercritical fluid chromatography (SFC) techniques as described in the literature. Still further, the stereoisomers can be obtained from stereoisomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0237] Geometric isomers may also be present in the compounds of this invention. This invention covers a variety of geometric isomers and mixtures thereof resulting from substituent arrangements around the carbon-carbon double bond or around the carbide ring. Substituents around the carbon-carbon double bond are designated as being in the “Z” or “E” configuration, wherein the terms “Z” and “E” are used according to IUPAC standards. Unless otherwise specified, the description of the double bond's structure covers both the “E” and “Z” isomers.

[0238] Substituents around a carbon-carbon double bond may alternatively be referred to as "cis" or "trans," where "cis" indicates that the substituent is on the same side of the double bond, and "trans" indicates that the substituent is on opposite sides of the double bond. The arrangement of substituents around the carbon ring is designated as "cis" or "trans." The term "cis" indicates that the substituent is on the same side of the ring plane, and the term "trans" indicates that the substituent is on opposite sides of the ring plane. A mixture of compounds in which substituents are placed on both sides of the ring plane is designated as "cis / trans."

[0239] This invention also includes isotopically labeled compounds of the invention, which are identical to the compounds described herein, except that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as... 2 H, 3 H, 13 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl.

[0240] Disclosed compounds labeled with certain isotopes (e.g., using...) 3 H and 14 C-labeled compounds can be used for the determination of compound and / or substrate tissue distribution. Deuteration (i.e. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Further, heavier isotopes (such as deuterium, i.e., 2 H) substitution can provide certain therapeutic advantages due to its greater metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in some cases. Isotope-labeled compounds of the present invention can generally be prepared by replacing non-isotope-labeled reagents with isotope-labeled reagents through a procedure similar to that disclosed, for example, in the examples herein.

[0241] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods of the present invention. Such an organism is preferably a mammal (e.g., a mouse, ape, horse, cow, pig, dog, cat, etc.), and more preferably a human.

[0242] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro.

[0243] As used herein, the term "pharmaceutically acceptable excipient" refers to any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and excipients, see Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.

[0244] As is known to those skilled in the art, the "salts" of the compounds of the present invention can be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids (such as oxalic acid), although not pharmaceutically acceptable on their own, can be used to prepare salts that can serve as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0245] Examples of alkalis include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and NW4. + Compounds (where W is C) 1-4 Alkyl groups, etc.

[0246] Examples of salts include, but are not limited to: acetates, adipicates, alginates, aspartates, benzoates, benzenesulfonates, hydrogen sulfates, butates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, disaccharides, dodecyl sulfates, ethanesulfonates, fumarates, flucoheptanoates, glycerophosphates, hemisulfates, heptanates, hexanoates, hydrochlorides, hydrobromide, hydroiodates, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, oxalates, palmitates, pectates, persulfates, phenylpropionates, picrates, neopentanoates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, etc. Other examples of salts include those with suitable cations such as Na+. + NH4 +and NW4 + (where W is C) 1-4 The anions of the compounds of the present invention are composed of alkyl groups, etc.

[0247] The abbreviations used in this article include diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); tetrabutylammonium iodide (TBAI); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate (PyBOP); 9-furoylmethoxycarbonyl (Fmoc); tetrabutyldimethylsilyl chloride (TBDMSCl); hydrogen fluoride (HF); phenyl (Ph); bis(trimethylsilyl)amine (HMDS); dimethylformamide (DMF); dichloromethane (DCM); tetrahydrofuran (THF); high performance liquid chromatography (HPLC); mass spectrometry (MS); evaporative light scattering detector (ELSD); electrospray ionization (ES); and nuclear magnetic resonance spectroscopy (NMR).

[0248] As used herein, the term "effective amount" refers to the amount of a compound (e.g., a nucleic acid, such as mRNA) sufficient to achieve a beneficial or desired outcome. An effective amount may be administered in one or more applications, doses, or administrations and is not intended to be limited to a particular formulation or route of administration. The term "effective amount" may be considered to include therapeutic and / or prophylactic effective amounts of a compound.

[0249] As used herein, the phrase “therapeutic effective amount” means an amount of a compound (e.g., nucleic acid, e.g., mRNA), material, or composition comprising a compound (e.g., nucleic acid, e.g., mRNA) that effectively produces some desired therapeutic effect in at least one cellular subpopulation in a mammal (e.g., human) or a subject (e.g., human subject) at a reasonable benefit / risk ratio suitable for any medical treatment.

[0250] As used herein, the phrase “preventative effective amount” means an amount of a compound (e.g., nucleic acid, e.g., mRNA), material, or composition containing a compound (e.g., nucleic acid, e.g., mRNA) that effectively produces some desired preventive effect in at least one cellular subpopulation of a mammal (e.g., a human) or a subject (e.g., a human subject) by reducing, minimizing, or eliminating the risk of developing a condition or reducing or minimizing the severity of a condition, at a reasonable benefit / risk ratio suitable for any medical treatment.

[0251] As used in this article, the term "treatment" includes any action that results in the improvement or enhancement of the symptoms of a condition, disease, disorder, etc., such as reducing, alleviating, regulating, improving, or eliminating them.

[0252] The phrase “pharmaceutically acceptable” is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the bounds of reasonable medical judgment, and in proportion to a reasonable benefit / risk ratio.

[0253] In this application, when an element or component is said to be included in and / or selected from the list of listed elements or components, it should be understood that the element or component may be any one of the listed elements or components, or the element or component may be selected from two or more of the listed elements or components.

[0254] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways, whether explicitly stated or implied herein, without departing from the spirit and scope of the invention. For example, unless otherwise understood from the context, when a particular compound is mentioned, that compound can be used in various embodiments of the compositions of the invention and / or the methods of the invention. In other words, within this application, embodiments have been described and depicted in a manner that enables clear and concise writing and drawing, but it is intended and will be understood that embodiments can be combined or separated in various ways without departing from the teachings of the invention and one or more of the inventions. For example, it should be understood that all features described and depicted herein are applicable to all aspects of one or more of the inventions described and depicted herein.

[0255] It should be understood that, unless otherwise understood from the context and usage, the expression "at least one of" includes each and every single one of the objects listed following the expression, as well as various combinations of two or more of the listed objects. Unless otherwise understood from the context, the expression "and / or" in combination with three or more of the listed objects should be understood to have the same meaning.

[0256] Unless otherwise explicitly stated or understood from the context, the use of the terms “include / includes / including,” “have / has / having,” and “contain / contains / containing” (including their grammatical equivalents) should generally be understood as open-ended and non-restrictive, e.g., not excluding additional unlisted elements or steps.

[0257] Unless otherwise explicitly stated, the invention also includes the specific numerical value itself when the term "about" is used before a numerical value. As used herein, unless otherwise indicated or inferred, the term "about" refers to a variation of ±10% from the nominal value.

[0258] As used herein, unless otherwise indicated, the term "antibody" means any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. It should be understood that the term covers intact antibodies (e.g., intact monoclonal antibodies) or fragments thereof such as the Fc fragment of an antibody (e.g., the Fc fragment of a monoclonal antibody) or the antigen-binding fragment of an antibody (e.g., the antigen-binding fragment of a monoclonal antibody), including modified or engineered intact antibodies, antigen-binding fragments, or Fc fragments. Examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), microantibodies, and biantibodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). The term also covers immunoglobulin monovariable domains, such as nanobodies (e.g., V...). HH ).

[0259] As used here, an "antibody that binds to X" (i.e., X is a specific antigen) or an "anti-X antibody" is an antibody that specifically recognizes antigen X.

[0260] As used herein, "embedded interchain disulfide bonds" or "interchain-embedded disulfide bonds" refers to disulfide bonds on a polypeptide that are not easily approached or effectively "embedded" in the hydrophobic region of the polypeptide by water-soluble reducing agents, making them unsuitable for use as reducing agents or for conjugation with other hydrophilic PEGs. Embedded interchain disulfide bonds are further described in WO 2017096361A1, which is incorporated herein by reference in its entirety.

[0261] As used herein, the specificity of targeted delivery of an LNP is defined as the ratio between the percentage of desired immune cell types that receive the delivered nucleic acid (e.g., on-target delivery) and the percentage of undesirable immune cell types that receive the delivered nucleic acid but are not intended to be the target of the delivery (e.g., off-target delivery). For example, higher specificity occurs when more desired immune cells receive the delivered nucleic acid and fewer undesirable immune cells receive it. The specificity of targeted delivery of an LNP can also be defined as the ratio of the amount of nucleic acid delivered to desired immune cells (e.g., on-target delivery) to the amount of nucleic acid delivered to undesirable immune cells (e.g., off-target delivery). Delivery specificity can be determined using any suitable method. As a non-limiting example, the expression level of the nucleic acid in the desired immune cell type can be measured and compared to the expression level of the nucleic acid in different immune cell types that are not intended to be the target of the delivery.

[0262] As used herein, in some embodiments, the reference LNP is an LNP that does not have an immune cell targeting group but is otherwise identical to the tested LNP. In some other embodiments, the reference LNP is an LNP that has a different ionizable cationic lipid but is otherwise identical to the tested LNP. In some embodiments, the reference LNP comprises ALC-0315 as an ionizable cationic lipid (which is different from the ionizable cationic lipid in the tested LNP) but is otherwise identical to the tested LNP.

[0263] As used herein, a humanized antibody is an antibody that is wholly or partially non-human in origin, and whose protein sequence has been modified to replace certain amino acids, such as amino acids appearing at one or more corresponding positions in the architecture regions of the VH and VL domains in antibody sequences derived from humans, to increase its similarity to antibodies naturally produced in humans, thereby avoiding or minimizing human immune responses. For example, using genetic engineering techniques, variable domains of a target non-human antibody can be combined with constant domains of a human antibody. The constant domains of humanized antibodies are often the human CH and CL domains.

[0264] As used in this article, the term "structural lipid" refers to sterols, and specifically to lipids containing sterol moieties.

[0265] It should be understood that the order of steps or the sequence of actions is irrelevant as long as the invention remains operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0266] Throughout this specification, alternative bases are disclosed in groups or ranges. In particular, it is intended that the description include each individual sub-combination of members of such groups and ranges. For example, the term "C" 1-6The term "alkyl" is specifically intended to separately disclose C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl. By way of other examples, integers in the range 0 to 40 are specifically intended to separately disclose 0, 1, 2, 3, 4, 5, 6, 7, 8 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 the integers in the range 1 to 20 are specifically intended to be disclosed separately as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0267] Unless otherwise claimed, the use of any and all instances or exemplary language (e.g., “such as” or “including”) herein is intended only to better illustrate the invention and does not impose any limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0268] Throughout this specification, where compositions and kits are described as having, including, or containing specific components, or where processes and methods are described as having, including, or containing specific steps, it is contemplated that there may also be compositions and kits of the invention that are substantially composed of or comprised of the listed components, as well as processes and methods of the invention that are substantially composed of or comprised of the listed processing steps.

[0269] Generally, unless otherwise specified, the percentage of a composition is by weight. Furthermore, if a variable is not accompanied by a definition, its previously defined meaning shall prevail.

[0270] Immunoglobulin monovariable domain

[0271] In some embodiments, the immune cell targeting group of the LNP as described herein includes an immunoglobulin monovariable domain, such as a nanobody.

[0272] The term "immunoglobulin single variable domain" ("ISV"), used interchangeably with "single variable domain," defines an immunoglobulin molecule in which an antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin ISVs from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V... H ) and light chain variable structural domain (V L The interaction between V and other antigens forms an antigen-binding site. In this case, V H and V L The complementarity-determining regions (CDRs) of both will facilitate the antigen-binding site; that is, a total of 6 CDRs will participate in the formation of the antigen-binding site. Given the above definition, the antigen-binding domains of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fab, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or biantibodies (all known in the art) derived from such conventional four-chain antibodies will generally not be considered as immunoglobulin monovariable domains, because in these cases, binding to the corresponding epitope of the antigen usually does not occur through a single immunoglobulin domain, but rather through a pair of (associated) immunoglobulin domains (such as light and heavy chain variable domains) that bind together to the epitope of the corresponding antigen, i.e., through the V... H -V L It happened.

[0273] Conversely, immunoglobulin monovariable domains (MMUs) can specifically bind to antigenic epitopes without pairing with other immunoglobulin MMUs. The binding site of an immunoglobulin MMU consists of a single V... H A single V HH Or a single V L Domain formation. Therefore, the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0274] Therefore, a single variable domain can be a sequence of light chain variable domains (e.g., V). L - sequence) or a suitable fragment thereof; or heavy chain variable domain sequence (e.g., V H -Sequence or V HH(sequence) or a suitable fragment thereof; as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0275] Immunoglobulin single variable domain (ISV) can be, for example, a heavy chain ISV, such as V. H V HH Including camel-like V H Or humanized V HH In one embodiment, it is V HH Including camel-like V H Or humanized V HH Heavy chain ISVs can be derived from conventional four-chain antibodies or heavy chain antibodies.

[0276] For example, the immunoglobulin single variable domain can be a (single) domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb), or Nanobody® ISV (as defined herein, and including but not limited to V). HH ); other single variable domains, or any suitable fragment of any of them.

[0277] In particular, the immunoglobulin single variable domain can be Nanobody® ISV (such as V... HH Including humanized V HH Or camel-like V H (or a suitable fragment thereof. [Note: Nanobody® is a registered trademark of Ablynx NV.])

[0278] “V HH Structural domain (also known as V) HH V HH Antibody fragments and V HH Antibodies were initially described as “heavy chain antibodies” (i.e., “antibodies without light chains”; Hamers-Casterman et al. 1993 Nature [Nature] 363: 446-448) antigen-binding immunoglobulin variable domains. The term “V” was chosen. HH The term "domain" is used to combine these variable domains with the heavy chain variable domains (referred to as "V" in this paper) present in conventional 4-chain antibodies. H The structural domain) and the light chain variable structural domain (referred to in this paper as "V") present in conventional 4-chain antibodies. L Distinguish between "structural domain" and "structural domain". Regarding V... HHFor further description, see Muyldermans' 2001 review article (Reviews in Molecular Biotechnology 74: 277-302).

[0279] For the terms “dAb” and “domain antibody”, see, for example, Ward et al. 1989 (Nature 341:544), Holt et al. 2003 (Trends Biotechnol. 21: 484); and other published patent applications such as WO 2004 / 068820, WO 2006 / 030220, WO 2006 / 003388 and Domantis Ltd. It should also be noted that, although less preferred in the context of this invention because they are not of mammalian origin, single variable domains can be derived from certain shark species (e.g., the so-called “IgNAR domain,” see, for example, WO 2005 / 18629).

[0280] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be produced by screening natural, immunoglobulin-producing, or synthetic libraries, such as through phage display.

[0281] The generation of immunoglobulin sequences (such as VHH) has been extensively described in various publications, including WO 1994 / 04678, Hamers-Casterman et al. 1993 (Nature 363: 446-448), and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74: 277-302, 2001). In these methods, camels are immunized with a target antigen to induce an immune response against the target antigen. Further screening of VHH obtained from this immunization... HH V of the library binding target antigen HH .

[0282] In these cases, antibody generation requires purified antigens for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production. Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0283] Immunoglobulin sequences of various origins may be used in this study, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. Furthermore, fully human, humanized, or chimeric sequences may be used in the methods described herein. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelidized domain antibodies, such as those 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), may be used herein. Additionally, ISV fusion to form multivalent and / or multispecific constructs (regarding those containing one or more V...) HH For information on multivalent and multispecific polypeptides with domains and their preparation, see also Conrath et al. 2001 (J. Biol. Chem., Vol. 276, 10. 7346-7350) and, for example, WO 1996 / 34103 and WO 1999 / 23221).

[0284] "Humanization V" HH "Contains V corresponding to natural occurrences" HH The amino acid sequence of the domain is the same as the amino acid sequence of the human body, but it has been "humanized," i.e., by using V, which is now present in conventional 4-chain antibodies from humans. H One or more amino acid residues at one or more corresponding positions in the domain (as shown above) replace the naturally occurring V. HH One or more amino acid residues in the amino acid sequence of the sequence (especially in the framework sequence). This can be done in a manner known per se, as will be clear to those skilled in the art, for example, based on existing techniques (e.g., WO 2008 / 020079). Furthermore, it should be noted that such humanized V can be obtained in any suitable manner known per se. HH Therefore, it is not strictly limited to peptides obtained using peptides that contain naturally occurring VHH domains as starting materials.

[0285] "Camelization V" H "Contains V corresponding to natural occurrences" H The amino acid sequence of the domain has been "camelized" (i.e., by using it in V-type heavy chain antibodies against camels). HH One or more amino acid residues appearing at one or more corresponding positions in the domain replace the naturally occurring V from a conventional 4-chain antibody. HThe amino acid sequence of one or more amino acid residues in the amino acid sequence of the domain. This can be done in a manner known per se, which should be clear to those skilled in the art, for example, based on descriptions of the prior art (e.g., Davies and Riechman 1994, FEBS [European Federation of Biochemical Societies Letters] 339: 285; 1995, Biotechnol. [Biotechnology] 13: 475; 1996, Prot. Eng [Protein Engineering]. 9: 531; and Riechman 1999, J. Immunol. Methods [Journal of Immunological Methods] 231: 25). As defined herein, such “camelization” substitution insertions are formed in and / or present in V H -V L The amino acid position of the interface and / or the so-called cameloid marker residue (see, for example, WO 1994 / 04678 and Davies and Riechmann (1994 and 1996, ibid.)). In one embodiment, it is used to generate or design cameloid V H V of the starting material or starting point H The sequence is derived from V in mammals. H Sequences, such as human V H Sequences, such as V H 3. Sequence. However, it should be noted that such camelified V can be obtained in any suitable manner known per se. H And therefore not strictly limited to those already using naturally occurring V H Peptides obtained from peptides whose structural domains are used as starting materials.

[0286] The structure of an immunoglobulin single variable domain sequence can be considered to consist of four frame regions (“FRs”), referred to in the art and herein as “frame region 1” (“FR1”); “frame region 2” (“FR2”); “frame region 3” (“FR3”); and “frame region 4” (“FR4”); these frame regions are interrupted by three complementarity-determining regions (“CDRs”), referred to in the art and herein as “complementarity-determining region 1” (“CDR1”); “complementarity-determining region 2” (“CDR2”); and “complementarity-determining region 3” (“CDR3”).

[0287] In such immunoglobulin sequences, the frame sequence can be any suitable frame sequence, and examples of suitable frame sequences will be clear to those skilled in the art, for example, based on standards manuals and further disclosures and the prior art mentioned herein.

[0288] A framework sequence is an immunoglobulin framework sequence or a suitable combination of framework sequences that have been (e.g., through humanization or camelification) derived from an immunoglobulin framework sequence. For example, the framework sequence could be derived from a light chain variable domain (e.g., V). L Sequence) and / or heavy-chain variable structural domains (e.g., V) H Sequence or V HH A frame sequence (often a sequence of frames). In one particular aspect, a frame sequence is derived from V. HH The sequence is a frame sequence (where the frame sequence may optionally be partially or fully humanized) or a camelified regular V. H Sequences (as defined in this article).

[0289] Specifically, the framework sequence present in the ISV sequence described herein may contain one or more marker residues (as defined herein) such that the ISV sequence is a Nanobody® ISV, such as V HH Including humanized V HH Or camel-like V H Non-limiting instances of such frame sequences (and suitable combinations thereof) will become clear from further disclosure herein.

[0290] V H Domain and V HH The total number of amino acid residues in the domain is typically in the range of 110 to 120, usually between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0291] However, it should be noted that the ISVs described herein are not limited in terms of their origin (or the nucleotide sequence used to express them) or the manner in which they are generated or obtained (or have been generated or obtained). Therefore, the ISV sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In a particular but non-limiting aspect, the ISV sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, particularly partially or fully humanized V...). HH Sequences), "camelized" (as defined in this article) immunoglobulin sequences (especially camelized V) HThe sequence), and ISVs obtained by techniques such as affinity maturation (e.g., starting from a synthetic, random or naturally occurring immunoglobulin sequence), CDR transplantation, faceting, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineered immunoglobulin sequences known to those skilled in the art; or any suitable combination of the foregoing.

[0292] Similarly, the nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated from a suitable naturally occurring template (e.g., DNA or RNA isolated from a cell) by PCR, a nucleotide sequence that has been isolated from a library (and especially an expression library), a nucleotide sequence that has been prepared by introducing a mutation into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0293] Typically, Nanobody® ISV (especially V) HH Sequence, including (partially) humanized V HH Sequence and Camelization V H The sequence may be characterized by the presence of one or more “marker residues” (as described herein) within one or more frame sequences (again, as further described herein). Therefore, generally, a Nanobody® ISV can be defined as an immunoglobulin sequence having the following (general) structure:

[0294] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0295] FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of the marker residues are further defined herein.

[0296] Specifically, Nanobody® ISV can be an immunoglobulin sequence having the following (general) structure:

[0297] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0298] FR1 to FR4 refer to frame regions 1 to 4, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, and the frame sequence is further defined in this paper.

[0299] More specifically, Nanobody® ISV can be an immunoglobulin sequence having the following (general) structure:

[0300] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4

[0301] FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively. Furthermore, 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 number are marker residues.

[0302] In one embodiment, the immunoglobulin single variable domain has certain amino acid substitutions in the architecture region that effectively prevent or reduce the binding of the polypeptide to so-called "pre-existing antibodies". Such an ISV has been described in WO 2015 / 173325, wherein (i) the amino acid residue at position 112 is one of K or Q; and / or (ii) the amino acid residue at position 89 is T; and / or (iii) the amino acid residue at position 89 is L and the amino acid residue at position 110 is one of K or Q; and (iv) in each of (i) to (iii), the amino acid at position 11 is preferably V.

[0303] polypeptide

[0304] Immunoglobulin monovariable domains can form part of a protein or polypeptide that may contain or consist substantially of one or more (at least one) immunoglobulin monovariable domains and may optionally further contain one or more additional amino acid sequences (all optionally linked via one or more suitable linkers). The term "immunoglobulin monovariable domain" may also cover such polypeptides. One or more immunoglobulin monovariable domains can serve as binding units in such proteins or polypeptides that may optionally contain one or more additional amino acids that can serve as binding units to provide, respectively, the monovalent, multivalent, or multispecific polypeptides of the present invention (for reference on multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al. 2001 (J. Biol. Chem. 276: 7346), and, for example, WO 1996 / 34103, WO1999 / 23221, and WO 2010 / 115998).

[0305] This polypeptide may contain or consist primarily of an immunoglobulin monovariable domain, as outlined above. Such polypeptides are also referred to herein as monovalent polypeptides.

[0306] The term "multivalent" indicates the presence of multiple ISVs in a polypeptide. In one embodiment, the polypeptide is "bivalent," meaning it contains or consists of two ISVs. In one embodiment, the polypeptide is "trivalent," meaning it contains or consists of three ISVs. In another embodiment, the polypeptide is "tetravalent," meaning it contains or consists of four ISVs. Therefore, the polypeptide can be "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," "octavalent," "nonavalent," etc., meaning the polypeptide contains or consists of two, three, four, five, six, seven, eight, nine, etc., ISVs, respectively. In one embodiment, the multivalent ISV polypeptide is trivalent. In another embodiment, the multivalent ISV polypeptide is tetravalent. In yet another embodiment, the multivalent ISV polypeptide is pentavalent.

[0307] In one embodiment, the multivalent ISV peptide can also be multispecific. The term "multispecific" refers to binding to multiple different target molecules (also called antigens). Therefore, the multivalent ISV peptide can be "bispecific," "trispecific," "quadrispecific," etc., meaning it can bind to two, three, four, or other different target molecules respectively.

[0308] For example, the peptide can be bispecific-trivalent, such as a peptide containing three ISVs or composed thereof, wherein two ISVs bind to a first target and one ISV binds to a second target different from the first target. In another example, the peptide can be trispecific-tetravalent, such as a peptide containing four ISVs or composed thereof, wherein one ISV binds to a first target, two ISVs bind to a second target different from the first target, and one ISV binds to a third target different from the first and second targets. In yet another example, the peptide can be trispecific-pentavalent, such as a peptide containing five ISVs or composed thereof, wherein two ISVs bind to a first target, two ISVs bind to a second target different from the first target, and one ISV binds to a third target different from the first and second targets.

[0309] In one embodiment, a multivalent ISV peptide can also be multi-complementary. The term "multi-complementary" refers to binding to multiple different epitopes on the same target molecule (also called an antigen). Therefore, a multivalent ISV peptide can be "bi-complementary," "tri-complementary," etc., meaning it can bind to two, three, or other different epitopes on the same target molecule, respectively.

[0310] In another aspect, the polypeptides described herein comprising one or more immunoglobulin monovariable domains (or suitable fragments thereof) or substantially composed of one or more immunoglobulin monovariable domains (or suitable fragments thereof) may further comprise one or more additional groups, residues, portions, or binding units. Such additional groups, residues, portions, binding units, or amino acid sequences may or may not impart additional function to the immunoglobulin monovariable domain (and / or the polypeptide in which it is located), and may or may not alter the properties of the immunoglobulin monovariable domain.

[0311] For example, such additional groups, residues, portions, 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, the one or more additional groups, residues, portions, or binding units are immunoglobulins. Even more preferably, the one or more additional groups, residues, portions, or binding units are selected from domain antibodies, amino acids suitable for use as domain antibodies, single-domain antibodies, amino acids suitable for use as single-domain antibodies, "dAbs," amino acids suitable for use as dAbs, or nanobodies.

[0312] Alternatively, such groups, residues, portions, or binding units may be, for example, chemical groups, residues, or portions that may or may not have biological and / or pharmacological activity. For example, but not limited to, such groups may be linked to one or more immunoglobulin monovariable domains to provide a “derivative” of the immunoglobulin monovariable domain.

[0313] In another embodiment, the additional residues can effectively prevent or reduce the binding of the polypeptide to a so-called "pre-existing antibody". For this purpose, the polypeptide and construct may contain a C-terminal extension (X)n (where 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 amino acid residue independently selected, preferably independently selected, from alanine (A), glycine (G), leucine (V), leucine (L), or isoleucine (I), preferably naturally occurring, with reference to WO 2012 / 175741). Therefore, the polypeptide may further contain a C-terminal extension (X)n, where n is 1 to 5, such as 1, 2, 3, 4, or 5, and where X is a naturally occurring amino acid, preferably not cysteine.

[0314] In the aforementioned polypeptide, the one or more immunoglobulin single variable domains and the one or more groups, residues, portions, or binding units may be directly linked to each other and / or linked via one or more suitable linkers or spacers. For example, when the one or more groups, residues, portions, or binding units are amino acids, the linker may also be an amino acid, such that the resulting polypeptide is a fusion protein or fusion polypeptide.

[0315] As used herein, the term "linker" refers to the fusion of two or more ISVs together to form a single peptide molecule. The use of linkers to connect two or more (multi)peptides is well known in the art. Other exemplary peptide linkers are shown in Table B. One commonly used class of peptide linkers is called "Gly-Ser" or "GS" linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues and typically contain one or more repetitions of a peptide motif such as the GGGGS (SEQ ID NO: 154) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 152), where n can be 1, 2, 3, 4, 5, 6, 7, or greater). Some common examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 157), the 15GS linker (n = 3) (SEQ ID NO: 158), and the 35GS linker (n = 7) (SEQ ID NO: 159). See, for example, Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27(10): 325-330).

[0316] It should generally be noted that the term nanobody, as used in its broadest sense herein, is not limited to a specific biological source or a specific preparation method. For example, as will be discussed in more detail below, nanobodies can be obtained by: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanizing” (as described below) a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by “camelizing” (as described below) a naturally occurring VH domain from any animal species, particularly mammalian species (such as humans), or by expressing a nucleic acid encoding such a camelized VH domain; (5) by “camelizing” a “domain antibody” or “Dab” as described by Ward et al. (ibid.), or by expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, peptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a nanobody using nucleic acid synthesis techniques and then expressing the resulting nucleic acid; and / or (8) by any combination of the foregoing. Based on the disclosure herein, suitable methods and techniques for carrying out the foregoing will be apparent to those skilled in the art, including, for example, the methods and techniques described in more detail below.

[0317] II. Ionizable cationic lipids

[0318] This article provides ionizable cationic lipids that can be used to generate lipid nanoparticle compositions to facilitate the delivery of payloads (e.g., nucleic acids, such as DNA, or RNA, such as mRNA) placed therein to cells, such as mammalian cells, like immune cells. Ionizable cationic lipids have been engineered to deliver nucleic acids (e.g., mRNA) intracellularly to the cytoplasmic compartment of target cell types and rapidly degrade into non-toxic components. The complex functions of ionizable cationic lipids are facilitated by the chemical and geometric interactions between the ionizable lipid head group, the hydrophobic "acyl tail" group, and the linker connecting the head group and the acyl tail group. Typically, the pK of the ionizable amine head group... aDesigned to be in the range of 6–8, such as between 6.2–7.4 or 6.7–7.2, it remains strongly cationic under acidic formulation conditions (e.g., pH 4–pH 5.5), neutral or weakly anionic at physiological pH (7.4), and cationic in early and late endosome compartments (e.g., pH 5.5–pH 7). The acyl tail group plays a crucial role in the fusion of the lipid nanoparticle with the endosome membrane and in membrane instability via structural perturbation. The three-dimensional structure of the acyl tail (determined by its length, unsaturation, and site) and the relative sizes of the head and tail groups are believed to play a role in promoting membrane fusion and thus in endosome escape from the lipid nanoparticle (a critical requirement for cytoplasmic delivery of nucleic acid payloads). The linker connecting the head and tail groups is designed to be degraded by physiologically ubiquitous enzymes (e.g., esterases or proteases) or by acid-catalyzed hydrolysis.

[0319] In some embodiments, the ionizable cationic lipid is present in the lipid blend in the range of 40-60 molar percentage.

[0320] III. Lipid-based immune cell-targeting conjugates

[0321] In one aspect, this document provides lipid nanoparticles (LNPs) for targeted delivery of nucleic acids to cells (e.g., immune cells), comprising lipid blends. The LNPs may comprise novel cationic lipids as described herein. In some embodiments, the lipid blend comprises a lipid-immune cell-targeting group conjugate comprising a compound having formula (II): [lipid] - [optional linker] - [immune cell-targeting group]. In some embodiments, the lipid blend comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid comprises an ionizable cationic lipid as described herein. In some embodiments, the LNP comprises a nucleic acid disposed therein.

[0322] In some embodiments, the immune cell targeting group comprises an antibody that binds to a T-cell antigen. In some embodiments, the T-cell antigen is CD3, CD4, CD7, or CD8, or a combination thereof (e.g., both CD3 and CD8, both CD4 and CD8, or both CD7 and CD8). In some embodiments, the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen. In some embodiments, the NK cell antigen is CD7, CD8, or CD56, or a combination thereof (e.g., both CD7 and CD8). In some embodiments, the antibody is a human or humanized antibody.

[0323] In some embodiments, the immune cell targeting group comprises a single antibody that binds to CD3 or CD7.

[0324] In some embodiments, the immune cell targeting group is covalently coupled to lipids in a lipid blend via a linker containing polyethylene glycol (PEG). In some embodiments, the lipid covalently coupled to the immune cell targeting group via the PEG-containing linker is distearylglycerol (DSG), distearyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglyceride (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), dialkylacetamide, or ceramide. In some embodiments, the PEG is PEG 2000. In some embodiments, the PEG is PEG 3400.

[0325] In some embodiments, the lipid-immune cell-targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 mol percent.

[0326] In some embodiments, the immune cell targeting group comprises an antibody, and the antibody is a Fab or an immunoglobulin single variable domain, such as a nanobody. In some embodiments, the immune cell targeting group comprises a Fab, F(ab')2, Fab'-SH, Fv, or scFv fragment. In some embodiments, the immune cell targeting group comprises a Fab engineered to knock out one or more natural interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment with C233S substitution, according to Kabat numbering; and / or a light chain fragment with C214S substitution, according to Kabat numbering. In some embodiments, the Fab comprises non-natural interchain disulfide bonds. In some embodiments, the immune cell targeting group comprises a Fab engineered to introduce one or more embedded interchain disulfide bonds. For example, in some embodiments, the Fab antibody comprises a heavy chain fragment with F174C substitution, according to Kabat numbering; and / or a light chain fragment with S176C substitution, according to Kabat numbering. In some embodiments, the Fab comprises a heavy chain segment substituted with F174C and C233S and a light chain segment substituted with S176C and C214S, according to Kabat numbering. In some embodiments, the immunocellular targeting group comprises a Fab engineered to knock out one or more native interchain disulfide bonds and introduce one or more embedded interchain disulfide bonds. In some embodiments, the immunocellular targeting group comprises a Fab containing a cysteine ​​residue at the C-terminus of the heavy chain or light chain segment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain segment of the Fab and the C-terminal cysteine ​​residue. In some embodiments, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immunocellular targeting group further comprises a single-chain variable segment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker. In some embodiments, the Fab antibody is a DS Fab (a Fab with wild-type (natural) interchain disulfide bonds), a NoDS Fab (a Fab with natural disulfide bonds knocked out, such as a Fab with C233S substitution on the heavy chain and / or C214S substitution on the light chain, according to Kabat numbering), a bDS Fab (a Fab without natural disulfide bonds and with non-natural interchain embedded disulfide bonds introduced, such as a Fab with F174C and C233S substitution on the heavy chain and / or S176C and C214S substitution on the light chain, according to Kabat numbering), or a bDS Fab-ScFv (a bDS Fab linked to ScFv via a linker (such as (G4S)x (SEQ ID NO: 154)).In some embodiments, the immune cell targeting group comprises a Fab having non-natural interchain disulfide bonds (e.g., engineered embedded interchain disulfide bonds).

[0327] In some embodiments, the ISV or nanobody further comprises a spacer contained within the V. HH One or more amino acids between the domain and the C-terminal cysteine ​​residue. In some embodiments, the immune cell targeting group comprises two or more V... HH Structural domain. In some embodiments, two or more V HH The domains are linked by amino acid linkers. In some embodiments, the immune cell targeting group includes a first V linked to the antibody CH1 domain. HH The domain and the second V linked to the constant domain of the antibody light chain HH The antibody CH1 domain and the antibody light chain constant domain are optionally linked by one or more disulfide bonds. In some embodiments, the immune cell targeting group comprises a V domain linked to the antibody CH1 domain. HH The antibody CH1 domain, and / or the antibody CH1 domain, is linked to the antibody light chain constant domain via one or more disulfide bonds. In some embodiments, the CH1 domain contains F174C and C233S substitutions, and / or the light chain constant domain contains S176C and C214S substitutions, according to Kabat numbering. In some embodiments, the antibody is ScFv, V HH (Nb), 2xV HH V HH -CH1 / empty Vk or V HH 1-CH1 / V HH -2-Nb bDS.

[0328] In some embodiments, the immune cell targeting group comprises Fab, which includes:

[0329] (a) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 1 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 2 or 3;

[0330] (b) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 4 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 5;

[0331] (c) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 7;

[0332] (d) Heavy chain fragment containing the amino acid sequence of SEQ ID NO: 8 and light chain fragment containing the amino acid sequence of SEQ ID NO: 9;

[0333] (e) Heavy chain fragment containing the amino acid sequence of SEQ ID NO: 10 and light chain fragment containing the amino acid sequence of SEQ ID NO: 11;

[0334] (f) Heavy chain fragments containing the amino acid sequence of SEQ ID NO: 12 and light chain fragments containing the amino acid sequence of SEQ ID NO: 13;

[0335] (g) Heavy chain fragment containing the amino acid sequence of SEQ ID NO: 14 and light chain fragment containing the amino acid sequence of SEQ ID NO: 15;

[0336] (h) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 16 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 17;

[0337] (i) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 18 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 19;

[0338] (j) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 20 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 21; or

[0339] (k) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 22 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 23.

[0340] In some embodiments, the immune cell targeting group comprises a bispecific targeting portion. In some embodiments, the bispecific targeting portion binds to two different types of immune cells. In some embodiments, the two different types of immune cells are CD4+ T cells and CD8+ T cells. In some embodiments, the bispecific targeting portion is a bispecific antibody. In some embodiments, the bispecific antibody is Fab-ScFv. In some embodiments, the LNP binds to a first antigen on the surface of a first type of immune cell and also binds to a second antigen on the surface of a second type of immune cell. In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises a first antibody that binds to the first antigen of the first type of immune cell, and the second conjugate comprises a second antibody that binds to the second antigen of the second type of immune cell. In some embodiments, the two different types of immune cells are T cells and NK cells. In some embodiments, the immune cell targeting group binds to (i) both CD3 and CD56; (ii) both CD8 and CD56; or (iii) both CD7 and CD56.

[0341] As discussed herein, the LNP can be targeted to specific cell types, such as immune cells, like T cells, B cells, or natural killer (NK) cells. This can be achieved using one or more lipids described herein. Furthermore, targeting can be enhanced by including a targeting group on the solvent-accessible surface of the LNP particle. For example, the targeting group may include a member of a specific binding pair (e.g., antibody-antigen pair, ligand-receptor pair, etc.). In some embodiments, the targeting group is an antibody. Targeting can be implemented, for example, by using lipid-immune cell targeting group conjugates described herein.

[0342] Optionally, the targeting portion is an antibody fragment without an Fc component. Previous attempts to target circulating immune cells with LNPs have employed whole antibodies (WO 2016 / 189532 A1). Liposomes or lipid-based particles with conjugated whole antibodies are cleared from circulation more quickly due to Fc conjugation, thus reducing their potential to reach target cells (Harding et al. (1997) Biochim Biophys. Acta [Chinese Journal of Biochemistry and Biophysics] 1327, 181-192; Sapra et al. (2004) Clin Cancer Res [Clinical Cancer Research] 10, 1100-1111; Aragnol et al., (1986) ProcNatl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 83, 2699-2703). Like liposomes targeting EGFR (Mamot et al., (2005) Cancer Res 65, 11631-11638), ErbB2 (Park et al., (2002) ClinCancer Res 8, 1172-1181), or EphA2 (Kamoun et al., 2019 Nat. Biomed.Eng 3, 264-280), liposomes targeted with antibody fragments retain their long-circulating properties. Furthermore, lipid-based loaders can be prepared using a micellar insertion process, which allows for near-quantitative incorporation after the antibody conjugate is manufactured separately (Nellis et al. (2005) Biotechnol Prog [Biotechnology Progress] 21, 221-232), compared to the extremely low insertion efficiency when conjugating intact IgG (Ishida et al. (1999) FEBSLett. [FEBS Letters] 460, 129-133) or the need to perform conjugation directly on intact LNPs (WO 2016 / 189532A1). scFv, Fab, or VHH fragments can also be directly conjugated with activated PEG-lipids to prepare insertable conjugates.

[0343] In some embodiments, PEG-(lipid) is equivalent to (lipid)-PEG.

[0344] In some embodiments, the targeting group may be a surface-binding antibody or a surface-binding antigen-binding fragment thereof, which can allow modulation of cell-targeting specificity. This is particularly useful because highly specific antibodies can be generated against target epitopes at desired target sites. In one embodiment, multiple different antibodies can be incorporated into and presented on the surface of the LNP, wherein each antibody binds to a different epitope on the same antigen or a different epitope on different antigens. Such an approach can increase the affinity and specificity of targeting interactions with specific target cells.

[0345] Target groups or combinations of target groups can be selected based on the desired localization, function, or structural characteristics of a given target cell. For example, to target T cells, T cell populations, or T cell subsets, one or more antibodies, or their antigen-binding fragments or antigen-binding derivatives, that target T cells (e.g., via T cell surface antigens) can be selected. Exemplary T cell surface antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD39, CD69, CD103, CD137, CD45, T cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, GL7, TLR2, TLR4, TLR5, and IL-15 receptors. To target NK cells or NK cell populations, one or more antibodies, their antigen-binding fragments, or antigen-binding derivatives that target NK cells (e.g., via NK cell surface antigens) can be selected. Exemplary NK cell surface antigens include, but are not limited to, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, and CD337 / NKP30. To target B cells or B cell populations, one or more antibodies, their antigen-binding fragments, or antigen-binding derivatives that target B cells (e.g., via B cell antigens) may be selected. Exemplary B cell antigens include, but are not limited to, CD19 (for all B cells except plasma cells), CD19, CD25, and CD30 (for activated B cells), CD27, CD38, CD78, CD138, and CD319 (for plasma cells), CD20, CD27, CD40, CD80, and PDL-2 (for memory cells), Notch2, CD1, CD21, and CD27 (for marginal zone B cells), CD21, CD22, and CD23 (for follicular B cells), and CD1, CD5, CD21, CD24, and TLR4 (for regulatory B cells).

[0346] In some embodiments, targeting may be implemented, for example, by using lipid-immune cell targeting conjugates described herein. Exemplary lipid-immune cell targeting conjugates may include compounds having formula (II), [lipid] - [optional linker] - [immune cell targeting group, such as a T cell targeting molecule, such as an anti-CD2 antibody, anti-CD3 antibody, anti-CD7 antibody, or anti-CD8 antibody] (Formula II).

[0347] In some embodiments, the immune cell targeting group is a polypeptide, and the lipid is conjugated to any position of the N-terminus, C-terminus, or middle portion of the polypeptide.

[0348] In some embodiments, the targeting group or targeting molecule is a T-cell target agent (e.g., an antibody) that binds to a T-cell antigen selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor (TCR) β, TCR-α, TCR-α / β, TCR-γ / δ, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor. In some embodiments, the T-cell antigen may be CD2, and the targeting group may be, for example, an anti-CD2 antibody. In some embodiments, the T-cell antigen may be CD3, and the targeting group may be, for example, an anti-CD3 antibody. In some embodiments, the T-cell antigen may be CD4, and the targeting group may be, for example, an anti-CD4 antibody. In some embodiments, the T-cell antigen may be CD5, and the targeting group may be, for example, an anti-CD5 antibody. In some embodiments, the T-cell antigen may be CD7, and the targeting group may be, for example, an anti-CD7 antibody. In some embodiments, the T-cell antigen may be CD8, and the targeting group may be, for example, an anti-CD8 antibody. In some embodiments, the T-cell antigen may be TCR β, and the targeting group may be, for example, an anti-TCR β antibody. In some embodiments, the antibody is a human or humanized antibody.

[0349] In some embodiments, the LNP comprises two conjugates. In some embodiments, the first conjugate comprises an antibody that binds CD3. In some embodiments, the second conjugate comprises an antibody that binds either CD11a or CD18. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD3 and CD11a. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD3 and CD18. In some embodiments, the LNP binds to CD7 and CD8 of the immune cell. In some embodiments, the first conjugate comprises an antibody that binds CD7, and the second conjugate comprises an antibody that binds CD8. In some embodiments, the LNP comprises one conjugate. In some embodiments, the conjugate comprises a bispecific antibody that binds both CD7 and CD8.

[0350] Exemplary CD2 binding agents may be antibodies selected from the group consisting of: 9.6 (academic.oup.com / intimm / article / 10 / 12 / 1863 / 744536), 9-1 (academic.oup.com / intimm / article / 10 / 12 / 1863 / 744536), TS2 / 18.1.1 (ATCC HB-195), Lo-CD2b (ATCC PTA-802), Lo-CD2a / BTI-322 (US Patent 6849258B1), Sipilzumab / MEDI-507 (US Patent 6849258B1 / en), 35.1 (ATCC HB-222), OKT11 (ATCC CRL-8027), RPA-2.1 (PCT Disclosure WO). 2020023559A1), AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 (R&D Systems), MAB1856 (R&D Systems), PAB30359 (Abnova Corporation), 10299-1 (Abnova Corporation), and their antigen-binding fragments. In some embodiments, the binder comprises a heavy chain variable domain (V) of an antibody selected from the group consisting of... H ) and light chain variable structural domain (V L): AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 (R&D Systems), MAB1856 (R&D Systems), PAB30359 (Annofa Biotech, Inc.), and 10299-1 (Annofa Biotech, Inc.). In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H and V L The heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 sequences are: AF1856 (R&D Systems), MAB18562 (R&D Systems), MAB18561 (R&D Systems), MAB1856 (R&D Systems), PAB30359 (Abnova Corporation), and 10299-1 (Abnova Corporation). These CDRs were generated by Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J.MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), and MacCallum (see, MacCallum). RM et al., (1996) J. MOL. BIOL. [Journal of Molecular Biology] 262: 732-745) or any other CDR determination method known in the art.

[0351] Exemplary CD2 binding agents may also be selected from antibodies or antibody fragments employing CDRs of the following clones: 9.6, 9-1, TS2 / 18.1.1, Lo-CD2b, Lo-CD2a, BTI-322, cipuzumab, 35.1, OKT11, RPA-2.1, SQB-3.21, LT2, TS1 / 8, UT329, 4F22, OX-34, UQ2 / 42, MU3, U7.4, NFN-76, or MOM-181-4-F(E).

[0352] Exemplary CD3 binding agents (CD3γ / δ / ε, CD3γ, CD3δ, CD3γ / ε, CD3δ / ε, or CD3ε) may be antibodies selected from the group consisting of: MEM-57 (CD3γ / δ / ε, EnzoLife Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Anopharm Biotechnology, Inc.), CD3-12 (CD3ε, Cell Signaling Technology), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, NovusBiologicals), 16669-1-AP (CD3δ, Invitrogen), and their antigen-binding fragments. In some embodiments, the binding agent comprises a V of an antibody selected from the group consisting of: H Domain and V L Domains: MEM-57 (CD3γ / δ / ε, Enzo Life Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Anapharma Biotech, Inc.), CD3-12 (CD3ε, Cell Signaling Technologies, Inc.), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, Vibio Biotechnology, Inc.), and 16669-1-AP (CD3δ, Ingenium Biotech, Inc.). In some embodiments, the binder comprises a V antibody selected from the group consisting of... H and V LThe heavy chains CDR1, CDR2, and CDR3, and the light chains CDR1, CDR2, and CDR3 are: MEM-57 (CD3γ / δ / ε, EnzoLife Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Anapharma Biotechnology, Inc.), CD3-12 (CD3ε, Cell Signaling Technology), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, NovusBiologicals), and 16669-1-AP (CD3δ, Invitrogen). These CDRs were derived by Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest], NIH Publication No. 91-3242. The CDR is determined by Bethesda, Chothia (see, for example, Chothia C & Lesk AM, (1987), J. MOL. BIOL. 196: 901-917), MacCallum (see, MacCallum RM et al., (1996) J. MOL. BIOL. [Journal of Molecular Biology] 262: 732-745), or any other CDR determination method known in the art.

[0353] Exemplary CD3 binding agents may also be selected from antibodies or antibody fragments employing CDRs of the following clones: hsp34, OKT-3, UCHT1, 38.1, HIT3a, RFT8, SK7, BC3, SP34-2, HU291, TRX4, Catuximab, Teplizumab, 3-106, 3-114, 3-148, 3-190, 3-271, 3-550, 4 -10, 4-48, H2C, F12Q, I2C, SP7, 3F3A1, CD3-12, 301, RIV9, JB38-29, JE17-74, GT0013, 4E2, 7A4, 4D10A6, SPV-T3b, M2AB, ICO-90, 30A1 or Hu38E4.v1 (US Patent Application 20200299409A1), REGN5458 (US Patent Application 20200024356 A1), bonnetumab ( go.drugbank.com / drugs / DB09052 / polypeptide_ sequences.fastaIn some embodiments, the conjugate comprises a Fab, wherein the Fab comprises (a) a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 1 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the LNP binds CD3 and also binds CD11a or CD18.

[0354] Exemplary CD4 binders may be antibodies selected from the group consisting of: ipalzumab (www.genome.jp / dbget-bin / www_bget?D09575), AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Annofa Biotech), CAL4 (Abcam), and antigen-binding fragments thereof. In some embodiments, the binder comprises a V-cell of an antibody selected from the group consisting of... H Domain and V L Domains: AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Annofa Biotech, Inc.), and CAL4 (Abogen Biosciences). In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H and V L The heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 sequences are: AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Annofa Biotech), and CAL4 (Abogen Biosciences). These CDRs were generated by Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J. MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), and MacCallum (see, MacCallum RM et al., (1996) J. MOL.). Determined by BIOL. [Journal of Molecular Biology] 262: 732-745 or any other CDR determination method known in the art.

[0355] Exemplary CD4 binding agents may also be selected from antibodies or antibody fragments employing CDRs of the following clones: ipalizumab, OKT4, RPA-T4, S3.5, SK3, N1UG0, RIV6, OTI18E3, MEM-241, B486A1, RFT-4g, 7E14, MDX.2, MEM-115, MEM-16, ICO-86, Edu-2, or ipalizumab.

[0356] Exemplary CD5 binders may be antibodies selected from the group consisting of: He3, MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Ebola), CD5 / 54 / F6 (Ebola), 65152 (Proteintech), and antigen-binding fragments thereof. In some embodiments, the binder comprises a V of an antibody selected from the group consisting of... H Domain and V L Domains: MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473+CD5 / 54 / F6 (Ebola), CD5 / 54 / F6 (Ebola), and 65152 (Protein Technologies). In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H and V LThe heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3 sequences are: MAB1636 (R&D Systems), AF1636 (R&D Systems), MAB115 (R&D Systems), C5 / 473 + CD5 / 54 / F6 (Embok), CD5 / 54 / F6 (Embok), and 65152 (Proteintech). These CDRs were generated by Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J. MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), and MacCallum (see, MacCallum). RM et al., (1996) J. MOL. BIOL. [Journal of Molecular Biology] 262: 732-745) or any other CDR determination method known in the art.

[0357] Exemplary CD5 binding agents may also be selected from antibodies or antibody fragments employing CDRs of the following clones: zolimomab, 5D7, L17F12, and UCHT2, 1D8, 3I21, 4H10, 8J23, 5O4, 4H2, 5G2, 8G8, 6M4, 2E3, 4E24, 4F10, 7J9, 7P9, 8E24, 6L18, 7H7, 1E7, 8J21, 7I11, 8M9, 1P21, 2H11, 3M22, 5M6, 5H8, 7I19, 1A2, 8E1 5. 8C10, 3P16, 4F3, 5M24, 5O24, 7B16, 1E8, 2H16, BLa1, 1804, DK23, Cris1, MEM-32, H65, 4C7, OX-19, Leu-1, 53-7.3, 4H8E6, T101, EP2952, D-9, H-3, HK231, N-20, Y2 / 178, H-300, CD5 / 54 / F6, Q-20, CC17, MOM-18539-S(P) or MOM-18885-S(P).

[0358] Exemplary CD7 binders may be antibodies selected from the group consisting of: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abogen), 1G10D8 (Protein Technologies), NBP2-32097 (Vibio Biotechnology), NBP2-38440 (Vibio Biotechnology), and antigen-binding fragments thereof. In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H Domain and V L Domains: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abogen), 1G10D8 (Protein Technologies), NBP2-32097 (Vibio Biotechnology Co., Ltd.), and NBP2-38440 (Vibio Biotechnology Co., Ltd.). In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H and V L The heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3 sequences are: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abogen), 1G10D8 (Protein Technologies), NBP2-32097 (Vibio Biotechnology), and NBP2-38440 (Vibio Biotechnology). These CDRs were derived by Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J. MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), and MacCallum (see, MacCallum RM et al., (1996)). The CDR is determined by J.MOL. BIOL. [Journal of Molecular Biology] 262: 732-745 or any other CDR determination method known in the art.

[0359] Exemplary CD7 binders may also be selected from antibodies or antibody fragments employing CDRs of the following clones: TH-69, 3Afl1, T3-3A1, 124-1D1, 3A1f, CD7-6B7, or VHH6.

[0360] Exemplary CD8 (CD8α, CD8α / α, CD8α / β, or CD8β) binders may be antibodies selected from the group consisting of: 2.43 (Ingenieur), Du CD8-1 (CD8α, Ingenieur), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abogen), C8 / 144B (CD8α, Vibio Biotechnology), YTS105.18 (CD8α, Vibio Biotechnology), TRX2 (patents.justia.com / patent / 20170198045), and antigen-binding fragments thereof. In some embodiments, the binder comprises a V of an antibody selected from the group consisting of... H Domain and V L Domains: 2.43 (Ingenieur), 51.1 (ATCC HB-230), Du CD8-1 (CD8α, Ingenieur), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abogen), C8 / 144B (CD8α, Vibio Biotechnology Co., Ltd.), and YTS105.18 (CD8α, Vibio Biotechnology Co., Ltd.). In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H and V LThe heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3 sequences are: 2.43 (Ingenieur), Du CD8-1 (CD8α, Ingenieur), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abogen), C8 / 144B (CD8α, Vibio Biotechnology Co., Ltd.), and YTS105.18 (CD8α, Vibio Biotechnology Co., Ltd.). These CDRs were derived by Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J. MOL. BIOL. [Journal of Molecular Biology] 196: The CDR is determined by (901-917), MacCallum (see MacCallum RM et al., (1996) J. MOL. BIOL. [Journal of Molecular Biology] 262: 732-745) or any other CDR determination method known in the art.

[0361] Exemplary CD8 binders may also be selected from antibodies or antibody fragments employing CDRs of the following clones: OKT-8, 51.1, S6F1, TRX2, and UCHT4, SP16, 3B5, C8-144B, HIT8a, RAVB3, LT8, 17D8, MEM-31, MEM-87, RIV11, DK-25, YTC141.1HL, or YTC182.20. In some embodiments, the conjugate comprises a Fab, wherein the Fab comprises a heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 7.

[0362] An exemplary CD8 binding agent is an antibody (e.g., an ISV) comprising three complementarity-determining domains CDR1, CDR2, and CDR3. In some embodiments, CDR1 comprises GSTFSDYG (SEQ ID NO: 100) or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or higher sequence identity therewith. In some embodiments, CDR2 comprises IDWNGEHT (SEQ ID NO: 101) or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or higher sequence identity therewith. In some embodiments, CDR3 comprises AADALPYTVRKYNY (SEQ ID NO: 102) or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 99%, or higher sequence identity therewith. In some embodiments: (1) any amino acid substitution is a conserved amino acid substitution; and / or (2) the amino acid sequence contains only amino acid substitutions and does not contain any associated amino acid deletions or insertions. In some embodiments, the ISVD comprises SEQ ID NO: 77, or is substantially composed of SEQ ID NO: 77. In some embodiments, the agent has at least 80%, at least 85%, such as 90% or 95% or higher sequence identity with SEQ ID NO: 77, or has 3, 2 or 1 amino acid differences from SEQ ID NO: 77; or any suitable combination thereof. In some embodiments, CD8 is derived from mammals, such as humans.

[0363] In one embodiment, the CD8 nanobody is BDSn:

[0364] Anti-CD8 BDSn Nb sequences (CDR1, CDR2, CDR3 underlined, based on IMGT names):

[0365] EVQLVESGGGLVQAGGSLRLSCAAS GSTFSYG VGWFRQAPGKGREFVAD IDWNGEHT SYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYC AADALPYTVRKYNY WGQGTQVTVSSGGCGGHHHHHH (SEQ ID NO:77)

[0366] In some embodiments, the CD8 nanobody described herein is at 10 -5 Up to 10 -12 mol / L (M) or less, 10 -7 Up to 10 -12 mol / L (M) or less, 10 -8 Up to 10 -12The dissociation constant (KD) of moles per liter (M), and / or at least 10 7 M -1 At least 10 8 M -1 At least 10 9 M -1 (e.g., at least 10) 12 M -1 The association constant (KA) of the nanobody for vWF is determined; and specifically, the nanobody binds to CD8 with a KD of less than 500 nM, less than 200 nM, and less than 10 nM (e.g., less than 500 μM). The KD and KA values ​​of the nanobody disclosed herein for vWF can be determined. More generally, the nanobody described herein may have a dissociation constant for vWF as described in this paragraph.

[0367] One class of CD8 nanobodies disclosed herein comprises nanobodies having an amino acid sequence corresponding to a naturally occurring VHH domain but which has been “humanized” (i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH domain with one or more amino acid residues appearing at one or more corresponding positions in the VH domain of a conventional human 4-chain antibody (e.g., indicated above). It should be noted that such humanized CD8 nanobodies disclosed herein can be obtained in any suitable manner known per se (i.e., as indicated under points (1)-(8) above), and are therefore not strictly limited to peptides already obtained using peptides containing a naturally occurring VHH domain as a starting material.

[0368] Another type of CD8 nanobodies disclosed herein comprises nanobodies having an amino acid sequence that has been “camelized” (i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VH domain of a conventional 4-chain antibody with one or more amino acid residues appearing at one or more corresponding positions in the VHH domain of a heavy chain antibody). This can be done in a manner known per se, as will be clear to those skilled in the art, for example, based on the further description below. Also refer to WO 94 / 04678. Such camelization can preferentially occur at amino acid positions present at the VH-VL interface and at so-called camelid marker residues (see also, for example, WO 94 / 04678), as also mentioned below. In some embodiments, the VH domain or sequence used as the starting material or starting point for generating or designing camelized nanobodies is a VH sequence from mammals, such as a human VH sequence. It should be noted that such camelized nanobodies disclosed herein can be obtained in any suitable manner known per se, and are therefore not strictly limited to peptides already obtained using peptides containing naturally occurring VH domains as starting materials.

[0369] For example, both "humanization" and "camelization" can be performed by: providing nucleotide sequences encoding naturally occurring VHH or VH domains, respectively; then altering one or more codons in these nucleotide sequences in a manner known per se, such that the new nucleotide sequences encode the humanized or camelized nanobodies disclosed herein, respectively; and then expressing the resulting nucleotide sequences in a manner known per se to provide the desired nanobodies. Alternatively, the amino acid sequences of the desired humanized or camelized nanobodies disclosed herein can be designed based on the amino acid sequences of naturally occurring VHH or VH domains, respectively, and then synthesized de novo using peptide synthesis techniques known per se. Furthermore, the nucleotide sequences encoding the desired humanized or camelized nanobodies can be designed based on the amino acid sequences or nucleotide sequences of naturally occurring VHH or VH domains, respectively; then synthesized de novo using nucleic acid synthesis techniques known per se; and then expressed in a manner known per se to provide the desired nanobodies.

[0370] Other suitable methods and techniques for obtaining nanobodies and / or the nucleotide sequences and / or nucleic acids encoding them (starting from a naturally occurring VH domain or preferably a VHH domain (amino acid sequence)) and / or from the nucleotide sequence and / or nucleic acid sequence encoding them) will be clear to those skilled in the art, and may, for example, include combining one or more amino acid sequences and / or nucleotide sequences from a naturally occurring VH domain (such as one or more FRs and / or CDRs) in a suitable manner with one or more amino acid sequences and / or nucleotide sequences from a naturally occurring VHH domain (such as one or more FRs or CDRs) to provide nanobodies (nucleotide sequences or nucleic acids encoding them). Compounds and constructs, particularly proteins and peptides, are also provided that comprise or substantially consist of at least one such amino acid sequence and / or nanobodies (or suitable fragments thereof) of this disclosure, and optionally further comprise one or more other groups, residues, portions, or binding units. In some embodiments, such other groups, residues, portions, binding units, or amino acid sequences may or may not provide additional functionality to the amino acid sequence and / or nanobody (and / or the compound or construct in which it is present), and may or may not alter the properties of the amino acid sequence and / or nanobody.

[0371] This disclosure also covers any polypeptide disclosed herein that has been glycosylated at one or more amino acid positions, typically depending on the host used to express the polypeptide. The polypeptide may comprise the amino acid sequence of the CD8 nanobody disclosed herein, fused to at least one additional amino acid sequence at its amino terminus, its carboxyl terminus, or both its amino terminus and its carboxyl terminus. Such additional amino acid sequences may comprise at least one other nanobody to provide a polypeptide comprising at least two (e.g., three, four, or five) nanobodies, wherein said nanobodies may optionally be linked via one or more linker sequences (as defined herein). A polypeptide comprising the CD8 nanobody disclosed herein and one or more additional nanobodies is a multivalent polypeptide. In a multivalent polypeptide, the two or more nanobodies may be the same or different. For example, two or more nanobodies in a multivalent polypeptide:

[0372] • It can target the same antigen, i.e., the same part or epitope of the antigen, or two or more different parts or epitopes of the antigen; and / or

[0373] • Can target different antigens;

[0374] • Or a combination thereof.

[0375] Therefore, divalent polypeptides, for example:

[0376] • It can contain two identical nanobodies;

[0377] • It may contain a first nanobody targeting a first portion or epitope of an antigen and a second nanobody targeting the same portion or epitope of the antigen or targeting another portion or epitope of the antigen;

[0378] Alternatively, it may include a first nanobody targeting a first antigen and a second nanobody targeting a second antigen different from the first antigen.

[0379] The trivalent polypeptide of the present invention is, for example:

[0380] • It can contain three identical or different nanobodies targeting the same or different parts or epitopes of the same antigen;

[0381] • It may comprise two identical or different nanobodies targeting the same or different portions or epitopes of a first antigen and a third nanobodies targeting a second antigen different from the first antigen; or

[0382] • It may include a first nanobody targeting a first antigen, a second nanobody targeting a second antigen different from the first antigen, and a third nanobody targeting a third antigen different from the first and second antigens.

[0383] Exemplary CD137 binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: 4B4-1, P566, or Urelumab. Exemplary CD28 binders may be selected from antibodies or antibody fragments employing the CDR of clone TAB08. Exemplary CD45 binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: BC8, 9.4, 4B2, Tu116, or GAP8.3. Exemplary CD18 binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: 1B4, TS1 / 18, MEM-48, YFC118-3, TA-4, MEM-148, or R3-3, 24. Exemplary CD11a binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: MHM24 or Efalizumab. Exemplary IL-2 receptor binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: YTH 906.9HL, IL2R.1, BC96, B-B10, 216, MEM-181, ITYV, MEM-140, ICO-105, Daclizumab, or selected from IL2 or IL2 fragments. Exemplary IL-15R binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: JM7A4 or OTI3D5, or selected from IL15 or IL15 fragments. Exemplary TLR2 binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: JM22-41, TL2.1, 11G7, or TLR2.45. Exemplary TLR4 binders may be selected from antibodies or antibody fragments employing the following cloned CDRs: HTA125 or 76B357-1. Exemplary TLR5 binders may be selected from antibodies or antibody fragments employing a CDR of the following clones: 85B152-5 or 9D759-2. Exemplary GL7 binders may be selected from antibodies or antibody fragments employing a CDR of clone GL7.

[0384] Exemplary PD-1 binders may be selected from antibodies or antibody fragments employing CDRs of the following clones: MIH4, J116, J150, OTIB11, OTI17B10, OTI3A1, or OTI16D4. Additionally, exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (Tecentriq®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0385] Exemplary CTLA-4 binders may be selected from antibodies or antibody fragments employing the CDRs of the following clones: ER4.7G.11[7G11], OTI9G4, OTI9F3, OTI3A5, A3.4H2.H12, 14D3, OTI3A12, OTI1A11, OTI1E8, OTI3B11, OTI3D2, OTI10C8, OTI2E9, OTI6F1, OTI7D3, OTI85B, OTI12C6. Exemplary anti-CTLA-4 antibodies are described in the following: U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910; 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984; International (PCT) Publications Nos. WO98 / 42752, WO00 / 37504, and WO01 / 14424; and European Patent No. EP1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0386] Exemplary TCR β binders may be antibodies selected from the group consisting of: H57-597 (Ingenieur), 8A3 (Vibio Biotechnology), R73 (TCRα / β, Abogen), E6Z3S (TRBC1 / TCRβ, Cell Signaling Technologies), and their antigen-binding fragments. In some embodiments, the binder comprises a V-cell of an antibody selected from the group consisting of... H Domain and V L Domains: H57-597 (Ingenieur), 8A3 (Vibio Biotechnology Co., Ltd.), R73 (TCRα / β, Abogen Biosciences), and E6Z3S (TRBC1 / TCRβ, Cell Signaling Technologies, Inc.). In some embodiments, the binder comprises V of an antibody selected from the group consisting of... H and V LThe heavy chain CDR1, CDR2, and CDR3 sequences and the light chain CDR1, CDR2, and CDR3 sequences are: H57-597 (Ingenieur), 8A3 (Vibio Biotechnology Co., Ltd.), R73 (TCRα / β, Abogen Biosciences), and E6Z3S (TRBC1 / TCRβ, Cell Signaling Technologies, Inc.). These CDRs were derived by Kabat (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J. MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), and MacCallum (see, MacCallum RM et al., (1996) J. MOL. BIOL. [Journal of Molecular Biology] 262: Determined by (732-745) or any other CDR determination method known in the art.

[0387] Exemplary CD137 binders may be selected from antibodies or antibody fragments employing the following clones of CDR: 4B4-1, P566, or Urelumab.

[0388] In some embodiments, the immune cell targeting group comprises an antibody selected from the group consisting of Fab, F(ab')2, Fab'-SH, Fv, and scFv fragments. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the immune cell targeting group comprises Fab or an immunoglobulin single variable domain, such as a nanobody. In some embodiments, the immune cell targeting group comprises Fab without native interchain disulfide bonds. For example, in some embodiments, the Fab comprises a heavy chain fragment with a C233S substitution and / or a light chain fragment with a C214S substitution, according to Kabat numbering. In some embodiments, the immune cell targeting group comprises Fab containing one or more non-native interchain disulfide bonds. In some embodiments, the interchain disulfide bond is located between two non-native cysteine ​​residues, respectively, on the light chain fragment and the heavy chain fragment. For example, in some embodiments, the Fab comprises a heavy chain fragment with an F174C substitution and / or a light chain fragment with an S176C substitution, according to Kabat numbering. In some embodiments, the Fab comprises a heavy chain segment substituted with F174C and C233S and / or a light chain segment substituted with S176C and C214S, according to Kabat numbering. In some embodiments, the immunocellular targeting group comprises a Fab lacking natural interchain disulfide bonds. In some embodiments, the Fab is engineered to replace one or two cysteine ​​residues forming natural interchain disulfide bonds on the natural constant light chain and the natural constant heavy chain with non-cysteine ​​amino acids, thereby removing natural interchain disulfide bonds in the Fab. In some embodiments, the immunocellular targeting group comprises a C-terminal cysteine ​​residue. In some embodiments, the immunocellular targeting group comprises a Fab containing a cysteine ​​residue at the C-terminus of the heavy chain or light chain segment. In some embodiments, the Fab further comprises one or more amino acids between the heavy chain of the Fab and the C-terminal cysteine ​​residue. For example, in some embodiments, the Fab comprises two or more amino acids derived from an antibody hinge region (e.g., a partial hinge sequence) between the C-terminus of the Fab and the C-terminal cysteine ​​residue. In some embodiments, the Fab comprises a heavy chain variable domain linked to the antibody CH1 domain and a light chain variable domain linked to the antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker. In some embodiments, the Fab antibody is DS Fab, NoDS Fab, bDS Fab, or bDS Fab-ScFv.

[0389] In some embodiments, the immune cell targeting group comprises an immunoglobulin monovariable domain, such as a nanobody (e.g., V). HHIn some embodiments, the nanobody contains a cysteine ​​residue at its C-terminus. In some embodiments, the nanobody further comprises a spacer contained in the V-terminus. HH One or more amino acids between the domain and the C-terminal cysteine ​​residue. In some embodiments, the spacer contains one or more glycine residues, such as two glycine residues. In some embodiments, the immune cell targeting group contains two or more V... HH Structural domain. In some embodiments, two or more V HH The domains are linked by amino acid linkers. In some embodiments, the amino acid linkers comprise one or more glycine and / or serine residues (e.g., one or more repeats of the sequence GGGGS (SEQ ID NO: 154)). In some embodiments, the immune cell targeting group comprises a first V linked to the antibody CH1 domain. HH The domain and the second V linked to the constant domain of the antibody light chain HH The antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds (e.g., interchain disulfide bonds). In some embodiments, the immune cell targeting group comprises a V domain linked to the antibody CH1 domain. HH The antibody CH1 domain is connected to an antibody light chain constant domain via one or more disulfide bonds. In some embodiments, the CH1 domain contains F174C and C233S substitutions, and the light chain constant domain contains S176C and C214S substitutions, according to Kabat numbering. In some embodiments, the antibody is ScFv, V HH 2xV HH V HH -CH1 / empty Vk or V HH 1-CH1 / V HH -2-Nb bDS.

[0390] An exemplary targeting portion may have the amino acid sequence described below:

[0391] Anti-CD3 hSP34-Fab sequence:

[0392] hSP34 heavy chain (HC) sequence (SEQ ID NO: 1):

[0393] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSSDKTHTC

[0394] Sequence of hSP34-mlam light chain (LC) (mouse λ) (SEQ ID NO: 2):

[0395] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADSS

[0396] SP34-hlam LC (human λ) (SEQ ID NO: 3):

[0397] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

[0398] Sequence of anti-CD3 Hu291-Fab:

[0399] Hu291 HC (SEQ ID NO: 4):

[0400] QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDK THTC

[0401] Hu 291 LC (SEQ ID NO: 5):

[0402] MDMRVPAQLLGLLLLWLPGAKCDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGES

[0403] Anti-CD8 TRX2-Fab sequence:

[0404] TRX2 HC (SEQ ID NO: 6):

[0405] QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSD KTHTC

[0406] TRX2 LC (SEQ ID NO: 7):

[0407] DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0408] Anti-CD8 OKT8-Fab sequence:

[0409] OKT8 HC (SEQ ID NO: 8):

[0410] QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGYGYYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0411] OKT8 LC (SEQ ID NO: 9):

[0412] DIVMTQSPSSLSASVGDRVTITCRTSRSISQYLAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0413] Anti-CD4 ibalizumab-Fab sequence:

[0414] Ibalizumab HC (SEQ ID NO: 10):

[0415] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSD KTHTC

[0416] Ibalizumab LC (SEQ ID NO: 11):

[0417] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0418] Anti-CD5 He3-Fab sequence:

[0419] He3 HC (SEQ ID NO: 12):

[0420] EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWINTHTGEPTYADSFKGRFTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0421] He3 LC (SEQ ID NO: 13):

[0422] DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0423] Anti-CD7 TH-69-Fab sequence:

[0424] TH-69 HC (SEQ ID NO: 14):

[0425] EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0426] TH-69 LC (SEQ ID NO: 15):

[0427] DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0428] Anti-CD2 TS2 / 18.1-Fab sequence:

[0429] TS2 / 18.1 HC (SEQ ID NO: 16):

[0430] EVQLVESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0431] TS2 / 18.1 LC (SEQ ID NO: 17):

[0432] DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHNFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0433] Anti-CD29.6-Fab sequence:

[0434] 9.6 HC (SEQ ID NO: 18):

[0435] QVQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0436] 9.6 LC (SEQ ID NO: 19):

[0437] NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0438] Anti-CD29-1-Fab sequence:

[0439] 9-1 HC (SEQ ID NO: 20):

[0440] QVQLQQPGTELVRPGSSVKLSCKASGYTFTSYWVNWVKQRPDQGLEWIGRIDPYDSETHYNQKFTDKAISTIDTSSNTAYMQLSTLTSDASAVYYCSRSPRDSSTNLADWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0441] 9-1 LC (SEQ ID NO: 21):

[0442] DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0443] mutOKT8-Fab sequence:

[0444] mutOKT8 HC (SEQ ID NO: 22):

[0445] QVQLVQSGAEDKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMGRIDPANDNTLYASKFQGRVTITADTSSNTAYMELSSLRSEDTAVYYCGRGAGAYVFDHWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0446] mutOKT8 LC (SEQ ID NO: 23):

[0447] DIVMTQSPSSLSASVGDRVTITCRTSRSISAALAWYQEKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNENPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES.

[0448] Sequence of anti-CD56 A1 Fab

[0449] A1 bDS HC (SEQ ID NO: 26):

[0450] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSNWIRQSPSGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARENIAAWTWAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0451] A1 bDS LC (SEQ ID NO: 27):

[0452] EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGLAPRLLIYDTSLRATDIPDRFSGSGSGTAFTLTISRLEPEDFAVYYCQQYGSSPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0453] Anti-CD56 A2 Fab Sequence

[0454] A2 bDS HC (SEQ ID NO: 28):

[0455] EVQLVQSGAEVKKPGSSVKVSCKASGGTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLSSGYSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0456] A2 bDS LC (SEQ ID NO: 29):

[0457] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLNWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEGEDVGDYYCMQALQSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0458] Anti-CD56 A3 Fab Sequence

[0459] A3 bDS HC (SEQ ID NO: 30):

[0460] EVQLVQSGAEVKKPGSSVKVSCKASGGTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDLSSGYSGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0461] A3 bDS LC (SEQ ID NO: 31):

[0462] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEADDVGVYYCMQSLQTPWTFGHGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0463] Fab sequence of anti-CD56 lorvotuzumab

[0464] Lorvotuzumab bDS HC (SEQ ID NO: 32):

[0465] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSFTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMRKGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0466] Lorvotuzumab bDS LC (SEQ ID NO: 33):

[0467] DVVMTQSPLSLPVTLGQPASISCRSSQIIIHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0468] Anti-CD2 RPA-2.10v1 Fab Sequence

[0469] RPA-2.10v1 bDS HC (SEQ ID NO: 34):

[0470] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYDMSWVRQTPEKRLEWVASISGGGFLYYLDSVKGRFTISRDNARNILYLHMTSLRSEDTAMYYCARSSYGEIMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0471] RPA-2.10v1 bDS LC (SEQ ID NO: 35):

[0472] DILLTQSPAILSVSPGERVSFSCRASQRIGTSIHWYQQRTTGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDVADYYCQQSHGWPFTFGGGTKLEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0473] Anti-CD137 4B4-1 Fab Sequence

[0474] 4B4-1 bDS HC (SEQ ID NO: 36):

[0475] QVQLQQPGAELVKPGASVKLSCKASGYTFSSYWMHWVKQRPGQVLEWIGEINPGNGHTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSFTTARGFAYWGQGTLVTVSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0476] 4B4-1 bDS LC(SEQ ID NO: 37):

[0477] DIVMTQSPATQSVTPGDRVSLSCRASQTISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQDGHSFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0478] hSP34-hlam NoDS HC(SEQ ID NO: 38):

[0479] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0480] hSP34-hlam NoDS LC(SEQ ID NO: 39):

[0481] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

[0482] hSP34-hlam DS Heavy Chain (SEQ ID NO: 40):

[0483] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0484] hSP34-hlam DS Light Chain (SEQ ID NO: 41):

[0485] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS

[0486] Anti-CD2 TS2 / 18.1 DS Fab

[0487] TS2 / 18.1 DS Heavy Chain (SEQ ID NO: 42):

[0488] EVQLVESGGGLVMPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISGGGFTYYPDTVKGRFTLSRDNAKNTLYLQMSSLKSEDTAMYYCARQGANWELVYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0489] TS2 / 18.1 DS LC (SEQ ID NO: 43):

[0490] DIVMTQSPATLSVTPGDRVFLSCRASQSISDFLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYFCQNGHNFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0491] Anti-CD29.6 DS Fab

[0492] 9.6 DS HC (SEQ ID NO: 44):

[0493] QVQLQQPGAELVRPGSSVKLSCKASGYTFTRYWIHWVKQRPIQGLEWIGNIDPSDSETHYNQKFKDKATLTVDKSSGTAYMQLSSLTSEDSAVYYCATEDLYYAMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[0494] 9.6 DS LC (SEQ ID NO: 45):

[0495] NIMMTQSPSSLAVSAGEKVTMTCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAVYYCHQYLSSHTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0496] hSP34-hlam bDS Heavy Chain (SEQ ID NO: 46):

[0497] EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0498] hSP34-hlam bDS Light Chain (SEQ ID NO: 47):

[0499] QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAACSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

[0500] Anti-CD3 TR66 bDS Fab sequence

[0501] TR66 bDS Heavy Chain (SEQ ID NO: 48):

[0502] QVQLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDNYSLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0503] TR66 bDS LC (SEQ ID NO: 49):

[0504] QIVLTQSPSSLSASLGEKVTMTCRASSSVSYMNWYQQKPGTSPKRWIYDTSKVASGVPDRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0505] Sequence of anti-CD3 TRX4 bDS Fab

[0506] TRX4 bDS HC (SEQ ID NO: 50):

[0507] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0508] TRX4 bDS LC (SEQ ID NO: 51):

[0509] DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAACSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTESS

[0510] Sequence of anti-CD3 HzUCHT1 bDS Fab

[0511] HzUCHT1 (Y59T) bDS HC (SEQ ID NO: 52):

[0512] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPTKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0513] HzUCHT1 bDS LC (SEQ ID NO: 53):

[0514] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0515] Sequence of anti-CD3 teplizumab bDS Fab

[0516] Teplizumab bDS HC (SEQ ID NO: 54):

[0517] QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0518] Tislelizumab bDS LC (SEQ ID NO: 55):

[0519] DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0520] Anti-CD8 TRX2 bDS Fab sequence

[0521] TRX2 bDS HC (SEQ ID NO: 56):

[0522] QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0523] TRX2 bDS LC (SEQ ID NO: 57):

[0524] DIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0525] Anti-CD2 Lo-CD2b bDS Fab sequence

[0526] Lo-CD2b bDS HC (SEQ ID NO: 58):

[0527] EVQLVESGGGLVQPGASLKLSCVASGFTFSDYWMSWVRQTPGKPMEWIGHIKYDGSYTNYAPSLKNRFTISRDNAKTTLYLQMSNVRSEDSATYYCAREAPGAASYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0528] Lo-CD2b bDS LC (SEQ ID NO: 59):

[0529] DVVLTQTPVAQPVTLGDQASISCRSSQSLVHSNGNTYLEWFLQKPGQSPQLLIYKVSNRFSGVPDRFIGSGSGSDFTLKISRVEPEDWGVYYCFQGTHDPYTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0530] Anti-CD235.1 bDS Fab sequence

[0531] 35.1 bDS HC (SEQ ID NO: 60):

[0532] EVQLQQSGAELVKPGASVKLSCRTSGFNIKDTYIHWVKQRPEQGLKWIGRIDPANGNTKYDPKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCVTYAYDGNWYFDVWGAGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0533] 35.1 bDS LC (SEQ ID NO: 61):

[0534] DIKMTQSPSSMYVSLGERVTITCKASQDINSFLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMEIYYCLQYDEFPYTFGGGTKLEMKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0535] Anti-CD2 OKT11 bDS Fab sequence

[0536] OKT11 bDS HC (SEQ ID NO: 62):

[0537] QVQLQQPGAELVRPGTSVKLSCKASGYTFTSYWMHWIKQRPEQGLEWIGRIDPYDSETHYNEKFKDKAILSVDKSSSTAYIQLSSLTSDDSAVYYCSRRDAKYDGYALDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0538] OKT11 bDS LC (SEQ ID NO: 63):

[0539] DIVMTQAAPSVPVTPGESVSISCRSSKTLLHSNGNTYLYWFLQRPGQSPQVLIYRMSNLASGVPNRFSGSGSETTFTLRISRVEAEDVGIYYCMQHLEYPYTFGGGTKLEIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0540] Anti-CD11a HzMHM24 bDS Fab Sequence

[0541] HzMHM24 bDS HC (SEQ ID NO: 64):

[0542] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWVGMIHPSDSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARGIYFYGTTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0543] HzMHM24 bDS LC (SEQ ID NO: 65):

[0544] DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0545] Anti-CD18 h1B4 bDS Fab Sequence

[0546] h1B4 bDS HC (SEQ ID NO: 66):

[0547] EVQLVESGGDLVQPGRSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVAAIDNDGGSISYPDTVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCARQGRLRRDYFDYWGQGTLVTVSTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0548] h1B4 bDS LC (SEQ ID NO: 67):

[0549] DIQMTQSPSSLSASVGDRVTITCRASESVDSYGNSFMHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQSNEDPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0550] bDS Fab sequence of anti-CD19 erlizumab

[0551] erlizumab bDS HC (SEQ ID NO: 68):

[0552] EVQLVESGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLEWVAGINPKNGGTSHNQRFMDRFTISVDKSTSTAYMQMNSLRAEDTAVYYCARWRGLNYGFDVRYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0553] erlizumab bDS LC (SEQ ID NO: 69):

[0554] DIQMTQSPSSLSASVGDRVTITCRASQDINNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0555] Anti-CD4 / CD8 ibalizumab / TRX2 bDS Fab-ScFv sequence

[0556] Ibalizumab / TRX2 bDS Fab-ScFv HC (SEQ ID NO: 70):

[0557] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0558] Ibalizumab / TRX2 bDS Fab-ScFv LC (SEQ ID NO: 71):

[0559] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIK

[0560] Anti-CD4 ibalizumab NoDS Fab sequence

[0561] Ibalizumab NoDS LC (SEQ ID NO: 72):

[0562] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTC

[0563] Ibalizumab NoDS HC (SEQ ID NO: 73):

[0564] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0565] Anti-CD4 OKT4 bDS Fab sequence

[0566] OKT4 bDS LC (SEQ ID NO: 74):

[0567] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVSAISDHSTNTYYPDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARKYGGDYDPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCHHHHHH

[0568] OKT4 bDS HC (SEQ ID NO: 75):

[0569] DIQMTQSPSSLSASVGDRVTITCQASQDINNYIAWYQHKPGKGPKLLIHYTSTLQPGIPSRFSGSGSGRDYTLTISSLQPEDFATYYCLQYDNLLFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0570] Anti-CD4 T023200008 Nb sequence (SEQ ID NO: 76)

[0571] CDR1, CDR2 and CDR3 are underlined, based on IMGT nomenclature:

[0572] EVQLVESGGGSVQPGGSLTLSCGTS GRTFNV MGWFRQAPGKEREFVAA VRWSSTGIYY TQYADSVKSRFTISRDNAKNTVYLEMNSLKPEDTAVYY CAADTYNSNPAR WDGYDFRGQGTLVTVSSGGCGGHHHHHH

[0573] Sequence of anti-CD8 BDSn Nb (SEQ ID NO: 77)

[0574] CDR1, CDR2 and CDR3 are underlined, based on IMGT nomenclature:

[0575] EVQLVESGGGLVQAGGSLRLSCAAS GSTFSYG VGWFRQAPGKGREFVAD IDWNGEHT SYADSVKGRFATSRDNAKNTAYLQMNSLKPEDTAVYYC AADALPYTVRKYNY WGQGTQVTVSSGGCGGHHHHHH

[0576] Sequence of anti-CD3 T0170117G03-A Nb (SEQ ID NO: 78) EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNIMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSAGGGSGGHHHHHHC

[0577] Sequence of anti-CD3 T0170060E11 Nb (SEQ ID NO: 79) EVQLVESGGGLVQPGGSLRLSCAASGDIYKSFDMGWYRQAPGKQRDLVAVIGSRGNNRGRTNYADSVKGRFTISRDGTGNTVYLLMNKLRPEDTAIYYCNTAPLVAGRPWGRGTLVTVSSGGGSGGHHHHHHC

[0578] Anti-CD7 V1 Nb sequence (SEQ ID NO: 80) DVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDTAMYYCAARFGPMGCVDLSTLSFGHWGQGTQVTVSITGGGCHHHHHHHH

[0579] Anti-TCR T017000700 Nb sequence (SEQ ID NO: 81)

[0580] CDR1, CDR2, and CDR3 are underlined, based on the IMGT name.

[0581] EVQLVESGGGVVQPGGSLRLSCVAS GYVHKINF YGWYRQAPGKEREKVAH ISIGDQT DYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYC RALSRIWPYDY WGQGTLVTVSSGGCGGHHHHHH

[0582] Anti-CD2828CD065G01 Nb sequence (SEQ ID NO: 82) EVQLVESGGGLVQPGGSLRLSCAASGSIFRLHTMEWYRRTPETQREWVATITSGGTTNYPDSVKGRFTISRDDTKKTVYLQMNSLKPEDTAVYYCHAVATEDAGFPPSNYWGQGTLVTVSSGGCGGHHHHHHH

[0583] Anti-CD3 T0170061C09 Nb sequence (SEQ ID NO: 83)

[0584] EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGREREFVAAIVWSDGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTLVTVSSGGCGGHHHHHHH

[0585] Anti-CD312D2 bDS Fab sequence

[0586] 12D2 bDS HC (SEQ ID NO: 84):

[0587] EVKLVESGGGLVQPGRSLRLSCAASGFNFYAYWMGWVRQAPGKGLEWIGEIKKDGTTINYTPSLKDRFTISRDNAQNTLYLQMTKLGSEDTALYYCAREERDGYFDYWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0588] 12D2 bDS LC (SEQ ID NO: 85):

[0589] QFVLTQPNSVSTNLGSTVKLSCKRSTGNIGSNYVNWYQQHEGRSPTTMIYRDDKRPDGVPDRFSGSIDRSSNSALLTINNVQTEDEADYFCQSYSSGIVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAACSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAESS

[0590] Anti-CD28 8G8A Fab sequence

[0591] 8G8A bDS HC (SEQ ID NO: 86):

[0592] EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSLTSEDSALYCARWGDGNYWGRGTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0593] 8G8A bDS LC (SEQ ID NO: 87):

[0594] DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISMEAEDAATYFCHQYHRSPTFGGGTKLETKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0595] Anti-CD28 2E12 Fab sequence

[0596] 2E12 bDS HC (SEQ ID NO: 88):

[0597] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSITKDNSKSQVFLKMNSLQTDDTARYYCARDGYSNFHYYVMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0598] 2E12 bDS LC (SEQ ID NO: 89):

[0599] DIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLISAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0600] Anti-CD28 CD28.9.3 Fab sequence

[0601] CD28.9.3 bDS HC (SEQ ID NO: 90):

[0602] QVKLQQSGPGLVTPSQSLSITCTVSGFSLSDYGVHWVRQSPGQGLEWLGVIWAGGGTNYNSALMSRKSISKDNSKSQVFLKMNSLQADDTAVYYCARDKGYSYYYSMDYW

[0603] GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0604] CD28.9.3 bDS LC (SEQ ID NO: 91):

[0605] DIVLTQSPASLAVSLGQRATISCRASESVEYYVTSLMQWYQQKPGQPPKLLIFAASNVESGVPARFSGSGSGTNFSLNIHPVDEDDVAMYFCQQSRKVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0606] Sequence of anti-CD28 HzTN228 Fab

[0607] HzTN228 bDS HC (SEQ ID NO: 92):

[0608] QVQLQESGPGLVKPSETLSLTCAVSGFSLTSYGVHWIRQPGKGLEWLGVIWPGTNFNSALMSRLTISEDTSKNQVSLKLSSVTAADTAVYCARDRAYGNYLYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0609] HzTN228 bDS LC (SEQ ID NO: 93):

[0610] DIQMTQSPSLSASVGDRVTITCRASESVEYVTSLMQWYQKPGKAPKLLIYAASNVDSGVPSRFSGSGTDFTLTISLQPEDIATYCQSRKVPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0611] Anti-CD28 TGN2122.C Fab Sequence

[0612] TGN2122.C bDS HC (SEQ ID NO: 94):

[0613] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYKIHWVRQAPGQGLEWIGYIYPYSGSSDYNQKFKSRATLTVDNSISTAYMELSRLRSDDTAVYYCARGGDAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0614] TGN2122.C bDS LC (SEQ ID NO: 95):

[0615] DIQMTQSPSSLSASVGDRVTITCGASENIYGALNWYQRKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTLTISSLQPEDFATYFCQNILGTWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0616] Anti-CD28 TGN2122.H Fab Sequence

[0617] TGN2122.H bDS HC (SEQ ID NO: 96):

[0618] EVQLVESGGGLVQPGGSLRLSCAASGFTFNIYYMSWVRQAPGKGLELVAAINPDGGNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARYGGPGFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0619] TGN2122.H bDS LC (SEQ ID NO: 97):

[0620] ENVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLWIYDTSKLASGIPARFSGSGSRNDYTLTISSLEPEDFAVYYCFPGSGFPFMYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES

[0621] Anti-CD8 TRX2 ScFv sequence (SEQ ID NO: 98):

[0622] QVQLVESGGGVVQPGRSLRLSCAASGFTFSDFGMNWVRQAPGKGLEWVALIYYDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPHYDGYYHFFDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKGSQDINNYLAWYQQKPGKAPKLLIYNTDILHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCYQYNNGYTFGQGTKVEIKGGGSGGCGGHHHHHH

[0623] V1 VHH-CH1 bDS HC (SEQ ID NO: 99):

[0624] DVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDTAMYYCAARFGPMGCVDLSTLSFGHWGQGTQV TVSITASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCGGHHHHHH

[0625] In some embodiments, the targeting portion comprises a polypeptide sequence as disclosed herein. In some embodiments, the targeting portion comprises all six CDRs of the polypeptide sequence as disclosed herein. In some embodiments, the targeting portion comprises CDR1, CDR2, and CDR3 of an immunoglobulin single variable domain (ISVD) as disclosed herein. In another embodiment, the targeting portion binds to the same epitope on the target molecule that the polypeptide sequence as disclosed herein binds to. In another embodiment, the targeting portion competes with the polypeptide sequence as disclosed herein for binding to the same epitope on the target molecule.

[0626] In some embodiments, the targeting group or immune cell targeting group (e.g., T cell target, B cell target, or NK cell target) may be covalently coupled to the lipid via a linker containing polyethylene glycol (PEG).

[0627] In other embodiments, the lipid used to generate the conjugate may be selected from distearate-phosphatidylethanolamine (DSPE):

[0628]

[0629] Dipalmitoylphosphatidylethanolamine (DPPE):

[0630]

[0631] Dimyristic phosphatidylethanolamine (DMPE):

[0632]

[0633] Distearatel-glycerol-glycerophosphate (DSPG):

[0634]

[0635] Dimyristic glycerol (DMG):

[0636]

[0637] Distearatelglycerol (DSG):

[0638] and

[0639] N-Palmitoyl-Sphingosine (C16-Ceramide)

[0640]

[0641] The immune cell targeting group can be covalently coupled to lipids directly or via a linker (e.g., a linker containing polyethylene glycol (PEG)). In some embodiments, the PEG is PEG 1000, PEG 2000, PEG 3400, PEG 3000, PEG 3450, PEG 4000, or PEG 5000. In some embodiments, the PEG is PEG 2000.

[0642] In some embodiments, the lipid-immune cell-targeting group conjugate is present in the lipid blend in the range of 0.001-0.5 mol%, 0.001-0.3 mol%, 0.002-0.2 mol%, 0.01-0.1 mol%, 0.1-0.3 mol%, or 0.1-0.2 mol%. The lipid-immune cell-targeting conjugate may be present in the lipid blend in the range of 0.001-0.5 mol%, 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%, 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%.

[0643] In some embodiments, the lipid-immune cell-targeting conjugate comprises DSPE, a PEG component, and a targeting antibody. In some embodiments, the antibody is a T-cell target, such as an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD7 antibody, an anti-CD8 antibody, or an anti-TCR β antibody.

[0644] Exemplary lipid-immune cell-targeting conjugates comprise DSPE and PEG 2000, as described, for example, in Ellis et al. (2005) BIOTECHNOL. PROG. [Progress in Biotechnology] 21, 205-220. Exemplary conjugates comprise a structure having formula (III), where scFv represents an engineered antibody binding site that binds to a target. In some embodiments, the engineered antibody binding site binds to any of the targets described above. In some embodiments, the engineered antibody binding site may be, for example, an engineered anti-CD3 antibody or an engineered anti-CD8 antibody. In some embodiments, the engineered antibody binding site may be, for example, an engineered anti-CD2 antibody or an engineered anti-CD7 antibody.

[0645] In some embodiments, the lipid-immune cell-targeting conjugate comprises DBPE or DAPE, a PEG component, and a targeting antibody. In some embodiments, the antibody is a T-cell target, such as an anti-CD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD7 antibody, an anti-CD8 antibody, or an anti-TCR β antibody.

[0646] In some embodiments, the lipid-immune cell targeting conjugate comprises DBPE or DAPE and PEG components, as shown below:

[0647]

[0648] DBPE-PEG2000-Mal;

[0649]

[0650] DAPE-PEG2000-Mal.

[0651] Examples of compounds having formula (III) (SEQ ID NO: 160) are shown below:

[0652] (III).

[0653] It is envisioned that the scFv in equation (III) can be replaced by a complete antibody or its antigen fragment (e.g., Fab).

[0654] Another example of a compound having formula (IV) (SEQ ID NO: 161) is shown below:

[0655]

[0656] Its generation is described in Nellis et al. (2005), ibid., or in U.S. Patent No. 7,022,336. It is envisioned that Fab in Formula (IV) can be replaced by an intact antibody or its antigen fragment (e.g., (Fab')2 fragment) or an engineered antibody binding site (e.g., scFv).

[0657] Other lipid-immune cell targeting group conjugates are described, for example, in U.S. Patent No. 7,022,336, wherein the targeting group can be replaced by a target targeting group (e.g., a targeting group that binds to T cell or NK cell surface antigens as described above).

[0658] In some embodiments, the lipid component of the exemplary conjugate having formula (II) can be any lipid described herein. In some instances, the lipid component of the conjugate having formula (II) is based on an ionizable cationic lipid described herein, such as an ionizable cationic lipid of formula (AI), (A-II), (BI), (B-II), (CI), (C-II), (C-III), (C-IV), (DI), (D-II), (E-1), or (E-II) or a salt thereof. For example, the exemplary ionizable cationic lipid can be selected from any one of Tables A, B, C, D, or E or a salt thereof.

[0659] In some embodiments, lipid conjugates based on the present disclosure may include:

[0660] scFv represents an engineered antibody binding site that binds to the targets described above (e.g., CD2, CD3, CD7, or CD8).

[0661] In some embodiments, the lipid blend may further comprise free PEG-lipids, for example, to reduce nonspecific binding via the targeting group. The free PEG-lipids may be the same as or different from the PEG-lipids included in the conjugate. In some embodiments, the free PEG-lipid is selected from the group consisting of: PEG-disteayl-phosphatidylethanolamine (PEG-DSPE) or PEG-myristoyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE-PEG), 1,2-dimyristoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DPG), 1,2-dioleoyl-rac-glycerol, methoxy polyethylene glycol (DOG-PEG), 1,2-disteayl-rac-glycerol-3-methylpolyoxyethylene (PEG-DSG), N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), DSPE-PEG-cysteine ​​or derivatives thereof. In some embodiments, the average PEG length is between 2000 and 5000, having lengths of 2000, 3400, or 5000. The final composition may contain a mixture of two or more of these PEGylated lipids. In some embodiments, the LNP composition comprises a mixture of PEG-lipids having myristoyl and stearyl chains. In some embodiments, the LNP composition comprises a mixture of PEG-lipids having palmitoyl and stearyl chains.

[0662] In some embodiments, the PEG-lipid derivatives have methoxy, hydroxy, or carboxylic acid end groups at the PEG ends.

[0663] The lipid-immune cell targeting group conjugate can be incorporated into LNPs as described below, for example, into LNPs containing, for example, ionizable cationic lipids, sterols, neutral phospholipids, and PEG-lipids. It is envisioned that in some embodiments, the LNP containing the lipid-immune cell targeting group may contain the ionizable cationic lipids described herein, or those described in, for example, U.S. Patent Nos. 10,221,127, 10,653,780, or U.S. Publication Nos. US 2018 / 0085474, US 2016 / 0317676, International Publication No. WO 2009 / 086558, or Miao et al. (2019) NATURE BIOTECH 37:1174-1185, or Jayaraman et al. (2012) ANGEW CHEM INT. 51: 8529-8533.

[0664] The LNP can be assigned using the methods described in the following sections and other group assignments.

[0665] IV. Lipid Nanoparticle Composition

[0666] This invention provides lipid nanoparticle (LNP) compositions comprising lipid blends containing ionizable cationic lipids and / or lipid-immune cell targeting conjugates as described herein. In some embodiments, the lipid blend may contain one or more of the ionizable cationic lipids described herein, as well as sterols, neutral phospholipids, PEG-lipids, and lipid-immune cell targeting group conjugates.

[0667] In some embodiments, the ionizable cationic lipids described herein may be present in the lipid blend in the range of 30-70 mol%, 30-60 mol%, 30-50 mol%, 40-70 mol%, 40-60 mol%, 40-50 mol%, 50-70 mol%, 50-60 mol%, or about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%.

[0668] Sterols

[0669] In some embodiments, the lipid blend of the lipid nanoparticles may include a sterol component, such as one or more sterols selected from the group consisting of: cholesterol, fucosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, stigmasterol, and rapeseed sterol. In some embodiments, the sterol is cholesterol.

[0670] The sterol (e.g., cholesterol) may be present in the lipid blend in the range of 20-70 mol%, 20-60 mol%, 20-50 mol%, 30-70 mol%, 30-60 mol%, 30-50 mol%, 40-70 mol%, 40-60 mol%, 40-50 mol%, 50-70 mol%, 50-60 mol%, or about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, or about 65 mol%.

[0671] In some embodiments, the sterol is present in the lipid blend in the range of 30-50 molar percentage. In some embodiments, the sterol is present in the lipid blend in the range of 20-70 molar percentage.

[0672] neutral phospholipids

[0673] In some embodiments, the lipid blend of the lipid nanoparticles may comprise one or more neutral phospholipids. The neutral phospholipid may be selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin (SM).

[0674] Other neutral phospholipids can be selected from the following groups: distearyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphocholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), dioleoyl-glycerol-phosphoethanolamine (DOPE), dilinoleoyl-glycerol-phosphocholine (DLPC), dimyristoyl-glycerol-phosphocholine (DMPC), dioleoyl-glycerol-phosphocholine (DOPC), dipalmitoyl-glycerol-phosphocholine (DPPC), docosanoyl-glycerol-phosphocholine (DUPC), palmitoyl-oleoyl-glycerol-phosphocholine (POPC), octadecenyl-glycerol-phosphocholine, oleoyl-cholesterolyl-hemisuccinoyl-glycerol-phosphocholine, hexadecyl-glycerol-phosphocholine, dilinonyl-glycerol-phosphocholine, arachidonicoyl-glycerol-3-phosphocholine, bis(docosahexaenoic)-glycerol-phosphocholine, and sphingomyelin.

[0675] The neutral phospholipid may be present in the lipid blend in the range of 1-10 mol%, 1-15 mol%, 1-12 mol%, 1-10 mol%, 3-15 mol%, 3-12 mol%, 3-10 mol%, 4-15 mol%, 4-12 mol%, 4-10 mol%, 4-8 mol%, 5-15 mol%, 5-12 mol%, 5-10 mol%, 6-15 mol%, 6-12 mol%, 6-10 mol%, or about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%.

[0676] In some embodiments, the neutral phospholipid is present in the lipid blend in the range of 1-15 molar percentage (e.g., about 5-15 molar percentage or about 5 to 10 molar percentage).

[0677] PEG-lipids

[0678] The lipid blend of the lipid nanoparticles may include one or more PEG or PEG-modified lipids. Such a class may alternatively be referred to as polyethylene glycol-modified lipids. PEG lipids are lipids modified with polyethylene glycol. As noted above, when lipid-immune cell targeting groups are included in the lipid blend, free PEG-lipids may be included in the lipid blend to reduce or eliminate nonspecific binding via the targeting groups. In some embodiments, the free PEG-lipids are a mixture of two or more distinct free PEG-lipids.

[0679] PEG lipids (e.g., free PEG-lipids) may be selected from the non-restrictive group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol. For example, PEG lipids can be PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearate glycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG and PEG-DSG), PEG-ceramide, PEG-distearate glycerol-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycerol-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide and PEG-distearate phosphatidylethanolamine (PEG-DSPE) lipids. In some embodiments, the free PEG-lipid comprises diacylphosphatidylethanolamine, dialkylphosphatidylethanolamine, diacylglycerol, ceramide, dialkylglycerol, or dialkylacetamide. In some embodiments, the alkyl chain is myristic acid, palmitic acid, oleic acid, linoleic acid, or stearic acid. In some embodiments, the free PEG-lipid is DMG-PEG. In some embodiments, the free PEG-lipid is DPG-PEG.

[0680] In some embodiments, the blend may contain free PEG-lipids selected from the group consisting of PEG-distearylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG, and PEG-DSG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-distearyl-phosphatidylethanolamine (PEG-DSPE), and PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE). In some embodiments, the free PEG-lipid contains diacylphosphatidylcholine comprising a dipalmitoyl (C16) chain or a distearyl (C18) chain.

[0681] PEG-lipids may be present in the lipid blend in the range of 1-10 mol%, 1-8 mol%, 1-7 mol%, 1-6 mol%, 1-5 mol%, 1-4 mol%, 1-3 mol%, 2-8 mol%, 2-7 mol%, 2-6 mol%, 2-5 mol%, 2-4 mol%, 2-3 mol%, or about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, or about 5 mol%. In some embodiments, the free PEG-lipids are present in the lipid blend in the range of 1-4 mol% (e.g., about 1-2 mol%, about 2-4 mol%, or about 1.5 mol%). In some embodiments, the free PEG-lipids are present in the lipid blend in the range of about 0.1-4 mol% (e.g., about 0.5 to 2.5 mol%, or about 1 to 2 mol%). In some embodiments, the PEG-lipids are free PEG-lipids.

[0682] In some embodiments, the PEG-lipid may be present in the lipid blend in the range of 0.01-10 mol%, 0.01-5 mol%, 0.01-4 mol%, 0.01-3 mol%, 0.01-2 mol%, 0.01-1 mol%, 0.1-10 mol%, 0.1-5 mol%, 0.1-4 mol%, 0.1-3 mol%, 0.1-2 mol%, 0.1-1 mol%, 0.5-10 mol%, 0.5-5 mol%, 0.5-4 mol%, 0.5-3 mol%, 0.5-2 mol%, 0.5-1 mol%, 1-2 mol%, 3-4 mol%, 4-5 mol%, 5-6 mol%, or 1.25-1.75 mol%. In some embodiments, the PET-lipid may be about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, or about 5.5 mol% of the lipid blend. In some embodiments, the PEG-lipid is free PEG-lipid.

[0683] In some embodiments, the lipid anchor length of the PEG-lipid is C14 (as in PEG-DMG). In some embodiments, the lipid anchor length of the PEG-lipid is C16 (as in DPG). In some embodiments, the lipid anchor length of the PEG-lipid is C18 (as in PEG-DSG). In some embodiments, the backbone or head group of the PEG-lipid is diacylglycerol or ethanolamine phosphate. In some embodiments, the PEG-lipid is a free PEG-lipid.

[0684] The LNP disclosed herein may comprise one or more free PEG-lipids not conjugated to an immune cell targeting group, as well as PEG-lipids conjugated to an immune cell targeting group. In some embodiments, the free PEG-lipids may comprise lipids that are the same as or different from the lipids in the lipid-immune cell targeting group conjugate.

[0685] In some embodiments, the free PEG lipid comprises PEG having a molecular weight of at least 2000 Daltons. In some embodiments, the PEG has a molecular weight of about 3000 to 5000 Daltons. In some embodiments, the Fab is an anti-CD3 antibody, and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 2000 Daltons. In some embodiments, the Fab is an anti-CD4 antibody, and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 3000 to 3500 Daltons.

[0686] Generation of lipid nanoparticles

[0687] In some embodiments, the LNP is generated by rapid mixing via an orbital vortex mixer or by microfluidic mixing. Orbital vortex mixing is performed by rapidly adding an ethanolic solution of lipids to an aqueous solution of the target nucleic acid, followed immediately by vortexing at 2,500 rpm. In some embodiments, the LNP is generated using a microfluidic mixing step. In some embodiments, microfluidic mixing is achieved by mixing an aqueous and organic stream at a controlled flow rate in a microfluidic channel using, for example, a NanoAssemblr device and microfluidic chip (Precision Nanosystems, Vancouver, BC) featuring an optimized mixing chamber geometry. In some embodiments, the LNP is generated using a microfluidic mixing step that rapidly mixes an ethanolic lipid solution and an aqueous nucleic acid solution, encapsulating the nucleic acid within the solid lipid nanoparticles. The nanoparticle suspension is then buffer-exchanged into a total water buffer using a selected membrane filtration device for ethanol removal and nanoparticle maturation.

[0688] In some embodiments, the resulting LNP composition comprises a lipid blend containing, for example, one or more ionizable cationic lipids described herein from about 40 mol percent to about 60 mol percent, one or more sterols from about 35 mol percent to about 50 mol percent, one or more neutral lipids from about 5 mol percent to about 15 mol percent, and one or more PEG-lipids from about 0.5 mol percent to about 5 mol percent.

[0689] In some embodiments, the resulting LNP composition comprises a lipid blend containing, for example, one or more ionizable cationic lipids described herein from about 20 mol percent to about 60 mol percent, one or more sterols from about 25 mol percent to about 50 mol percent, one or more neutral lipids from about 15 mol percent to about 60 mol percent, and one or more PEG-lipids from about 0 mol percent to about 5 mol percent.

[0690] In some embodiments, the resulting LNP composition comprises a lipid blend containing, for example, one or more ionizable cationic lipids described herein from about 30 mol percent to about 40 mol percent, one or more sterols from about 20 mol percent to about 40 mol percent, one or more neutral lipids from about 30 mol percent to about 50 mol percent, and one or more PEG-lipids from about 0 mol percent to about 5 mol percent.

[0691] In some embodiments, the resulting LNP composition comprises a lipid blend containing, for example, one or more ionizable cationic lipids described herein from about 30 mol percent to about 40 mol percent, one or more sterols from about 20 mol percent to about 40 mol percent, one or more neutral lipids from about 30 mol percent to about 50 mol percent, and one or more PEG-lipids from about 0 mol percent to about 5 mol percent.

[0692] In some embodiments, the resulting LNP composition comprises a lipid blend comprising, for example, one or more ionizable cationic lipids described herein from about 30 mol percent to about 40 mol percent, one or more sterols from about 20 mol percent to about 40 mol percent, one or more neutral lipids from about 30 mol percent to about 50 mol percent, one or more PEG-lipids from about 0 mol percent to about 5 mol percent, PEG-targeting lipids from about 0.01 to 0.5 mol percent, and PEG click handles (i.e., azides, DBCO-azides, and Tz-TCO) from 0 to 0.5 mol percent.

[0693] In some embodiments, the resulting LNP composition comprises a lipid blend comprising, for example, one or more ionizable cationic lipids described herein from about 30 mol percent to about 40 mol percent, one or more sterols from about 20 mol percent to about 40 mol percent, one or more neutral lipids from about 30 mol percent to about 50 mol percent, and one or more PEG-lipids from about 0 mol percent to about 5 mol percent, about 0.03 to 0.05 PEG-targeted lipids, or about 0.1 to 0.2 PEG clickers (i.e., azides, DBCO-azides, and Tz-TCO).

[0694] In some embodiments, the resulting LNP composition comprises a lipid blend comprising, for example, from about 30 mol percent to about 40 mol percent of one or more ionizable cationic lipids described herein, from about 20 mol percent to about 40 mol percent of one or more sterols, from about 30 mol percent to about 50 mol percent of one or more neutral lipids, from about 0 mol percent to about 5 mol percent of one or more DSPE PEG FABs, and about 0.03 to 0.05 or about 0.1 to 0.2 mol percent of DSPE PEG azides.

[0695] In some embodiments, the resulting LNP composition comprises ionizable lipids: structural lipids: sterol lipids: DSG-PEG2000:DSPE-PEG2000-maleimide in a ratio of 58:7:33.5:1.4:0.1 or 58:10:30.5:1.4:0.1.

[0696] In some embodiments, the resulting LNP composition comprises ionizable lipids: structural lipids: sterol lipids: DPG-PEG2000:DSPE-PEG2000-maleimide in a ratio of 58:7:33.5:1.4:0.1 or 58:10:30.5:1.4:0.1.

[0697] In some embodiments, the resulting LNP composition comprises ionizable lipids: structural lipids: sterol lipids: PEG-lipids: DSPE PEG FAB: DSPE PEG azide, in molar ratios of approximately 33.0: 39.9: 25.4: 1.54: 0.05: 0.11.

[0698] In some embodiments, the resulting LNP composition comprises ionizable lipids: structural lipids: sterol lipids: PEG-lipids: DSPE PEG FAB: DSPE PEG azide, in a molar ratio of approximately 33.5: 40.5: 25.8: 0.0: 0.05: 0.11.

[0699] In some embodiments, the lipids containing the target moiety are present at less than 2 mol%.

[0700] In some embodiments, the lipids containing the targeting portion are present at less than 1.5 mol%.

[0701] In some embodiments, the lipids containing the target moiety are present at less than 1.0 mol%.

[0702] In some embodiments, the lipids containing the targeting portion are present at less than 0.5 mol%.

[0703] Physical properties of lipid nanoparticles

[0704] The characteristics of the LNP composition can depend on the components contained in the lipid nanoparticle (LNP) composition and their absolute or relative amounts. The characteristics can also vary depending on the preparation method and conditions of the LNP composition.

[0705] LNP compositions can be characterized using a variety of methods. For example, microscopic examination (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the LNP composition. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size.

[0706] Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure various characteristics of LNP compositions, such as particle size, polydispersity index, and zeta potential. RNA encapsulation efficiency is determined by a combination of RNA-binding dyes (ribogreen and cybergreen used to determine the proportion of RNA accessible to the dye) and LNP de-formulation, followed by HPLC analysis of the total RNA content.

[0707] In some embodiments, the LNP has an average diameter in the range of 1-250 nm, 1-200 nm, 1-150 nm, 1-100 nm, 50-250 nm, 50-200 nm, 50-150 nm, 50-100 nm, 75-250 nm, 75-200 nm, 75-150 nm, 75-100 nm, 100-250 nm, 100-200 nm, and 100-150 nm. In some embodiments, the LNP composition may have an average diameter of about 1 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. In some embodiments, the LNP has an average diameter of about 100 nm. In some embodiments, the LNP has an average diameter in the range of 50-200 nm.

[0708] In some embodiments, LNPs containing the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, showed an average diameter change of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% after freeze-thaw. In some embodiments, LNPs containing the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, showed an average diameter change of less than 30% after freeze-thaw. In some embodiments, freeze-thaw and diameter measurements were performed using 10% sucrose in MES pH 6.5 buffer.

[0709] In some embodiments, LNPs containing ionizable cationic lipids prepared and characterized using the methods described herein showed a mean diameter change of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% after insertion of the targeting antibody. In some embodiments, LNPs containing ionizable cationic lipids prepared and characterized using the methods described herein showed a mean diameter change of less than 15% after insertion of the targeting antibody. In some embodiments, the diameter change after insertion of the targeting antibody was measured in a MES at pH 6.5 after incubation at 37°C for 4 hours.

[0710] In some embodiments, LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have an average LNP diameter of less than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. In some embodiments, LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have an average LNP diameter of less than 100 nm.

[0711] Alternatively or additionally, the LNP composition may have a polydispersity index in the range of 0.05-1, 0.05-0.75, 0.05-0.5, 0.05-0.4, 0.05-0.3, 0.05-0.2, 0.08-1, 0.08-0.75, 0.08-0.5, 0.08-0.4, 0.08-0.3, 0.08-0.2, 0.1-1, 0.1-0.75, 0.1-0.5, 0.1-0.4, 0.1-0.3, or 0.1-0.2. In some embodiments, the polydispersity index is in the range of 0.1-0.25, 0.1-0.2, 0.1-0.19, 0.1-0.18, 0.1-0.17, 0.1-0.16, or 0.1-0.15. In some embodiments, the LNP has a polydispersity index in the range of 0.05 to 1.

[0712] In some embodiments, the LNP composition or LNP comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, has a polydispersity of less than 0.4, 0.3, 0.25, 0.2, 0.15, 0.1, or 0.05. In some embodiments, the LNP comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, has a polydispersity of less than 0.25.

[0713] Alternatively or additionally, the LNP composition may have a zeta potential of about -30 mV to about +30 mV. In some embodiments, the LNP composition has a zeta potential of about -10 mV to about +20 mV. The zeta potential may vary with pH. Thus, in some embodiments, the LNP composition may have a zeta potential of about 0 mV to about +30 mV, or about +10 mV to about +30 mV, or about +20 mV to about +30 mV at pH 5.5 or pH 5, and / or may have a zeta potential of about -30 mV to about +5 mV, or about -20 mV to about +15 mV at pH 7.4. In some embodiments, the LNP has a zeta potential of from about +5 mV to about +50 mV at pH 5 (e.g., from about +10 mV to about +30 mV at pH 5). In some embodiments, the LNP has a zeta potential of from about -10 mV to about +10 mV at pH 7.4.

[0714] In some embodiments, LNP compositions or LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have a zeta potential greater than -10, -9, -8, -7, -6, -5.5, -5, -4.5, -4, -3.5, -3, -2.5, -2, -1.5, -1, or -0.5 mV at pH 7.4. In some embodiments, LNP compositions or LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have a zeta potential greater than -10 mV at pH 7.4. In some embodiments, LNP compositions or LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have a zeta potential greater than -1 mV at pH 7.4. In some embodiments, LNP compositions or LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have a zeta potential greater than -1, 0, 1, 2, 3, 4, 4.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 mV at pH 5.5. In some embodiments, LNP compositions or LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have a zeta potential greater than 5 mV at pH 5.5. In some embodiments, LNP compositions or LNPs comprising the ionizable cationic lipids described herein, prepared and characterized using the methods described herein, have a zeta potential greater than 15 mV at pH 5.5.

[0715] V. Payload

[0716] LNP compositions may contain agents, such as nucleic acid molecules, for delivery to cells (e.g., immune cells) or tissues (e.g., cells or tissues of a subject). In some embodiments, the number of nucleotides in the nucleic acid ranges from about 400 to about 6000.

[0717] The LNP compositions of the present invention may include nucleic acids, such as DNA or RNA, such as mRNA, tRNA, microRNA, siRNA, gRNA (guide RNA), circRNA (circular RNA), ribozymes, decoy RNA, or dicer substrate siRNA. In some embodiments, the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine. In some embodiments, the mRNA encodes a polypeptide capable of regulating an immune response in immune cells. In some embodiments, the mRNA encodes a polypeptide capable of reprogramming immune cells. In some embodiments, the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR). In some embodiments, the CAR is TTR-023 anti-CD20 (Leu-16). In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the mRNA encoding the CAR comprises a 25-nucleotide sequence. TTR-023 anti-CD20 (Leu-16) CAR sequence (including leader sequence) (SEQ ID NO: 24):

[0718] METDTLLLWVLLLWVPGSTGDYKAKEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARS NYYGSSYWFFDVWGAGTTVTVSSGGGSGGGSGGGGSSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQ QWSFNPPTFGGGTKLEIKGGGGSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRSRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 24)

[0719] The corresponding nucleic acid sequence (SEQ ID NO: 25):

[0720]

[0721] It is envisioned that nucleic acids may contain naturally occurring components, such as naturally occurring bases, sugars, or linker groups (e.g., phosphodiester linker groups); or they may contain non-naturally occurring components or modifications (e.g., thioester linker groups). For example, the nucleic acid may be synthesized to contain bases, sugars, or linker modifications known to those skilled in the art. Furthermore, the nucleic acid may be linear or cyclic, or have any desired conformation. The LNP composition may include a variety of nucleic acid molecules (e.g., a variety of RNA molecules), which may be the same or different.

[0722] In some embodiments, the payload is mRNA. In some embodiments, a particular LNP composition may contain a number of mRNA molecules, which may be the same or different. In some embodiments, one or more LNP compositions comprising one or more different mRNAs may be combined with and / or simultaneously contacted with cells. It is envisioned that the mRNA may comprise one or more of stem-loop, chain-terminating nucleosides, polyA sequences, polyadenylation signals, and / or 5' cap structures. The mRNA may encode a receptor, such as a chimeric antigen receptor (CAR), for use in, for example, immune disorders, inflammatory disorders, or cancer. Additionally, the mRNA may encode an antigen for use in therapeutic or prophylactic vaccines, for example, for treating or preventing infection by pathogens (e.g., microbial or viral pathogens), or for reducing or improving side effects directly or indirectly caused by such infections.

[0723] In some embodiments, CAR is selected from:

[0724]

[0725] In some embodiments, CAR is selected from:

[0726]

[0727] In some embodiments, the LNP composition may include one or more other components, including but not limited to one or more pharmaceutically acceptable excipients, hydrophobic small molecules, therapeutic agents, carbohydrates, polymers, permeability-enhancing molecules, and surface modifiers.

[0728] In some embodiments, the wt / wt ratio of the lipid component to the payload (e.g., mRNA) in the resulting LNP composition is from about 1:1 to about 50:1. In some embodiments, the wt / wt ratio of the lipid component to the payload (e.g., mRNA) in the resulting composition is from about 5:1 to about 50:1. In some embodiments, the wt / wt ratio is from about 5:1 to about 40:1. In some embodiments, the wt / wt ratio is from about 10:1 to about 40:1. In some embodiments, the wt / wt ratio is from about 15:1 to about 25:1.

[0729] In some embodiments, the encapsulation efficiency of the payload (e.g., mRNA) in these lipid nanoparticles is at least 50%. In other embodiments, the encapsulation efficiency is at least 80%, at least 90%, or greater than 90%.

[0730] In some embodiments, LNPs containing ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit encapsulation efficiencies greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 99%. In some embodiments, LNPs containing ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit encapsulation efficiencies greater than 87.5%. In some embodiments, LNPs containing ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit dye-accessible RNA less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1%. In some embodiments, LNPs containing ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit dye-accessible RNA less than 12.5%.

[0731] In some embodiments, LNPs containing ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit total mRNA recoveries greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, LNPs containing ionizable cationic lipids described herein, prepared and characterized using the methods described herein, exhibit total mRNA recoveries greater than 80%.

[0732] RNA payload

[0733] In some embodiments, the RNA payload is an mRNA, tRNA, microRNA, or siRNA payload.

[0734] In some embodiments, the lipid nanoparticle composition is optimized for delivering RNA (e.g., mRNA) to target cells for translation within those cells. The mRNA may be naturally occurring or non-naturally occurring. The mRNA may include one or more modified nucleobases, nucleosides, or nucleotides.

[0735] The nucleobase may be selected from the non-restricted group consisting of: adenine, guanine, uracil, cytosine, 7-methylguanine, 5-methylcytosine, 5-hydroxymethylcytosine, thymine, pseudouracil, dihydrouracil, N1-methylpseudorazine, hypoxanthine, and xanthine. In some embodiments, the nucleobase is N1-methylpseudorazine.

[0736] Nucleosides of mRNA are compounds comprising a combination of a sugar molecule (e.g., a 5- or 6-carbon sugar, such as pentose, ribose, arabinose, xylose, glucose, galactose, or their deoxygenated derivatives) and a nucleobase. Nucleosides can be classical nucleosides (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine) or analogues thereof, and may include one or more substitutions or modifications.

[0737] The nucleotides of mRNA are compounds containing a nucleoside and a phosphate group or a substitutional group (e.g., borate phosphate, thiophosphate, selenophosphate, phosphonate, alkyl, amidation, and glycerol). The nucleotide can be a classical nucleotide (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine monophosphate) or analogs thereof, and may include one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused or open rings; oxidation and / or reduction of nucleobases, sugars, and / or phosphates or substitutional components. A nucleotide may include one or more phosphates or substitutional groups. For example, a nucleotide may include a nucleoside and a triphosphate group. "Nucleoside triphosphate" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) can refer to a classical nucleoside triphosphate or its analogs or derivatives, and may include one or more substitutions or modifications as described herein.

[0738] mRNA may include a 5' untranslated region, a 3' untranslated region, and / or a coding or translated sequence. mRNA may include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of nucleosides, nucleosides, or nucleotides may be classically substituted, modified, or otherwise non-naturally occurring analogs. In some embodiments, all of a particular nucleobase type may be modified. For example, all cytosines in the mRNA may be 5-methylcytosine. In some embodiments, one or more or all uridine bases may be N1-methylpseudouridine.

[0739] In some embodiments, the mRNA may include a 5' cap structure, a chain-terminating nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal.

[0740] A cap structure or cap type is a compound comprising two nucleoside moieties linked by a linker, and can be selected from naturally occurring caps, non-natural caps, or cap analogs. A cap type can include one or more modified nucleoside and / or linker moieties. For example, a natural mRNA cap can include a guanine nucleotide linked by a triphosphate bond at its 5' position and a guanine (G) nucleotide methylated at its 7' position, such as m7G(5')ppp(5')G, commonly written as m7GpppG. A cap type can also be an anti-reverse cap analog. A non-limiting list of possible cap types includes m7GpppG, m7Gpppm7G, m73'dGpppG, m7Gpppm7G, m73'dGpppG, and m2702'GppppG.

[0741] Alternatively or additionally, mRNA may include chain-terminating nucleosides. For example, chain-terminating nucleosides may include those deoxygenated at the 2' and / or 3' positions of their glycosyl groups. Such a class may include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymidine, and 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymidine.

[0742] Alternatively or additionally, mRNA may include stem loops, such as histone stem loops. A stem loop may include 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs. The stem loop can be located in any region of the mRNA. For example, the stem loop can be located in, before, or after an untranslated region (5' untranslated region or 3' untranslated region), a coding region, or a polyA sequence or tail.

[0743] Alternatively or additionally, the mRNA may include a polyA sequence and / or a polyadenylation signal. The polyA sequence may consist entirely or primarily of adenine nucleotides or their analogues or derivatives. The polyA sequence may be a tail located near the 3' untranslated region of the mRNA.

[0744] mRNA can encode any target polypeptide, including any naturally occurring or non-naturally occurring polypeptide or otherwise modified polypeptide. Polypeptides encoded by mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the polypeptide encoded by mRNA can have a therapeutic effect when expressed in cells. In some embodiments, mRNA can encode antibodies, enzymes, growth factors, hormones, cytokines, viral proteins (e.g., viral capsid proteins), antigens, vaccines, or receptors. In some embodiments, mRNA can encode engineered receptors (such as CARs) or antigens used in therapeutic vaccines (e.g., cancer vaccines) or prophylactic vaccines (e.g., vaccines used to minimize the risk or severity of infection by microbial or viral pathogens). In some embodiments, mRNA encodes polypeptides capable of modulating immune responses in immune cells. In some embodiments, mRNA encodes polypeptides capable of reprogramming immune cells. In some embodiments, mRNA encodes synthetic T-cell receptors (synTCRs) or chimeric antigen receptors (CARs).

[0745] Lipid compositions can be engineered for one or more specific applications or targets. For example, LNP compositions can be engineered to deliver mRNA to specific cells, tissues, organs, or systems or groups thereof in a mammalian body (e.g., the renal system). The physicochemical properties of LNP compositions can be modified to increase selectivity for specific target sites within the subject. For example, particle size can be adjusted based on the fenestration size of different organs. The mRNA included in the LNP composition can also be dependent on one or more desired delivery targets. For example, the mRNA can be selected for a specific indication, condition, disease, or disorder, and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery).

[0746] The amount of mRNA in a lipid composition can depend on the size, sequence, and / or other characteristics of the mRNA. The amount of mRNA in an LNP can also depend on the size, composition, desired target, and / or other characteristics of the LNP composition. The relative amounts of mRNA and other elements (e.g., lipids) can also vary. The amount of mRNA in an LNP composition can be measured, for example, using absorption spectroscopy (e.g., UV-Vis spectroscopy).

[0747] In some embodiments, one or more mRNAs, lipids, and polymers, and their amounts, can be selected to provide a specific N:P ratio (the ratio of positively charged lipid or polymeric amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups). The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the mRNA. Generally, a lower N:P ratio is preferred. The N:P ratio can depend on the specific lipid and its pKa. In some embodiments, the mRNA and LNP composition and / or their relative amounts can be selected to provide an N:P ratio from about 1:1 to about 30:1 or from about 1:1 to about 20:1. In some embodiments, the N:P ratio can be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. In some embodiments, the N:P ratio can be from about 2:1 to about 5:1. In some embodiments, the N:P ratio may be about 4:1. In other embodiments, the N:P ratio ranges from about 4:1 to about 8:1. For example, the N:P ratio may be about 4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, about 5.0:1, about 5.1:1, about 5.2:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.6:1, about 5.7:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0748] The amount of mRNA in the nanoparticle composition can depend on the size, sequence, and / or other characteristics of the mRNA. The amount of mRNA in the nanoparticle composition can also depend on the size, composition, desired target, and / or other characteristics of the nanoparticle composition. The relative amounts of mRNA and other elements (e.g., lipids) can also vary. In some embodiments, the wt / wt ratio of lipid components to mRNA in the nanoparticle composition can be from about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the wt / wt ratio of lipid components to mRNA can be from about 10:1 to about 40:1. The amount of mRNA in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-Vis spectroscopy).

[0749] The encapsulation efficiency of mRNA describes the amount of mRNA that is encapsulated or otherwise associated with the lipid composition after preparation, relative to the initial amount provided. Encapsulation efficiency is ideally high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of mRNA in a solution containing the lipid composition before and after degradation with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free mRNA in solution. For the LNP compositions of the present invention, the mRNA encapsulation efficiency can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%.

[0750] VI. Preparation and Delivery Methods

[0751] The LNP compositions of the present invention can be formulated, in whole or in part, into pharmaceutical compositions. Such pharmaceutical compositions may further comprise one or more pharmaceutically acceptable excipients or adjuvants, as described herein. General guidance for the formulation and manufacture of pharmaceutical compositions and agents can be obtained, for example, from Remington's (2006), ibid. Conventional excipients and adjuvants may be used in any pharmaceutical composition of the present invention, unless any conventional excipient or adjuvant may be incompatible with one or more components of the LNP composition of the present invention. An excipient or adjuvant may be incompatible with a component if its combination with a component of the LNP composition could result in any undesirable biological effect or otherwise harmful effect.

[0752] In some embodiments, one or more excipients or adjuvants may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the LNP composition of the present invention. For example, the one or more excipients or adjuvants may constitute 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. In some embodiments, the excipients are, for example, approved by the U.S. Food and Drug Administration for use in humans and for veterinary purposes. In some embodiments, the excipients are pharmaceutical grade. In some embodiments, the excipients conform to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0753] The relative amounts of one or more lipids or LNPs, one or more pharmaceutically acceptable excipients and / or any other ingredients in the pharmaceutical composition will vary depending on the identity, body size and / or condition of the subject to be treated and further depending on the route of administration of the composition.

[0754] Lipid compositions and / or pharmaceutical compositions comprising one or more LNP compositions may be administered to any subject, including human patients who may benefit from therapeutic effects provided by the delivery of nucleic acids, such as RNA (e.g., mRNA, tRNA, or siRNA), to one or more specific cells, tissues, organs, or systems or groups thereof (e.g., the renal system). While the descriptions of LNP compositions and pharmaceutical compositions comprising LNP compositions provided herein are primarily directed toward compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other mammal. It is understood that compositions suitable for administration to humans may be modified to make them suitable for administration to a variety of animals.

[0755] The pharmaceutical compositions disclosed herein may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient (e.g., payload).

[0756] The pharmaceutical compositions of the present invention can be prepared in various forms suitable for a variety of routes and methods of administration. For example, the pharmaceutical compositions of the present invention can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal application (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and patches), suspensions, powders, and other forms.

[0757] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitol, and mixtures thereof. Besides inert diluents, oral compositions may also include excipients such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or aromatizers.

[0758] Injectable formulations can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques, such as sterile injectable aqueous or oily suspensions. Sterile injectable formulations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic, parenteral-acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Acceptable media and solvents that can be used are, in particular, water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile non-volatile oils are routinely used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injections.

[0759] Injectable preparations can be sterilized, for example, by filtering through a bacterial retention filter and / or incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0760] Other components

[0761] In addition, it is envisioned that the pharmaceutical composition may include one or more components other than those described above.

[0762] The pharmaceutical composition may also include one or more permeability enhancer molecules, carbohydrates, polymers, therapeutic agents, surface modifiers, or other components. Permeability enhancer molecules may be those described, for example, in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogs).

[0763] The pharmaceutical composition may also contain surface modifiers, including, for example, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyloctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carboxycysteine, ipramone, mesna, ambroxol, sobrorilol, domiocol, letostan, stipronin, thiopronin, gelling agents, thymosin β4, alfa-chain enzyme, netticine, and erdosteine), and DNases (e.g., rhDNase). The surface modifiers may be placed within and / or on the surface of the composition described herein.

[0764] In addition to these components, pharmaceutical compositions containing the LNP compositions of the present invention may also include any substances that can be used in pharmaceutical compositions. For example, the pharmaceutical composition may include one or more pharmaceutically acceptable excipients or adjuvants, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersants, suspending agents, granulation agents, disintegrants, fillers, flow aids, liquid media, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other types. Excipients such as waxes, butters, colorants, coating agents, flavoring agents, and aromatizers may also be included. Pharmaceutically acceptable excipients are well known in the art (see, for example, Remington's [Remington] (2006) ibid.).

[0765] The dispersant may be selected from a non-limiting list of the following: potato starch, corn starch, cassava starch, sodium starch glycolate, clay, alginate, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crosspovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium dodecyl sulfate, quaternary ammonium compounds and / or combinations thereof.

[0766] Surfactants and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth gum, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers and carboxylated ethylene polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), and sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®]). 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80], polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene dodecyl ether [BRIJ® 45]). 30]), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC® F 68, POLOXAMER® 188, cetrimonium bromide, cetrimonium chloride, benzalkonium chloride, sodium docusate and / or combinations thereof.

[0767] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, α-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzyl alcohol, bromonitrile glycol, trimethylammonium bromophthalamide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, xylene, cresol, ethanol, glycerol, hexadiazine, imidazoline, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcoholic preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and / or phenethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deferoxamine mesylate, bromobrown trimethylammonium, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, metabisulfite, potassium sulfite, and potassium metabisulfite.

[0768] Examples of buffers include, but are not limited to, citrate buffers, acetate buffers, phosphate buffers, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lacturonate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, glycerol, aminosulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free water, isotonic saline, Ringer's solution, ethanol and / or combinations thereof.

[0769] In some embodiments, these lipid nanoparticle compositions and formulations thereof are suitable for intravenous, intramuscular, intradermal, subcutaneous, intraarterial, intratumoral, or inhalation administration. In some embodiments, doses of about 0.001 mg / kg to about 10 mg / kg are administered to the subject. The compositions according to this disclosure can be formulated in dosage units for ease of administration and dosage consistency. However, it should be understood that the total daily dose of the compositions disclosed will be determined by the attending physician within a reasonable medical judgment.

[0770] For any given patient, the appropriate dose level for a particular treatment, prevention, or otherwise (e.g., for imaging) will depend on a number of factors, including the severity and identity of the disorder being treated (if any); one or more mRNAs used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific drug composition used; the duration of treatment; drugs used in combination with or concurrently with the specific drug composition used; and similar factors well known in the medical field.

[0771] VII. Methods

[0772] This disclosure provides methods for delivering a payload to target cells or tissues (e.g., target cells or tissues of a subject) and LNPs or pharmaceutical compositions containing such LNPs for use in such methods. Any disclosure herein relating to methods such as treating a disease or disorder, or delivering nucleic acids to cells, or generating a target polypeptide in cells, should also be interpreted as a disclosure relating to LNPs or pharmaceutical compositions containing such LNPs for use in such methods.

[0773] In another aspect, this article provides a method for delivering a nucleic acid to an immune cell (e.g., a T cell), the method comprising exposing the immune cell to an LNP containing the nucleic acid as described herein, under conditions that allow the nucleic acid to enter the immune cell.

[0774] In another aspect, this article provides a method for delivering nucleic acids to the cells (e.g., immune cells, such as T cells) of a subject in need, the method comprising administering to the subject a composition containing an LNP containing nucleic acids as described herein, thereby delivering the nucleic acid to the immune cells.

[0775] In another aspect, this article provides a method for targeting the delivery of a nucleic acid (e.g., mRNA) to the cells (e.g., immune cells (e.g., T cells)) of a subject, the method comprising administering to the subject an LNP containing the nucleic acid as described herein to facilitate targeted delivery of the nucleic acid to the immune cells.

[0776] In some embodiments, the present invention provides a method for generating a target polypeptide (e.g., a target protein) in mammalian cells, and an LNP or pharmaceutical composition containing the LNP for use in such a method. The method for generating a polypeptide in such cells involves contacting the cells with an LNP composition containing a target RNA (e.g., mRNA encoding the target polypeptide (e.g., the target protein)). After contacting the cells with the LNP composition, the mRNA can be absorbed and translated within the cells to produce the target polypeptide.

[0777] Typically, the process of contacting mammalian cells with an LNP composition comprising mRNA encoding a target polypeptide can be performed in vivo, in vitro, or in vitro. The amount of LNP composition contacted with the cells and / or the amount of mRNA therein can depend on the type of cells or tissues contacted, the method of administration, the physicochemical characteristics of the LNP composition and the mRNA therein (e.g., size, charge, and chemical composition), and other factors. Generally, an effective amount of LNP composition will allow for efficient polypeptide production in the cells. Efficiency measures may include polypeptide translation (indicated by polypeptide expression), mRNA degradation levels, and indicators of immune response.

[0778] The step of contacting an LNP composition comprising mRNA with a cell can involve or cause transfection, wherein the LNP composition can fuse with the cell membrane to allow delivery of the mRNA into the cell. After introduction into the cell's cytoplasm, the mRNA is then translated into a protein or peptide via the cell's intracytoplasmic protein synthesis machinery.

[0779] In some embodiments, the LNP compositions described herein can be used to deliver therapeutic or preventative agents to a subject. For example, the mRNA included in the LNP composition may encode a polypeptide and produce a therapeutic or preventative polypeptide upon contact with and / or entry into (e.g., transfection) cells. In some embodiments, the mRNA included in the LNP compositions of the present invention may encode a polypeptide that can improve or increase the immunity of a subject.

[0780] In some embodiments, contacting cells with an LNP composition comprising mRNA can reduce the cell's innate immune response to exogenous nucleic acids. Cells may be contacted with a first LNP composition comprising a first amount of a first exogenous mRNA including a translatable region, and the level of the cell's innate immune response to this first exogenous mRNA can be determined. Subsequently, the cells may be contacted with a second composition comprising a second amount of the first exogenous mRNA, which is a smaller amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition may comprise a first amount of a second exogenous mRNA different from the first exogenous mRNA. The steps of contacting the cells with the first composition and the second composition may be repeated once or multiple times.

[0781] Additionally, the efficiency of peptide production in the cell can be optionally determined, and the cell can be repeatedly contacted with the first composition and / or the second composition until the target protein production efficiency is achieved.

[0782] This disclosure provides a method for delivering nucleic acids (e.g., mRNA) to mammalian cells or tissues (e.g., mammalian cells or tissues of a subject). Delivering mRNA to such cells or tissues involves administering an LNP composition comprising the mRNA to the subject, for example by injection (e.g., via intramuscular injection) or intravascular delivery. After administration, the LNP can target and / or contact cells, such as immune cells, like T cells. Upon contacting the cell with the LNP composition, the translateable mRNA can be translated within the cell to produce a target polypeptide.

[0783] In some embodiments, the LNP compositions of the present invention can target specific types or classes of cells. Such targeting can be facilitated using lipids described herein to form LNPs, which may also include a targeting group for targeting target cells. In some embodiments, specific delivery can result in a greater than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold increase in the amount of mRNA reaching the target target (e.g., cells expressing or expressing at high levels the target receptor that binds to the immune cell targeting group of the LNP) compared to other targets (e.g., cells that do not express or only express the target receptor at low levels).

[0784] The LNP compositions of the present invention can be used to treat diseases, disorders, or conditions characterized by the absence or abnormality of protein or polypeptide activity. Upon delivery of mRNA encoding the absent or abnormal polypeptide to a cell, translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the absence of the polypeptide or the abnormal activity caused by the polypeptide. Because translation can occur rapidly, the methods and compositions of the present invention can be used to treat acute diseases, disorders, or conditions such as sepsis, stroke, and myocardial infarction. The mRNA included in the LNP compositions of the present invention can also alter the transcription rate of a given species, thereby affecting gene expression.

[0785] The compositions applicable to this invention are characterized by diseases, disorders, and / or conditions involving dysfunction or abnormality of protein or peptide activity, including but not limited to cancer and proliferative disorders, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. Many diseases, disorders, and / or conditions may be characterized by a lack of protein activity (or a significant reduction, rendering the protein unable to perform its proper function). Such proteins may be absent, or they may be substantially nonfunctional. A specific example of a dysfunctional protein is a missense mutation variant of the cystic fibrosis transmembrane conduction regulator (CFTR) gene, which produces a dysfunctional variant of the CFTR protein, leading to cystic fibrosis. This disclosure provides a method for treating such diseases, disorders, and / or conditions in a subject by administering an LNP composition comprising an mRNA and a lipid component comprising KL10, phospholipids (optionally unsaturated), PEG lipids, and structural lipids, wherein the mRNA encodes a peptide that antagonizes or otherwise overcomes the abnormal protein activity present in the subject's cells.

[0786] The therapeutic and / or prophylactic compositions described herein may be administered to a subject in any reasonable amount and via any route of administration for the purpose of effectively preventing, treating, diagnosing, or imaging diseases, disorders, and / or conditions and / or any other purpose. The specific amount administered to a given subject may vary depending on the subject's species, age and general condition, the purpose of administration, the specific composition, the method of administration, etc. The compositions according to this disclosure may be formulated in dosage units to facilitate consistency of administration and dosage. However, it should be understood that the total daily dosage of the compositions disclosed herein will be determined by the attending physician within the bounds of reasonable medical judgment.

[0787] LNP compositions comprising one or more mRNAs can be administered via various routes, such as oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, percutaneous or intradermal, intradermal, rectal, vaginal, intraperitoneal, local, transmucosal, nasal, or intratumoral administration. In some embodiments, the LNP composition can be administered intravenously, intramuscularly, intradermally, intraarterially, intratumorally, or subcutaneously. However, this disclosure covers delivery of the LNP compositions of the present invention via any suitable route (given possible advances in pharmaceutical delivery science). Generally, the most suitable route of administration will depend on a variety of factors, including the nature of the LNP composition comprising one or more mRNAs (e.g., its stability in various bodily environments such as the bloodstream and gastrointestinal tract), the patient's condition (e.g., whether the patient can tolerate the particular route of administration), etc.

[0788] LNP compositions comprising one or more mRNAs may be used in combination with one or more other therapeutic, preventative, diagnostic, or imaging agents. The phrase “in combination with” is not intended to imply that the agents must be administered simultaneously and / or formulated for co-delivery, but such delivery methods are within the scope of this disclosure. For example, one or more LNP compositions comprising one or more different mRNAs may be administered in combination. The compositions may be administered simultaneously, before, or after one or more other desired therapeutic agents or medical procedures. Typically, each agent will be administered at a dose and / or schedule determined for that agent. In some embodiments, this disclosure covers the delivery of the compositions of the present invention or their imaging, diagnostic, or preventative compositions in combination with such agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution in the body.

[0789] It should be further understood that the combined therapeutic, preventative, diagnostic, or imaging active agents can be administered together in a single composition or individually in different compositions. Generally, it is desired that the combined agents be utilized at a level not exceeding that when used alone. In some embodiments, the level of combined utilization may be lower than that when used alone.

[0790] The specific combination of therapies (treatment agents or procedures) employed in a combination regimen will take into account the compatibility of the desired treatment agents and / or procedures and the desired therapeutic effect to be achieved. It should also be understood that the therapies employed may achieve the desired effect on the same obstacle (e.g., a composition that can be used to treat cancer may be administered concurrently with a chemotherapy agent), or they may achieve different effects (e.g., control of any adverse effects).

[0791] In some embodiments, no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 30%, no more than 35%, no more than 40%, no more than 45%, or no more than 50% of cells not intended to be the target of delivery are transfected with the LNP. In some embodiments, cells not intended to be the target of delivery are non-immune cells of the subject. In some embodiments, no more than 5% of non-immune cells are transfected with the LNP. In some embodiments, cells not intended to be the target of delivery are cells not targeted by the method. In some embodiments, cells not intended to be the target of delivery are subject cells not targeted by the method.

[0792] In some embodiments, the half-life of the nucleic acid delivered to the immune cell by the LNP described herein, or the peptide encoded by the nucleic acid delivered by the LNP and expressed in the immune cell, is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2 times, at least 3 times, at least 4 times, or at least 5 times longer than the half-life of the nucleic acid delivered by the LNP or the peptide encoded by the nucleic acid delivered by the LNP. In some embodiments, the half-life of the nucleic acid delivered by the LNP or the peptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by the reference LNP or the peptide encoded by the nucleic acid delivered by the reference LNP.

[0793] In some embodiments, the composition of the LNP differs from the composition of the reference LNP in the following aspects: the type of ionizable cationic lipid, the relative amount of ionizable cationic lipid, the length of the lipid anchor in the PEG lipid, the backbone or head group of the PEG lipid, the relative amount of the PEG lipid, or the type of immune cell targeting group, or any combination thereof. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only in the type of ionizable cationic lipid. In some embodiments, the composition of the LNP differs from the composition of the reference LNP only in the amount of PEG lipid. In some embodiments, the reference LNP contains the cationic lipid DLin-KC3-DMA, but is otherwise identical to the tested LNP. In some embodiments, the reference LNP contains the cationic lipid DLin-KC2-DMA, but is otherwise identical to the tested LNP. In some embodiments, the reference LNP contains the cationic lipid ALC-0315, but is otherwise identical to the tested LNP. In some embodiments, the reference LNP contains the cationic lipid SM-102, but is otherwise identical to the tested LNP. In some embodiments, the PEG lipid is a free PEG lipid.

[0794] In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of immune cells are transfected with the LNP. In some embodiments, at least 10% of immune cells are transfected with the LNP. In some embodiments, the immune cells are the subject's immune cells. In some embodiments, the immune cells are immune cells targeted by the method. In some embodiments, the immune cells are the subject's immune cells targeted by the method.

[0795] In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the expression level of the nucleic acid delivered by the reference LNP. In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by the reference LNP. In some embodiments, the expression level is measured and compared using the methods described herein. In some embodiments, the expression level is measured by the ratio of cells expressing the encoded polypeptide. In some embodiments, the expression level is measured using FACS. In some embodiments, the expression level is measured by the average amount of the encoded polypeptide expressed in cells. In some embodiments, the expression level is measured as average fluorescence intensity. In some embodiments, the expression level is measured by the amount of the encoded polypeptide or other material secreted by cells.

[0796] In another aspect, this article provides a method for targeting the delivery of nucleic acids to the immune cells of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise ionizable cationic lipids. In some embodiments, the LNPs comprise conjugates containing compounds of the following formula (II):

[0797] [Lipids] - [Optional Linker] - [Immune Cell Targeting Group].

[0798] In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises the nucleic acid.

[0799] In some embodiments, one aspect of this disclosure relates to an LNP or a pharmaceutical composition containing the LNP as disclosed herein for use in a method of delivering nucleic acid to the immune cells of a subject. Such a method can be used to treat a disease or disorder as disclosed herein. In some embodiments, the method as disclosed herein may include contacting the immune cells of a subject in vitro or ex vivo with lipid nanoparticles (LNPs). In some embodiments, the LNP in this disclosure is the LNP as described herein.

[0800] In some embodiments, the LNP provides at least one of the following benefits: (i) increased specificity for targeted delivery to the immune cell compared to a reference LNP;

[0801] (ii) The half-life of the nucleic acid or the polypeptide encoded by the nucleic acid is increased in the immune cell compared with the reference LNP;

[0802] (iii) The transfection rate increased compared to the reference LNP; and

[0803] (iv) Low levels of dye-accessible mRNA (< 15%) and high RNA encapsulation efficiency, wherein at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0804] In some aspects, methods are provided for expressing a target peptide in targeted immune cells of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise ionizable cationic lipids. In some embodiments, the LNPs comprise conjugates having a structure containing the following formula (II):

[0805] [Lipids] - [Optional Linker] - [Immune Cell Targeting Group].

[0806] In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises nucleic acids encoding the polypeptide. In some embodiments, one aspect of this disclosure relates to the use of an LNP or pharmaceutical composition containing the LNP as disclosed herein in a method for expressing a target polypeptide in targeted immune cells of a subject. Such a method can be used to treat diseases or disorders as disclosed herein. In some embodiments, the method as disclosed herein may include contacting the subject's immune cells in vitro or ex vivo with lipid nanoparticles (LNPs).

[0807] In some embodiments, LNPs provide at least one of the following benefits: (i) increased expression levels in the immune cell compared to a reference LNP;

[0808] (ii) Increased specificity of expression in the immune cell compared to the reference LNP; (iii) Increased half-life of the nucleic acid or the polypeptide encoded by the nucleic acid in the immune cell compared to the reference LNP.

[0809] (iv) Compared with the reference LNP, the transfection rate increased; and

[0810] (v) Low levels of dye-accessible mRNA (< 15%) and high RNA encapsulation efficiency, wherein at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0811] In some aspects, methods for modulating the cellular function of target immune cells in a subject are provided. In some embodiments, the method includes administering lipid nanoparticles (LNPs) to the subject. In some embodiments, the LNPs comprise ionizable cationic lipids. In some embodiments, the LNPs comprise conjugates having a structure containing the following formula (II):

[0812] [Lipids] - [Optional Linker] - [Immune Cell Targeting Group].

[0813] In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises nucleic acids encoding polypeptides for modulating the cellular function of the immune cells. In some embodiments, one aspect of this disclosure relates to the use of LNPs as disclosed herein or pharmaceutical compositions containing them in methods for modulating the cellular function of targeted immune cells in a subject. Such methods can be used to treat diseases or disorders as disclosed herein. In some embodiments, methods as disclosed herein may include contacting the subject's immune cells in vitro or ex vivo with lipid nanoparticles (LNPs).

[0814] In some embodiments, LNPs provide at least one of the following benefits: (i) increased expression levels in the immune cell compared to a reference LNP;

[0815] (ii) Increased specificity of expression in this immune cell compared to the reference LNP;

[0816] (iii) The half-life of the nucleic acid or the polypeptide encoded by the nucleic acid is increased in the immune cell compared with the reference LNP;

[0817] (iv) The transfection rate increased compared to the reference LNP;

[0818] (v) This LNP can be administered at a lower dose than the reference LNP to achieve the same biological effect in the immune cells; and

[0819] (vi) Low levels of dye-accessible mRNA (< 15%) and high RNA encapsulation efficiency, wherein at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0820] In some embodiments, the regulation of cell function includes reprogramming the immune cell to initiate an immune response. In some embodiments, the regulation of cell function includes modulating the antigen specificity of the immune cell.

[0821] In some aspects, methods are provided for treating, improving, or preventing symptoms of a disorder or disease in a subject of need. In any embodiment of the methods described herein for treating, improving, and / or preventing symptoms of a disorder or disease by administering, for example, an LNP of the present invention, this disclosure is also intended to be construed as providing, for example, an LNP for use in said methods for treating, improving, and / or preventing symptoms of a disorder or disease. In some embodiments, the method includes administering lipid nanoparticles (LNPs) to the subject to deliver nucleic acids to the subject's immune cells. In some embodiments, the LNP comprises an ionizable cationic lipid. In some embodiments, the LNP comprises a conjugate having a structure containing the following formula (II):

[0822] [Lipids] - [Optional Linker] - [Immune Cell Targeting Group].

[0823] In some embodiments, the LNP comprises sterols or other structural lipids. In some embodiments, the LNP comprises neutral phospholipids. In some embodiments, the LNP comprises free polyethylene glycol (PEG) lipids. In some embodiments, the LNP comprises the nucleic acid.

[0824] In some embodiments, nucleic acids modulate the immune response of the immune cells, thereby treating or improving the symptoms. In some embodiments, one aspect of this disclosure relates to the use of lipid nanoparticles (LNPs) or pharmaceutical compositions containing them as disclosed herein in methods of treating, improving, or preventing symptoms of a disorder or disease in a subject of need. The disease or disorder may be as disclosed herein. In some embodiments, methods as disclosed herein may include contacting the subject's immune cells in vitro or ex vivo with lipid nanoparticles (LNPs).

[0825] In some embodiments, the LNP provides at least one of the following benefits: (i) increased specificity of the nucleic acid delivery to the immune cell compared to a reference LNP;

[0826] (ii) The half-life of the nucleic acid or the polypeptide encoded by the nucleic acid is increased in the immune cell compared with the reference LNP;

[0827] (iii) The transfection rate increased compared to the reference LNP;

[0828] (iv) This LNP can be administered at a lower dose than the reference LNP to achieve the same therapeutic effect;

[0829] (v) Compared with the reference LNP, the level of immune cell functional gain was increased; and

[0830] (vi) Low levels of dye-accessible mRNA (< 15%) and high RNA encapsulation efficiency, wherein at least 80% of the mRNA is recovered in the final formulation relative to the total RNA used in LNP batch preparation.

[0831] In some embodiments, the barrier is an immune disorder, an inflammatory disorder, or cancer. In some embodiments, the nucleic acid encodes an antigen used in therapeutic or preventative vaccines for treating or preventing pathogen infection.

[0832] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of non-immune cells are transfected with the LNP. In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of undesirable immune cells not intended to be the target of the delivery are transfected with the LNP. In some embodiments, the half-life of the nucleic acid delivered to the immune cell by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, or longer than the half-life of the nucleic acid delivered to the immune cell by a reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP.

[0833] In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of the immune cells intended to be the target of the delivery are transfected with the LNP.

[0834] In some embodiments, the expression level of the nucleic acid delivered by the LNP is at least 5%, at least 10%, 1.5, 2, 3, 4, 5, 10, 15, 20 or more higher than the expression level of the nucleic acid delivered by the reference LNP in the same immune cells.

[0835] In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to NK cells of a subject. The LNPs contain:

[0836] (a) Ionizable cationic lipids, (b) conjugates comprising the structure of formula (II):

[0837] [Lipids] - [Optional Linker] - [Immune Cell Targeting Group];

[0838] (c) Sterols or other structural lipids;

[0839] (d) Neutral phospholipids;

[0840] (e) Free polyethylene glycol (PEG) lipids; and

[0841] (f) The nucleic acid. In some embodiments, the immune cell targeting group comprises an antibody that binds to CD56.

[0842] In one aspect, lipid nanoparticles (LNPs) are provided for delivering nucleic acids to immune cells of a subject. The LNPs comprise:

[0843] (a) Ionizable cationic lipids;

[0844] (b) Conjugates comprising the following structure: [lipid] - [optional linker] - [immune cell targeting group];

[0845] (c) Sterols or other structural lipids;

[0846] (d) Neutral phospholipids;

[0847] (e) Free polyethylene glycol (PEG) lipids; and

[0848] (f) The nucleic acid.

[0849] In some embodiments, the immune cell targeting group comprises an antibody that binds to CD7 or CD8, and the free PEG lipid is DMG-PEG or DPG-PEG.

[0850] In some aspects, methods are provided for targeting the delivery of nucleic acids to immune cells of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs) provided herein. In some embodiments, the method is used to target NK cells. In some embodiments, an immune cell targeting group binds to CD56. In some embodiments, the method is used to simultaneously target both T cells and NK cells. In some embodiments, the immune cell targeting group binds to CD7, CD8, or both CD7 and CD8. In some embodiments, the method is used to simultaneously target both CD4+ and CD8+ T cells. In some embodiments, the immune cell targeting group comprises a polypeptide that binds to CD3 or CD7.

[0851] In some aspects, methods are provided for expressing a target peptide in targeted immune cells of a subject. In some embodiments, the method includes contacting the immune cells with lipid nanoparticles (LNPs) provided herein.

[0852] In some aspects, methods for modulating the cellular function of target immune cells in a subject are provided. In some embodiments, the method includes administering lipid nanoparticles (LNPs) provided herein to the subject.

[0853] In some respects, methods are provided for treating, improving, or preventing symptoms of a disorder or disease in a subject in need. In some embodiments, the method includes administering lipid nanoparticles (LNPs) provided herein to the subject.

[0854] In some aspects, methods for treating a subject with a CD8-related disease or disorder are provided. In some embodiments, the method includes administering the pharmaceutical composition described herein to the subject. In some embodiments, the disease or disorder is cancer.

[0855] The LNPs disclosed and claimed in this disclosure are applicable to the methods described above.

[0856] VIII. Reagent kits for use in medical applications

[0857] Another aspect of the invention provides a kit for treating medical disorders. The kit comprises: an ionizable cationic lipid, a lipid-immune cell-targeting conjugate, a lipid nanoparticle composition comprising an ionizable cationic lipid and / or a lipid-immune cell-targeting conjugate (with or without encapsulated payload (e.g., mRNA)), and instructions for treating medical disorders such as cancer or microbial or viral infections. Example

[0858] This disclosure illustrates, through examples, the compositions, formulations, preparations, nanoparticles, and / or nanomaterials described herein. This disclosure also illustrates, through examples, methods for preparing, characterizing, and validating the compositions, formulations, preparations, nanoparticles, and / or nanomaterials described herein.

[0859] Example 1: Materials and Methods

[0860] This example provides exemplary materials and methods for preparing, characterizing, and validating the compositions, formulations, nanoparticles, and / or nanomaterials described herein.

[0861] LNP formulations

[0862] In particular, this example provides an exemplary LNP formulation.

[0863] The lipid nanoparticle component is dissolved in 100% ethanol at a specified lipid component molar ratio. The nucleic acid (NA) cargo is dissolved in 10 mM citrate and 100 mM NaCl (pH 4.0), resulting in an NA cargo concentration of approximately 0.22 mg / mL. In some embodiments, the NA cargo comprises functional NA and a reporter DNA barcode, both mixed at a functional NA to barcode mass ratio of 1:10 to 10:1. As described herein, the NA can be siRNA, antisense, expressed DNA, or mRNA.

[0864] LNPs were prepared using a total lipid to NA mass ratio of 11.7. LNPs were formed via microfluidic mixing of lipid and NA solutions using a Precision Nanosystems NanoAssemblr Spark or Benchtop series instrument, according to the manufacturer's protocol. During mixing at varying flow rates, the aqueous solvent to organic solvent ratio was maintained at approximately 2:1 or 3:1. After mixing, the LNPs were collected and diluted with PBS (approximately 1:1 v / v). Further buffer exchange was performed by dialyzing in PBS at 4°C for 4 to 24 hours using a 20 kDa filter. Following initial dialysis, individual LNP formulations were characterized by dynamic light scattering (DLS) to measure size (e.g., diameter) and polydispersity. Furthermore, the pKa of the LNP subsets was measured via 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) assay. LNPs within a specific diameter and polydispersity range were pooled and further dialyzed with phosphate-buffered saline (PBS) for 1 to 4 hours at 4°C using a 100 kDa dialysis cartridge. After the second dialysis, the LNPs were aseptically filtered using a 0.22 μM filter and stored at 4°C for further use.

[0865] LNP characterization

[0866] DLS-LNP hydrodynamic diameter and polydispersity index (PDI) were measured using high-throughput dynamic light scattering (DLS) (DynaPro microplate reader II, Wyatt). LNPs were diluted to appropriate concentrations with IX PBS and analyzed. NA concentration was determined using the Qubit microRNA kit (for siRNA) or the HS RNA kit (for mRNA) according to the manufacturer's instructions. Encapsulation efficiency was determined by measuring the nucleic acid concentration in uncleaved and cleaved LNPs.

[0867] pKa

[0868] Stock solutions of 10 mM HEPES (Sigma Aldrich), 10 mM MES (Sigma Aldrich), 10 mM sodium acetate (Sigma), and 140 nM sodium chloride (Sigma Aldrich) were prepared, and the pH was adjusted to approximately pH 4–10 using hydrogen chloride and sodium hydroxide. Each pH value was repeated four times. 140 pL of the pH-adjusted buffer was added to each well of a 96-well plate, followed by 5 pL of 2-(p-toluidine)-6-naphthalenesulfonic acid (60 pg / mL). 5 pL of LNP was added to each well. After incubation for 5 minutes with gentle shaking, fluorescence was measured using an excitation wavelength of 325 nm and an emission wavelength of 435 nm (BioTek Synergy H4 Hybrid).

[0869] LNP application

[0870] Male and female mice aged approximately 8–12 weeks were used in the study described in this example. Each mouse was temporarily restrained and the combined LNP was administered intravenously (IV) via tail vein injection, with up to five animals per experiment. Age-matched mice were also used to administer a carrier (IX PBS) via tail vein injection, with up to three animals per experiment. Tissue samples, including liver, spleen, bone marrow, kidney, lung, muscle, and blood, were collected 72 hours after administration for analysis.

[0871] flow

[0872] Liver, kidney, lung, and muscle tissues were mechanically digested, followed by enzymatic digestion using a protease mixture, and then filtered through a 70 μM filter to generate a single-cell suspension. Spleen tissue was mechanically digested to produce a single-cell suspension. All tissues were treated with (ammonium-chlorine-potassium) ACK buffer to lyse red blood cells, and then stained with fluorescently labeled antibodies for flow cytometry and fluorescence-activated cell sorting (FACS). Commercially available antibodies were used. Samples were collected via flow cytometry using BD FACSMelody (Becton Dickinson) to generate gating prior to sorting. Generally, the gating structure was size -> single cell -> live cell -> target cell. T cells were defined as CD45+CD3+, monocytes as CD45+CD11b+, and B cells as CD45+CD19+. Endothelial cells were defined as CD31+, monocytes and Kupffer cells as CD45+CD11b+, and hepatocytes as CD31- / CD45-. For siRNA studies, downregulation of the target gene was gated. For mRNA studies, upregulation of the target gene was gated. Tissue from mice treated with the drug was used to set the sorting gate. Up to one million cells with the correct phenotype from each cell subpopulation were sorted into PBS. After sorting, the cells were pelleted by centrifugation, and DNA was extracted using Quick Extract DNA extraction solution (Lucigen) according to the manufacturer's protocol. After DNA extraction, the DNA was stored at -20°C.

[0873] Concentration and Encapsulation Efficiency

[0874] According to the manufacturer's instructions, NA concentration is determined using the Qubit microRNA kit (for siRNA) or the HS RNA kit (for mRNA). Encapsulation efficiency is determined by measuring uncleaved and cleaved LNPs.

[0875] hEPO expression

[0876] For human EPO (hEPO) protein expression, mice were temporarily restrained and blood was collected (via tail vein) 6 hours after administration. The blood was collected in heparinized tubes, processed into plasma, and stored at -80°C until ready for use. The appropriately diluted plasma was used to measure hEPO protein using an R&D Systems ELISA kit (DuoSet; DY286-05) according to the manufacturer's instructions.

[0877] Tolerance ALT / AST quantification

[0878] For quantification of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in rats, rats were temporarily restrained and blood was collected at 2, 4, 6, 24, 48, and 72 hours after administration. Blood was collected in heparinized tubes, processed into plasma, and stored at -80°C until ready for use. AST was quantified using the AST / GOT reagent (Thermo Fisher Scientific, TR70121) and ALT was quantified using the ALT / GPT reagent (Thermo Fisher Scientific, TR71121) according to the manufacturer's instructions.

[0879] RatMCP-I ELISA

[0880] To assess rat monocyte chemoattractant protein-1 (MCP-I) expression, rats were temporarily restrained and blood was collected at 2, 4, 6, 24, 48, and 72 hours post-administration. Blood was collected in heparinized tubes, processed into plasma, and stored at -80°C until ready for use. Appropriately diluted plasma was used to measure MCP-I protein using an R&D Systems ELISA kit (DuoSet; DY3144-05) according to the manufacturer's instructions.

[0881] Example 2: Effectiveness

[0882] An exemplary LNP formulation with efficient delivery to various cell types can be determined as described in this example.

[0883] This example provides exemplary LNP compositions, formulations, nanoparticles, and / or nanomaterials for efficient delivery to the various cell types described herein.

[0884] Based on results derived from Screen, several exemplary lipid LNP formulations were identified for spleen delivery, and particularly for B-cell delivery. These identified lipids were formulated into LNP formulations and screened using the Cre reporter system. Three Ai 14 mice were used in each group. The payload contained 0.3 mg / kg Cre mRNA. Data were collected 168 hours post-injection. Results were compared with the ALC-315 LNP formulation as a control.

[0885] Example 3: Preparation of lipid nanoparticle compositions

[0886] LNP compositions were prepared to generate ionizable lipids: structural lipids : sterol lipids : PEG-lipids in a molar ratio of 33 : 39.9 : 25.4 : 1.5. The lipids were mixed and dissolved in ethanol (organic phase) in the following ratio. An mRNA phase (aqueous phase) was prepared using RNase-free water and 500 mM citrate pH 4 buffer to achieve a final concentration of 10 mM citrate and 0.12 mg / ml mRNA.

[0887] For each LNP composition, lipids and mRNA were mixed on NanoAssemblr Ignite at a 3:1 volume ratio at a flow rate of 12 ml / min. Samples were loaded into a Slide-a-Lyzer G3 dialysis cartridge (10 kWh MWCO) and dialyzed at room temperature to 200 times the sample volume in 1x TBS (diluted from 20x stock solution (Thermo Fisher Scientific)) for 2 hours. The dialysis buffer was then replaced with fresh 1x TBS buffer and dialyzed overnight at 4°C for at least 12 hours. Dialysis samples were then collected and concentrated to 2000 g using an Amicon ultracentrifuge filter (100 kWh MWCO). The concentrated samples were then sterilely filtered using a 0.2 μm mPES filter. Samples were then analyzed using a Ribogreen RNA assay kit (Thermo Fisher Scientific) for measurements including size and polydispersity (Malvern Panalytical) and mRNA for encapsulation.

[0888] Example 4: Targeted LNP Formulation Scheme

[0889] LNP compositions were prepared to generate ionizable lipids: structural lipids : sterol lipids : PEG-lipids : DSPEPEG FAB : DSPE PEG azide, in molar ratios of 33 : 39.9 : 25.4 : 1.5 : 0.05 : 0.1. mRNA was suspended in 10 mM citrate pH 4 buffer (aqueous phase). Ionizable lipids, structural lipids, and cholesterol were dissolved in an ethanol stream (organic phase). DSPE PEG Fab containing small amounts of DSPE PEG azide and DMG PEG 2000 was dissolved in 1x TBS in a dilution stream (dilution phase). The LNPs were then mixed at a water-to-organic-to-dilution-phase ratio of 3 : 1 : 4.

[0890] Example 5: Ribogreen for LNP analysis

[0891] The mRNA used in the preparation was diluted to 2 μg / mL with TE buffer, and then serially diluted to 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. Two standard curves were generated, one in TE buffer and the other in 4% Triton buffer (TX). The concentrated and sterile filtered LNP was diluted twice with TE to fit the TE and TX standard curves. The samples were loaded into 96-well plates, and either TE or 4% Triton buffer was added to the wells. Ribogreen dye was diluted 1:200 with TE buffer and added to all wells. The plates were then incubated on a microplate reader with shaking at 37°C for 3 minutes, and the readings were taken on the microplate reader.

[0892] Example 6: Flow Scheme

[0893] Eight to ten-week-old C57BL / 6 animals (stock number: 000664) were obtained from JAX and allowed at least three days to acclimatize. The formulation, consisting of lipid nanoparticles (LNPs) containing a dose range of eGFP mRNA, was administered intravenously at a dose volume of 10 μL / g body weight. Sixteen hours after administration, the animals were euthanized, and peripheral blood and spleen were collected and processed for flow cytometry.

[0894] FlowJo was used to analyze the streaming data, and gating was performed for each sample at the individual live cell and T cell subset (CD4 and CD8). GFP expression was calculated for each experimental group, and GFP+ cells were reported as the percentage of total T cells, T cell subtypes, and non-T cells. GFP expression for all live cells and MFI across different cell populations were also calculated.

[0895] Example 7: Targeting lipids

[0896] A targeting agent (e.g., Ab, scFv, or Fab) is associated with a targeting LNP, thereby functionalizing the LNP surface. The click stem on the targeting agent, complementary to the click chemistry present on the LNP, is activated according to methods known in the art. For example, an LNP containing 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-PEG-tetraazine (DSPE-PEG-Tz) is associated with a targeting agent specifically or non-specifically modified with a complementary trans-cyclooctene (TCO) click stem. 1 μM of the targeting molecule (e.g., anti-CD5Fab, scFV, or Ab, or anti-CD8Fab, scFv, or Ab) is added to the LNP, and click chemistry association is performed at room temperature for 16 hours according to methods known in the art. Unassociated antibodies are removed from the LNP by size exclusion chromatography (SEC) using a Sephacryl 400 resin column.

[0897] SPAAC adjoining:

[0898] Fab'-DBCO:

[0899] Fab' was conjugated to DBCO-PEG3-maleimide via covalent coupling between the maleimide group and the C-terminal cysteine ​​residue in the light chain (LC). The protein was neutralized with 1 M sodium bicarbonate buffer (pH 9.0) (5–7 mg / mL in sodium acetate buffer (pH 5.5), followed by the addition of 1.2 molar equivalents of DBCO-PEG3-Mal, and the reaction was carried out at room temperature for 2 hours. Excess DBCO-PEG3-Mal was removed using a PD-10 desalting column.

[0900] DSPE-PEG2000-Fab:

[0901] The conjugation reaction was carried out using 3 molar equivalents of DBCO-PEG3-N3 (Avanti Polar Lipids) and Fab-DBCO in phosphate buffer at pH 7.4 at room temperature for 12–16 hours. The formation of the conjugates was monitored by SDS-PAGE and LC-MS. The conjugates were separated by filtration through a 100 kDa Millipore regenerated cellulose membrane using phosphate buffer at pH 7.4 and stored at 4°C before use.

[0902] Example 8: Synthesis of Ionizable Lipids

[0903] This example provides exemplary materials and methods for preparing, characterizing, and validating the ionizable lipids described herein. As described in the examples below, in some exemplary embodiments, compounds are prepared according to the following general procedures. It should be understood that while the general methods depict the synthesis of certain compounds disclosed herein, the following general methods and other methods known to one of ordinary skill in the art can be applied to all compounds and subclasses and species of each of these compounds as described herein.

[0904] The following example lipids were prepared according to the following synthetic protocol.

[0905] Option 1 (Intermediate)

[0906]

[0907] Scheme 2 - Representative Synthesis of Class A Compounds

[0908] Lipid A-4

[0909]

[0910] Lipid A-31

[0911]

[0912] Option 2a

[0913]

[0914] Scheme 3 - Representative Synthesis of Class B Compounds

[0915] Lipid B-9

[0916]

[0917] Lipids B-11, B-12 and B-13

[0918]

[0919] Lipid B-10

[0920]

[0921] Scheme 4 - Representative Synthesis of Class C Compounds

[0922] Lipid C-15

[0923]

[0924] Lipid C-26

[0925]

[0926] Scheme 5 - Representative Synthesis of Class E Compounds

[0927] Lipid E-1

[0928]

[0929] Lipid E-3

[0930]

[0931] General procedure for preparing compound 2

[0932]

[0933] To a solution of compound 1 (100 g, 780 mmol) in MeOH (300 mL) and H₂O (600 mL), NaOAc (128 g, 1.56 mol) and NH₂OH·HCl (108 g, 1.56 mol) were added. The mixture was stirred at 70°C for 6 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.49, Starting material: R f = 0.59) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to remove MeOH. The residue was diluted with H2O (500 mL) and extracted with EtOAc (300 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1). The crude product was used for the next step without further purification. Compound 2 (108 g, crude product) was obtained as a yellow oil.

[0934] 1 H NMR: ET89868-12-P1A1 (400 MHz, DMSO-d6)

[0935] δ = 10.61 (s, 1H), 3.62 (s, 1H), 3.11 (s, 3H), 2.97 (br d, J = 14.3Hz, 1H), 1.99 (s, 1H), 1.59 - 1.77 (m, 3H), 1.48 (br s, 2H), 1.25 (br s, 1H)

[0936] General procedure for preparing compound 3

[0937]

[0938] SOCl2 (108 g, 905 mmol) and MeOH (1.00 L) were added to a solution of compound 2 (108 g, 754 mmol) in CCl4 (1.00 L). The mixture was stirred at 0–5°C for 2 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.55, Starting material: R f = 0.59) indicates that the starting material has been completely consumed. The reaction mixture was diluted with 3.00 L of EtOAc and washed with 2.00 L of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product was used for the next step without further purification. Compound 3 (107 g, crude product) was obtained as a brown oil.

[0939] General procedure for preparing compound 4

[0940]

[0941] PPTS (9.60 g, 38.2 mmol) was added to a solution of compound 3 (40.0 g, 254 mmol) in compound 3A (133 g, 1.01 mol). The mixture was stirred at 120°C for 5 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.63, Starting material: R f = 0.28) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 1 to 80 / 1). Compound 4 was given as a colorless oil (30.0 g, 84.9 mmol, 33.4% yield).

[0942] 1 H NMR: ET89868-26-P1A1 (400 MHz, CDCl3)

[0943] δ = 4.46 (s, 1H), 3.56 (s, 2H), 3.42 (s, 2H), 2.35 (s, 2H), 1.56 (brd, J = 7.0 Hz, 10H), 1.24 - 1.37 (m, 20H), 0.89 (s, 6H)

[0944] General procedure for preparing compound 5

[0945]

[0946] KOH (38.1 g, 679 mmol) was added to a solution of compound 4 (30.0 g, 84.9 mmol) in EtOH (150 mL) and H₂O (150 mL). The mixture was stirred at 110°C for 5 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.53, Starting material: R f= 0.76) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to remove EtOH. The residue was diluted with 300 mL of H2O and acidified to pH = 5 with 1M HCl. The mixture was then extracted with 900 mL of EtOAc (300 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was used for the next step without further purification. Compound 5 (31.6 g, crude product) was given as a yellow oil.

[0947] 1 H NMR: ET89868-32-P1B1 (400 MHz, DMSO-d6)

[0948] δ = 4.39 (br s, 1H), 3.27 - 3.52 (m, 4H), 2.10 (br s, 2H), 1.46 (brs, 8H), 1.25 (br s, 22H), 0.85 (br s, 6H)

[0949] General procedure for preparing compound 6

[0950]

[0951] Compound 5A (3.47 g, 38.6 mmol), EDCI (22.2 g, 116 mmol), and DMAP (1.18 g, 9.64 mmol) were added to a solution of compound 5 (31.6 g, 84.8 mmol) in DCM (300 mL). The mixture was stirred at 25°C for 10 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.63, Starting material: R f = 0.51) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was washed with 200 mL of H2O and then extracted with 900 mL of petroleum ether (300 mL x 3). The combined organic layers were washed with 200 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The crude product was used for the next step without further purification. Compound 6 (33.0 g, crude product) was given as a yellow oil.

[0952] 1 H NMR: ET89868-35-P1A2 (400 MHz, CDCl3)

[0953] δ = 4.75 (s, 4H), 4.46 (s, 2H), 3.55 (s, 4H), 3.41 (s, 4H), 2.44 (s,4H), 1.56 (br s, 18H), 1.28 (br s, 42H), 0.89 (s, 12H)

[0954] General procedure for preparing compound 7

[0955]

[0956] Compound 6A (2.05 g, 20.0 mmol), NaBH(OAc)3 (6.36 g, 30.0 mmol), and HOAc (90.2 mg, 1.50 mmol) were added to a solution of compound 6 (8.00 g, 10.0 mmol) in DCM (80.0 mL). The mixture was stirred at 25°C for 5 hours. LCMS (product RT = 1.701 min) indicated that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to remove DCM, washed with 200 mL of H2O, and extracted with 900 mL of EtOAc (300 mL x 3). The combined organic layers were washed with 200 mL of NaHCO3 and 200 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 50 / 1). Compound 7 was obtained as a yellow oil (4.60 g, 5.20 mmol, 51.9% yield).

[0957] General procedure for preparing compound 9

[0958]

[0959] DMAP (7.72 g, 63.2 mmol), pyridine (9.99 g, 126 mmol), and tetrahydrofuran-2,5-dione (12.6 g, 126 mmol) were added to a solution of compound 8 (10.0 g, 63.2 mmol) in THF (10.0 mL). The mixture was stirred at 50°C for 8 hours. The mixture was then stirred at 70°C for 6 hours. LCMS (product RT = 1.717 min) showed that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to remove THF, and the residue was washed with 150 mL of H2O and extracted with 150 mL of MTBE (50 mL x 3). The combined organic layers were washed with 50 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1). Compound 9 was obtained as a yellow oil (10.0 g, 38.7 mmol, 61.3% yield).

[0960] 1 H NMR: ET89868-6-P1A1 (400 MHz, CDCl3)

[0961] δ = 12.17 (br s, 1H), 4.79 (s, 1H), 2.46 (s, 4H), 1.41 - 1.50 (m,4H), 1.23 (br s, 10H), 0.85 (s, 6H)

[0962] General procedure for preparing A-31

[0963]

[0964] Compound 9 (1.97 g, 7.62 mmol), EDCI (1.95 g, 10.2 mmol), and DMAP (31.1 mg, 254 μmol) were added to a solution of compound 7 (4.50 g, 5.08 mmol) in DCM (45.0 mL). The mixture was stirred at 25°C for 2 hours. LCMS (product RT = 2.545 min) indicated that the starting material was completely consumed. The reaction mixture was diluted with 30 mL of DCM, washed with 80 mL of brine, and extracted with 150 mL of DCM (50 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 10 / 1). A-31 (601 mg, 534 μmol, 10.5% yield) was given as a yellow oil.

[0965] 1 H NMR: ET89868-44-P1A1 (400 MHz, CDCl3)

[0966] δ = 4.88 (br s, 1H), 4.44 (s, 3H), 4.10 - 4.37 (m, 3H), 3.86 (br s,1H), 3.46 - 3.61 (m, 4H), 3.24 - 3.45 (m, 5H), 2.44 - 2.76 (m, 10H), 2.26 -2.43 (m, 6H), 1.89 (br d, J = 6.9 Hz, 2H), 1.42 - 1.76 (m, 22H), 1.27 (br s,54H), 0.88 (s, 18H)

[0967] LCMS: Product ET89868-44-P1B1: RT = 3.439 min, [M+H] + = 1126.0, Purity = 97.0%

[0968] General procedure for preparing compound 7b

[0969]

[0970] Compound 6B (2.57 g, 20.0 mmol), NaBH(OAc)3 (6.36 g, 30.0 mmol), and HOAc (90.2 mg, 1.50 mmol) were added to a solution of compound 6 (8 g, 10.0 mmol) in DCM (80.0 mL). The mixture was stirred at 25°C for 5 hours. LCMS (product RT = 1.773 min) indicated that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to remove DCM, washed with 200 mL of H2O, and extracted with 900 mL of EtOAc (300 mL x 3). The combined organic layers were washed with 200 mL of NaHCO3 and 200 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 50 / 1). Compound 7b was obtained as a yellow oil (4.00 g, 4.39 mmol, 43.84% yield).

[0971] General procedure for preparing A-6

[0972]

[0973] Compound 9 (1.66 g, 6.42 mmol), EDCI (1.64 g, 8.56 mmol), and DMAP (26.1 mg, 214 μmol) were added to a solution of compound 7b (3.90 g, 4.28 mmol) in DCM (39.0 mL). The mixture was stirred at 25°C for 1 hour. LCMS (product RT = 2.559 min) indicated that the starting material was completely consumed. The reaction mixture was diluted with 30 mL of DCM, washed with 80 mL of brine, and extracted with 150 mL of DCM (50 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 10 / 1). A-6 (604 mg, 524 μmol, 12.3% yield) was given as a yellow oil.

[0974] 1 H NMR: ET89868-45-P1A1 (400 MHz, CDCl3)

[0975] δ = 4.87 (br d, J = 1.9 Hz, 1H), 4.44 (s, 3H), 4.10 - 4.37 (m, 3H), 3.86 (br d, J = 5.1 Hz, 1H), 3.55 (br d, J = 9.3 Hz, 4H), 3.39 (br d, J = 9.3Hz, 6H), 2.48 - 3.01 (m, 10H), 2.31 (br d, J = 7.3 Hz, 4H), 1.77 - 2.07 (m,6H), 1.55 (br d, J = 7.1 Hz, 22H), 1.27 (br s, 54H), 0.88 (s, 18H)

[0976] LCMS: Product ET89868-45-P1B2: RT = 3.529 min, [M+H] + = 1151.9, purity = 99.6%

[0977] General procedure for preparing compound 4c

[0978]

[0979] PPTS (2.40 g, 9.54 mmol) was added to a solution of compound 3 (10.0 g, 63.61 mmol) in compound 3C (32.6 g, 254 mmol). The mixture was stirred at 120°C for 5 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.63, Starting material: R f = 0.28) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 1 to 70 / 1). Compound 4c was given as a colorless oil (6.00 g, 17.2 mmol, 26.98% yield).

[0980] 1 H NMR: ET89868-21-P1A1 (400 MHz, CDCl3)

[0981] δ = 5.23 - 5.51 (m, 4H), 4.46 (s, 1H), 3.57 (s, 2H), 3.42 (s, 2H), 2.35 (s, 2H), 2.05 (br d, J = 3.6 Hz, 8H), 1.57 (br d, J = 2.3 Hz, 10H), 1.42(s, 4H), 0.96 (s, 6H)

[0982] General procedure for preparing compound 5c

[0983]

[0984] KOH (7.70 g, 137 mmol) was added to a solution of compound 4c (6.00 g, 17.2 mmol) in EtOH (30.0 mL) and H₂O (30.0 mL). The mixture was stirred at 110°C for 5 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) f = 0.45, Starting material: R f = 0.70) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to remove EtOH. The residue was diluted with 100 mL of H2O and acidified to pH = 5 with 1M HCl. The mixture was then extracted with 450 mL of EtOAc (150 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was used for the next step without further purification. Compound 5c (6.10 g, crude) was given as a yellow oil.

[0985] 1 H NMR: ET89868-34-P1B1 (400 MHz, DMSO-d6)

[0986] δ = 5.22 - 5.49 (m, 4H), 4.40 (t, J = 5.6 Hz, 1H), 3.48 (td, J = 6.4,9.4 Hz, 2H), 3.34 (td, J = 6.4, 9.4 Hz, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.99(quin, J = 6.9 Hz, 8H), 1.43 - 1.52 (m, 8H), 1.24 - 1.39 (m, 6H), 0.91 (t, J= 7.6 Hz, 6H)

[0987] General procedure for preparing compound 6c

[0988]

[0989] Compound 5A (678 mg, 7.52 mmol), EDCI (4.33 g, 22.6 mmol), and DMAP (230 mg, 1.88 mmol) were added to a solution of compound 5c (6.10 g, 16.6 mmol) in DCM (60.0 mL). The mixture was stirred at 25°C for 10 hours. TLC (petroleum ether / ethyl acetate = 3 / 1, product: R) was performed. f = 0.61, Starting material: R f = 0.51) indicates that the starting material has been completely consumed. The reaction mixture was concentrated under reduced pressure to remove DCM. The residue was washed with 100 mL of H2O and then extracted with 450 mL of petroleum ether (150 mL x 3). The combined organic layers were washed with 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The crude product was used for the next step without further purification. Compound 6c (6.40 g, crude product) was given as a yellow oil.

[0990] 1 H NMR: ET89868-36-P1A2 (400 MHz, CDCl3)

[0991] δ = 5.28 - 5.45 (m, 8H), 4.46 (t, J = 5.7 Hz, 2H), 3.57 (td, J = 6.5,9.2 Hz, 4H), 3.41 (td, J = 6.6, 9.2 Hz, 4H), 2.44 (t, J = 7.6 Hz, 4H), 1.99 -2.09 (m, 16H), 1.55 - 1.72 (m, 16H), 1.37 - 1.47 (m, 12H), 0.96 (t, J = 7.5Hz, 12H)

[0992] General procedure for preparing compound 7c

[0993]

[0994] Compound 6A (1.65 g, 16.2 mmol), NaBH(OAc)3 (5.14 g, 24.3 mmol), and HOAc (72.9 mg, 1.21 mmol) were added to a solution of compound 6c (6.40 g, 8.09 mmol) in DCM (64.0 mL). The mixture was stirred at 25°C for 5 hours. LCMS (product RT = 1.701 min) indicated that the starting material was completely consumed. The reaction mixture was concentrated under reduced pressure to remove DCM, washed with 100 mL of H2O, and extracted with 450 mL of EtOAc (150 mL x 3). The combined organic layers were washed with 100 mL of NaHCO3 and 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 50 / 1). Compound 7c (3.80 g, 4.33 mmol, 53.5% yield) was obtained as a yellow oil.

[0995] General procedure for preparing A-4

[0996]

[0997] Compound 9 (1.63 g, 6.33 mmol), EDCI (1.62 g, 8.43 mmol), and DMAP (25.8 mg, 211 μmol) were added to a solution of compound 7c (3.70 g, 4.22 mmol) in DCM (37.0 mL). The mixture was stirred at 25°C for 2 hours. TLC (DCM / MeOH = 5 / 1, product: R) was performed. f = 0.59, Starting material: R f= 0.56) indicates that the starting material has been completely consumed. The reaction mixture was diluted with 30 mL of DCM, washed with 80 mL of brine, and extracted with 150 mL of DCM (50 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, DCM / MeOH = 100 / 1 to 10 / 1). A-4 (615 mg, 550 μmol, 13.1% yield) was given as a yellow oil.

[0998] 1 H NMR: ET89868-45-P1A1 (400 MHz, CDCl3)

[0999] δ = 5.20 - 5.42 (m, 8H), 4.77 - 4.85 (m, 1H), 4.32 - 4.41 (m, 3H), 4.03 - 4.29 (m, 3H), 3.67 - 3.92 (m, 1H), 3.44 - 3.53 (m, 4H), 3.16 - 3.39(m, 6H), 2.28 - 2.59 (m, 10H), 2.21 - 2.31 (m, 6H), 1.92 - 2.02 (m, 14H),1.80 (br d, J = 6.9 Hz, 2H), 1.46 - 1.61 (m, 18H), 1.29 - 1.38 (m, 12H), 1.14- 1.26 (m, 14H), 0.80 - 0.91 (m, 18H)

[1000] LCMS: Product ET89868-46-P1B2: RT = 3.131 min, [M+H] + = 1117.9, purity = 96.1%

[1001] General procedure for preparing compounds

[1002]

[1003] PPTS (2.40 g, 9.54 mmol) was added to a solution of compound 3 (10.0 g, 63.6 mmol) in compound 3D (24.1 g, 191 mm...

Claims

1. A compound having formula (A-II): (A–II) Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein L is an optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; L1 is optionally a substituted C1-6 alkylene or C2-6 heteroalkylene; Each L2 is independently a C1-6 alkylene group; L3 is an optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; L4 is a non-existent, optionally substituted C1- 10 Alkylene or optionally substituted C2- 10 Heteroalkyl; L5 is an optionally substituted C1- 10 Alkyl, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Alkyne group or optionally substituted C2- 10 Heteroalkyl; X is non-existent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-; Each X2 is non-existent, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, or -OC(O)O-; Each R is independently hydrogen, OR6, or optionally substituted with a group selected from C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl; R1 is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered aliphatic ring, optionally substituted 3- to 7-membered heterocyclic group containing 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -CN, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, - NR2C(O)N(R2)2, -NR2C(S)N(R2)2, -NR2C(NR2)N(R2)2, -NR2C(CHR2)N(R2)2, -N(OR 2)C(O)R2, -N(OR2)S(O)2R2, -N(OR2)C(O)OR2, -N(OR2)C(O)N(R2)2, -N(OR2)C(S)N( R2)2, -N(OR2)C(NR2)N(R2)2, -N(OR2)C(CHR2)N(R2)2, -C(NR2)N(R2)2, -C(NR2)R2, -C(O)N(R2)OR2, -C(R2)N(R2)2C(O)OR2, -CR2(R3)2, -OP(O)(OR2)2 or -P(O)(OR2)2; or R1 is selected from the following rings: 3- to 7-membered aliphatic rings and 3- to 7-membered heterocyclic groups containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen or sulfur, wherein the aliphatic or heterocyclic ring is optionally substituted by 1 to 4 R2 or R3 groups. Each R2 group is independently hydrogen, oxo, -CN, -NO2, -OR4, -S(O)2R4, -S(O)2N(R4)2, or -(CH2). n -R4 or optionally substituted groups selected from: C1-6 aliphatic, phenyl, 3- to 7-membered aliphatic, 5- to 6-membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and 3- to 7-membered heterocyclic groups containing 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or two R2s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups, the heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen, or sulfur; each R3 is independently -(CH2)n-R4; or two R3s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups, the heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen, or sulfur; each R3 is independently -(CH2)n-R4; or two R3s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups. One or more atoms together form an optionally substituted 5- to 6-membered heterocyclic group, the heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen, or sulfur; each R4 is independently hydrogen, -OR5, -N(R5)2, -OC(O)R5, -OC(O)OR5, -CN, -C(O)N(R5)2, -NR5C(O)R5, -OC(O)N(R5)2, -N(R5)C(O)OR5, -NR5S(O)2R5, -NR5C(O)N(R5)2, -NR5C(S)N(R5)2, or -NR5C(NR5)N(R5)2; and Each R5 is independently hydrogen or optionally substituted C. 1-6 Aliphatic; or the two R5s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups, which contain 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R6 is independently C 4-12 Aliphatic; and each n is independently 0 to 4.

2. The compound of claim 1, wherein R1 is -N(R2)2.

3. The compound of claim 1, wherein L1-R1 are selected from the group consisting of: 。 4. The compound of claim 1, wherein... Choose from the following groups: .

5. The compound of claim 1, wherein... yes .

6. The compound of claim 1, wherein... yes .

7. The compound of claim 1, wherein... yes .

8. A compound having the formula (BI): (B-I) Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein Each R' is independently a non-existent, optionally substituted C1-6 alkyl or optionally substituted C1-6 heteroalkyl; Each L is independently an optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; Each X is independently O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O; Each Z is independent , or The condition is that at least one or two of Z are or ; Each of m is independently 1-9; t is 1, 2, or 3; X 10 It is non-existent, O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O; "R" is H, , or ; L 10 It is an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; Each R 11 Independently, it is C2- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, C2-C substituted bicyclic compounds with 7- to 12-membered bridging substitutions containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 10 Aliphatic, 1-adamantyl, 2-adamantyl, sterol, and phenyl; and Each R 10 Independently hydrogen, OR6, or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol and phenyl.

9. The compound of claim 8, wherein Z is .

10. The compound of claim 8, wherein Z is .

11. The compound of claim 8, wherein R' is H or CH3.

12. The compound of claim 8, represented by formula (B-II): (B-II) Z and R are each independent of each other. , or The condition is that at least two or three of Z and R are or .

13. The compound according to claims 8-12, wherein... Choose from the following groups: 。 14. The compound according to claims 8-12, wherein Choose from the following groups: , , ,and .

15. A compound having the formula (CI): (C-I) Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein X 20 Each of these is independent of the following: O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O; L 20 Each of them is independently and optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; Z 20 Each of them is independently , or The condition is Z. 20 At least one of them is or ; Each of m is 1-9; t is 1, 2, or 3; L 10 It is an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; R 11 Each of them is independently C2- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, C2-C substituted bicyclic compounds with 7- to 12-membered bridging substitutions containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 10 Aliphatic, 1-adamantyl, 2-adamantyl, sterol or phenyl; and R 10 Each of these is independently hydrogen, OR6, or optionally substituted groups selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9; It is a 4- to 8-membered ring or a heterocyclic ring; L1 is absent, a C1-6 alkylene group, or a C2-6 heteroalkylene group; R1 is hydrogen, optionally substituted phenyl, optionally substituted 3- to 7-membered aliphatic ring, optionally substituted 3- to 7-membered heterocyclic group containing 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted 5- to 6-membered monocyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, optionally substituted 8- to 10-membered bicyclic heteroaryl group containing 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -CN, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, - NR2C(O)N(R2)2, -NR2C(S)N(R2)2, -NR2C(NR2)N(R2)2, -NR2C(CHR2)N(R2)2, -N(OR 2)C(O)R2, -N(OR2)S(O)2R2, -N(OR2)C(O)OR2, -N(OR2)C(O)N(R2)2, -N(OR2)C(S)N( R2)2, -N(OR2)C(NR2)N(R2)2, -N(OR2)C(CHR2)N(R2)2, -C(NR2)N(R2)2, -C(NR2)R2, -C(O)N(R2)OR2, -C(R2)N(R2)2C(O)OR2, -CR2(R3)2, -OP(O)(OR2)2 or -P(O)(OR2)2; or R1 is selected from the following rings: 3- to 7-membered aliphatic rings and 3- to 7-membered heterocyclic groups containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen or sulfur, wherein the aliphatic or heterocyclic ring is optionally substituted by 1 to 4 R2 or R3 groups. Each R2 is independently hydrogen, oxo, -CN, -NO2, -OR4, -S(O)2R4, -S(O)2N(R4)2, -(CH2)n-R4, or optionally substituted with a group selected from: C1-6 aliphatic, phenyl, 3- to 7-membered aliphatic rings, 5- to 6-membered monocyclic heteroaryl groups containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and 3- to 7-membered heterocyclic groups containing 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or R2 appearing twice together with one or more atoms to which they are attached to form optionally substituted 4- to 7-membered heterocyclic groups, which contain 0-1 additional heteroatoms selected from nitrogen, oxygen, and sulfur; each R3 is independently -(CH2). n -R4; or two R3s together with one or more atoms to which they are attached form optionally substituted 5- to 6-membered heterocyclic groups, the heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen, or sulfur; each R4 is independently hydrogen, -OR5, -N(R5)2, -OC(O)R5, -OC(O)OR5, -CN, -C(O)N(R5)2, -NR5C(O)R5, -OC(O)N(R5)2, -N(R5)C(O)OR5, -NR5S(O)2R5, -NR5C(O)N(R5)2, -NR5C(S)N(R5)2, or -NR5C(NR5)N(R5)2; and Each R5 is independently hydrogen or optionally substituted C. 1-6 Aliphatic; or the two R5s together with one or more atoms attached to them form optionally substituted 4- to 7-membered heterocyclic groups, the heterocyclic group containing 0-1 additional heteroatoms selected from nitrogen, oxygen, or sulfur; each R6 is independently C 4-12 Aliphatic; and each n is independently 0 to 4.

16. The compound of claim 15, wherein... Choose from the following groups: 。 17. The compound of claim 15, wherein... Choose from the following groups: 。 18. The compound of claim 15, wherein yes or .

19. The compound of claims 15-18, wherein L1 is absent, and R1 is -OR2, -C(O)OR2, -C(O)SR2, -OC(O)R2, -OC(O)OR2, -N(R2)2, -C(O)N(R2)2, -S(O)2N(R2)2, -NR2C(O)R2, -OC(O)N(R2)2, -N(R2)C(O)OR2, -NR2S(O)2R2, or -NR2C(O)N(R2)2; and R2 is as defined in claim 15.

20. The compound of claim 15, wherein R1 is -OR2 or -C(O)OR2.

21. The compound of claim 15, wherein L1-R1 are selected from the group consisting of: and .

22. The compound of claim 15, represented by formula (C-II): (C-II) Where L1 is non-existent, R1 is OR2, q is 2-9, and X is... 20 It is OC(O) or C(O)O, R2, L 20 and Z 20 As defined in claim 15.

23. The compound of claim 15, represented by formula (C-III): (C-III) Where L1 is non-existent, R1 is OR2, q is 2-9, and X is... 20 It is OC(O) or C(O)O, R2, L 20 and Z 20 As defined in claim 15.

24. The compound of claim 15, wherein -L 20 -Z 20 Choose from the following groups: 。 25. The compound of claim 15, represented by formula (C-IV): (C-IV) Where L1 is non-existent, R1 is OR2, q is 2-9, and X is... 20 It is OC(O) or C(O)O, R2, L 20 and Z 20 As defined in claim 15.

26. The compound according to claims 15-25, wherein... Choose from the following groups: 。 27. The compound according to claims 15-25, wherein... Choose from the following groups: , , ,and .

28. A compound having formula (EI): (EI) Or the N-oxide, isomer, or pharmaceutically acceptable salt thereof of the compound, wherein X 30 Each of these is independent of the following: O, S, N(R”), SS, OC(O), C(O)O, C(O)NH, NHC(O), OC(O)O, OC(O)NH or NHC(O)O; L 30 Each of them is independently and optionally substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; X 31 Each of them is independently NR”; where R” is H, , or The condition is Z. 30 At least one of them is or ; Z 30 Each of them is independently , or The condition is Z. 30 At least one of them is or ; Each of m is independently 1 to 9; t is 1, 2, or 3; L 10 It is an optional substituted C1- 10 Alkylene, optionally substituted C1- 10 alkenyl, optionally substituted C1- 10 Ethyne or optionally substituted C2- 10 Heteroalkyl; R 11 Each of them is independently C2- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, C2-C substituted bicyclic compounds with 7- to 12-membered bridging substitutions containing 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 10 Aliphatic, 1-adamantyl, 2-adamantyl, sterol or phenyl; and R 10 Each of the groups in the group is independently and optionally substituted with a group selected from the following: C6- 20 Aliphatic, 3- to 12-membered cyclic aliphatic, 7- to 12-membered bridging bicyclic containing 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, 1-adamantyl, 2-adamantyl, sterol or phenyl.

29. The compound of claim 28, represented by formula (E-II). (E-II) Where X 30 L 30 X 31 and Z 30 As defined in claim 20.

30. The compound of claim 28, wherein X 30 It is C(O)O, OC(O), C(O)NH, NHC(O) or O.

31. The compound according to claims 28-30, wherein... Choose from the following groups: 。 32. The compound according to claims 28-30, wherein... Choose from the following groups: , , ,and .

33. The compound of claim 28, wherein X 30 It is C(O)NH.

34. The compound of claim 33, wherein X 30 It is C(O)O.

35. The compound of claim 28, wherein X 31 It's NR.

36. The compound of claim 28, wherein Z 30 Choose from the following groups: 。 37. The compound according to any one of claims 1-36, wherein the pKa of the protonated form of the compound is about 4.5 to about 8.

0.

38. The compound of claim 1, having one of the following structures: 。 39. The compound of claim 8, having one of the following structures: 。 40. The compound of claim 15, having one of the following structures: 。 41. The compound of claim 28, having one of the following structures: 。 42. A compound having one of the following structures: 。 43. A lipid nanoparticle (LNP) for targeted delivery of nucleic acids to immune cells, the lipid nanoparticle comprising a lipid blend comprising: (a) A lipid-immune cell-targeting group conjugate comprising a compound having formula (II): [lipid] - [optional linker] - [immune cell-targeting group]; and (b) An ionizable cationic lipid comprising any one of the compounds of claims 1 to 42, wherein the LNP further comprises a nucleic acid disposed therein.

44. The LNP of claim 43, wherein the immune cell targeting group comprises an antibody that binds to a T cell antigen.

45. The LNP of claim 44, wherein the T cell antigen is CD3, CD4, CD7, CD8 or a combination thereof (e.g., both CD3 and CD8, both CD4 and CD8, or both CD7 and CD8).

46. ​​The LNP of any one of claims 43 to 45, wherein the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen.

47. The LNP of claim 46, wherein the NK cell antigen is CD7, CD8, CD56 or a combination thereof (e.g., both CD7 and CD8).

48. The LNP of any one of claims 43 to 47, wherein the immune cell targeting group is covalently coupled to the lipids in the lipid blend via a linker containing polyethylene glycol (PEG).

49. The LNP of claim 48, wherein the lipid covalently coupled to the immune cell targeting group via a PEG-containing linker is distearylglycerol (DSG), distearyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglyceride (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), or a ceramide.

50. The LNP as claimed in claim 48 or 49, wherein the PEG is PEG 2000 or PEG 3400.

51. The LNP of any one of claims 43 to 50, wherein the lipid-immune cell-targeting group conjugate is present in the lipid blend in the range of 0.001 to 0.5 mol percent (e.g., 0.002-0.2 mol percent).

52. The LNP of any one of claims 43 to 51, wherein the lipid blend further comprises one or more of structural lipids (e.g., sterols), neutral phospholipids, and free PEG-lipids.

53. The LNP of any one of claims 43 to 52, wherein the ionizable cationic lipid is present in the lipid blend in a range of 30-70 (e.g., 40-60) molar percentage.

54. The LNP of claim 52, wherein the sterol is present in the lipid blend in a range of 20-70 (e.g., 30-50) molar percentage.

55. The LNP as claimed in claim 52 or 54, wherein the sterol is cholesterol.

56. The LNP according to any one of claims 52 to 55, wherein the neutral phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin.

57. The LNP of any one of claims 52 to 56, wherein the neutral phospholipid is present in the lipid blend in the range of 5-15 molar percentage.

58. The LNP of any one of claims 52 to 57, wherein the free PEG-lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, or wherein the PEG lipid is PEG-dioleoylglycerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-dimyristoyl-glycerol, etc. Acyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG and PEG-DSG), PEG-ceramide, PEG-distearyl-glycerol-phosphate (PEG-DSPG), PEG-dioleoyl-glycerol-phosphate ethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide or PEG-distearyl-phosphatidylethanolamine (PEG-DSPE) lipids.

59. The LNP of any one of claims 52 to 57, wherein the free PEG-lipid comprises diacylphosphatidylethanolamine containing a dipalmitoyl (C16) chain or a distearate (C18) chain, and optionally the free PEG-lipid comprises PEG-DPG and PEG-DMG.

60. The LNP of any one of claims 52 to 59, wherein the free PEG-lipid is present in the lipid blend in the range of 1-4 molar percentage.

61. The LNP of any one of claims 52 to 60, wherein the free PEG-lipid comprises lipids that are the same as or different from the lipids in the lipid-immune cell targeting group conjugate.

62. The LNP as claimed in any one of claims 43 to 61, wherein the LNP has an average diameter in the range of 50 nm to 200 nm.

63. The LNP of claim 62, wherein the LNP has an average diameter of about 100 nm.

64. The LNP as claimed in any one of claims 43 to 63, wherein the LNP has a polydispersity index in the range of 0.05 to 1.

65. The LNP of any one of claims 43 to 64, wherein the LNP has a zeta potential from about +10 mV to about +30 mV at pH 5.

66. The LNP according to any one of claims 43 to 65, wherein the nucleic acid is DNA or RNA.

67. The LNP of claim 66, wherein the RNA is mRNA.

68. The LNP of claim 67, wherein the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine.

69. The LNP of claim 67, wherein the mRNA encodes a polypeptide capable of regulating the immune response in the immune cell.

70. The LNP of claim 67, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

71. The LNP of claim 69, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

72. The LNP of any one of claims 43 to 71, wherein the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin monovariable domain (e.g., a nanobody).

73. The LNP of any one of claims 43 to 71, wherein the immune cell targeting group comprises a Fab, F(ab')2, Fab'-SH, Fv, or scFv fragment.

74. The LNP of claim 72 or claim 73, wherein the immune cell targeting group comprises a Fab engineered to knock out the natural interchain disulfide bond at the C-terminus.

75. The LNP of claim 74, wherein the Fab comprises a heavy chain segment containing C233S substitution and a light chain segment containing C214S substitution, according to the Kabat number.

76. The LNP of any one of claims 73 to 75, wherein the immune cell targeting group comprises a Fab having non-natural interchain disulfide bonds (e.g., engineered embedded interchain disulfide bonds).

77. The LNP of claim 76, wherein the Fab contains F174C substitution in the heavy chain segment and S176C substitution in the light chain segment, according to the Kabat designation.

78. The LNP of claims 73 to 77, wherein the immune cell targeting group comprises a Fab containing a cysteine ​​residue at the C-terminus of the heavy or light chain segment.

79. The LNP of claim 78, wherein the Fab further comprises one or more amino acids between the heavy chain segment of the Fab and the C-terminal cysteine.

80. The LNP of claim 72, wherein the immune cell targeting group comprises an immunoglobulin monovariable domain.

81. The LNP as claimed in claim 72 or 80, wherein the immunoglobulin monovariable domain comprises a cysteine ​​residue at its C-terminus.

82. The LNP of claim 81, wherein the immunoglobulin single variable domain comprises V HH The structural domain, and further includes spacers contained within the V. HH One or more amino acids between the domain and the C-terminal cysteine ​​residue.

83. The LNP of any one of claims 73 and 80 to 82, wherein the immune cell targeting group comprises two or more V HH Structural domain.

84. The LNP as claimed in claim 83, wherein the two or more V HH The domains are linked by amino acid linkers.

85. The LNP of claim 83, wherein the immune cell targeting group comprises a first V linked to the antibody CH1 domain. HH The domain and the second V linked to the constant domain of the antibody light chain HH The antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds.

86. The LNP of any one of claims 72 and 80 to 82, wherein the immune cell targeting group comprises a V domain linked to the antibody CH1 domain. HH The antibody CH1 domain is connected to the antibody light chain constant domain via one or more disulfide bonds.

87. The LNP as claimed in claim 85 or 86, wherein the CH1 domain comprises F174C and C233S substitutions, and the light chain constant domain comprises S176C and C214S substitutions, according to the Kabat designation.

88. The LNP of any one of claims 43 to 69, wherein the immune cell targeting group comprises Fab, the Fab comprising: (a) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 1 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 2 or 3; or (b) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO:

7.

89. The LNP of any one of claims 43 to 88, wherein the ionizable cationic lipid is selected from lipids A-1 to A-10.

90. The LNP of any one of claims 43 to 88, wherein the ionizable cationic lipid is selected from lipids A-11 to A-20.

91. The LNP of any one of claims 43 to 88, wherein the ionizable cationic lipid is selected from lipids A-21 to A-31.

92. The LNP as claimed in any one of claims 43 to 91, wherein the LNP comprises: (a) The ionizable cationic lipid; (b) The conjugate comprises a compound of the following formula: [Lipids] - [Optional Linker] - [Immune Cell Targeting Group]; (c) Sterols or other structural lipids; (d) Neutral phospholipids; (e) Free polyethylene glycol (PEG) lipids; and (f) The nucleic acid.

93. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to an immune cell, and wherein the immune cell is an NK cell, and the immune cell targeting group comprises an antibody that binds to CD56.

94. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to immune cells, and wherein the immune cell targeting group comprises an antibody that binds to CD7 or CD8, and the free PEG lipid is DMG-PEG or PEG-DPG.

95. The LNP of any one of claims 43 to 92, wherein the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin monovariable domain.

96. The LNP of claim 95, wherein the Fab is engineered to knock out the natural interchain disulfide bonds at the C-terminus.

97. The LNP of claim 96, wherein the Fab comprises a heavy chain segment containing C233S substitution and a light chain segment containing C214S substitution.

98. The LNP of claim 96, wherein the Fab comprises non-natural interchain disulfide bonds.

99. The LNP of claim 96, wherein the Fab contains F174C substitution in the heavy chain segment and S176C substitution in the light chain segment.

100. The LNP of claim 95, wherein the antibody is an immunoglobulin single variable (ISV) domain, and the ISV domain is Nanobody® ISV.

101. The LNP of claim 100, wherein the free PEG lipid comprises PEG having a molecular weight of at least 2000 Daltons.

102. The LNP of claim 101, wherein the PEG has a molecular weight of about 3,000 to 5,000 Daltons.

103. The LNP of claim 95, wherein the antibody is Fab.

104. The LNP of claim 103, wherein the Fab binds CD3, and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 2000 Daltons.

105. The LNP of claim 103, wherein the Fab is an anti-CD4 antibody, and the free PEG lipid in the LNP comprises PEG having a molecular weight of about 3,000 to 3,500 Daltons.

106. The LNP of claim 95, wherein the immune cell targeting group comprises two or more V HH Structural domain.

107. The LNP as claimed in claim 106, wherein the two or more V HH The domains are linked by amino acid linkers.

108. The LNP of claim 107, wherein the immune cell targeting group comprises a first V linked to the antibody CH1 domain. HH The domain and the second V linked to the constant domain of the antibody light chain HH Structural domain.

109. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to immune cells, and wherein the LNP binds CD3 and also binds CD11a or CD18.

110. The LNP of claim 109, wherein the LNP comprises two conjugates, wherein the first conjugate comprises an antibody binding to CD3, and the second conjugate comprises an antibody binding to CD11a or CD18.

111. The LNP of claim 109, wherein the LNP comprises a conjugate and the conjugate comprises a bispecific antibody that binds both CD3 and CD11a.

112. The LNP of claim 109, wherein the LNP comprises a conjugate and the conjugate comprises a bispecific antibody that binds both CD3 and CD18.

113. The LNP as claimed in claim 111 or 112, wherein the bispecific antibody is an immunoglobulin with a single variable domain or Fab-ScFv.

114. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to immune cells, and wherein the LNP binds to CD7 and CD8 of the immune cells.

115. The LNP of claim 114, wherein the LNP comprises two conjugates, wherein the first conjugate comprises an antibody that binds to CD7, and the second conjugate binds to CD8.

116. The LNP of claim 114, wherein the LNP comprises a conjugate, wherein the conjugate comprises a bispecific antibody binding CD7 and CD8.

117. The LNP of claim 116, wherein the bispecific antibody is an immunoglobulin with a single variable domain or Fab-ScFv.

118. The LNP as claimed in any one of claims 43 to 92, wherein the LNP binds to a first antigen on the surface of an immune cell of a first type and also binds to a second antigen on the surface of a second type of immune cell.

119. The LNP as described in claim 118, wherein the two different types of immune cells are CD4+ T cells and CD8+ T cells.

120. The LNP of claim 118, wherein the LNP comprises two conjugates, and the first conjugate comprises a first antibody that binds to a first antigen of the first type of immune cell, and the second conjugate comprises a second antibody that binds to a second antigen of the second type of immune cell.

121. The LNP of claim 118, wherein the LNP comprises a conjugate, and the conjugate comprises a bispecific antibody, and the bispecific antibody binds to both a first antigen on the first type of immune cell and a second antigen on the second type of immune cell.

122. The LNP of any one of claims 43 to 92, wherein the bispecific antibody is an immunoglobulin with a single variable domain or Fab-ScFv.

123. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to immune cells, and wherein the immune cell targeting group comprises a single antibody that binds to CD3 or CD7.

124. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to immune cells, and wherein the immune cell targeting group binds to CD7, CD8, or both CD7 and CD8.

125. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to both T cells and NK cells, wherein the immune cell targeting group is bound to: (a) Both CD3 and CD56; (b) Both CD8 and CD56; or (c) Both CD7 and CD56.

126. The LNP as claimed in any one of claims 93 to 125, wherein the LNP has an average diameter in the range of 50 nm to 200 nm.

127. The LNP of claim 126, wherein the LNP has an average diameter of about 100 nm.

128. The LNP as claimed in any one of claims 93 to 127, wherein the LNP has a polydispersity index in the range of 0.05 to 1.

129. The LNP of any one of claims 93 to 128, wherein the LNP has a zeta potential from about +10 mV to about +30 mV at pH 5.

130. The LNP of any one of claims 93 to 129, wherein the nucleic acid is DNA or RNA.

131. The LNP of claim 130, wherein the RNA is mRNA.

132. The LNP of claim 131, wherein the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine.

133. The LNP of claim 131, wherein the mRNA encodes a polypeptide capable of regulating the immune response in the immune cell.

134. The LNP of claim 133, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

135. The LNP of claim 134, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

136. The LNP of any one of claims 43 to 92, wherein the LNP is used to deliver nucleic acids to immune cells, and wherein the immune cell targeting group comprises Fab lacking natural interchain disulfide bonds.

137. The LNP of claim 136, wherein the Fab is engineered to replace one or two cysteine ​​residues forming natural interchain disulfide bonds on the natural constant light chain and the natural constant heavy chain with non-cysteine ​​amino acids, thereby removing natural interchain disulfide bonds in the Fab.

138. A method for targeting the delivery of a nucleic acid to the immune cells of a subject, the method comprising contacting the immune cells with an LNP as claimed in any one of claims 43 to 137, wherein the LNP contains the nucleic acid.

139. A method for expressing a target polypeptide in a subject's targeted immune cells, the method comprising contacting the immune cells with an LNP as claimed in any one of claims 43 to 137, wherein the LNP contains a nucleic acid encoding the polypeptide.

140. A method for modulating the cellular function of target immune cells in a subject, the method comprising administering to the subject an LNP as described in any one of claims 43 to 137, wherein the LNP comprises a nucleic acid that modulates the cellular function of the immune cells.

141. A method for treating, improving, or preventing symptoms of a disorder or disease in a subject in need, the method comprising administering an LNP to the subject to deliver a nucleic acid to the subject's immune cells, wherein the LNP is as described in any one of claims 43 to 137, wherein the LNP contains the nucleic acid.

142. The method of claim 141, wherein the barrier is an immune barrier, an inflammatory barrier, or cancer.

143. The method of claim 141, wherein the nucleic acid encodes an antigen for use in a therapeutic or preventive vaccine for treating or preventing pathogen infection.

144. The method of any one of claims 138 to 143, wherein the ionizable cationic lipid is 。 145. The method of any one of claims 138 to 143, wherein the ionizable cationic lipid is 。 146. The method of any one of claims 138 to 143, wherein the ionizable cationic lipid is 。 147. The method of any one of claims 138 to 146, wherein the immune cell targeting group comprises an antibody that binds to a T-cell antigen.

148. The method of claim 147, wherein the T-cell antigen is CD3, CD8, or both CD3 and CD8.

149. The method of any one of claims 138 to 146, wherein the immune cell targeting group comprises an antibody that binds to a natural killer (NK) cell antigen.

150. The method of claim 149, wherein the NK cell antigen is CD7, CD8, or CD56.

151. The method of any one of claims 138 to 150, wherein the antibody is a human antibody or a humanized antibody.

152. The method of any one of claims 138 to 151, wherein the immune cell targeting group is covalently coupled to the lipids in the lipid blend via a linker containing polyethylene glycol (PEG).

153. The method of claim 152, wherein the lipid covalently coupled to the immune cell targeting group via a PEG-containing linker is distearylglycerol (DSG), distearyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglyceride (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), or a ceramide.

154. The method of claim 152 or 153, wherein the PEG is PEG 2000.

155. The method of any one of claims 138 to 154, wherein the lipid-immune cell-targeting group conjugate is present in the lipid blend in the range of 0.002-0.2 mol percent.

156. The method of any one of claims 138 to 155, wherein the ionizable cationic lipid is present in the lipid blend in the range of 40-60 molar percentage.

157. The method of claims 138 to 156, wherein the sterol is cholesterol.

158. The method of any one of claims 49 to 157, wherein the sterol is present in the lipid blend in the range of 30-50 molar percentage.

159. The method of claims 138 to 158, wherein the neutral phospholipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin (SM).

160. The method of claims 138 to 159, wherein the neutral phospholipid is present in the lipid blend in the range of 5-15 molar percentage.

161. The method of any one of claims 138 to 160, wherein the free PEG-lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, wherein the PEG lipid may be PEG-dioleoylglycerol (PEG-DOG), PEG-myristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycerol-phosphatidylethanolamine (PEG-DLPE), PEG-myristoyl-glycerol, etc. Acyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-distearylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG, such as PEG-DMG, PEG-DPG and PEG-DSG), PEG-ceramide, PEG-distearyl-glycerol-phosphate (PEG-DSPG), PEG-dioleoyl-glycerol-phosphate ethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide or PEG-distearyl-phosphatidylethanolamine (PEG-DSPE) lipids.

162. The method of claims 138 to 160, wherein the free PEG-lipid comprises diacylphosphatidylethanolamine, which comprises a dimyristoyl (C14) chain, a dipalmitoyl (C16) chain, or a distearate (C18) chain.

163. The method of any one of claims 138 to 162, wherein the free PEG-lipid is present in the lipid blend in the range of 0.5-2.5 mol percent.

164. The method of any one of claims 138 to 163, wherein the free PEG-lipid comprises lipids that are the same as or different from the lipids in the lipid-immune cell targeting group conjugate.

165. The method of claims 138 to 164, wherein the LNP has an average diameter in the range of 50 nm to 200 nm.

166. The method of claim 165, wherein the LNP has an average diameter of about 100 nm.

167. The method of claims 138 to 166, wherein the LNP has a polydispersity index in the range of 0.05 to 1.

168. The method of claims 138 to 167, wherein the LNP has a zeta potential from about +10 mV to about +30 mV at pH 5.

169. The method of claims 138 to 168, wherein the nucleic acid is DNA or RNA.

170. The method of claim 169, wherein the RNA is mRNA, tRNA, siRNA, gNRA, or microRNA.

171. The method of claim 170, wherein the mRNA encodes a receptor, growth factor, hormone, cytokine, antibody, antigen, enzyme, or vaccine.

172. The method of claim 170, wherein the mRNA encodes a polypeptide capable of regulating the immune response in the immune cell.

173. The method of claim 170, wherein the mRNA encodes a polypeptide capable of reprogramming the immune cell.

174. The method of claim 170, wherein the mRNA encodes a synthetic T-cell receptor (synTCR) or a chimeric antigen receptor (CAR).

175. The method of any one of claims 138 to 174, wherein the immune cell targeting group comprises an antibody, and the antibody is a Fab or immunoglobulin monovariable domain.

176. The method of any one of claims 138 to 174, wherein the immune cell targeting group comprises an antibody fragment selected from the group consisting of: Fab, F(ab')2, Fab'-SH, Fv, and scFv fragments.

177. The method of claim 175 or 176, wherein the immune cell targeting group comprises a Fab containing one or more interchain disulfide bonds.

178. The method of claim 177, wherein the Fab comprises heavy chain segments substituted with F174C and C233S and light chain segments substituted with S176C and C214S, according to the Kabat number.

179. The method of any one of claims 175 to 178, wherein the immune cell targeting group comprises a Fab containing a cysteine ​​residue at the C-terminus of the heavy or light chain segment.

180. The method of claim 175, wherein the Fab further comprises one or more amino acids between the heavy chain segment of the Fab and the C-terminal cysteine.

181. The method of any one of claims 176 to 180, wherein the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single-chain variable fragment (scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker.

182. The method of claim 175, wherein the immune cell targeting group comprises an immunoglobulin monovariable domain.

183. The method of claim 175 or 182, wherein the immunoglobulin monovariable domain comprises a cysteine ​​residue at its C-terminus.

184. The method of claim 183, wherein the immunoglobulin monovariable domain comprises V HH The structural domain, and further includes spacers contained within the V. HH One or more amino acids between the domain and the C-terminal cysteine ​​residue.

185. The method of any one of claims 175 and 182 to 184, wherein the immune cell targeting group comprises two or more V HH Structural domain.

186. The method of claim 185, wherein the two or more V HH The domains are linked by amino acid linkers.

187. The method of claim 185, wherein the immune cell targeting group comprises a first V linked to the antibody CH1 domain. HH The domain and the second V linked to the constant domain of the antibody light chain HH The antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds.

188. The method of any one of claims 175 and 182 to 184, wherein the immune cell targeting group comprises a V domain linked to the antibody CH1 domain. HH The antibody CH1 domain is connected to the antibody light chain constant domain via one or more disulfide bonds.

189. The method of claim 185 or 186, wherein the CH1 domain comprises F174C and C233S substitutions, and the light chain constant domain comprises S176C and C214S substitutions, according to the Kabat designation.

190. The method of any one of claims 138 to 174, wherein the immune cell targeting group comprises Fab, the Fab comprising: (a) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 1 and a light chain fragment containing the amino acid sequence of SEQ ID NO: 2 or 3; (b) A heavy chain fragment containing the amino acid sequence of SEQ ID NO: 6 and a light chain fragment containing the amino acid sequence of SEQ ID NO:

7.

191. The method of any one of claims 138 to 190, wherein no more than 5% of non-immune cells are transfected with the LNP.

192. The method of any one of claims 138 to 191, wherein the half-life of the nucleic acid delivered by the LNP or the polypeptide encoded by the nucleic acid delivered by the LNP is at least 10% longer than the half-life of the nucleic acid delivered by the reference LNP or the polypeptide encoded by the nucleic acid delivered by the reference LNP.

193. The method of any one of claims 138 to 192, wherein at least 10% of immune cells are transfected with the LNP.

194. The method of any one of claims 138 to 193, wherein the expression level of the nucleic acid delivered by the LNP is at least 10% higher than the expression level of the nucleic acid delivered by the reference LNP.

195. A compound having one of the following structures: 。

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