Conjugation processes

Abstract

Provided herein are methods of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, in which the antibody addition and conjugation steps are incorporated into the downstream processing stage, resulting in a continuous one-pot process for the production and processing of antibody-lipid nanoparticle conjugates. Also provided herein are antibody-lipid nanoparticle conjugates prepared by such methods. Further provided herein are tangential flow filtration (TFF) apparatuses configured to circulate a composition comprising the lipid nanoparticle during addition of the antibody, and TFF apparatuses comprising an antibody-lipid nanoparticle conjugate.

Description

[0001] CONJUGATION PROCESSES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] This application claims the benefit of and priority under 35 U. S. C. § 119(e) to U. S.

[0004] Provisional Application Number 63 / 761,807, filed February 21, 2025, the contents of which are incorporated herewith by reference.

[0005] BACKGROUND OF THE INVENTION

[0006]

[0002] Lipid nanoparticles (LNPs) have become a crucial delivery vehicle for a variety of gene / mRNA therapy. However, LNPs have been primarily used for drug delivery to the liver and local muscles, limiting applications primarily to liver-related disease and vaccines. As such, the need for extrahepatic delivery of LNPs is paramount to broadening the therapeutic application of this platform.

[0007]

[0003] Antibody targeting is an effective strategy to achieve this goal, making antibody-LNP conjugates a promising approach for applications such as in vivo CAR-T cell therapy and in vivo stem cell editing. However, the complexity of producing antibody-LNP conjugates presents significant challenges. An extra conjugation step between the LNP and antibody and extra purification steps are required to remove unwanted materials such as the unreacted antibodies from the antibody-LNP conjugate products, significantly increasing the time, cost, and risk of failure of the production process. Therefore, novel processes are required to streamline the production process of antibody-LNP conjugate.

[0008] SUMMARY OF THE INVENTION

[0009]

[0004] The present disclosure addresses these challenges by detailing a strategy for formulating antibody-conjugated LNPs in which the antibody addition and conjugation steps are incorporated into the downstream processing stage, specifically during tangential flow filtration (TFF), resulting in a continuous one-pot process for the production and processing of antibody-lipid nanoparticle conjugates.

[0010]

[0005] Accordingly, in one aspect, the present disclosure provides a method of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, the method comprising:

[0011] coupling a lipid nanoparticle with an antibody in a filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0012] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing the purified composition comprising the antibody-lipid nanoparticle conjugate.

[0013]

[0006] In another aspect, the present disclosure provides a method of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, the method comprising:

[0014] D0957.70000WQ00 1 / 51

[0015] #14937867vlpurifying a composition comprising a lipid nanoparticle in a filtration apparatus; coupling the lipid nanoparticle with an antibody in the filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0016] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing the purified composition comprising the antibody-lipid nanoparticle conjugate.

[0017]

[0007] In another aspect, the present disclosure provides an antibody-lipid nanoparticle conjugate prepared by any of the methods described herein.

[0018]

[0008] In another aspect, the present disclosure provides an antibody-lipid nanoparticle conjugate prepared by a method comprising:

[0019] coupling a lipid nanoparticle with an antibody in a filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0020] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing a purified composition comprising the antibody-lipid nanoparticle conjugate.

[0021]

[0009] In another aspect, the present disclosure provides an antibody-lipid nanoparticle conjugate prepared by a method comprising:

[0022] purifying a composition comprising a lipid nanoparticle in a filtration apparatus; coupling the lipid nanoparticle with an antibody in the filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0023] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing a purified composition comprising the antibody-lipid nanoparticle conjugate.

[0024]

[0010] In another aspect, the present disclosure provides a tangential flow filtration (TFF) apparatus comprising:

[0025] a composition comprising a lipid nanoparticle; and

[0026] means for adding an antibody to the TFF apparatus;

[0027] wherein the TFF apparatus is configured to circulate the composition comprising the lipid nanoparticle during addition of the antibody.

[0028]

[0011] In another aspect, the present disclosure provides a tangential flow filtration (TFF) apparatus comprising an antibody-lipid nanoparticle conjugate.

[0029]

[0012] It should be appreciated that the foregoing concepts, and the additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying drawings.

[0030] D0957.70000WQ00 2 / 51

[0031] #14937867vlBRIEF DESCRIPTION OF THE DRAWINGS

[0032]

[0013] FIG. 1 shows a representative schematic of antibody addition during TFF.

[0033] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0034]

[0014] The aspects described herein are not limited to specific embodiments, systems, compositions, methods, or configurations, and as such can, of course, vary. The terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0035]

[0015] The present disclosure provides methods for producing antibody-conjugated LNPs through a streamlined process that incorporates antibody conjugation during a tangential flow filtration (TFF) step. In a typical LNP process, after the lipid components and an agent (e.g., mRNA) are mixed to formulate the initial immature LNPs, a significant amount of ethanol (e.g., 25%) remains, and the buffer is acidic. LNPs are not stable for storage under these conditions, and a subsequent dilution step with a neutral buffer is often required to lower the amount of ethanol while increasing the pH closer to neutral. However, doing so decreases the concentration of the LNP solution significantly, such that performing the conjugation step immediately afterward could lead to incomplete conjugation. In addition, even after dilution, a significant amount of ethanol (e.g., 8.3-12.5%) remains, which may impair the functionality of an antibody added directly to the diluted LNPs.

[0036]

[0016] Therefore, desirable features of an antibody-LNP conjugation process include: (1) removal of a majority of ethanol as a first purification step prior to antibody addition and conjugation; (2) concentration of diluted LNPs prior to antibody addition and conjugation; and (3) removal of unreacted antibodies from the antibody-LNP conjugates following antibody addition and conjugation in the same apparatus.

[0037]

[0017] Accordingly, in one aspect, the present disclosure provides a method of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, the method comprising:

[0038] coupling a lipid nanoparticle with an antibody in a filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0039] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing the purified composition comprising the antibody-lipid nanoparticle conjugate.

[0040]

[0018] In some embodiments, the method further comprises purifying a composition comprising the lipid nanoparticle in the filtration apparatus prior to the coupling step.

[0041]

[0019] In another aspect, the present disclosure provides a method of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, the method comprising:

[0042] purifying a composition comprising a lipid nanoparticle in a filtration apparatus; coupling the lipid nanoparticle with an antibody in the filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0043] D0957.70000WQ00 3 / 51

[0044] #14937867vlpurifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing the purified composition comprising the antibody-lipid nanoparticle conjugate.

[0045]

[0020] In some embodiments, purifying the composition comprising the lipid nanoparticle comprises reducing an amount of ethanol in the composition comprising the lipid nanoparticle. In some embodiments, the amount of ethanol in the composition is reduced from between about 15% v / v and about 35% v / v to between about 0% v / v and about 5% v / v. In some embodiments, the amount of ethanol in the composition is reduced from about 25% v / v to between about 0% v / v and about 5% v / v. In some embodiments, the amount of ethanol in the composition is reduced from between about 15% v / v and about 35% v / v to between about 0% v / v and about 10% v / v, about 0% v / v and about 7.5% v / v, about 0% v / v and about 5% v / v, about 0% v / v and about 4% v / v, about 0% v / v and about 3% v / v, about 0% v / v and about 2% v / v, or about 0% v / v and about 1% v / v. In some embodiments, the amount of ethanol in the composition is reduced from between about 15% v / v and about 35% v / v to about 10% v / v, about 9% v / v, about 8% v / v, about 7% v / v, about 6% v / v, about 5% v / v, about 4% v / v, about 3% v / v, about 2% v / v, about 1% v / v, or about 0% v / v. In some embodiments, the amount of ethanol in the composition is reduced to about 10% v / v, about 9% v / v, about 8% v / v, about 7% v / v, about 6% v / v, about 5% v / v, about 4% v / v, about 3% v / v, about 2% v / v, about 1% v / v, or about 0% v / v. In some embodiments, purifying the composition comprising the lipid nanoparticle comprises removing substantially all ethanol in the composition comprising the lipid nanoparticle.

[0046]

[0021] In some embodiments, purifying the composition comprising the lipid nanoparticle comprises increasing a concentration of the composition comprising the lipid nanoparticle. In some embodiments, the concentration refers to the concentration of the lipid nanoparticle in the composition (e.g., the amount of lipid nanoparticle in the volume of the composition). In some embodiments, the concentration of the composition comprising the lipid nanoparticle is increased by a factor of between about 1.1 and about 10.0, about 1.1 and about 5.0, about 1.1 and about 3.0, about 1.5 and about 10.0, about 1.5 and about 5.0, about 1.5 and about 3.0, about 2.0 and about 10.0, about 2.0 and about 5.0, or about 2.0 and about 3.0. In some embodiments, the concentration of the composition comprising the lipid nanoparticle is increased by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0. In some embodiments, the concentration of the composition comprising the lipid nanoparticle is increased by a factor of about 3.0.

[0047]

[0022] In some embodiments, purifying the composition comprising the lipid nanoparticle comprises diafiltration of the composition comprising the lipid nanoparticle. In some embodiments, the diafiltration is performed for between about 2 and about 20, about 2 and about 16, about 2 and about 10, about 2 and about 9, about 2 and about 8, about 2 and about 7, about 2 and about 6, about 2 and about 5, about 2 and about 4, about 2 and about 3, about 3 and about 20, about 3 and about 16, about 3 and about 10, about 3 and about 9, about 3 and about 8, about 3 and about 7, about 3 and about 6, about 3 and about 5, about 3 and about 4, about 4 and about 10, about 4 and about 9, about 4 and D0957.70000WQ00 4 / 51

[0048] #14937867vlabout 8, about 4 and about 7, about 4 and about 6, or about 4 and about 5 diafiltration volumes. In some embodiments, the diafiltration is performed for between 2 and 10 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 10 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 9 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 8 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 7 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 6 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 5 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 4 and about 10 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 4 and about 8 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 4 and about 6 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 2 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 3 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 4 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 5 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 6 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 7 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 8 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 9 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 10 diafiltration volumes.

[0049]

[0023] In some embodiments, the filtration apparatus is a tangential flow filtration (TFF) apparatus. In some embodiments, coupling the lipid nanoparticle with the antibody in the filtration apparatus comprises closing a permeate line of the TFF apparatus. In some embodiments, the method further comprises reducing a flow rate of the TFF apparatus. In some embodiments, the method further comprises adding the antibody to the TFF apparatus. In some embodiments, the composition comprising the lipid nanoparticle is circulated throughout the TFF apparatus during addition of the antibody. In some embodiments, the lipid nanoparticle and antibody are maintained within the filtration apparatus during the coupling step. In some embodiments, the lipid nanoparticle and antibody are maintained within the TFF apparatus during the coupling step.

[0050]

[0024] In some embodiments, the coupling step comprises a reaction time of between about 5 minutes and about 24 hours. In some embodiments, the reaction time is between about 5 minutes and D0957.70000WQ00 5 / 51

[0051] #14937867vlabout 20 hours, about 5 minutes and about 16 hours, about 5 minutes and about 12 hours, about 5 minutes and about 8 hours, about 5 minutes and about 6 hours, about 5 minutes and about 4 hours, about 5 minutes and about 3 hours, about 5 minutes and about 2 hours, about 5 minutes and about 1 hour, about 5 minutes and about 30 minutes, about 5 minutes and about 15 minutes, about 15 minutes and about 8 hours, about 15 minutes and about 4 hours, about 15 minutes and about 2 hours, about 15 minutes and about 1 hour, about 30 minutes and about 8 hours, about 30 minutes and about 4 hours, about 30 minutes and about 2 hours, about 30 minutes and about 1 hour, about 1 hour and about 8 hours, about 1 hour and about 4 hours, about 1 hour and about 2 hours, about 2 hours and about 8 hours, or about 2 hours and about 4 hours. In some embodiments, the reaction time is between about 5 minutes and about 8 hours, about 5 minutes and about 4 hours, about 5 minutes and about 2 hours, about 5 minutes and about 1 hour, about 5 minutes and about 30 minutes, about 5 minutes and about 15 minutes, about 30 minutes and about 8 hours, about 30 minutes and about 4 hours, about 30 minutes and about 1 hour, about 1 hour and about 8 hours, about 1 hour and about 4 hours, about 1 hour and about 2 hours, or about 2 hours and about 4 hours. In some embodiments, the reaction time is between about 5 minutes and about 4 hours.

[0052]

[0025] In some embodiments, purifying the composition comprising the antibody-lipid nanoparticle further comprises opening the permeate line of the TFF apparatus. In some embodiments, purifying the composition comprising the antibody-lipid nanoparticle further comprises increasing a flow rate of the TFF apparatus. In some embodiments, the antibody-lipid nanoparticle conjugate is maintained within the filtration apparatus during the purification step.

[0053]

[0026] In some embodiments, purifying the composition comprising the antibody-lipid nanoparticle conjugate comprises increasing the concentration of the composition comprising the antibody-lipid nanoparticle conjugate. In some embodiments, the concentration refers to the concentration of the antibody-lipid nanoparticle conjugate in the composition (e.g., the amount of antibody-lipid nanoparticle conjugate in the volume of the composition). In some embodiments, the concentration of the composition comprising the antibody-lipid nanoparticle conjugate is increased by a factor of between about 1.1 and about 10.0, about 1.1 and about 5.0, about 1.1 and about 3.0, about 1.5 and about 10.0, about 1.5 and about 5.0, about 1.5 and about 3.0, about 2.0 and about 10.0, about 2.0 and about 5.0, or about 2.0 and about 3.0. In some embodiments, the concentration of the composition comprising the antibody-lipid nanoparticle conjugate is increased by a factor of about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 5.0.

[0054]

[0027] In some embodiments, purifying the composition comprising the antibody-lipid nanoparticle conjugate comprises diafiltration of the composition comprising the antibody-lipid nanoparticle conjugate. In some embodiments, the diafiltration is performed for between about 2 and about 20, about 2 and about 16, about 2 and about 10, about 2 and about 9, about 2 and about 8, about 2 and about 7, about 2 and about 6, about 2 and about 5, about 2 and about 4, about 2 and about 3, about 3 and about 20, about 3 and about 16, about 3 and about 10, about 3 and about 9, about 3 and about 8, D0957.70000WQ00 6 / 51

[0055] #14937867vlabout 3 and about 7, about 3 and about 6, about 3 and about 5, about 3 and about 4, about 4 and about 10, about 4 and about 9, about 4 and about 8, about 4 and about 7, about 4 and about 6, or about 4 and about 5 diafiltration volumes. In some embodiments, the diafiltration is performed for between 2 and 10 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 10 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 9 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 8 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 7 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 6 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 3 and about 5 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 4 and about 10 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 4 and about 8 diafiltration volumes. In some embodiments, the diafiltration is performed for between about 4 and about 6 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 2 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 3 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 4 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 5 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 6 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 7 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 8 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 9 diafiltration volumes. In some embodiments, the diafiltration is performed for at least about 10 diafiltration volumes.

[0056]

[0028] In some embodiments, the purified composition comprising the antibody-lipid nanoparticle conjugate comprises between about 0 molar % and about 5 molar %, about 0 molar % and about 4 molar %, about 0 molar % and about 3 molar %, about 0 molar % and about 2 molar %, about 0 molar % and about 1 molar %, about 0 molar % and about 0.5 molar %, or about 0 molar % and about 0.1 molar % of the antibody (z.e., unreacted antibody). In some embodiments, the purified composition comprising the antibody-lipid nanoparticle conjugate comprises about 5.0 molar %, about 4.0 molar %, about 3.0 molar %, about 2.0 molar %, about 1.0 molar %, about 0.5 molar %, about 0.1 molar %, or about 0.0 molar % of the antibody. In some embodiments, the purified composition comprising the antibody-lipid nanoparticle conjugate is substantially free of the antibody.

[0057] D0957.70000WQ00 7 / 51

[0058] #14937867vlTangential Flow Filtration (TFF) Apparatuses

[0059]

[0029] In another aspect, the present disclosure provides a tangential flow filtration (TFF) apparatus comprising:

[0060] a composition comprising a lipid nanoparticle; and

[0061] means for adding an antibody to the TFF apparatus;

[0062] wherein the TFF apparatus is configured to circulate the composition comprising the lipid nanoparticle during addition of the antibody.

[0063]

[0030] In another aspect, the present disclosure provides a tangential flow filtration (TFF) apparatus comprising an antibody-lipid nanoparticle conjugate, optionally comprising a lipid nanoparticle and an antibody.

[0064]

[0031] Generally, the TFF apparatus comprises a cassette housing a tangential flow filter (e.g., a column) and configured to permit a flow of fluid to contact and pass tangent to the filter. The tangential flow filter may be a membrane (e.g., a porous membrane), according to some embodiments. In some embodiments, the tangential flow filter may be a flat sheet membrane. In some embodiments, the tangential flow filter is configured to retain or permit permeation of species based on their size (e.g., by allowing smaller species to pass through pores of the filter while retaining larger species that cannot pass through the pores in the tangential flow of fluid). After filtration, fluid retained in the flow that passed tangentially to the filter is the retentate, while fluid that passed through the filter is the permeate.

[0065]

[0032] Any of a variety of suitable tangential flow filter materials may be used. For example, in some embodiments, the tangential flow filter comprises a polymer membrane. The polymer membrane may be hydrophilic, in some embodiments. According to some embodiments, for example, the polymer membrane comprises polyethersulfone (PES). For example, the polymer membrane may be a Pall Omega ™ (PES) membrane or a generic equivalent thereof. In some embodiments, the polymer membrane is hydrophobic. According to some embodiments, for example, the polymer membrane comprises polyvinylidene fluoride (PVDF). For example, the polymer membrane may be a Planova™ membrane (e.g., a Planova ™ 35N membrane) or a generic equivalent thereof. Different flow conditions (e.g., different pressures, loadings, and flow-rates) may be suitable for different tangential flow filters, e.g., depending on the hydrophilicity or hydrophobicity of the tangential flow filter.

[0066]

[0033] A tangential flow filter may have any of a variety of suitable areas, depending on the embodiment. In some embodiments, a tangential flow filter has an area of greater than or equal to 0.1 m2, greater than or equal to 0.2 m2, greater than or equal to 0.5 m2, greater than or equal to 1 m2, greater than or equal to 2 m2, greater than or equal to 3 m2, greater than or equal to 4 m2, greater than or equal to 5 m2, greater than or equal to 6 m2, greater than or equal to 7 m2, greater than or equal to 8 m2, or greater than or equal to 9 m2. In some embodiments, a tangential flow filter has an area of less than or equal to 10 m2, less than or equal to 9 m2, less than or equal to 8 m2, less than or equal to 7 m2, D0957.70000WQ00 8 / 51

[0067] #14937867vlless than or equal to 6 m2, less than or equal to 5 m2, less than or equal to 4 m2, less than or equal to 3 m2, less than or equal to 2 m2, less than or equal to 1 m2, less than or equal to 0.5 m2, or less than or equal to 0.2 m2. Combinations of these ranges are also possible (e.g., greater than or equal to 0.1 m2and less than or equal to 10 m2, greater than or equal to 1 m2and less than or equal to 8 m2, or greater than or equal to 0.1 m2and less than or equal to 0.5 m2).

[0068]

[0034] A tangential flow filter may be configured for size exclusion filtration of species with any of a variety of suitable molecular weights. In some embodiments, the TFF apparatus comprises a column (e.g., a tangential flow filter) having a molecular weight cut-off (MWCO) of between about 10 kDa and about 500 kDa, about 50 kDa and about 500 kDa, about 100 kDa and about 500 kDa, about 200 kDa and about 500 kDa, about 300 kDa and about 500 kDa, about 400 kDa and about 500 kDa, about 100 kDa and about 400 kDa, about 100 kDa and about 300 kDa, about 100 kDa and about 200 kDa, about 200 kDa and about 400 kDa, about 200 kDa and about 300 kDa, or about 300 kDa and about 400 kDa. In some embodiments, the column has a MWCO of at least about 100 kDa, at least about 200 kDa, at least about 300 kDa, at least about 400 kDa, or at least about 500 kDa. In some embodiments, the column has a MWCO of at least about 100 kDa. In some embodiments, the column has a MWCO of at least about 200 kDa. In some embodiments, the column has a MWCO of at least about 300 kDa. In some embodiments, the column has a MWCO of at least about 400 kDa. In some embodiments, the column has a MWCO of at least about 500 kDa.

[0069]

[0035] A tangential flow filtration cassette may generally include any of a variety of appropriate number of tangential flow filters (e.g., adjacent filters configured to operate in parallel by filtering the same fluid flow tangential to all the filters). Each filter may independently have physical properties (e.g., an area and / or a molecular-weight cut-off) described within the above ranges. In some embodiments, a tangential flow filtration cassette comprises greater than or equal to 1 filter, greater than or equal to 2 filters, greater than or equal to 3 filters, or greater than or equal to 4 filters. In some embodiments, a tangential flow filtration cassette comprises less than or equal to 5 filters, less than or equal to 4 filters, less than or equal to 3 filters, or less than or equal to 2 filters. Combinations of these ranges are also possible (e.g., greater than or equal to 1 filter and less than or equal to 5 filters, or greater than or equal to 1 filter and less than or equal to 3 filters).

[0070]

[0036] Any of appropriate pressures may be used for tangential flow filtration. In some embodiments, tangential flow filtration is performed with a pressure of greater than or equal to 1 psi, greater than or equal to 2 psi, greater than or equal to 3 psi, greater than or equal to 4 psi, greater than or equal to 5 psi, greater than or equal to 6 psi, greater than or equal to 7 psi, greater than or equal to 8 psi, greater than or equal to 9 psi, greater than or equal to 10 psi, greater than or equal to 11 psi, greater than or equal to 12 psi, greater than or equal to 13 psi, greater than or equal to 14 psi, or greater. In some embodiments, tangential flow filtration is performed with a pressure of less than or equal to 15 psi, less than or equal to 14 psi, less than or equal to 13 psi, less than or equal to 12 psi, less than or equal to 11 psi, less than or equal to 10 psi, less than or equal to 9 psi, less than or equal to D0957.70000WQ00 9 / 51

[0071] #14937867vl8 psi, less than or equal to 7 psi, less than or equal to 6 psi, less than or equal to 5 psi, less than or equal to 4 psi, less than or equal to 3 psi, or less than or equal to 2 psi. Combinations of these ranges are also possible (e.g., greater than or equal to 1 psi and less than or equal to 15 psi, greater than or equal to 10 psi and less than or equal to 15 psi, or greater than or equal to 3 psi and less than or equal to 5 psi).

[0072]

[0037] Tangential flow filtration may be performed with any of a variety of suitable filter loading volumes (referring to the volume of mixture which may be loaded per square meter of total filter area of the tangential flow filter(s)). In some embodiments, a tangential flow filter is loaded to a capacity of greater than or equal to 5 L / m2, greater than or equal to 10 L / m2, greater than or equal to 20 L / m2, greater than or equal to 30 L / m2, greater than or equal to 40 L / m2, greater than or equal to 50 L / m2, greater than or equal to 60 L / m2, greater than or equal to 70 L / m2, greater than or equal to 80 L / m2, greater than or equal to 90 L / m2, greater than or equal to 100 L / m2, greater than or equal to 110 L / m2, greater than or equal to 120 L / m2, greater than or equal to 130 L / m2, or greater than or equal to 140 L / m2. In some embodiments, tangential flow filter is loaded to a capacity of less than or equal to 150 L / m2, less than or equal to 140 L / m2, less than or equal to 130 L / m2, less than or equal to 120 L / m2, less than or equal to 110 L / m2, less than or equal to 100 L / m2, less than or equal to 90 L / m2, less than or equal to 80 L / m2, less than or equal to 70 L / m2, less than or equal to 60 L / m2, less than or equal to 50 L / m2, less than or equal to 40 L / m2, less than or equal to 30 L / m2, or less than or equal to 20 L / m2. Combinations of these ranges are also possible (e.g., greater than or equal to 10 L / m2and less than or equal to 150 L / m2, greater than or equal to 10 L / m2and less than or equal to 100 L / m2, or greater than or equal to 10 L / m2and less than or equal to 50 L / m2).

[0073]

[0038] Any of a variety of suitable flow rates may be used for tangential flow filtration. In some embodiments, tangential flow filtration is performed with a flow rate of greater than or equal to 5 L / hr, greater than or equal to 10 L / hr, greater than or equal to 50 L / hr, greater than or equal to 100 L / hr, greater than or equal to 200 L / hr, greater than or equal to 300 L / hr, greater than or equal to 400 L / hr, greater than or equal to 500 L / hr, greater than or equal to 600 L / hr, greater than or equal to 700 L / hr, greater than or equal to 800 L / hr, greater than or equal to 900 L / hr, greater than or equal to 1000 L / hr, greater than or equal to 1100 L / hr, greater than or equal to 1200 L / hr, greater than or equal to 1300 L / hr, or greater than or equal to 1400 L / hr. In some embodiments, tangential flow filtration is performed with a flow rate of less than or equal to 1500 L / hr, less than or equal to 1400 L / hr, less than or equal to 1300 L / hr, less than or equal to 1200 L / hr, less than or equal to 1100 L / hr, less than or equal to 1000 L / hr, less than or equal to 900 L / hr, less than or equal to 800 L / hr, less than or equal to 700 L / hr, less than or equal to 600 L / hr, less than or equal to 500 L / hr, less than or equal to 400 L / hr, less than or equal to 300 L / hr, less than or equal to 200 L / hr, less than or equal to 100 L / hr, or less than or equal to 50 L / hr. Combinations of these ranges are also possible (e.g., greater than or equal to 100 L / hr and less than or equal to 1500 L / hr, greater than or equal to 1000 L / hr and less than or equal to 1500 L / hr, greater than or equal to 400 L / hr and less than or equal to 700 L / hr, greater than or equal D0957.70000WQ00 10 / 51

[0074] #14937867vlto 100 L / hr and less than or equal to 200 L / hr, or greater than or equal to 5 L / hr and less than or equal to 100 L / hr).

[0075]

[0039] Tangential flow filtration may be performed using a suitable buffer. Any of a variety of suitable buffers may be used in the one or more solvent solutions of the tangential flow filtration buffer. For example, the buffer may comprise a tris(hydroxymethyl)aminomethane (Tris), a bis-tris-propane (BTP) buffer, a biological buffer (e.g., MOPS, HEPES, PIPES, etc.), a phosphate buffer (e.g., phosphate buffered saline) or a mixture or combination thereof. In some embodiments, a tangential flow filtration buffer has a concentration of greater than or equal to 10 mM, greater than or equal to 20 mM, greater than or equal to 50 mM, greater than or equal to 100 mM, greater than or equal to 200 mM, greater than or equal to 500 mM, greater than or equal to 1.0 M, greater than or equal to 2.0 M, greater than or equal to 5.0 M, or greater than or equal to 8.0 M. In some embodiments, a tangential flow filtration buffer has a concentration of less than or equal to 10.0 M, less than or equal to 8.0 M, less than or equal to 5.0 M, less than or equal to 2.0 M, less than or equal to 1.0 M, less than or equal to 500 mM, less than or equal to 200 mM, less than or equal to 100 mM, less than or equal to 50 mM, or less than or equal to 20 mM. Combinations of these ranges are also possible (e.g., greater than or equal to 10 mM and less than or equal to 10.0 M, or greater than or equal to 100 mM and less than or equal to 2.0 M).

[0076]

[0040] In some embodiments, the TFF apparatus comprises a permeate line configured to close before addition of the antibody. In some embodiments, the TFF apparatus comprises a permeate line configured to open after conjugation of the lipid nanoparticle to the antibody. In some embodiments, the antibody-lipid nanoparticle conjugate is maintained within the TFF apparatus during conjugation of the lipid nanoparticle to the antibody.

[0077]

[0041] The schematic of the TFF apparatus during addition of the antibody shown in FIG. 1 is exemplary and is non-limiting as to the specific location at which the antibody is added. As such, the antibody may be added to the TFF apparatus at any suitable location. For instance, in some embodiments, the antibody may be added through a T or Y valve of the TFF apparatus, to the concentrate, etc. The means for adding an antibody to the TFF apparatus may be any suitable means. For instance, in some embodiments, the means for adding an antibody to the TFF apparatus may be manual addition, addition using a syringe pump, addition using a peristaltic pump, etc.

[0078] Lipid Nanoparticles

[0079]

[0042] The term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids.

[0080]

[0043] The term “particle” refers to a small object, fragment, or piece of a substance that may be a single element, inorganic material, organic material, or mixture thereof. Examples of particles include

[0081] D0957.70000WQ00 11 / 51

[0082] #14937867vlpolymeric particles, single-emulsion particles, double-emulsion particles, coacervates, liposomes, microparticles, nanoparticles (e.g., lipid nanoparticles), macroscopic particles, pellets, crystals, aggregates, composites, pulverized, milled or otherwise disrupted matrices, and cross-linked protein or polysaccharide particles, each of which have an average characteristic dimension of about less than about 1 mm and at least 1 nm, where the characteristic dimension, or “critical dimension,” of the particle is the smallest cross-sectional dimension of the particle. A particle may be composed of a single substance or multiple substances. In some embodiments, the particle is a nanoparticle.

[0083]

[0044] The term “nanoparticle” refers to a particle having a characteristic dimension of less than about 1 micrometer and at least about 1 nanometer, where the characteristic dimension of the particle is the smallest cross-sectional dimension of the particle.

[0084]

[0045] In some embodiments, the average diameter of a nanoparticle is at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at least about 170 nm, at least about 180 nm, at least about 190 nm, at least about 200 nm, at least about 210 nm, at least about 220 nm, at least about 230 nm, at least about 240 nm, at least about 250 nm, at least about 260 nm, at least about 270 nm, at least about 280 nm, at least about 290 nm, at least about 300 nm, at least about 350 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 600 nm, at least about 700 nm, at least about 800 nm, or at least about 900 nm. In some embodiments, the average diameter of the nanoparticle is less than about 1 μm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 290 nm, less than about 280 nm, less than about 270 nm, less than about 260 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm. Combinations of the above ranges (e.g., at least about 100 nm and less than about 1 μm) are also within the scope of the present invention.

[0085]

[0046] The nanoparticles described herein may include additional materials such as polymers (e.g., synthetic polymers (e.g., PEG, PLGA) and natural polymers (e.g., phospholipids)). In some embodiments, the additional materials are approved by a regulatory agency, such as the U. S. FDA, for human and veterinary use.

[0086]

[0047] The nanoparticles (e.g., lipid nanoparticles) may be prepared using any method known in the art, such as precipitation, milling, spray drying, single and double emulsion solvent evaporation, D0957.70000WQ00 12 / 51

[0087] #14937867vlsolvent extraction, phase separation, and simple and complex coacervation. In some embodiments, methods of preparing the nanoparticles are the double emulsion process and spray drying. The conditions used in preparing the nanoparticles may be altered to yield particles of a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, “stickiness”, shape, polydispersity, etc.). The method of preparing the nanoparticle and the conditions (e.g., solvent, temperature, concentration, and air flow rate, etc.) used may also depend on the agent being complexed, encapsulated, or mixed, and / or the composition of the matrix. Methods developed for making nanoparticles for delivery of agents that are included in the nanoparticles are described in the literature. See, e.g., Doubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release 5: 13-22, 1987; Mathiowitz et al., Reactive Polymers 6:275-283, 1987; Mathiowitz et al., J. Appl. Polymer Sci. 35:755-774, 1988.

[0088]

[0048] If the nanoparticles prepared by any of the above methods have a size range outside of the desired range, the nanoparticles can be sized, for example, using a sieve. The nanoparticles may also be coated. In some embodiments, the nanoparticles are coated with a surface-altering agent. In some embodiments, the nanoparticles are coated to achieve desirable surface properties (e.g., a particular charge).

[0089]

[0049] In some embodiments, the polydispersity index (PDI, determined by dynamic light scattering) of the nanoparticles described herein (e.g., lipid nanoparticles) is between 0.01 and 0.9, between 0.1 and 0.9, between 0.1 and 0.7, between 0.1 and 0.5, between 0.01 and 0.4, between 0.03 and 0.4, between 0.1 and 0.4, between 0.01 and 0.3, between 0.03 and 0.3, or between 0.1 and 0.3.

[0090]

[0050] In some embodiments, the lipid nanoparticle comprises one or more of an ionizable lipid, phospholipid, sterol, PEG-lipid, or functionalized lipid. In some embodiments, the lipid nanoparticle comprises an ionizable lipid. In some embodiments, the lipid nanoparticle comprises a phospholipid. In some embodiments, the lipid nanoparticle comprises a sterol. In some embodiments, the lipid nanoparticle comprises a PEG-lipid. In some embodiments, the lipid nanoparticle comprises an ionizable lipid, phospholipid, sterol, and PEG-lipid. In some embodiments, the lipid nanoparticle comprises an ionizable lipid, phospholipid, sterol, PEG-lipid, and functionalized lipid.

[0091]

[0051] The term “click chemistry” refers to a chemical synthesis technique introduced by K. Barry Sharpless of The Scripps Research Institute, describing chemistry tailored to generate covalent bonds quickly and reliably by joining small units comprising reactive groups together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40: 2004–2021; Evans, Australian Journal of Chemistry (2007) 60: 384–395). Exemplary coupling reactions (some of which may be classified as “click chemistry”) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide–alkyne Huisgen cycloaddition; thiol–yne addition; imine formation; Michael D0957.70000WQ00 13 / 51

[0092] #14937867vladditions (e.g., maleimide addition); and Diels–Alder reactions (e.g., tetrazine [4 + 2] cycloaddition). Exemplary click chemistry reactions include, but are not limited to, azide–alkyne Huisgen cycloaddition; and Diels–Alder reactions (e.g., tetrazine [4 + 2] cycloaddition). In some embodiments, click chemistry reactions are modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force > 84 kJ / mol to favor a reaction with a single reaction product, and / or can be carried out under physiological conditions. In some embodiments, a click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, use readily available starting materials and reagents, uses no toxic solvents or use a solvent that is benign or easily removed (preferably water), and / or provides simple product isolation by non-chromatographic methods (crystallization or distillation).

[0093]

[0052] The terms “click chemistry handle” and “click handle” refer to a reactant, or a reactive group, that can partake in a click chemistry reaction. For example, a strained alkyne, e.g., a cyclooctyne, is a click chemistry handle, since it can partake in a strain-promoted cycloaddition (see, e.g., Table 1). In general, click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other. Such click chemistry handle pairs that are reactive with each other are sometimes referred to herein as partner click chemistry handles. For example, an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne. Exemplary click chemistry handles suitable for use according to some aspects of this invention are described herein, for example, in Tables 1 and 2. In some embodiments, click chemistry handles are used that can react to form covalent bonds in the presence of a metal catalyst, e.g., copper (II). In some embodiments, click chemistry handles are used that can react to form covalent bonds in the absence of a metal catalyst. Additional suitable click chemistry handles are well known to those of skill in the art, and such click chemistry handles include, but are not limited to, the click chemistry reaction partners, groups, and handles described in Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900 - 4908 and PCT / US2012 / 044584 and references therein, which references are incorporated herein by reference for click chemistry handles and methodology.

[0094] D0957.70000WQ00 14 / 51

[0095] #14937867vlTable 1: Exemplary click chemistry handles and reactions.

[0096] 1,3-d ipolar cycloaddition terminal alkyne azide

[0097] Strain-promoted cycloaddition

[0098]

[0099] strained alkyne

[0100] Diels-Alder reaction

[0101] Thiol-ene reaction

[0102]

[0103] Table 2: Exemplary click chemistry handles and reactions (from Becer, Hoogenboom, and Schubert, Click Chemistry Beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900 – 4908.).

[0104] Reagent Reagent B Mechanism Notes on reactiona,1Reference A

[0105] azide alkyne Cu-catalyzed [3+2] azide- 2 h at 60°C in H2O [9]

[0106] alkyne cycloaddition

[0107] (CuAAC)

[0108] azide cyclooctyne strain-promoted [3+2] 1 h at RT [6- azide-alkyne 8,10,11] cycloaddition (SPAAC)

[0109] azide activated [3+2] Huisgen 4 h at 50°C

[0012] alkyne cycloaddition

[0110] azide electron- [3+2] cycloaddition 1 h at RT in H₂O

[0013] deficient

[0111] alkyne

[0112] azide aryne [3+2] cycloaddition 4 h at RT in THF with [14,15] crown ether or 24 h at RT

[0113] in CH3CN

[0114] tetrazine alkene Diels-Alder retro- [4+2] 40 min at 25 °C (100% [36-38] cycloaddition yield)

[0115] N2 is the only by-product tetrazole alkene 1,3 -dipolar cycloaddition few min UV irradiation [39,40]

[0116] (photoclick) and then overnight at 4°C

[0117] D0957.70000WQ00 15 / 51

[0118] #14937867vlReagent Reagent B Mechanism Notes on reactiona,1Reference A

[0119] dithioester diene hetero-Diels-Alder 10 min at RT

[0043]

[0120] cycloaddition

[0121] anthracene maleimide [4+2] Diels-Alder reaction 2 days at reflux in

[0041]

[0122] toluene

[0123] thiol alkene radical addition 30 min UV (quantitative [19-23] (thio click) conv.) or

[0124] 24 h UV irradiation

[0125] (>96%)

[0126] thiol enone Michael addition 24 h at RT in CH₃CN

[0027] thiol maleimide Michael addition 1 h at 40°C in THF or [24-26]

[0127] 16 h at RT in dioxane

[0128] thiol para-fluoro nucleophilic substitution overnight at RT in DMF

[0032]

[0129] or

[0130] 60 min at 40°C in DMF

[0131] amine para-fluoro nucleophilic substitution 20 min MW at 95°C in

[0030]

[0132] NMP as solvent

[0133] [a] RT=room temperature, DMF=N,N-dimethylformamide, NMP=N-methylpyrolidone, THF=tetrahydrofuran, CH₃CN=acetonitrile

[0134]

[0053] In some embodiments, the lipid nanoparticle comprises a first click handle. In some embodiments, the first click handle is selected from those shown in Tables 1-2. In some embodiments, the first click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans-cyclooctene), terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety. In some embodiments, the first click handle comprises an activated alkene (e.g., maleimide). In some embodiments, the first click handle comprises a maleimide. In some embodiments, the first click handle comprises a strained alkene (e.g., trans -cyclooctene). In some embodiments, the first click handle comprises trans -cyclooctene (TCO). In some embodiments, the first click handle comprises a terminal alkyne. In some embodiments, the first click handle comprises a strained alkyne (e.g., cyclooctyne). In some embodiments, the first click handle comprises a cyclooctyne. In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl-less cyclooctyne (ALO). In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN). In some embodiments, the first click handle comprises a tetrazine. In some embodiments, the first click handle comprises a thiol. In some embodiments, the first click handle comprises an azide.

[0135] Antibody-Lipid Nanoparticle Conjugates

[0136]

[0054] In another aspect, the present disclosure provides an antibody-lipid nanoparticle conjugate prepared by any of the methods described herein.

[0137] D0957.70000WQ00 16 / 51

[0138] #14937867vl

[0055] In another aspect, the present disclosure provides an antibody-lipid nanoparticle conjugate prepared by a method comprising:

[0139] coupling a lipid nanoparticle with an antibody in a filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0140] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the fdtration apparatus, thereby providing a purified composition comprising the antibody-lipid nanoparticle conjugate.

[0141]

[0056] In some embodiments, the antibody-lipid nanoparticle conjugate is prepared by a method further comprising purifying a composition comprising the lipid nanoparticle in the filtration apparatus prior to the coupling step.

[0142]

[0057] In another aspect, the present disclosure provides an antibody-lipid nanoparticle conjugate prepared by a method comprising:

[0143] purifying a composition comprising a lipid nanoparticle in a filtration apparatus; coupling the lipid nanoparticle with an antibody in the filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; and

[0144] purifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing a purified composition comprising the antibody-lipid nanoparticle conjugate.

[0145]

[0058] In some embodiments, the antibody-lipid nanoparticle conjugate comprises one or more of an ionizable lipid, phospholipid, sterol, PEG-lipid, or functionalized lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises an ionizable lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises a phospholipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises a sterol. In some embodiments, the antibody-lipid nanoparticle conjugate comprises a PEG-lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises an ionizable lipid, phospholipid, sterol, and PEG-lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises an ionizable lipid, phospholipid, sterol, PEG-lipid, and functionalized lipid.

[0146] Ionizable Lipid

[0147]

[0059] The term “ionizable lipid” refers to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. An ionizable lipid may be positively charged, in which case it can be referred to as “cationic lipid”. In certain embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipid. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged).

[0148] Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or

[0149] D0957.70000WQ00 17 / 51

[0150] #14937867vltertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In some embodiments, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. Ionizable lipids can also be the compounds disclosed in International Publication Nos.: WO 2017 / 075531, WO 2015 / 199952, WO 2013 / 086354, or WO 2013 / 116126, or selected from formulae CLI-CLXXXXII of US Patent No. 7,404,969.

[0151]

[0060] In some embodiments, the ionizable lipid is an unsaturated ionizable lipid, a multi-tail ionizable lipid, a polymeric ionizable lipid, a biodegradable ionizable lipid and / or a branched-tail ionizable lipid. In some embodiments, the ionizable lipid is selected from Fig. 1 or Fig. 2 of Han, X., et al., Nat Commun 12, 7233 (2021). Figs. 1 and 2 of Han, X., et al., Nat Commun 12, 7233 (2021) are incorporated herein by reference in their entirety. In some embodiments, the unsaturated ionizable lipid is Dlin-MC3-DMA (i.e., MC3), OF-O2, A6, or A18-Iso2DC18. In some embodiments, the multi-tail ionizable lipid is 98N12-5, C12-200, CKK-E12, or 9A1P9. In some embodiments, the ionizable polymer-lipid is 7C1 or G0-C14. In some embodiments, the biodegradable ionizable lipid is L319, 304013, C12-200, OF-Deg-Lin, or 306-O12B. In some embodiments, the branched-tail ionizable lipid is 306O10 or FTT5. In some embodiments, the ionizable lipid is SM-102, ALC-0315, Acuitas A9, Lipid 2,2 (8,8) 4C CH3, Genevant CL1, LP000001, LP01, or MC3. In some embodiments, the ionizable lipid is SM-102 or LP01.

[0152]

[0061] In some embodiments, the lipid nanoparticle comprises between about 20 and about 80, about 30 and about 70, about 35 and about 65, about 40 and about 60, or about 45 and about 55 molar % of an ionizable lipid. In some embodiments, the lipid nanoparticle comprises about 30, about 35, about 40, about 45, about 47.5, about 49, about 50, about 51, about 52.5, about 55, about 60, about 65, or about 70 molar % of an ionizable lipid. In some embodiments, the lipid nanoparticle comprises about 50 molar % of an ionizable lipid. In some embodiments, the lipid nanoparticle comprises between about 20 and about 80, about 30 and about 70, about 35 and about 65, about 40 and about 60, or about 45 and about 55 molar % of SM-102 or LP01. In some embodiments, the lipid nanoparticle comprises about 30, about 35, about 40, about 45, about 47.5, about 49, about 50, about 51, about 52.5, about 55, about 60, about 65, or about 70 molar % of SM-102 or LP01. In some embodiments, the lipid nanoparticle comprises about 50 molar % of SM-102 or LP01.

[0153] Phospholipid

[0154]

[0062] In some embodiments, the phospholipid is a phosphoethanolamine or phosphatidylcholine. As used herein, the terms “phosphorylethanolamine” and “phosphoethanolamine” are used

[0155] D0957.70000WQ00 18 / 51

[0156] #14937867vlinterchangeably. In some embodiments, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), or 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In some embodiments, the phospholipid is a phosphoethanolamine. In some embodiments, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE) or l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In some embodiments, the phospholipid is l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC). In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE). In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0157]

[0063] In some embodiments, the lipid nanoparticle comprises between about 0 and about 75, about 0 and about 50, about 5 and about 40, about 10 and about 30, or about 15 and about 25 molar % of a phospholipid. In some embodiments, the lipid nanoparticle comprises about 5, about 10, about 15, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, or about 50 molar % of a phospholipid. In some embodiments, the lipid nanoparticle comprises about 22 molar % of a phospholipid. In some embodiments, the lipid nanoparticle comprises between about 0 and about 75, about 0 and about 50, about 5 and about 40, about 10 and about 30, or about 15 and about 25 molar % of DSPC. In some embodiments, the lipid nanoparticle comprises about 5, about 10, about 15, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, or about 50 molar % of DSPC. In some embodiments, the lipid nanoparticle comprises about 22 molar % of DSPC.

[0158] Sterol

[0159]

[0064] The term “sterol” refers to a subgroup of steroids also known as steroid alcohols, i. e., a steroid containing at least one hydroxyl group. Sterols are usually divided into two classes: (1) plant sterols also known as “phytosterols,” and (2) animal sterols also known as “zoosterols.” The term “sterol” includes, but is not limited to, cholesterol, sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, and all natural or synthesized forms and derivatives thereof, including isomers.

[0160]

[0065] In some embodiments, the sterol is cholesterol, sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol,

[0161] D0957.70000WQ00 19 / 51

[0162] #14937867vlfecosterol, pollinastasterol, or a derivative thereof. In some embodiments, the sterol is cholesterol, or a derivative thereof. In some embodiments, the sterol is cholesterol.

[0163]

[0066] In some embodiments, the lipid nanoparticle comprises between about 0 and about 75, about 0 and about 50, about 5 and about 50, about 10 and about 40, about 15 and about 35, or about 20 and about 30 molar % of a sterol. In some embodiments, the lipid nanoparticle comprises about 5, about 10, about 15, about 20, about 22.5, about 24, about 25, about 26, about 27.5, about 30, about 35, about 40, about 45, or about 50 molar % of a sterol. In some embodiments, the lipid nanoparticle comprises about 25 molar % of a sterol. In some embodiments, the lipid nanoparticle comprises about 25.5 molar % of a sterol. In some embodiments, the lipid nanoparticle comprises between about 0 and about 75, about 0 and about 50, about 5 and about 50, about 10 and about 40, about 15 and about 35, or about 20 and about 30 molar % of cholesterol. In some embodiments, the lipid nanoparticle comprises about 5, about 10, about 15, about 20, about 22.5, about 24, about 25, about 26, about 27.5, about 30, about 35, about 40, about 45, or about 50 molar % of cholesterol. In some embodiments, the lipid nanoparticle comprises about 25 molar % of cholesterol. In some embodiments, the lipid nanoparticle comprises about 25.5 molar % of cholesterol.

[0164] PEG-Lipid

[0165]

[0067] The term “PEG-lipid” refers to a PEGylated lipid. In some embodiments, the PEG-lipid is a PEG-phospholipid or PEG-glyceride lipid.

[0166]

[0068] In some embodiments, the PEG-lipid is a PEG-phospholipid. In some embodiments, the PEG-phospholipid is a PEG-phosphoethanolamine. In some embodiments, the PEG-phospholipid is a PEG-phosphatidylcholine. In some embodiments, the PEG-lipid is l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14PEG2000).

[0167]

[0069] In some embodiments, the PEG component of the PEG-lipid has a molecular weight of about 350, about 550, about 750, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, or about 10000. In some embodiments, the PEG component of the PEG-lipid has a molecular weight of about 750, about 1000, about 2000, about 3000, about 4000, or about 5000. In some embodiments, the PEG component of the PEG-lipid has a molecular weight of 500-1000, 1000-2000, 2000-3000, 3000-4000, 4000-5000, 5000-6000, 6000-7000, 7000-8000, 8000-9000, or 9000-10000. In some embodiments, the PEG component of the PEG-lipid has a molecular weight of about 1000, about 2000, or about 3000. In some embodiments, the PEG component of the PEG-lipid has a molecular weight of about 2000.

[0168]

[0070] In some embodiments, the PEG-lipid is stearoyl-substituted (C₁₈). In some embodiments, the PEG-lipid is palmitoyl-substituted (C₁₆). In some embodiments, the PEG-lipid is myristoyl-substituted (C₁₄).

[0169]

[0071] In some embodiments, the PEG-lipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (C₁₈PEG5000), 1,2-

[0170] D0957.70000WQ00 20 / 51

[0171] #14937867vldipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (C₁₈PEG5000), 1,2-dimyristoyl-sn-glycero-3 -phosphoethanolamine-N- [methoxy(polyethylene glycol)-5000] (C₁₄PEG5000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (C₁₈PEG3000), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (C₁₆PEG3000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (C14PEG3000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₈PEG2000), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₆PEG2000), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₄PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (C₁₈PEG1000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (C₁₆PEG1000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 1000] (C₁₄PEG1000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (C₁₈PEG750), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (C₁₆PEG750), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (C14PEG750). In some embodiments, the PEG-lipid is selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₈PEG2000), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₆PEG2000), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₄PEG2000). In some embodiments, the PEG-lipid is selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (C₁₄PEG5000), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (C14PEG3000), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₄PEG2000), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (C₁₄PEG1000), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750] (C14PEG750). In some embodiments, the PEG-phospholipid is l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C₁₄PEG2000). In some embodiments, the PEG-lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG).

[0172]

[0072] In some embodiments, the PEG-lipid is a PEG-glyceride lipid. In some embodiments, the PEG-lipid is l,2-distearoyl-rac-glycero-3 -methoxypolyethylene glycol or 1,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol. In some embodiments, the PEG-lipid is l,2-distearoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DSG-PEG2000), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (DMG-PEG2000 or DMG-PEG2k), or 1,2-dipalmitoyl-rac- D0957.70000WQ00 21 / 51

[0173] #14937867vlglycero-3 -methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the PEG-lipid is 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DSG-PEG2000). In some embodiments, the PEG-lipid is l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2000 or DMG-PEG2k). In some embodiments, the PEG-lipid is l,2-dipalmitoyl-rac-glycero-3-methylpoly oxyethylene.

[0174]

[0073] In some embodiments, the lipid nanoparticle comprises between about 0.0 and about 20.0, about 0.0 and about 15.0, about 0.0 and about 10.0, about 0.0 and about 5.0, about 1.0 and about 20.0, about 1.0 and about 15.0, about 1.0 and about 10.0, about 1.0 and about 5.0, or about 1.0 and about 3.0 molar % of a PEG-lipid. In some embodiments, the lipid nanoparticle comprises about 0.5, about 1.0, about 1.25, about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.5, about 4.0, or about 5.0 molar % of a PEG-lipid. In some embodiments, the lipid nanoparticle comprises about 2.0 molar % of a PEG-lipid. In some embodiments, the lipid nanoparticle comprises between about 0.0 and about 20.0, about 0.0 and about 15.0, about 0.0 and about 10.0, about 0.0 and about 5.0, about 1.0 and about 20.0, about 1.0 and about 15.0, about 1.0 and about 10.0, about 1.0 and about 5.0, or about 1.0 and about 3.0 molar % of DMG-PEG2k. In some embodiments, the lipid nanoparticle comprises about 0.5, about 1.0, about 1.25, about 1.5, about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.5, about 4.0, or about 5.0 molar % of DMG-PEG2k. In some embodiments, the lipid nanoparticle comprises about 2.0 molar % of DMG-PEG2k.

[0175] Functionalized Lipid

[0176]

[0074] In some embodiments, the functionalized lipid comprises a PEG component. In some embodiments, the PEG component of the functionalized lipid has a molecular weight of about 350, about 550, about 750, about 1000, about 2000, about 3000, about 4000, or about 5000. In some embodiments, the PEG component of the functionalized lipid has a molecular weight of about 500, about 750, about 1000, about 2000, about 3000, about 4000, or about 5000. In some embodiments, the PEG component of the functionalized lipid has a molecular weight of 500-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000. In some embodiments, the PEG component of the functionalized lipid has a molecular weight of about 1000, about 2000, or about 3000. In some embodiments, the PEG component of the functionalized lipid has a molecular weight of about 2000.

[0177]

[0075] In some embodiments, the functionalized lipid comprises a first click handle. In some embodiments, the functionalized lipid comprises a first click handle selected from those shown in Tables 1-2. In some embodiments, the first click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans -cyclooctene), terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety. In some embodiments, the first click handle comprises an activated alkene (e.g., maleimide). In some embodiments, the first click handle comprises a maleimide. In some embodiments, the first click handle comprises a strained alkene (e.g., trans-cyclooctene). In some embodiments, the first click handle comprises trans -cyclooctene (TCO). In

[0178] D0957.70000WQ00 22 / 51

[0179] #14937867vlsome embodiments, the first click handle comprises a terminal alkyne. In some embodiments, the first click handle comprises a strained alkyne (e.g., cyclooctyne). In some embodiments, the first click handle comprises a cyclooctyne. In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl -less cyclooctyne (ALO). In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN). In some embodiments, the first click handle comprises a tetrazine. In some embodiments, the first click handle comprises a thiol. In some embodiments, the first click handle comprises an azide.

[0180]

[0076] In some embodiments, the functionalized lipid comprises a PEG component and an activated alkene (e.g., maleimide), strained alkene (e.g., trans-cyclooctene). terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety (e.g., the functionalized lipid is a PEG with a first click handle).

[0181]

[0077] In some embodiments, the lipid nanoparticle comprises between about 0.0 and about 5.0, about 0.0 and about 2.5, about 0.0 and about 1.0, about 0.0 and about 0.5, about 0.25 and about 0.75, or about 0.5 and about 1.0 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises about 0.5 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises between about 0.0 and about 5.0, about 0.0 and about 2.5, about 0.0 and about 1.0, about 0.0 and about 0.5, about 0.25 and about 0.75, or about 0.5 and about 1.0 molar % of PEG with a first click handle. In some embodiments, the lipid nanoparticle comprises about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 molar % of PEG with a first click handle. In some embodiments, the lipid nanoparticle comprises about 0.5 molar % of PEG with a first click handle.

[0182] Antibody

[0183]

[0078] An “amino acid” refers to natural and unnatural D / L alpha-amino acids, as well as natural and unnatural beta- and gamma- amino acids. A “peptide” refers to two amino acids joined by a peptide bond. A “polypeptide” refers to three or more amino acids joined by peptide bonds. An “amino acid side chain” refers to the group(s) pended to the alpha carbon (if an alpha amino acid), alpha and beta carbon (if a beta amino acid), or the alpha, beta, and gamma carbon (if a gamma amino acid).

[0184] Exemplary amino acid side chains are depicted herein.

[0185]

[0079] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of

[0186] D0957.70000WQ00 23 / 51

[0187] #14937867vlany size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and / or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these.

[0188]

[0080] As used herein, the term “antibody” refers to a polypeptide that comprises at least one immunoglobulin variable domain, which comprises at least one distinct antigen-specific binding site, or a portion of an immunoglobulin variable domain (such as a paratope or portion thereof) that comprises at least one distinct antigen-specific binding site. In some embodiments, an antibody is a full-length antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence. In another embodiment, an antibody comprises a heavy chain constant region selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant regions. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and / or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant region comprising, e.g., an Fc region. An immunoglobulin constant regions refers to a heavy or light chain constant region. Human IgG heavy chain and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (s), gamma (y) or mu (p) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (s), gamma (y) or mu (p) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CHI, CH2, and / or CH3 domain. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U. S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra. In some embodiments, an antibody comprises a heavy chain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any

[0189] D0957.70000WQ00 24 / 51

[0190] #14937867vlof the variable chain constant regions provided herein. In some embodiments, an antibody comprises a light chain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the light chain constant regions provided herein. In some embodiments, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecule are conjugated to the antibody viaN-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, or a phospholipid unit. In some embodiments, an antibody is aglycosylated (e.g., afucosylated). In some embodiments, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Still further, an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

[0191]

[0081] In some embodiments, the antibody is a full-length antibody, chimeric antibody, Fab fragment, F(ab')2 fragment, Fv fragment, scFv fragment, multispecific antibody, bispecific antibody, nanobody, or diabody.

[0192]

[0082] In some embodiments, the antibody comprises a second click handle. In some embodiments, the second click handle is selected from those shown in Tables 1-2. In some embodiments, the second click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans-cyclooctene). terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety. In some embodiments, the second click handle comprises an activated alkene (e.g., maleimide). In some embodiments, the second click handle comprises a maleimide. In some embodiments, the second click handle comprises a strained alkene (e.g., trans -cyclooctene). In some embodiments, the second click handle comprises trans -cyclooctene (TCO). In some embodiments, the second click handle comprises a terminal alkyne. In some embodiments, the second click handle comprises a strained alkyne (e.g., D0957.70000WQ00 25 / 51

[0193] #14937867vlcyclooctyne). In some embodiments, the second click handle comprises a cyclooctyne. In some embodiments, the second click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl-less cyclooctyne (ALO). In some embodiments, the second click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN). In some embodiments, the second click handle comprises a tetrazine. In some embodiments, the second click handle comprises a thiol. In some embodiments, the second click handle comprises an azide.

[0194]

[0083] The second click handle may be connected to (e.g., bonded to) the antibody at any residue, optionally wherein a linker connects the second click handle and the antibody. The term “linker” refers to a divalent moiety connected to (i.e., bonded to) two monovalent moieties, such as the second click handle and the antibody. In some embodiments, the second click handle is connected to the antibody at the C terminus, optionally wherein a linker connects the second click handle and the C terminus. In some embodiments, the second click handle is connected to the antibody at the N terminus, optionally wherein a linker connects the second click handle and the N terminus. In some embodiments, the second click handle is connected to the antibody at a side chain of an amino acid residue (e.g., at a side chain of a tryptophan, tyrosine, asparagine, cysteine, glutamine, serine, threonine, aspartic acid, glutamic acid, arginine, histidine, or lysine residue), optionally wherein a linker connects the second click handle and the side chain of the amino acid.

[0195]

[0084] As alternative means to target a particular cell, collection of cells, or tissue, the antibody used in the methods of the present disclosure may be replaced with a targeting agent, thereby forming a targeting agent-lipid nanoparticle conjugate. The targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, or polynucleotide, etc. The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the targeting agent-lipid nanoparticle conjugate. In some embodiments, the targeting agent is a protein or peptide.

[0196] Agent

[0197]

[0085] The term “gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5 ’ non-coding sequences) and following (3 ’ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” or “chimeric construct” refers to any gene or a construct, not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene or chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene

[0198] D0957.70000WQ00 26 / 51

[0199] #14937867vlnot normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0200]

[0086] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. The oligonucleotide may comprise a modified base moiety which is selected from the group including, but not limited to, 5 -fluorouracil, 5 -bromouracil, 5 -chlorouracil, 5 -iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 -(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl -2 -thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5- methoxyaminomethyl-2 -thiouracil, beta-D-mannosylqueosine, 5 ’-methoxycarboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5 -methyl -2 -thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil- 5-oxyacetic acid methylester, uracil-5 -oxyacetic acid, 5 -methyl-2- thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, a thio-guanine, and 2,6-diaminopurine. The oligonucleotide may comprise one or more locked nucleic acid (LNA) moieties. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single -stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNAs) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing carbohydrate or lipids. Exemplary DNAs include single-stranded DNA (ssDNA), double -stranded DNA (dsDNA), plasmid DNA (pDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), a provirus, a lysogen, repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single -stranded RNA (ssRNA), double -stranded RNA (dsRNA), small interfering RNA (siRNA), messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, signal recognition particle D0957.70000WQ00 27 / 51

[0201] #14937867vlRNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), a polyinosinic acid, a ribozyme, a flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, viral satellite RNA, single guide RNA (sgRNA), prime editing guide RNA (pegRNA), and a ribonucleoprotein (RNP) complex.

[0202]

[0087] Polynucleotides described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those that are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., Nucl. Acids Res., 16, 3209, (1988), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U. S. A. 85, 7448-7451, (1988)). A number of methods have been developed for delivering antisense DNA or RNA to cells, e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art.

[0203] Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bemoist et al., Nature, 290, 304-310, (1981); Yamamoto etal., Cell, 22, 787-797, (1980); Wagner et al., Proc. Natl. Acad. Sci. U. S. A. 78, 1441-1445, (1981); Brinster et al., Nature 296, 39-42, (1982)). Any type of plasmid, cosmid, yeast artificial chromosome or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site. Alternatively, viral vectors can be

[0204] D0957.70000WQ00 28 / 51

[0205] #14937867vlused which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).

[0206]

[0088] The polynucleotides may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.

[0207]

[0089] A “recombinant nucleic acid molecule” is a nucleic acid molecule that has undergone a molecular biological manipulation, i. e., non-naturally occurring nucleic acid molecule or genetically engineered nucleic acid molecule. Furthermore, the term “recombinant DNA molecule” refers to a nucleic acid sequence which is not naturally occurring, or can be made by the artificial combination of two otherwise separated segments of nucleic acid sequence, i.e., by ligating together pieces of DNA that are not normally continuous. By “recombinantly produced” is meant artificial combination often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques using restriction enzymes, ligases, and similar recombinant techniques as described by, for example, Sambrook et al., Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N. Y.; (1989), or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover) IREL Press, Oxford, (1985); each of which is incorporated herein by reference.

[0208]

[0090] Such manipulation may be done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it may be performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in nature. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, open D0957.70000WQ00 29 / 51

[0209] #14937867vlreading frames, or other useful features may be incorporated by design. Examples of recombinant nucleic acid molecule include recombinant vectors, such as cloning or expression vectors which contain DNA sequences encoding Ror family proteins or immunoglobulin proteins which are in a 5' to 3' (sense) orientation or in a 3' to 5' (antisense) orientation.

[0210]

[0091] The term “pDNA,” “plasmid DNA,” or “plasmid” refers to a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell.

[0211] Plasmids can be found in all three major domains: Archaea, Bacteria, and Eukarya. In nature, plasmids carry genes that may benefit survival of the subject (e.g., antibiotic resistance) and can frequently be transmitted from one bacterium to another (even of another species) via horizontal gene transfer. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host subjects. Plasmid sizes may vary from 1 to over 1,000 kbp. Plasmids are considered replicons, capable of replicating autonomously within a suitable host.

[0212]

[0092] A sequence “complementary” to a portion of an RNA, refers to a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double -stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[0213]

[0093] The terms “nucleic acid” or “nucleic acid sequence”, “nucleic acid molecule”, “nucleic acid fragment” or “polynucleotide” may be used interchangeably with “gene”, “mRNA encoded by a gene” and “cDNA”.

[0214]

[0094] The term “mRNA” or “mRNA molecule” refers to messenger RNA, or the RNA that serves as a template for protein synthesis in a cell. The sequence of a strand of mRNA is based on the sequence of a complementary strand of DNA comprising a sequence coding for the protein to be synthesized.

[0215]

[0095] The term “siRNA” or “siRNA molecule” refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway, where the siRNA interferes with the expression of specific genes with a complementary nucleotide sequence. siRNA molecules can vary in length (e.g., between 18-30 or 20-25 basepairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some siRNA have unpaired overhanging bases on the 5’ or 3’ end of the sense strand and / or the antisense strand. The term siRNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.

[0216] D0957.70000WQ00 30 / 51

[0217] #14937867vl

[0096] A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In some embodiments, the nonhuman animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In some embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease.

[0218]

[0097] The term “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.

[0219]

[0098] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0220]

[0099] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.

[0221]

[0100] The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease.

[0222]

[0101] An “effective amount” of a compound or agent described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a compound or agent described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular compound or agent, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject.

[0223]

[0102] In some embodiments, the lipid nanoparticle further comprises an agent. In some embodiments, the antibody-lipid nanoparticle conjugate further comprises an agent.

[0224]

[0103] Agents that are delivered by the lipid nanoparticles or antibody-lipid nanoparticle conjugates described herein may be pharmaceutical (e.g., therapeutic or prophylactic), diagnostic, cosmetic, or nutraceutical agents. Any chemical compound to be administered to a subject may be delivered using the lipid nanoparticles or antibody-lipid nanoparticle conjugates described herein. In some embodiments, the agent is an organic molecule, inorganic molecule, polynucleotide, protein, peptide, targeting agent, an isotopically labeled chemical compound, vaccine, an immunological agent, or an agent useful in bioprocessing (e.g., intracellular manufacturing of proteins, such as a cell’s bioprocessing of a commercially useful chemical or fuel). For example, intracellular delivery of an agent may be useful in bioprocessing by maintaining the cell’s health and / or growth, e.g., in the manufacturing of proteins. Any chemical compound to be administered to a subject or contacted with

[0225] D0957.70000WQ00 31 / 51

[0226] #14937867vla cell may be delivered to the subject or cell using the lipid nanoparticles or antibody-lipid nanoparticle conjugates.

[0227]

[0104] Exemplary agents that may be included in a lipid nanoparticle or antibody-lipid nanoparticle conjugate described herein include, but are not limited to, small molecules, organometallic compounds, polynucleotides, proteins, peptides, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, small molecules linked to proteins, glycoproteins, steroids, nucleotides, oligonucleotides, polynucleotides, nucleosides, antisense oligonucleotides, lipids, hormones, vitamins, cells, metals, targeting agents, isotopically labeled chemical compounds, drugs (e.g., compounds approved for human or veterinary use by the U. S. Food and Drug Administration as provided in the Code of Federal Regulations), vaccines, immunological agents, agents useful in bioprocessing, and mixtures thereof. In some embodiments, the agents are nutraceutical agents. In some embodiments, the agents are pharmaceutical agents (e.g., a therapeutic or prophylactic agent). In some embodiments, the agent is an antibiotic agent (e.g., an anti-bacterial, anti-viral, or anti-fungal agent), anesthetic, steroidal agent, anti-proliferative agent, anti-inflammatory agent, anti-angiogenesis agent, anti-neoplastic agent, anti-cancer agent, anti-diabetic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, immunosuppressant, anti-depressant, anti-psychotic, (3-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal, nutritional agent, anti-allergic agent, or pain-relieving agent. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, polynucleotide (e.g., mRNA), and cell extracts. Therapeutic and prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, and Freund’s adjuvant, etc.

[0228]

[0105] In some embodiments, an agent to be delivered or used in a lipid nanoparticle or antibody-lipid nanoparticle conjugate described herein is a polynucleotide. In some embodiments, the agent is DNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the agent is plasmid DNA (pDNA). In some embodiments, the agent is single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, or viral DNA. In some embodiments, the agent is RNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the agent is messenger RNA (mRNA). In some embodiments, the agent is small interfering RNA (siRNA). In some embodiments, the agent is messenger RNA (mRNA), singlestranded RNA (ssRNA), double -stranded RNA (dsRNA), small interfering RNA (siRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long D0957.70000WQ00 32 / 51

[0229] #14937867vlncRNA or IncRNA), satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, viral satellite RNA, single guide RNA (sgRNA), prime editing guide RNA (pegRNA), or ribonucleoprotein (RNP) complex. In some embodiments, the agent is an RNA that carries out RNA interference (RNAi). The phenomenon of RNAi is discussed in greater detail, for example, in the following references: Elbashir et al., 2001, Genes Dev., 15:188; Fire et al., 1998, Nature, 391:806; Tabara et al., 1999, Cell, 99:123; Hammond et al., Nature, 2000, 404:293; Zamore et al., 2000, Cell, 101:25; Chakraborty, 2007, Curr. Drug Targets, 8:469; and Morris and Rossi, 2006, Gene Ther., 13:553. In some embodiments, upon delivery of an RNA into a subject, tissue, or cell, the RNA is able to interfere with the expression of a specific gene in the subject, tissue, or cell. In some embodiments, the agent is a pDNA, siRNA, mRNA, or a combination thereof.

[0230]

[0106] In some embodiments, the DNA is plasmid DNA (pDNA). In some embodiments, the DNA is single -stranded DNA (ssDNA), double -stranded DNA (dsDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, or viral DNA. In some embodiments, the RNA is messenger RNA (mRNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, viral satellite RNA, single guide RNA (sgRNA), prime editing guide RNA (pegRNA), or ribonucleoprotein (RNP) complex. In some embodiments, the RNA is coding RNA or non-coding RNA. In some embodiments, the coding RNA is messenger RNA (mRNA). In some embodiments, the RNA is precursor messenger RNA. In some embodiments, the non-coding RNA is doublestranded RNA, short hairpin RNA, microRNA, guide RNA, transfer RNA, antisense RNA, long noncoding RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA, ribosomal RNA, Piwi-interacting RNA, small nucleolar RNA, or spliced leader RNA. In some embodiments, the non-coding RNA is small interfering RNA. In some embodiments, the RNA is single -stranded RNA, heterogeneous nuclear RNA, satellite RNA, viral RNA, or viral satellite RNA. In some embodiments, the RNA is single guide RNA (sgRNA). In some embodiments, the RNA is prime editing guide RNA (pegRNA). In some embodiments, the RNA is a ribonucleoprotein (RNP) complex. In some embodiments, the RNP complex comprises Cas9 protein and sgRNA.

[0231] D0957.70000WQ00 33 / 51

[0232] #14937867vl

[0107] In some embodiments, the polynucleotide may be provided as an antisense agent or RNAi. See, e.g., Fire et al., Nature 391:806-811, 1998. Antisense therapy is meant to include, e.g., administration or in situ provision of single- or double-stranded polynucleotides, or derivatives thereof, which specifically hybridize, e.g., bind, under cellular conditions, with cellular mRNA and / or genomic DNA, or mutants thereof, so as to inhibit the expression of the encoded protein, e.g., by inhibiting transcription and / or translation. See, e.g., Crooke, “Molecular mechanisms of action of antisense drugs,” Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke, “Evaluating the mechanism of action of anti-proliferative antisense drugs,” Antisense Nucleic Acid Drug Dev. 10(2): 123-126, discussion 127, 2000; Methods in Enzymology volumes 313-314, 1999. The binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix (i.e., triple helix formation). See, e.g., Chan etal., J. Mol. Med. 75(4):267-282, 1997.

[0233]

[0108] In some embodiments, pDNA, siRNA, dsRNA, shRNA, miRNA, mRNA, tRNA, asRNA, and / or RNAi can be designed and / or predicted using one or more of a large number of available algorithms. To give but a few examples, the following resources can be utilized to design and / or predict polynucleotides: algorithms found at Alnylam Online; Dharmacon Online; OligoEngine Online; Molecula Online; Ambion Online; BioPredsi Online; RNAi Web Online; Chang Bioscience Online; Invitrogen Online; LentiWeb Online GenScript Online; Protocol Online; Reynolds et al., 2004, Nat. Biotechnol., 22:326; Naito et al., 2006, Nucleic Acids Res., 34: W448; Li et al., 2007, RNA, 13:1765; Yiu et al., 2005, Bioinformatics, 21: 144; and Jia et al., 2006, BMC Bioinformatics, 7: 271.

[0234]

[0109] The polynucleotide included in a lipid nanoparticle or antibody-lipid nanoparticle conjugate may be of any size or sequence, and it may be single- or double-stranded. In some embodiments, the polynucleotide includes at least about 30, at least about 100, at least about 300, at least about 1,000, at least about 3,000, or at least about 10,000 base pairs. In some embodiments, the polynucleotide includes less than about 10,000, less than about 3,000, less than about 1,000, less than about 300, less than about 100, or less than about 30 base pairs. Combinations of the above ranges (e.g., at least about 100 and less than about 1,000) are also within the scope of the invention. The polynucleotide may be provided by any means known in the art. In some embodiments, the polynucleotide is engineered using recombinant techniques. See, e.g., Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989). The polynucleotide may also be obtained from natural sources and purified from contaminating components found normally in nature. The polynucleotide may also be chemically synthesized in a laboratory. In some embodiments, the polynucleotide is synthesized using standard solid phase chemistry. The polynucleotide may be isolated and / or purified. In some embodiments, the polynucleotide is substantially free of impurities. In some embodiments, the polynucleotide is at least about 50%, at

[0235] D0957.70000WQ00 34 / 51

[0236] #14937867vlleast about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% free of impurities.

[0237]

[0110] The polynucleotide may be modified by physical, chemical, and / or biological means. The modifications include methylation, phosphorylation, and end-capping, etc. In some embodiments, the modifications lead to increased stability of the polynucleotide.

[0238]

[0111] Wherever a polynucleotide is employed in the lipid nanoparticle or antibody-lipid nanoparticle conjugate, a derivative of the polynucleotide may also be used. These derivatives include products resulted from modifications of the polynucleotide in the base moieties, sugar moieties, and / or phosphate moieties of the polynucleotide. Modified base moieties include, but are not limited to, 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 -bromouridine, C5-fluorouridine, C5 -iodouridine, C5-propynyl -uridine,

[0239] C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugar moieties include, but are not limited to, 2'-fluororibose, ribose, 2'-deoxyribose, 3 '-azido-2', 3 '-dideoxyribose, 2', 3'-dideoxyribose, arabinose (the 2'-epimer of ribose), acyclic sugars, and hexoses. The nucleosides may be strung together by linkages other than the phosphodiester linkage found in naturally occurring DNA and RNA. Modified linkages include, but are not limited to, phosphorothioate and

[0240] 5'-N-phosphoramidite linkages. Combinations of the various modifications may be used in a single polynucleotide. These modified polynucleotides may be provided by any means known in the art; however, as will be appreciated by those of skill in the art, the modified polynucleotides may be prepared using synthetic chemistry in vitro.

[0241]

[0112] The polynucleotide described herein may be in any form, such as a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, and an artificial chromosome.

[0242]

[0113] The polynucleotide described herein may be of any sequence. In some embodiments, the polynucleotide encodes a protein or peptide. The encoded protein may be an enzyme, structural protein, receptor, soluble receptor, ion channel, active (e.g., pharmaceutically active) protein, cytokine, interleukin, antibody, antibody fragment, antigen, coagulation factor, albumin, growth factor, hormone, and insulin, etc. The polynucleotide may also comprise regulatory regions to control the expression of a gene. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA boxes, ribosomal binding sites, and stop sites for transcription, etc. In some embodiments, the polynucleotide is not intended to encode a protein. For example, the polynucleotide may be used to fix an error in the genome of the cell being transfected.

[0243]

[0114] In some embodiments, the polynucleotide is immunomodulatory, e.g., immunostimulatory, or immunosuppressive. In some embodiments, the polynucleotide described herein comprises a sequence encoding an antigenic peptide or protein. A lipid nanoparticle or antibody-lipid nanoparticle conjugate containing the polynucleotide can be delivered to a subject to induce an immunologic response

[0244] D0957.70000WQ00 35 / 51

[0245] #14937867vlsufficient to decrease the chance of a subsequent infection and / or lessen the symptoms associated with such an infection. The polynucleotide of these vaccines may be combined with interleukins, interferon, cytokines, and / or adjuvants described herein.

[0246]

[0115] The antigenic protein or peptides encoded by the polynucleotide may be derived from bacterial organisms, such as Streptococccus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynehacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans, Borrelia burgdorferi, and Camphylobacter jejuni,' from viruses, such as coronavirus (e.g., SARS-CoV-2), smallpox virus, influenza A virus, influenza B virus, respiratory syncytial virus, parainfluenza virus, measles virus, HIV virus, varicella-zoster virus, herpes simplex 1 virus, herpes simplex 2 virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps virus, rabies virus, rubella virus, coxsackieviruses, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus; and from fungal, protozoan, or parasitic organisms, such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0247]

[0116] In some embodiments, the RNA is an mRNA for inducing functional protein expression. In some embodiments, the RNA is an mRNA that encodes one or more antigens. In some embodiments, the antigen selected from a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, a tumor-associated antigen, a tumor-specific antigen, or any combination thereof. In some embodiments, the antigen induces an adaptive immune response.

[0248]

[0117] In some embodiments, the agent in a lipid nanoparticle or antibody-lipid nanoparticle conjugate that is delivered to a subject in need thereof may be a mixture of two or more agents that may be useful as, e.g., combination therapies. The lipid nanoparticles or antibody-lipid nanoparticle conjugates including the two or more agents can be administered to achieve a synergistic effect. In some embodiments, the lipid nanoparticles or antibody-lipid nanoparticle conjugates including the two or more agents can be administered to improve the activity and / or bioavailability, reduce and / or modify the metabolism, inhibit the excretion, and / or modify the distribution within the body of a subject, of each one of the two or more agents. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and / or it may achieve different effects.

[0249] D0957.70000WQ00 36 / 51

[0250] #14937867vl

[0118] The lipid nanoparticles or antibody-lipid nanoparticle conjugates can be administered concurrently with, prior to, or subsequent to the one or more agents (e.g., pharmaceutical agents). The two or more agents may be useful for treating and / or preventing a same disease or different diseases described herein. Each one of the agents may be administered at a dose and / or on a time schedule determined for that agent. The agents may also be administered together with each other and / or with the lipid nanoparticle or antibody-lipid nanoparticle conjugate described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the agents and / or the desired therapeutic and / or prophylactic effect to be achieved. In general, it is expected that the agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

[0251]

[0119] In some embodiments, the agent is provided in an effective amount in the lipid nanoparticle. In some embodiments, the agent is provided in an effective amount in the antibody-lipid nanoparticle conjugate.

[0252]

[0120] In some embodiments, the agent and the lipid nanoparticle are not covalently attached. In some embodiments, the agent and the antibody-lipid nanoparticle conjugate are not covalently attached. In some embodiments, the lipid nanoparticle encapsulates the agent. In some embodiments, the antibody-lipid nanoparticle conjugate encapsulates the agent. In some embodiments, the lipid nanoparticle portion of the antibody-lipid nanoparticle conjugate encapsulates the agent.

[0253] Additional Embodiments of Lipid Nanoparticles and Antibody-Lipid Nanoparticle Conjugates

[0121] In some embodiments, the lipid nanoparticle comprises an ionizable lipid, phospholipid, sterol, PEG-lipid, and functionalized lipid. In some embodiments, the lipid nanoparticle comprises SM-102, DSPC, cholesterol, DMG-PEG2k, and a functionalized lipid. In some embodiments, the lipid nanoparticle comprises SM-102, DSPC, cholesterol, DMG-PEG2k, and PEG with a first click handle. In some embodiments, the lipid nanoparticle comprises LP01, DSPC, cholesterol, DMG-PEG2k, and a functionalized lipid. In some embodiments, the lipid nanoparticle comprises LP01, DSPC, cholesterol, DMG-PEG2k, and PEG with a first click handle.

[0254]

[0122] In some embodiments, the antibody-lipid nanoparticle conjugate comprises an ionizable lipid, phospholipid, sterol, PEG-lipid, and functionalized lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises SM-102, DSPC, cholesterol, DMG-PEG2k, and a functionalized lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises SM-102, DSPC, cholesterol, DMG-PEG2k, and PEG with a first click handle. In some embodiments, the antibody-lipid nanoparticle conjugate comprises LP01, DSPC, cholesterol, DMG-PEG2k, and a functionalized lipid. In some embodiments, the antibody-lipid nanoparticle conjugate comprises LP01, DSPC, cholesterol, DMG-PEG2k, and PEG with a first click handle.

[0255] D0957.70000WQ00 37 / 51

[0256] #14937867vl

[0123] In some embodiments, the lipid nanoparticle comprises between about 45 and about 55 molar % of an ionizable lipid, about 15 and about 25 molar % of a phospholipid, about 20 and about 30 molar % of a sterol, about 1.0 and about 3.0 molar % of a PEG-lipid, and about 0.25 and about 0.75 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises between about 45 and about 55 molar % of SM-102, about 15 and about 25 molar % of DSPC, about 20 and about 30 molar % of cholesterol, about 1.0 and about 3.0 molar % of DMG-PEG2k, and about 0.25 and about 0.75 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises between about 45 and about 55 molar % of LP01, about 15 and about 25 molar % of DSPC, about 20 and about 30 molar % of cholesterol, about 1.0 and about 3.0 molar % of DMG-PEG2k, and about 0.25 and about 0.75 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises between about 45 and about 55 molar % of SM-102, about 15 and about 25 molar % of DSPC, about 20 and about 30 molar % of cholesterol, about 1.0 and about 3.0 molar % of DMG-PEG2k, and about 0.25 and about 0.75 molar % of PEG with a first click handle. In some embodiments, the lipid nanoparticle comprises between about 45 and about 55 molar % of LP01, about 15 and about 25 molar % of DSPC, about 20 and about 30 molar % of cholesterol, about 1.0 and about 3.0 molar % of DMG-PEG2k, and about 0.25 and about 0.75 molar % of PEG with a first click handle.

[0257]

[0124] In some embodiments, the lipid nanoparticle comprises about 50 molar % of an ionizable lipid, about 22 molar % of a phospholipid, about 25.5 molar % of a sterol, about 2.0 molar % of a PEG-lipid, and about 0.5 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises about 50 molar % of SM-102, about 22 molar % of DSPC, about 25.5 molar % of cholesterol, about 2.0 molar % of DMG-PEG2k, and about 0.5 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises about 50 molar % of LP01, about 22 molar % of DSPC, about 25.5 molar % of cholesterol, about 2.0 molar % of DMG-PEG2k, and about 0.5 molar % of a functionalized lipid. In some embodiments, the lipid nanoparticle comprises about 50 molar % of SM-102, about 22 molar % of DSPC, about 25.5 molar % of cholesterol, about 2.0 molar % of DMG-PEG2k, and about 0.5 molar % of PEG with a first click handle. In some embodiments, the lipid nanoparticle comprises about 50 molar % of LP01, about 22 molar % of DSPC, about 25.5 molar % of cholesterol, about 2.0 molar % of DMG-PEG2k, and about 0.5 molar % of PEG with a first click handle.

[0258]

[0125] In some embodiments, the functionalized lipid comprises a first click handle selected from those shown in Tables 1-2, and the antibody comprises a second click handle comprising a complementary moiety (i.e., a moiety that undergoes a click reaction with the first click handle). In some embodiments, the first click handle is selected from those shown in Tables 1-2, and the second click handle comprises a complementary moiety ( / . e., a moiety that undergoes a click reaction with the first click handle).

[0259] D0957.70000WQ00 38 / 51

[0260] #14937867vl

[0126] In some embodiments, the first click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans-cyclooctene). terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety, and the second click handle comprises a complementary moiety. In some embodiments, the first click handle comprises an activated alkene (e.g., maleimide), and the second click handle comprises a thiol. In some embodiments, the first click handle comprises a maleimide, and the second click handle comprises a thiol. In some embodiments, the first click handle comprises a strained alkene (e.g., trans-cyclooctene). and the second click handle comprises a tetrazine. In some embodiments, the first click handle comprises trans -cyclooctene (TCO), and the second click handle comprises a tetrazine (e.g., methyltetrazine). In some embodiments, the first click handle comprises a terminal alkyne, and the second click handle comprises an azide. In some embodiments, the first click handle comprises a strained alkyne (e.g., cyclooctyne), and the second click handle comprises an azide. In some embodiments, the first click handle comprises a cyclooctyne, and the second click handle comprises an azide. In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl-less cyclooctyne (ALO), and the second click handle comprises an azide. In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN), and the second click handle comprises an azide. In some embodiments, the first click handle comprises a strained alkyne (e.g., cyclooctyne), and the second click handle comprises a tetrazine. In some embodiments, the first click handle comprises a cyclooctyne, and the second click handle comprises a tetrazine. In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl -less cyclooctyne (ALO), and the second click handle comprises a tetrazine. In some embodiments, the first click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN), and the second click handle comprises a tetrazine.

[0261]

[0127] In some embodiments, the antibody comprises a second click handle selected from those shown in Tables 1-2, and the functionalized lipid comprises a first click handle comprising a complementary moiety (i.e., a moiety that undergoes a click reaction with the first click handle). In some embodiments, the second click handle is selected from those shown in Tables 1-2, and the first click handle comprises a complementary moiety (i. e., a moiety that undergoes a click reaction with the first click handle).

[0262] D0957.70000WQ00 39 / 51

[0263] #14937867vl

[0128] In some embodiments, the second click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans -cyclooctene), terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety, and the first click handle comprises a complementary moiety. In some embodiments, the second click handle comprises an activated alkene (e.g., maleimide), and the first click handle comprises a thiol. In some embodiments, the second click handle comprises a maleimide, and the first click handle comprises a thiol. In some embodiments, the second click handle comprises a strained alkene (e.g., trans-cyclooctene). and the first click handle comprises a tetrazine. In some embodiments, the second click handle comprises trans -cyclooctene (TCO), and the first click handle comprises a tetrazine (e.g., methyltetrazine). In some embodiments, the second click handle comprises a terminal alkyne, and the first click handle comprises an azide. In some embodiments, the second click handle comprises a strained alkyne (e.g., cyclooctyne), and the first click handle comprises an azide. In some embodiments, the second click handle comprises a cyclooctyne, and the first click handle comprises an azide. In some embodiments, the second click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl-less cyclooctyne (ALO), and the first click handle comprises an azide. In some embodiments, the second click handle comprises strained cyclooct-2-yn-1 -methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN), and the first click handle comprises an azide. In some embodiments, the second click handle comprises a strained alkyne (e.g., cyclooctyne), and the first click handle comprises a tetrazine. In some embodiments, the second click handle comprises a cyclooctyne, and the first click handle comprises a tetrazine. In some embodiments, the second click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), biarylazacyclooctynone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), bicyclononyne (BCN), dimethoxyazacyclooctyne (DIMAC), monofluorinated cyclooctyne (MOFO), cyclooctyne (OCT), or aryl -less cyclooctyne (ALO), and the first click handle comprises a tetrazine. In some embodiments, the second click handle comprises strained cyclooct-2-yn-1-methylcarbamate (SCO), dibenzoazacyclooctyne (DIBAC or DBCO), or bicyclononyne (BCN), and the first click handle comprises a tetrazine.

[0264] EXAMPLES

[0265]

[0129] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate

[0266] D0957.70000WQ00 40 / 51

[0267] #14937867vlthe compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.

[0268] Example 1: Preparation of Antibody-Lipid Nanoparticle Conjugates in Tangential Flow Filtration (TFF) Apparatus

[0269]

[0130] Lipids were dissolved in ethanol at molar ratios of 50:22:25.5:2:0.5 (ionizable lipid (SM-102 or LP01) / DSPC / Cholesterol / DMG-PEG2k / PEG with a first click handle). The lipid mixture in ethanol was combined with a 25 mM sodium citrate buffer (pH 4) containing mRNA cargo (eGFP mRNA) made in-house at a ratio of 3: 1 (aqueous: ethanol) using a microfluidic mixer (XGen / Micro& Nano). Beginning waste is 2 mL while end waste is 0 mL. Formulations were diluted with 2X volume of 1XDPBS buffer, and were neutralized with 1% v / v IM pH 8 Tris buffer after dilution. The now neutralized LNP was allowed to mature at room temperature for 1.5 hrs prior to TFF processing.

[0270]

[0131] All TFF related tubing, adaptors and inserts were soaked with 0.1 N NaOH overnight a day prior to remove endotoxin. TFF system with a column of 235 cm2MWCO 300K was installed and washed sequentially with 500 mL of RNAase-free water (RFW), 0.5M NaOH, RFW and lastly 1XDPBS.

[0271]

[0132] ENPs were loaded onto the TFF and were subjected to 3X concentration and 3X diafiltration volume (DV) exchange with 1XDPBS sequentially. To start the conjugation reaction, the permeate line of the TFF system is closed, and the flow rate is reduced, and an antibody modified with a complementary click ligand (e.g., an antibody with a second click handle) is added gradually to the TFF system while the ENPs circulate within the TFF system at a rate to ensure adequate mixing. The conjugation proceeds at room temperature for 1.5 hours.

[0272]

[0133] Subsequently, the permeate line is reopened, and ENP solution was concentrated 2X and exchanged 5X DV to fully remove unreacted antibody. All TFF lines were drained into the feed bottle at the end for maximal recovery.

[0273]

[0134] The LNPs were then sterile filtered through 0.2 μm filter. RNA encapsulation and concentration are measured with QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, CA). Based on the results, the LNPs are diluted to the desired final concentration with sucrose and 1XDPBS buffer, and stored at -80 °C until use. One-time freeze-thaw was performed to evaluate the freeze-thaw stability of the LNP-antibody conjugate. As seen in Table 3, the size of the LNP remains very stable throughout the TFF / conjugation process, indicating the robustness of the process.

[0274] Table 3. Size and polydispersity index (PDI) of LNPs during one-pot TFF / conjugation process.

[0275] Size (nm) PDI

[0276] Size Post 1:2 PBS dilution 71.13 0.063

[0277]

[0278] size post tris 80.84 0.064

[0279] D0957.70000WQ00 41 / 51

[0280] #14937867vlSize (nm) PDI

[0281] post tris Ihr 80.78 0.091

[0282] size post conc4X 79.5 0.05

[0283] post 3DV 79.09 0.082

[0284] post reaction 1.5hr 79.29 0.077

[0285] post 2Xconc, 5DV 81.04 0.107

[0286] post filtering 78.11 0.09

[0287]

[0288] 1FT 78.98 0.063

[0289]

[0135] The methods of the present disclosure provide several advantages over existing methods of preparing antibody-lipid nanoparticle conjugates: (1) simplified production of antibody-conjugated LNPs by integrating antibody addition into the downstream processing step; (2) quality of the LNP-antibody conjugates ensured by full removal of ethanol prior to conjugation; (3) increased LNP concentration enhances the conjugation reaction rate and enables the use of a wider range of less reactive conjugation partners; (4) reduced manufacturing complexity and enhanced scalability, as the entire process can be done within 12 hrs; (5) efficient removal of unreacted antibodies to improve product purity; and (6) compatibility with large-scale production and storage for future use.

[0290] INCORPORATION BY REFERENCE

[0291]

[0136] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

[0292] EQUIVALENTS AND SCOPE

[0293]

[0137] When a range of values is listed, it is intended to encompass each value and sub-range within the range. A range is inclusive of the values at the two ends of the range unless otherwise provided.

[0294]

[0138] Use of the phrase “at least one” instance refers to 1, 2, 3, 4, or more instances, but also encompasses a range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.

[0295]

[0139] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” “About” and “about” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, or more typically, within 5%, 4%, 3%, 2%, or 1% of a given value or range of values.

[0296] D0957.70000WQ00 42 / 51

[0297] #14937867vl

[0140] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0298]

[0141] In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0299]

[0142] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, some embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0300]

[0143] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

[0301]

[0144] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but D0957.70000WQ00 43 / 51

[0302] #14937867vlrather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0303] D0957.70000WQ00 44 / 51

[0304] #14937867vl

Claims

CLAIMSWhat is claimed is:

1. A method of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, the method comprising:coupling a lipid nanoparticle with an antibody in a filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; andpurifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing the purified composition comprising the antibody-lipid nanoparticle conjugate.

2. The method of claim 1, further comprising purifying a composition comprising the lipid nanoparticle in the filtration apparatus prior to the coupling step.

3. A method of preparing a purified composition comprising an antibody-lipid nanoparticle conjugate, the method comprising:purifying a composition comprising a lipid nanoparticle in a filtration apparatus; coupling the lipid nanoparticle with an antibody in the filtration apparatus to provide a composition comprising the antibody-lipid nanoparticle conjugate; andpurifying the composition comprising the antibody-lipid nanoparticle conjugate in the filtration apparatus, thereby providing the purified composition comprising the antibody-lipid nanoparticle conjugate.

4. The method of claim 2 or 3, wherein purifying the composition comprising the lipid nanoparticle comprises reducing an amount of ethanol in the composition comprising the lipid nanoparticle.

5. The method of claim 4, wherein the amount of ethanol in the composition is reduced from between about 15% v / v and about 35% v / v to between about 0% v / v and about 5% v / v.

6. The method of claim 4 or 5, wherein the amount of ethanol in the composition is reduced from about 25% v / v to between about 0% v / v and about 5% v / v.

7. The method of any one of claims 2-6, wherein purifying the composition comprising the lipid nanoparticle comprises removing substantially all ethanol in the composition comprising the lipid nanoparticle.

8. The method of any one of claims 2-7, wherein purifying the composition comprising the lipid nanoparticle comprises increasing a concentration of the composition comprising the lipid nanoparticle.D0957.70000WQ00 45 / 51#14937867vl9. The method of claim 8, wherein the concentration of the composition comprising the lipid nanoparticle is increased by a factor of about 3.0.

10. The method of any one of claims 2-9, wherein purifying the composition comprising the lipid nanoparticle comprises diafiltration of the composition comprising the lipid nanoparticle.

11. The method of claim 10, wherein the diafiltration is performed for between about 2 and about 20 diafiltration volumes.

12. The method of claim 10 or 11, wherein the diafiltration is performed for between about 3 and about 5 diafiltration volumes.

13. The method of any one of claims 1-12, wherein the filtration apparatus is a tangential flow filtration (TFF) apparatus.

14. The method of claim 13, wherein the TFF apparatus comprises a column having a molecular weight cut-off (MWCO) of between about 10 kDa and about 500 kDa.

15. The method of claim 14, wherein the column has a molecular weight cut-off (MWCO) of about 300 kDa or about 500 kDa.

16. The method of any one of claims 13-15, wherein coupling the lipid nanoparticle with the antibody in the filtration apparatus comprises closing a permeate line of the TFF apparatus.

17. The method of any one of claims 13-16, further comprising reducing a flow rate of the TFF apparatus.

18. The method of any one of claims 13-17, further comprising adding the antibody to the TFF apparatus.

19. The method of any one of claims 13-18, wherein the composition comprising the lipid nanoparticle is circulated throughout the TFF apparatus during addition of the antibody.

20. The method of any one of claims 1-19, wherein the coupling step comprises a reaction time of between about 5 minutes and about 24 hours.

21. The method of claim 20, wherein the reaction time is between about 5 minutes and about 4 hours.

22. The method of any one of claims 1-21, wherein the lipid nanoparticle comprises a first click handle.D0957.70000WQ00 46 / 51#14937867vl23. The method of claim 22, wherein the first click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans -cyclooctene), terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety.

24. The method of claim 22 or 23, wherein the first click handle comprises a trans-cyclooctene.

25. The method of any one of claims 1-24, wherein the antibody comprises a second click handle.

26. The method of claim 25, wherein the second click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans -cyclooctene), terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety.

27. The method of claim 25 or 26, wherein the second click handle comprises a tetrazine.

28. The method of any one of claims 1-27, wherein the lipid nanoparticle and antibody are maintained within the filtration apparatus during the coupling step.

29. The method of any one of claims 13-28, wherein purifying the composition comprising the antibody-lipid nanoparticle further comprises opening the permeate line of the TFF apparatus.

30. The method of any one of claims 1-29, wherein purifying the composition comprising the antibody-lipid nanoparticle conjugate comprises increasing a concentration of the composition comprising the antibody-lipid nanoparticle conjugate.

31. The method of any one of claims 1-30, wherein purifying the composition comprising the antibody-lipid nanoparticle conjugate comprises diafiltration of the composition comprising the antibody-lipid nanoparticle conjugate.

32. The method of claim 31, wherein the diafiltration is performed for between 2 and 20 diafiltration volumes.

33. The method of claim 31 or 32, wherein the diafiltration is performed for between 4 and 6 diafiltration volumes.

34. The method of any one of claims 1-33, wherein the purified composition comprising the antibody-lipid nanoparticle conjugate is substantially free of the antibody.

35. The method of any one of claims 1-34, wherein the antibody-lipid nanoparticle conjugate is maintained within the filtration apparatus during the purification step.D0957.70000WQ00 47 / 51#14937867vl36. The method of any one of claims 1-35, wherein the lipid nanoparticle further comprises an agent.

37. The method of any one of claims 1-36, wherein the antibody-lipid nanoparticle conjugate further comprises an agent.

38. The method of claim 36 or 37, wherein the agent is an organic molecule, inorganic molecule, polynucleotide, protein, peptide, targeting agent, an isotopically labeled chemical compound, vaccine, an immunological agent, or an agent useful in bioprocessing.

39. The method of any one of claims 36-38, wherein the agent is a polynucleotide.

40. The method of claim 38 or 39, wherein the polynucleotide is RNA.

41. The method of claim 40, wherein the RNA is coding RNA or non-coding RNA.

42. The method of claim 40 or 41, wherein the RNA is messenger RNA (mRNA), single -stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, or viral satellite RNA.

43. The method of claim 41, wherein the coding RNA is messenger RNA (mRNA).

44. The method of claim 41, wherein the non-coding RNA is double-stranded RNA, short hairpin RNA, microRNA, guide RNA, transfer RNA, antisense RNA, long non-coding RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA, ribosomal RNA, Piwi-interacting RNA, small nucleolar RNA, or spliced leader RNA.

45. The method of claim 38 or 39, wherein the polynucleotide is DNA.

46. The method of claim 45, wherein the DNA is plasmid DNA (pDNA).

47. The method of claim 45, wherein the DNA is single -stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, or viral DNA.D0957.70000WQ00 48 / 51#14937867vl48. The method of any one of claims 1-47, wherein the lipid nanoparticle comprises one or more of an ionizable lipid, phospholipid, sterol, PEG-lipid, or functionalized lipid.

49. The method of any one of claims 1-48, wherein the antibody-lipid nanoparticle conjugate comprises one or more of an ionizable lipid, phospholipid, sterol, PEG-lipid, or functionalized lipid.

50. The method of claim 48 or 49, wherein the ionizable lipid is SM-102 or LP01.

51. The method of any one of claims 48-50, wherein the phospholipid is 1,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC).

52. The method of any one of claims 48-51, wherein the sterol is cholesterol.

53. The method of any one of claims 48-52, wherein the PEG-lipid is 1,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col-2000 (DMG-PEG2k).

54. The method of any one of claims 48-53, wherein the functionalized lipid comprises a first click handle.

55. The method of claim 54, wherein the first click handle comprises an activated alkene (e.g., maleimide), strained alkene (e.g., trans -cyclooctene), terminal alkyne, strained alkyne (e.g., cyclooctyne), tetrazine, thiol, or azide moiety.

56. The method of claim 54 or 55, wherein the first click handle comprises a trans-cyclooctene.

57. The method of any one of claims 48-56, wherein the functionalized lipid comprises a PEG component.

58. The method of any one of claims 48-57, wherein the functionalized lipid is PEG with a first click handle.

59. The method of any one of claims 36-58, wherein the agent and the lipid nanoparticle are not covalently attached.

60. The method of any one of claims 36-59, wherein the agent and the antibody-lipid nanoparticle conjugate are not covalently attached.

61. The method of any one of claims 36-60, wherein the lipid nanoparticle encapsulates the agent.

62. The method of any one of claims 1-61, wherein the antibody is a full-length antibody, chimeric antibody, Fab fragment, F(ab')2 fragment, Fv fragment, scFv fragment, multispecific antibody, bispecific antibody, nanobody, or diabody.D0957.70000WQ00 49 / 51#14937867vl63. An antibody-lipid nanoparticle conjugate prepared by the method of any one of claims 1-62.

64. A tangential flow filtration (TFF) apparatus comprising:a composition comprising a lipid nanoparticle; andmeans for adding an antibody to the TFF apparatus;wherein the TFF apparatus is configured to circulate the composition comprising the lipid nanoparticle during addition of the antibody.

65. A tangential flow filtration (TFF) apparatus comprising an antibody-lipid nanoparticle conjugate, optionally comprising a lipid nanoparticle and an antibody.

66. The TFF apparatus of claim 64 or 65, comprising a column having a molecular weight cut-off (MWCO) of between about 10 kDa and about 500 kDa.

67. The TFF apparatus of claim 66, wherein the column has a molecular weight cut-off (MWCO) of about 300 kDa or about 500 kDa.

68. The TFF apparatus of any one of claims 64-67, comprising a permeate line configured to close before addition of the antibody.

69. The TFF apparatus of any one of claims 64-68, wherein the antibody-lipid nanoparticle conjugate is maintained within the TFF apparatus during conjugation of the lipid nanoparticle to the antibody.

70. The TFF apparatus of any one of claims 64-69, comprising a permeate line configured to open after conjugation of the lipid nanoparticle to the antibody.D0957.70000WQ00 50 / 51#14937867vl

Images