Fusion proteins and their use in the preparation of t cell-targeted lnps

Abstract

The present application relates to the technical field of nucleic acid gene therapy, and particularly relates to a fusion protein and application thereof in preparation of T cell targeted LNP. The present application provides a fusion protein, which comprises CD3ScFv, a CD86 extracellular segment N terminal and a G4H12 fragment. The protein has a T cell targeting function, and the LNP prepared by using the fusion protein and PEGylated lipids to prepare PEGylated proteins can improve the endocytosis of T cells, and does not excessively activate T cells.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid gene therapy technology, and in particular to fusion proteins and their application in the preparation of T cell-targeting LNPs. Background Technology

[0002] Chimeric antigen receptors (CARs) are proteins expressed on the surface of immune cells. These receptors have the ability to recognize and bind to specific proteins on the surface of malignant cells, thereby triggering the activation and cytotoxicity of immune cells to eliminate the targeted malignant cells.

[0003] In the field of modern medicine, chimeric antigen receptor (CAR) cell therapy is gradually emerging as a highly promising immunotherapy approach. Current chimeric antigen receptor cell therapies primarily utilize viral vectors or non-viral vectors (such as transposons) to perform a series of complex editing operations on immune cells such as T cells and NK cells. After editing, cell production is required, which involves culturing and expanding the modified immune cells in large quantities to meet therapeutic standards. Finally, these edited immune cells are reinfused into the patient, hoping they will recognize and attack tumor cells, thereby achieving the goal of treating the disease.

[0004] However, this method of in vitro editing to express CARs in immune cells has some significant drawbacks. Firstly, its production cost is extremely high. From vector selection and preparation to immune cell editing, culture, and quality testing, each step requires substantial investment of human, material, and financial resources. Secondly, the entire preparation cycle is lengthy. From the initial collection of the patient's immune cells to the final reinfusion of the modified cells into the patient, it often takes weeks or even months. This not only greatly increases the financial burden on patients but also causes many patients' conditions to worsen during the long waiting period. Therefore, there is a need to develop a new type of CAR product that can rapidly prepare CARs and reduce production costs.

[0005] Existing technologies utilize targeted delivery media such as LNP particles to encapsulate CAR genes and then deliver them directly into the body. This approach avoids complex in vitro cell editing and culture processes, potentially significantly shortening treatment cycles and reducing costs. However, in vivo preparation methods typically use CD3 ScFv as the targeting protein, which has limited targeted transduction efficiency. This means that CAR genes encapsulated in LNP particles may not be efficiently delivered to target immune cells, thus affecting the final efficacy of CAR cell therapy. Therefore, developing novel targeted fusion proteins is essential to further optimize in vivo preparation methods. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a fusion protein and its application in the preparation of T cell-targeting LNPs.

[0007] The fusion protein provided by the present invention includes CD3 ScFv, the N-terminus of the extracellular segment of CD86, and the G4H12 fragment.

[0008] The antibody targeting CD3 molecules used in this invention is a single-chain variable region (ScFv) derived from an OKT3 antibody. In some embodiments, the CD3 ScFv has an amino acid sequence as shown in SEQ ID NO:1, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:1, or has an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:1.

[0009] CD86 is a cell surface membrane protein belonging to the B7 family. It is highly expressed on the surface of antigen-presenting cells and contains two extracellular immunoglobulin domains. This invention utilizes the N-terminus of the CD86 extracellular segment, which participates in interactions with other ligands (such as CD28 or CTLA-4). In some embodiments, the N-terminus of the CD86 extracellular segment has the amino acid sequence shown in SEQ ID NO:2, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:2, or has an amino acid sequence with more than 80% identity to the amino acid sequence shown in SEQ ID NO:2.

[0010] In this invention, the role of G4H12 is to provide a disulfide bond at the tail of the fusion protein, which is then reduced by TCEP to form a free thiol group. This thiol group binds to the MAL group on DSPE-PEG-MAL. In some embodiments, the G4H12 fragment has the amino acid sequence shown in SEQ ID NO:3, or has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:3, or has an amino acid sequence with more than 80% identity to the amino acid sequence shown in SEQ ID NO:3.

[0011] In this invention, the "identity of more than 80%" means that the identity is not less than 80%, not less than 85%, not less than 90%, not less than 95%, not less than 98%, not less than 99%, not less than 99.5%, not less than 99.8%, or not less than 99.9%.

[0012] In this invention, the linking order of the fragments in the fusion protein is not limited. For example, from the N-terminus to the C-terminus, the sequence may be CD3 ScFv, the N-terminus of the CD86 extracellular segment, and the G4H12 fragment; or the N-terminus of the CD86 extracellular segment, CD3 ScFv, and the G4H12 fragment; or the G4H12 fragment, CD3 ScFv, and the N-terminus of the CD86 extracellular segment; or the G4H12 fragment, the N-terminus of the CD86 extracellular segment, and CD3 ScFv. In a specific embodiment, CD3 ScFv is linked to the N-terminus of the CD86 extracellular segment by 7 amino acids, and G4H12 is linked to the C-terminus to form the fusion protein.

[0013] In the fusion protein described in this invention, the various fragments may or may not contain linkers, and this invention does not impose any limitations on this. For example, a linker may be present between the N-terminus of the CD3 ScFv and CD86 extracellular segments.

[0014] More specifically, the fusion protein of the present invention comprises, from the N-terminus to the C-terminus, CD3 ScFv, linker, the N-terminus of the extracellular segment of CD86, and the G4H12 fragment.

[0015] As a feasible example, the linker is G4S, (G4S) n(n>1) GSTSGSGKPGSGEGSTKG, etc.

[0016] In some specific embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO:4. In this invention, the fusion protein as described above is denoted as: CD3 ScFv-CD86-G4H12. Specifically, CD3 ScFv is coupled to the N-terminus of the extracellular segment of CD86 via amino acids, and G4H12 is linked to the C-terminus of the extracellular segment of CD86. The fusion protein described in this application includes a CD3-targeting ScFv (OKT3) linked to the extracellular segment of CD86 via a linker, and a G4H12 amino acid sequence is designed after CD86 to form the CD3ScFv-CD86-G4H12 structure, with the amino acid sequence shown in SEQ ID NO:4. This achieves targeted function, enhances T cell endocytosis without over-activating T cells.

[0017] This invention also provides some biological materials, including at least one of the following:

[0018] I) Nucleic acid encoding the fusion protein as described above;

[0019] II) Expression units containing the nucleic acid described in I);

[0020] III) A recombinant vector containing the nucleic acid described in I) or the expression unit described in II);

[0021] IV) Transformants that have been transformed or transfected with the expression vector described in III);

[0022] The culture products of the transformants described in (V) and (IV).

[0023] The method for preparing the fusion protein described in this invention employs genetic engineering techniques, specifically including culturing the transformants as described above to obtain a product containing the fusion protein as described above.

[0024] This invention also provides a PEGylated protein, comprising the fusion protein as described above and PEGylated lipids, wherein the PEGylated lipids are selected from at least one of the following: ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide (also known as DSPE-PEG-MAL or DSPE-PEG-maleimide), DMG-PEG-MAL (DMG-PEG-maleimide), DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, and DMG-PEG2000-MAL. In a specific embodiment, the average molecular weight of PEG in the PEGylated protein is 2000.

[0025] The fusion protein in the PEGylated protein provided by this invention is linked to PEGylated lipids via thioether bonds.

[0026] In this invention, the C-terminus of the fusion protein is reduced to form a -SH group, which then undergoes an alkylation reaction with the double bond in the PEGylated lipid to form a thioether bond.

[0027] The -SH group is formed when the target protein carrying G4H12 reduces the disulfide bond of G4H12 with the TCEP reducing agent to form -SH.

[0028] In this invention, the preparation method of the PEGylated protein as described above includes: mixing the fusion protein as described above with a TCEP solution for reaction, adding a solution of PEGylated lipids, and obtaining the PEGylated protein after the reaction.

[0029] This invention optimizes the targeted LNP synthesis process by using the TCEP method to modify the fusion protein, forming a fusion protein-PEG2000-DSPE targeted PEGylated protein. The PEGylated protein fusion protein-PEG2000-DSPE and LNP lipid components are directly synthesized into target nanoparticles through microfluidics. The synthesis process is a one-step process, which is simpler and solves the problem of dimer formation in SATS synthesis. It also improves the encapsulation efficiency, coupling efficiency and transduction efficiency of targeted LNP synthesis.

[0030] In this embodiment of the invention, the molar ratio of the fusion protein, TCEP, and PEGylated lipids as described above is 1:(25-50):(5-10). As a feasible example, the molar ratio of the fusion protein, TCEP, and PEGylated lipids as described above is 1:25:10.

[0031] Specifically, the preparation of the PEGylated protein includes:

[0032] 1) Preparation of targeted protein solutions.

[0033] 2) Add an appropriate amount of 0.5M TCEP solution to the targeted protein solution and react at room temperature in the dark.

[0034] 3) Weigh an appropriate amount of DSPE-PEG-MAL and dissolve it in DMSO to prepare a 5mM (14.7mg / ml) DSPE-PEG-MAL working solution.

[0035] 4) Add an appropriate amount of 5mM DSPE-PEG-MAL working solution to the solution in step 2), with the target protein:TCEP:DSPE-PEG-MAL ratio being 1:25:10. React at room temperature in the dark.

[0036] 5) Add the solution obtained in step 4) to an equilibrated desalting column to remove impurities. This yields a PEGylated lipid CD3 ScFv-CD86-PEG-DSPE linked to the target protein.

[0037] In a specific embodiment, a step can also be added whereby solutions obtained from different steps are added to an equilibrated desalting column for solution replacement or impurity removal.

[0038] Furthermore, the present invention also provides the application of the fusion protein or the PEGylated protein as described above in the preparation of T cell-targeting LNPs.

[0039] In this invention, the T cells are either T cells or CAR-T cells, and this invention does not limit the specific type of T cells.

[0040] In this invention, the packaging material for the T-cell targeted LNP includes cationic lipids, neutral lipids, PEG or PEGylated lipids, and cholesterol.

[0041] The T-cell-targeting LNP described in this invention is used to encapsulate nucleic acids, which are biological macromolecules with nucleotides as their basic building blocks, capable of carrying and transmitting genetic information. Nucleic acid is a general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); it can be mRNA, small interfering RNA, tRNA, miRNA, aiRNA, ssRNA, short hairpin RNA (shRNA), dsRNA, gene sequences, ssDNA, dsDNA, or plasmids. The mRNA can be mRNA encoding fluorescent signals, mRNA encoding chimeric antigen receptors, or mRNA sequences of antigenic polypeptides that can elicit an immune response in the body; this invention does not limit this.

[0042] The present invention also provides a method for preparing targeted LNPs, wherein cationic lipids, neutral lipids, PEG or PEGylated lipids and cholesterol or its derivatives are dissolved in ethanol to obtain an alcohol phase;

[0043] Nucleic acid and the PEGylated protein as described above were dissolved in citrate buffer to obtain the citrate phase; the alcohol phase and the citrate phase were then prepared into lipid nanoparticles via microfluidic control.

[0044] In some embodiments, the cationic lipids are selected from at least one of SM102, DLin-MC3-DMA, ALC-0315, or ATX-126;

[0045] In some embodiments, the neutral lipids are selected from at least one of DSPC, DOPE, DPPC, PC, PE, PG, PI, or PS;

[0046] In some embodiments, the PEG has an average molecular weight of 500 to 5000;

[0047] In some embodiments, the PEGylated lipids are ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DMG-PEG2000-MAL, aminated-PEG-DMG, and hydroxylated-PEG-DMG.

[0048] In a specific embodiment, the molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%). More specifically, the molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are 33.33%: 13.33%: 2%: 51.33%.

[0049] In a specific embodiment, the citrate buffer solution has a pH of 4.0 and a concentration of 0.02M;

[0050] In a specific embodiment, the ratio of alcohol phase flow rate to citric acid phase flow rate in the microfluidic step is 1:3.

[0051] This invention provides a fusion protein comprising CD3 ScFv, the N-terminus of the CD86 extracellular domain, and a G4H12 fragment. This protein possesses T cell targeting capabilities. The preparation of LNPs using PEGylated proteins derived from this fusion protein and PEGylated lipids enhances T cell endocytosis without excessively activating T cells. Attached Figure Description

[0052] Figure 1 The CD3 ScFv-CD86-PEG2000-DSPE TCEP modification process is shown;

[0053] Figure 2 The CD3 ScFv-CD86-PEG2000-DSPE SATA modification process is shown;

[0054] Figure 3 A flowchart of the synthesis of specifically targeted LNPs is shown;

[0055] Figure 4 The in vitro transduction efficiency verification of synthesis process 1 is shown; Figure 4 In the middle, A represents the GFP positivity rate of T cells transfected with different synthetic schemes, and the vertical axis represents the transduced GFP gene expression rate; Figure 4 In the middle B, the average fluorescence intensity of GFP transfected into T cells using different synthesis schemes is represented by the MFI, and the vertical axis represents the transduced GFP gene expression intensity.

[0056] Figure 5 The effects of different target proteins on transduction efficiency were shown;

[0057] Figure 6 The effects of different proteins on T cell activation were demonstrated.

[0058] Figure 7 The encapsulation efficiency of targeted LNPs synthesized using different synthetic processes is shown.

[0059] Figure 8 The in vitro transduction validation of targeted LNPs synthesized using different synthetic processes is shown; Figure 8 In the middle section, A represents the in vitro transduction positivity rate of targeted LNPs synthesized from PEGylated lipids from different processes; Figure 8 In the middle B, the average fluorescence intensity of targeted LNPs synthesized from PEGylated lipids from different processes is represented by B.

[0060] Figure 9 The in vivo transduction efficiency of targeted LNPs synthesized using different synthetic processes is demonstrated.

[0061] Figure 10 This demonstrates the in vivo efficacy validation of the targeted fusion protein LNP particles;

[0062] Figure 11 The encapsulation efficiency and transduction efficiency of different protein modification systems were demonstrated; among them Figure 11 A represents the encapsulation rate detection. Figure 11 B represents the transduction efficiency test. Detailed Implementation

[0063] This invention provides a fusion protein and its application in the preparation of T-cell-targeted LNPs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0064] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0065] Unless otherwise stated herein, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless otherwise expressly indicated, the singular forms “a” and “this” include plural indicators.

[0066] In this invention, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions "consisting of...".

[0067] As used herein, “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0068] "At least one" means one or more, and "more than" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0069] Lipid nanoparticles (LNPs), also known as lipid nanoparticles, are the most common drug delivery carriers besides viral vectors. Their main components include cationic lipids, accessory lipids (or neutral lipids), PEG / PEG derivatives / PEGylated lipids, and cholesterol. With a well-defined composition, they can be mass-produced using ethanol hydration in specific proportions. Their primary mechanism of action involves intracellular drug delivery via endocytosis or pinocytosis and binding to cellular LDLRs. LNPs dissociate based on the pH of the intracellular lysosomes, releasing the drug and allowing it to exert its function.

[0070] Antibody: Commonly referred to as "immunoglobulin," it encompasses antibodies with the structural features of natural antibodies and antibody-like molecules with structural features different from natural antibodies but exhibiting specificity for binding to antigen molecules. In this application, the term "antibody" has its broadest meaning, encompassing immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites or domains, and can be used to refer to antigen-binding structural fragments (e.g., antigen-binding fragments) or complexes of one or more antigen-binding fragments (e.g., scFv). Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), type (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0071] ScFv: Single-chain antibody, a synthetic antibody formed by linking the variable regions of the antibody heavy chain and light chain through a short peptide of 15-20 amino acids (sometimes referred to as a linker). It retains the antibody's activity against the antigen, has a small molecular weight, strong penetrability, and weak immunogenicity. The anti-CD3 antibody described in this application is an anti-CD3 ScFv, represented as CD3ScFv in the specific structure. Anti-CD3 ScFv can be derived from publicly disclosed antibodies targeting CD3, such as OKT3 antibody, UCHT1, and SP34.

[0072] Fusion Protein: The fusion protein described in this application refers to the expression product of two recombined genes obtained through DNA recombination technology. In this application, the fusion protein can be a protein complex formed by the above-mentioned antibody binding to one or more other proteins, peptides, or protein functional domains in any manner. In some embodiments, the binding is a direct and / or indirect connection between the antibody and the other one or more proteins, peptides, or protein functional domains. In some embodiments, the carbon (C) terminus of the antibody in the fusion protein is connected to the nitrogen (N) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the antibody in the fusion protein is connected to the carbon (C) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the nitrogen (N) terminus of the antibody in the fusion protein is connected to the nitrogen (N) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the carbon (C) terminus of the antibody in the fusion protein is connected to the carbon (C) terminus of the other protein, peptide, or protein functional domain. In some embodiments, the antibody in the fusion protein is tandem with one or more other proteins, peptides, or protein functional domains. In some embodiments, the C-terminus and / or N-terminus of the antibody in the fusion protein is linked to at least two or more other proteins, peptides, or protein functional domains, and these other proteins, peptides, or protein functional domains are not linked in tandem. In some embodiments, the other one or more proteins, peptides, or protein functional domains bind the same or different antigens or different antigenic epitopes to the antibody.

[0073] Cationic lipids are a class of biomacromolecules containing cationic hydrophilic groups. Common cationic polymers include polyethyleneimine (PEI), polylysine (PLL), dendritic macromolecules, and cationic lipids. Cationic lipids are widely used due to their good biocompatibility, simple and well-defined structure, reproducibility, and large-scale production capabilities. Cationic lipid components mainly consist of amphiphilic organic small molecules linked by nonpolar hydrophobic tails and polar hydrophilic heads via linkages. The hydrophilic head is generally composed of a single or multiple protonated amino groups, ensuring electrostatic interaction with negatively charged nucleic acids. The hydrophobic structure is usually composed of steroidal compounds such as cholesterol and alkyl chains. Based on the different linkage methods of the hydrophilic head and hydrophobic tail, cationic lipids can be structurally classified into three types: head-tail, geminal, and dumbbell-shaped, with the head-tail structure being the most common. Cationic lipids constitute the most important part of LNP formulations; they are key components of LNPs, facilitating binding to negatively charged cell membranes and serving as a core component for nucleic acid drug delivery. Common cationic lipids include: DLin-MC3-DMA, ALC-0315, SM102, ATX-126, etc.

[0074] Neutral lipids are structural components of LNPs, also known as accessory lipids. Common examples include DOPE (dioleoylphosphatidylethanolamine), DSPC (dispalmitoylphosphatidylcholine), and DOPC (dioleoyllecithin). Accessory lipids often serve as structural lipids in LNP formulations, spontaneously forming a bilayer membrane structure. This enhances the membrane stability of LNPs, preventing their rapid metabolism and excretion in the bloodstream. Simultaneously, they can disrupt endosome stability, improving nucleic acid delivery efficiency.

[0075] PEG (polyethylene glycol) lipids are polymers in which PEG is chemically bonded to the ends of lipids. They are crucial components in lipid peroxides (LNPs), regulating half-life and cellular uptake, influencing LNP population size and dispersibility, preventing LNP aggregation, and ensuring particle stability during preparation and storage. The length and structure of the PEG chain significantly impact its role in LNPs. Longer PEG chains provide better protection but may result in slower PEG removal from the LNP surface, affecting cellular uptake. Shorter PEG chains are easier to remove but may be less stable. In LNP applications, PEG with an average molecular weight of 500-5000 is commonly chosen, with PEG2000 (average molecular weight 2000) being preferred, to ensure LNP stability and effective PEG removal from the LNP.

[0076] PEGylated proteins: These are polymers formed by chemically modifying single-chain antibodies, antibodies, peptides, proteins, or fusion proteins expressed through gene recombination, to react with PEG or PEG derivatives or PEGylated lipids (such as DSPE-PEG-MAL, DMG-PEG-MAL, etc.). In some embodiments, these proteins may be proteins or fusion proteins that target biological tissues, organs, or specific cells, such as CD3ScFv-CD86-PEG-DSPE and CD3ScFv-CD86-PEG-DMG as described in this application.

[0077] Cholesterol is an abundant cell membrane component and is often used as a structural lipid in LNP formulations. Cholesterol is primarily found in the outer shell of LNPs, and modifications to its sterol structure can cause tissue changes on the LNP surface. When bound to high Tm (low gel-liquid crystal phase transition) lipids, cholesterol increases membrane fluidity and narrows the bilayer. In both cases, cholesterol pulls the lipids towards a liquid state.

[0078] Molar ratio: Under specific preparation conditions, cationic lipids and nucleic acids bind effectively via electrostatics. The molar concentration of protonated nitrogen ions in the cationic lipids and the molar concentration of phosphorus atoms in the nucleic acids are in a specific ratio, such as an N / P ratio ranging from 2:1 to 10:1. LNPs are synthesized simultaneously through ethanol hydration in specific molar ratios. Different lipid proportions result in different LNP assemblies, such as a bilayer membrane structure formed on the surface of PEG polymerization, or, when the cationic lipid ratio is high, an LNP bilayer membrane encapsulating cationic lipid and nucleic acid polymer particles. Lipid nanoparticles can be formed within a certain range of lipid molar ratios and can effectively package LNPs.

[0079] SATA protein modification method: also known as SATA-maleimide chemical coupling method, its working principle is as follows: the primary amine group of the target protein is modified by SATA (N-succinimide-S-acetyl thioacetate) to introduce an active group. Then the reactant reacts with hydroxylamine to make the antibody carry a free thiol group. Finally, the active thiol group on the target protein is linked to the MAL group on the DSPE-PEG-MAL or DMG-PEG-MAL lipid by using a thioether coupling chemical reaction to form a PEGylated protein.

[0080] TCEP protein modification method: also known as antibody site-directed modification of thiol-maleimide coupling method, its working principle is as follows: disulfide bonds are designed at specific sites of the target protein to be modified, and the disulfide bonds are reduced by TCEP reducing agent, so that the modified protein has free thiol groups. Then, through a thioether coupling chemical reaction, the free thiol groups on the antibody are linked to the MAL groups on DSPE-PEG-MAL or DMG-PEG-MAL lipids to form PEGylated proteins.

[0081] The LNP synthesis process involved in this case includes:

[0082] The current industry-standard LNP synthesis process involves dissolving ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in ethanol at a molar ratio of 50%:10%:1.5%:38.5%. The target mRNA is dissolved in 0.02M citrate buffer at pH 4.0 at a P:N ratio of 1:3 with the ionizable cationic lipids. The LNP solution is synthesized using a microfluidic system (Aitesen) with the ethanol-to-citrate phase flow rate ratio at 1:3. The solution is then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research2i[KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer.

[0083] Current targeted LNP synthesis process: The final LNP synthesis process of the above LNP synthesis process contains LNP particles. The target protein or target protein-PEG-DSPE is added to the solution, and the targeted LNP is formed by shaking or sonication.

[0084] Precision biological targeted LNP synthesis process (synthesis process 1): Ionizable cationic lipids such as SM102, neutral lipids, PEGylated lipids, and cholesterol or other modified cholesterol are dissolved in ethanol at a molar ratio of 50%:10%:1.5%:38.5%. PEGylated proteins (CD3 ScFv-CD86-PEG-DSPE or CD3 ScFv-PEG-DSPE synthesized using SATA or TCEP protein modification methods) and target nucleic acids are dissolved in citrate buffer, with the target nucleic acid dissolved at a P:N ratio of 1:3 to the ionizable cationic lipids. The citrate buffer is pH 4.0, with a concentration of 0.02M. Microfluidics (Aitesen) is used, with the equipment settings being an ethanol-citric acid phase flow rate ratio of 1:3. The targeted LNP solution is synthesized through the microfluidic device and dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research2i[KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer.

[0085] Precision biological targeted LNP synthesis process (synthesis process 2): Ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol are dissolved in ethanol at a molar ratio of 33%:13%:2%:51%. PEGylated proteins (CD3ScFv-CD86-PEG-DSPE or CD3ScFv-PEG-DSPE synthesized using SATA or TCEP protein modification methods) and target nucleic acids are dissolved in citrate buffer. The target nucleic acid is dissolved at a P:N ratio of 1:3 with the ionizable cationic lipids. The citrate buffer is pH 4.0 with a concentration of 0.02M. Using a microfluidic device (Aitesen), the device settings are ethanol phase flow rate:citrate phase flow rate ratio of 1:3. The targeted LNP solution is synthesized through the microfluidic device. Solution replacement is performed by dialysis with PBS buffer or TFF tangential flow. Finally, the LNP is stored in 0.01M PBS buffer.

[0086] In vitro transduction efficiency verification: T cells or peripheral blood mononuclear cells (PBMCs) 3e5 cells were placed in 48-well plates. Synthetic LNPs or protein-targeted LNPs (targeted LNPs) were taken and, according to mRNA quantification (mRNA quantification was performed using an RNA nucleic acid quantification kit (Thermo, catalog number: R11490) (probe method)), 1 μg of mRNA particles were added to the wells. The wells were then brought to a final volume of 200 μL with 1640 medium + 10% FBS. After culturing at 37°C and 5% CO2 for 24 h, cells were collected, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. GFP did not require labeling; the positivity rate and mean fluorescence intensity (MFI) of GFP were directly detected using flow cytometry. CD19 CAR was detected using anti-antibody B937D4-AF647. 1 μg of antibody was added, and the cells were labeled at 4°C for 30 min. After washing with 1 mL of PBS, the cells were centrifuged at 350g for 5 min and then resuspended in 100 μL of PBS. The CAR positivity rate and MFI were then detected.

[0087] Encapsulation rate detection principle:

[0088] Assessing the encapsulation effect of LNP on mRNA by measuring total mRNA and free mRNA in the formulation is a key indicator of the biological activity of nucleic acid drugs. Encapsulation efficiency is the core indicator of this. Encapsulation efficiency is measured using the RiboGreen fluorescent dye quantitative detection kit (manufacturer: Invitrogen, catalog number R11490). After the fluorescent dye binds to nucleic acid, fluorescence is excited at a specific wavelength, and the signal values ​​are analyzed and compared. First, the concentration of free RNA in the LNP-RNA solution is measured. Then, the LNP structure is destroyed using Triton-100, and the concentration of all RNA in the solution is measured. The fluorescence value of the test sample is assigned according to the concentration of the standard. The encapsulation efficiency is calculated as: (Total RNA amount - Free RNA amount) / Total RNA amount * 100%. The specific operating steps are as follows:

[0089] Preparation of 1XTF Buffer: Stock solution: 1M Tris-HCl (pH 7.4), 0.5M EDTA (pH 8.0), prepare 100mL.

[0090] The 50ul sample to be tested was gradually diluted 100-fold using 1X TF Buffer;

[0091] 0.5% Triton 100 preparation: Add 5 ml of Triton 100 to 995 ml of TE buffer (V / V) and shake gently.

[0092] Total LNP-RNA assay: Add 100 μL of a 100-fold diluted sample to 900 μL of 0.5% Triton 100 (named Sample 1). Cell-free LNP-RNA assay: Add 100 μL of a 100-fold diluted sample to 900 μL of 1*TE buffer (named Sample 2).

[0093] Preparation of standard: Add 10 μL of standard to 990 μL of 1*TE buffer (the original concentration of the standard is 1 mg / mL), and then dilute the above sample with TE buffer to 200, 100, 20, 4, 0 ng / mL;

[0094] Add 100 μL of standard, sample 1, or sample 2 to each well, and repeat for three replicates.

[0095] Fluorescent dye preparation (2000-fold dilution): Add 5 μL of fluorescent dye to 10m LTE buffer;

[0096] After adding 100 μL of dye to each well, the OD value was measured in the dark under excitation light of 485 nm and emission light of 520 nm.

[0097] A standard curve was constructed based on the standard curve. Sample 1, representing the OD value of total RNA, was substituted into the standard curve. The value was multiplied by 1000 (the original solution was diluted 1000 times) to obtain the total RNA OD value. Similarly, Sample 2 was recorded as the free RNA OD value. Encapsulation efficiency = (total RNA OD value - free RNA OD value) / total RNA OD value * 100%. mRNA quantification was performed using an RNA nucleic acid quantification kit (manufacturer: Thermo, catalog number: R11490) (probe method).

[0098] The existing lipid ratio range is as follows: ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in a molar ratio range of 50%:10%:1.5%:38.5%.

[0099] The lipid ratio range described in this application is as follows: ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in a molar ratio range of 33.33%: 13.33%: 2%: 51.33%.

[0100] Preparation of 0.02M citric acid reagent:

[0101] 1) Weigh 19.213g of citric acid (manufacturer SPECTRUM Lot No.1IE0871 FW192.13) and dissolve it in a certain amount of ultrapure water. After it is completely dissolved, dilute it to 1L in a 1L volumetric flask to obtain a 0.1M / L mother liquor.

[0102] 2) Weigh 25.807g of sodium citrate (manufacturer SPECTRUM Lot No.1HK0873 FW258.07) and dissolve it in a certain amount of ultrapure water. After it is completely dissolved, dilute it to 1L in a 1L volumetric flask to obtain a 0.1M / L mother liquor.

[0103] 3) Prepare 50ml of 0.02M citrate buffer: 40ml ultrapure water + 6.55ml 0.1M / L citrate stock solution + 3.45ml 0.1M / L sodium citrate stock solution.

[0104] The fusion protein used in this application is CD3 ScFv-CD86-G4H12, with the amino acid sequence shown in SEQ ID NO.4. The PEGylated protein is CD3 ScFv-CD86-PEG-DSPE or CD3 ScFv-CD86-PEG-DMG, where PEG is PEG2000. The PEGylated protein can be synthesized using the SATA protein modification method or the TCEP protein modification method.

[0105] Synthesizing PEGylated proteins using the SATA protein modification method:

[0106] 1) After removing the desalting column preservative solution from the Thermofisher 89889 desalting column, equilibrate the desalting column with 1 ml of 0.1M PB, 0.15M NaCl pH7.4 solution.

[0107] 2) 512 μl of aqueous solution of CD3 ScFv-CD86-G4H12 fusion protein, with a concentration of 3.90 mg / ml.

[0108] 3) Weigh 6.3 mg SATA (sigmaaldrich A9043) and dissolve it in 1364 μl DMSO (Origen CP-70) to prepare a 20 mM (4.62 mg / ml) SATA working solution.

[0109] 4) Add 4.5 μl of 20 mM SATA working solution to 256 μl of fusion protein solution (the molar ratio of fusion protein to SATA is 1:5), and react at room temperature in the dark for 0.5 h.

[0110] 5) Add the solutions obtained in step 4) to the desalting column after equilibration in step 1), centrifuge at 1000g for 2 min, and collect the flow-through liquid. Collect 340 μl of each solution.

[0111] 6) Add 34 μl of 0.5 M hydroxylamine, 25 mM EDTA, 0.1 M PB, and 0.15 M NaCl pH 7.4 solution (1 / 10 of the liquid volume) to the above solution and react at room temperature in the dark for 2 h.

[0112] 7) Weigh 6.0 mg of DSPE-PEG-MAL (WH-0010205 from Huasheng Biotechnology) and dissolve it in 408 μl of DMSO (OrigenCP-70) to prepare a 5 mM (14.7 mg / ml) DSPE-PEG-MAL working solution.

[0113] 8) Add 17.8 μl of 5 mM DSPE-PEG-MAL working solution to each of the solutions in 6), wherein the fusion protein:SATA:DSPE-PEG-MAL = 1:5:5. Incubate at room temperature in the dark for 0.5 h.

[0114] 9) Add 370 μl of the solution obtained in step 8) to the equilibrated desalting column, centrifuge at 1000 g for 2 min, and collect the flow-through. This yields the PEGylated protein CD3 ScFv-CD86-PEG-DSPE.

[0115] In some embodiments, step 4 is: take 1.8 μl of 20 mM SATA working solution and add it to 256 μl of fusion protein solution (the molar ratio of fusion protein to SATA is 1:2), react at room temperature in the dark for 0.5 h, and the fusion protein:SATA:DSPE-PEG-MAL in step 8 is 1:2:5.

[0116] PEGylated protein CD3 ScFv-CD86-PEG-DSPE was synthesized using the TCEP protein modification method.

[0117] 1) After removing the desalting column preservative solution from the Thermofisher 89889 desalting column, equilibrate the desalting column with 1 ml of 0.1M PB, 0.15M NaCl pH7.4 solution.

[0118] 2) 660 μl of aqueous solution of CD3 ScFv-CD86-G4H12 fusion protein, with a concentration of 5.05 mg / ml.

[0119] 3) Add 1.6 μl of 0.5 M TCEP (Aladdin T107252-25g) aqueous solution to 350 μl of fusion protein solution, and react at room temperature in the dark for 1 h. (The molar ratio of fusion protein to TCEP is 1:25).

[0120] 4) Add the solution obtained in step 3) to the equilibrated desalting column, centrifuge at 1000g for 2 min, and collect the flow-through liquid.

[0121] 5) Weigh 13.3 mg of DSPE-PEG-MAL (WH-0010205 from Huasheng Biotechnology) and dissolve it in 905 μl of DMSO (OrigenCP-70) to prepare a 5 mM (14.7 mg / ml) DSPE-PEG-MAL working solution.

[0122] 6) Add 63.2 μl of 5 mM DSPE-PEG-MAL working solution to the flow-through solution obtained in step 4), with the fusion protein:TCEP:DSPE-PEG-MAL ratio being 1:25:10. Incubate at room temperature in the dark for 0.5 h.

[0123] 7) Add 400 μl of the solution obtained in step 6) to the equilibrated desalting column, centrifuge at 1000g for 2 min, and collect the flow-through. This yields the PEGylated protein CD3 ScFv-CD86-PEG-DSPE.

[0124] CD3 ScFv-CD86-PEG-DMG was synthesized according to the above scheme, except that DSPE-PEG-MAL was replaced with DMG-PEG-MAL (manufacturer: Aivito, product number 160743-62-4).

[0125] The sequence involved in this case:

[0126]

[0127]

[0128]

[0129] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0130] The present invention will be further illustrated below with reference to the embodiments:

[0131] Example 1: Synthesis process 1, compared with the current targeted LNP synthesis process, improves the transfection efficiency of T cells.

[0132] CD3 ScFv-CD86-PEG-DSPE is a PEGylated protein synthesized from the CD3 ScFv-CD86-G4H12 fusion protein described in this application using a SATA modification process, wherein the PEG is PEG2000. The molar ratio of protein:SATA:PEG2000-DSPE-MAL is 1:5:5. The target mRNA is GFP mRNA, with the sequence shown in SEQ ID NO.5. The GFP gene was transduced in vitro using the Precision Biosynthesis Process 1 described above, with 150 μg of CD3 ScFv-CD86-PEG2000-DSPE added to citrate buffer. The synthesis system consisted of 1.5 mL of alcohol and 4.5 mL of citrate. The synthesis rate was 12 mL / min for the alcohol phase and 36 mL / min for the citrate phase. After microfluidic synthesis, the particles were dialyzed, with PBS replaced every 1 hour for a total of 4 times. Samples were then collected and quantitatively analyzed using an RNA quantification kit. Labeled as Precision Biosynthesis Process 1. The ionizable cationic lipids are SM102, the neutral lipids are DSPC, and the PEGylated lipids are DSPE-PEG and cholesterol (CHOL). In the targeted LNP synthesis process, DSPE-PEG and the target protein (CD3 ScFv-CD86-G4H12 in this example) are modified by different protein modification methods, SATA or TCEP, to form a PEGylated protein (CD3 ScFv-CD86-PEG-DSPE in this example).

[0133] The targeted LNP was synthesized according to the current targeted LNP synthesis process (first synthesizing LNP according to the aforementioned industry-standard LNP synthesis process and then synthesizing the targeted LNP with the targeted PEGylated protein), wherein the targeted PEGylated protein was CD3ScFv-CD86-PEG-DSPE, labeled with the current process in the literature.

[0134] Synthesis process 1 is a one-step synthesis method, where the target protein and LNP synthetic particles are synthesized in one step. Current literature uses a two-step synthesis method, first synthesizing the LNP particles, and then conjugating the target protein to them. In this embodiment, synthesis process 1 and the current literature process use the same LNP lipid ratio; the only difference is the targeted synthesis step. Validation was performed according to the aforementioned in vitro transduction efficiency verification method. T cells from different donors were transfected multiple times, and statistical analysis was conducted. The target mRNA was GFP-expressing RNA, and it was directly detected by flow cytometry. The results are as follows... Figure 4 As shown, paired t-test analysis revealed that the particles synthesized using the precise biosynthesis process 1 exhibited significantly higher in vitro transfection efficiency than those reported in the literature. In conclusion, the one-step synthesis method described in this application represents a superior targeted LNP synthesis protocol.

[0135] Example 2: The fusion protein CD3ScFv-CD86 improves T cell transfection efficiency compared to CD3ScFv.

[0136] The ability of a targeted LNP to transduce a target gene into a target cell is inseparable from the design of its target protein. Currently, CD3 antibodies are used as the source of the targeted protein in the field. This application optimizes the target protein and designs a CD3ScFv-CD86 fusion protein. This embodiment verifies the fusion protein described in this application. Using the target protein CD3ScFv-CD86-G4H12 or CD3 ScFv-G4H12, the amino acid sequence of which is shown in SEQ ID NO.4, PEGylated proteins CD3 ScFv-CD86-PEG-DSPE or CD3 ScFv-PEG-DSPE were synthesized using the SATA modification process, where PEG was PEG2000. The targeted LNP was synthesized using synthesis process 1, with the target mRNA being an mRNA expressing GFP, the sequence of which is shown in SEQ ID NO.2. The specific steps were the same as in Example 1, wherein the ionizable cationic lipid was SM102, the neutral lipid was DSPC, and the PEGylated lipids were DMG-PEG and cholesterol (CHOL). Non-targeted LNP particles, or LNP groups, were synthesized using synthesis process 1: ionizable cationic lipid SM102, neutral lipid DSPC, PEGylated lipid DMG-PEG, and cholesterol CHOL were dissolved in the alcohol phase at a molar ratio of 50%:10%:1.5%:38.5%. Target mRNA and PEGylated protein were dissolved in citrate buffer, with the target mRNA dissolved at a P:N ratio of 1:3 to the ionizable cationic lipid. The citrate buffer was pH 4.0 and had a concentration of 0.02 M. A microfluidic system (Aitesen) was used, with the alcohol phase flow rate to citrate phase flow rate ratio at 1:3. Targeted LNP solutions were synthesized using the microfluidic system and then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research2i[KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer.

[0137] In the non-targeted LNP synthesis component, DMG-PEG is a simple DMG-PEG compound without the target protein, manufactured by: Weihua Biotechnology WH-0010205; in the targeted LNP synthesis process, the PEGylated lipid DSPE-PEG and the target protein CD3 ScFv-CD86-G4H12 or CD3 ScFv-G4H12 are modified by different protein modification methods, SATA method or TCEP method, to obtain the PEGylated protein: CD3 ScFv-CD86-PEG-DSPE or CD3ScFv-PEG-DSPE.

[0138] GFP gene expression efficiency was detected using in vitro transduction efficiency verification methods, such as... Figure 5 As shown in Table 1, Figure 5 The x-axis represents different fusion proteins, and the y-axis represents the GFP positivity rate. Specifically, the x-axis represents the LNP group (without the target protein), the CD3-LNP group (containing the CD3 ScFv target protein), and the CD3-CD86-LNP group (containing the CD3ScFv-CD86 fusion protein as the target protein).

[0139] Table 1. GFP gene expression efficiency transduced by different target proteins.

[0140]

[0141] The results showed that, using the unpaired T assay, there was no significant difference between the LNP group and the CD3ScFv-LNP group. Compared with the CD3ScFv-CD86-LNP group (hereinafter referred to as CD3-CD86-LNP), the CD3-CD86-LNP group containing the CD3ScFv-CD86-G4H12 fusion protein described in this application had a significantly higher GFP transduction efficiency (P < 0.0001). These results indicate that the designed fusion protein significantly improved T cell transduction efficiency.

[0142] Example 3: CD3 ScFv-CD86 fusion protein CD86 signaling does not activate T cells

[0143] CD3 ScFv is a primary signal for T cell activation, activating the T cell receptor (TCR). In the human body, when infected by cells or viruses, T cells are stimulated to activate the CD3 primary activation signal. Simultaneously, the binding of CD86 on APC cells to the CD28 ligand on T cells activates the secondary activation signal, thereby enhancing the T cell's cytotoxic and cytokine secretion functions to clear viruses and other pathogens. The CD28 signaling pathway activates NF-κB signaling; the expression level of NF-κB is typically used to assess the activation status of CD28 signaling.

[0144] Theoretically, CD86 can promote T cell activation through the CD28 signaling pathway, thereby enhancing T cell transduction efficiency. However, this activation requires a functional CD86 sequence structure. The fusion protein CD3ScFv-CD86 in this application recombines CD3ScFv and CD86 proteins into a single expression cassette. In this structure, CD86 functions more like an extracellular support structure and does not perform CD28 signaling activation. To verify whether the increased transduction efficiency of the fusion protein CD3ScFv-CD86 is related to T cell activation by the CD86-CD28 signaling pathway, Jukart cells were used. Jukart cells were transfected with a lentiviral vector expressing the NF-Kb-Luc-GFP gene at a multiplicity of infection (MOI) of 3. Flow cytometry analysis confirmed successful gene construction. Jukart-NF-Kb-Luc-GFP cells, constructed from 1e6 cells, were seeded in 24-well plates. Different amounts of CD3 ScFv and the fusion protein CD3 ScFv-CD86-G4H12 were added, and after 48 h of culture, the cells were lysed, and Luc expression was detected at 450 nm using a microplate reader. The expression of the NF-Kb-Luc-GFP gene in T cells after stimulation with CD3 ScFv and the fusion protein CD3 ScFv-CD86-G4H12 was compared to verify whether the CD86 molecule functions. Results are as follows: Figure 6 As shown, the vertical axis represents the absorbance of Luc expression; higher absorbance indicates higher NF-Kb gene expression.

[0145] The results showed that, based on Luc expression, there was no significant difference between the CD3 ScFv and CD3 ScFv-CD86 fusion protein across different dose groups. This suggests that the CD3 ScFv-CD86 fusion protein is a truncated CD86 structure, lacking the complete CD86-CD28 signaling sequence, and therefore has almost no activating effect on cells. The improved transduction efficiency of the LNP containing the CD3 ScFv-CD86 fusion protein compared to the LNP using CD3 ScFv as the target protein is likely due to the novel structure formed by the truncated CD86 and CD3 ScFv, combined with its conformation after being displayed on the LNP, and cannot be explained by the known CD86-CD28 signaling mechanism in this field.

[0146] Example 4: Comparison of the performance of targeted LNPs synthesized by different synthesis processes

[0147] Example 1 demonstrates the superiority of the one-step synthesis method by comparing synthesis process 1 with existing processes in the literature. In some examples, the molar ratios of the components in LNP synthesis may vary during the synthesis process. The applicant designed synthesis process 2, which differs from synthesis process 1 only in the molar ratios of the LNP synthesis components. In this example, the transduction efficiency and encapsulation efficiency of the one-step synthesis process 2 with varying molar ratios of the LNP synthesis components were verified. Furthermore, for the one-step synthesis method, PEGylated proteins with targeting proteins can be prepared using various methods. Whether PEGylated proteins with targeting proteins prepared by different methods affect the properties of LNP particles has not been studied in the prior art. In this example, we verified and compared the transduction efficiency and encapsulation efficiency of targeted LNPs synthesized using PEGylated proteins with targeting proteins prepared by the SATA method and the TCEP method as synthetic raw materials.

[0148] 4.1 Encapsulation efficiency detection of targeted LNPs synthesized by different synthesis processes

[0149] Currently, existing technologies for preparing targeted LNPs all use the SATA method to modify fusion proteins, but the SATA method can lead to dimer formation. We have optimized the targeted LNP synthesis process to develop the TCEP method for fusion protein modification. The specific method is described above, and the fusion protein is CD3ScFv-CD86-G4H12 as described in this application.

[0150] Based on the aforementioned precise biosynthesis process 2, PEGylated proteins synthesized using either the SATA method or the TCEP method were used as raw materials to synthesize targeted LNP particles. The specific component ratios of the targeted LNP particles were SM102 (33.33%), DSPC (13.33%), PEG-DMG (2%), and CHOL (51.33%). Targeted LNP particles synthesized using the SATA method were labeled using the SATA method, and those synthesized using the TCEP method were labeled using the TCEP method. 150 μg ScFv or a fusion protein conjugate was added to the citrate buffer. The synthesis system consisted of 1.5 mL of alcohol phase and 4.5 mL of citrate. The synthesis rate was 12 mL / min for the alcohol phase and 36 mL / min for the citrate phase. After microfluidic synthesis of the particles, dialysis was performed, with PBS replaced every 1 hour for a total of 4 times before sample collection.

[0151] The encapsulation efficiency of targeted LNP particles was detected using an RNA quantification kit, as described previously. Results are as follows: Figure 7As shown, the results indicate that, through t-test and unpaired test, the TCEP-modified fusion protein PEGylated protein significantly improves the particle encapsulation efficiency, meeting the particle encapsulation rate requirements for industrial-scale production.

[0152] 4.2 Validation of In Vitro Transduction of Targeted LNPs Synthesized by Different Synthetic Processes

[0153] Precision biosynthesis process 2 synthesizes targeted LNPs (see 1 for details). In vitro transfection was validated according to the above-described in vitro transduction efficiency verification method. T cells from different donors were repeatedly transfected and statistically analyzed. The target nucleic acid was RNA expressing CD19 CAR, with the sequence shown in SEQ ID NO.6. CAR-positive detection antibody was added at 1 μg, labeled at 4℃ for 30 min, washed with 1 mL of PBS, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. CAR positivity rate and MFI were detected. Results are as follows... Figure 8 As shown, Figure 8 In the middle section, A represents the positive rate of in vitro transduction of PEGylated proteins from different sources that target LNP synthesis. Figure 8 In the middle B, the average fluorescence intensity of the targeted LNP in vitro transduction of PEGylated protein from different process sources is represented.

[0154] The results showed that the targeted PEGylated protein containing the targeted fusion protein CD3ScFv-CD86-G4H12 described in this application, synthesized using the TCEP method, as a raw material for the PEG derivative of the targeted LNP, significantly improved the mean fluorescence intensity (MFI) of the target gene expression. Paired t-test analysis showed that P = 0.0021.

[0155] 4.3 Verification of in vivo transduction efficiency using different synthesis processes

[0156] Human PBMCs were injected via tail vein into severely immunodeficient mice, such as NCG mice. The PBMC dosage ranged from 5e6 cells / mouse to 3e7 cells / mouse. Between 10 and 20 days after injection, blood samples were collected from the orbital sinus to measure the proportion of hCD45+ cells in the peripheral blood nucleated cells, which ranged from 5% to 70%. Mice were randomly assigned to receive 40 μg / mouse of PEGylated protein synthesized using different processes. The target LNP was synthesized using LNP synthesis process 2, with the target mRNA being the CD19 CAR gene and the target cells being T cells. Mice were euthanized 24 hours after infusion of the targeted LNP. Spleens were harvested, and erythrocytes were lysed. Cell counts of 1e6-2e6 cells were used to detect the CAR expression positivity rate. CD19 CAR was performed using the in-house produced anti-antibody B937D4-AF647 from Precision Biotechnology. 1 μg of antibody was added, along with 1 μg of commercially available CD45 and CD3 antibodies. After labeling at 4°C for 30 min, cells were washed with 1 mL of PBS, centrifuged at 350g for 5 min, and resuspended in 100 μL of PBS. CAR positivity and MFI were then detected. Results are as follows: Figure 9 As shown, the vertical axis represents the transduced CAR gene expression efficiency.

[0157] The results indicate that the fusion protein of this application, the TCEP site-modified fusion protein PEGylated protein significantly improved the in vivo target cell transduction efficiency of targeted LNPs, and the result was P = 0.0303 through one-way ANOVA.

[0158] Example 5: In vivo preparation and functional verification of CAR-T cells using targeted LNP particles

[0159] Human PBMCs (1e7 cells) were injected via tail vein into severely immunodeficient mice, such as NCG mice. Twenty-four hours later, 2e6 cells of Nalm6-Luc-GFP were injected via tail vein. Mice were then intraperitoneally injected with 15 mg / kg of Luceferase substrate. In vivo imaging was performed using a live imaging system. After in vivo imaging, the targeted LNP prepared according to the protocol in Example 3 was injected via tail vein at a rate of 40 μg / mouse. Subsequent in vivo imaging and particle injection were performed approximately once a week, and the efficacy of the particles was evaluated based on changes in fluorescence values. Results are as follows: Figure 10 As shown, the experiment was divided into three groups: the Nalm6 group, the LNP group, and the targeted particle group. Compared with the LNP group, the targeted particle group had a significant inhibitory effect on tumor proliferation. The results indicate that the targeted particles can achieve in vivo CAR-T cell self-production, thereby achieving the goal of tumor treatment.

[0160] In conjunction with Examples 1, 2, and 3, the targeted fusion protein CD3ScFv-CD86-G4H12 described in this application exhibits superior targeted transduction capabilities under different modification methods (SATA or TCEP) and different synthesis processes (synthesis process 1 or synthesis process 2), promoting the transduction of target nucleic acids encapsulated by targeted LNPs into target cells. The targeted fusion protein CD3ScFv-CD86-G4H12 described in this application is used in a one-step synthesis method for targeted LNPs. The LNP components are: ionizable cationic lipids such as SM102, neutral lipids, PEG or PEGylated lipids, and cholesterol or other modified cholesterol in a molar ratio ranging from (33%-50%):(10%-13.33%):(1.5%-2%):(38.5%-51.33%), with a preferred molar ratio of 33.33%:13.33%:2%:51.33%. The synthesized targeted LNP has a more efficient ability to transduce the target gene into the target cell. Preferably, the PEGylated protein linked to the target protein synthesized using the TCEP method has higher transduction efficiency and encapsulation rate.

[0161] Example 6: Detection of Targeted PEGylated Protein Modification Conditions for Fusion Protein Modification

[0162] The fusion protein was modified with PEG using the TCEP method described above. Two TCEP concentrations were set, with the fusion protein:TCEP:DSPE-PEG-MAL molar ratios of 1:50:10 and 1:25:10, respectively, to compare the effects of different TCEP concentrations on the target particle encapsulation efficiency and transfection efficiency. CD3ScFv-CD86-PEG-DSPE was synthesized according to the above scheme. The formulation components SM102 (33.33%), DSPC (13.33%), DMG-PEG (2%), and CHOL (51.33%) were dissolved in ethanol to form the ethanol phase. The target nucleic acid and CD3ScFv-CD86-PEG-DSPE were dissolved in citrate buffer to form the citrate phase. The target nucleic acid was CD19 CAR mRNA, dissolved at a P:N ratio of 1:3 with ionizable cationic lipids. The citrate buffer in the citrate phase was a pH 4.0, 0.02M citrate buffer. Using a microfluidic system (Aitesen), with the device settings at an alcohol-to-citric acid flow rate ratio of 1:3, LNP solutions were synthesized via the microfluidic device and then dialyzed using PBS buffer or TFF tangential flow (Replekin). The solution was replaced using the Research 2i [KR2i]TFF system, and the LNP was finally stored in 0.01M PBS buffer. mRNA quantification was performed using an RNA nucleic acid quantification kit.

[0163] Encapsulation efficiency was assessed using an RNA quantification kit (Thermo, catalog number R11490). Different batches of T cells were simultaneously transfected to verify the transfection efficiency and encapsulation efficiency of the targeting particles. Results are as follows: Figure 11 As shown, the encapsulation efficiency and transduction efficiency of different protein modification systems are validated; among them Figure 11 A represents the encapsulation rate detection. Figure 11 B represents the transduction efficiency test.

[0164] The results show that the molar ratios of TCEP:DSPE-PEG-MAL of 1:50:10 and 1:25:10, respectively, do not affect the encapsulation efficiency and transduction efficiency, thus achieving the objectives of this application. Different concentrations of TCEP have no effect on the encapsulation efficiency, nor on the expression and fluorescence intensity of CAR after different batches of T transfection. Therefore, in the synthesis of PEGylated lipids modified with fusion proteins, the molar ratio of fusion protein, TCEP, or PEGylated lipids should be 1:(25-50):(5-10).

[0165] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, comprising CD3 ScFv, the N-terminus of the extracellular domain of CD86, and the G4H12 fragment.

2. The fusion protein according to claim 1, characterized in that, The CD3 ScFv has an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:

1.

3. The fusion protein according to claim 1, characterized in that, The extracellular segment of CD86 has an N-terminus with an amino acid sequence as shown in SEQ ID NO:2, or an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that has more than 80% identity with the amino acid sequence shown in SEQ ID NO:

2.

4. The fusion protein according to claim 1, characterized in that, The G4H12 fragment has an amino acid sequence as shown in SEQ ID NO:3, or has an amino acid sequence obtained by deleting, substituting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO:3, or has an amino acid sequence that is more than 80% identical to the amino acid sequence shown in SEQ ID NO:

3.

5. The fusion protein according to any one of claims 1 to 4, characterized in that, From the N-terminus to the C-terminus, it includes CD3ScFv, linker, the N-terminus of the extracellular segment of CD86, and the G4H12 fragment.

6. The fusion protein according to claim 5, characterized in that, It has the amino acid sequence shown in SEQ ID NO:

4.

7. Biomaterials, including at least one of the following: I) Nucleic acid encoding the fusion protein according to any one of claims 1 to 6; II) Expression units containing the nucleic acid described in I); III) A recombinant vector containing the nucleic acid described in I) or the expression unit described in II); IV) Transformants that have been transformed or transfected with the expression vector described in III); The culture products of the transformants described in (V) and (IV).

8. A PEGylated protein comprising the fusion protein and PEGylated lipids as described in any one of claims 1 to 6.

9. The PEGylated protein according to claim 8, characterized in that, The PEGylated lipids include at least one of ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, and DMG-PEG2000-MAL.

10. The PEGylated protein according to claim 8 or 9, characterized in that, The average molecular weight of PEG is 2000.

11. A method for preparing the PEGylated protein according to any one of claims 8 to 10, comprising: The fusion protein according to any one of claims 1 to 6 is mixed with TCEP solution and reacted, PEGylated lipids are added, and after reaction, the PEGylated protein according to any one of claims 8 to 10 is obtained.

12. The preparation method according to claim 11, characterized in that, The molar ratio of the fusion protein, TCEP and PEGylated lipids according to any one of claims 1 to 6 is 1:(25 to 50):(5 to 10).

13. The use of the fusion protein according to any one of claims 1 to 6 or the PEGylated protein according to any one of claims 8 to 11 in the preparation of T cell-targeting LNPs.

14. A method for preparing targeted LNPs, characterized in that, Cationic lipids, neutral lipids, PEG or PEGylated lipids, and cholesterol or its derivatives are dissolved in ethanol to obtain the alcohol phase; The nucleic acid and the PEGylated protein according to any one of claims 8 to 11 are dissolved in citrate buffer to obtain the citrate phase; the alcohol phase and the citrate phase are prepared into lipid nanoparticles by microfluidic control.

15. The preparation method according to claim 14, characterized in that, The cationic lipids are selected from at least one of SM102, DLin-MC3-DMA, ALC-0315 or ATX-126; The neutral lipids are selected from at least one of DSPC, DOPE, DPPC, PC, PE, PG, PI or PS; The PEG has an average molecular weight of 500 to 5000. The PEGylated lipids are ALC-0159, DMG-PEG, PEG2000-C-DMG, DSPE-PEG, DPPE-MPEG(2000), DSPE-PEG-Amine, DSPE-PEG-NHS,MW 2000, DSPE-PEG-Maleimide, DMG-PEG-MAL, DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DMG-PEG2000-MAL, Aminated-PEG-DMG, and Hydroxylated-PEG-DMG.

16. The preparation method according to claim 14 or 15, characterized in that, The molar percentages of the cationic lipids, neutral lipids, PEG or PEGylated lipids, cholesterol or their derivatives are (33%–50%): (10%–13.33%): (1.5%–2%): (38.5%–51.33%).

17. The preparation method according to any one of claims 14 to 16, characterized in that, The citrate buffer solution has a pH of 4.0 and a concentration of 0.02M. In the microfluidic step, the ratio of alcohol phase flow rate to citric acid phase flow rate is 1:3.

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