Lipid nanoparticles for immunotherapy

Abstract

Disclosed herein are compositions and methods relating to lipid nanoparticles (LNPs) comprising ionizable lipids. The LNP may comprise a nucleic acid sequence encoding a therapeutic peptide (e.g., a bispecific antibody or antigen-binding fragment thereof) for use in immunotherapy.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 392,800, filed July 27, 2022; and U.S. Provisional Application No. 63 / 510,616, filed June 27, 2023; each of which is incorporated herein by reference in its entirety. Background Technology

[0003] Synthetic mRNA provides a template for the synthesis of any given peptide, protein, or protein fragment and is suitable for a wide range of pharmaceutical applications, including various forms of cancer immunotherapy. For mRNA to function in vivo, a safe, efficient, and stable delivery system is required that protect the nucleic acid from degradation and allow cellular uptake and release of the mRNA. Lipid nanoparticle-mRNA formulations have been developed and are undergoing clinical evaluation for the prevention and treatment of viral infections, cancer, and genetic diseases. Summary of the Invention

[0004] This document provides pharmaceutical compositions and methods relating to lipid nanoparticles for delivering nucleic acid molecules that encode therapeutic peptides configured to bind B-cell antigens. In some embodiments, the pharmaceutical composition comprises: a) a synthetic nucleic acid molecule encoding a therapeutic peptide configured to bind B-cell antigens; and b) a plurality of lipid nanoparticles, wherein the synthetic nucleic acid molecule is encapsulated within at least one of the plurality of lipid nanoparticles.

[0005] In some embodiments, the B-cell antigen is selected from the group consisting of: memory B-cell antigen, naive B-cell antigen, plasmablast antigen, or plasma cell antigen, and any combination thereof. In some embodiments, the B-cell antigen is selected from the group consisting of: differentiation cluster (CD)10, CD19, CD20, CD22, CD27, CD32b, CD38, CD40, B-cell maturation antigen (BCMA), B-cell activating factor receptor (BAFFR), CD138, CD5, and any combination thereof. In some embodiments, the B-cell antigen includes CD19. In some embodiments, the therapeutic peptide is capable of binding the B-cell antigen with a higher affinity than other antigens. In some embodiments, the therapeutic peptide binds to a second antigen. In some embodiments, the second antigen includes an immune cell antigen or a tumor antigen. In some embodiments, the immune cell antigen includes a T-cell antigen, a Treg cell antigen, or a natural killer cell (NK cell) antigen. In some embodiments, the second antigen includes CD3 and the therapeutic peptide is configured to bind CD3.

[0006] In some embodiments, the therapeutic peptide comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises a bispecific antibody, a multispecific antibody, or a chimeric antigen receptor (CAR). In some embodiments, the bispecific antibody or antigen-binding fragment thereof comprises a bispecific T-cell connective (BiTE). In some embodiments, the BiTE is configured to bind CD19 and CD3. In some embodiments, the therapeutic peptide comprises an FDA-approved drug, wherein the FDA-approved drug is configured to bind a B-cell antigen. In some embodiments, the FDA-approved drug includes blinatumomab, inebilizumab, loncastuximab, or tafasitamab. In some embodiments, the synthetic nucleic acid molecule further comprises a regulatory nucleic acid sequence. In some embodiments, the regulatory nucleic acid sequence comprises a promoter or signal peptide. In some embodiments, the promoter comprises a tissue-specific promoter.

[0007] In some embodiments, the synthetic nucleic acid molecule comprises a synthetic ribonucleic acid sequence (RNA). In some embodiments, the synthetic RNA comprises a chemically modified nucleic acid, and wherein the synthetic RNA comprises improved stability. In some embodiments, the synthetic RNA comprises N6-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), or N4-acetylcytidine (ac4C), or any combination thereof. In some embodiments, the synthetic RNA comprises a 5' cap.

[0008] In some embodiments, the synthetic RNA comprises, in the 5' to 3' orientation: a) a 5' untranslated region (5'UTR) coding region; b) a signal peptide coding region; c) a therapeutic peptide coding region; d) a 3' untranslated region (3'UTR) coding region; and e) a poly-A tail coding region. In some embodiments, the signal peptide coding region comprises the sequence of SEQ ID NO:11. In some embodiments, the synthetic RNA further comprises an adapter coding region. In some embodiments, the adapter coding region comprises the sequence of SEQ ID NO:12 or SEQ ID NO:13. In some embodiments, the therapeutic peptide coding region encodes a variable light (VL) chain by a sequence that is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO:1, 3, 5, 7, or 9; and encodes a variable heavy (VH) chain by a sequence that is at least 90%, 95%, 98%, or 99% identical to SEQ ID NO:2, 4, 6, 8, or 10. In some embodiments, the therapeutic peptide coding region encodes the VL and VH chains via the following sequences: 1) SEQ ID NO: 1 and 2; 2) SEQ ID NO: 3 and 4; 3) SEQ ID NO: 5 and 6; 4) SEQ ID NO: 7 and 8; or 5) SEQ ID NO: 9 and 10. In some embodiments, the therapeutic peptide coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with sequences selected from the group consisting of: 1) SEQ ID NO: 1 and 2; 2) SEQ ID NO: 5 and 6; or 3) SEQ ID NO: 9 and 10, wherein these nucleic acid sequences encode an anti-CD19 antibody or an anti-CD19 binding fragment thereof. In some embodiments, the therapeutic peptide coding region comprises a nucleic acid sequence consisting of: 1) SEQ ID NO: 3 and 4; or 2) SEQ ID NO: 7 and 8, wherein these nucleic acid sequences encode an anti-CD3 antibody or an anti-CD3 binding fragment thereof. In some embodiments, the therapeutic peptide coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with the following: 1) SEQ ID NO: 3 and 4; or 2) SEQ ID NO: 7 and 8, wherein these nucleic acid sequences encode an anti-CD3 antibody or an anti-CD3 binding fragment thereof.

[0009] In some embodiments, the synthesized RNA further encodes a protein tag. In some embodiments, the protein tag includes a histidine tag, a flag tag, or a hemagglutinin tag. In some embodiments, the therapeutic peptide coding region comprises, in a 5' to 3' orientation: a) an anti-CD19 light chain coding region; b) an anti-CD19 heavy chain coding region; c) an anti-CD3 heavy chain coding region; and d) an anti-CD3 light chain coding region. In some embodiments, the anti-CD19 light chain coding region comprises the sequence of SEQ ID NO: 1, 3, 5, or 9. In some embodiments, the anti-CD19 heavy chain coding region comprises the sequence of SEQ ID NO: 2, 4, 6, or 10. In some embodiments, the anti-CD19 light chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 1, 3, 5, or 9. In some embodiments, the anti-CD19 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:2, 4, 6, or 10. In some embodiments, the anti-CD3 light chain coding region comprises the sequence of SEQ ID NO:7. In some embodiments, the anti-CD3 light chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:7. In some embodiments, the anti-CD3 heavy chain coding region comprises the sequence of SEQ ID NO:8. In some embodiments, the anti-CD3 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:8. In some embodiments, the anti-CD19 light chain coding region comprises SEQ ID NO:1 and the anti-CD19 heavy chain coding region comprises SEQ ID NO:2; wherein the anti-CD3 light chain coding region comprises SEQ ID NO:3 and the anti-CD3 heavy chain coding region comprises SEQ ID NO:4. In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:5 and the anti-CD19 heavy chain coding region includes SEQ ID NO:6; wherein the anti-CD3 light chain coding region includes SEQ ID NO:7 and the anti-CD3 heavy chain coding region includes SEQ ID NO:8. In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:9 and the anti-CD19 heavy chain coding region includes SEQ ID 10; wherein the anti-CD3 light chain coding region includes SEQ ID NO:7 and the anti-CD3 heavy chain coding region includes SEQ ID NO:8. In some embodiments, the anti-CD19 heavy chain coding region includes a nucleotide sequence encoding the light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.In some embodiments, the anti-CD19 heavy chain coding region includes a nucleotide sequence encoding the heavy chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.

[0010] In some embodiments, the synthetic RNA comprises single-stranded synthetic RNA. In some embodiments, the lipid nanoparticles target a tissue or organ when administered to a subject. In some embodiments, the tissue or organ includes the liver. In some embodiments, the tissue or organ includes a lymphoid organ. In some embodiments, the lymphoid organ includes bone marrow, spleen, or lymph nodes. In some embodiments, the lipid nanoparticles target target cells when administered to a subject. In some embodiments, the target cells include hepatocytes, lymphocytes, leukocytes, myeloid cells, or hematopoietic stem cells. In some embodiments, the target cells include B cells, and wherein the B cells include plasmablasts, plasma cells, or memory B cells. In some embodiments, the target cells include T cells. In some embodiments, the target cells include cells circulating in the subject's systemic circulation. In some embodiments, the target cells include cells within the subject's tissues or organs.

[0011] In some embodiments, the lipid nanoparticles exhibit a faster clearance rate compared to other lipid nanoparticles when administered to a subject. In some embodiments, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition has a longer half-life compared to a corresponding therapeutic peptide contained in another pharmaceutical composition when administered to a subject. In some embodiments, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition has a half-life of at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, or at least 12 hours when administered to a subject. Half-life of at least 16 hours, at least 18 hours, at least 24 hours, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 8.5 days, at least 9 days, at least 9.5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days or more.

[0012] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence has a larger area under the curve (AUC) compared to the corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

[0013] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence has an AUC that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more greater than that of a corresponding therapeutic peptide in an equivalent dose of another pharmaceutical composition.

[0014] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence results in prolonged B-cell depletion compared to a corresponding therapeutic peptide in an equivalent dose of another pharmaceutical composition.

[0015] In some implementations, the prolonged B-cell depletion includes B-cell depletion for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 17.5 days, at least 20 days, at least 25 days, at least 27.5 days, at least 30 days, at least 35 days, at least 37.5 days, at least 40 days, at least 45 days, at least 47.5 days, at least 50 days, at least 55 days, at least 57.5 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, or at least 120 days or more.

[0016] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition causes depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject. In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition causes depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject. In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition causes depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the naïve B cells in the subject. In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition causes depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the mature B cells in the subject. In some embodiments, the mature B cells include memory B cells, plasma cells, or plasmablasts.

[0017] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B cell depletion compared to a corresponding therapeutic peptide in an equivalent dose of another pharmaceutical composition.

[0018] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a solution suitable for injection into a subject. In some embodiments, the pharmaceutical composition further comprises a small molecule drug. In some embodiments, the small molecule drug comprises an agent for chemotherapy. In some embodiments, the subject comprises a mammal. In some embodiments, the mammal comprises a human, a non-human primate, or a rodent. In some embodiments, the pharmaceutical composition has zero to minimal toxicity when administered to the subject. In some embodiments, the toxicity includes a transient increase in cytokines or liver enzymes. In some embodiments, the toxicity includes mild inflammation or mild hepatotoxicity. In some embodiments, after administration to the subject, the pharmaceutical composition leads to activation of CD69+ T cells.

[0019] In some embodiments, the lipid nanoparticles comprise a lipid composition; wherein the lipid composition comprises an ionizable lipid or a pharmaceutically acceptable salt thereof; wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail R lipid having the following structure: Or its pharmaceutically acceptable salt; wherein

[0020] Rk1 is independently a C1-C12 divalent aliphatic or heteroaliphatic group;

[0021] Rk3 is independently a C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3-C12 heterocycloalkyl, aryl or heteroaryl;

[0022] Rk2 can be independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl or heteroaryl;

[0023] Rk4 and Rk5 are each independently H or C1-C12 divalent aliphatic groups; and

[0024] M is O or NRk6, where Rk6 is H or a C1-C12 aliphatic group.

[0025] In some embodiments, the amine head group is represented by the following:

[0026]

[0027] Wherein Ra, Ra', Ra” and Ra”' are each independently H, C1-20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, or R lipid; and Z is a C1-C20 divalent aliphatic group, a C1-C20 divalent heteroaliphatic group, a divalent aryl group or a divalent heteroaryl group.

[0028] In some embodiments, the ionizable lipid is represented by formula (II):

[0029]

[0030] Or its pharmaceutically acceptable salt, wherein:

[0031] Rb is a substituted or unsubstituted alkyl group;

[0032] n1 and n2 are each independently 1, 2, 3, 4, 5, or 6; and

[0033] Rb1, Rb2, Rb3, and Rb4 are each independently H or R lipids.

[0034] At least one of Rb1, Rb2, Rb3, and Rb4 is not H.

[0035] In some embodiments, the amine head group is selected from the group consisting of:

[0036]

[0037] In some embodiments, the at least one hydrophobic tail includes

[0038] In some embodiments, the ionizable lipid includes

[0039]

[0040] In some embodiments, the lipid composition further comprises a steroid. In some embodiments, the steroid comprises cholesterol or a cholesterol derivative. In some embodiments, the lipid composition further comprises an accessory lipid. In some embodiments, the accessory lipid comprises a phospholipid or a zwitterionic lipid comprising 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In some embodiments, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises a polyethylene glycol (PEG) conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(PEG)-2000 (DSPE-PEG2k)] or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). In some embodiments, the lipid composition further comprises a steroid, an auxiliary lipid, and a polymer-conjugated lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage of about 30% to about 90%. In some embodiments, the steroid is present in the lipid composition at a weight percentage of about 10% to about 40%. In some embodiments, the auxiliary lipid is present in the lipid composition at a weight percentage of about 1% to about 20%. In some embodiments, the polymer-conjugated lipid is present in the lipid composition at a weight percentage of about 1% to about 20%. In some embodiments, the weight ratio of the ionizable lipid / steroid / auxiliary lipid / polymer-conjugated lipid is about 16 / 4 / 1 / 1. In some embodiments, the weight ratio of the pharmaceutical formulation / lipid composition is about 1:200 to about 1:5. In some embodiments, the lipid composition further comprises a steroid and an auxiliary lipid. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage of about 30% to about 90%. In some embodiments, the auxiliary lipid is present in the lipid composition at a weight percentage of about 5% to about 40%. In some embodiments, the steroid is present in the lipid composition at a weight percentage of about 5% to about 40%. In some embodiments, the weight ratio of the ionizable lipid / steroid / auxiliary lipid is about 2 / 1 / 1. In some embodiments, the lipid composition further comprises a pharmaceutically acceptable carrier.

[0041] In some embodiments, the pharmaceutically acceptable carrier comprises a sugar, including mannitol, sucrose, maltose, or trehalose. In some embodiments, the carrier is present in the composition at a weight percentage of about 5% to about 60%. In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein not all hydrophobic tails are identical. In some embodiments, the ionizable lipid comprises at least two hydrophobic tails, wherein two or more hydrophobic tails are identical. In some embodiments, the pharmaceutical composition further comprises a polynucleotide, oligonucleotide, polypeptide, oligopeptide, small molecule compound, or any combination thereof. In some embodiments, the small molecule compound comprises a drug for chemotherapy.

[0042] In some embodiments, the physical properties of the lipid composition are more stable compared to other lipid compositions. In some embodiments, the physical properties of the lipid composition are stable for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months or longer when stored at 4°C, -20°C, or -80°C. In some embodiments, the physical properties of the lipid composition are stable for at least 5 months when stored at 4°C, -20°C, or -80°C. In some embodiments, the physical properties of the lipid composition include its size, polydispersity index (PDI), encapsulation efficiency (EE%), or pKa.

[0043] In some aspects, this disclosure provides a method for depleting B cells, comprising administering the pharmaceutical composition provided herein to a subject in need, wherein the subject in need suffers from a disease requiring B-cell depletion therapy (BCDT). In some embodiments, the disease requiring B-cell depletion therapy (BCDT) includes B-cell malignancies or autoimmune diseases. In some embodiments, the B-cell malignancies include B-cell lymphomas, wherein the B-cell lymphomas include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Walter's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma). In some embodiments, the B-cell lymphoma affects the spleen or lymph nodes. In some embodiments, the B-cell malignancy includes multiple myeloma. In some embodiments, the autoimmune disease includes allergic diseases, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), anti-myelin oligodendrocyte glycoprotein (anti-MOG) spectrum disorders, neuromyelitis optica spectrum disorders (NMOSD), anti-NMDAR encephalitis, or myasthenia gravis. In some embodiments, the disease requiring B-cell depletion therapy (BCDT) further includes pemphigus vulgaris or Sjögren's syndrome.

[0044] In some aspects, this disclosure provides a method for treating hematologic malignancies, comprising administering the pharmaceutical composition provided herein to a subject in need, wherein the subject in need suffers from a hematologic malignancy. In some embodiments, the hematologic malignancy includes B-cell lymphoma, wherein the B-cell lymphoma includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, or Burkitt lymphoma. In some embodiments, the administration includes local administration, oral administration, or administration by injection. In some embodiments, the administration includes administration by intravenous injection or by intramuscular injection. In some embodiments, this method results in the expression of the therapeutic peptide having a longer half-life compared to any other method comprising administering an equivalent dose of the therapeutic peptide.

[0045] In some embodiments, the method results in the expression of therapeutic peptides having the following half-lives: at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 12 hours, at least 16 hours, at least 18 hours, at least 24 hours, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 8.5 days, at least 9 days, at least 9.5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days or more.

[0046] In some embodiments, this method results in the expression of a therapeutic peptide with a larger area under the curve (AUC) compared to other methods. In some embodiments, this method results in the expression of a therapeutic peptide with an AUC of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more compared to any other method including the administration of an equivalent dose of the therapeutic peptide.

[0047] In some embodiments, the method results in prolonged B-cell depletion compared to any other method that includes administering an equivalent dose of the therapeutic peptide. In some embodiments, this prolonged B-cell depletion includes B-cell depletion for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 17.5 days, at least 20 days, at least 25 days, at least 27.5 days, at least 30 days, at least 35 days, at least 37.5 days, at least 40 days, at least 45 days, at least 47.5 days, at least 50 days, at least 55 days, at least 57.5 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, or at least 120 days or more. In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject. In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject. In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of naïve B cells in the subject. In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the mature B cells in the subject. In some embodiments, the mature B cells include memory B cells, plasma cells, or plasmablasts. In some embodiments, the method results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B-cell depletion compared to any other method that includes administering an equivalent dose of the therapeutic peptide.

[0048] In some embodiments, the method further includes administering a small molecule drug. In some embodiments, the small molecule drug includes agents used for chemotherapy. In some embodiments, the subject of need includes a mammal. In some embodiments, the mammal includes a human, a non-human primate, or a rodent. In some embodiments, the drug formulation is administered in one or more doses. In some embodiments, the drug formulation is administered at a dose not exceeding 3 mg / kg (mg of nucleic acid per kg of body weight). In some embodiments, the drug formulation is administered at a dose of about 0.007 mg / kg to about 0.2 mg / kg (mg of nucleic acid per kg of body weight).

[0049] In some embodiments, the pharmaceutical preparation is administered at a dose of about 0.014 mg / kg to about 0.1 mg / kg (mg of nucleic acid per kg of body weight).

[0050] By incorporating via reference

[0051] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually incorporated by reference. Attached Figure Description

[0052] The novel features of this disclosure are set forth in the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of this disclosure, in which:

[0053] Figure 1 An exemplary synthetic scheme for lipid tails is described.

[0054] Figures 2A-2C The characterization and physicochemical properties of the leading L88-mRNA were described compared to the reference ALC-0315-mRNA. hEPO mRNA was used as a surrogate cargo to be formulated with specified active lipids. Figure 2A The size and polydispersity index (PDI) were determined by dynamic light scattering (DLS). Figure 2B The percentage of encapsulation efficiency (EE%) determined by RiboGreen assay is shown. Figure 2C The surface pKa values ​​of the LNP, determined by TNS measurements, are shown. Data points are presented as mean ± SD.

[0055] Figures 3A-3CThe characterization and physicochemical properties of L88-003 were described in the long-term stability study. The size, PD, EE%, and mRNA integrity of L88-003 were detected at the fresh preparation time or after 5 months of storage at 4°C or -80°C.

[0056] Figures 4A-4B The tissue distribution of L88 in WT mice was described. Firefly luciferase mRNA was used as an alternative cargo to be formulated with L88 and intravenously injected into CD1 mice (n=2). Figure 4A Representative results of whole-body and organ luciferase imaging performed by IVIS 6 hours post-injection are shown. Figure 4B The luciferase protein in tissue lysates from designated organs was quantified by luciferase activity assay. Data are presented as mean ± SD.

[0057] Figure 5 Cell tropism analysis of L88 in the liver, spleen, and bone marrow, as determined by flow cytometry, was depicted in an Ai9 mouse model. Cre recombinase mRNA was delivered to Ai9 mice (n=2) via IV administration of an L88 formulation. The percentage of Tdtomato+ cells in the specified cell type was calculated by flow cytometry 7 days after treatment in each mouse. Data are presented as mean ± SD.

[0058] Figure 6 Protein expression levels of L88-hEPO mRNA were depicted after repeated administration. CD1 mice (n=5) were intravenously injected weekly with 0.5 mg / kg LNP containing human erythropoietin (hEPO) mRNA for 5 weeks. Plasma hEPO levels in each mouse were quantified by ELISA 6 h after each administration, and each data point is presented as mean ± SD.

[0059] Figures 7A-7B The liver clearance rate of L88 after a single IV dose is shown in CD1 mice. Figure 7A The study described the lipid levels of L88 in mouse livers (n=2 per group) determined by LC-MS 6 h after treatment with a specified dose of L88-luciferase mRNA via IV injection. Figure 7B In this study, the amounts of L88 and ALC-0315 lipids in the liver were analyzed by LC-MS at specified time points following intravenous injection of 0.5 mg / kg LNP-Luc mRNA. Relative amounts were normalized to individual tissue weights and expressed as mean ± SD.

[0060] Figure 8A This is a schematic diagram of the mRNA molecule encoding CD19 / CD3 BiTE. Figures 8B-8CThis study demonstrates the delivery of mRNA encoding CD19 / CD3 BiTE, encapsulated by multiple LNPs, into cell cultures. Soluble BiTE protein was expressed in all tested groups, and BiTE protein was also expressed in the cell supernatant. Figure 8B ) and purified BiTE protein ( Figure 8C The mRNA was bound to CD19 on an ELISA plate. At least three mRNA designs were tested. HTX-01-001 mRNA contained SEQ ID NO:1-4. HTX-01-002 mRNA contained SEQ ID NO:5-8. HTX-01-003 mRNA contained SEQ ID NO:7-10.

[0061] Figures 9A-9B This demonstrates that CD19 / CD3 BiTE mediates T cell killing of cancer cells. (See FACs analysis). Figure 9A ) and the calculated results after normalization for the untreated control ( Figure 9B As shown in the figure, CD19-CD3 BiTE generated by transfecting mRNA encoding BiTE in vitro can induce strong T cell killing activity against CD19-positive Raji cells.

[0062] Figure 10A The size distribution of various LNPs containing mRNAs encoding CD19 / CD3 BiTE was depicted. Figure 10B The encapsulation efficiency of LNPs containing mRNA encoding CD19 / CD3 BiTE was depicted.

[0063] Figures 11A-11B The in vitro efficacy of BiTE against CD19-expressing cancer cells was demonstrated. LNP containing mRNA encoding CD19 / CD3 BiTE was intravenously injected into BALB / c mice. Plasma from the treated mice was used for tumor killing assays. Figure 11A The in vitro tumor-killing activity of BiTE was demonstrated. Figure 11B The plasma concentration of BiTE was quantified by ELISA. Both L88 and L93 lipid nanoparticles were used.

[0064] Figures 12A-12B The pharmacokinetic profile of single-dose LNP-CD19 / CD3 BiTE mRNA in Balb / c mice is shown. Figure 12A This is a schematic diagram of the experimental design for a single-dose pharmacokinetic (PK) study. For example... Figure 12BThe concentration of CD19-CD3 BiTE protein in the plasma of Balb / c mice after intravenous administration of LNP-mRNA is depicted. Mice were treated with a total of 5 μg of BiTE mRNA. Blood was drawn from 5 mice at each time point for quantification by CD3 ELISA assay. Concentrations from duplicate replicates of the technical ELISA are shown. Data are presented as mean ± SD.

[0065] Figures 13A-13H The results of a efficacy study of L88-003 in a Raji-luciferase xenograft mouse model derived from hPBMC reconstruction are presented. Figure 13A This is a schematic diagram of the research design, in which tumor cells are seeded and regimented into 3 treatment groups (n=5 per group), including LNP-mRNA (L88-003, 1.6 μg per mouse), recombinant BiTE protein (rPR003, 10 μg per mouse) and vector. Figure 13B Images depicting the total photon flux of a single mouse in each group captured by IVIS at specified time points are shown. Figures 13C-13G As depicted, for each treatment group, the bioluminescence signal was quantified to photons per second using Living Image 4.7 software. Individual total flux (TF) at the end of the study was analyzed. Each point represents the bioluminescence signal of a single mouse, and statistical analysis was compared with the G1_vector control group using the Kruskal-Wallis test and Dunn's multiple comparison test. ***p<0.01. E, F, G) analyzed the changes in TF for each mouse in each group during the treatment process. Figure 13H The relative weight changes of each mouse during the treatment were normalized to the baseline time point (t=0), and each point is represented as mean ± SD.

[0066] Figures 14A-14B The p-p ... Figure 14A This is a schematic diagram of a three-stage dose progression and blood collection at specified time points for PK spectrum analysis. Figure 14B The concentrations of CD19-CD3 BiTE protein in cynomolgus monkey plasma were quantified by CD3 ELISA at specified time points following intravenous infusion of L88-003. Data are presented as mean ± SD of two replicates of the technical ELISA, and the dashed line represents the LLOQ at 2 ng / ml.

[0067] Figures 15A-15HThe study showed circulating B cell exhaustion and T cell dynamics in cynomolgus monkeys after three weekly doses of L88-003 (e.g., LNP-CD19 BiTE mRNA). Figure 15A This is a schematic diagram of a three-stage dose progression and blood collection at specified times for phenotypic analysis of peripheral B cells and T cells using flow cytometry. Figure 5 C shows that treatment with LNP-CD19 BiTE mRNA depletes circulating plasmablasts and plasma cells. Figure 15B and Figure 15D The absolute cell counts of circulating CD20+ B cells and circulating CD3+ T cells at specified time points are displayed respectively. Figure 15E and Figure 15F The absolute cell counts of circulating CD3+CD4+ T helper cells and CD3+CD8+ CTLs are displayed at specified time points. Figure 15G and Figure 15H The percentage of CD69+ activated T cells in the CD4 and CD8 T cell subgroups are shown separately.

[0068] Figure 16 The relative weight changes were depicted by monitoring twice a week and normalized to baseline (t=0).

[0069] Figures 17A-17I The results of a non-clinical tolerability study in cynomolgus monkeys were described after three weekly doses of L88-003 with progressively increasing dosage. Figure 17A It is a schematic diagram of a three-stage dose progression and blood collection at specified time points for clinical hematology, clinical chemistry, and cytokine analysis. Figures 17B-17D This displays the hematological analysis of whole blood CBCs evaluated at specified time points. The absolute counts of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) are shown at [time points missing]. Figure 17B , Figure 17C and Figure 17D The dashed lines depict reference ranges and / or pre-drug readings. Figures 17E-17G The clinical chemistry analyses measured at specified time points are shown. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were at [missing data]. Figure 17E , Figure 17F and Figure 17G The dashed lines depict reference ranges and / or pre-drug readings. Figure 17H and Figure 17I Plasma cytokine levels, including IL6, IL2, IL4, IFNγ, and TNFα, were quantified using multiplex luminex assays. Detailed Implementation

[0070] Messenger RNA (mRNA) offers numerous advantages over other therapies as a platform for efficient protein expression in vivo. The large size and negative charge of mRNA are obstacles to its efficient delivery to the cytosol. Naked mRNA can be spontaneously taken up by various cell types, but this typically leads to degradation in acidic lysosome compartments. Lipid nanoparticles (LNPs) have been identified as an effective way to protect mRNA from the effects of ubiquitous ribonucleases, protect it from immune cells, deliver it to cells, and allow it to escape from endosomes. LNPs generally consist of four main components: an ionizable compound, a sterol, a phospholipid, and a lipid-anchored polyethylene glycol (PEG). The phospholipid and sterol work together to stabilize the LNP, the lipid-anchored PEG provides vial and storage stability, and the ionizable lipid is crucial for cellular uptake and endosome escape, allowing the mRNA to be released into the cytosol. By altering the ratio and properties of these lipid components, the potency and tolerability of the formulation can be modified. Importantly, careful process control is required for mRNA / LNP formulations to ensure reproducibility and stability in production.

[0071] While full-length antibodies have achieved significant success in targeting cancer, immune diseases, and infectious diseases, they are relatively large and exhibit lower tissue penetration than small molecules. Conversely, therapeutic peptides derived from antibody fragments, such as bispecific T-cell connectives (e.g., BiTE), can be very small, allowing for better tissue penetration. However, the use of these molecules is hampered by their short half-life of several hours and their tendency to aggregate. Both of these properties can be improved or avoided through mRNA-mediated expression, as the antibody will be continuously expressed from the mRNA until it is degraded. This approach also prevents aggregation, often observed during protein purification. LNP formulations encapsulating mRNA for delivery offer advantages over recombinant proteins because LNPs can provide greater biodistribution and tissue penetration than recombinant antibody proteins. LNPs optimized for mRNA delivery can lead to higher expression of the therapeutic peptide through more convenient administration routes, such as subcutaneous or intramural administration.

[0072] This disclosure provides pharmaceutical compositions and methods relating to lipid nanoparticles (LNPs) comprising ionizable lipids and nucleic acid sequences encoding therapeutic peptides. In some embodiments, the pharmaceutical composition comprises: a) a synthetic nucleic acid sequence encoding a therapeutic peptide configured to bind a B-cell antigen; and b) a plurality of lipid nanoparticles, wherein the synthetic nucleic acid molecule is encapsulated within at least one of the plurality of lipid nanoparticles. The pharmaceutical compositions and methods provided herein can result in high levels of therapeutic peptide expression in subjects. In some embodiments, the pharmaceutical compositions and methods result in severe and prolonged B-cell exhaustion due to in vivo expression of these therapeutic peptides. In some embodiments, the pharmaceutical compositions and methods result in zero to minimal toxicity in subjects.

[0073] Therapeutic peptides

[0074] Some aspects of this disclosure relate to a pharmaceutical composition comprising a synthetic nucleic acid molecule encoding a therapeutic peptide. The nucleic acid molecule may comprise a nucleic acid sequence. The synthetic nucleic acid molecule or the nucleic acid sequence may be DNA. The synthetic nucleic acid molecule or the nucleic acid sequence may be RNA. In some embodiments, the synthetic nucleic acid molecule or the nucleic acid sequence comprises messenger RNA (mRNA) encoding the therapeutic peptide. In some embodiments, the therapeutic peptide is configured to bind or be able to bind antigens on B lymphocytes (e.g., B cells or B-cells). In some embodiments, the therapeutic peptide preferably binds B cell antigens. In some embodiments, the therapeutic peptide is configured to bind B cell antigens with a higher affinity than other antigens. The ability of the therapeutic peptide to bind any antigen can be measured by a dissociation constant (Kd). In some embodiments, the Kd of the therapeutic peptide for binding B cell antigens is smaller than for any other antigen. In some embodiments, the Kd of the therapeutic peptide for B cell antigens is in the nanomolar or micromolar range.

[0075] The therapeutic peptides provided herein can be configured to bind any B-cell antigen, or can be capable of binding any B-cell antigen. A B-cell antigen can refer to any macromolecule expressed on the surface of a B cell. In some embodiments, the B-cell antigen includes surface proteins of the B cell. In some embodiments, the B-cell antigen includes membrane-bound proteins of the B cell. In some embodiments, the B-cell antigen includes membrane-associated proteins of the B cell. In some embodiments, the B-cell antigen includes glycosylated proteins of the B cell. In some embodiments, the B-cell antigen includes peptides, glycans, or combinations thereof.

[0076] The therapeutic peptide can be configured to bind to or be able to bind to B cell antigens expressed on B cells at any stage of cell development. In some embodiments, the B cell antigen can be expressed by any subtype of B cell. In some embodiments, the B cell antigen includes memory B cell antigen, naive B cell antigen, plasmablast antigen, or plasma cell antigen. In some embodiments, the B cell antigen is selected from the group consisting of: CD10, CD19, CD20, CD22, CD27, CD32b, CD38, CD40, B cell maturation antigen (BCMA), B cell activating factor receptor (BAFFR), CD138, and CD5. In some embodiments, the B cell antigen includes CD10, CD19, CD20, CD22, CD27, CD32b, CD38, CD40, B cell maturation antigen (BCMA), B cell activating factor receptor (BAFFR), CD138, or CD5, or any combination thereof. In some embodiments, the B cell antigen includes CD19.

[0077] The therapeutic peptides provided herein can be configured to bind one or more antigens. In some embodiments, the therapeutic peptides provided herein are configured to bind two or more antigens. In some embodiments, the therapeutic peptide is configured to bind two or more B-cell antigens. In some embodiments, the therapeutic peptide is configured to bind a first antigen (e.g., a B-cell antigen) and a second antigen. In some embodiments, the second antigen is different from the first antigen (e.g., a B-cell antigen). In some embodiments, the second antigen is also a B-cell antigen. In some embodiments, the second antigen is not a B-cell antigen.

[0078] In some embodiments, the therapeutic peptide is configured to bind to a second antigen, wherein the second antigen includes an immune cell antigen or a tumor antigen. An immune cell antigen can refer to any macromolecule expressed on the surface of an immune cell. In some embodiments, the immune cell antigen includes a surface protein of the immune cell. In some embodiments, the immune cell antigen includes a membrane-bound protein of the immune cell. In some embodiments, the immune cell antigen includes a membrane-associated protein of the immune cell. In some embodiments, the immune cell antigen includes a glycosylated protein of the immune cell. In some embodiments, the immune cell antigen includes a peptide, a glycan, or a combination thereof. A tumor cell antigen can refer to any macromolecule expressed on the surface of a tumor cell. In some embodiments, the tumor cell antigen includes a surface protein of the tumor cell. In some embodiments, the tumor cell antigen includes a membrane-bound protein of the tumor cell. In some embodiments, the tumor cell antigen includes a membrane-associated protein of the tumor cell. In some embodiments, the tumor cell antigen includes a glycosylated protein of the tumor cell. In some embodiments, the tumor cell antigen includes a peptide, a glycan, or a combination thereof. In some embodiments, the therapeutic peptide is capable of binding to immune cell antigens, including T cell antigens, Treg cell antigens, or natural killer cell (NK cell) antigens. In some embodiments, the therapeutic peptide is capable of binding to CD3.

[0079] In some embodiments, the therapeutic peptide comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment comprises a bispecific antibody, a multispecific antibody, a chimeric antigen receptor (CAR), or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment may comprise a light chain or a heavy chain. In some embodiments, the antigen-binding fragment may comprise a fragment antigen-binding (Fab or F(ab)) domain or a variant thereof, such as F(ab') or F(ab')2. In some embodiments, the antigen-binding fragment may comprise Fab, a fragment variable region (Fv), recombinant immunoglobulin (rIgG), a single-chain variable fragment (scFv), a heavy chain antibody (hcAb), a single-domain antibody, a heavy chain variable heavy domain (VHH), a neoantigen receptor variable domain (VNAR), a single-domain antibody (sdAb), or a nanobody. In some embodiments, its antigen-binding fragment comprises scFv. In some embodiments, the antibody or its antigen-binding fragment comprises a bispecific antibody or a multispecific antibody.

[0080] In some embodiments, the therapeutic peptide comprises a bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the therapeutic peptide comprises an scFv bispecific antibody or an antigen-binding fragment. In some embodiments, the therapeutic peptide comprises a bispecific T-cell connective (BiTE). In some embodiments, the therapeutic peptide comprises a BiTE that binds to both CD19 and CD3.

[0081] In some embodiments, the therapeutic peptide includes an FDA-approved drug configured to bind to a B-cell antigen. In some embodiments, the FDA-approved drug includes belintolimab, inelolizumab, rontoxicumab, or tancituzumab.

[0082] In some embodiments, the therapeutic peptide can be operatively linked to another peptide. In some embodiments, the therapeutic peptide can be produced by a cell from another peptide. In some embodiments, the therapeutic peptide can be produced by a cell from a peptide containing a signal peptide. The signal peptide can be cleaved in the cell to produce the therapeutic peptide. In some embodiments, the therapeutic peptide can be operatively linked to a protein tag. In some embodiments, the protein tag includes a histidine tag, a flag tag, or a hemagglutinin tag. In some embodiments, the protein tag is selected from the group consisting of: CBP tag, flag tag, GST tag, HA tag, HBH tag, MBP tag, myc tag, his tag, S tag, SUMO tag, TAP tag, TRX tag, E tag, E2 tag, KT3, T7, VSVG, OLLAS, protein C, NE tag, Xpress tag, Avi and V5 tags, and any combination thereof. In some embodiments, the protein tag includes a CBP tag, a flag tag, a GST tag, a HA tag, an HBH tag, an MBP tag, a myc tag, a his tag, an S tag, a SUMO tag, a TAP tag, a TRX tag, an E tag, an E2 tag, a KT3 tag, a T7 tag, a VSVG tag, an OLLAS tag, a protein C tag, an NE tag, an Xpress tag, an Avi tag, or a V5 tag, or a portion thereof, or any combination thereof.

[0083] In some embodiments, the therapeutic peptide comprises a heavy chain and a light chain. In some embodiments, the therapeutic peptide comprises an anti-CD19 heavy chain and an anti-CD19 light chain. In some embodiments, the therapeutic peptide further comprises an anti-CD3 heavy chain and an anti-CD3 light chain. In some embodiments, the anti-CD19 heavy chain has the amino acid sequence of SEQ ID NO: 30, 34, or 38. In some embodiments, the anti-CD19 light chain has the amino acid sequence of SEQ ID NO: 29, 33, or 37. In some embodiments, the anti-CD3 heavy chain has the amino acid sequence of SEQ ID NO: 32 or 36. In some embodiments, the anti-CD3 light chain has the amino acid sequence of SEQ ID NO: 31 or 35.

[0084] In some embodiments, the anti-CD19 light chain is encoded by a nucleic acid sequence of SEQ ID NO: 1, 3, 5, or 9. In some embodiments, the anti-CD19 heavy chain is encoded by a nucleic acid sequence of SEQ ID NO: 2, 4, 6, or 10. In some embodiments, the anti-CD19 light chain is encoded by a nucleic acid sequence having 90% sequence identity with SEQ ID NO: 1, 3, 5, or 9. In some embodiments, the anti-CD19 heavy chain is encoded by a nucleic acid sequence having 90% sequence identity with SEQ ID NO: 2, 4, 6, or 10.

[0085] In some embodiments, the anti-CD3 light chain is encoded by the nucleic acid sequence of SEQ ID NO:7. In some embodiments, the anti-CD3 light chain is encoded by a nucleic acid sequence having 90% sequence identity with SEQ ID NO:7. In some embodiments, the anti-CD3 heavy chain is encoded by the nucleic acid sequence of SEQ ID NO:8. In some embodiments, the coding region of the anti-CD3 heavy chain contains a nucleic acid sequence having 90% sequence identity with SEQ ID NO:8.

[0086] In some embodiments, the anti-CD19 light chain comprises SEQ ID NO:29 and the anti-CD19 heavy chain comprises SEQ ID NO:30; wherein the anti-CD3 light chain comprises SEQ ID NO:31 and the anti-CD3 heavy chain comprises SEQ ID NO:32.

[0087] In some embodiments, the anti-CD19 light chain comprises SEQ ID NO:33 and the anti-CD19 heavy chain comprises SEQ ID NO:34; wherein the anti-CD3 light chain comprises SEQ ID NO:35 and the anti-CD3 heavy chain comprises SEQ ID NO:36.

[0088] In some embodiments, the anti-CD19 light chain comprises SEQ ID NO:37 and the anti-CD19 heavy chain comprises SEQ ID NO:38; wherein the anti-CD3 light chain comprises SEQ ID NO:35 and the anti-CD3 heavy chain comprises SEQ ID NO:36.

[0089] In some embodiments, the anti-CD19 heavy chain comprises, or contains, the light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.

[0090] In some embodiments, after the pharmaceutical composition is administered to the subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition leads to the activation of CD69+ T cells.

[0091] The therapeutic peptide encoded by the nucleic acid sequence (e.g., mRNA) of the composition provided herein may have a better pharmacokinetic profile than a corresponding recombinant therapeutic peptide bound to the same antigen. After administration of the pharmaceutical composition to the subject, the therapeutic peptide encoded by the nucleic acid sequence may be expressed in vivo by the subject. The resulting therapeutic peptide may have better potency and / or efficacy than a recombinant therapeutic peptide bound to the same antigen. In some embodiments, after administration of the pharmaceutical composition to the subject, the resulting therapeutic peptide has a longer half-life compared to a corresponding therapeutic peptide of another pharmaceutical composition. In some embodiments, after administration of the pharmaceutical composition to the subject, the therapeutic peptide expressed by the subject has a half-life of at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 12 hours, or longer. Half-life of at least 16 hours, at least 18 hours, at least 24 hours, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 8.5 days, at least 9 days, at least 9.5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days or more.

[0092] In some embodiments, after the pharmaceutical composition is administered to a subject, the therapeutic peptide expressed by the subject has a larger area under the curve (AUC) compared to the corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

[0093] In some embodiments, after the pharmaceutical composition is administered to a subject, the therapeutic peptide expressed by the subject has an AUC that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more greater than that of a corresponding therapeutic peptide in an equivalent dose of another pharmaceutical composition.

[0094] In some embodiments, after the pharmaceutical composition is administered to a subject, the therapeutic peptide expressed by the subject results in prolonged B-cell depletion compared to the corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

[0095] The therapeutic peptide encoded by the nucleic acid sequence (e.g., mRNA) of the compositions provided herein may have a better pharmacodynamic profile than the corresponding recombinant therapeutic peptide that binds to the same antigen. Following administration of the pharmaceutical composition to a subject, the therapeutic peptide encoded by the nucleic acid sequence of these compositions may result in prolonged and / or more severe B-cell depletion in vivo. In some implementations, the prolonged B-cell depletion includes B-cell depletion for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 17.5 days, at least 20 days, at least 25 days, at least 27.5 days, at least 30 days, at least 35 days, at least 37.5 days, at least 40 days, at least 45 days, at least 47.5 days, at least 50 days, at least 55 days, at least 57.5 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, or at least 120 days or more.

[0096] In some embodiments, when administered to the subject, the therapeutic peptide expressed by the subject results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject. In some embodiments, after administration of the pharmaceutical composition to the subject, the therapeutic peptide expressed by the subject results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject.

[0097] In some embodiments, upon administration to the subject, the therapeutic peptide expressed by the subject results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the subject's initial B cells. In some embodiments, after administration of the pharmaceutical composition to the subject, the therapeutic peptide expressed by the subject results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the subject's mature B cells.

[0098] In some embodiments, after administration to the subject, the therapeutic peptide expressed by the subject results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B-cell depletion compared to a corresponding therapeutic peptide in an equivalent dose of another pharmaceutical composition.

[0099] In some embodiments, the therapeutic peptide expressed by a subject has zero to minimal toxicity after administration of the pharmaceutical composition to the subject. In some embodiments, the toxicity includes a transient increase in cytokines or liver enzymes. In some embodiments, the toxicity includes mild inflammation or mild hepatotoxicity.

[0100] Nucleic acid

[0101] The nucleic acid molecules and sequences provided herein may include ribonucleic acid (RNA) molecules and RNA sequences. The nucleic acid molecules and sequences provided herein may include deoxyribonucleic acid (DNA) molecules and DNA sequences. In some embodiments, the nucleic acids provided herein include RNA, DNA, DNA / RNA hybrids, nucleic acid analogs, chemically modified nucleic acids, chimeras composed of two or more nucleic acids or nucleic acid analogs, or any combination thereof. In some embodiments, the nucleic acid molecule and / or nucleic acid sequence is selected from the group consisting of: RNA, DNA, DNA / RNA hybrids, nucleic acid analogs, chemically modified nucleic acids, chimeras composed of two or more nucleic acids or nucleic acid analogs, and any combination thereof.

[0102] The pharmaceutical compositions provided herein comprise a synthetic nucleic acid molecule (e.g., mRNA) encoding a therapeutic peptide. In some cases, the mRNA encapsulated by the lipid compositions provided herein further comprises regulatory sequences that may contribute to and / or promote the expression of the therapeutic peptide. These regulatory sequences may comprise a 5' cap, a 5' untranslated region, a promoter, a signal peptide sequence, a 3' untranslated region, and a poly-A tail. In some cases, these regulatory sequences comprise enhancers (e.g., CMV enhancers) to further enhance the expression of the therapeutic peptide.

[0103] In some embodiments, the pharmaceutical composition comprises a synthetic nucleic acid sequence further containing a regulatory nucleic acid sequence. In some embodiments, the regulatory nucleic acid sequence comprises a promoter or signal peptide. In some embodiments, the promoter comprises a tissue-specific promoter. In some embodiments, the synthetic nucleic acid sequence comprises a synthetic ribonucleic acid sequence (RNA). In some embodiments, the synthetic RNA comprises chemically modified nucleic acids, wherein these chemical modifications improve the stability of the synthetic RNA. In some embodiments, the synthetic RNA comprises N6-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), or N4-acetylcytidine (ac4C), or any combination thereof. In some embodiments, the synthetic RNA comprises a 5' cap.

[0104] In some embodiments, the synthetic RNA comprises, in the 5' to 3' orientation: a) a 5' untranslated region (5'UTR) coding region; b) a signal peptide coding region; c) a therapeutic peptide coding region; d) a 3' untranslated region (3'UTR) coding region; and e) a poly-A tail coding region. Table 1 lists the nucleic acid sequence (RNA or DNA sequence) encoding CD19-CD3 BiTE and the amino acid sequence of CD19-CD3 BiTE.

[0105] Table 1. Nucleic acid sequence and amino acid sequence.

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] In some embodiments, the signal peptide coding region comprises the sequence of SEQ ID NO:11. In some embodiments, the synthetic RNA further comprises an adapter coding region. In some embodiments, the adapter coding region comprises the sequence of SEQ ID NO:12 or SEQ ID NO:13. In some embodiments, the therapeutic peptide coding region comprises a nucleic acid sequence selected from the group consisting of: 1) SEQ ID NO:1 and 2; 2) SEQ ID NO:5 and 6; and 3) SEQ ID NO:9 and 10, wherein these nucleic acid sequences encode an anti-CD19 antibody or an anti-CD19 binding fragment thereof.

[0115] In some embodiments, the therapeutic peptide coding region comprises a nucleic acid sequence having 90% sequence identity with a sequence selected from the group consisting of: 1) SEQ ID NO: 1 and 2; 2) SEQ ID NO: 5 and 6; or 3) SEQ ID NO: 9 and 10, wherein these nucleic acid sequences encode an anti-CD19 antibody or an anti-CD19 binding fragment thereof.

[0116] In some embodiments, the therapeutic peptide coding region comprises nucleic acid sequences consisting of: 1) SEQ ID NO:3 and 4; or 2) SEQ ID NO:7 and 8, wherein these nucleic acid sequences encode an anti-CD3 antibody or an anti-CD3 binding fragment thereof.

[0117] In some embodiments, the therapeutic peptide coding region comprises nucleic acid sequences having 90% sequence identity with the following: 1) SEQ ID NO:3 and 4; or 2) SEQ ID NO:7 and 8, wherein these nucleic acid sequences encode an anti-CD3 antibody or an anti-CD3 binding fragment thereof.

[0118] In some embodiments, the synthetic nucleic acid sequence includes a therapeutic peptide coding region. In some embodiments, the therapeutic peptide coding region includes, in a 5' to 3' orientation: a) an anti-CD19 light chain coding region; b) an anti-CD19 heavy chain coding region; c) an anti-CD3 heavy chain coding region; and d) an anti-CD3 light chain coding region.

[0119] In some embodiments, the anti-CD19 light chain coding region comprises the sequence of SEQ ID NO:1, 3, 5, or 9. In some embodiments, the anti-CD19 light chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:1, 3, 5, or 9. In some embodiments, the anti-CD19 light chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, 3, 5, or 9.

[0120] In some embodiments, the anti-CD19 heavy chain coding region comprises the sequence of SEQ ID NO:2, 4, 6, or 10. In some embodiments, the anti-CD19 heavy chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:2, 4, 6, or 10. In some embodiments, the anti-CD19 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2, 4, 6, or 10.

[0121] In some embodiments, the anti-CD3 light chain coding region comprises the sequence of SEQ ID NO:7. In some embodiments, the anti-CD3 light chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:7. In some embodiments, the anti-CD3 light chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7.

[0122] In some embodiments, the anti-CD3 heavy chain coding region comprises the sequence of SEQ ID NO:8. In some embodiments, the anti-CD3 heavy chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:8. In some embodiments, the anti-CD3 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8.

[0123] In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:1 and the anti-CD19 heavy chain coding region includes SEQ ID NO:2; wherein the anti-CD3 light chain coding region includes SEQ ID NO:3 and the anti-CD3 heavy chain coding region includes SEQ ID NO:4.

[0124] In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:5 and the anti-CD19 heavy chain coding region includes SEQ ID NO:6; wherein the anti-CD3 light chain coding region includes SEQ ID NO:7 and the anti-CD3 heavy chain coding region includes SEQ ID NO:8.

[0125] In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:9 and the anti-CD19 heavy chain coding region includes SEQ ID NO:10; wherein the anti-CD3 light chain coding region includes SEQ ID NO:7 and the anti-CD3 heavy chain coding region includes SEQ ID NO:8.

[0126] In some embodiments, the anti-CD19 heavy chain coding region comprises, or contains, a nucleotide sequence encoding the light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab. In some embodiments, the anti-CD19 heavy chain coding region comprises, or contains, a nucleotide sequence encoding the heavy chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab. In some embodiments, the anti-CD3 light chain coding region comprises, or contains, a nucleotide sequence encoding the light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab. In some embodiments, the anti-CD3 heavy chain coding region comprises, or contains, a nucleotide sequence encoding the heavy chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.

[0127] In some implementations, the synthetic RNA comprises single-stranded synthetic RNA.

[0128] In some embodiments, the synthesized RNA further encodes a protein tag. In some embodiments, the protein tag includes a histidine tag, a flag tag, or a hemagglutinin tag.

[0129] In some cases, the mRNA encoding the therapeutic peptide may contain a signal peptide, which may be cleaved during post-translational processing.

[0130] In some embodiments, the synthetic nucleic acid sequence includes a therapeutic peptide coding region. In some embodiments, the therapeutic peptide coding region includes, in a 5' to 3' orientation: a) an anti-CD19 light chain coding region; b) an anti-CD19 heavy chain coding region; c) an anti-CD3 heavy chain coding region; and d) an anti-CD3 light chain coding region.

[0131] In some embodiments, the anti-CD19 light chain coding region comprises the sequence of SEQ ID NO:1, 3, 5, or 9. In some embodiments, the anti-CD19 light chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:1, 3, 5, or 9. In some embodiments, the anti-CD19 light chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:1, 3, 5, or 9.

[0132] In some embodiments, the anti-CD19 heavy chain coding region comprises the sequence of SEQ ID NO:2, 4, 6, or 10. In some embodiments, the anti-CD19 heavy chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:2, 4, 6, or 10. In some embodiments, the anti-CD19 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2, 4, 6, or 10.

[0133] In some embodiments, the anti-CD3 light chain coding region comprises the sequence of SEQ ID NO:7. In some embodiments, the anti-CD3 light chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:7. In some embodiments, the anti-CD3 light chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:7.

[0134] In some embodiments, the anti-CD3 heavy chain coding region comprises the sequence of SEQ ID NO:8. In some embodiments, the anti-CD3 heavy chain coding region comprises a nucleic acid sequence having 90% sequence identity with SEQ ID NO:8. In some embodiments, the anti-CD3 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:8.

[0135] In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:1 and the anti-CD19 heavy chain coding region includes SEQ ID NO:2; wherein the anti-CD3 light chain coding region includes SEQ ID NO:3 and the anti-CD3 heavy chain coding region includes SEQ ID NO:4.

[0136] In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:5 and the anti-CD19 heavy chain coding region includes SEQ ID NO:6; wherein the anti-CD3 light chain coding region includes SEQ ID NO:7 and the anti-CD3 heavy chain coding region includes SEQ ID NO:8.

[0137] In some embodiments, the anti-CD19 light chain coding region includes SEQ ID NO:9 and the anti-CD19 heavy chain coding region includes SEQ ID NO:10; wherein the anti-CD3 light chain coding region includes SEQ ID NO:7 and the anti-CD3 heavy chain coding region includes SEQ ID NO:8.

[0138] In some embodiments, the anti-CD19 heavy chain coding region comprises, or contains, a nucleotide sequence encoding the light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab. In some embodiments, the anti-CD19 heavy chain coding region comprises, or contains, a nucleotide sequence encoding the heavy chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab. In some embodiments, the anti-CD3 light chain coding region comprises, or contains, a nucleotide sequence encoding the light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab. In some embodiments, the anti-CD3 heavy chain coding region comprises, or contains, a nucleotide sequence encoding the heavy chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.

[0139] In some cases, the mRNA contains a hepatocyte-specific promoter, and these LNPs contain mRNA encoding a soluble therapeutic peptide (e.g., BiTE). In other cases, the mRNA contains a strong promoter that enhances the expression of the therapeutic peptide for immunotherapy. This strong promoter may include a human cytomegalovirus (hCMV), chicken β-actin / CMV enhancer (CAG), elongation factor 1α (EF1α), or glycerol phosphokinase (PGK) promoter.

[0140] In some cases, the mRNA encapsulated by the LNP of this disclosure contains a naturally occurring or artificial promoter. In some cases, the mRNA contains a promoter specific for expression in immune cells compared to non-immune cells. In some cases, the mRNA contains a T cell-specific promoter, and these LNPs containing the mRNA encoding a therapeutic peptide can be used for CAR-T therapy. In some cases, the T cell-specific promoter contains a promoter that drives the endogenous expression of T cell-specific proteins, including CD3 (e.g., CD3γ, CD3δ, CD3ε, CD3ζ), CD4, CD8, CD28, TCRB, or TRAC. In some cases, the promoter can lead to stronger expression of the therapeutic peptide in immune cells, including lymphocytes, T cells, CD4+ T cells, CD8+ T cells, etc. T cells, αβT cells, γδT cells, regulatory T cells (Tregs), cytotoxic T lymphocytes, Th1 cells, Th2 cells, Th17 cells, Th9 cells, naive T cells, memory T cells, effector T cells, effector-memory T cells (TEM), central memory T cells (TCMs), resident memory T cells (TRMs), follicular helper T cells (TFHs), natural killer T cells (NKTs), tumor-infiltrating lymphocytes (TILs), natural killer cells (NKs), innate lymphocytes (ILCs), ILC1 cells, ILC 2 cells, ILC3 cells, lymphotissue-induced (LTi) cells, B cells, B1 cells, B1a cells, B1b cells, B2 cells, plasma cells, B regulatory cells, memory B cells, marginal zone B cells, follicular B cells, germinal center B cells, antigen-presenting cells (APCs), monocytes, macrophages, M1 macrophages, M2 macrophages, tissue-associated macrophages, dendritic cells, plasmacytoid dendritic cells, neutrophils, mast cells, basophils, eosinophils, common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof.

[0141] In some cases, the mRNA encoding the therapeutic peptide disclosed herein contains natural, synthetic, and / or artificial nucleotide analogs or bases. In some cases, the synthetic or artificial nucleotide analog or base contains modifications at one or more of the following locations: a deoxyribose moiety, a ribose moiety, a phosphate moiety, a nucleoside moiety, or a combination thereof.

[0142] In some cases, the nucleotide analog or artificial nucleotide base includes a nucleic acid with a modified 2' hydroxyl group at the ribose moiety. In some cases, this modification includes H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Illustrative alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. In some cases, the alkyl moiety further comprises a modification. In some cases, this modification includes an azo group, ketone group, aldehyde group, carboxyl group, nitro group, nitroso group, nitrile group, heterocyclic group (e.g., imidazolyl, hydrazyl, or hydroxyamino), isocyanate or cyanate group, or sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some cases, the alkyl moiety further comprises a heterosubstituted group. In some cases, the carbon atom of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some cases, the heterocyclic substitution includes, but is not limited to, morpholino, imidazolyl, and pyrrolidinyl.

[0143] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2'-O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.

[0144] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, which includes an extended amino group at the propyl linker that binds the amino group to the 2' oxygen. In some cases, this modification neutralizes the total negative charge derived from phosphate in the oligonucleotide molecule by introducing a positive charge from the amino group of each sugar, thereby improving cellular uptake properties due to its zwitterionic nature.

[0145] In some cases, the modification at the 2' hydroxyl group is a locked ribonucleotide modification or a bridged ribonucleotide modification (e.g., locked nucleic acid or LNA), in which the oxygen molecule bound at the 2' carbon is linked to the 4' carbon via a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene linked bicyclic ribonucleotide monomer.

[0146] In some cases, additional modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy-2'-fluorine, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA).

[0147] In some cases, nucleotide analogs contain modified bases, such as N1-methylpseuuridine, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides modified at the 5 position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenine, 2-methyladenine, 3-methylcytidine, 6-methyluridine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine. Glycosides, 5-methylaminoethyluridine, 5-methyloxyuridine, denitronucleotides (such as 7-deazo-adenosine, 6-azauridine, 6-azacytidine, or 6-azathymidine), 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, braided glycosides, archapurinol, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups (such as aminophenol or 2,4,6-trimethoxybenzene), as G-clamp nucleotides (G-clamp Nucleotides are nucleotides modified with cytosine, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those with modifications to the sugar moiety, and nucleotides having a sugar or analogue that is not a ribosyl group. For example, in some cases, these sugar moieties are or are based on mannose, arabinose, pyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbon rings. The term "nucleotide" also includes universal bases. For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0148] In some cases, one or more modifications optionally occur at the internucleotide bond. In some cases, the modified internucleotide bond may include, but is not limited to, thiophosphates; dithiophosphates; methylphosphonates; 5'-alkylphosphonates; 5'-methylphosphonates; 3'-alkylphosphonates; trifluoroborates; 3'-5' or 2'-5' bonded boron phosphates and selenophosphates; triphosphates; thioalkylphosphonates; hydrogen phosphonate bonds; alkylphosphonates; alkylthiophosphonates; arylthiophosphonates; selenophosphates; diselenophosphates; hypophosphonates; phosphoramide esters; 3'-alkylphosphatamide esters; aminoalkylphosphatamide esters; thiophosphatamide esters; piperazine phosphoramides. Esters; aniline thiophosphates; aniline phosphates; ketones; sulfonates; sulfonamides; carbonates; carbamates; methylenehydrazine; methylenedimethylhydrazine; methyl acetal; thiomethyl acetal; oximes; methyleneimino; methylenemethylimino; thioamides; bonds with ribose acetyl groups; aminoethylglycine; silyl or siloxane bonds; alkyl or cycloalkyl bonds, for example, of 1 to 10 carbons, with or without heteroatoms, which are saturated or unsaturated and / or substituted and / or contain heteroatoms; bonds with morpholino structures, amides or polyamides, wherein the bases are directly or indirectly connected to a nitrogen atom in the main chain; and combinations thereof.

[0149] In some cases, one or more modifications include a modified phosphate backbone, wherein the modification produces a neutral or uncharged backbone. In some cases, the phosphate backbone is modified by alkylation to produce an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, as used herein in the context of alkylation, alkyl refers to a straight-chain or branched saturated hydrocarbon group containing 1 to 6 carbon atoms. In some cases, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl. In some cases, the modified phosphate ester is a phosphate ester group as described in U.S. Patent No. 9,481,905.

[0150] In some embodiments, the additional modified phosphate backbone includes methylphosphonate, ethylphosphonate, methylthiophosphate, or methoxyphosphonate. In some cases, the modified phosphate is a methylphosphonate. In some cases, the modified phosphate is an ethylphosphonate. In some cases, the modified phosphate is a methylthiophosphate. In some cases, the modified phosphate is a methoxyphosphonate.

[0151] In some cases, one or more modifications may further optionally include modifications to the ribose moiety, the phosphate backbone, and the nucleoside, or modifications to a nucleotide analog at the 3' or 5' end. For example, the 3' end may optionally contain a 3' cationic group, or the nucleoside may be inverted by a 3'-3' bond at the 3' end. In another alternative, the 3' end may optionally be conjugated to an aminoalkyl group (e.g., 3'C5-aminoalkyl dT). In yet another alternative, the 3' end may optionally be conjugated to a debasement site (e.g., a purine- or pyrimidine-free site). In some cases, the 5' end may be conjugated to an aminoalkyl group (e.g., a 5'-O-alkylamino substituent). In some cases, the 5' end may be conjugated to a debasement site (e.g., a purine- or pyrimidine-free site).

[0152] lipid compositions

[0153] In one respect, this article provides a pharmaceutical composition comprising a synthetic nucleic acid sequence assembled with a lipid composition, wherein the lipid composition comprises ionizable lipids.

[0154] In some embodiments, the composition comprises a synthetic nucleic acid sequence assembled with a lipid composition, and the lipid composition comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail having the following structure. 脂质 :

[0155] Or its pharmaceutically acceptable salt; wherein

[0156] R k1 Independently constitutes a C1-C12 divalent aliphatic or heteroaliphatic group;

[0157] R k3 It is independently a C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3-C12 heterocycloalkyl, aryl or heteroaryl;

[0158] R k2 It is independently C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl or heteroaryl;

[0159] R k4 and R k5 Each is independently an H or C1-C12 divalent aliphatic group;

[0160] M is O or NR k6 , where R k6It is an H or C1-C12 aliphatic group; and the synthetic nucleic acid sequence can encode one or more therapeutic peptides, wherein the therapeutic peptide binds to B cell antigens.

[0161] In some embodiments, the ionizable lipid comprises an amine head group and at least one hydrophobic tail having the structure of formula (I). 脂质 :

[0162]

[0163] Or its pharmaceutically acceptable salt;

[0164] in:

[0165] *Indicates the nitrogen connection point in the amine head group;

[0166] R1 and R2 are each independently a C1-C12 divalent aliphatic or heteroaliphatic group;

[0167] X is L1, L2, L3, and L4 are each independently a bond, O, S, or NR. c G represents O, S, or NR. d Q is OR e SR f or NR g R h Furthermore, r and t are each independently 1-6;

[0168] R c R d R e R f R g and R h Each is independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl;

[0169] Y and U are each independently a bond, O, S, NR. 10 Or Se;

[0170] n is 0 or 1;

[0171] R3 and R4 are each independently H, C1-C10 alkyl, C1-C10 heteroalkyl, aryl, or heteroaryl; or R3 and R4 together with the atoms they are attached to form C=O;

[0172] R5 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl; and

[0173] The synthetic nucleic acid sequence can encode one or more therapeutic peptides, wherein the therapeutic peptide binds to B cell antigens.

[0174] In some implementations, n is 0. In some implementations, n is 1.

[0175] In some embodiments, n is 1, and R3 and R4, along with their bonded atoms, together form C=O. In some embodiments, Y is CH2 and U is O. In some embodiments, both Y and U are O. In some embodiments, U is CH2 and Y is O. In some embodiments, Y and U are NR. 10 In some implementations, Y is 0 and U is NR. 10 In some implementations, Y is S and U is NR. 10 .

[0176] In some implementations, n is 0, and both Y and U are S.

[0177] In some implementations, n is 0, and one of Y and U is 0.

[0178] In some implementations, n is 0, and one of Y and U is Se.

[0179] In some embodiments, R1 is a straight-chain or branched C1-C12 alkyl group. In some embodiments, R1 is a straight-chain or branched C1-C10 alkyl group. In some embodiments, R1 is a straight-chain or branched C1-C8 alkyl group. In some embodiments, R1 is a straight-chain or branched C1-C6 alkyl group. In some embodiments, R1 is a straight-chain or branched C1-C4 alkyl group. In some embodiments, R1 is a C2 alkyl group, for example, In some embodiments, R1 is a C3 alkyl group, for example, In some embodiments, R1 is a C4 alkyl group. In some embodiments, R1 is a C1-C12 heteroaliphatic group.

[0180] In some embodiments, R2 is a straight-chain or branched C1-C12 alkyl group. In some embodiments, R2 is a straight-chain or branched C1-C10 alkyl group. In some embodiments, R2 is a straight-chain or branched C1-C8 alkyl group. In some embodiments, R2 is a straight-chain or branched C1-C6 alkyl group. In some embodiments, R2 is a straight-chain or branched C1-C4 alkyl group. In some embodiments, R2 is a C2 alkyl group, for example... In some embodiments, R2 is a C3 alkyl group, for example, In some embodiments, R2 is a C4 alkyl group. In some embodiments, R2 is a C1-C12 heteroaliphatic group.

[0181] In some embodiments, the amine head group is represented by the following:

[0182]

[0183] Wherein Ra, Ra', Ra” and Ra”' are each independently H, C1-20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, or R 脂质 , Furthermore, Z is a C1-C20 divalent aliphatic group, a C1-C20 divalent heteroaliphatic group, a divalent aryl group, or a divalent heteroaryl group.

[0184] In some embodiments, the ionizable lipid is represented by formula (II):

[0185]

[0186] Or its pharmaceutically acceptable salt, wherein:

[0187] i)R b It can be a substituted or unsubstituted alkyl, hydroxyalkyl, alkoxyalkyl, or aryl group;

[0188] ii) n1 and n2 are each independently 1, 2, 3, 4, 5, or 6; and

[0189] iii)R b1 R b2 R b3 and R b4 Each independently is H or R 脂质

[0190]

[0191] Where R b1 R b2 R b3 and R b4 At least one of them is not H.

[0192] In some implementations, R b It is a straight-chain or branched C1-C6 alkyl group. In some embodiments, R b The substituent is a substituted straight-chain or branched C1-C6 alkyl group. In some embodiments, the substituent includes hydroxyl, carbonyl, thiocarbonyl, alkoxy, phosphoryl, phosphate ester, phosphonate, hypophosphonate, amino, amide, cyclic amine, amino, imine, cyano, nitro, azide, mercapto, alkylthio, sulfate ester, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, aralkyl, or aromatic or heteroaromatic moiety. In some embodiments, the substituent includes hydroxyl, NH-Boc, ... Or any suitable substituent thereof.

[0193] In some implementations, n1 is 1, 2, 3, 4, 5, or 6. In some implementations, n1 is 2 or 3.

[0194] In some implementations, n2 is 1, 2, 3, 4, 5, or 6. In some implementations, n2 is 2 or 3.

[0195] In some implementations, n1 and n2 are the same. In some implementations, n1 and n2 are different. In some implementations, both n1 and n2 are 2. In some implementations, both n1 and n2 are 3. In some implementations, both n1 and n2 are 4.

[0196] In some implementations, R b1 Not H. In some implementations, R b2 Not H. In some implementations, R b3 Not H. In some implementations, R b4 Not H.

[0197] In some implementations, R b1 R b2 R b3 and R b4 At least two of them are not H. In some implementations, R b1 R b2 R b3 and R b4 At least three of them are not H. In some implementations, R b1 R b2 R b3 and R b4 None of them are H.

[0198] In some embodiments, the amine head group is selected from the group consisting of:

[0199]

[0200] In some embodiments, the at least one hydrophobic tail has the following structure:

[0201]

[0202] Where R k1 and R k3 Each is independently a C1-C10 alkyl group;

[0203] R k2It can be C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl or heteroaryl;

[0204] R k4 and R k5 Each is independently H or C1-C10 alkyl.

[0205] In some implementations, R k1 It is a straight-chain or branched C1-C10 alkyl group. In some embodiments, R k1 It is a straight-chain or branched C1-C4 alkyl group. In some embodiments, R k1 It is a C2 alkyl group, for example, In some implementations, R k1 It is a C3 alkyl group, for example, In some implementations, R k1 It is a C4 alkyl group.

[0206] In some implementations, R k3 It is a straight-chain or branched C1-C10 alkyl group. In some embodiments, R k3 It is a straight-chain or branched C1-C4 alkyl group. In some embodiments, R k3 It is a C2 alkyl group, for example, In some implementations, R k3 It is a C3 alkyl group, for example, In some implementations, R k3 It is a C4 alkyl group.

[0207] In some implementations, R k4 For H. In some implementations, R k4 It is a C1-C10 alkyl group. In some embodiments, R k4 It is a C1-C4 alkyl group. In some embodiments, R k4 It is a C4-C10 alkyl group.

[0208] In some implementations, R k5 For H. In some implementations, R k5 It is a C1-C10 alkyl group. In some embodiments, R k5 It is a C1-C4 alkyl group. In some embodiments, R k5 It is a C4-C10 alkyl group.

[0209] In some implementations, R k2 It is a C1-C20 alkyl group. In some embodiments, R k2 It is a C2-C20 alkenyl group. In some embodiments, R k2It is a C2-C20 acetylene group. In some embodiments, R k2 It is a C3-C20 cycloalkyl group. In some embodiments, R k2 It is a C1-C20 heteroalkyl group. In some embodiments, R k2 It is a C3-C20 heterocyclic alkyl, aryl, or heteroaryl compound.

[0210] In some embodiments, the at least one hydrophobic tail includes

[0211] In some embodiments, the at least one hydrophobic tail is selected from Table 2.

[0212] Table 2. Exemplary hydrophobic tails

[0213]

[0214]

[0215]

[0216] In some embodiments, the ionizable lipid comprises:

[0217]

[0218]

[0219] In some embodiments, the ionizable lipid comprises at least two hydrophobic tails. In some embodiments, the at least two hydrophobic tails are independently structural. In some embodiments, the at least two hydrophobic tails are identical. In some embodiments, the at least two hydrophobic tails are dissimilar. In some embodiments, one of the at least two hydrophobic tails differs from the others.

[0220] In some embodiments, the lipid composition comprises at least three hydrophobic tails. In some embodiments, the at least three hydrophobic tails are independently structurally distinct. In some embodiments, the at least three hydrophobic tails are identical. In some embodiments, the at least three hydrophobic tails are dissimilar. In some embodiments, one of the at least three hydrophobic tails differs from the others.

[0221] In some embodiments, the lipid composition comprises two hydrophobic tails. In some embodiments, the two hydrophobic tails are independently structurally distinct. In some embodiments, the two hydrophobic tails are identical. In other embodiments, the two hydrophobic tails are different.

[0222] In some embodiments, the lipid composition comprises three hydrophobic tails. In some embodiments, the three hydrophobic tails are independently structurally distinct. In some embodiments, the three hydrophobic tails are identical. In some embodiments, two of the three hydrophobic tails are identical, while the third hydrophobic tail is different. In some embodiments, all three hydrophobic tails are different.

[0223] In some embodiments, the lipid composition comprises four hydrophobic tails. In some embodiments, the four hydrophobic tails are independently structurally distinct. In some embodiments, the four hydrophobic tails are identical. In some embodiments, three of the four hydrophobic tails are identical, while the fourth hydrophobic tail is different. In some embodiments, two of the four hydrophobic tails are identical, the other two are identical, and these two are different from the other two. In some embodiments, two of the four hydrophobic tails are identical, while the other two are different from each other and different from these two. In some embodiments, all four hydrophobic tails are different.

[0224] In some embodiments, the ionizable lipid is selected from Table 3.

[0225] Table 3. Exemplary ionizable lipids

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] In some embodiments, the compositions provided herein further comprise a steroid. In some embodiments, the steroid comprises cholesterol or a cholesterol derivative. In some embodiments, the compositions provided herein further comprise an accessory lipid. In some embodiments, the accessory lipid comprises a phospholipid, such as 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In some embodiments, the compositions provided herein further comprise a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k)] or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2k).

[0248] In some embodiments, the lipid composition comprises ionizable lipids, steroids, auxiliary lipids, and polymer-conjugated lipids disclosed in this application.

[0249] In some embodiments, the ionizable lipid is present in the lipid composition in the following weight percentages: about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 80%, about 10% to about 90%, about 20% to about 30%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 30% to about 40%, about 30% to about 40%. % to 50%, about 30% to 60%, about 30% to 70%, about 30% to 80%, about 30% to 90%, about 40% to 50%, about 40% to 60%, about 40% to 70%, about 40% to 80%, about 40% to 90%, about 50% to 60%, about 50% to 70%, about 50% to 80%, about 50% to 90%, about 60% to 70%, about 60% to 80%, about 60% to 90%, about 70% to 80%, about 70% to 90%, or about 80% to 90%.

[0250] In some embodiments, the auxiliary lipid is present in the lipid composition in the following weight percentages: about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%.

[0251] In some embodiments, the steroid is present in the lipid composition in the following weight percentages: about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, or about 30% to about 40%.

[0252] In some embodiments, the polymer-conjugated lipid is present in the lipid composition in the following weight percentages: about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%.

[0253] In some embodiments, the weight ratio of the ionizable lipid / steroid / auxiliary lipid / polymer conjugated lipid is about 14 / 4 / 1 / 1, about 15 / 4 / 1 / 1, about 16 / 4 / 1 / 1, about 17 / 4 / 1 / 1, about 18 / 4 / 1 / 1, about 19 / 4 / 1 / 1, about 20 / 4 / 1 / 1, about 14 / 4 / 2 / 1, about 15 / 4 / 2 / 1, about 16 / 4 / 2 / 1, about 16.8 / 4 / 2 / 1, about 17 / 4 / 2 / 1, about 18 / 4 / 2 / 1, about 19 / 4 / 2 / 1, or about 20 / 4 / 2 / 1.

[0254] In some embodiments, the lipid composition comprises an ionizable lipid, a steroid, and a cofactor lipid disclosed in this application. In some embodiments, the ionizable lipid is present in the lipid composition at a weight percentage of about 30% to about 90%. In some embodiments, the cofactor lipid is present in the lipid composition at a weight percentage of about 5% to about 40%. In some embodiments, the steroid is present in the lipid composition at a weight percentage of about 5% to about 40%. In some embodiments, the weight ratio of the ionizable lipid / steroid / cofactor lipid is about 1 / 1 / 1, 2 / 1 / 1, about 3 / 1 / 1, about 4 / 1 / 1, about 5 / 1 / 1, about 6 / 1 / 1, about 2 / 2 / 1, about 3 / 2 / 1, about 4 / 2 / 1, about 5 / 2 / 1, or about 6 / 2 / 1.

[0255] In some embodiments, the lipid composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the carrier comprises a pharmaceutically acceptable excipient. The carrier may comprise a sugar, including mannitol, sucrose, maltose, or trehalose. The carrier may comprise EC-16, (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), stearic acid, perfluoroundecanoic acid, saponins, mannitol, borneol, amikacin-EC16, kanamycin-EC16, neomycin-EC16, or bile salts. In some embodiments, the carrier is present in the composition at a weight percentage of about 5% to about 60%. In some embodiments, the excipient is present in the composition at the following weight percentages: about 1% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, or about 10% to about 20%.

[0256] In some embodiments, the pharmaceutical compositions provided herein may further comprise polynucleotides, oligonucleotides, polypeptides, oligopeptides, small molecule compounds, or any combination thereof. In some embodiments, the polynucleotide is messenger ribonucleic acid (mRNA).

[0257] In some embodiments, the pharmaceutical composition comprises a polynucleotide that encodes its genetic product.

[0258] In some embodiments, the nucleic acid sequences provided herein are assembled in the lipid composition in the following weight ratios of the pharmaceutical formulation / lipid composition: about 1:200 to about 1:100, about 1:200 to about 1:50, about 1:200 to about 1:40, about 1:200 to about 1:30, about 1:200 to about 1:20, about 1:200 to about 1:10, about 1:200 to about 1:5, about 1:200 to about 1:1, about 1:100 to about 1:50, about 1:100 to about 1:40, about 1:100 to about 1:25, about 1:100 to about 1:20, about 1:100 to about 1:15, about 1:100 to about 1:10, about 1:100 to about 1:5, or about 1:100 to about 1:1.

[0259] In some embodiments, the composition is formulated for systemic or local administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for intramuscular administration.

[0260] In some embodiments, the lipid nanoparticles target a tissue or organ when administered to a subject. In some embodiments, the tissue or organ includes the liver. In some embodiments, the tissue or organ includes a lymphatic organ. In some embodiments, the lymphatic organ includes bone marrow, spleen, or lymph nodes.

[0261] In some embodiments, when administered to a subject, the lipid nanoparticles target target cells. In some embodiments, the target cells include hepatocytes, lymphocytes, leukocytes, myeloid cells, or hematopoietic stem cells. In some embodiments, the target cells include B cells, and wherein the B cells include plasmablasts, plasma cells, or memory B cells. In some embodiments, the target cells include T cells. In some embodiments, the target cells include mature B cells. In some embodiments, the mature B cells include memory B cells, plasma cells, or plasmablasts.

[0262] In some embodiments, the target cells include cells circulating in the subject's system. In some embodiments, the target cells include cells within the subject's tissues or organs.

[0263] In some embodiments, the lipid nanoparticles exhibit a faster clearance rate compared to other compositions when administered to a subject.

[0264] Additional lipids

[0265] In some embodiments, the lipid composition further comprises additional lipids, including steroids or steroid derivatives, PEG lipids, and auxiliary lipids (e.g., phospholipids or other zwitterionic lipids).

[0266] In some embodiments, the lipid composition further comprises an auxiliary lipid. In some embodiments, the auxiliary lipid comprises lipids that contribute to the stability or delivery efficiency of the lipid composition. In some embodiments, the auxiliary lipid comprises zwitterionic lipids. In some embodiments, the auxiliary lipid comprises phospholipids. In some embodiments, the phospholipid may contain one or two long chains (e.g., C6-C). 24 The phospholipid may contain an alkyl or alkenyl group, glycerol or sphingosine, one or two phosphate groups, and optionally a small organic molecule. The small organic molecule may be an amino acid, a sugar, or an amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, wherein zwitterionic lipids define lipids and lipid-like molecules that are both positively and negatively charged. In some embodiments of these lipid compositions, the phospholipid is not ethylphosphocholine. In some embodiments, the auxiliary lipid may include 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC).

[0267] In some embodiments, the composition may further comprise the phospholipid in a molar percentage of about 5 to about 30% of the total lipid composition.

[0268] In some embodiments, the auxiliary lipid is present in the lipid composition in the following weight percentages: about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%.

[0269] In some embodiments, the lipid composition comprises a molar percentage of about 8% to about 23% of the phospholipid. In some embodiments, the lipid composition comprises a molar percentage of about 10% to about 20% of the phospholipid. In some embodiments, the lipid composition comprises a molar percentage of about 15% to about 20% of the phospholipid. In some embodiments, the lipid composition comprises a molar percentage of about 8% to about 15% of the phospholipid. In some embodiments, the lipid composition comprises a molar percentage of about 10% to about 15% of the phospholipid. In some embodiments, the lipid composition comprises a molar percentage of about 12% to about 18% of the phospholipid. In some embodiments, the lipid composition comprises a molar percentage of the phospholipid at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, or at least about 23%. In some embodiments, the lipid composition comprises the phospholipid in the following molar percentages: up to about 8%, up to about 10%, up to about 12%, up to about 15%, up to about 18%, up to about 20%, or up to about 23%.

[0270] In some embodiments, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure, which may further comprise one or more substitutions, including alkyl, alkoxy, hydroxyl, oxo, acyl, or a double bond between two or more carbon atoms. In one aspect, the ring structure of the steroid comprises three fused cyclohexyl rings and one fused cyclopentyl ring, as shown in the following formula: In some embodiments, the steroid derivative comprises the above-mentioned ring structure having one or more non-alkyl substituted elements. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as: In some embodiments, the steroid or steroid derivative is a cholesterane or a cholesterane derivative. In cholesteranes, the ring structure is further defined by the following formula: As described above, the cholesterane derivative comprises one or more non-alkyl substitutions in the above-described ring system. In some embodiments, the cholesterane or cholesterane derivative is a cholesterane or a cholesterane derivative or a sterol or a sterol derivative. In other embodiments, the cholesterane or cholesterane derivative is cholesterol and sterol or derivatives thereof.

[0271] In some embodiments, the composition may further comprise the steroid in a molar percentage of about 20 to about 60% of the total lipid composition. In some embodiments, the steroid is present in the lipid composition in the following weight percentages: about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 20% to about 30%, about 20% to about 40%, or about 30% to about 40%.

[0272] In some embodiments, the lipid composition comprises a molar percentage of about 15% to about 46% of the steroid or steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of about 20% to about 40% of the steroid or steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of about 25% to about 35% of the steroid or steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of about 30% to about 40% of the steroid or steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of about 20% to about 30% of the steroid or steroid derivative. In some embodiments, the lipid composition comprises a molar percentage of the steroid or steroid derivative in the following molar percentages: at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative in the following molar percentages: up to about 15%, up to about 20%, up to about 25%, up to about 30%, up to about 35%, up to about 40%, up to about 45%, or up to about 46%.

[0273] In some embodiments, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride that further comprises a PEG chain linked to the glycerol group. In other embodiments, the PEG lipid contains one or more C6-C groups linked to a connector. 24 Long-chain alkyl or alkenyl or C6-C 24Compounds containing fatty acid groups and having a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropyl-3-amine, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearate phosphatidylethanolamine or PEG-modified myristoyl-sn-glycerol is used. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component in the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG-modified lipid is from about 100, 200, 400, 500, 600, 800, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 12500 to about 15000. Some non-limiting examples of lipids that may be used in this application are taught by U.S. Patent 5,820,873, WO 2010 / 141069 or U.S. Patent 8,450,298, which are incorporated herein by reference.

[0274] In some embodiments, the PEG lipid has the following structural formula: Where: R 12 and R 13 Each is independently an alkyl group (C≤24) alkenyl (C≤24) Or a substituted version of any of these groups; R e Hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) And x is 1-250. In some implementations, R e alkyl (C≤8) , such as methyl. R 12 and R 13 Each is independently an alkyl group (C≤4-20) In some embodiments, x is 5-250. In one embodiment, x is 5-125 or x is 100-250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol.

[0275] In some embodiments, the PEG lipid has the following structural formula: Wherein: n1 is an integer between 1 and 100, and n2 and n3 are each independently selected from integers between 1 and 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range thereof. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0276] In some embodiments, the polymer-conjugated lipid comprises 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k)] or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2k).

[0277] In some embodiments, the lipid composition comprises about 0.5% to about 20% of the polymer-conjugated lipid in molar percentage. In some embodiments, the lipid composition comprises about 1% to about 8% of the polymer-conjugated lipid in molar percentage. In some embodiments, the lipid composition comprises about 2% to about 7% of the polymer-conjugated lipid in molar percentage. In some embodiments, the lipid composition comprises about 3% to about 5% of the polymer-conjugated lipid in molar percentage. In some embodiments, the lipid composition comprises about 5% to about 10% of the polymer-conjugated lipid in molar percentage. In some embodiments, the lipid composition comprises the polymer-conjugated lipid in the following molar percentages: at least about 0.5%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5%, or at least about 10%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid in the following molar percentages: up to about 0.5%, up to about 1%, up to about 1.5%, up to about 2%, up to about 2.5%, up to about 3%, up to about 3.5%, up to about 4%, up to about 4.5%, up to about 5%, up to about 5.5%, up to about 6%, up to about 6.5%, up to about 7%, up to about 7.5%, up to about 8%, up to about 8.5%, up to about 9%, up to about 9.5%, up to about 10%, up to about 15%, or up to 20%.

[0278] In some embodiments, the polymer-conjugated lipid is present in the lipid composition in the following weight percentages: about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 5% to about 10%, about 5% to about 20%, or about 10% to about 20%.

[0279] Cancer immunotherapy

[0280] This invention provides a pharmaceutical composition comprising a nucleic acid sequence encapsulated by a lipid composition provided herein, wherein the nucleic acid sequence encodes one or more therapeutic peptides. In some embodiments, these therapeutic peptides can be used for cancer immunotherapy (e.g., B-cell malignancies). In some embodiments, the cancer immunotherapy includes T-cell-based therapies. In some embodiments, the one or more therapeutic peptides for cancer immunotherapy comprise antibodies or fragments thereof.

[0281] In some embodiments, the antibody encoded by the mRNA comprises a multispecific antibody. In some embodiments, the multispecific antibody comprises a bispecific T-cell adaptor (BiTE). In some embodiments, the BiTE binds to a surface protein on an immune cell (e.g., CD3 on a T cell) and a tumor antigen. The tumor antigen is a protein derived from a tumor or cancer cell. The tumor antigen may be overexpressed or uniquely expressed on or within a tumor or cancer cell, allowing tumor targeting. In some embodiments, the BiTE binds to both CD3 and CD19. In some embodiments, the compositions and methods provided herein involve a nucleic acid sequence encoding a CD19 / CD3 BiTE. In some embodiments, the nucleic acid sequence encoding a CD19 / CD3 BiTE comprises an RNA sequence encoding a CD19 BiTE. In some embodiments, the nucleic acid sequence comprises a DNA sequence that can be translated into an RNA sequence encoding a CD19 BiTE. In some embodiments, the nucleic acid sequence encoding a portion of a CD19 BiTE comprises one or more nucleic acid sequences encoding a portion of a CD19 BiTE as listed in Table 1. In some embodiments, the nucleic acid sequence encoding CD19 BiTE or a portion thereof further comprises one or more nucleic acid sequences encoding a signal peptide, one or more adapter regions, or one or more protein tags.

[0282] In some cases, the therapeutic peptides encoded by the mRNA encapsulated by the LNP of this disclosure contain a protein tag (e.g., a 6X His tag). In some cases, this protein tag may aid in the identification or purification of the therapeutic peptide.

[0283] In some cases, the mRNA encapsulated by the LNP of this disclosure may encode more than one therapeutic peptide. In some cases, the LNP of this disclosure contains mRNA encoding at least 1, 2, 3, 4, 5, 6 or more therapeutic peptides.

[0284] In some embodiments, these LNPs contain mRNA encoding a multispecific antigen-binding peptide for immunotherapy. In some cases, the multispecific antigen-binding peptide includes a first domain that binds to antigens expressed by immune cells and a second domain that binds to antigens expressed by tumor cells. This multispecific antigen-binding peptide can bind to any of the antigens expressed by the immune cells provided herein.

[0285] In some embodiments, the LNP of this disclosure comprises mRNA encoding a chimeric antigen receptor derived from a neutralizing antibody, which includes the antigen-binding fragment, transmembrane domain, and intracellular signaling domain described herein. The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within the cell to act as an effector by generating a second messenger or by responding to such a messenger, thereby regulating cellular activity through a defined signaling pathway.

[0286] As used herein, an "intracellular signaling domain" refers to the intracellular portion of a molecule. This intracellular signaling domain generates signals that promote the immune effector functions of CAR-containing cells (e.g., CART cells). Examples of immune effector functions (e.g., in CART cells) include cytolytic activity and cofactor activities, including the secretion of cytokines. In some cases, the intracellular signaling domain may contain a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those domains derived from molecules responsible for primary or antigen-dependent stimulation. In some cases, the intracellular signaling domain may contain a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those domains derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. The primary intracellular signaling domain may contain a signaling motif called an immune receptor tyrosine activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12.

[0287] The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that can be used for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0288] Co-stimulatory intracellular signal transduction domains can originate from the intracellular portion of co-stimulatory molecules. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte activating molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83, etc.

[0289] This intracellular signal transduction domain may contain the entire intracellular portion of its source molecule or the entire natural intracellular signal transduction domain or a functional fragment thereof.

[0290] Regarding the transmembrane domain, in various embodiments, the CAR can be designed to include a transmembrane domain attached to an extracellular domain of the CAR. The transmembrane domain may contain one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the transmembrane source protein (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 15 of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the transmembrane source protein (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or up to 15 of the intracellular region). In one aspect, the transmembrane domain is a transmembrane domain associated with one of the other domains of the CAR used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to prevent such a domain from binding to the transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex. In some cases, this transmembrane domain can be isodimated with another CAR on the surface of the CAR T cell. In some cases, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of the natural binding partner present in the same CAR T cell.

[0291] This transmembrane domain can be of natural or recombinant origin. When the origin is natural, the domain can be derived from any membrane-binding or transmembrane protein. In some cases, the transmembrane domain can transduce signals to the intracellular domain(s) whenever the CAR has bound to its target. In some cases, the transmembrane domain(s) may include at least the following transmembrane domains: for example, the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0292] In some cases, the transmembrane domain may be connected to the extracellular region of the CAR, such as the antigen-binding domain, via a hinge or spacer (e.g., a hinge from a human protein). In some cases, the hinge may be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge or a CD8a hinge. In one aspect, the hinge or spacer includes an IgG4 hinge.

[0293] The cytoplasmic domain or cytoplasmic region of the CAR contains an intracellular signal transduction domain. This intracellular signal transduction domain is generally responsible for activating at least one normal effector function of the immune cell to which the CAR is introduced. The term "effector function" refers to a specific function of the cell. Effector functions of T cells, for example, can be cytolytic or helper activities, including cytokine secretion. The term "intracellular signal transduction domain" refers to a protein portion that transduces effector function signals and directs the cell to perform its specific function. While the entire intracellular signal transduction domain can generally be used, in many cases it is not necessary to use the entire strand. A truncated portion of the intracellular signal transduction domain can be used to replace the entire strand to the extent necessary for transducing effector function signals. Therefore, the term "intracellular signal transduction domain" is intended to include any truncated portion of the intracellular signal transduction domain sufficient to transduce effector function signals.

[0294] Examples of intracellular signal transduction domains used in this disclosure include cytoplasmic sequences of T-cell receptors (TCRs) and co-receptors that cooperate to initiate signal transduction after antigen receptor engagement, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities.

[0295] It is known that the signal generated by the TCR alone is insufficient to fully activate the T cell, and secondary and / or co-stimulatory signals can also be involved. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domains) and those sequences that function in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling domains, e.g., co-stimulatory domains).

[0296] Primary signaling domains regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary intracellular signaling domains that function in a stimulatory manner may contain signaling motifs called immune receptor tyrosine activation motifs or ITAMs.

[0297] Examples of ITAMs containing primary intracellular signal transduction domains, as specifically used herein, include those ITAMs such as TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In some cases, the CAR contains an intracellular signal transduction domain of CD3ζ, for example, a primary signal transduction domain. In one embodiment, the primary signal transduction domain contains a modified ITAM domain, for example, a mutated ITAM domain having altered (e.g., increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signal transduction domain contains a modified ITAM-containing primary intracellular signal transduction domain, for example, an optimized and / or truncated ITAM-containing primary intracellular signal transduction domain. In one embodiment, the primary signal transduction domain contains one, two, three, four, or more ITAM motifs.

[0298] Co-stimulatory molecules are cell surface molecules, in addition to antigen receptors or their ligands, that contribute to the effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, among others. For example, CD27 co-stimulation has been shown to enhance the expansion, effector function, and survival of human CAR-T cells in vitro and to enhance the persistence and antitumor activity of human T cells in vivo (Song et al. Blood. 2012; 119(3):696-706).

[0299] Short oligopeptide or short polypeptide linkers with lengths between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can form bonds between intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In another embodiment, a single amino acid (e.g., alanine, glycine) can be used as a suitable linker.

[0300] In one aspect, the intracellular signal transduction domain is designed to contain two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signal transduction domains. In some cases, the two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signal transduction domains are separated by a connector molecule (e.g., the connector molecule described herein). In some cases, the intracellular signal transduction domain contains two co-stimulatory signal transduction domains. In some cases, the connector molecule is a glycine residue. In some embodiments, the connector is an alanine residue.

[0301] In some embodiments, the mRNA-encoded therapeutic peptides for cancer immunotherapy include immunomodulators. In some cases, immunomodulators include cytokines, cytokine receptors, chemokines, chemokine receptors, immune co-receptors, or immune co-receptor ligands. The expression of the immunomodulator may be driven by the expression regulatory regions disclosed herein.

[0302] In some cases, these therapeutic peptides used in cancer immunotherapy include cytokines or functional fragments thereof, such as G-CSF, GITRL, GM-CSF, IFN-α, IFN-β, IFN-γ, IL-1RA, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-23, LIF, LIGHT, LT-β, ​​M-CSF, MSP, OSM, OX40L, SCF, TALL-1, TGF-β, TGF-β1, TGF-β2, TGF-β3, TNF-α, TNF-β, TRAIL, TRANCE, or TWEAK.

[0303] In some cases, these therapeutic peptides used in cancer immunotherapy include cytokine receptors or functional fragments thereof, such as shared γ-chain receptors, shared β-chain receptors, interferon receptors, TNF family receptors, TGF-β receptors, Apo3, CD114, CD115, CD116, CD117, CD118, CD120, CD120a, CD120b, CD121, CD121a, CD121b, CD122, CD123, CD124, CD126, CD127, CD130, CD131, CD132, CD212, CD213, CD213a1, CD213a13, CD213a2, CD25, CD27, CD30, CD4, CD40, and C. D95(Fas), CDw119, CDw121b, CDw125, CDw131, CDw136, CDw137(41BB), CDw210, CDw217, GITR, HVEM, IL-11R, IL-11Ra, IL-14R, IL-15R, IL-15Ra, IL-18R, IL-1 8Rα, IL-18Rβ, IL-20R, IL-20Rα, IL-20Rβ, IL-9R, LIFR, LTβR, OPG, OSMR, OX40, RANK, TACI, TGF-βR1, TGF-βR2, TGF-βR3, TRAILR1, TRAILR2, TRAILR3, or TRAILR4.

[0304] In some cases, these therapeutic peptides used in cancer immunotherapy include chemokines or functional fragments thereof, such as ACT-2, AMAC-a, ATAC, BLC, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL3, CCL4, CCL5, CCL7, CCL8, CKb-6, CKb-8, CTACK, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL2, and CXCL3. CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, DC-CK1, ELC, ENA-78, eotaxin, eotaxin-2, eotaxin-3, Eskine, exodus-1, exodus-2, exodus-3, fractalkine, GCP-2, GROa, GROb, GROg, HCC-1, HCC-2, HCC-4, I-309, IL-8, ILC, IP-10, I-TAC, LAG-1, LARC, LCC-1, LD78α, LEC, Lkn-1, LMC, lymphoactin, lymphoactin b, MCAF, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MDNCF, MGSA-a, MGSA-b, MGSA-g, Mig, MIP-1d, MIP-1α, MIP-1β, MIP-2a, MIP-2b, MIP-3, MIP-3α, MIP-3β, MIP-4, MIP -4a, MIP-5, MPIF-1, MPIF-2, NAF, NAP-1, NAP-2, oncostatin, PARC, PF4, PPBP, RANTES, SCM-1a, SCM-1b, SDF-1α / β, SLC, STCP-1, TARC, TECK, XCL1 or XCL2.

[0305] In some cases, these therapeutic peptides used in cancer immunotherapy include chemokine receptors or functional fragments thereof, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, XCR1, or XCR1.

[0306] In some cases, these therapeutic peptides used in cancer immunotherapy include co-stimulatory immune receptors or functional fragments thereof, such as CD28, 2B4 (CD244, SLAMF4), 4-1BB (CD137), CD2 (LFA2, OX34), CD21, CD226 (DNAM1), CD27 (TNFRSF7), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), C RTAM(CD355), DcR3, DR3(TNFRSF25), GITR(CD357), HVEM(CD270), ICOS(CD278), LIGHT, LTβR(TNFRSF3), Ly108 (NTBA, CD352, SLAMF6), Ly9 (CD229, SLAMF3), OX40 (CD134), SLAM (CD150, SLAMF1), TIM1 (HAVCR1, KIM1) or TIM2.

[0307] In some cases, these therapeutic peptides used in cancer immunotherapy include activating NK receptors or functional fragments thereof, such as CD100 (SEMA4D), CD16 (FcgRIIIA), CD160 (BY55), CD244 (2B4, SLAMF4), CD27, CD94-NKG2C, CD94-NKG2E, CD94-NKG2H, CD96, CRTAM, DAP12, DNAM1 (CD226), KIR2DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, Ly49, NCR, NKG2D (KLRK1, CD314), NKp30 (NCR3), NKp44(NCR2), NKp46(NCR1), NKp80(KLRF1, CLEC5C), NTB-A(SLAMF6), PSGL1 or SLAMF7(CRACC, CS1, CD319).

[0308] In some cases, these therapeutic peptides used in cancer immunotherapy include Fcγ receptor (FcγR), Fcε receptor (FcεR), Fcα receptor (FcαR), Fcμ receptor (FcμR), neonatal Fc receptor (FcRn), CD4, CD5, CD8, CD21, CD22, CD27, CD28, CD32, CD40, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (ICOS), CD247ζ, CD247η, 41BB, DAP10, DAP12, FYN, LAT, Lck, MAPK, MHC complex, NFAT, NF-κB, PLC-γ, iC3b, C3dg, C3d, Zap70, MyD88, their functional fragments, or combinations thereof.

[0309] In some cases, these therapeutic peptides used in cancer immunotherapy include domains derived from transcription factors, interacting with transcription factors, increasing the expression of transcription factors, or activating transcription factors, such as E2A, Pax5, EBF, PU.1, Ikaros, GATA3, Th-POK, Tbet, Bcl6, NF-κB, NFAT, AP-1, NFAT, STAT1, STAT2, STAT3, STAT4, STAT5, STAT5A, STAT5B, STAT6, STAT7, IRF1, IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, AP-1, Eomes, FoxP3, Id2, PLZF, ROR-γ-T, TCF7, ThPOK, or any combination thereof.

[0310] method

[0311] In some aspects, this document provides methods for depleting B cells, which include administering the pharmaceutical compositions provided herein to a subject in need. B cells have been found to be involved in the etiology of many diseases. It is well known that B cells can produce antibodies against pathogens, a process tightly controlled by the immune system. Diseases involving dysregulation of B cell maturation, differentiation, and proliferation can be treated with B cell depletion therapy (BCDT). In some embodiments, the subject in need may have a disease requiring BCDT. In some embodiments, these methods for depleting B cells may be used within the BCDT. In some embodiments, these methods for depleting B cells may be part of the BCDT.

[0312] In some embodiments, BCDT includes B-cell malignancies or autoimmune diseases. In some embodiments, the B-cell malignancy includes B-cell lymphoma, which includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Wald's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma). In some embodiments, the B-cell lymphoma affects the spleen or lymph nodes.

[0313] B-cell malignancies can be present in the circulation or in localized tissues, such as bone marrow or secondary lymphoid tissue. In some embodiments, the B-cell malignancy includes multiple myeloma.

[0314] In some embodiments, BCDT includes autoimmune diseases. In some embodiments, the autoimmune diseases include allergic diseases, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), anti-myelin oligodendrocyte glycoprotein (anti-MOG) spectrum diseases, neuromyelitis optica spectrum diseases (NMOSD), anti-NMDAR encephalitis, or myasthenia gravis.

[0315] In some implementations, diseases requiring B-cell depletion therapy (BCDT) further include pemphigus vulgaris or Sjögren's syndrome.

[0316] In some respects, this article provides a method for treating hematologic malignancies, comprising administering the pharmaceutical composition provided herein to a subject in need, wherein the subject in need suffers from a hematologic malignancy.

[0317] In some embodiments, the hematologic malignancy includes B-cell lymphoma. In some embodiments, the B-cell lymphoma includes diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, or Burkitt lymphoma.

[0318] The application described herein may include topical application, oral application, or injection. In some embodiments, the application includes intravenous injection or intramuscular injection.

[0319] In some embodiments, the pharmaceutical compositions provided herein can be used for the approved indications or off-label indications of an FDA-approved drug that binds to a B-cell antigen. In some embodiments, the pharmaceutical compositions provided herein can be used for the approved indications or off-label indications of belintolimab, inelolizumab, rontoxicumab, or tancituzumab.

[0320] In some embodiments, the methods provided herein result in the expression of the therapeutic peptide having a longer half-life compared to any other method that includes administering an equivalent dose of the therapeutic peptide.

[0321] In some embodiments, the method results in the expression of therapeutic peptides having the following half-lives: at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, at least 12 hours, at least 16 hours, at least 18 hours, at least 24 hours, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 8.5 days, at least 9 days, at least 9.5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days or more.

[0322] In some implementations, this method results in the expression of therapeutic peptides with a larger area under the curve (AUC) compared to other methods.

[0323] In some embodiments, the method results in an AUC greater than that of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more of the expression of the therapeutic peptide compared to any other method that includes administering an equivalent dose of the therapeutic peptide.

[0324] In some embodiments, the method results in prolonged B-cell depletion compared to any other method that includes administering an equivalent dose of the therapeutic peptide. In some embodiments, this prolonged B-cell depletion includes B-cell depletion for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 17.5 days, at least 20 days, at least 25 days, at least 27.5 days, at least 30 days, at least 35 days, at least 37.5 days, at least 40 days, at least 45 days, at least 47.5 days, at least 50 days, at least 55 days, at least 57.5 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, or at least 120 days or more.

[0325] In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject. In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject.

[0326] In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the naïve B cells in the subject. In some embodiments, the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the mature B cells in the subject. In some embodiments, the mature B cells include memory B cells, plasma cells, or plasmablasts.

[0327] In some embodiments, the method results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B-cell depletion compared to any other method that includes administering an equivalent dose of the therapeutic peptide.

[0328] In some embodiments, the method further includes administering a second pharmaceutical composition comprising an active agent. The pharmaceutical compositions provided herein can be used in combination with another active agent. In some embodiments, the method further includes administering a small molecule drug. In some embodiments, the small molecule drug includes agents used in chemotherapy.

[0329] In some embodiments, the desired subject includes mammals. In some embodiments, the mammal includes humans, non-human primates, or rodents.

[0330] In some embodiments, the method for delivering a pharmaceutical formulation to a target organ of a subject in need provides a greater amount or activity of the pharmaceutical formulation in that target organ of the subject compared to the absence of the lipid composition. In some embodiments, the method for delivering a pharmaceutical formulation to a target organ of a subject in need provides a greater amount or activity of the pharmaceutical formulation in that target organ of the subject compared to a non-target organ.

[0331] In some embodiments of this method, the composition of this application may be administered by any suitable route, including parenteral delivery (e.g., injection), such as intravenous, intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intracardiac, intraperitoneal, intranasal, or intraocular injection.

[0332] In some embodiments, the method provides efficient delivery of the pharmaceutical composition to the cells of a subject. In some embodiments, the method including administration of the pharmaceutical composition provided herein results in targeted delivery of the pharmaceutical composition to a target organ. In some embodiments, the target organ includes the liver.

[0333] In some embodiments, the method delivers a pharmaceutical composition to a target organ (e.g., liver or spleen) or target cell (e.g., immune cell) of a subject, thereby providing an effective amount or activity of the pharmaceutical composition in the target organ or target cell that is at least 1.1 times greater than the corresponding amount or activity of the pharmaceutical composition achieved in a non-target organ or non-target cell of the subject. In some embodiments, the effective amount or activity of the pharmaceutical composition in the target organ or target cells is at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 18 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, or at least 300 times greater than the corresponding amount or activity of the pharmaceutical composition achieved in the non-target organs or non-target cells of the subject.

[0334] In some embodiments, these delivery methods include administering the pharmaceutical composition described herein, which provides an effective amount or activity of the pharmaceutical composition that is at least 1.1 times greater than the corresponding amount or activity of the pharmaceutical composition achieved by administering other compositions. In some embodiments, the effective amount or activity of the pharmaceutical composition produced by administering the lipid composition described herein is at least 1.1 times greater, at least 1.5 times greater, at least 2 times greater, at least 2.5 times greater, at least 3 times greater, at least 3.5 times greater, at least 4 times greater, at least 4.5 times greater, at least 5 times greater, at least 5.5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 15 times greater, at least 18 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 75 times greater, at least 100 times greater, at least 200 times greater, or at least 300 times greater than the corresponding amount or activity of the pharmaceutical composition achieved by administering other compositions.

[0335] In some embodiments, these delivery methods include administering the lipids described herein, which provide an effective amount or activity of the pharmaceutical composition that is at least 1.1 times greater than the corresponding amount or activity of the pharmaceutical composition achieved by administering other lipids. In some embodiments, the effective amount or activity of the pharmaceutical composition produced by administering the lipids described herein is at least 1.1 times, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 18 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 75 times, at least 100 times, at least 200 times, or at least 300 times greater than the corresponding amount or activity of the pharmaceutical composition achieved by administering other lipids.

[0336] In some embodiments, delivery of the pharmaceutical composition to cells can lead to cell death, such as apoptosis.

[0337] In one aspect, this article provides a high-potency dosage form of a pharmaceutical composition formulated with ionizable lipids, the dosage form comprising a pharmaceutical composition assembled with lipid compositions as described herein (e.g., mRNA encoding a therapeutic peptide for cancer immunotherapy).

[0338] The dosage of the pharmaceutical composition provided herein can be measured in mg / kg, where mg / kg refers to the mg of total nucleic acid (total mRNA) used to prepare these LNPs per kg of subject body weight. In some embodiments, the pharmaceutical composition is present in the dosage form at doses of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1.0, 0.5, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002, or 0.001 mg / kg (or mpk) of body weight, or in the range between any two of the foregoing values. In some embodiments, the pharmaceutical composition is present in the dosage form at a dose not exceeding about 10 mg / kg (or mpk) of body weight. In some embodiments, the pharmaceutical composition is present in the dosage form at doses not exceeding about 9 mg / kg, not exceeding about 8 mg / kg, not exceeding about 7 mg / kg, not exceeding about 6 mg / kg, not exceeding about 5 mg / kg, not exceeding about 4 mg / kg, not exceeding about 3 mg / kg, not exceeding about 2 mg / kg, not exceeding about 1 mg / kg, not exceeding about 0.5 mg / kg, not exceeding about 0.2 mg / kg, not exceeding about 0.1 mg / kg, not exceeding about 0.05 mg / kg, or not exceeding about 0.01 mg / kg. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 5 mg / mL.

[0339] In some embodiments, the pharmaceutical composition is present in the dosage form at concentrations of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 mg / mL, or in the range of any two of the foregoing values.

[0340] In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 5 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 2 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 1 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 0.5 mg / mL. In some embodiments, the pharmaceutical composition is present in the dosage form at a concentration not exceeding about 0.1 mg / mL.

[0341] In some embodiments, the pharmaceutical composition is present in the dosage form at concentrations of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 micrograms per milliliter (μg / mL), or in the range of any two of the foregoing values. In some embodiments, the pharmaceutical composition is present in the dosage form at concentrations not exceeding about 10, not exceeding about 9, not exceeding about 8, not exceeding about 7, not exceeding about 6, not exceeding about 5, not exceeding about 4, not exceeding about 3, not exceeding about 2, not exceeding about 1, not exceeding about 0.5, not exceeding about 0.2, or not exceeding about 0.1 micrograms per milliliter (μg / mL).

[0342] Any suitable dosage form can be prepared for delivery, for example, by oral, rectal, vaginal, mucosal, pulmonary (including tracheal or inhalation) or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intracardiac, intravenous, intraperitoneal, intranasal or intraocular injection.

[0343] In some embodiments, the pharmaceutical composition is administered at doses not exceeding about 10 mg / kg (or mpk) of body weight, not exceeding about 9 mg / kg, not exceeding about 8 mg / kg, not exceeding about 7 mg / kg, not exceeding about 6 mg / kg, not exceeding about 5 mg / kg, not exceeding about 4 mg / kg, not exceeding about 3 mg / kg, not exceeding about 2 mg / kg, not exceeding about 1 mg / kg, not exceeding about 0.5 mg / kg, not exceeding about 0.2 mg / kg, not exceeding about 0.1 mg / kg, not exceeding about 0.05 mg / kg, or not exceeding about 0.01 mg / kg of body weight. In some embodiments, the pharmaceutical composition is administered at doses from about 1 μg / kg of body weight to about 3 mg / kg of body weight.

[0344] In some embodiments, a single dose of the lipid composition provided herein may be repeated. If desired, the effective dose of the active lipid composition may be administered at appropriate intervals throughout the treatment course as one, two, three, four, five, six, or more doses. In some embodiments, the lipid composition may be administered two or three times daily. In some embodiments, the lipid composition will be administered once daily. In some embodiments, the lipid composition may be administered approximately every 1 week, approximately every 2 weeks, approximately every 3 weeks, approximately every 4 weeks, approximately every 5 weeks, approximately every 6 weeks, approximately every 7 weeks, approximately every 8 weeks, approximately every 9 weeks, approximately every 10 weeks, approximately every 11 weeks, approximately every 12 weeks, approximately every 13 weeks, approximately every 14 weeks, approximately every 15 weeks, approximately every 16 weeks, approximately every 17 weeks, or approximately every 18 weeks. In some embodiments, the lipid composition is administered approximately every 1 month, approximately every 2 months, approximately every 3 months, approximately every 4 months, approximately every 5 months, approximately every 6 months, approximately every 7 months, approximately every 8 months, approximately every 9 months, approximately every 10 months, approximately every 11 months, approximately every 12 months, approximately every 13 months, approximately every 14 months, approximately every 15 months, approximately every 16 months, approximately every 17 months, approximately every 18 months, approximately every 2 years, approximately every 2.5 years, approximately every 3 years, approximately every 3.5 years, approximately every 4 years, approximately every 4.5 years, or approximately every 5 years. Any subject in need can be treated using the methods of this application.

[0345] In some implementations, the subject has been identified as having a mutation in the target gene. In some implementations, the mutation in the target gene is associated with cancer or tumor.

[0346] In some embodiments, the subject has been identified as exhibiting abnormal expression or activity of a protein or polynucleotide corresponding to a target gene. In some embodiments, this abnormal expression or activity of the protein or polynucleotide is associated with cancer or tumor.

[0347] In some embodiments, this document provides a method for targeted delivery of a pharmaceutical formulation to a cell type, comprising contacting the cell with a composition of this application. In some embodiments of this method, the pharmaceutical composition comprises a pharmaceutical formulation (e.g., mRNA) assembled with a lipid composition as described in this application, wherein the lipid composition comprises any head or tail group disclosed herein.

[0348] In some embodiments, the contact is ex vivo. In some embodiments, the contact is in vitro. In some embodiments, the contact is in vivo. In some embodiments, the contact includes administering to a subject the composition comprising the therapeutic agent assembled with the lipid composition.

[0349] In some embodiments, the method results in zero to minimal toxicity. In some embodiments, the toxicity includes a transient increase in cytokines or liver enzymes. In some embodiments, the toxicity includes mild inflammation or mild hepatotoxicity.

[0350] Pharmaceutical Composition

[0351] The compositions and methods disclosed herein can be used to treat individuals in need. The pharmaceutical compositions described herein may comprise therapeutic or prophylactic compositions or any combination thereof. In some embodiments, these lipid compositions may be assembled using a nucleic acid sequence encoding a therapeutic peptide (e.g., BiTE). In some embodiments, the individual is a mammal (e.g., a human or a non-human mammal). When administered to an animal (e.g., a human or a non-human animal), the composition or the lipid composition is preferably administered as a pharmaceutical composition comprising, for example, the lipid composition of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline; or other solvents or carriers such as glycols, glycerol, oils (e.g., olive oil) or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for human administration, particularly for invasive routes of administration (i.e., routes that circumvent transport or diffusion across the epithelial barrier, such as intravenous or intramuscular injection), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of the drug or selective targeting of one or more cells, tissues, or organs. The pharmaceutical composition can be in dosage unit form such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophiles for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system (e.g., a skin patch). The composition can also be present in a solution suitable for topical application (e.g., a lotion, cream, or ointment).

[0352] In some embodiments, the pharmaceutical composition targets a tissue or organ when administered to a subject. In some embodiments, the tissue or organ includes the liver. In some embodiments, the tissue or organ includes a lymphatic organ. In some embodiments, the lymphatic organ includes bone marrow, spleen, or lymph nodes.

[0353] In some embodiments, when administered to a subject, the pharmaceutical composition targets target cells. In some embodiments, the target cells include hepatocytes, lymphocytes, leukocytes, myeloid cells, or hematopoietic stem cells. In some embodiments, the target cells include B cells, and wherein the B cells include plasmablasts, plasma cells, or memory B cells. In some embodiments, the target cells include T cells.

[0354] In some embodiments, the target cells include cells circulating in the subject's system. In some embodiments, the target cells include cells within the subject's tissues or organs.

[0355] In some embodiments, the pharmaceutical composition has improved storage stability compared to other compositions.

[0356] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition has a longer half-life compared to the corresponding therapeutic peptide of another pharmaceutical composition.

[0357] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition has a larger area under the curve (AUC) compared to the corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

[0358] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in prolonged B-cell depletion compared to a corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

[0359] In some embodiments, upon administration to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject. In some embodiments, after administration to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject.

[0360] In some embodiments, upon administration to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition causes depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the initial B cells in the subject. In some embodiments, after administration to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition causes depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the mature B cells in the subject.

[0361] In some embodiments, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B cell depletion compared to a corresponding therapeutic peptide in an equivalent dose of another pharmaceutical composition.

[0362] In some embodiments, the pharmaceutical composition exhibits zero to minimal toxicity when administered to a subject. In some embodiments, the toxicity includes a transient increase in cytokines or liver enzymes. In some embodiments, the toxicity includes mild inflammation or mild hepatotoxicity.

[0363] In some embodiments, the pharmaceutical composition exhibits a faster clearance rate compared to other compositions when administered to a subject.

[0364] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a solution suitable for injection into a subject. In some embodiments, the pharmaceutical composition further comprises a small molecule drug. In some embodiments, the small molecule drug comprises an agent for chemotherapy.

[0365] The pharmaceutical composition provided herein may comprise 1) an anti-CD19 light chain coding region comprising a nucleic acid sequence having 90% sequence identity with SEQ ID NO:1 and an anti-CD19 heavy chain coding region comprising a nucleic acid sequence having 90% sequence identity with SEQ ID NO:2; and 2) an anti-CD3 light chain coding region comprising a nucleic acid sequence having 90% sequence identity with SEQ ID NO:3 and an anti-CD3 heavy chain coding region comprising a nucleic acid sequence having 90% sequence identity with SEQ ID NO:4.

[0366] In some embodiments, after administration to the subject, the pharmaceutical composition leads to the activation of CD69+ T cells.

[0367] In some embodiments, the physical properties of the pharmaceutical composition are stable for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months or longer when stored at 4°C, -20°C, or -80°C. In some embodiments, the physical properties of the pharmaceutical composition are stable for at least 5 months when stored at 4°C, -20°C, or -80°C. In some embodiments, these physical properties of the pharmaceutical composition include the size, polydispersity index (PDI), encapsulation efficiency (EE%), or pKa of the lipid composition.

[0368] Pharmaceutically acceptable carriers may contain physiologically acceptable agents that function, for example, to stabilize lipid compositions (such as the lipid compositions of the present invention), increase their solubility, or increase their absorption. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers or excipients. The choice of pharmaceutically acceptable carriers (including physiologically acceptable agents) depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymer matrix into which it may be incorporated, for example, the lipid compositions of the present invention. Liposomes (e.g., containing phospholipids or other lipids) are non-toxic, physiologically acceptable, and metabolizable carriers, and their manufacture and administration are relatively simple.

[0369] The phrase “pharmaceutically acceptable” is used in this article to refer to lipid compositions, materials, compositions and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reactions or other problems or complications, and that meet a reasonable benefit / risk ratio.

[0370] As used herein, the phrase “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is harmless to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, mannose, trehalose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, etc. (10) Corn oil and soybean oil; (11) Diols, such as propylene glycol; (12) Polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (13) Esters, such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers, such as magnesium hydroxide and aluminum hydroxide; (16) Alginate; (17) Pyrothermic water; (18) Isotonic saline; (19) Ringer's solution; (20) Ethanol; (21) Phosphate buffer; and (22) Other non-toxic and compatible substances used in pharmaceutical preparations.

[0371] The pharmaceutical composition can be administered to a subject via any of a variety of routes of administration, including, for example, oral (e.g., in the form of an aqueous or non-aqueous solution, drench or suspension, tablets, capsules (including microcapsules and gelatin capsules), bolus, powder, granules, or paste for application to the tongue); absorption through the oral mucosa (e.g., sublingual); subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment, or spray applied to the skin). The lipid composition can also be formulated for inhalation. In some embodiments, the lipid composition can be simply dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for such routes of administration can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0372] These formulations can be conveniently presented in unit dosage forms and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific route of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the lipid composition that produces the therapeutic effect. Generally, within one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0373] Methods for preparing these formulations or compositions include the steps of associating an active composition (such as a lipid (e.g., nanoparticle) composition as described herein) with a carrier and optionally one or more auxiliary ingredients. Generally, these formulations are prepared by associating a lipid (e.g., nanoparticle) composition as described herein with a liquid carrier or a finely pulverized solid carrier, or both, uniformly and tightly, and then, if necessary, shaping the product.

[0374] As used herein, the phrases “parenteral administration” and “administered parenterally” refer to administration methods other than enteral and local administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, and intrasternal injections and infusions. In some embodiments, pharmaceutical compositions comprising the mRNA / LNPs provided herein are administered via a parenteral route (e.g., intravenous or intramuscular injection). Pharmaceutical compositions suitable for parenteral administration comprise one or more active lipid compositions in combination with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions, dispersants, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersants prior to use, which may contain antioxidants, buffers, bacteriostatic agents, solutes or suspending agents or thickeners that make the formulation isotonic with the blood of the intended recipient.

[0375] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Suitable flowability can be maintained, for example, by using coating materials (such as lecithin), maintaining the desired particle size in the case of dispersants, and using surfactants.

[0376] These compositions may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. Protection against microbial activity can be ensured by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). It may also be desirable to include isotonic agents (such as sugars, sodium chloride, etc.) in these compositions. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0377] In some cases, to prolong the effect of a drug, it is desirable to slow its absorption after subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. Therefore, the absorption rate of the drug depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, delayed absorption of the parenteral drug form can be achieved by dissolving or suspending the drug in an oil carrier.

[0378] Injectable depot formulations are manufactured by forming a microencapsulated matrix of the lipid composition within a biodegradable polymer, such as polylactide-polyglycolic acid. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydride). Injectable depot formulations are also prepared by encapsulating the drug in a tissue-compatible liposome or microemulsion.

[0379] For use in the methods of the present invention, the active lipid composition may be administered on its own or as a pharmaceutical composition comprising, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of an active ingredient in combination with a pharmaceutically acceptable carrier.

[0380] The delivery method can also be provided by rechargeable or biodegradable devices. In recent years, various sustained-release polymeric devices have been developed and tested in vivo for the controlled release of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels) (both biodegradable and non-degradable) can be used to form implants at specific target sites for the sustained release of lipid compositions.

[0381] The actual dose levels of these active ingredients in these pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to that patient.

[0382] The selected dose level will depend on a variety of factors, including the specific lipid composition or combination of lipid compositions used, or the activity of its esters, salts or amides, the route of administration, the time of administration, the excretion rate of the specific lipid composition used, the duration of treatment, other drugs, lipid compositions and / or materials used in combination with the specific lipid composition used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical field.

[0383] A physician or veterinarian with ordinary skill in the art can readily determine the desired therapeutically effective amount of the pharmaceutical composition and prescribe it. For example, the physician or veterinarian may begin administration of the pharmaceutical composition or lipid composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. “Therapeutically effective amount” means the concentration of the lipid composition sufficient to elicit the desired therapeutic effect. It is widely understood that this effective amount of the lipid composition will vary depending on the subject’s weight, sex, age, and medical history. Other factors affecting this effective amount may include, but are not limited to, the severity of the patient’s condition, the disease being treated, the stability of the lipid composition, and (if necessary) another type of therapeutic agent administered with the lipid composition of the present invention. A larger total dose can be delivered by administering the agent multiple times. Methods for determining potency and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine, 13th edition, 1814-1882, incorporated herein by reference).

[0384] Generally, the appropriate daily dose of the active lipid composition used in the compositions and methods of the present invention will be the amount of the lipid composition at which the minimum therapeutic effect is effectively produced. This effective dose will generally depend on the factors mentioned above.

[0385] If desired, the effective dose of the active lipid composition may be administered at appropriate intervals throughout the treatment process as one, two, three, four, five, six, or more doses, optionally in unit dose form. In some embodiments of the invention, the active lipid composition may be administered two or three times daily. In some embodiments, the active lipid composition will be administered once daily.

[0386] Patients or subjects receiving this treatment are any animals in need, including primates, especially humans; and other mammals such as horses, cattle, pigs, sheep, cats, and dogs; poultry; and pets in general.

[0387] In some embodiments, the lipid compositions of the present invention can be used alone or in combination with another type of therapeutic agent.

[0388] This disclosure includes pharmaceutically acceptable salts for use in the lipid compositions of the present invention in the compositions and methods of the present invention. In some embodiments, the salts of the present invention contemplated include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In some embodiments, the salts of the present invention contemplated include, but are not limited to, L-arginine, benzylamine, benzathine, betaine, calcium hydroxide, choline, dianophenate, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazolium, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In some embodiments, the salts of the present invention contemplated include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In some embodiments, the salts of the present invention considered include, but are not limited to, 1-hydroxy-2-naphtholic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetaminobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, L-ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, decanoic acid (n-decanoic acid), hexanoic acid (n-hexanoic acid), octanoic acid (n-octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclohexane, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, etc. Acids, galactobionic acid, gentic acid, D-glucoheptanoic acid, D-gluconic acid, D-glucuronic acid, glutamic acid, glutamate, glutaric acid, glycerophosphate, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, L-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, L-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, L-tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid.

[0389] These pharmaceutically acceptable acid addition salts can also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates can also be prepared. The source of such solvates can be from the crystallization solvent, inherent in the solvent used for preparation or crystallization, or foreign to this solvent.

[0390] Wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents and flavor enhancers, preservatives and antioxidants may also be present in these compositions.

[0391] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0392] Reagent test kit

[0393] On the other hand, a kit is provided that comprises the pharmaceutical composition described herein and / or lipid nanoparticle (LNP) formulation. In some embodiments, the kit may further comprise a means for administering the composition provided herein, such as a suitable syringe for intravenous or intramuscular administration.

[0394] In any of the methods disclosed herein, the method further includes providing instructions for use (IFU) that include instructions for administering the LNP compositions to a subject. In some embodiments, the user instructions direct a user to intravenous or intramuscular injection of the LNP compositions containing mRNA for immunotherapy.

[0395] definition

[0396] Before describing embodiments of this disclosure, it will be understood that such embodiments are provided by way of example only, and various alternatives to the embodiments of this disclosure described herein may be employed in the practice of this disclosure. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from this disclosure.

[0397] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While similar or equivalent methods and materials described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, these materials, methods, and embodiments are illustrative only and are not intended to be limiting. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from this disclosure.

[0398] In the context of this application, unless otherwise stated, the following terms shall have the meanings assigned to them:

[0399] As used throughout the specification and claims, the terms “a” or “an” and “the” are generally used in the sense that they refer to “at least one” or “at least one,” “at least first,” “one or more,” or “multiple” or “a plurality of” the components or steps mentioned herein, unless an upper limit is expressly specified thereafter. For example, as used herein, “cleavage sequence” means “at least a first cleavage sequence,” but includes multiple cleavage sequences. The operative limits and parameters of combinations according to this application, as well as the amount of any single reagent, will be known to those skilled in the art.

[0400] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and generally refer to a polymer of amino acids of any length. This polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. These terms also cover amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation (such as conjugation with a labeled component).

[0401] As used herein, the term "antibody" refers to a naturally occurring, partially synthesized, or fully synthesized immunoglobulin (Ig). The term also covers any polypeptide or protein having a binding domain that is an antigen-binding domain or homologous to an antigen-binding domain. The term further includes "antigen-binding fragment" or "functional fragment thereof," or "fraction of antibody," "antibody fragment," "functional fragment of antibody," and other interchangeable terms for similar binding fragments, as described below. Antibodies include, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, chemically engineered antibodies, deimmunized antibodies, affinity-matured antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), heteroconjugate antibodies, antibody fragments, and combinations thereof (e.g., simultaneously deimmunized monoclonal antibodies, simultaneously deimmunized humanized antibodies, etc.). Antibodies can be, for example, mouse antibodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies, bispecific antibodies, diabody, triabody, or tetrabody. The antigen-binding fragment may include, for example, Fab', F(ab')2, Fab, Fv, rlgG, scFv, hcAb (heavy chain antibody), single-domain antibody, VHH, VNAR, sdAb, or nanobody.

[0402] The term "antigen" refers to a molecule that is bound by an antibody or a fragment thereof. In some cases, the antigen may be called a "ligand" of the antibody. Antigens can originate from surface proteins of cells (such as immune cells or cancer cells). In some cases, antigens can originate from surface proteins of tumor cells. The antigen can be targeted by the antibody to kill the cell.

[0403] As used herein, the terms “treatment,” “treating,” “palliating,” or “ameliorating” are used interchangeably. These terms generally refer to methods used to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. A therapeutic benefit means the elimination or relief of the underlying disease being treated. Furthermore, a therapeutic benefit is achieved by eliminating or relieving one or more physiological symptoms associated with the underlying disease or by improving one or more clinical parameters, resulting in an observed improvement in the subject, even if the subject may still have the underlying disease. For preventive benefits, these compositions may be administered to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of a disease, even if the disease may not yet be diagnosed.

[0404] As used herein, "therapeutic effect" or "therapeutic benefit" generally refers to physiological effects resulting from administration of the disclosed peptides other than the ability to induce the production of antibodies against an antigenic epitope of the bioactive protein, including but not limited to reducing, alleviating, or preventing disease in humans or other animals, or improving one or more clinical parameters associated with the underlying disease, or otherwise enhancing the physical or mental health of humans or animals. For preventative benefits, these compositions may be administered to subjects at risk of developing a specific disease, a recurrence of a previous disease, symptoms or signs of the disease, or subjects who report one or more of the physiological symptoms of the disease, even if the disease may not yet be diagnosed.

[0405] As used herein, the terms "therapeutic effective amount" and "therapeutic effective dose" generally refer to an amount of a drug or bioactive protein, alone or as part of a polypeptide composition, that, when administered to a subject in a single dose or repeated administration, is capable of having any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such effect does not necessarily have to be absolutely beneficial. The determination of a therapeutically effective amount is entirely within the competence of those skilled in the art, especially based on the detailed disclosure provided herein.

[0406] For chemical groups and compound categories, the number of carbon atoms in that group or category is indicated as follows: "Cn" defines the exact number of carbon atoms (n) in that group / category. "C≤n" defines the maximum number of carbon atoms (n) that can be in that group / category, with the minimum number being as small as possible for the relevant group / category; for example, it is understood that the minimum number of carbon atoms in the group "alkenyl (C≤8)" or the category "alkene (C≤8)" is two. This is in contrast to "alkoxy (C≤10)", which specifies alkoxy groups having 1 to 10 carbon atoms. "Cm-n" or "Cm-Cn" defines both the minimum (m) and maximum number of carbon atoms (n) in that group. Thus, "C1-C10 alkyl" specifies those alkyl groups having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or category they modify, and may or may not be enclosed in parentheses, without implying any change in meaning. Therefore, the terms "C5 olefin," "C5-olefin," and "olefin" are used interchangeably. (C5) "and "olefins" C5 "They are all synonyms."

[0407] When used to modify compounds or chemical groups, the term "saturated" means that, unless otherwise stated below, the compound or chemical group does not have carbon-carbon double or triple bonds. When the term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substituted versions of saturated groups, one or more carbon-oxygen double bonds or one carbon-nitrogen double bond may be present. And when such bonds are present, carbon-carbon double bonds that may appear as part of a keto-enol tautomerism or an imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that no more of the substance can dissolve in the solution.

[0408] The term "aliphatic" generally refers to a compound or chemical group that is acyclic or cyclic but non-aromatic hydrocarbon compound or group. In aliphatic compounds / groups, these carbon atoms can be linked together in straight chains, branched chains, or non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, i.e., linked by single carbon-carbon bonds (alkane / alkyl), or unsaturated, having one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkynyl / alkynyl).

[0409] When used to modify atoms in a compound or chemical group, the term "aromatic" indicates that the compound or chemical group contains a planar unsaturated atomic ring that is stabilized by the interactions of the bonds that form the ring.

[0410] When used without the modifier "substituted," the term "alkyl" refers to a monovalent saturated aliphatic group that has a carbon atom as a linker, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. Examples of alkyl groups include -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), and -CH(CH3)2(i-Pr). i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-butyl, t-Bu or tBu) and -CH2C(CH3)3 (neopentyl) are non-limiting examples of alkyl groups. When used without the modifier "substituted," the term "alkanediyl" refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as connecting points, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. "Alkane" refers to a class of compounds having the formula HR, where R is an alkyl group (as defined above). When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is a subset of substituted alkyl groups where the hydrogen atom substitution is limited to halogens (i.e., -F, -Cl, -Br, or -I), such that no atoms other than carbon, hydrogen, and halogens exist. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups where the hydrogen atom substitution is limited to fluorine, such that no atoms other than carbon, hydrogen, and fluorine exist. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0411] When used without the modifier "substituted," the term "cycloalkyl" refers to a monovalent saturated aliphatic group having a carbon atom as a connecting point that forms part of one or more non-aromatic ring structures, without carbon-carbon double or triple bonds, and without atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). When used without the modifier "substituted," the term "cycloalkyldiyl" refers to a divalent saturated aliphatic group having two carbon atoms as connecting points, without carbon-carbon double or triple bonds, and without atoms other than carbon and hydrogen. Atom. The group is a non-limiting example of a cycloalkanediyl group. “Cycloalkane” refers to a class of compounds having the formula HR, where R is a cycloalkyl group (as defined above). When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0412] When used without the modifier "substituted," the term "alkenyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as a linking point, a linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. When used without the modifier "substituted," the term "alkenidyl" refers to a divalent unsaturated aliphatic group having two carbon atoms as linking points, a linear or branched, linear or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkene dienes. It is noteworthy that although the alkene diene is aliphatic, it is not excluded that the group can form part of an aromatic structure when attached at both ends. The terms "alkene" and "olefin" are synonymous and refer to a class of compounds having the formula HR, where R is an alkenyl group (as defined above). Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene having only one carbon-carbon double bond, where the bond is part of a vinyl group at the end of the molecule. When any of these terms is used with the modifier “substituted,” one or more hydrogen atoms have been independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0413] When used without the modifier "substituted," the term "alkynyl" refers to a monovalent unsaturated aliphatic group having a carbon atom as a linker, a linear or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term "alkynyl" does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alynyl" refers to a class of compounds having the formula HR, where R is an alkynyl group. When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH or -S(O)2NH2.

[0414] When used without the modifier "substituted," the term "aryl" refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a connecting point, which forms part of one or more six-membered aromatic ring structures, wherein all ring atoms are carbon, and wherein the group is not composed of atoms other than carbon and hydrogen. If more than one ring is present, these rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl or aralkyl groups (carbon number restrictions allow) connected to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and monovalent groups derived from biphenyl. When used without the modifier "substituted," the term "aryldiaryl" refers to a divalent aromatic group having two aromatic carbon atoms as connecting points, which form part of one or more six-membered aromatic ring structures, wherein all ring atoms are carbon, and wherein the monovalent group is not composed of atoms other than carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number restrictions allow) connected to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, these rings may be fused or unfused. Unfused rings may be connected by one or more of the following: covalent bonds, alkyldiyl, or alkylenediyl groups (carbon number restrictions allow). Non-limiting examples of aryldiyl groups include:

[0415]

[0416] When used without the modifier "substituted," the term "aralkyl" refers to the monovalent group -alkyldiyl-aryl, wherein the terms "alkyldiyl" and "aryl" are each used in a manner consistent with the definitions provided above. Non-limiting examples are: benzyl (benzyl, Bn) and 2-phenyl-ethyl. When the term "aralkyl" is used with the modifier "substituted," one or more hydrogen atoms from the alkyl diel and / or the aryl group have been independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Non-limiting examples of substituted aralkyl groups are (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-ethyl-1-yl.

[0417] When used to modify compounds or chemical groups, the term "hetero" indicates that the compound or chemical group has at least one atom that is not carbon, such as N, O, S, Se, P, Si, B, or any other heteroatom. For example, a heteroaliphatic can be any aliphatic moiety containing at least one heteroatom selected from N, O, P, B, S, Si, Sb, Al, Sn, As, Se, and Ge. A heterocyclic can be any ring containing a ring atom that is not carbon. A heterocyclic can be substituted with any number of substituents (e.g., alkyl and halogen atoms). A heterocyclic can be aromatic (heteroaryl) or non-aromatic. Non-limiting examples of heterocyclics include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.

[0418] When used without the modifier "substituted," the term "heteroaryl" refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as a linking point, which forms part of one or more aromatic ring structures, wherein at least one of these ring atoms is nitrogen, oxygen, or sulfur, and wherein the heteroaryl group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. A heteroaryl ring may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, and sulfur. If more than one ring is present, these rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number restrictions allow) connected to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazole (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridinyl (pyridyl), pyrroleyl, pyrimidinyl, pyrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thiopheneyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a connecting point. When used without the modifier "substituted," the term "heteroaryldiyl" refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two connecting points, these atoms forming part of one or more aromatic ring structures, wherein at least one of these ring atoms is nitrogen, oxygen, or sulfur, and wherein the divalent group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. If more than one ring is present, these rings can be fused or unfused. Unfused rings can be linked by one or more of the following: covalent bonds, alkyl dienes, or alkenyl dienes (carbon number restrictions allow). As used herein, this term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number restrictions allow) linked to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include:

[0419]

[0420] When used without the modifier "substituted," the term "heterocyclic alkyl" refers to a monovalent non-aromatic group having a carbon or nitrogen atom as a linking point, which forms part of one or more non-aromatic ring structures, wherein at least one of these ring atoms is nitrogen, oxygen, or sulfur, and wherein the heterocyclic alkyl group is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. The heterocyclic alkyl ring may contain one, two, three, or four ring atoms selected from nitrogen, oxygen, or sulfur. If more than one ring is present, these rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups attached to the ring or ring system (as permitted by carbon number restrictions). Furthermore, the term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocyclic alkyl groups include aziridinyl, aziridine, pyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocyclic alkyl" refers to a heterocyclic alkyl group having a nitrogen atom as a connecting point. N-pyrrolyl is an example of such a group. When used without the modifier "substituted," the term "heterocyclic alkyl dieryl" refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two connecting points, these atoms forming part of one or more ring structures, wherein at least one of these ring atoms is nitrogen, oxygen, or sulfur, and wherein the divalent group is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. If more than one ring is present, these rings can be fused or unfused. Unfused rings can be linked by one or more of the following: covalent bonds, alkyl dienes, or alkenyl dienes (as permitted by carbon number restrictions). As used herein, this term does not exclude the presence of one or more alkyl groups (as permitted by carbon number restrictions) attached to the ring or ring system. Furthermore, this term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocyclic alkyl dienes include:

[0421]

[0422] When these terms are used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0423] When used without the modifier "substituted," the term "acyl" refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl (as these terms are defined above). The groups -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)CH2C6H5, and -C(O) (imidazolyl) are non-limiting examples of acyl groups. "Thioacyl" is defined similarly, except that the oxygen atom in the group -C(O)R has been replaced with a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkane as defined above, where at least one of these hydrogen atoms has been replaced with a -CHO group. When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms (including hydrogen atoms directly attached to carbon atoms in a carbonyl or thiocarbonyl group, if present) have been independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (carboxymethyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0424] When used without the modifier "substituted," the term "alkoxy" refers to the group -OR, where R is an alkyl group (as defined above). Non-limiting examples include: -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert-butoxy), -OCH(CH2)2, -O-cyclopentyl, and -O-cyclohexyl. When used without the modifier "substituted," the terms "cycloalkoxy," "alkenoxy," "alkynoxy," "aryloxy," "arylalkoxy," "heteroaryloxy," "heterocyclic alkoxy," and "acyloxy" refer to groups defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heterocyclic alkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent group -O-alkadiyl-, -O-alkadiyl-O-, or -alkadiyl-O-alkadiyl-. When used without the modifier "substituted," the terms "alkathio" and "acylthio" refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane as defined above, wherein at least one of these hydrogen atoms has been replaced with a hydroxyl group. The term "ether" corresponds to an alkane as defined above, wherein at least one of these hydrogen atoms has been replaced with an alkoxy group. When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms have been independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH or -S(O)2NH2.

[0425] When used without the modifier "substituted," the term "alkylamino" refers to the group -NHR, where R is an alkyl group (as defined above). Non-limiting examples include -NHCH3 and -NHCH2CH3.

[0426] When used without the modifier "substituted," the term "dialkylamino" refers to the group -NRR', where R and R' can be the same or different alkyl groups, or R and R' can be combined to represent an alkyldiyl group. Non-limiting examples of dialkylamino include -N(CH3)2 and -N(CH3)(CH2CH3). When used without the modifier "substituted," the terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocyclic alkylamino," "alkoxyamino," and "alkylsulfonamideamino" refer to groups defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocyclic alkyl, alkoxy, and alkylsulfonamide, respectively. A non-limiting example of arylamino is -NHC6H5. The term "alkylaminodiyl" refers to the divalent group -NH-alkyldiyl, -NH-alkyldiyl-NH-, or -alkyldiyl-NH-alkyldiyl-. When used without the modifier "substituted," the term "amido" (acylamino) refers to the group -NHR, where R is an acyl group (as defined above). A non-limiting example of an amide group is -NHC(O)CH3. When used without the modifier "substituted," the term "alkylimino" refers to the divalent group =NR, where R is an alkyl group (as defined above). When any of these terms is used with the modifier “substituted”, one or more hydrogen atoms attached to the carbon atom have been independently replaced with -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting examples of substituted amide groups.

[0427] The term "substituted" refers to a portion of the main chain in which a substituent replaces a hydrogen atom on one or more carbons. It will be understood that "substitution" or "substituted with" includes the implicit restriction that such substitution satisfies the permissible valence of the substituted atom and the substituent, and that the substitution results in a stable compound, for example, which does not spontaneously undergo transformations (such as by rearrangement, cyclization, elimination, etc.). As used herein, the term "substituted" is considered to include all permissible substituents in organic compounds. In a broad sense, these permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents in organic compounds. For suitable organic compounds, these permissible substituents may be one or more, and may be the same or different. For the purposes of this invention, these heteroatoms (such as nitrogen) may have hydrogen substituents satisfying the valence of these heteroatoms and / or any permissible substituents in the organic compounds described herein. Substituents may include any substituents described herein, such as halogens, hydroxyl groups, carbonyl groups (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (e.g., thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonates, hypophosphonates, amino groups, amide groups, amidine groups, imine groups, cyano groups, nitro groups, azide groups, mercapto groups, alkylthio groups, sulfate groups, sulfonates, sulfonamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that, where appropriate, the substituted portion of the hydrocarbon chain may itself be substituted. Unless otherwise stated, the terms aliphatic, heteroaliphatic, oxyaliphatic, alkyl, alkylene, alkenyl, alkenylene, ynyl, alkenylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenyl, cycloalkenyl, cycloalkenyl, cycloalkenyl, hydroxyalkyl, heterocycloalkyl, heterocycloalkylene, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenyl, aryl and heteroaryl include substituted and unsubstituted portions.

[0428] Unless otherwise stated, all figures representing amounts of components, reaction conditions, etc., used in this specification and claims are to be understood to be modified by the term "about" in all cases. Therefore, unless otherwise stated, the numerical parameters listed in this specification and appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained in this application. Generally, when referring to measurable values ​​(such as amounts of weight, time, dosage, etc.), the term "about," as used herein, is intended to cover variations from the specified amount such that, in one example, ±20% or ±10%, in another example, ±5%, in yet another example, ±3%, in another example, ±1%, and in yet another example, ±0.1%, such variations are suitable for carrying out the disclosed method.

[0429] As used herein, the term "average molecular weight" refers to the relationship between the number of moles of each polymer species and the molar mass of that species. Specifically, each polymer molecule can have different levels of polymerization and therefore different molar masses. Average molecular weight can be used to represent the molecular weight of multiple polymer molecules. Average molecular weight is generally synonymous with average molar mass. Specifically, there are three main types of average molecular weight: number-average molar mass, weight-average molar mass, and Z-average molar mass. In the context of this application, unless otherwise stated, the average molecular weight represents the number-average molar mass or weight-average molar mass of the formula. In some embodiments, the average molecular weight is a number-average molar mass. In some embodiments, the average molecular weight can be used to describe the PEG component present in lipids.

[0430] The terms “comprise,” “have,” and “include” are open-ended conjunctions. Any form or tense of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” is also open-ended. For example, any method involving one or more steps of “comprises,” “has,” and “includes” is not limited to having only those steps and also covers other steps not listed.

[0431] As used in the specification and / or claims, the term "effective" means sufficient to achieve the desired, intended, or planned result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutic effective amount," or "pharmaceutically effective amount" means an amount of compound sufficient to achieve such treatment of a disease when administered to a subject or patient for the treatment of that disease.

[0432] As used herein, the terms "patient" or "subject" refer to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In some embodiments, the patient or subject is a primate (e.g., a non-human primate). In some embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.

[0433] In the context of delivering a payload to one or more target cells, as used herein, the terms “assemble” or “assembled” generally refer to one or more covalent or non-covalent interactions or associations, such as causing a therapeutic or preventative agent to be compounded with or encapsulated in a lipid composition.

[0434] As used herein, the term "lipid composition" generally refers to a composition comprising one or more lipid compounds, including but not limited to cationic lipid complexes, liposomes, and lipid particles. Examples of lipid compositions include suspensions, emulsions, and vesicle compositions.

[0435] As used generally in this article, “pharmaceutically acceptable” means, to a reasonable extent of medical judgment, those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues, organs and / or body fluids of humans and animals without excessive toxicity, irritation, allergic reactions or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0436] "Pharmaceutically acceptable salt" means a salt of the compound of this application that is pharmaceutically acceptable (as defined above) and has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-en-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-en-1-carboxylic acid, acetic acid, aliphatic monocarboxylic acids and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, etc. Camphor sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptanoic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthyl acid, lactic acid, lauroyl sulfate, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, benzoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the present acidic protons can react with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucosamine, etc. It should be recognized that the specific anion or cation forming part of any salt of this disclosure is not essential, as long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts, their preparation methods, and uses are presented in Handbook of Pharmaceutical Salts: Properties, and Use (edited by PHStahl and CGWermuth, Verlag Helvetica Chimica Acta, 2002).

[0437] As used herein, the term “pharmaceuticalally acceptable carrier” refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting a chemical reagent.

[0438] As used in this disclosure, the term "accessory lipid" refers to a lipid that contributes to the stability or delivery efficiency of a lipid composition. Accessory lipids can be zwitterionic lipids, such as phospholipids. Accessory lipids can be phosphatidylcholine, distearate phosphatidylcholine, dioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In some cases, the term "accessory lipid" refers to phospholipids or other zwitterionic lipids in the LNP composition. In some cases, when a formulation of an LNP is described using the weight ratios of these lipid components (e.g., lipids, steroids, accessory lipids, and polymer-conjugated lipids), accessory lipids refer to phospholipids or another zwitterionic lipid. For example, the weight ratio of lipids / steroids / accessory lipids / polymer-conjugated lipids is about 4 / 1 / 1. "Accessory lipid" can refer to any class of lipid molecules that improve the particle stability and flowability of lipid nanoparticles (LNPs). Several classes of molecules can be used as accessory lipids, such as phospholipids (e.g., ethanolamine phosphate, choline phosphate), zwitterionic lipids, steroid derivatives, and polymer-conjugated lipids (e.g., PEGylated lipids). Representative accessory lipids include cholesterol, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), phosphatidylcholine (PC), methoxy-polyethylene glycol (MW 2k)-distearate phosphatidylethanolamine (mPEG2k-DSPE), and 1,2-dimyristoyl-rac-glycerol-3-methoxy-polyethylene glycol-2000 (DMG-PEG2k).

[0439] Example

[0440] The present disclosure provides the following embodiments to further illustrate some implementations of the present disclosure, but is not intended to limit the scope of the present disclosure; and it will be understood, by its exemplary nature, that other procedures, methods or techniques known to those skilled in the art may be used alternatively.

[0441] Example 1: Formulation and Characterization of Lipid Nanoparticles (LNPs)

[0442] Table 4 lists some of the lipids tested. All tested lipids were synthesized via a solvent-free Michael addition reaction between an amine head and an alkyl-acrylate lipid tail.

[0443] In the case of homogenous tailed lipids containing a hydrophobic tail, an aliphatic amine head (3,3'-diamino-N-methyldipropylamine) and the corresponding acrylate tail were mixed at 1 to 5 molar ratio in Teflon-lined screw-cap vials for 48 h at 70 °C. The crude product was purified using a mobile phase of methanol / DCM on a Teledyne Isco chromatographic system. The purified lipids were characterized by electrospray ionization mass spectrometry (ESI-MS).

[0444] In the case of asymmetric heterogeneous lipids, the amine head was mixed with the major lipid tail at a molar ratio of 1 to 3.5. After stirring at 70°C for 48 hours, the three-tailed lipid was similarly purified and subsequently reacted with the fourth tail at a molar ratio of 1:1.5. The final product was separated and verified by ESI-MS.

[0445] In the case of symmetrical heterogeneous lipids, a boc-protected amine head is reacted with a lipid tail at a molar ratio of 1 to 2.5. After mixing at 70°C for 48 hours, the semi-assembled lipids are purified by removing the boc protecting group with a solution of trifluoroacetic acid in DCM, and the semi-lipids are further reacted with a second lipid tail to obtain a fully heterogeneous but symmetrical lipid product.

[0446] Figure 1 The synthesis of lipid L0088 tail was described. Under argon atmosphere, in a round-bottom flask with a magnetic stir bar, 4-nitrophenyl chloroformate was added to a solution of 2-butyl-1-octanol and triethylamine in THF (500 ml). The reaction mixture was stirred for 2–3 hours until complete, monitored by TLC. The reaction mixture was then diluted with ethyl acetate and washed successively with 1N HCl solution, saturated NaHCO3 solution, and brine. The organic layer was dried over MgSO4, filtered, and concentrated. The residue was purified by rapid column chromatography on silica (0–10% ethyl acetate solution in hexane) to give 3 (88% yield).

[0447] Reagent 3 was added to a solution of 2-hydroxyethyl acrylate in DMF under argon atmosphere. The reaction mixture was heated to 50°C, and K2CO3 was added to the reaction mixture after 10 min. The entire reaction mixture was then heated at 80°C for 2–3 h until complete, as examined by TLC. DMF was removed by rotary evaporation, and the residue was separated by adding water and ethyl acetate. The aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with water and brine, dried over Na2SO4, filtered, and concentrated. The residue was carefully purified by rapid column chromatography on silica (10–30% ethyl acetate in hexane) to give 4 (70%).

[0448] Example 2. Formulation of mRNA encoding BiTE using cationic lipid nanoparticles

[0449] The test lipids in Table 4 were prepared using cholesterol, DOPC, DMG-PEG, and mRNA encoding CD19-CD3 BiTE. Generally, the component lipids (ionizable cationic lipids, cholesterol, DOPC, and DMG-PEG) were dissolved in ethanol at an appropriate molar ratio of 46 / 42 / 10 / 1.6. Each lipid was dissolved in ethanol and mixed to achieve the molar ratio specified in the organic phase. The nucleic acid was dissolved in 25 mM sodium acetate pH 5.2 buffer to achieve a concentration of 0.134 mg / mL. The aqueous and organic solutions were then mixed... The mixture was mixed in a Precision NanoSystems container at a flow rate of 3:1 (v / v; separately) and a total flow rate of 20 mL / min. The resulting mixture was dialyzed overnight against at least 1000 volumes of 10 mM Tris-HCl pH 7.5 buffer using a Slide-A-Lyzer MINI dialysis unit (MWCO, 3.5 kDa). Sucrose was then added to the formulation to a final concentration of 1.32 mg / mL, followed by 0.22 μm filtration and storage at -4 °C and -80 °C for short-term and long-term storage, respectively.

[0450] Table 4. Non-limiting exemplary chemical structures of the tested ionizable lipids

[0451]

[0452]

[0453]

[0454] Example 3. Characterization of cationic lipid nanoparticles

[0455] LNPs were formulated with different cationic lipids (test lipids and reference lipids) and mRNA encoding luciferase, and the physical properties of these LNPs were evaluated. The particle size, polydispersity index (PDI), and encapsulation of all lipid nanoparticles were characterized. LNPs were diluted in PBS pH 7.4 and transferred to quartz cuvettes (ZEN2112) for size measurement by dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern Panalytical) with a particle RI of 1.45 and an absorbance of 0.001 in PBS (viscosity 0.888 cP and RI 1.335) at 25 °C. Measurements were performed using a 173° backscatter detection angle, previously equilibrated to 25 °C for 30 s, in duplicate, with five runs per run, each run lasting 10 s, with no delay between measurements. Each measurement was performed at a fixed position of 4.65 mm in the quartz cuvette with automatic attenuation selection. Measurements were performed using commercially available Invitrogen. TM Quant-it TM Encapsulation efficiency (EE%) was measured using the RiboGreen RNA Assay Kit. LNP preparations were diluted 250-fold with TE buffer or Triton / TE (0.5% v / v Triton in TE buffer) and incubated at room temperature for 30 min to extract LNPs with Triton, then added to 96-well plates. The microplates were then immediately introduced into the microplate. The iD3 plate reader was used to read fluorescence at Ex485 / Em530. Encapsulation percentage was calculated as [total mRNA – free mRNA] / [total mRNA] × 100%. The surface pKa value of LNP was determined based on TNS assay. Briefly, a solution of 20 mM sodium phosphate, 25 mM citrate, 20 mM ammonium acetate, and 150 mM NaCl was titrated to pH values ​​varying by 0.5 from 2.0 to 12.0 and aliquoted into black 96-well plates. LNP and 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS, Sigma Aldrich) were diluted in these solutions to final concentrations of 20 μM and 6 μM, respectively. Fluorescence intensity was read on the plate reader with excitation at 322 nm and emission at 431 nm. Assuming that the minimum and maximum fluorescence values ​​correspond to zero and 100% protonation, respectively, the pKa value was calculated at the pH corresponding to 50% protonation of LNP.

[0456] like Figure 2A As shown, the particle size and PDI of the tested LNP and the reference LNP are comparable, with no significant difference. Figure 2B As shown, the encapsulation efficiency (EE%) of the test LNP and the reference LNP are comparable, with no significant difference. Figure 2CAs shown, the pKa of the test LNP and the reference LNP are comparable, with no significant difference. Overall, the test LNP and the reference LNP are comparable in physical properties.

[0457] The size, PDI, pKa, and EE% of these LNPs were measured over time to evaluate their stability under different storage conditions. mRNA integrity was analyzed over time by fragment analysis. We observed that the physical and chemical properties of the LNPs formulated with these ionizable lipids were stable over time when stored at 4°C or -80°C. Figure 3A , Figure 3B and Figure 3C The LNPs (e.g., L88-CD19 / CD3 BiTE) depicted, containing L0088 and mRNA encoding CD19 / CD3 BiTE, maintained stable size, PDI, pKa, EE% and mRNA integrity for up to 5 months.

[0458] Example 4: Characterization of LNP in mice

[0459] Tissue and cell tropism

[0460] All animal experiments were performed according to approved animal protocols. LNP (Luc LNP) was formulated with lipids and mRNA encoding luciferase and administered intravenously (iv) to CD1 mice at 0.5 mg / kg body weight. Six hours post-injection, mice were administered 100 μL of luciferin K salt (15 mg / mL in PBS) via intraperitoneal injection. Mice were then imaged using an in vivo imaging system (IVIS), and the total flux to regions of interest was quantified. Various mouse organs were harvested 6 hours after the iv injection of Luc mRNA / LNP. Tissues were homogenized and proteins extracted. Luciferase levels were measured using a luciferase assay. Figure 4A The described process involves intravenous injection of L88 LNPs containing Luc mRNA (e.g., L88-Luc LNPs) into mice, resulting in the production of luciferase, primarily observed in the liver and spleen. Figure 4B The distribution of tissue lysates toward the liver and spleen was also observed.

[0461] L88 LNPs containing Cre mRNA (L88-Cre LNPs) were administered intravenously to Ai9 mice at a dose of 0.5 mg / kg. One week later, mouse organs were harvested, and bone marrow was also extracted. Tissues were treated with an enzyme, such as papain, to obtain single-cell suspensions. The isolated cells were stained with an antibody mixture and analyzed by flow cytometry to identify the distribution of L88-Cre LNPs. Figure 5The cell tropism of L88 LNPs was demonstrated. L88 LNPs specifically target some cells but not others.

[0462] Protein expression over time following repeated administration of L88LNP.

[0463] L88 LNPs (e.g., L88-hEPO LNPs) containing mRNA encoding human erythropoietin (hEPO) were intravenously injected into CD1 mice at a dose of 0.5 mg / kg. One dose was administered to the same mouse weekly for at least 5 weeks. Plasma hEPO protein levels were quantified by ELISA at the same time after each injection. Figure 6 The changes in plasma hEPO over time are shown.

[0464] Pharmacokinetics of LNP.

[0465] Luc LNP was administered intravenously to CD1 mice at a dose of 0.5 mg / kg body weight. Liver tissue was obtained at 6 hours, 7 days, 14 days, and 21 days post-injection. Lipids were extracted and analyzed by liquid chromatography-mass spectrometry (LC-MS). Figure 7A and Figure 7B The graph depicts the amount of lipids (ng of lipids per kg of liver) over time. The clearance rate of L88-LNP is faster than that of the reference LNP.

[0466] Example 5. L88-CD19 / CD3 BiTE: Construct Design, In Vivo Expression, and Testing

[0467] Human codon-optimized DNA sequences were generated through gene synthesis. VH and VL sequences derived from different anti-CD19 and anti-CD3 clones were fused via a 3x glycine-serine (GS) linker. The anti-CD19 and anti-CD3 scFv sequences were linked via a 5-residue GS linker. All bispecific T-cell adaptor (BiTE) constructs contained a secretion signal and a C-terminal 6xHis tag. RNA sequences are listed in Table 1. Multiple mRNA constructs containing the anti-CD19 coding region of SEQ ID NO:1-2, SEQ ID NO:5-6, or SEQ ID NO:9-10 were synthesized. These mRNA constructs contained the anti-CD3 coding region of SEQ ID NO:3-4 or SEQ ID NO:7-8.

[0468] The mRNA encoding BiTE was synthesized at TriLink Biotechnology. Figure 8AThe structure represents the mRNA encoding BiTE. All mRNAs have a 5'-cap (cap 1), a 3' poly-A tail, and are modified with pseudouridine. In each mRNA, the 5'-UTR and 3'-UTR are located flanking the coding sequence of the CD19-CD3 BiTE mRNA.

[0469] In vitro expression of CD19-CD3 BiTE encoded by mRNA was analyzed by lipofection of HEK293 cells and subsequently determined by Western blotting. Generally, HEK293 cells were seeded in 24-well TC plates containing 1 ml DMEM + 10% FBS one day prior to lipofection. For lipofection, a 1:2 mixture of mRNA and lipofectamine was prepared under sterile, ribonuclease-free conditions and applied to HEK293 cells at approximately 60-70% confluence. After incubation at 37°C and 5% CO2 for 48 h, the supernatant was collected and stored at -20°C until further use. Western blotting was used to measure cell supernatants containing secreted CD19-CD3 BiTE using an antibody conjugated to 6xHis horseradish peroxidase (HRP).

[0470] To evaluate the binding affinity of mRNA-encoded CD19-CD3 BiTE to human CD19 and CD3 proteins, as well as the dual binding of both arms, an ELISA assay was performed using supernatant from mRNA-transfected HEK293 cells and the corresponding purified recombinant CD19-CD3 BiTE protein. Ninety-six wells of the ELISA plate were coated overnight at 4°C with the specified capture antigen protein. After brief washing and blocking, 100 μl of appropriately diluted sample was added to the plate and incubated at room temperature for 2 h. After three aspirations and washes, 100 μl of the prepared assay antibody-anti-6xHis-HRP mixture was added to each well and incubated at room temperature for 1 h. After three aspirations and washes / soaks, TMB substrate solution was added, and the HRP reaction was stopped using 2N H2SO4. The absorbance was read at 450 nm using a SpectraMax iD3 reader. The binding curves of the purified recombinant CD19-CD3 BiTE protein to the secreted protein in the supernatant were analyzed using Prism software, with the corresponding protein as a concentration standard.

[0471] CD19 ELISA binding results are shown in Figure 8B and Figure 8CIn summary, although the three CD19-CD3 BiTE recombinant proteins have different CD19 binding affinities, similar binding abilities to secreted BiTE were determined after transfection of Hek293 cells with 1 μg BiTE mRNA for 48 h. At least three mRNA designs were tested. HTX-01-001 mRNA contained SEQ ID NO: 1-4. HTX-01-002 mRNA contained SEQ ID NO: 5-8. HTX-01-003 mRNA contained SEQ ID NO: 7-10.

[0472] Analysis of CD19-CD3 BiTE-mediated T cell killing of cancer cells encoded by mRNA.

[0473] For the CD3 adaptor BsAb, the induced T cell lytic activity against tumor cells is the most important characteristic for assessing its in vitro titer and specificity. As described in this article, for the mRNA-encoded CD19-CD3 BiTE, we performed a killing assay against CD19-positive tumor cells using the collected supernatant. For the CD19-positive tumor cell line Raji, we evaluated T cell lytic activity using a flow cytometry-based killing assay. Generally, Raji cells were first labeled with CellTrace Violet dye according to the manufacturer's instructions, and 20,000 cells were seeded per well in 96-well round-bottom tissue culture plates. Resting human PBMCs were added to each well at an E / T ratio of 5:1. Then, 100 μl of supernatant collected from Hek293 cells transfected with BiTE mRNA was added to the co-culture system and incubated for 20 h. After treatment, the cells were stained with the immobilizable viability dye eFluor 780 (FVD-eFluor780) according to the manufacturer's instructions. The number of live target cells was measured using the gates of CellTrace Violet+FVD-eFluor780. Killing activity was calculated as ([number of untreated live target cells – number of treated live target cells] / [number of untreated live target cells] x 100%).

[0474] Such as FACs analysis ( Figure 9A ) and the calculated results after normalization for the untreated control ( Figure 9B As shown in both cases, CD19-CD3BiTE generated by transfecting mRNA in vitro can induce strong T cell killing activity against CD19-positive Raji cells.

[0475] In vivo screening of mRNA encoding CD19-CD3 BiTE prepared by cationic LNP.

[0476] Nine types of lipid nanoparticles (e.g., LNP-CD19 / CD3 mRNA) encoding CD19 BiTE mRNA were formulated as described in Example 2 and characterized as described in Example 3. The particle size, polydispersity index (PDI), and encapsulation of the nine nanoparticles using different cationic lipids but with the same mRNA clone HTX-01-003 were characterized. LNP was diluted in PBS pH 7.4 and transferred to a quartz cuvette (ZEN2112) for size measurement by dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern Panalytical). The particle RI used was 1.45, and the absorbance in PBS at 25°C (viscosity 0.888 cP, RI 1.335) was 0.001. Measurements were performed using a 173° backscatter detection angle previously equilibrated to 25°C for 30 s, in duplicate, with five runs per run, each lasting 10 s, with no delay between measurements. Each measurement is taken at a fixed position of 4.65 mm in the quartz cuvette and features automatic attenuation selection. For example... Figure 10A As shown, the particle sizes of the nine different LNPs using the same mRNA cargo varied, but all were in the range of 80-150 nm. Furthermore, all LNPs uniformly had a PDI of less than 0.15.

[0477] By using commercial Invitrogen TM Quant-it TM The RiboGreen RNA Assay Kit was designed to achieve high encapsulation efficiency. The LNP preparation was diluted 250-fold with TE buffer or Triton / TE (0.5% v / v Triton in TE buffer) and incubated at room temperature for 30 min to extract LNPs with Triton, then added to 96-well plates. The microplate was then immediately introduced into the microplate. The iD3 reader is used to read fluorescence (Ex485 / Em530). Please add the formula for calculating the encapsulation percentage. Figure 10B As shown, under the same conditions, an ideal encapsulation rate was determined for most LNPs, especially for LNPs using HTX-88 (e.g., L88-CD19 / CD3 mRNA), which showed an optimal encapsulation rate of over 90%.

[0478] To evaluate the expression level of CD19-CD3 BiTE generation in vivo by the cationic LNP formulation, Balb / c WT mice were used in this study. 10 μg of the formulation described in Example 6 was administered intravenously (iv) via the tail vein. Plasma levels of CD19-CD3 BiTE were detected 5 h after iv administration using a CD19-based ELISA assay. Experiments were performed as described in Example 3, and purified HTX-01-003BiTE protein was used as a standard for analysis.

[0479] like Figure 11B As shown, despite preparing the same amount of mRNA, different cationic LNPs exhibited significant differences in in vivo mRNA delivery efficiency 5 hours after injection. Figure 11B As shown, the HTX-88-formulated mRNA encoding CD19-CD3 BiTE (e.g., L88-CD19 / CD3 mRNA) exhibited the highest endogenous protein expression among all nine different HTX LNPs (including the clinically approved LNP for Pfizer's Covid-19 vaccine). Sufficient amounts of protein were also translated from the CD19-CD3 BiTE-encoding mRNA delivered from the HTX-93 LNP (e.g., L93-CD19 / CD3 mRNA). These two cationic LNPs were selected for delivering different mRNA cargoes for in-depth in vitro analysis.

[0480] LNP-prepared mRNA encoding CD19-CD3BiTE (LNP-CD19 / CD3mRNA In vitro evaluation

[0481] To evaluate the pharmacological activity of functional CD19-CD3BiTE, an endogenous translation of CD19-CD3BiTE mRNA formulated from LNP, plasma samples were collected in BALB / c mice at 5 h, 24 h, 96 h, and 168 h post-injection after a single intravenous administration of L88-CD19 / CD3 mRNA or L93-CD19 / CD3 mRNA, as specified. Both in vitro killing activity and plasma concentration were evaluated.

[0482] like Figure 11BAs shown, plasma levels of CD19-CD3 BiTE protein, endogenously translated from the administered mRNA, were detectable at 6 h and persisted for several days, especially in the 5 μg mRNA administration group. Therefore, the in vitro cytotoxic activity of plasma from treated mice showed maximum killing at 24 h, with no significant difference between the 5 and 1 μg dose groups. However, unlike the sharp decline in plasma protein expression levels 4 days after injection, a single dose of LNP-formulated mRNA at the same time point achieved strong in vitro tumor cell killing activity. Moreover, four of the five treatment groups maintained above the half-maximum killing activity on day 4, and a single dose of HTX-CH-L88 formulated with 5 μg HTX-01-002 mRNA maintained above the half-maximum killing activity for 7 days.

[0483] Example 7. Efficacy of L88-CD19 / CD3 mRNA in animals

[0484] The study design is shown in Table 5.

[0485] Table 5. Research Design.

[0486]

[0487]

[0488] L88-003 (or LNP-CD19 / CD3 mRNA, hereinafter referred to as LNP-CD19 / CD3 mRNA) was used in this study. Figures 12A-12B The pharmacokinetic profile of a single dose of LNP-CD19 / CD3 mRNA in Balb / c mice is shown. Figure 12A This is a schematic diagram of the experimental design for a single-dose pharmacokinetic (PK) study. For example... Figure 12B The concentrations of CD19-CD3 BiTE protein in the plasma of Balb / c mice following intravenous administration of LNP mRNA are depicted. Mice were treated intravenously with a single dose of L88-CD19 / CD3 mRNA containing 5 μg of CD19 BiTE mRNA. Blood was drawn from five mice at each time point for quantification by CD3 ELISA. Concentrations from duplicate replicates of the technical ELISA are shown. Data are presented as mean ± SD. PK parameters of plasma BiTE protein expression after administration of 5 μg L88-CD19 / CD3 mRNA were calculated by WinNonlin (New Jersey, US) and are included in Table 6 below.

[0489]

[0490] Figures 13A-13HResults from efficacy studies of L88-003 (e.g., LNP-CD19 / CD3 mRNA) in a Raji-luciferase xenograft mouse model reconstituted in hPBMCs are presented. Figure 13A This is a schematic diagram of the research design, in which tumor cells were inoculated and arranged into 3 treatment groups (n=5 per group), including LNP-CD19 / CD3 mRNA (L88-003, 1.6 μg mRNA was injected intravenously into each mouse), recombinant BiTE protein (rPR003, 10 μg recombinant BiTE protein was injected intravenously into each mouse) and vector. Figure 13B Images of the total photon flux of individual mice in each group, captured at specified time points using an in vivo imaging system (IVIS), are depicted. Figures 13C-13G As shown, LNP-CD19 / CD3 mRNA achieved comparable results in xenograft tumor killing compared to recombinant BiTE protein. Furthermore, the administration frequency of LNP-CD19 / CD3 mRNA was lower than that of recombinant BiTE protein due to its longer half-life. For each treatment group, bioluminescence signals were quantified to photons per second using LivingImage 4.7 software. Individual total flux (TF) at the end of the study was analyzed. Each point represents the bioluminescence signal of a single mouse, and statistical analysis was compared with the G1_vector control group using the Kruskal-Wallis test and Dunn's multiple comparison test. ***p<0.01. E, F, G) analyzed the changes in TF per mouse in each group during treatment. Figure 13H The relative weight changes of each mouse during the treatment were similar. The mouse weights were normalized to the baseline time point (t=0), and each point represents the mean ± SD. Figures 14A-14B This study presents a plasma CD19-CD3 BiTE protein pharmacokinetic profile analysis in cynomolgus monkeys following stepwise escalation of IV infusion of L88-003 (e.g., an LNP containing mRNA encoding CD19 / CD3BiTE). Figure 14A This is a schematic diagram illustrating the three-stage dose escalation and blood collection at specified time points for PK profile analysis. L88-003 was administered based on the body weight of non-human primates. The first dose of L88-003 was 0.014 mg / kg. The second dose was 0.07 mg / kg, and the third dose was 0.1 mg / kg. Figure 14BThe concentrations of CD19 / CD3 BiTE protein in cynomolgus monkey plasma were quantified by CD3 ELISA at specified time points following intravenous infusion of L88-003 protein. Data are presented as mean ± SD of duplicate replicates of the technical ELISA, with the dashed line representing the LLOQ at 2 ng / ml. Table 7 lists the pharmacokinetic parameters of expressed BiTE protein as analyzed by WinNoLin after administration of L88-003.

[0491] Table 7. Dose-dependent protein expression of LNP-BiTE mRNA in NHP

[0492]

[0493] Figures 15A-15G The study showed circulating B cell exhaustion and T cell dynamics in cynomolgus monkeys after three weekly doses of L88-003 (e.g., LNP-CD19 BiTE mRNA). Figure 15A This is a schematic diagram of a three-stage dose progression and blood collection at designated time points for phenotypic analysis of peripheral B cells and T cells using flow cytometry. Figure 15B and Figure 15D The absolute cell counts of circulating CD20+ B cells and circulating CD3+ T cells at specified time points are shown separately. Circulating B cells were observed after administration and persisted for over 100 days. Prolonged 80% depletion of circulating B cells was observed after the last dose of L88-003, lasting at least 13 weeks compared to pre-dose levels. Circulating B cell counts began to recover 5 weeks after the last dose. However, at 13 weeks post-dose, only about 20% of the pre-dose B cell count was captured, indicating prolonged in-situ depletion of CD19+ memory B cells and plasmablasts in secondary lymphoid tissues. These CD19+ cells cannot recover in the short term after complete depletion. Moreover, as... Figure 15C The study described a 17-fold decrease in circulating plasmablasts / plasma cells from the L88-003 treatment group at 105 days post-treatment compared to untreated groups, indicating effective depletion of long-term persistent CD19+ plasmablasts (generally considered more difficult to target and kill). All of this suggests that, in addition to the liver, the LNP-CD19 / CD3 mRNA composition was successfully delivered to secondary lymphoid tissues (e.g., spleen, lymph nodes, bone marrow, etc.) and resulted in the in situ production of BiTE with enhanced potency for killing all CD19+ cell types, including tissue-resident memory B cells and plasmablasts. Figure 15E and Figure 15F The absolute cell counts of circulating CD3+CD4+ T helper cells and CD3+CD8+ CTLs are displayed at specified time points. Figure 15G and Figure 15HThe percentages of CD69+ activated T cells in the CD4 and CD8 T cell subsets are shown separately. L88-003 treatment was associated with transient fluctuations in both circulating CD4+ and CD8+ T cells. In contrast to the persistent decrease in B cells, T lymphocytes recovered rapidly after each infusion and reached or exceeded baseline levels before the next dose. L88-003 treatment was also associated with transient upregulation of CD69, a T cell activation marker, in the CD4+ and CD8+ T cell subsets. Therefore, L88-003 is capable of mediating T cell activation in vivo.

[0494] Figure 16 Relative body weight changes were depicted, monitored twice weekly and normalized to baseline (t=0). No significant weight loss was observed in the animals, and the LNP composition was well tolerated.

[0495] Figures 17A-17I The results of a non-clinical tolerability study in cynomolgus monkeys were described after three weekly doses of L88-003 (e.g., LNP-CD19 BiTEMRNA). Figure 17A It is a schematic diagram of a three-stage dose progression and blood collection at specified time points for clinical hematology, clinical chemistry, and cytokine analysis. Figures 17B-17D This displays the hematological analysis of whole blood CBCs evaluated at specified time points. The absolute counts of white blood cells (WBC), red blood cells (RBC), and platelets (PLT) are shown at [time points missing]. Figure 17B , Figure 17C and Figure 17D The dashed lines depict reference ranges and / or pre-drug readings. Figures 17E-17G The clinical chemistry analyses measured at specified time points are shown. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were at [missing data]. Figure 17E , Figure 17F and Figure 17G The figure is plotted in the middle. Only transient increases in liver enzymes were observed, and the LNP composition was well tolerated. Dashed lines depict reference ranges and / or pre-dose readings. Figure 17H and Figure 17I Plasma cytokine levels, including IL6, IL2, IL4, IFNγ, and TNFα, were quantified by multiplex Luminex assays. Only transient increases in cytokines were observed, and the LNP composition was well tolerated.

Claims

1. A pharmaceutical composition comprising: a) A synthetic nucleic acid molecule encoding a therapeutic peptide, wherein the therapeutic peptide is configured to bind a B-cell antigen; and b) Multiple lipid nanoparticles; The synthetic nucleic acid molecule is encapsulated within at least one of the plurality of lipid nanoparticles.

2. The pharmaceutical composition of claim 1, wherein the B cell antigen is selected from the group consisting of: memory B cell antigen, naive B cell antigen, plasmablast antigen or plasma cell antigen and any combination thereof.

3. The pharmaceutical composition according to claim 1, wherein the B cell antigen is selected from the group consisting of: differentiation cluster (CD)10, CD19, CD20, CD22, CD27, CD32b, CD38, CD40, B cell maturation antigen (BCMA), B cell activating factor receptor (BAFFR), CD138, CD5, and any combination thereof.

4. The pharmaceutical composition according to claim 1, wherein the B-cell antigen comprises CD19.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the therapeutic peptide is capable of binding the B cell antigen with a higher affinity than other antigens.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the therapeutic peptide binds to the second antigen.

7. The pharmaceutical composition according to claim 6, wherein the second antigen comprises an immune cell antigen or a tumor antigen.

8. The pharmaceutical composition according to claim 7, wherein the immune cell antigen comprises T cell antigen, Treg cell antigen, or natural killer cell (NK cell) antigen.

9. The pharmaceutical composition according to any one of claims 6-8, wherein the second antigen comprises CD3, and the therapeutic peptide is configured to bind CD3.

10. The pharmaceutical composition according to any one of claims 1-9, wherein the therapeutic peptide comprises an antibody or an antigen-binding fragment thereof.

11. The pharmaceutical composition of claim 10, wherein the antibody or its antigen-binding fragment comprises a bispecific antibody, a multispecific antibody, or a chimeric antigen receptor (CAR).

12. The pharmaceutical composition of claim 10, wherein the bispecific antibody or its antigen-binding fragment comprises a bispecific T-cell connector (BiTE).

13. The pharmaceutical composition of claim 12, wherein the BiTE is configured to bind CD19 and CD3.

14. The pharmaceutical composition according to any one of claims 1-13, wherein the therapeutic peptide comprises an FDA-approved drug, wherein the FDA-approved drug is configured to bind to a B-cell antigen.

15. The pharmaceutical composition of claim 14, wherein the FDA-approved pharmaceutical product includes belintolimab, inelolizumab, rontoxicumab, or tancituzumab.

16. The pharmaceutical composition according to any one of claims 1-15, wherein the synthetic nucleic acid molecule further comprises a regulatory nucleic acid sequence.

17. The pharmaceutical composition of claim 16, wherein the regulatory nucleic acid sequence comprises a promoter or a signal peptide.

18. The pharmaceutical composition of claim 17, wherein the promoter comprises a tissue-specific promoter.

19. The pharmaceutical composition according to any one of claims 1-18, wherein the synthetic nucleic acid molecule comprises a synthetic ribonucleic acid sequence (RNA).

20. The pharmaceutical composition of claim 19, wherein the synthetic RNA comprises a chemically modified nucleic acid, and wherein the synthetic RNA comprises improved stability.

21. The pharmaceutical composition according to claim 19 or 20, wherein the synthetic RNA comprises N6-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), or N4-acetylcytidine (ac4C), or any combination thereof.

22. The pharmaceutical composition according to any one of claims 19-21, wherein the synthetic RNA comprises a 5' cap.

23. The pharmaceutical composition according to any one of claims 19-22, wherein the synthetic RNA comprises, in the 5' to 3' orientation: a) 5' Untranslated Region (5'UTR) encoding area; b) Signal peptide coding region; c) Therapeutic peptide coding region; d) 3' Untranslated Region (3'UTR) encoding area; and e) Clustered A-tail coding area.

24. The pharmaceutical composition of claim 23, wherein the signal peptide coding region comprises the sequence of SEQ ID NO:

11.

25. The pharmaceutical composition of claim 23, wherein the synthetic RNA further comprises an adapter coding region.

26. The pharmaceutical composition of claim 25, wherein the connector coding region comprises the sequence of SEQ ID NO:12 or SEQ ID NO:

13.

27. The pharmaceutical composition according to any one of claims 23-26, wherein the therapeutic peptide coding region encodes a variable light (VL) chain by at least 90%, 95%, 98%, or 99% of a sequence identical to SEQ ID NO: 1, 3, 5, 7, or 9; and encodes a variable heavy (VH) chain by at least 90%, 95%, 98%, or 99% of a sequence identical to SEQ ID NO: 2, 4, 6, 8, or 10. The pharmaceutical composition according to claim 23, wherein the therapeutic peptide coding region encodes the VL and VH chains by the following sequences: 1) SEQ ID NO: 1 and 2; 2) SEQ ID NO: 3 and 4; 3) SEQ ID NO: 5 and 6; 4) SEQ ID NO: 7 and 8; or 5) SEQ ID NO: 9 and 10.

28. The pharmaceutical composition according to any one of claims 23-26, wherein the therapeutic peptide encoding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with a sequence selected from the group consisting of: 1) SEQ ID NO: 1 and 2; 2) SEQ ID NO: 5 and 6; or 3) SEQ ID NO: 9 and 10, wherein the nucleic acid sequence encodes an anti-CD19 antibody or an anti-CD19 binding fragment thereof.

29. The pharmaceutical composition according to any one of claims 23-28, wherein the therapeutic peptide encoding region comprises a nucleic acid sequence consisting of: 1) SEQ ID NO:3 and 4; or 2) SEQ ID NO:7 and 8, wherein the nucleic acid sequence encodes an anti-CD3 antibody or an anti-CD3 binding fragment thereof.

30. The pharmaceutical composition according to any one of claims 23-28, wherein the therapeutic peptide coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with the following: 1) SEQ ID NO: 3 and 4; or 2) SEQ ID NO: 7 and 8, wherein the nucleic acid sequence encodes an anti-CD3 antibody or an anti-CD3 binding fragment thereof.

31. The pharmaceutical composition of claim 23, wherein the synthetic RNA further encodes a protein tag.

32. The pharmaceutical composition of claim 31, wherein the protein tag includes a histidine tag, a flag tag, or a hemagglutinin tag.

33. The pharmaceutical composition according to any one of claims 23-32, wherein the therapeutic peptide coding region comprises, in a 5' to 3' orientation: a) Anti-CD19 light chain coding region; b) Anti-CD19 heavy chain coding region; c) Anti-CD3 heavy chain coding region; and d) Anti-CD3 light chain coding region.

34. The pharmaceutical composition of claim 33, wherein the anti-CD19 light chain coding region comprises the sequence of SEQ ID NO: 1, 3, 5 or 9.

35. The pharmaceutical composition of claim 33, wherein the anti-CD19 heavy chain coding region comprises the sequence of SEQ ID NO: 2, 4, 6 or 10.

36. The pharmaceutical composition of claim 33, wherein the anti-CD19 light chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 1, 3, 5, or 9.

37. The pharmaceutical composition of claim 33, wherein the anti-CD19 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 2, 4, 6, or 10.

38. The pharmaceutical composition according to any one of claims 33-37, wherein the anti-CD3 light chain coding region comprises the sequence of SEQ ID NO:

7.

39. The pharmaceutical composition according to any one of claims 33-37, wherein the anti-CD3 light chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:

7.

40. The pharmaceutical composition of claim 33, wherein the anti-CD3 heavy chain coding region comprises the sequence of SEQ ID NO:

8.

41. The pharmaceutical composition of claim 33, wherein the anti-CD3 heavy chain coding region comprises a nucleic acid sequence having at least 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO:

8.

42. The pharmaceutical composition according to any one of claims 34-41, wherein the anti-CD19 light chain coding region comprises SEQ ID NO:1 and the anti-CD19 heavy chain coding region comprises SEQ ID NO:2; wherein the anti-CD3 light chain coding region comprises SEQ ID NO:3 and the anti-CD3 heavy chain coding region comprises SEQ ID NO:

4.

43. The pharmaceutical composition according to any one of claims 34-41, wherein the anti-CD19 light chain coding region comprises SEQ ID NO:5 and the anti-CD19 heavy chain coding region comprises SEQ ID NO:6; wherein the anti-CD3 light chain coding region comprises SEQ ID NO:7 and the anti-CD3 heavy chain coding region comprises SEQ ID NO:

8.

44. The pharmaceutical composition according to any one of claims 34-41, wherein the anti-CD19 light chain coding region comprises SEQ ID NO:9 and the anti-CD19 heavy chain coding region comprises SEQ ID NO:10; wherein the anti-CD3 light chain coding region comprises SEQ ID NO:7 and the anti-CD3 heavy chain coding region comprises SEQ ID NO:

8.

45. The pharmaceutical composition of claim 33, wherein the anti-CD19 heavy chain coding region comprises a nucleotide sequence encoding a light chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.

46. ​​The pharmaceutical composition of claim 33, wherein the anti-CD19 heavy chain coding region comprises a nucleotide sequence encoding the heavy chain of belintolimumab, inelizumab, rontoxicumab, or tancituzumab.

47. The pharmaceutical composition according to any one of claims 19-46, wherein the synthetic RNA comprises single-stranded synthetic RNA.

48. The pharmaceutical composition according to any one of claims 1-47, wherein the lipid nanoparticles target tissues or organs when administered to a subject.

49. The pharmaceutical composition of claim 48, wherein the tissue or organ comprises the liver.

50. The pharmaceutical composition of claim 48, wherein the tissue or organ comprises a lymphatic organ.

51. The pharmaceutical composition of claim 50, wherein the lymphatic organ comprises bone marrow, spleen, or lymph node.

52. The pharmaceutical composition according to any one of claims 1-47, wherein the lipid nanoparticles target target cells when administered to a subject.

53. The pharmaceutical composition of claim 52, wherein the target cells include hepatocytes, lymphocytes, leukocytes, myeloid cells, or hematopoietic stem cells.

54. The pharmaceutical composition of claim 53, wherein the target cells comprise B cells, and wherein the B cells comprise plasmablasts, plasma cells, or memory B cells.

55. The pharmaceutical composition of claim 53, wherein the target cells comprise T cells.

56. The pharmaceutical composition according to any one of claims 52-55, wherein the target cells comprise cells in the systemic circulation of the subject.

57. The pharmaceutical composition according to any one of claims 52-55, wherein the target cells comprise cells within the tissues or organs of the subject.

58. The pharmaceutical composition according to any one of claims 1-57, wherein when administered to a subject, the lipid nanoparticles have a faster clearance rate compared to other lipid nanoparticles.

59. The pharmaceutical composition according to any one of claims 1-58, wherein, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition has a longer half-life compared to a corresponding therapeutic peptide contained in another pharmaceutical composition.

60. The pharmaceutical composition according to any one of claims 1-Error! No reference source found, when administered to a subject, wherein the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition has a duration of at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, Half-life of at least 12 hours, at least 16 hours, at least 18 hours, at least 24 hours, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 8.5 days, at least 9 days, at least 9.5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days or more.

61. The pharmaceutical composition according to any one of claims 1-60, wherein, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence has a larger area under the curve (AUC) compared to the corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

62. The pharmaceutical composition according to any one of claims 1-60, when administered to a subject, wherein the therapeutic peptide encoded by the synthetic nucleic acid sequence has an AUC at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more greater than that of a corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

63. The pharmaceutical composition according to any one of claims 1-62, when administered to a subject, results in prolonged B-cell depletion when the therapeutic peptide encoded by the synthetic nucleic acid sequence is compared with the corresponding therapeutic peptide of an equivalent dose of another pharmaceutical composition.

64. The pharmaceutical composition of claim 63, wherein the prolonged B-cell depletion comprises B-cell depletion for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 17.5 days, at least 20 days, at least 25 days, at least 27.5 days, at least 30 days, at least 35 days, at least 37.5 days, at least 40 days, at least 45 days, at least 47.5 days, at least 50 days, at least 55 days, at least 57.5 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, or at least 120 days or more.

65. The pharmaceutical composition according to any one of claims 1-64, when administered to a subject, wherein the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject.

66. The pharmaceutical composition according to any one of claims 1-64, when administered to a subject, wherein the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject.

67. The pharmaceutical composition according to any one of claims 1-64, when administered to a subject, wherein the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the initial B cells in the subject.

68. The pharmaceutical composition according to any one of claims 1-64, wherein, when administered to a subject, the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of mature B cells in the subject.

69. The pharmaceutical composition of claim 68, wherein the mature B cells comprise memory B cells, plasma cells, or plasmablasts.

70. The pharmaceutical composition according to any one of claims 1-69, when administered to a subject, wherein the therapeutic peptide encoded by the synthetic nucleic acid sequence of the pharmaceutical composition results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B-cell depletion compared to an equivalent dose of a corresponding therapeutic peptide of another pharmaceutical composition.

71. The pharmaceutical composition according to any one of claims 1-70, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

72. The pharmaceutical composition of claim 71, wherein the pharmaceutically acceptable excipient comprises a solution suitable for injection into a subject.

73. The pharmaceutical composition according to any one of claims 1-72, wherein the pharmaceutical composition further comprises a small molecule drug.

74. The pharmaceutical composition of claim 73, wherein the small molecule drug comprises an agent for chemotherapy.

75. The pharmaceutical composition according to any one of claims 1-74, wherein the subject comprises a mammal.

76. The pharmaceutical composition of claim 75, wherein the mammal includes humans, non-human primates, or rodents.

77. The pharmaceutical composition according to any one of claims 1-76, wherein the pharmaceutical composition has zero to minimal toxicity when administered to a subject.

78. The pharmaceutical composition of claim 77, wherein the toxicity comprises a transient increase in cytokines or liver enzymes.

79. The pharmaceutical composition of claim 77, wherein the toxicity includes mild inflammation or mild hepatotoxicity.

80. The pharmaceutical composition according to any one of claims 1-76, wherein, after administration to the subject, the pharmaceutical composition results in the activation of CD69+ T cells.

81. The pharmaceutical composition according to any one of claims 1-80, wherein the lipid nanoparticles comprise a lipid composition; The lipid composition thereof comprises an ionizable lipid or a pharmaceutically acceptable salt thereof; The ionizable lipid comprises an amine head group and at least one hydrophobic tail R having the following structure 脂质 : Or its pharmaceutically acceptable salt; wherein R k1 Independently constitutes a C1-C12 divalent aliphatic or heteroaliphatic group; R k3 It is independently a C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3-C12 heterocycloalkyl, aryl or heteroaryl; R k2 It is independently C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl or heteroaryl; R k4 and R k5 Each is independently an H or C1-C12 divalent aliphatic group; and M is O or NR k6 , where R k6 It is an H or C1-C12 aliphatic group.

82. The pharmaceutical composition according to any one of claims 1-81, wherein the amine head group is represented by the following: Wherein Ra, Ra', Ra” and Ra”' are each independently H, C1-20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, or R 脂质 Furthermore, Z is a C1-C20 divalent aliphatic group, a C1-C20 divalent heteroaliphatic group, a divalent aryl group, or a divalent heteroaryl group.

83. The pharmaceutical composition according to claim 81 or 82, wherein the ionizable lipid is represented by formula (II): Or its pharmaceutically acceptable salt, wherein: R b Alkyl groups, whether substituted or unsubstituted; n1 and n2 are each independently 1, 2, 3, 4, 5, or 6; and R b1 R b2 R b3 and R b4 Each independently is H or R 脂质 , Where R b1 R b2 R b3 and R b4 At least one of them is not H.

84. The pharmaceutical composition according to claim 81 or 82, wherein the amine head group is selected from the group consisting of:

85. The pharmaceutical composition according to any one of claims 81-84, wherein the at least one hydrophobic tail comprises 86. The pharmaceutical composition according to any one of claims 81-85, wherein the ionizable lipid comprises 87. The pharmaceutical composition according to any one of claims 81-86, wherein the lipid composition further comprises a steroid.

88. The pharmaceutical composition of claim 87, wherein the steroid comprises cholesterol or a cholesterol derivative.

89. The pharmaceutical composition according to any one of claims 1-88, wherein the lipid composition further comprises an auxiliary lipid.

90. The pharmaceutical composition of claim 89, wherein the auxiliary lipid comprises a phospholipid or a zwitterionic lipid, including 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC).

91. The pharmaceutical composition according to any one of claims 1-900, wherein the lipid composition further comprises polymer-conjugated lipids.

92. The pharmaceutical composition of claim 91, wherein the polymer-conjugated lipid comprises polyethylene glycol (PEG)-conjugated lipid.

93. The pharmaceutical composition of claim 91, wherein the polymer-conjugated lipid comprises 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k)] or 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2k).

94. The pharmaceutical composition according to any one of claims 1-93, wherein the lipid composition further comprises steroids, auxiliary lipids, and polymer-conjugated lipids.

95. The pharmaceutical composition according to any one of claims 1-94, wherein the ionizable lipid is present in the lipid composition in a weight percentage of about 30% to about 90%.

96. The pharmaceutical composition according to any one of claims 1-95, wherein the steroid is present in the lipid composition in a weight percentage of about 10% to about 40%.

97. The pharmaceutical composition according to any one of claims 89-96, wherein the auxiliary lipid is present in the lipid composition at a weight percentage of about 1% to about 20%.

98. The pharmaceutical composition according to any one of claims 91-97, wherein the polymer-conjugated lipid is present in the lipid composition at a weight percentage of about 1% to about 20%.

99. The pharmaceutical composition according to any one of claims 94-98, wherein the weight ratio of the ionizable lipid / steroid / auxiliary lipid / polymer conjugated lipid is about 16 / 4 / 1 / 1.

100. The pharmaceutical composition according to any one of claims 14-98, wherein the weight ratio of the pharmaceutical formulation / lipid composition is from about 1:200 to about 1:

5.

101. The pharmaceutical composition according to any one of claims 1-91, wherein the lipid composition further comprises a steroid and an auxiliary lipid.

102. The pharmaceutical composition of claim 101, wherein the ionizable lipid is present in the lipid composition in a weight percentage of about 30% to about 90%.

103. The pharmaceutical composition according to claim 101 or 102, wherein the auxiliary lipid is present in the lipid composition at a weight percentage of about 5% to about 40%.

104. The pharmaceutical composition according to any one of claims 101-103, wherein the steroid is present in the lipid composition in a weight percentage of about 5% to about 40%.

105. The pharmaceutical composition according to any one of claims 101-104, wherein the weight ratio of the ionizable lipid / steroid / auxiliary lipid is about 2 / 1 / 1.

106. The pharmaceutical composition according to any one of claims 1-105, wherein the lipid composition further comprises a pharmaceutically acceptable carrier.

107. The pharmaceutical composition of claim 106, wherein the pharmaceutically acceptable carrier comprises a sugar, and the sugar comprises mannitol, sucrose, maltose, or trehalose.

108. The pharmaceutical composition according to claims 106-107, wherein the carrier is present in the composition at a weight percentage of about 5% to about 60%.

109. The pharmaceutical composition according to any one of claims 1-108, wherein the ionizable lipid comprises at least two hydrophobic tails, wherein not all of the hydrophobic tails are identical.

110. The pharmaceutical composition according to any one of claims 1-108, wherein the ionizable lipid comprises at least two hydrophobic tails, wherein the two or more hydrophobic tails are identical.

111. The pharmaceutical composition according to any one of claims 1-110, wherein the pharmaceutical composition further comprises polynucleotides, oligonucleotides, polypeptides, oligopeptides, small molecule compounds, or any combination thereof.

112. The pharmaceutical composition of claim 111, wherein the small molecule compound comprises a drug for chemotherapy.

113. The pharmaceutical composition according to any one of claims 1-112, wherein the physical properties of the lipid composition are more stable compared to other lipid compositions.

114. The pharmaceutical composition according to any one of claims 1-113, wherein the physical properties of the lipid composition are stable for at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months or longer when stored at 4°C, -20°C, or -80°C.

115. The pharmaceutical composition according to any one of claims 1-113, wherein the physical properties of the lipid composition are stable for at least 5 months when stored at 4°C, -20°C, or -80°C.

116. The pharmaceutical composition according to any one of claims 113-115, wherein the physical properties of the lipid composition include the size, polydispersity index (PDI), encapsulation efficiency (EE%), or pKa of the lipid composition.

117. A method for depleting B cells, comprising administering a pharmaceutical composition according to any one of claims 1-116 to a subject in need, wherein the subject in need suffers from a disease requiring B cell depletion therapy (BCDT).

118. The method of claim 117, wherein the disease requiring B-cell depletion therapy (BCDT) includes B-cell malignancies or autoimmune diseases.

119. The method of claim 118, wherein the B-cell malignancy comprises B-cell lymphoma, wherein the B-cell lymphoma comprises diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Wald's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma).

120. The method of claim 119, wherein the B-cell lymphoma affects the spleen or lymph nodes.

121. The method of claim 118, wherein the B-cell malignancy comprises multiple myeloma.

122. The method of claim 118, wherein the autoimmune disease includes allergic diseases, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), anti-myelin oligodendrocyte glycoprotein (anti-MOG) spectrum diseases, neuromyelitis optica spectrum diseases (NMOSD), anti-NMDAR encephalitis, or myasthenia gravis.

123. The method of claim 118, wherein the disease requiring B-cell depletion therapy (BCDT) further includes pemphigus vulgaris or Sjögren's syndrome.

124. A method for treating hematologic malignancies, comprising administering a pharmaceutical composition according to any one of claims 1-115 to a subject in need, wherein the subject in need suffers from a hematologic malignancy.

125. The method of claim 124, wherein the hematologic malignancy comprises B-cell lymphoma, wherein the B-cell lymphoma comprises diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, or Burkitt lymphoma.

126. The method according to any one of claims 116-125, wherein the application comprises topical application, oral application, or injection application.

127. The method according to any one of claims 116-125, wherein the administration comprises administration by intravenous injection or administration by intramuscular injection.

128. The method according to any one of claims 116-127, wherein the method results in the expression of the therapeutic peptide having a longer half-life compared to any other method comprising administering an equivalent dose of the therapeutic peptide.

129. The method according to any one of claims 116-128, wherein the method results in the expression of a therapeutic peptide having the following half-lives: at least 2 hours, at least 2.5 hours, at least 3 hours, at least 3.5 hours, at least 4 hours, at least 4.5 hours, at least 5 hours, at least 5.5 hours, at least 6 hours, at least 6.5 hours, at least 7 hours, at least 7.5 hours, at least 8 hours, at least 8.5 hours, at least 9 hours, at least 9.5 hours, at least 10 hours, to Less than 12 hours, at least 16 hours, at least 18 hours, at least 24 hours, at least 1.5 days, at least 2 days, at least 2.5 days, at least 3 days, at least 3.5 days, at least 4 days, at least 4.5 days, at least 5 days, at least 5.5 days, at least 6 days, at least 6.5 days, at least 7 days, at least 7.5 days, at least 8 days, at least 8.5 days, at least 9 days, at least 9.5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or at least 15 days or more.

130. The method according to any one of claims 116-129, wherein the method results in the expression of a therapeutic peptide with a larger area under the curve (AUC) compared to other methods.

131. The method according to any one of claims 116-130, wherein the method, compared with any other method comprising administering an equivalent dose of the therapeutic peptide, results in the expression of the therapeutic peptide having an AUC of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% or more.

132. The method according to any one of claims 116-131, wherein the method results in prolonged B-cell depletion compared to any other method comprising administering an equivalent dose of the therapeutic peptide.

133. The method of claim 132, wherein the prolonged B-cell exhaustion comprises B-cell exhaustion for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 17.5 days, at least 20 days, at least 25 days, at least 27.5 days, at least 30 days, at least 35 days, at least 37.5 days, at least 40 days, at least 45 days, at least 47.5 days, at least 50 days, at least 55 days, at least 57.5 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, or at least 120 days or more.

134. The method of any one of claims 116-133, wherein the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of circulating B cells in the subject.

135. The method of any one of claims 116-133, wherein the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of tissue B cells in the subject.

136. The method of any one of claims 116-135, wherein the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of the initial B cells in the subject.

137. The method of any one of claims 116-135, wherein the method results in the depletion of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more of mature B cells in the subject.

138. The method of claim 137, wherein the mature B cell comprises memory B cells, plasma cells, or plasmablasts.

139. The method of any one of claims 116-138, wherein the method results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200% or more of B-cell depletion compared to any other method comprising administering an equivalent dose of the therapeutic peptide.

140. The method according to any one of claims 116-139, wherein the method further comprises administering a small molecule drug.

141. The method of claim 140, wherein the small molecule drug comprises an agent for chemotherapy.

142. The method according to any one of claims 116-141, wherein the subject in need comprises a mammal.

143. The method of claim 142, wherein the mammal includes humans, non-human primates, or rodents.

144. The method according to any one of claims 116-143, wherein the pharmaceutical preparation is administered in one or more doses.

145. The method according to any one of claims 116-144, wherein the pharmaceutical preparation is administered at a dose not exceeding 3 mg / kg (mg nucleic acid per kg body weight).

146. The method of claim 145, wherein the pharmaceutical preparation is administered at a dose of about 0.007 mg / kg to about 0.2 mg / kg (mg nucleic acid per kg body weight).

147. The method of claim 146, wherein the pharmaceutical preparation is administered at a dose of about 0.014 mg / kg to about 0.1 mg / kg (mg nucleic acid per kg body weight).

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