WO2025006613A1 - Nanoparticules lipidiques pour immunothérapie anticancéreuse - Google Patents

Nanoparticules lipidiques pour immunothérapie anticancéreuse Download PDF

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WO2025006613A1
WO2025006613A1 PCT/US2024/035626 US2024035626W WO2025006613A1 WO 2025006613 A1 WO2025006613 A1 WO 2025006613A1 US 2024035626 W US2024035626 W US 2024035626W WO 2025006613 A1 WO2025006613 A1 WO 2025006613A1
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composition
lipid
pharmaceutical agent
cell
independently
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Kate ZHANG
Haishan LI
Xin KAI
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Hopewell Therapeutics Inc
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Hopewell Therapeutics Inc
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    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
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    • A61K9/5123—Organic compounds, e.g. fats, sugars
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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Definitions

  • Synthetic mRNA provides a template for the synthesis of any given peptide, protein or protein fragment and lends itself to a broad range of pharmaceutical applications, including different modalities of cancer immunotherapy.
  • mRNA requires safe, effective, and stable delivery systems that protect the nucleic acid from degradation and that allow cellular uptake and mRNA release.
  • Lipid nanoparticle-mRNA formulations have been developed and are under clinical evaluation for the prevention and treatment of viral infections, cancer, and genetic diseases.
  • compositions and methods related to delivering nucleic acid molecules e.g., mRNA
  • nucleic acid molecules e.g., mRNA
  • BiTE bispecific T-cell engagers
  • PLC3 glypican-3
  • the present disclosure provides a composition comprising a pharmaceutical agent assembled with a lipid composition, wherein the lipid composition comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least two hydrophobic tail groups R Lipid , wherein each R L1 P ld independently comprises a structure selected from the group consisting of or a pharmaceutically acceptable salt thereof, wherein: Rki and Rk3 are each independently Cl -CIO alkyl; and each R k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; wherein the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE).
  • the pharmaceutical agent comprises one or more
  • the present disclosure provides a composition comprising a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least two hydrophobic tail groups R Lipid , wherein each R L1 P ld independently comprise a structure of Formula (I/a): or a pharmaceutically acceptable salt thereof; wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl; and each R k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; wherein the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTEise each Rk2 is independently a C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl. In some embodiments, each Rk2 is independently a C1-C20 alkyl or C2-C20 alkenyl.
  • each Rki is independently C1-C4 alkyl and each Rk3 is independently C1-C4 alkyl.
  • the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein:
  • R b is a substituted or unsubstituted Cl -CIO alkyl; nl and n2 are each independently 1, 2, 3, 4, 5, or 6; and
  • R bl , R b2 , R b3 and R b4 are each independently H or R Lipid ; wherein at least one of R bl , R b2 , R b3 and R b4 is not H.
  • R b is a C1-C4 alkyl which is optionally substituted with 1 to 4 groups selected from -OH; and nl and n2 are each independently 1, 2, 3, or 4.
  • each of R bl , R b2 , R b3 and R b4 is not H.
  • the amine head group is selected from the group consisting of
  • the ionizable lipid comprises L702 some embodiments, the ionizable lipid comprises
  • the lipid composition further comprises a steroid.
  • the steroid comprises cholesterol or a cholesterol derivative.
  • the lipid composition further comprises a helper lipid.
  • the helper lipid comprises phospholipids or zwitterionic lipids comprising l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
  • DOPE dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • the lipid composition further comprises a polymer conjugated lipid.
  • the polymer conjugated lipid comprises a polyethylene glycol (PEG) conjugated lipid.
  • the polymer conjugated lipid comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000 (DSPE-PEG2k) or 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k).
  • the lipid composition further comprises a steroid, a helper lipid, and a polymer conjugated lipid.
  • the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.
  • the steroid is present in the lipid composition at a weight percentage from about 10% to about 40%.
  • the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.
  • the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 20%.
  • the weight ratio of the ionizable lipid/steroid/helper lipid/polymer conjugated lipid is about 4/1/I/1.
  • the weight ratio of the pharmaceutical agent/lipid composition is from about 1 :200 to about 1 :5.
  • the lipid composition further comprises a steroid and a helper lipid.
  • the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.
  • the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%.
  • the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%.
  • the weight ratio of the ionizable lipid/steroid/helper lipid is about 2/1/1.
  • the lipid composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier comprises a sugar, and wherein the sugar comprises mannitol, sucrose, maltose, or trehalose.
  • the carrier is present in the composition at a weight percentage from about 5% to about 60%.
  • the ionizable lipid comprises at least two hydrophobic tails, wherein not all hydrophobic tails are identical.
  • the ionizable lipid comprises at least two hydrophobic tails, wherein two or more hydrophobic tails are identical.
  • the pharmaceutical agent comprises one or more mRNA molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE) or optionally the pharmaceutical agent comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1, or optionally the pharmaceutical agent comprises SEQ ID NO: 1.
  • the pharmaceutical agent further comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof.
  • the small molecule compound comprises a drug for chemotherapy.
  • 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 more 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.
  • the physical properties of the lipid composition comprise size, poly dispersity index (PDI), encapsulation efficacy (EE%), or pKa of the lipid composition.
  • the lipid composition targets a target organ after being administered to a subject.
  • the target organ comprises a liver, lung or spleen.
  • the composition targets a target cell after being administered to a subject.
  • the target cell comprises a hepatocyte, a Kupffer cell, a liver sinusoidal endothelial cell (LSEC), a B cell, a T cell, a dendritic cell, a macrophage, a myeloid cell, a hematopoietic stem cells, or a leukocyte.
  • the composition after being administered to a subj ect the composition has a faster rate of clearance compared to other compositions.
  • the composition after being administered to a subject the composition has a longer half-life compared to other compositions.
  • after being administered to a subject the composition has a half-life of at least 2 hours, at least 3 hours, at least 4 hours, or at least 5 hours.
  • the composition after the composition is administered to a subject the composition has a larger area under curve (AUC) compared to other compositions. In some embodiments, after the composition is administered to a subject the composition has an AUC of that is at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60% or larger compared to other compositions.
  • AUC area under curve
  • the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering an effective amount of the composition provided herein.
  • the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising: administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition provided herein; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to that achieved absent the lipid composition.
  • the present disclosure provides a method for delivering a pharmaceutical agent to a target organ in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical agent assembled with a lipid composition provided herein; and thereby providing a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to a non-target organ.
  • the administering is through systemic administration. In some embodiments, the administering is through intramuscular administration. In some embodiments, the administering is through intravenous administration. In some embodiments, the pharmaceutical agent is delivered at a dosage of no more than 3 mg/kg body weight. In some embodiments, the pharmaceutical agent is delivered at a dosage from about 0.25 mg/kg to about 1 mg/kg body weight. In some embodiments, the pharmaceutical agent is delivered in one or more doses. In some embodiments, the subject is a mammal, and wherein the mammal comprises a rodent or a nonhuman primate. In some embodiments, the activity of the pharmaceutical agent comprises cancer cell killing.
  • he amount of the pharmaceutical agent is measured by pharmacokinetic parameters, and wherein the pharmacokinetic parameters comprises Cmax, Tmax, half-life, or area under curve (AUC).
  • the greater amount of the pharmaceutical agent comprises a larger Cmax, a longer half-life, or a larger area under curve (AUC).
  • the method results in greater cancer cell killing.
  • the method results in greater the pharmacokinetic parameters of the pharmaceutical agent, and wherein the pharmacokinetic parameters of the pharmaceutical agent comprise Cmax, Tmax, half-life, or area under curve (AUC).
  • the half-life of the pharmaceutical agent is at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours or more.
  • the target organ comprises a liver, lung or spleen.
  • the present disclosure provides a method for treating a solid-state tumor in a subject in need thereof, comprising administering an effective amount of the composition provided herein.
  • the solid-state tumor comprises hepatocellular carcinoma.
  • the solid-state tumor comprises lung cancer.
  • the lung cancer comprises non-small cell lung cancer.
  • the administering comprises administering the composition intravenously or intramuscularly to a subject in need thereof.
  • the subject comprises a mammal.
  • FIG. 1 is a schematic representation of the constructs of glypican-3 (GPC3)/CD3 encoding mRNA.
  • FIG. 2 depicts a schematic diagram of dose regiments and blood collection at indicated timepoints for PK profiling of plasma GPC3/CD3 BiTE protein, BiTE mRNA and ionizable lipid.
  • FIGs. 3A-3B depicts protein PK profiling of single dose intravenous (i.v.) administration of LNP-GPC3/CD3 BiTE mRNA in CD1 mice.
  • FIG. 3A shows GPC3-CD3 BiTE protein concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg LNP- GPC3/CD3 BiTE mRNA (L88-008), and 0.25 mg/kg of recombinant GPC3/CD3 BiTE protein (rPR008).
  • FIG. 3B shows ex vivo concentration- dependent cytotoxicity against GPC3+ HepG2-luc cells induced by BiTE protein produced in vivo, which was collected at 48h post-dose of 0.5 mg/kg L88-008, were measured by luminescence-based assay.
  • hPBMCs were used as effectors at effector target (E:T) cell ratio of 5: 1 and detect luminescence single of target cell following 24h incubation.
  • % of specific lysis were calculated as ([number of live target cells without treatment - number of live target cells with treatment] / [number of live target cells without treatment] x 100%.
  • FIG. 4 depicts mRNAPK profiling of single dose intravenous (i.v.) administration of LNP- GPC3/CD3 BiTE mRNA in CD1 mice.
  • GPC3-CD3 BiTE mRNA concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg L88-008 were quantified by bDNA assay.
  • Data are presented as means ⁇ SD and dotted line depicts the LLOQ of 1 pg/ml.
  • FIG. 5 depicts ionizable lipid L88 PK profiling of single dose intravenous (i.v.) administration of LNP-GPC3/CD3 BiTE mRNA in CD1 mice.
  • L88 lipid concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg L88-008 were quantified by LC-MS.
  • Data are presented as means ⁇ SD and dotted line depicts the LLOQ of 1 ng/ml.
  • FIG. 6 is a schematic diagram of an efficacy study design.
  • FIGs 7A-7E depicts the results of the efficacy study. Efficacy study of L88-008 was performed in in hPBMC-reconstituted Hep3B-Luciferase orthotopic hepatocellular carcinomas (HCC) mouse model.
  • FIG. 1 Hep3B-Luciferase orthotopic hepatocellular carcinomas
  • FIG. 7A shows the total photon flux images of individual mice in each group captured at indicated timepoints by IVIS.
  • FIG. 7B shows the bioluminescence signal quantitated as photons/sec using living Image 4.7 software for each treatment group. Each dot represent mean bioluminescence signal of each group and statistical analysis were compared to G1 PBS control group by 2-way ANOVA test with multiple comparisons analysis at D38 after tumor inoculation. **** p ⁇ o 0001.
  • FIGs 7C-7E show total flux (TF) changes of each mouse in individual groups during the treatment were analysis.
  • the statistical analysis was compared to G1_PBS control group by 2-way ANOVA test with multiple comparisons analysis at D38 after tumor inoculation. *** p ⁇ 0.001.
  • FIGs. 9A-9C show the liver and spleen tissue images and weight at end-timepoint (D38 after tumor inoculation).
  • FIG. 9A depicts the terminal liver and spleen tissues from each mouse collected.
  • FIG. 9B and FIG. 9C show liver including tumor tissue and spleen weighted at endtimepoint.
  • Each dots represent 1 mouse data and statistical analysis were compared to G1 PBS control by paired two-tailed t-test. ns, not significant; * P ⁇ 0.05, ** P ⁇ 0.01.
  • FIG. 10 is a bar graph of GPC3-CD3 BiTE protein expression in lung cancer cell lines.
  • LNPs Lipid nanoparticles
  • the phospholipid and sterol work together to stabilize the LNP, the lipid-anchored PEG provides vial and storage stability and the ionizable lipid is critical for cellular uptake and endosomal escape, allowing for release of the mRNA into the cytosol.
  • the efficacy and tolerability of the formulation can be altered.
  • mRNA/LNP formulation requires careful process controls to ensure reproducibility of manufacturing and stability.
  • full-length antibodies have engendered significant success targeting cancers, immune disorders and infectious diseases, they are relatively large and exhibit less tissue penetrance than small molecules.
  • therapeutic peptides derived from fragments of antibodies such as bispecific T-cell engagers (e.g., BiTE)
  • BiTE bispecific T-cell engagers
  • the use of these molecules has been hampered by their short halflives of several hours and propensity to aggregate. Both properties could be improved or avoided through mRNA-mediated expression since antibody will be continuously expressed from the mRNA until the mRNA is degraded. This approach would also prevent the aggregation often observed during protein purification.
  • LNP formulations that encapsulate mRNA for delivery can offer advantages for this technology over recombinant proteins as LNPs can offer greater biodistribution, with more tissue penetration than recombinant antibody proteins.
  • LNPs optimized for mRNA delivery can result in higher expression of therapeutic peptides via more convenient routes of administration, such as subcutaneous or IM.
  • the present disclosure relates to compositions and methods comprising LNPs assembled with an mRNA encoding GPC3-CD3 BiTE.
  • Glypican-3 (GPC3) is an oncofetal heparan sulfate (HS) glycoprotein attached to the cell membrane via a glycosylphos-phatidylinositol (GPI) anchors.
  • HS oncofetal heparan sulfate
  • GPC3 is highly expressed in a wide variety of tissues during embryonic development; however, its expression is hardly detectable in normal adult tissues at both mRNA and protein level. Recent discoveries have shown that GPC3 is highly expressed in a certain tumor tissue.
  • GPC3 has been studied as a molecular marker for the detection and treatment of hepatocellular carcinoma (HCC), a malignant tumor with a fairly poor prognosis (5-year survival of less than 50%). Elevated protein expression of GPC3 has also been reported in lung cancer especially in squamous cell carcinomas by IHC. The overall high protein expression of GPC3 is 45% (21/60) in lung cancer, positive expression of GPC3 protein in lung squamous cell carcinoma is higher than in lung adenocarcinoma (70% vs 20%).
  • GPC3-CD3 BiTE or mRNA encoding GPC3-CD3 BiTE may be an option for treating HCC or various forms of lung cancer.
  • the present disclosure provides compositions and methods related to lipid nanoparticles (LNPs) comprising ionizable lipids and nucleic acid molecules encoding therapeutic peptides for cancer immunotherapy.
  • the LNPs comprise mRNA that encode an antibody or a fragment thereof.
  • the LNPs comprise mRNA that encode a bispecific T-cell engager (BiTE).
  • the composition provided herein comprises LNPs encapsulating an mRNA encoding GPC3-CD3 BiTE.
  • the mRNA encoding the GPC3-CD3 BiTE is codon-optimized to increase expression in a subject.
  • the subject comprises a mammal.
  • the subject comprises a rodent, human or a non-human primate.
  • the mRNA encoding the GPC3- CD3 BiTE comprises the nucleic acid sequence of SEQ ID NO: 1.
  • the LNPs of the composition may deliver the mRNA payload specially to one or more organs (e.g., organ targeting LNPs).
  • the LNPs are liver-targeting LNPs and the LNPs encapsulating the GPC3-CD3 BiTE mRNA can be delivered preferentially to the liver.
  • the LNPs are lung-targeting LNPs and the LNPs encapsulating the GPC3- CD3 BiTE mRNA can be delivered preferentially to the lung.
  • the LNPs are lymphoid tissue-targeting LNPs and the LNPs encapsulating the GPC3-CD3 BiTE mRNA can be delivered preferentially to the lymphoid tissues.
  • the lipid compositions provided herein result in higher expression of therapeutic peptides (e.g., GPC3-CD3 BiTE) than other lipid compositions.
  • the lipid compositions have a longer half-life than other compositions after administered to a subject in need thereof.
  • the lipid compositions have a longer half-life than chimeric BiTE proteins.
  • the methods provided herein relates to enhanced exposure of BiTE in a subject in need thereof due to administration of the lipid composition comprising mRNA encoding the BiTE.
  • the methods comprise administering the lipid compositions via convenient routes of administration, such as subcutaneous or IM.
  • cleavage sequence means “at least a first cleavage sequence” but includes a plurality of cleavage sequences.
  • polypeptide “peptide”, and “protein” are used interchangeably herein to generally refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • the term “antibody” refers to an immunoglobulin (Ig) whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen-binding domain.
  • the term further includes “antigen-binding fragments” or “functional fragment thereof, or “fragment of an antibody”, “antibody fragment”, “functional fragment of an antibody” and other interchangeable terms for similar binding fragments such as described below.
  • An antibody includes, for example, monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, recombinant antibodies, chemically engineered antibodies, deimmunized antibodies, affinity- matured antibodies, multispecific antibodies (for example, bispecific antibodies and polyreactive antibodies), heteroconjugate antibodies, antibody fragments, and combinations thereof (e.g., a monoclonal antibody that is also deimmunized, a humanized antibody that is also deimmunized, etc.).
  • An antibody can be, for example, murine, chimeric, humanized, heteroconjugate, bispecific, diabody, triabody, or tetrabody.
  • the antigen binding fragment can include, for example, Fab’, F(ab’)2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, or nanobody.
  • the term “antigen” refers to a molecule bound by an antibody or a fragment thereof.
  • the antigen can be referred to as a “ligand” of the antibody.
  • An antigen can be derived from a surface protein of a cell, such as an immune cell or a cancer cell.
  • an antigen can be derived from a surface protein of a tumor cell.
  • An antigen can be targeted by the antibody for killing of a cell.
  • the terms “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms generally refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms or improvement in one or more clinical parameters associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • a “therapeutic effect” or “therapeutic benefit,” as used herein, generally refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease or an improvement in one or more clinical parameters associated with the underlying disorder in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a polypeptide of the disclosure other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein.
  • compositions may be administered to a subject at risk of developing a particular disease, a recurrence of a former disease, condition or symptom of the disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • terapéuticaally effective amount and “therapeutically effective dose”, as used herein, generally refer to an amount of a drug or a biologically active protein, either alone or as a part of a polypeptide composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the number of carbon atoms in the group or class is as indicated as follows: “Cn” defines the exact number (n) of carbon atoms in the group/class. “C ⁇ n” defines the maximum number (n) of carbon atoms that can be in the group/class, with the minimum number as small as possible for the group/class in question, e.g., it is understood that the minimum number of carbon atoms in the group “alkenyl(C ⁇ 8)” or the class “alkene(C ⁇ 8)” is two. Compare with “alkoxy(C ⁇ 10)”, which designates alkoxy groups having from 1 to 10 carbon atoms.
  • Cm-n or “Cm-Cn” defines both the minimum (m) and maximum number (n) of carbon atoms in the group.
  • Cl -CIO alkyl designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning.
  • C5 olefin “C5-olefin”, “olefines)”, and “olefines” are all synonymous.
  • saturated when used to modify a compound or chemical group means the compound or chemical group has no carbon-carbon double and no carbon-carbon triple bonds, except as noted below.
  • the term when used to modify an atom, it means that the atom is not part of any double or triple bond.
  • substituted versions of saturated groups one or more carbon oxygen double bond or a carbon nitrogen double bond may be present. And when such a bond is present, then carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine/enamine tautomerism are not precluded.
  • saturated when used to modify a solution of a substance, it means that no more of that substance can dissolve in that solution.
  • aliphatic generally signifies that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group.
  • the carbon atoms can be joined together in straight chains, branched chains, or non-aromatic rings (alicyclic).
  • Aliphatic compounds/groups can be saturated, that is joined by single carbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkenes/ alkenyl) or with one or more carbon-carbon triple bonds (alkynes/alkynyl).
  • aromatic when used to modify a compound or a chemical group atom means the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by an interaction of the bonds forming the ring.
  • alkyl when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen.
  • the groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (zz-Pr or propyl), -CH(CH3)2 (z-Pr, 'Pr or isopropyl), -CH2CH2CH2CH3 (zz-Bu), -CH(CH3)CH2CH3 ( ec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl, /-butyl, Z-Bu or 'Bu), and -CH2C(CH3)3 (zzeo-pentyl) are non-limiting examples of alkyl groups.
  • alkanediyl when used without the “substituted” modifier refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
  • the groups -CEfe- (methylene), -CH2CH2-, -CEEC CEE ⁇ CEE-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups.
  • An “alkane” refers to the class of compounds having the formula H-R, wherein R is alkyl as this term is defined above.
  • haloalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e.
  • -F, -Cl, -Br, or -I such that no other atoms aside from carbon, hydrogen and halogen are present.
  • the group, -CH2CI is a nonlimiting example of a haloalkyl.
  • fluoroalkyl is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present.
  • the groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.
  • cycloalkyl when used without the “substituted” modifier refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, the carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
  • Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy).
  • cycloalkanediyl when used without the “substituted” modifier refers to a divalent saturated aliphatic group with two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen.
  • the group is a non-limiting example of cycloalkanediyl group.
  • a “cycloalkane” refers to the class of compounds having the formula H-R, wherein R is cycloalkyl as this term is defined above.
  • alkenyl when used without the “substituted” modifier refers to an monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a linear or branched, acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
  • alkenediyl when used without the “substituted” modifier refers to a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, a linear or branched acyclic structure, at least one nonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen.
  • alkenediyl group is aliphatic, once connected at both ends, this group is not precluded from forming part of an aromatic structure.
  • alkene and olefin are synonymous and refer to the class of compounds having the formula H-R, wherein R is alkenyl as this term is defined above.
  • terminal alkene and a-olefin are synonymous and refer to an alkene having just one carbon-carbon double bond, wherein that bond is part of a vinyl group at an end of the molecule.
  • alkynyl when used without the “substituted” modifier refers to a monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, 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 preclude the presence of one or more nonaromatic carbon-carbon double bonds.
  • An “alkyne” refers to the class of compounds having the formula H-R, wherein R is alkynyl.
  • one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH 3 , -NHCH3, -NHCH2CH3, -N(CH 3 )2, -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 )2, -OC(O)CH3, -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 .
  • aryl when used without the “substituted” modifier refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the point of attachment, the carbon atom forming part of a one or more six-membered aromatic ring structure, wherein the ring atoms are all carbon, and wherein the group consists of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present.
  • Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and a monovalent group derived from biphenyl.
  • the term “arenediyl” when used without the “substituted” modifier refers to a divalent aromatic group with two aromatic carbon atoms as points of attachment, the carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein the ring atoms are all carbon, and wherein the monovalent group consists of no atoms other than carbon and hydrogen.
  • the term does not preclude the presence of one or more alkyl, aryl or aralkyl groups (carbon number limitation permitting) attached to the first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting).
  • arenediyl groups include:
  • aralkyl when used without the “substituted” modifier refers to the monovalent group -alkanediyl-aryl, in which the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above.
  • Non-limiting examples are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl.
  • aralkyl When the term aralkyl is used with the “substituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl group has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH 3 , -NHCH3, -NHCH2CH3, -N(CH 3 )2, -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -0C(0)CH3, -NHC(0)CH3, -S(O) 2 OH or -S(O) 2 NH 2 .
  • substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phen
  • hetero when used to modify a compound or chemical group means the compound or chemical group has at least an atom that is not carbon, for example, N, O, S, Se, P, Si, B, or any other heteroatom.
  • 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 heterocycle can be any ring containing a ring atom that is not carbon.
  • a heterocycle can be substituted with any number of substituents, for example, alkyl groups and halogen atoms.
  • a heterocycle can be aromatic (heteroaryl) or non-aromatic.
  • heterocycles include pyrrole, pyrrolidine, pyridine, piperidine, succinamide, maleimide, morpholine, imidazole, thiophene, furan, tetrahydrofuran, pyran, and tetrahydropyran.
  • heteroaryl when used without the “substituted” modifier refers to a monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur.
  • Heteroaryl rings may contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen, and sulfur. If more than one ring is present, the rings may be fused or unfused.
  • heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl.
  • A-heteroaryl refers to a heteroaryl group with a nitrogen atom as the point of attachment.
  • heteroaryl when used without the “substituted” modifier refers to an divalent aromatic group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the two points of attachment, the atoms forming part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused.
  • Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting). As used herein, the term does not preclude the presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation permitting) attached to the aromatic ring or aromatic ring system.
  • heteroarenediyl groups include:
  • heterocycloalkyl when used without the “substituted” modifier refers to a monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of attachment, the carbon atom or nitrogen atom forming part of one or more non-aromatic ring structures wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur.
  • Heterocycloalkyl rings may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. If more than one ring is present, the rings may be fused or unfused.
  • the term does not preclude the presence of one or more alkyl groups (carbon number limitation permitting) attached to the ring or ring system. Also, the term does not preclude 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 heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl.
  • A-heterocycloalkyl refers to a heterocycloalkyl group with a nitrogen atom as the point of attachment.
  • A-pyrrolidinyl is an example of such a group.
  • heterocycloalkanediyl when used without the “substituted” modifier refers to a divalent cyclic group, with two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the two points of attachment, the atoms forming part of one or more ring structure(s) wherein at least one of the ring atoms is nitrogen, oxygen or sulfur, and wherein the divalent group consists of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur.
  • the rings may be fused or unfused.
  • Unfused rings may be connected via one or more of the following: a covalent bond, alkanediyl, or alkenediyl groups (carbon number limitation permitting).
  • a covalent bond alkanediyl, or alkenediyl groups (carbon number limitation permitting).
  • alkanediyl or alkenediyl groups (carbon number limitation permitting).
  • alkyl groups carbon number limitation permitting
  • the term does not preclude 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 heterocycloalkanediyl groups include: When these terms are used with the “substituted” modifier one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH 3 , -NHCH3, -NHCH2CH3, -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 .
  • acyl when used without the “substituted” modifier refers to the group -C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as those terms are defined above.
  • acyl groups are non-limiting examples of acyl groups.
  • a “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group -C(O)R has been replaced with a sulfur atom, -C(S)R.
  • aldehyde corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a -CHO group.
  • one or more hydrogen atom (including a hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH 3 , -NHCH3, -NHCH2CH3, -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 .
  • the groups, -C(O)CH 2 CF 3 , -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH 2 (carbamoyl), and -CON(CH3) 2 are non-limiting examples of substituted acyl groups.
  • alkoxy when used without the “substituted” modifier refers to the group -OR, in which R is an alkyl, as that term is defined above.
  • R is an alkyl
  • 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.
  • cycloalkoxy when used without the “substituted” modifier, refers to groups, defined as -OR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively.
  • alkoxydiyl refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-
  • alkylthio and acylthio when used without the “substituted” modifier refers to the group -SR, in which R is an alkyl and acyl, respectively.
  • alcohol corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with a hydroxy group.
  • ether corresponds to an alkane, as defined above, wherein at least one of the hydrogen atoms has been replaced with an alkoxy group.
  • substituted one or more hydrogen atom has been independently replaced by -OH, -F, -Cl, -Br, -I, -NH 2 , -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH 3 , -NHCH3, -NHCH2CH3, -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 )2, -0C(0)CH3, -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 .
  • alkylamino when used without the “substituted” modifier refers to the group -NHR, in which R is an alkyl, as that term is defined above.
  • Non-limiting examples include: -NHCH3 and -NHCH2CH3.
  • dialkylamino when used without the “substituted” modifier refers to the group -NRR', in which R and R' can be the same or different alkyl groups, or R and R' can be taken together to represent an alkanediyl.
  • Non-limiting examples of dialkylamino groups include: - N(CH3)2 and -N(CH3)(CH2CH3).
  • cycloalkylamino when used without the “substituted” modifier, refers to groups, defined as -NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively.
  • R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively.
  • arylamino group is -NHQHs.
  • alkylaminodiyl refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-
  • acylamino when used without the “substituted” modifier, refers to the group -NHR, in which R is acyl, as that term is defined above.
  • a non-limiting example of an amido group is -NHC(O)CH3.
  • substituted refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.
  • Substituents can include any substituents described herein, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic mo
  • aliphatic, heteroaliphatic, oxyaliphatic, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkyl, cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, cycloalkynylene, hydroxyalkyl, heterocycloalkyl, heterocycloalkylene, heterocycloalkenyl, heterocycloalkenylene, aryl, and heteroaryl mentioned herein include both substituted and unsubstituted moieties.
  • 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.
  • each polymer molecule may have different levels of polymerization and thus a different molar mass.
  • the average molecular weight can be used to represent the molecular weight of a plurality of polymer molecules.
  • Average molecular weight is typically synonymous with average molar mass.
  • the average molecular weight represents either the number average molar mass or weight average molar mass of the formula.
  • the average molecular weight is the number average molar mass.
  • the average molecular weight may be used to describe a PEG component present in a lipid.
  • the term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.
  • the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof.
  • the patient or subject is a primate (e.g., non-human primate).
  • the patient or subject is a human.
  • Non-limiting examples of human subjects are adults, juveniles, infants and fetuses.
  • assemble in context of delivery of a payload to target cell(s) generally refers to covalent or non-covalent interaction(s) or association(s), for example, such that a therapeutic or prophylactic agent be complexed with or encapsulated in a lipid composition.
  • lipid composition generally refers to a composition comprising lipid compound(s), including but not limited to, a lipoplex, a liposome, a lipid particle.
  • lipid compositions include suspensions, emulsions, and vesicular compositions.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable salts” means salts of compounds of the present application which are pharmaceutically acceptable, as defined above, and which possess 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, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2 -hydroxy ethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene- 1 -carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-l -carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, c
  • Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting 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, V-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
  • pharmaceutically acceptable carrier means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
  • prevention includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
  • helper lipid refers to a lipid that contributes to the stability or delivery efficacy of a lipid composition.
  • a helper lipid can be a zwitterionic lipid, such as a phospholipid.
  • a helper lipid can be phosphatidylcholine, distearoylphosphatidylcholine, di oleoylphosphatidylethanolamine, l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
  • DOPE dioleoyl-sn-glycero-3 -phosphocholine
  • helper lipid refers to phospholipids or other zwitterionic lipids in the LNP composition.
  • a helper lipid refers to a phospholipid or another zwitterionic lipid.
  • the weight ratio of the lipidoid/steroid/helper lipid/polymer conjugated lipid is about 4/1/1/1.
  • Helper lipid can refer to any class of lipid molecules that improves the particle stability and fluidity of lipid nanoparticles (LNP).
  • helper lipids such as phospholipids (e.g., phosphoethanolamine, phosphocholine), zwitterionic lipids, steroid derivatives, and polymer conjugated lipids (e.g., PEGylated lipid).
  • phospholipids e.g., phosphoethanolamine, phosphocholine
  • zwitterionic lipids e.g., steroid derivatives
  • polymer conjugated lipids e.g., PEGylated lipid
  • helper lipids include cholesterol, 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), Phosphatidylcholine (PC), Methoxy-Polyethyleneglycol (MW 2k)- distearoylphosphatidylethanolamine (mPEG2k-DSPE), and l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2k).
  • DOPE 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • PC Phosphatidylcholine
  • PC Methoxy-Polyethyleneglycol
  • mPEG2k-DSPE Methoxy-Polyethylenegly
  • composition comprising a pharmaceutical agent assembled with a lipid composition, wherein the lipid composition comprises an ionizable lipid.
  • the composition comprises a pharmaceutical agent assembled with a lipid composition, the lipid composition comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail group R Lipid , wherein each R L1 P ld independently comprises a structure of a structure of: or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently a Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl;
  • R k4 and Rks are each independently H or Cl -CIO alkyl.
  • the composition comprises a pharmaceutical agent assembled with a lipid composition
  • the lipid composition comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail group R Lipid , wherein each R L1 P ld independently comprises a structure of: or a pharmaceutically acceptable salt thereof; wherein:
  • Rki is independently a C1-C12 bivalent aliphatic or heteroaliphatic radical
  • Rk3 is independently a Cl -Cl 2 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3- C12 heterocycloalkyl, aryl, or heteroaryl;
  • Rk2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl;
  • R k4 and Rks are each independently H or C1-C12 bivalent aliphatic radical; M is O or NRk6, wherein
  • R k6 is H or C1-C12 bivalent aliphatic radical; and the pharmaceutical agent comprises one or more nucleic acid molecules encoding one or more therapeutic peptides for cancer immunotherapy.
  • composition comprising a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least two hydrophobic tail groups R Lipid , wherein each R L1 P ld independently comprises a structure selected from the group consisting of: or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl; and each R k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; wherein the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE).
  • the composition comprises a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least one hydrophobic tail group R Lipid , wherein each R L1 P ld independently comprises a structure of Formula (Va): or a pharmaceutically acceptable salt thereof; wherein:
  • R kl is independently a C1-C12 bivalent aliphatic or heteroaliphatic radical
  • R k3 is independently a Cl -Cl 2 alkyl
  • R k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl,
  • the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE).
  • the composition comprises a pharmaceutical agent assembled with a lipid composition that comprises an ionizable lipid, wherein the ionizable lipid comprises an amine head group and at least two hydrophobic tail groups R Lipid , wherein each R L1 P ld independently comprises a structure of Formula (Fa): or a pharmaceutically acceptable salt thereof; wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl; and each R k2 is independently a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; wherein the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE).
  • the ionizable lipid comprises at least two hydrophobic tail groups R Lipid . In some embodiments, the ionizable lipid comprises at least three hydrophobic tail groups R Lipid . In some embodiments, the ionizable lipid comprises three or four hydrophobic tail groups R Lipid . In some embodiments, the ionizable lipid comprises four hydrophobic tail groups R Lipid .
  • each R L1 P ld is independently selected from the group consisting of or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl.
  • each R L1 P ld is independently selected from the group consisting of or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl.
  • each R L1 P ld is independently selected from the group consisting of or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl,
  • each R L1 P ld is independently selected from or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl.
  • each R L1 P ld is independently selected from or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl,
  • each R L1 P ld is independently selected from or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3-C20 heterocycloalkyl, aryl, or heteroaryl.
  • each R L1 P ld is independently selected from or a pharmaceutically acceptable salt thereof, wherein:
  • Rki and Rk3 are each independently Cl -CIO alkyl
  • Rk2 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl.
  • Rk2 is selected from C1-C20 alkyl, C2-C20 alkenyl, C2- C20 alkynyl, and C1-C20 heteroalkyl. In some embodiments, Rk2 is selected from C1-C20 alkyl, C2-C20 alkenyl, and C1-C20 heteroalkyl. In some embodiments, Rk2 is selected from C1-C20 alkyl and C2-C20 alkenyl. In some embodiments, Rk2 is C1-C20 alkyl. In some embodiments, Rk2 is C2-C20 alkenyl.
  • the ionizable lipid comprises an amine head group and at least one hydrophobic tail R L1 P ld having a structure of Formula (I): or a pharmaceutically acceptable salt thereof; wherein:
  • Ri and R2 are each independently a C1-C12 bivalent aliphatic or heteroaliphatic radical; in which each of Li, L2, L3, and L4 is, independently, a bond, O, S, orNR c ; G is O, S, or NR d ; Q is OR e , SR f , orNR g R h ; and each of r and t is independently 1-6; each of R c , R d , R e , R f , R g , and R h is independently H, Cl -CIO alkyl, Cl -CIO heteroalkyl, aryl, or heteroaryl;
  • Y and U are each independently a bond, O, S, NR 10 , or Se; n is 0 or 1;
  • R 5 is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C1-C20 heteroalkyl, C3- C20 heterocycloalkyl, aryl, or heteroaryl; and the pharmaceutical agent comprises one or more nucleic acid molecules encoding one or more therapeutic peptides for cancer immunotherapy.
  • n is 0. In some embodiments, n is 1.
  • Y is CH2 and U is O.
  • Y and U are both O.
  • U is CH2 and Y is O.
  • Y and U are NR 10 .
  • Y is O and U is NR 10 .
  • Y is S and U is NR 10 .
  • n is 0, and Y and U are both S.
  • n is 0, and one of Y and U is Se.
  • Ri is a C1-C12 alkyl, linear or branched. In some embodiments, Ri is a Cl -CIO alkyl, linear or branched. In some embodiments, Ri is a C1-C8 alkyl, linear or branched. In some embodiments, Ri is a C1-C6 alkyl, linear or branched. In some embodiments, Ri is a C1-C4 alkyl, linear or branched. In some embodiments, Ri is a C2 alkyl, e.g., , yl. In some embodiments,
  • Ri is a C1-C12 heteroaliphatic radical.
  • R2 is a C1-C12 alkyl, linear or branched. In some embodiments, R2 is a Cl -CIO alkyl, linear or branched. In some embodiments, R2 is a C1-C8 alkyl, linear or branched. In some embodiments, R2 is a C1-C6 alkyl, linear or branched. In some embodiments, R2 is a C1-C4 alkyl, linear or branched. In some embodiments, R2 is a C2 alkyl, e.g.,
  • R2 is a C3 alkyl, e.g., In some embodiments, R2 is a C4 alkyl. In some embodiments, R2 is a Cl -Cl 2 heteroaliphatic radical.
  • Rki is a Cl -CIO alkyl, linear or branched. In some embodiments,
  • Rki is a C1-C4 alkyl, linear or branched. In some embodiments, Rki is a C2 alkyl, e.g.,
  • Rki is a C3 alkyl, e.g., In some embodiments, Rki is a C4 alkyl.
  • Rk3 is a Cl -CIO alkyl, linear or branched. In some embodiments, Rk3 is a C1-C4 alkyl, linear or branched. In some embodiments, Rk3 is a C2 alkyl, e.g., .
  • Rk3 is a C3 alkyl, e.g., , or . In some embodiments, Rk3 is a C4 alkyl.
  • Rk4 is H. In some embodiments, Rk4 is Cl -CIO alkyl. In some embodiments, Rk4 is C1-C4 alkyl. In some embodiments, Rk4 is C4-C10 alkyl.
  • Rks is H. In some embodiments, Rks is C1-C10 alkyl. In some embodiments, Rks is C1-C4 alkyl. In some embodiments, Rks is C4-C10 alkyl.
  • Rk2 is a C1-C20 alkyl. In some embodiments, Rk2 is a C2- C20 alkenyl. In some embodiments, Rk2 is a C2-C20 alkynyl. In some embodiments, Rk2 is a C3- C20 cycloalkyl. In some embodiments, Rk2 is a C1-C20 heteroalkyl. In some embodiments, Rk2 is a C3- C20 heterocycloalkyl, aryl, or heteroaryl.
  • the at least one hydrophobic tail comprises
  • the amine head group is represented by wherein Ra, Ra’, Ra”, and Ra’” are each independently, H, Cl -20 alkyl, C2-C20 alkenyl, C2- C20 alkynyl, C3-C20 cycloalkyl or heterocycloalkyl, C1-C20 heteroalkyl, C3-C20 aryl or heteroaryl, bivalent aliphatic radical, a C1-C20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical.
  • the ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein:
  • R b is a substituted or unsubstituted alkyl, hydroxyalkyl, alkoxyalkyl, or aryl; nl and n2 are each independently 1, 2, 3, 4, 5, or 6; and
  • R bl , R b2 , R b3 and R b4 are each independently H, or R L1 P ld wherein at least one of R bl , R b2 , R b3 and R b4 is not H.
  • ionizable lipid is represented by Formula (II):
  • R bl , R b2 , R b3 and R b4 are each independently H or R Lipid ; wherein at least one of R bl , R b2 , R b3 and R b4 is not H.
  • ionizable lipid is represented by Formula (II): or a pharmaceutically acceptable salt thereof, wherein:
  • R b is a C1-C4 alkyl which is optionally substituted with 1 to 4 groups selected from -OH; nl and n2 are each independently 1, 2, 3, or 4;
  • R bl , R b2 , R b3 and R b4 are each independently H or R Lipid ; wherein at least one of R bl , R b2 , R b3 and R b4 is not H.
  • R b is a C1-C6 alkyl which is optionally substituted with 1 to 4 groups selected from -OH; nl and n2 are each independently 1, 2, 3, or 4; and R bl , R b2 , R b3 and R b4 are each independently R L1 P ld .
  • R b is unsubstituted C1-C6 alkyl; nl and n2 are each independently 1, 2, 3, or 4; and R bl , R b2 , R b3 and R b4 are each independently R L1 P ld
  • R b is a C1-C6 alkyl, linear or branched. In some embodiments, R b is a substituted C1-C6 alkyl, linear or branched. In some embodiments, a substituent comprises hydroxyl, carbonyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, cyclic amine, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. In some embodiments, the substituent comprises hydroxyl,
  • nl is 1, 2, 3, 4, 5, or 6. In some embodiments, nl is 2 or 3.
  • n2 is 1, 2, 3, 4, 5, or 6. In some embodiments, n2 is 2 or 3.
  • nl and n2 are identical. In some embodiments, nl and n2 are different. In some embodiments, both nl and n2 are 2. In some embodiments, both nl and n2 are 3. In some embodiments, both nl and n2 are 4.
  • R bl is not H. In some embodiments, R b2 is not H. In some embodiments, R b3 is not H. In some embodiments, R b4 is not H.
  • R bl , R b2 , R b3 and R b4 are not H. In some embodiments, at least three of R bl , R b2 , R b3 and R b4 are not H. In some embodiments, none of R bl , R b2 , R b3 and R b4 is H.
  • the amine head group is selected from the group consisting of:
  • the amine head group is selected from the group consisting of:
  • the amine head group is selected from the group consisting of:
  • the at least one hydrophobic tail is selected from the TABLE 1.
  • the ionizable lipid comprises at least two hydrophobic tails.
  • the at least two hydrophobic tails are independently of structure . In some embodiments, the at least two hydrophobic tails are identical. In some embodiments, the at least two hydrophobic tails are not identical. In some embodiments, one of the at least two hydrophobic tails is different from the rest.
  • the lipid composition comprises at least three hydrophobic tails. In some embodiments, the at least three hydrophobic tails are independently of structure in some embodiments, the at least three hydrophobic tails are identical. In some embodiments, the at least three hydrophobic tails are not identical. In some embodiments, one of the at least three hydrophobic tails is different from the rest. [00121] In some embodiments, the lipid composition comprises two hydrophobic tails. In some embodiments, the two hydrophobic tails are independently of structure . In some embodiments, the two hydrophobic tails are identical. In some embodiments, the two hydrophobic tails are not identical.
  • the lipid composition comprises three hydrophobic tails.
  • the three hydrophobic tails are independently of structure .
  • the three hydrophobic tails are identical.
  • two of the three hydrophobic tails are identical and the third hydrophobic tail is different.
  • all three hydrophobic tails are different.
  • the lipid composition comprises four hydrophobic tails.
  • the four hydrophobic tails are independently of structure .
  • the four hydrophobic tails are identical.
  • three of the four hydrophobic tails are identical and the fourth hydrophobic tail is different.
  • two of the four hydrophobic tails are identical, the other two hydrophobic tails are identical, and the two are different from other two.
  • two of the four hydrophobic tails are identical while the other two are different from each other and are different from the two. In some embodiments, all four hydrophobic tails are different.
  • the ionizable lipid comprises:
  • the ionizable lipid is selected from TABLE 2.
  • the composition provided herein further comprises a steroid.
  • the steroid comprises a cholesterol or a cholesterol derivative.
  • the composition provided herein further comprises a helper lipid.
  • the helper lipid comprises a phospholipid, such as l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
  • DOPE dioleoyl-sn-glycero-3- phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • the composition provided herein further comprises a polymer conjugated lipid.
  • the polymer conjugated lipid comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000 (DSPE-PEG2k) or 1,2- dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG2k).
  • the lipid composition comprises an ionizable lipid disclosed in this application, a steroid, a helper lipid, and a polymer conjugated lipid.
  • the ionizable lipid is present in the lipid composition at a weight percentage from 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%, from about 20% to about 30%, from about 20% to about 30%, from about 20% to about 40%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 90%, from about 30% to about 40%, from about 30% to about 50%, from about 30% to about 60%, from about 30% to about 70%, from about 30% to about 80%, from about 30% to about 90%, from about 40% to about 50%, from about 40% to about 60%, from about 40% to about 70%, from about 40% to about 80%, from about 40% to about 90%, from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 60% to about 70%, from about 60% to about 80%, from about from about 20% to about 80%,
  • the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.
  • the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.
  • the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.
  • the weight ratio of the ionizable lipid/steroid/helper 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.
  • the lipid composition comprises an ionizable lipid disclosed in this application, a steroid and a helper lipid.
  • the ionizable lipid is present in the lipid composition at a weight percentage from about 30% to about 90%.
  • the helper lipid is present in the lipid composition at a weight percentage from about 5% to about 40%.
  • the steroid is present in the lipid composition at a weight percentage from about 5% to about 40%.
  • the weight ratio of the ionizable lipid/steroid/helper 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.
  • the lipid composition further comprises a pharmaceutically acceptable carrier.
  • a carrier comprises a pharmaceutically acceptable excipient.
  • the carrier can comprise a sugar, wherein the sugar comprises mannitol, sucrose, maltose, or trehalose.
  • the carrier can comprise EC- 16, (2-hydroxypropyl)-P-cyclodextrin ((HP-P- CD), stearic acid, Perfluoroundecanoic, Saponin, Mannitol, Borneol, Amikacin-EC16, Kanamycin-EC16, Neomycin-EC16, or Bile salts.
  • the carrier is present in the composition at a weight percentage from about 5% to about 60%.
  • the excipient is present in the composition at a weight percentage from about 1% to about 70%, from about 5% to about 60%, from about 5% to about 50%, from about 5% to about 40%, from about 5% to about 30%, from about 10% to about 50%, from about 10% to about 40%, from about 10% to about 30%, or from about 10% to about 20%.
  • the pharmaceutical agent comprises a polynucleotide, an oligonucleotide, a polypeptide, an oligopeptide, a small molecule compound, or any combination thereof.
  • the polynucleotide is a messenger ribonucleic acid (mRNA).
  • mRNA messenger ribonucleic acid
  • the pharmaceutical agent comprises a polynucleotide that encodes a gene product thereof.
  • the pharmaceutical agent comprises an mRNA encoding at least one therapeutic peptide for cancer immunotherapy.
  • the pharmaceutical agent comprises an mRNA encoding an antibody or a fragment thereof.
  • the mRNA encodes a bispecific T cell engager (BiTE).
  • the mRNA encoding the GPC3-CD3 BiTE is codon-optimized to increase expression in a subject.
  • the subject comprises a mammal.
  • the subject comprises a rodent, human or a non-human primate.
  • the mRNA encoding the GPC3-CD3 BiTE comprises the nucleic acid sequence of SEQ ID NO: 1.
  • the pharmaceutical agent is used for cancer immunotherapy.
  • the cancer immunotherapy is T-cell mediated immunotherapy.
  • the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE).
  • the pharmaceutical agent comprises an mRNA encoding the GPC3/CD3 BiTE.
  • pharmaceutical agent is assembled in the lipid composition at a weight ratio of the pharmaceutical agent/lipid composition of from about 1 :200 to about 1 : 100, from about 1 :200 to about 1 :50, from about 1 :200 to about 1 :40, from about 1 :200 to about 1 :30, from about 1 :200 to about 1 :20, from about 1 :200 to about 1 : 10, from about 1 :200 to about 1 :5, from about 1 :200 to about 1 : 1, from about 1 : 100 to about 1 :50, from about 1 : 100 to about 1 :40, from about 1 : 100 to about 1 :25, from about 1 : 100 to about 1 :20, from about 1 : 100 to about 1 : 15, from about 1 : 100 to about 1 : 10, from about 1 : 100 to about 1 :5 or from about 1 : 100 to about 1 : 1.
  • 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.
  • the lipid composition further comprises an additional lipid comprising a steroid or a steroid derivative, a PEG lipid, and a helper lipid (e.g., phospholipids or other zwitterionic lipids).
  • an additional lipid comprising a steroid or a steroid derivative, a PEG lipid, and a helper lipid (e.g., phospholipids or other zwitterionic lipids).
  • the lipid composition further comprises a helper lipid.
  • the helper lipid comprises a lipid that contributes to the stability or delivery efficiency of the lipid compositions.
  • the helper lipid comprises a zwitterionic lipid.
  • the helper lipid comprises a phospholipid.
  • the phospholipid may contain one or two long chain (e.g., C6-C24) alkyl or alkenyl groups, a glycerol or a 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.
  • the phospholipid is a phosphatidylcholine.
  • the phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.
  • other zwitterionic lipids are used, where zwitterionic lipid defines lipid and lipid-like molecules with both a positive charge and a negative charge.
  • the phospholipid is not an ethylphosphocholine.
  • the helper lipid can comprise 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE) or l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC).
  • DOPE 1,2-dioleoyl-sn- glycero-3 -phosphoethanolamine
  • DOPC l,2-dioleoyl-sn-glycero-3 -phosphocholine
  • compositions may further comprise a molar percentage of the phospholipid to the total lipid composition from about 5 to about 30.
  • the helper lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.
  • the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 23%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 20%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 15% to about 20%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 8% to about 15%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 10% to about 15%. In some embodiments, the lipid composition comprises the phospholipid at a molar percentage from about 12% to about 18%.
  • the lipid composition comprises the phospholipid at a molar percentage of 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 at a molar percentage of at most about 8%, at most about 10%, at most about 12%, at most about 15%, at most about 18%, at most about 20%, or at most about 23%.
  • the lipid composition further comprises a steroid or steroid derivative.
  • the steroid or steroid derivative comprises any steroid or steroid derivative.
  • the term “steroid” is a class of compounds with a four ring 17 carbon cyclic structure which can further comprises one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or a double bond between two or more carbon atoms.
  • the ring structure of a steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring as shown in the formula:
  • a steroid derivative comprises the ring structure above with one or more non-alkyl substitutions.
  • the steroid or steroid derivative is a sterol wherein the formula is further defined as: .
  • the steroid or steroid derivative is a cholestane or cholestane derivative.
  • the ring structure is further defined by the formula: .
  • a cholestane derivative includes one or more non-alkyl substitution of the above ring system.
  • the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or a sterol derivative.
  • the cholestane or cholestane derivative is both a cholesterol and a sterol or a derivative thereof.
  • the compositions may further comprise a molar percentage of the steroid to the total lipid composition from about 20 to about 60.
  • the steroid is present in the lipid composition at a weight percentage from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 20% to about 30%, from about 20% to about 40%, or from about 30% to about 40%.
  • the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 15% to about 46%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 25% to about 35%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 30% to about 40%. In some embodiments, the lipid composition comprises the steroid or steroid derivative at a molar percentage from about 20% to about 30%.
  • the lipid composition comprises the steroid or steroid derivative at a molar percentage of 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 at a molar percentage of at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 46%. [00150] In some embodiments, the lipid composition further comprises a polymer conjugated lipid. In some embodiments, the polymer conjugated lipid is a PEG lipid.
  • the PEG lipid is a diglyceride which also comprises a PEG chain attached to the glycerol group.
  • the PEG lipid is a compound which contains one or more C6-C24 long chain alkyl or alkenyl group or a C6-C24 fatty acid group attached to a linker group with a PEG chain.
  • Some non-limiting examples of a PEG lipid includes a PEG modified phosphatidylethanolamine and phosphatidic acid, a PEG ceramide conjugated, PEG modified dialkylamines and PEG modified l,2-diacyloxypropan-3 -amines, PEG modified diacylglycerols and dialkylglycerols.
  • PEG modified diastearoylphosphatidylethanolamine or PEG modified dimyristoyl- w-glycerol is measured by the molecular weight of PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000.
  • the molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about 15,000.
  • Some non-limiting examples of lipids that may be used in the present application are taught by U.S. Patent 5,820,873, WO 2010/141069, or U.S. Patent 8,450,298, which is incorporated herein by reference.
  • the PEG lipid has a structural formula: , wherein: R12 and R13 are each independently alkyl(c ⁇ 24), alkenyl(c ⁇ 24), or a substituted version of either of these groups; R e is hydrogen, alkyl(c ⁇ 8), or substituted alkyl(c ⁇ 8); and x is 1-250.
  • R e is hydrogen, alkyl(c ⁇ 8), or substituted alkyl(c ⁇ 8); and x is 1-250.
  • Re is alkyl(c ⁇ 8) such as methyl.
  • R12 and R13 are each independently alkyl(c ⁇ 4-20).
  • x is 5-250. In one embodiment, x is 5-125 or x is 100-250.
  • the PEG lipid is 1,2-dimyristoyl-sw-glycerol, methoxypolyethylene glycol.
  • the PEG lipid has a structural formula: , wherein: ni is an integer between 1 and 100 and n2 and ns are each independently selected from an integer between 1 and 29.
  • m 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 derivable therein.
  • n2 is from 5 to 23.
  • n2 is 11 to about 17.
  • ns is from 5 to 23.
  • ns is 11 to about 17.
  • the polymer conjugated lipid comprises 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2k) or 1,2- dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG2k).
  • the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 0.5% to about 20%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 1% to about 8%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 2% to about 7%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 3% to about 5%. In some embodiments, the lipid composition comprises the polymer-conjugated lipid at a molar percentage from about 5% to about 10%.
  • the lipid composition comprises the polymer- conjugated lipid at a molar percentage of 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%.
  • the lipid composition comprises the polymer-conjugated lipid at a molar percentage of at most about 0.5%, at most about 1%, at most about 1.5%, at most about 2%, at most about 2.5%, at most about 3%, at most about 3.5%, at most about 4%, at most about 4.5%, at most about 5%, at most about 5.5%, at most about 6%, at most about 6.5%, at most about 7%, at most about 7.5%, at most about 8%, at most about 8.5%, at most about 9%, at most about 9.5%, at most about 10%, at most about 15%, or at most 20%.
  • the polymer conjugated lipid is present in the lipid composition at a weight percentage from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 5% to about 10%, from about 5% to about 20%, or from about 10% to about 20%.
  • Cancer immunotherapy is the science of modulating the immune system against cancer.
  • Immune checkpoint inhibitors targeting the CTLA-4 and PD1/PD-L1 pathways emerged as a revolutionary cancer treatment strategy, due to impressive clinical responses and overall outcome benefits in certain tumor types.
  • Novel immunotherapies that redirect T-cells against tumor antigens through antibody fragments independent of major histocompatibility complex (MHC) presentation have been under investigation.
  • chimeric antigen-receptor- modified (CAR) T cells and bispecific T-cell engagers (BiTEs) have demonstrated remarkable clinical responses in hematologic malignancies.
  • BiTEs are recombinant proteins composed of two different single-chain variable fragment (scFv) regions from two different monoclonal antibodies.
  • the scFv constructs are covalently connected by a flexible small peptide linker, altogether comprising a ⁇ 55 kDa polypeptide chain.
  • One scFv-binding domain is engineered to target a select tumor-associated antigen (TAA) and the other scFv domain is typically specific for CD3, the invariant component of the T-cell receptor (TCR) complex.
  • TAA tumor-associated antigen
  • TCR TCR
  • the length of the inter-scFv linker varies depending on construct, but linker size does not directly impact tumor-killing activity.
  • BiTEs have a 100-to- 10,000-fold higher efficacy in tumor cell lysis with a low ratio of T-cells to target tumor cells in cellular models. Subsequent to BiTE-induced T-cell activation, the diffusion of released cytokines in the immune synapse also plays a role in upregulation of cell surface molecules of the surrounding cells, further contributing to the antitumor activity of BiTEs, commonly named the “bystander effect”.
  • BiTEs can be produced in large quantities by mammalian cell lines, minimizing interpatient variability and providing “off-the-shelf’ therapies that now are undergoing investigation in a multitude of tumor types.
  • first-generation compounds have failed to demonstrate significant antitumor activity in solid tumors.
  • Challenges of implementing BiTEs in solid tumors include the lack of target antigen expression, tumor inaccessibility, and the impact of an immunosuppressive tumor microenvironment.
  • Glypican-3 GPC3 is highly expressed in hepatocellular carcinoma (HCC) and various types of lung cancers in human.
  • lipid nanoparticle compositions comprising a pharmaceutical agent encapsulated by the lipid composition provided herein, wherein the pharmaceutical agent comprises one or more nucleic acid molecules encoding one or more therapeutic peptides targeting the tumor marker GPC3 for cancer immunotherapy.
  • the cancer immunotherapy comprises T-cell based therapies.
  • the one or more therapeutic peptides for cancer immunotherapy comprise an antibody or a fragment thereof.
  • the antibody encoded by the mRNA comprises a multispecific antibody.
  • the multispecific antibody comprises a bispecific T-cell engager (BiTE).
  • the BiTE binds to a surface protein on an immune cell (e.g., CD3 on a T-cell) and a tumor antigen.
  • a tumor antigen is a protein derived from a tumor or a cancer cell.
  • a tumor antigen can be over-expressed or uniquely expressed on or in a tumor or a cancer cell, which allows tumor targeting.
  • the BiTE binds to CD3 and glypican-3 (GPC3).
  • the pharmaceutical agent comprises one or more nucleic acid molecules encoding a bispecific T-cell engager targeting glypican-3 and CD3 (GPC3/CD3 BiTE).
  • the pharmaceutical agent comprises an mRNA encoding the GPC3/CD3 BiTE.
  • the mRNA encoding the GPC3/CD3 BiTE comprises a sequence having at least 70% sequence identity to SEQ ID NO: 1.
  • the mRNA encoding the GPC3/CD3 BiTE comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1.
  • the mRNA encoding the GPC3/CD3 BiTE comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1.
  • the mRNA encoding the GPC3/CD3 BiTE comprises the sequence of SEQ ID NO: 1.
  • the therapeutic peptides encoded by the mRNA encapsulated by the LNPs of the present disclosure comprise a protein tag (e.g., a 6X His tag).
  • the protein tag can facilitate identification or purification of the therapeutic peptide.
  • more than one therapeutic peptide can be encoded by the mRNA encapsulated by the LNPs of the present disclosure.
  • the LNPs of the present disclosure comprises mRNA encoding at least 1, 2, 3, 4, 5, 6 or more therapeutic peptides.
  • the LNPs comprises mRNAs that encode a multispecific antigen-binding peptide for immunotherapy.
  • a multispecific antigen-binding peptide comprises a first domain that binds to an antigen expressed by an immune cell and a second domain that binds to an antigen expressed by a tumor cell.
  • the multispecific antigen-binding peptide can bind to an antigen expressed by any one of the immune cells provided herein.
  • the LNPs of the disclosure comprises mRNAs that encode a chimeric antigen receptor derived from a neutralizing antibody comprising an antigen-binding fragment, a transmembrane domain, and an intracellular signaling domain described herein.
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell.
  • immune effector function e.g., in a CART cell
  • the intracellular signaling domain can comprise a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP 12.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that can be used for an efficient immune response.
  • Costimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la/CD18) and 4-1BB (CD137).
  • a costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region).
  • the transmembrane domain is one that is associated with one of the other domains of the CAR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another CAR on the CAR T-cell surface.
  • the amino acid sequence of the transmembrane domain can be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR T-cell.
  • the transmembrane domain can be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some cases, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target.
  • the transmembrane domain can include at least the transmembrane region(s) of e.g., the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge or a spacer, e.g., a hinge from a human protein.
  • the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • the hinge or spacer comprises an IgG4 hinge.
  • the cytoplasmic domain or region of the CAR includes an intracellular signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • Examples of intracellular signaling domains for use in the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • TCR T cell receptor
  • co-receptors that act in concert to initiate signal transduction following antigen receptor engagement
  • any derivative or variant of these sequences and any recombinant sequence that has the same functional capability are known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and/or costimulatory signal can also be involved.
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling domain, e.g., a costimulatory domain).
  • a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • Examples of IT AM containing primary intracellular signaling domains that are of particular use herein include those of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • a CAR comprises an intracellular signaling domain, e.g., a primary signaling domain, of CD3-zeta.
  • a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
  • a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
  • a primary signaling domain comprises one, two, three, four or more IT AM motifs.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that can play a role for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3) :696-706).
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequence.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains.
  • the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein.
  • the intracellular signaling domain comprises two costimulatory signaling domains.
  • the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.
  • the mRNA-encoded therapeutic peptides for cancer immunotherapy comprises an immunomodulator.
  • an immunomodulator comprises a cytokine, cytokine receptor, chemokine, chemokine receptor, immune co-receptor, or immune co-receptor ligand. Expression of the immunomodulator can be driven by an expression regulatory region disclosed herein.
  • the therapeutic peptides for cancer immunotherapy comprises a cytokine or a functional fragment thereof, for example, G-CSF, GITRL, GM-CSF, IFN-a, IFN-P, IFN-y, IL-IRA, IL-la, IL-ip, 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-P, M-CSF, MSP, OSM, OX40L, SCF, TALL-1, TGF-P, TGF-pi, TGF-P2, TGF-P3, TNF-a, TNF-P, TRAIL, TRANCE, or TWEAK.
  • a cytokine or a functional fragment thereof for example, G-CSF, GITRL, GM-CSF
  • the therapeutic peptides for cancer immunotherapy comprises a cytokine receptor or a functional fragment thereof, for example, a common gamma chain receptor, a common beta chain receptor, an interferon receptor, a TNF family receptor, a TGF-B receptor, Apo3, CD114, CD115, CD116, CD117, CD118, CD120, CD120a, CD120b, CD121, CD121a, CD121b, CD122, CD123, CD124, CD126, CD127, CD130, CD131, CD132, CD212, CD213, CD213al, CD213al3, CD213a2, CD25, CD27, CD30, CD4, CD40, CD95 (Fas), CDwl l9, CDwl21b, CDwl25, CDwl31, CDwl36, CDwl37 (41BB), CDw210, CDw217, GITR, HVEM, IL-11R, IL-l lRa, IL-14R, IL-15
  • the therapeutic peptides for cancer immunotherapy comprises a chemokine or a functional fragment thereof, for example, ACT-2, AMAC-a, ATAC, 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, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, DC-CK1, ELC, ENA- 78, eotaxin, eotaxin-2, eotaxin-3, Eskin
  • the therapeutic peptides for cancer immunotherapy comprises a chemokine receptor or a functional fragment thereof, for example, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, XCR1, or XCRL
  • the therapeutic peptides for cancer immunotherapy comprises an activating NK receptor, or a functional fragment thereof, for example, CD 100 (SEMA4D), CD 16 (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).
  • CD 100 SEMA4D
  • CD 16 FcgRIIIA
  • the therapeutic peptides for cancer immunotherapy comprises an Fey receptor (FcyR), an Fes receptor (FcsR), an Fea receptor (FcaR), an Fcp receptor (FcpR), neonatal Fc receptor (FcRn), CD4, CD5, CD8, CD21, CD22, CD27, CD28, CD32, CD40, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (ICOS), CD247 ⁇ CD247q, 4 IBB, DAP 10, DAP12, FYN, LAT, Lek, MAPK, MHC complex, NF AT, NF-KB, PLC-y, iC3b, C3dg, C3d, Zap70, MyD88, a functional fragment thereof, or a combination thereof.
  • the therapeutic peptides for cancer immunotherapy comprises a domain that is, is derived from, interacts with, increases expression of, or activates a transcription factor, such as, for example, E2A, Pax5, EBF, PU.1, Ikaros, GATA3, Th-POK, Tbet, Bcl6, NF- KB, NFAT, AP-1, NFAT, STAT1, STAT2, STAT3, STAT4, STAT5, STAT5A, STAT5B, STAT6, STAT7, IRF I , IRF2, IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, IRF9, AP-1, Eomes, FoxP3, Id2, PLZF, ROR-gamma-T, TCF7, ThPOK, or any combination thereof.
  • a transcription factor such as, for example, E2A, Pax5, EBF, PU.1, Ikaros, GATA3, Th-POK, Tbet, Bcl6, NF- KB, NF
  • the nucleic acid molecules comprise a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 1.
  • the mRNA comprises the nucleic acid sequence of SEQ ID NO: 1.
  • the mRNA encoding the GPC3/CD3 BiTE comprises a nucleic acid sequence encoding a signal peptide, wherein the signal peptide can be cleaved during post- translational processing.
  • the mRNA comprises a promoter sequence.
  • the promoter sequence comprises a tissue-specific promoter.
  • the tissue specific promoter comprises a hepatocyte-specific promoter or a lung-specific promoter.
  • the mRNA comprises a strong promoter that enhances the expression of the therapeutic peptide for immunotherapy (e.g., GPC3/CD3 BiTE).
  • the strong promoter can comprise a Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor- 1 alpha (EFla), or phosphoglycerokinase (PGK) promoter.
  • hCMV Human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EFla elongation factor- 1 alpha
  • PGK phosphoglycerokinase
  • the mRNA encapsulated by the LNPs of the present disclosure comprises a naturally occurring or an artificial promoter.
  • the mRNA comprises a promoter that is specific for expression in immune cells as compared to non-immune cells.
  • the mRNA comprises a T-cell specific promoter and the LNPs comprising the mRNA encoding a therapeutic peptide can be used for CAR-T therapy.
  • the T-cell specific promoter comprises promoters that drive endogenous expression of T-cell specific proteins comprising CD3 (e.g., CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta), CD4, CD8, CD28, TCRB or TRAC.
  • CD3 e.g., CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta
  • CD4 e.g., CD8 CD28, TCRB or TRAC.
  • the promoter can result in stronger expression of therapeutic peptides in immune cells comprising lymphocytes, T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, T regulatory cells (Tregs), cytotoxic T lymphocytes, Thl cells, Th2 cells, Th 17 cells, Th9 cells, naive T cells, memory T cells, effector T cells, effector-memory T cells (TEM), central memory T cells (TCM), resident memory T cells (TRM), follicular helper T cells (TFH), Natural killer T cells (NKTs), tumor-infiltrating lymphocytes (TILs), Natural killer cells (NKs), Innate Lymphoid Cells (ILCs), ILC1 cells, ILC2 cells, ILC3 cells, lymphoid tissue inducer (LTi) cells, B cells, Bl cells, Bia cells, Bib cells, B2 cells, plasma cells, B regulatory cells, memory B cells, marginal zone B
  • an mRNA encoding the GPC3/CD3 BiTE comprises natural, synthetic, and/or artificial nucleotide analogues or bases.
  • the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of a deoxyribose moiety, ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.
  • a nucleotide analogue or artificial nucleotide base comprises a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety.
  • the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety.
  • Illustrative alkyl moiety includes, but are not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen.
  • the alkyl moiety further comprises a modification.
  • the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide).
  • the alkyl moiety further comprises a hetero substitution.
  • the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur.
  • the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.
  • the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2 '-O-m ethoxy ethyl (2’-0-M0E) modification.
  • the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety whereas the 2'0- methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.
  • the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2' oxygen.
  • this modification neutralizes the phosphate-derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties.
  • the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxy-methylene- linked bicyclic ribonucleotide monomer.
  • a locked or bridged ribose modification e.g., locked nucleic acid or LNA
  • additional modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy -2 '-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O- dimethylaminopropyl (2'-O-DMAP), T-O- dimethylaminoethyloxyethyl (2'-0-DMAE0E), or 2'- O-N-methylacetamido (2'-0-NMA).
  • a nucleotide analogue comprises a modified base, for example, Nl- methylpseudouridine, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6- methylguanine, N, N, -dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1- methylinosine, 3 -methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5- (2- amino) propyl uridine, 5-halocytidine, 5-halouridine, 4- acetylcytidine, 1- methyladenosine, 2-methyladenosine, 3 -methylcytidine, 6-m ethyluridine, 2- methylguanosine, 7-methylguanosine, 2, 2-dimethylguanosine, 5- methylaminoeth
  • Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
  • the sugar moieties in some cases are or are based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
  • nucleotide also includes universal bases.
  • universal bases include but are not limited to 3 -nitropyrrole, 5-nitroindole, or nebularine.
  • a modified intemucleotide linkages can include, but is not limited to, phosphorothioates; phosphorodithioates; methylphosphonates; 5'- alkylenephosphonates; 5'- methylphosphonate; 3 '-alkylene phosphonates; borontrifluoridates; borano phosphate esters and selenophosphates of 3'-5'linkage or 2'-5'linkage; phosphotriesters; thionoalkylphosphotriesters; hydrogen phosphonate linkages; alkyl phosphonates; alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3'- alkylphosphoramidates; aminoalkylphosphoramidates; thionophosphorami
  • one or more modifications comprise a modified phosphate backbone in which the modification generates a neutral or uncharged backbone.
  • the phosphate backbone is modified by alkylation to generate an uncharged or neutral phosphate backbone.
  • alkylation includes methylation, ethylation, and propylation.
  • an alkyl group refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms.
  • 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 groups.
  • a modified phosphate is a phosphate group as described in U.S. Patent No. 9481905.
  • additional modified phosphate backbones comprise methylphosphonate, ethylphosphonate, methylthiophosphonate, or methoxyphosphonate.
  • the modified phosphate is methylphosphonate.
  • the modified phosphate is ethylphosphonate.
  • the modified phosphate is methylthiophosphonate.
  • the modified phosphate is methoxyphosphonate.
  • one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3' or the 5' terminus.
  • the 3' terminus optionally include a 3' cationic group, or by inverting the nucleoside at the 3 '-terminus with a 3 '-3' linkage.
  • the 3 '-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT.
  • the 3 '-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.
  • the 5 '-terminus is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent.
  • the 5 '-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.
  • the present disclosure provides methods for treating a cancer using the lipid nanoparticle (LNP) compositions described herein, wherein the LNPs comprise nucleic acid molecules (e.g., mRNA) encoding one or more therapeutic peptides (e.g., BiTE) for cancer immunotherapy.
  • LNP lipid nanoparticle
  • the present disclosure also provides a method for delivering a pharmaceutical agent (e.g., mRNA encoding BiTE) to a target organ in a subject in need thereof, the method comprising administering an effective amount of the composition provided herein.
  • the target organ comprises a liver, lung or secondary lymphoid tissue (e.g., spleen or lymph nodes).
  • the target organ is a liver.
  • the target organ is a lung.
  • the cancer comprises a leukemia.
  • the cancer comprises a solid-state tumor.
  • the leukemia comprises acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, T-cell prolymphocytic leukemia, lymphoid leukemia, large granular lymphocytic leukemia, B-cell prolymphocytic leukemia, myeloproliferative neoplasm, or mastocytosis.
  • the present disclosure provides a method for treating a solid- state tumor in a subject in need thereof, comprising administering an effective amount of the composition provided herein.
  • the solid-state tumor comprises hepatocellular carcinoma.
  • the solid-state tumor comprises lung cancer.
  • the lung cancer comprises non-small cell lung cancer.
  • the administering comprises administering the composition intravenously or intramuscularly to a subject in need thereof.
  • the subject comprises a mammal.
  • the subject comprises a human or a non-human primate.
  • the subject comprises a rodent.
  • the solid-state tumor expresses glypican-3 (GPC3).
  • the solid-state tumor comprises breast cancer, liver cancer, lung cancer, brain cancer, lymphoma, colorectal cancer, prostate cancer, sarcoma, melanoma, pancreatic cancer, kidney cancer, thyroid cancer, Kaposi sarcoma, uterine cancer, bladder cancer, soft-tissue sarcoma, oropharyngeal cancer, non-small cell lung carcinoma, or multiple myeloma.
  • the cancer comprises small cell lung carcinoma or non-small cell lung carcinoma.
  • the non-small cell carcinoma comprises adenocarcinoma, squamous cell carcinoma, or large cell carcinoma.
  • the cancer comprises hepatocellular carcinomas (HCC).
  • the composition of the present application can be administrated through any suitable routes comprising parenteral delivery (e.g., injections), such as intravenous, intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery e.g., injections
  • the method for delivering a pharmaceutical agent to a target organ in a subject in need thereof provides a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to that achieved absent the lipid composition.
  • the method for delivering a pharmaceutical agent to a target organ in a subject in need thereof provides a greater amount or activity of the pharmaceutical agent in the target organ in the subject as compared to a non-target organ.
  • the amount of the pharmaceutical agent is measured by pharmacokinetic parameters.
  • pharmacokinetic parameters comprise Cmax, Tmax, half-life, or area under curve (AUC).
  • the greater amount of the pharmaceutical agent comprises a larger Cmax, a longer half-life, or a larger area under curve (AUC).
  • the methods provided herein result in greater the pharmacokinetic parameters of the pharmaceutical agent, and wherein the pharmacokinetic parameters of the pharmaceutical agent comprise Cmax, Tmax, half-life, or area under curve (AUC).
  • the half-life of the pharmaceutical agent is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours or more.
  • the AUC of the pharmaceutical agent is 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 150%, at least 200% or more higher than the AUC of the pharmaceutical agent delivered in other compositions.
  • the Cmax of the pharmaceutical agent is 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 150%, at least 200% or more higher than the Cmax of the pharmaceutical agent delivered in other compositions.
  • the method provides potent delivery of the pharmaceutical agent to a cell of a subject.
  • the method comprising administering the composition provided herein results in targeted delivery of the pharmaceutical agent to a target organ.
  • the target organ comprises liver.
  • the target organ comprises lung.
  • the method delivers a pharmaceutical agent to a target organ (e.g., liver or lung) or a target cell (e.g., a cell of the target organ or an immune cell) of a subject, wherein the method comprises administering a composition described herein, and thereby providing an effective amount or activity of the pharmaceutical agent in the target organ or target cell that is at least 1.1 -fold greater than a corresponding amount or activity of the pharmaceutical agent achieved in a non-target organ or non-target cell of the subject.
  • a target organ e.g., liver or lung
  • a target cell e.g., a cell of the target organ or an immune cell
  • the effective amount or activity of the pharmaceutical agent in the target organ or target cell is at least 1.1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3- fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, at least 18-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 75-fold greater, at least 100-fold greater, at least 200-fold greater, or at least 300-fold greater, than a corresponding amount or activity of the pharmaceutical agent achieved in non-target organ or non-target cell of the subject.
  • the methods of delivery comprise administering a lipid composition described herein provides an effective amount or activity of a pharmaceutical agent at least 1.1 -fold greater than a corresponding amount or activity of the pharmaceutical agent achieved by administering other compositions.
  • the effective amount or activity of the pharmaceutical agent results from administering a lipid composition described herein is at least 1.1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, at least 18-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 75-fold greater, at least 100-fold greater, at least 200-fold greater, or at least 300-fold greater, than a corresponding amount or activity of the pharmaceutical agent achieved by administering other compositions.
  • the methods of delivery comprise administering a lipid described herein provides an effective amount or activity of a pharmaceutical agent at least 1.1- fold greater than a corresponding amount or activity of the pharmaceutical agent achieved by administering other lipids.
  • the effective amount or activity of the pharmaceutical agent results from administering a lipid described herein is at least 1.1-fold greater, at least 1.5-fold greater, at least 2-fold greater, at least 2.5-fold greater, at least 3-fold greater, at least 3.5-fold greater, at least 4-fold greater, at least 4.5-fold greater, at least 5-fold greater, at least 5.5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 15-fold greater, at least 18-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 75-fold greater, at least 100-fold greater, at least 200-fold greater, or at
  • the delivery of the pharmaceutical agent to a cell may cause cell death, such as apoptosis of the cell.
  • a pharmaceutical agent e.g., mRNA encoding the GPC3/CD3 BiTE
  • the pharmaceutical agent is present in the dosage form at a dose 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, .005, 0.002, or 0.001 milligram per kilogram (mg/kg, or mpk) body weight, or of a range between (inclusive) any two of the foregoing values.
  • the pharmaceutical agent is present in the dosage form at a dose of no more than about 10 milligram per kilogram (mg/kg, or mpk) body weight. In some embodiments, the pharmaceutical agent is present in the dosage form at a dose of no more than about 9 mg/kg, no more than about 8 mg/kg , no more than about 7 mg/kg, no more than about 6 mg/kg, no more than about 5 mg/kg, no more than about 4 mg/kg, no more than about 3 mg/kg, no more than about 2 mg/kg, no more than about 1 mg/kg, no more than about 0.5 mg/kg, no more than about 0.2 mg/kg, no more than about 0.1 mg/kg, no more than about 0.05 mg/kg, or no more than about 0.01 mg/kg. In some embodiments, the pharmaceutical agent is present in the dosage form at a concentration of no more than about 5 milligram per milliliter (mg/mL).
  • the pharmaceutical agent is present in the dosage form at a concentration of about 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 milligram per milliliter (mg/mL), or of a range between (inclusive) any two of the foregoing values.
  • the pharmaceutical agent is present in the dosage form at a concentration of no more than about 5 milligram per milliliter (mg/mL). In some embodiments, the pharmaceutical agent is present in the dosage form at a concentration of no more than about 2 milligram per milliliter (mg/mL). In some embodiments, the pharmaceutical agent is present in the dosage form at a concentration of no more than about 1 milligram per milliliter (mg/mL). In some embodiments, the pharmaceutical agent is present in the dosage form at a concentration of no more than about 0.5 milligram per milliliter (mg/mL). In some embodiments, the pharmaceutical agent is present in the dosage form at a concentration of no more than about 0.1 milligram per milliliter (mg/mL).
  • the pharmaceutical agent e.g., nucleic acids, mRNA
  • the pharmaceutical agent is present in the dosage form at a concentration of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, or 0.1 microgram per milliliter (pg/mL), or of a range between (inclusive) any two of the foregoing values.
  • the pharmaceutical agent is present in the dosage form at a concentration of no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about 6, no more than about 5, no more than about 4, no more than about 3, no more than about 2, no more than about 1, no more than about 0.5, no more than about 0.2, no more than about 0.1 microgram per milliliter (pg/mL).
  • Any suitable dosage form can be prepared for delivery, for example, via oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the pharmaceutical agent is administered at a dosage of no more than about 10 milligram per kilogram (mg/kg, or mpk) body weight, no more than about 9 mg/kg, no more than about 8 mg/kg , no more than about 7 mg/kg, no more than about 6 mg/kg, no more than about 5 mg/kg, no more than about 4 mg/kg, no more than about 3 mg/kg, no more than about 2 mg/kg, no more than about 1 mg/kg, no more than about 0.5 mg/kg, no more than about 0.2 mg/kg, no more than about 0.1 mg/kg, no more than about 0.05 mg/kg, or no more than about 0.01 mg/kg body weight. In some embodiments, the pharmaceutical agent is administered at a dosage from about 1 pg/kg body weight to about 3 mg/kg body weight.
  • the administration of a dose of the lipid composition provided here can be repeated.
  • the effective dose of the active lipid composition can be administered as one, two, three, four, five, six or more doses administered separately at appropriate intervals throughout the course of treatment.
  • the lipid composition can be administered two or three times daily.
  • the lipid composition will be administered once daily.
  • the lipid composition is administered about every 1 week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 9 weeks, about every 10 weeks, about every 11 weeks, about every 12 weeks, about every 13 weeks, about every 14 weeks, about every 15 weeks, about every 16 weeks, about every 17 weeks, or about every 18 weeks.
  • the lipid composition is administered about every 1 month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every 12 months, about every 13 months, about every 14 months, about every 15 months, about every 16 months, about every 17 months, about every 18 months, about every 2 years, about every 2.5 years, about every 3 years, about every 3.5 years, about every 4 years, about every 4.5 years, or about every 5 years. Any subject in need thereof can be treated with the method of the present application.
  • the subject has been determined to have a mutation in a target gene.
  • the mutation in the target gene is associated with a cancer or a tumor.
  • the subject has been determined to exhibit an aberrant expression or activity of a protein or polynucleotide that corresponds to a target gene.
  • the aberrant expression or activity of the protein or polynucleotide is associated with a cancer or a tumor.
  • the subject comprises a mammal.
  • the mammal comprises a rodent, a non-human primate, or human.
  • the subject is a non-human primate.
  • the pharmaceutical composition comprises a pharmaceutical agent (e.g., mRNA) assembled with a lipid composition as described in the present application, e.g., wherein the lipid composition comprises any of the head or tail groups disclosed herein.
  • a pharmaceutical agent e.g., mRNA
  • the contacting is ex vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the contacting comprises administering to a subject the composition comprising the therapeutic agent assembled with the lipid composition.
  • compositions and methods of the present disclosure may be utilized to treat an individual in need thereof.
  • the pharmaceutical composition described herein may comprise a therapeutic or prophylactic composition, or any combination thereof.
  • the lipidoid compositions can be assembled with mRNA encoding a therapeutic peptide (e.g., BiTE) for cancer immunotherapy.
  • the individual is a mammal (e.g., a human or a non-human mammal).
  • the composition or the lipidoid composition is preferably administered as a pharmaceutical composition comprising, for example, a lipidoid composition of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like.
  • 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 administration, such as a lotion, cream, or ointment.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a lipidoid composition such as a lipidoid composition of the invention.
  • physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent depends, for example, on the route of administration of the composition.
  • the preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system.
  • the pharmaceutical composition also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a lipidoid composition of the invention.
  • Liposomes for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • compositions, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, mannose, trehalose, and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (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, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin).
  • the lipidoid composition may also be formulated for inhalation.
  • a lipidoid composition may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the lipidoid composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety -nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an active composition, such as a lipidoid (e.g., nanoparticle) composition as described herein, with the carrier and, optionally, one or more accessory ingredients.
  • an active composition such as a lipidoid (e.g., nanoparticle) composition as described herein
  • the formulations are prepared by uniformly and intimately bringing into association a lipidoid (e.g., nanoparticle) composition as described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the pharmaceutical compositions comprising the mRNA/LNPs provided herein are administered through parenteral routes (e.g., intravenous injection or intramuscular injection).
  • compositions suitable for parenteral administration comprise one or more active lipidoid compositions in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsulated matrices of the subject lipidoid compositions in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • active lipidoid compositions can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Methods of introduction may also be provided by rechargeable or biodegradable devices.
  • Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non- degradable polymers, can be used to form an implant for the sustained release of a lipidoid composition at a particular target site.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular lipidoid composition or combination of lipidoid compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular lipidoid composition(s) being employed, the duration of the treatment, other drugs, lipidoid compositions and/or materials used in combination with the particular lipidoid composition(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. [00246] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the pharmaceutical composition or lipidoid composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • therapeutically effective amount is meant the concentration of a lipidoid composition that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the lipidoid composition will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the lipidoid composition, and, if desired, another type of therapeutic agent being administered with the lipidoid composition of the invention.
  • a suitable daily dose of an active lipidoid composition used in the compositions and methods of the invention will be that amount of the lipidoid composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective dose of the active lipidoid composition may be administered as one, two, three, four, five, six or more doses administered separately at appropriate intervals throughout the course of treatment, optionally, in unit dosage forms.
  • the active lipidoid composition may be administered two or three times daily. In some embodiments, the active lipidoid composition will be administered once daily.
  • the patient or subject receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
  • lipidoid compositions of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts.
  • contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts.
  • contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.
  • contemplated salts of the invention include, but are not limited to, l-hydroxy-2-naphthoic acid, 2, 2-di chloroacetic acid, 2-hydroxy ethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1-ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)- camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, e
  • the pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared.
  • the source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BEIT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BEIT), lec
  • kits comprising the pharmaceutical composition and/or lipid nanoparticle (LNP) formulations described herein.
  • the kit can further comprise an apparatus for administering the compositions provided herein, such as appropriate syringes for intravenous or intramuscular administration.
  • the method further comprises providing instructions for use (IFU), the IFU including instructions for administering the LNP compositions to a subject.
  • the user instruction directs a user to inject the LNP compositions comprising mRNA intravenously or intramuscularly for use in cancer immunotherapy.
  • Example 1 Lipid nanoparticle (LNP) formulation and characterization
  • test lipids were synthesized through a solvent free Michael Addition reaction between an amine head and an alkyl-acrylate lipid tail.
  • the aliphatic amine head (3,3’-Diamino-N-methyldipropylamine) and the corresponding acrylate tail were mixed at 1 to 5 molar ratio in Teflon-lined glass screw-top vials at 70 °C for 48 h.
  • the crude products were purified using a Teledyne Isco Chromatography system using the mobile phase of methanol/DCM.
  • the purified lipidiods were characterized by electrospray ionization mass spectrometry (ESI-MS).
  • the amine head was mixed with the major lipid tail at 1 to 3.5 molar ratio. After 48 hours stirring at 70 °C, three tailed lipids were purified similarly and reacted with the fourth tail subsequently at 1 : 1.5 molar ratio. The final products were isolated and verified by ESI-MS.
  • Test lipids in TABLE 3 were formulated with cholesterol, DOPC, DMG-PEG, and the mRNA encoding GPC3-CD3 BiTE (SEQ ID NO: 1).
  • component lipids ionizable cationic lipids, cholesterol, DOPC, and DMG-PEG
  • Each lipid was dissolved in ethanol and mixed to reach the specified molar ratios in the organic phase.
  • Nucleic acids were dissolved in 25 mM sodium acetate pH 5.2 buffer to reach 0.134 mg/mL.
  • aqueous and organic solutions were mixed in the NanoAssemblr® (Precision NanoSystems) at a flow rate ratio of 3: 1 (v/v; respectively) and a total flow rate of 20 mL/min.
  • the resultant mixture was dialyzed directly against at least 1000-fold volume of 10 mM Tris-HCl pH 7.5 buffer overnight using a Slide- A-Lyzer MINI Dialysis Device (MWCO, 3.5 KDa). Sucrose was then added to formulations to the final concentration of 1.32 mg/mL followed by 0.22 pm filtration and stored in -4 °C, -80 °C for short-term and long-term storage, respectively.
  • MWCO Slide- A-Lyzer MINI Dialysis Device
  • DLS Dynamic Light Scattering
  • Measurements were made using a 173° backscatter angle of detection previously equilibrated to 25 °C for 30 s in duplicates, each with 5 runs and 10 s run duration, without delay between measurements. Each measurement had a fixed position of 4.65mm in the quartz cuvette with an automatic attenuation selection. Encapsulation efficiency (EE%) was tested using the RiboGreen Assay.
  • FIG. 1 is a schematic representation of the constructs of glypican-3 (GPC3)/CD3 encoding mRNA. TABLE 4. Shows the 3 groups in the study.
  • FIG. 2 depicts a schematic diagram of dose regiments and blood collection at indicated timepoints for PK profiling of plasma GPC3/CD3 BiTE protein, BiTE mRNA and ionizable lipid.
  • FIGs. 3A-3B depicts protein PK profiling of single dose IV administration of LNP-
  • FIG. 3A shows GPC3-CD3 BiTE protein concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg LNP-GPC3/CD3 BiTE mRNA (L88-008), and 0.25 mg/kg of recombinant GPC3/CD3 BiTE protein (rPR008).
  • 5 mice in each treatment group were blood draw of indicated timepoints for plasma BiTE quantification by CD3 ELISA assay. Concentrations from technical ELISA duplicates are shown.
  • FIG. 3A shows GPC3-CD3 BiTE protein concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg LNP-GPC3/CD3 BiTE mRNA (L88-008), and 0.25 mg/kg of recombinant GPC3/CD3 BiTE protein (rPR008).
  • 5 mice in each treatment group were blood draw of indicated timepoints for plasma BiTE quantification by CD3 ELISA assay. Concentrations from technical ELISA duplicates are
  • 3B shows ex vivo concentration-dependent cytotoxicity against GPC3+ HepG2-luc cells induced by BiTE protein produced in vivo, which was collected at 48h post-dose of 0.5 mg/kg L88-008, were measured by luminescence-based assay.
  • hPBMCs were used as effectors at effector target (E:T) cell ratio of 5: 1 and detect luminescence single of target cell following 24h incubation.
  • % of specific lysis were calculated as ([number of live target cells without treatment - number of live target cells with treatment] / [number of live target cells without treatment] x 100%.
  • FIG. 4 depicts mRNA PK profiling of single dose IV administration of LNP- GPC3/CD3 BiTE mRNA in CD1 mice.
  • GPC3-CD3 BiTE mRNA concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg L88-008 were quantified by bDNA assay.
  • Data are presented as means ⁇ SD and dotted line depicts the LLOQ of 1 pg/ml
  • FIG. 5 depicts ionizable lipid L88 PK profiling of single dose IV administration of LNP-GPC3/CD3 BiTE mRNA in CD1 mice.
  • L88 lipid concentration in the plasma of CD1 mice after i.v. administration of 0.5 and 0.1 mg/kg L88-008 were quantified by LC-MS.
  • Data are presented as means ⁇ SD and dotted line depicts the LLOQ of 1 ng/ml.
  • FIG. 6 is a schematic diagram of an efficacy study design.
  • HCC Hep3B-Luciferase orthotopic hepatocellular carcinomas
  • FIGs 7A-7E depicts the results of the efficacy study.
  • Efficacy study of L88-008 was performed in in hPBMC -reconstituted Hep3B-Luciferase orthotopic HCC mouse model.
  • FIG. 7A shows the total photon flux images of individual mice in each group captured at indicated timepoints by IVIS.
  • FIG. 7B shows the bioluminescence signal quantitated as photons/sec using living Image 4.7 software for each treatment group. Each dot represent mean bioluminescence signal of each group and statistical analysis were compared to G1 PBS control group by 2-way ANOVA test with multiple comparisons analysis at D38 after tumor inoculation. **** p ⁇ 0.0001.
  • FIGs 7C-7E show total flux (TF) changes of each mouse in individual groups during the treatment were analysis.
  • FIGs 9A-9C show the liver and spleen tissue images and weight at end-timepoint (D38 after tumor inoculation).
  • FIG. 9A depicts the terminal liver and spleen tissues from each mouse collected.
  • FIG. 9B and FIG. 9C show liver including tumor tissue and spleen weighted at end-timepoint.
  • Each dots represent 1 mouse data and statistical analysis were compared to G1 PBS control by paired two-tailed t-test. Ns, not significant; * P ⁇ 0.05, ** P ⁇ 0.01. Example 5.
  • Lung-targeting LNPs were formulated with the GPC3-CD3 BiTE mRNA and lungtargeting lipids (e.g., L202, L618 and L669) and characterized as described in Examples 1-3.
  • the lung-targeting GPC3-CD3 BiTE LNPs were added to the lung cancer cell lines at 200 ng LNP- BiTE mRNA per 10,000 cells.
  • the following lung cancer cell lines were transfected: Lung Adenocarcinoma (A549), Lung squamous cell carcinoma (SK-MES-1, NCI-H226) and Small Cell Lung Cancer (NCI-H446).
  • BiTE protein expression in the cell media was quantified by ELISA.
  • the concentration of the BiTE protein (ng/ml) in the cell media of the lung cancer cell lines transfected by the LNPs e.g., L202-GPC3/CD3 BiTE, L618-GPC3/CD3 BiTE and L669-GPC3/CD3 BiTE.
  • the lung-targeting GPC3-CD3 BiTE LNPs will also be tested in animal models of various types of human lung cancers.
  • the immunodeficient B-NDG B2M mice female, 5 or 6 weeks old
  • H446- Luc human lung cancer cells with high GPC3 and luciferase expression
  • Tumor growth will be assessed by measuring the luminescence signal from H446-Luc cells using IVIS spectrum CT biophotonic imager.
  • the H446-Luc orthotopic tumor model will be established and will be groups by the values of luminescence and body weight.
  • mice Each group of mice will be intravenously (i.v.) infused with human PBMCs and followed by repeat i.v. injection of lung LNP formulated with the GPC3-CD3 mRNA or a corresponding recombinant BiTE protein control.
  • IVIS In vivo luciferase signal measured by IVIS will be quantified for monitoring tumor growth until the humane endpoints.
  • Body weight will also be monitored to assess tolerability of the LNP treatment during the study.

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Abstract

Sont divulgués des compositions, des méthodes et des kits associés à des nanoparticules lipidiques (LNP) comprenant des lipides ionisables. Les LNP peuvent comprendre des ARNm codant des peptides thérapeutiques pour l'immunothérapie anticancéreuse. Dans certains modes de réalisation, les LNP comprennent des ARNm codant un mobilisateur bispécifique de lymphocytes T (BiTE) GPC3/CD3.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022117050A1 (fr) * 2020-12-02 2022-06-09 康诺亚生物医药科技（成都）有限公司 Développement d'un nouveau médicament thérapeutique d'engageur de tumeur et son utilisation
WO2022155598A2 (fr) * 2021-01-15 2022-07-21 Trustees Of Tufts College Nanoparticules lipidiques pour l'administration ciblée d'arnm
WO2022261490A2 (fr) * 2021-06-10 2022-12-15 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2023107648A2 (fr) * 2021-12-09 2023-06-15 Massachusetts Institute Of Technology Synthèse d'ester, de carbonate et de nouveaux lipides ionisables biodégradables dérivés de carbamate à partir de ricinoléate de méthyle ou de 12-hydroxystéarate de méthyle et ses applications
WO2024026026A1 (fr) * 2022-07-27 2024-02-01 Trustees Of Tufts College Criblage in vivo à haut débit de nanoparticules lipidiques

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Publication number Priority date Publication date Assignee Title
WO2022117050A1 (fr) * 2020-12-02 2022-06-09 康诺亚生物医药科技（成都）有限公司 Développement d'un nouveau médicament thérapeutique d'engageur de tumeur et son utilisation
WO2022155598A2 (fr) * 2021-01-15 2022-07-21 Trustees Of Tufts College Nanoparticules lipidiques pour l'administration ciblée d'arnm
WO2022261490A2 (fr) * 2021-06-10 2022-12-15 Orna Therapeutics, Inc. Méthodes et compositions d'arn circulaire
WO2023107648A2 (fr) * 2021-12-09 2023-06-15 Massachusetts Institute Of Technology Synthèse d'ester, de carbonate et de nouveaux lipides ionisables biodégradables dérivés de carbamate à partir de ricinoléate de méthyle ou de 12-hydroxystéarate de méthyle et ses applications
WO2024026026A1 (fr) * 2022-07-27 2024-02-01 Trustees Of Tufts College Criblage in vivo à haut débit de nanoparticules lipidiques

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