Patent Application
20230067722
The present invention relates to the field of lipid nanoparticles (LNP); more specifically comprising an ionizable lipid, a phospholipid, a sterol, a PEG lipid and one or more nucleic acids. The LNP's of the present invention are characterized in comprising less than about 1 mol % of a PEG lipid (such as diC18-PEG2000 lipid). The present invention provides use of the LNP's for immunogenic delivery of nucleic acid molecules, specifically mRNA; thereby making them highly suitable for use in vaccines, such as for the treatment of cancer or infectious diseases. Finally, methods are provided for preparing such LNP's.
One of the major challenges in the field of targeted delivery of biologically active substances is often their instability and low cell penetrating potential. This is specifically the case for the delivery of nucleic acid molecules, in particular (m)RNA molecules. Therefore, proper packaging is crucial for adequate protection and delivery. Hence, there is a continuous need for methods and compositions for packaging biologically active substances, such as nucleic acids.
In that respect, lipid-based nanoparticle compositions such as lipoplexes and liposomes have been used as packaging vehicles for biologically active substances to allow transport into cells and/or intracellular compartments. These lipid-based nanoparticle compositions typically comprise a mixture of different lipids such as cationic lipids, ionizable lipids, phospholipids, structural lipids (such as sterols or cholesterol), PEG (polyethylene glycol) lipids, . . . (as reviewed in Reichmuth et al., 2016).
Lipid based nanoparticles composed of a mixture of 4 lipids—a cationic or ionizable lipid, a phospholipid, a sterol and a PEGylated lipid—have been developed for the non-immunogenic delivery of siRNA and mRNA to the liver after systemic administration. While many of such lipid compositions are known in the art, the ones used in mRNA delivery in vivo, typically comprise a level of PEG lipids of at least 1.5 mol %, and very often contain a diC14 based PEG lipid (DMG-PEG lipids).
We have now surprisingly found however, that PEG lipids, which are present at low amounts (i.e. less than about 1 mol %) in the LNP's, give rise to nanoparticles which are highly suitable for immunogenic delivery of mRNA upon systemic injection of the LNP's. These effects are moreover even more pronounced for the longer chain PEG lipids such as diC18-PEG lipids.
In a first aspect, the present invention provides an mRNA vaccine comprising one or more lipid nanoparticles which comprise:
In a further aspect, the present invention provides a lipid nanoparticle (LNP) for use in mRNA vaccination, said LNP comprising:
In yet a further aspect, the present invention provides a lipid nanoparticle (LNP) comprising:
In a specific embodiment of the present invention, said diC18-PEG2000 lipid is selected from the list comprising: a (distearoyl-based)-PEG2000 lipid such as DSG-PEG2000 lipid or DSPE-PEG2000 lipid; or a (dioleolyl-based)-PEG2000 lipid such as DOG-PEG2000 lipid or DOPE-PEG2000 lipid.
In a further specific embodiment of the present invention, said LNP comprises about 0.5 mol % of said PEG lipid.
In another particular embodiment of the present invention, said ionizable lipid is selected from the list comprising:
wherein:
RCOO is selected from the list comprising: myristoyl, α-D-Tocopherolsuccinoyl, linoleoyl and oleoyl; and
X is selected from the list comprising:
In a preferred embodiment, said ionizable lipid is a lipid of formula (I) wherein RCOO is α-D-Tocopherolsuccinoyl and X is
In yet a further embodiment of the present invention, said phospholipid is selected from the list comprising: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and mixtures thereof.
In yet a further embodiment of the present invention, said sterol is selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol.
In a further specific embodiment, said LNP comprises between 30-70 mol % of said ionizable lipid; preferably between 45-65 mol %.
In yet a further embodiment of the present invention, said LNP comprises about or less than 45 mol % of said sterol.
In a further embodiment, said LNP comprises between 5-25 mol % of a phospholipid; preferably between 4-15 mol %.
In a particular embodiment of the present invention, said LNP comprises:
In a very specific embodiment of the present invention, said LNP comprises:
In another very specific embodiment of the present invention, said LNP comprises:
In another very specific embodiment of the present invention, said LNP comprises:
In another very specific embodiment of the present invention, said LNP comprises:
In another very specific embodiment of the present invention, said LNP comprises:
In another embodiment of the present invention, said one or more nucleic acid molecules are selected from the list comprising mRNA and DNA, preferably mRNA.
In a more specific embodiment, said one or more mRNA molecules are selected from the list comprising immunomodulatory polypeptide-encoding mRNA and/or antigen-encoding mRNA. Said immunomodulatory-encoding mRNA may for example be selected from a list comprising mRNA molecules encoding for CD40L, CD70 and caTLR4.
In yet a further aspect, the present invention provides a pharmaceutical composition or a vaccine comprising one or more lipid nanoparticles as defined herein and an acceptable pharmaceutical carrier.
The present invention also provides the lipid nanoparticles, pharmaceutical compositions or vaccines as defined herein for use in human or veterinary medicine; in particular for use in the treatment of cancer or infectious diseases.
With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
As already detailed herein above, the present invention provides an mRNA vaccine comprising one or more lipid nanoparticles comprising:
In a particular embodiment, the present invention provides an mRNA vaccine comprising one or more lipid nanoparticles comprising:
The present invention also provides a lipid nanoparticle (LNP) for use in mRNA vaccination, said LNP comprising:
In a particular embodiment, the present invention provides a lipid nanoparticle (LNP) for use in mRNA vaccination, said LNP comprising:
Accordingly, the present invention provides LNP's comprising PEG lipids, present at a relatively low amount (e.g. less than about 1 mol %; in particular about and between 0.5-0.9 mol %), for which we have surprisingly found that these are highly suitable for immunogenic delivery of nucleic acids, specifically mRNA. Particularly, this effect was found to be even more pronounced for LNPs comprising long chain PEG lipids such as C18-PEG lipids, even more specifically C18-PEG2000 lipids. “immunogenic delivery of nucleic acid molecules” means delivery of nucleic acid molecules to cells whereby contact with cells, internalization and/or expression inside the cells of said nucleic acids molecules result in induction of an immune response.
Therefore, in a further aspect, the present invention provides a lipid nanoparticle (LNP) comprising:
In a specific embodiment, the present invention provides a lipid nanoparticle (LNP) comprising:
A lipid nanoparticle (LNP) is generally known as a nanosized particle composed of a combination of different lipids. While many different types of lipids may be included in such LNP, the LNP's of the present invention are typically composed of a combination of an ionizable lipid, a phospholipid, a sterol and a PEG lipid.
Wherever in the context of the current application, particular embodiments are provided in respect of lipid nanoparticles as disclosed herein, the limitations provided in such embodiments equally apply to the lipid nanoparticles as part of the claimed mRNA vaccines or intended for use in mRNA vaccination.
As used herein, the term “nanoparticle” refers to any particle having a diameter making the particle suitable for systemic, in particular intravenous administration, of, in particular, nucleic acids, typically having a diameter of less than 1000 nanometers (nm), preferably less than 500 nm, even more preferably less than 200 nm, such as for example between 50 and 200 nm; preferably between 80 and 160 nm.
In the context of the present invention, the term “PEG lipid” or alternatively “PEGylated lipid” is meant to be any suitable lipid modified with a PEG (polyethylene glycol) group. Particularly suitable PEG lipids in the context of the present invention are characterized in being diC18-PEG lipids. Where in the context of the invention, the term C18-PEG lipids is used, this is meant to be diC18-PEG lipids, i.e. lipids having 2 C18 lipid tails. However, also shorter chain PEG lipids, such as dC14-PiEG lipids (e.g. DMG-PEG, more in particular DMG-PEG2000; or DMPE-PEG, more in particular DMPE-PEG2000) or diC16-PEG lipids can suitably be used. diC18-PEG lipids contain a polyethylene glycol moiety, which defines the molecular weight of the lipids, as well as a fatty acid tail comprising 18 C-atoms. In a particular embodiment, said diC18-PEG2000 lipid is selected from the list comprising: a (distearoyl-based)-PEG2000 lipid such as DSG-PEG2000 lipid (2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or a (dioleolyl-based)-PEG2000 lipid such as DOG-PEG2000 lipid (1,2-Dioleolyl-rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000])
In the context of the present invention the term “ionizable” (or alternatively cationic) in the context of a compound or lipid means the presence of any uncharged group in said compound or lipid which is capable of dissociating by yielding an ion (usually an H+ ion) and thus itself becoming positively charged. Alternatively, any uncharged group in said compound or lipid may yield an electron and thus becoming negatively charged.
In the context of the present invention any type of ionizable lipid can suitably be used. Specifically, suitable ionizable lipids are ionizable amino lipids which comprise 2 identical or different tails linked via an S—S bond, each of said tails comprising an ionizable amine such as represented by
In a specific embodiment, said ionizable lipid is a compound of formula (I):
wherein:
RCOO is selected from the list comprising: myristoyl, α-D-Tocopherolsuccinoyl, linoleoyl and oleoyl; and
X is selected from the list comprising:
Such ionizable lipids may specifically be represented by anyone of the following formulae:
More specifically, said ionizable lipid is a lipid of formula (I) wherein RCOO is α-D-Tocopherolsuccinoyl and X is
such as represented by
Other suitable ionizable lipids may be selected from 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl) piperazin-1-yl)ethyl) azanediyl) bis(dodecan-2-ol) (C12-200); and dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA).
Hence, in a specific embodiment, the present invention provides a lipid nanoparticle comprising:
In the context of the present invention, the term “phospholipid” is meant to be a lipid molecule consisting of two hydrophobic fatty acid “tails” and a hydrophilic “head” consisting of a phosphate groups. The two components are most often joined together by a glycerol molecule, hence, in the phospholipid of the present invention is preferably a glycerol-phospholipid. Furthermore, the phosphate group is often modified with simple organic molecules such as choline (i.e. rendering a phosphocholine) or ethanolamine (i.e. rendering a phosphoethanolamine).
Suitable phospholipids within the context of the invention can be selected from the list comprising: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.
In a more specific embodiment, said phospholipid is selected from the list comprising: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and mixtures thereof.
Hence, in a specific embodiment, the present invention provides a lipid nanoparticle comprising:
In the context of the present invention, the term “sterol”, also known as steroid alcohol, is a subgroup of steroids that occur naturally in plants, animal and fungi, or can be produced by some bacteria. In the context of the present invention, any suitable sterol may be used, such as selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol and stigmasterol; preferably cholesterol.
Hence, in a specific embodiment, the present invention provides a lipid nanoparticle comprising:
In a very specific embodiment of the present invention, said lipid nanoparticle comprises:
In another very specific embodiment of the present invention, said lipid nanoparticle comprises:
In a specific embodiment of the present invention, said LNP comprises a ratio of ionizable lipid to phospholipid of about 8:1, alternatively about 6:1, about 4:1 or about 2:1.
In a further specific embodiment, said LNP comprises about and between 30-70 mol % of said ionizable lipid; preferably about and between 45 and 65 mol %; such as about 65 mol % about or above 45 mol %, about or above 50 mol %, about or above 55 mol %, about or above 60 mol %.
In a further embodiment, said LNP comprises between 4-25 mol % of a phospholipid; preferably between 4-15 mol %; such as for example about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, or about 15 mol %; preferably about and between 6 mol % and 9 mol %.
Hence, in a specific embodiment of the present invention one or more of the following applies:
Therefore, in a very specific embodiment of the present invention, said LNP comprises:
Where in the context of the present invention mol % is used, it is meant to be the mol % of the specified component with respect to the empty nanoparticle, i.e. without nucleic acids. This means that the mol % of a component is calculated with respect to the total amount of ionizable lipids, phospholipids, sterols and PEG lipids, present in said LNP.
In yet a further specific embodiment, the present invention provides a lipid nanoparticle comprising:
More in particular, the present invention provides a lipid nanoparticle comprising:
More in particular, the present invention provides a lipid nanoparticle comprising:
In a very specific embodiment of the present invention, said LNP comprises:
Therefore, in a very specific embodiment of the present invention, said LNP comprises:
The composition of other particularly suitable LNP's in the context of the invention is represented in table 1.
Other particularly suitable LNP's are characterized by an ionizable lipid/phospholipid/sterol/C18-PEG2000 lipid ratio of:
The inventors have found that the LNP's of the present invention are particularly suitable for the immunogenic delivery of nucleic acids. Hence the present invention provides LNP's comprising one or more nucleic acid molecules, such as DNA or RNA, more specifically mRNA.
The amount of nucleic acid in said LNP's is typically represented by the N/P ratio, i.e. the ratio of nitrogen atoms in ionizable lipids to phosphate groups in the nucleic acids. In the context of the present invention, the N/P ratio of the LNP's is about and between 4:1 and 16:1.
A “nucleic acid” in the context of the invention is a deoxyribonucleic acid (DNA) or preferably a ribonucleic acid (RNA), more preferably mRNA. Nucleic acids include according to the invention genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may according to the invention be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle. A nucleic acid can be employed for introduction into, i.e. transfection of cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and/or polyadenylation.
In the context of the present invention, the term “RNA” relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2′-position of a β-D-ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs. Nucleic acids may be comprised in a vector. The term “vector” as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial or analogs of naturally-occurring RNA.
According to the present invention, the term “RNA” includes and preferably relates to “mRNA” which means “messenger RNA” and relates to a “transcript” which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5′ untranslated region (5′-UTR), a protein or peptide coding region and a 3′ untranslated region (3′-UTR). mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.
In a specific embodiment of the present invention, said mRNA molecules are mRNA molecules encoding immune modulating proteins.
In the context of the present invention, the term “mRNA molecules encoding immune modulating proteins” is meant to be mRNA molecules encoding proteins that modify the functionality of antigen presenting cells; more in particular dendritic cells. Such molecules may be selected from the list comprising CD40L, CD70, caTLR4, IL-12p70, EL-selectin, CCR7, and/or 4-1 BBL, ICOSL, OX40L, IL-21; more in particular one or more of CD40L, CD70 and caTLR4. A preferred combination of immunostimulatory factors used in the methods of the invention is CD40L and caTLR4 (i.e. “DiMix”). In another preferred embodiment, the combination of CD40L, CD70 and caTLR4 immunostimulatory molecules is used, which is herein also named “TriMix”.
In another specific embodiment, said mRNA molecules are mRNA molecules encoding antigen- and/or disease-specific proteins.
According to the present invention, the term “antigen” comprises any molecule, preferably a peptide or protein, which comprises at least one epitope that will elicit an immune response and/or against which an immune response is directed; accordingly, the term antigen is also meant to encompass minimal epitopes from antigens. A “minimal epitope” as defined herein is meant to be the smallest structure which is capable of eliciting an immune response. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune response, which is preferably specific for the antigen or cells expressing the antigen. In particular, an “antigen” relates to a molecule which, optionally after processing, is presented by MHC molecules and reacts specifically with T lymphocytes (T cells).
In a specific embodiment, the antigen is a target-specific antigen which can be a tumor antigen, or a bacterial, viral or fungal antigen. Said target-specific antigen can be derived from either one of: total mRNA isolated from (a) target cell(s), one or more specific target mRNA molecules, protein lysates of (a) target cell(s), specific proteins from (a) target cell(s), or a synthetic target-specific peptide or protein and synthetic mRNA or DNA encoding a target-specific antigen or its derived peptides.
To avoid any misunderstanding, the LNP's of the present invention may comprise a single mRNA molecules, or they may comprise multiple mRNA molecules, such as a combination of one or more mRNA molecules encoding immune modulating proteins and/or one or more mRNA molecules encoding antigen- and/or disease-specific proteins.
In a very specific embodiment, said mRNA molecules encoding immunomodulatory molecules may be combined with one or more mRNA molecules encoding antigen- and/or disease-specific proteins. For example, the LNP's of the present invention may comprise mRNA molecules encoding the immunostimulatory molecules CD40L, CD70 and/or caTLR4 (such as Dimix or Trimix); in combination with one or more mRNA molecules encoding antigen- and/or disease-specific proteins. Thus, in a very specific embodiment, the LNP's of the present invention comprise an mRNA molecule encoding CD40L, CD70 and/or caTLR4; in combination with one or more mRNA molecules encoding antigen- and/or disease-specific proteins.
In a further aspect, the present invention provides a pharmaceutical composition comprising one or more LNP's as defined herein. Such pharmaceutical compositions are particularly suitable as a vaccine. Thus, the invention also provides a vaccine comprising one or more LNP's according to the present invention.
In the context of the present invention, the term “vaccine” as used herein is meant to be any preparation intended to provide adaptive immunity (antibodies and/or T cell responses) against a disease. To that end, a vaccine as meant herein contains at least one mRNA molecule encoding an antigen to which an adaptive immune response is mounted. This antigen can be present in the format of a weakened or killed form of a microbe, a protein or peptide, or an antigen encoding a nucleic acid. An antigen in the context of this invention is meant to be a protein or peptide recognized by the immune system of a host as being foreign, thereby stimulating the production of antibodies against is, with the purpose of combating such antigens. Vaccines can be prophylactic (example: to prevent or ameliorate the effects of a future infection by any natural or “wild” pathogen), or therapeutic (example, to actively treat or reduce the symptoms of an ongoing disease). The administration of vaccines is called vaccination.
The vaccine of the invention may be used for inducing an immune response, in particular an immune response against a disease-associated antigen or cells expressing a disease-associated antigen, such as an immune response against cancer. Accordingly, the vaccine may be used for prophylactic and/or therapeutic treatment of a disease involving a disease-associated antigen or cells expressing a disease-associated antigen, such as cancer. Preferably said immune response is a T cell response. In one embodiment, the disease-associated antigen is a tumor antigen. The antigen encoded by the RNA comprised in the nanoparticles described herein preferably is a disease-associated antigen or elicits an immune response against a disease-associated antigen or cells expressing a disease-associated antigen.
The LNP's and vaccines of the present invention are specifically intended for intravenous administration, i.e. the infusion of liquid substance directly into a vein. The intravenous route is the fastest way to deliver fluids and medications throughout the body, i.e. systemically. The present invention thus provides intravenous vaccines, as well as the use of the disclosed vaccines and LNP's for intravenous administration. The vaccines and LNP's of the present invention can thus be administered intravenously. The present invention also provides the use of the vaccines and LNP's according to the present invention; wherein the vaccine is administered intravenously.
The present invention also provides the LNP's, pharmaceutical compositions and vaccines according to this invention for use in human or veterinary medicine. The use of the LNP's, pharmaceutical compositions and vaccines according to this invention for human or veterinary medicine is also intended. Finally, the invention provides a method for the prophylaxis and treatment of human and veterinary disorders, by administering the LNP's, pharmaceutical compositions and vaccines according to this invention to a subject in need thereof.
The present invention further provides the use of an LNP, a pharmaceutical composition or a vaccine according to the present invention for the immunogenic delivery of said one or more nucleic acid molecules. As such the LNP's, pharmaceutical compositions and vaccine of the present invention are highly useful in the treatment several human and veterinary disorders. Thus, the present invention provides the LNP's, pharmaceutical compositions and vaccines of the present invention for use in the treatment of cancer or infectious diseases.
The lipid nanoparticles of the present invention may be prepared in accordance with the protocols as specified in the Examples part. More generally, the LNP's may be prepared using a method comprising:
In further detail, the lipid components are combined in suitable concentrations in an alcoholic vehicle such as ethanol. Thereto, an aqueous composition comprising the nucleic acid is added, and subsequently loaded in a microfluidic mixing device.
The aim of microfluidic mixing is to achieve thorough and rapid mixing of multiple samples (i.e. lipid phase and nucleic acid phase) in a microscale device. Such sample mixing is typically achieved by enhancing the diffusion effect between the different species flows. Thereto several microfluidic mixing devices can be used, such as for example reviewed in Lee et al., 2011. A particularly suitable microfluidic mixing device according to the present invention is the NanoAssemblr from Precision Nanosystems.
Other technologies suitable for preparing the LNP's of the present invention include dispersing the components in a suitable dispersing medium, for example, aqueous solvent and alcoholic solvent, and applying one or more of the following methods: ethanol dilution method, a simple hydration method, sonication, heating, vortex, an ether injecting method, a French press method, a cholic acid method, a Ca2+ fusion method, a freeze-thaw method, a reversed-phase evaporation method, T-junction mixing, Microfluidic Hydrodynamic Focusing, Staggered Herringbone Mixing, and the like.
Female C57BL/6 Mice were purchased from Charles River Laboratories (France) and housed in individually vented cages with standard bedding material and cage enrichment. The animals were maintained and treated in accordance to the institutional (Vrije Universiteit Brussel) and European Union guidelines for animal experimentation. Mice had ad libitum access to food and water. Experiments started when mice were 6 to 10 weeks old. Weight of mice was monitored every 2 days.
In case of vaccination with ADPGK Synthetic Long Peptide (SLP), mice were injected intraperitoneally with a combination of 50 μg ADPGK SLP (GIPVHLELASMTNMELMSSIVHQQVFPT (SEQ ID No. 3), Genscript) , 50 μg anti-CD40 Mab (Clone FJK45, BioXCell) and 100 μg pIC HMW (InvivoGen) in 200 μl of PBS at identical time intervals.
Capped, non-nucleoside modified E7 and ADPGK mRNA was prepared by eTheRNA by in vitro transcription (IVT) from the eTheRNA plasmid pEtherna, in accordance with the protocol as described in W02015071295. The sequence encoding the HPV16-E7 or ADPGK protein was cloned in-frame between the signal sequence and the transmembrane and cytoplasmic regions of human DC-LAMP. This chimeric gene was cloned in the pEtherna plasmid that was enriched with a translation enhancer at the 5′ end and an RNA stabilizing sequence at the 3′ end. After IVT, dsRNA was removed by cellulose purification. Cellulose powder was purchased from Sigma and washed in 1xSTE (Sodium Chloride-Tris-EDTA) buffer with 16% ethanol. IVT mRNA (in 1xSTE buffer with 16% ethanol) was added to the washed cellulose pellet and shaken at room temperature for 20 minutes. This solution is then brought over a vacuum filter (Corning). The eluate contains the ssRNA fraction and was used for all experiments. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium).
Lipid based nanoparticles are produced by microfluidic mixing of an mRNA solution in sodium acetate buffer (100 mM, pH4) and lipid solution in a 2:1 volume ratio at a speed of 9 mL/min using the NanoAssemblr Benchtop (Precision Nanosystems). The lipid solution contained a mixture of Coatsome-EC (NOF corporation), DOPE (Avanti), Cholesterol (Sigma) and one of the following PEG lipids: DMG-PEG2000 (C14 lipid) (Sunbright GM-020, NOF corporation), DPG-PEG2000 (C16 lipid) (Sunbright GP-020, NOF corporation), DSG-PEG2000 (C18 lipid) (Sunbright GS-020, NOF corporation). The 4 lipids were mixed at different molar ratios. LNP's were dialyzed against TBS (10000 times more TBS volume than LNP volume) using slide-a-lyzer dialysis cassettes (20K MWCO, 3 mL, ThermoFisher). Size, polydispersity and zeta potential were measured with a Zetasizer Nano (Malvern). % mRNA encapsulation was measured by ribogreen assay (ThermoFisher).
Blood was collected from treated and control mice approximately 6 days after immunization. Red blood cells were lysed and the remaining white blood cells were stained with APC labelled E7RAHYNIVTF)-tetramer (SEQ ID N° 1) or ADPGK (ASMTNMELM)-tetramer (SEQ ID N° 2) according to the manufacturer's instructions (MBL International). Excess tetramer was washed away. Hereafter, an antibody mixture for surface molecules (listed in table 2) was added to the cells and incubated for 30 minutes at 4° C. Data was acquired on an LSR Fortessa or Attune cytometer and analyzed with Flow Jo Software.
Mice received a single (
Mice received four intravenous administrations with 10 μg ADPGK mRNA packaged in a low percentage PEG LNP (50/10/39.5/0.5 ionizable lipid/DOPE/cholesterol/PEG-lipid) or with 50 μg ADPGK synthetic long peptide (SLP). Percentage of ADPGK-specific CD8+ T cells in blood was determined 6 days after the fourth immunization. DSG-PEG2000 (C18) LNP's are superior to DMG-PEG2000 (C14) LNP's in eliciting an antigen specific immune response (
All mice experiments were performed with approval from the Utrecht Animal Welfare Body of the UMC Utrecht or by the Animal Ethics Committee of Ghent University. Animal care was according to established guidelines. All mice had unlimited access to water and standard laboratory animal chow. Female C57BI/6J mice were obtained from Charles River Laboratories, Inc. (Germany/France). μMT mice were obtained from The Jackson Laboratory (USA). Non-GLP study in non-human primates were performed at Charles River Laboratories (France) according to local regulations.
Codon optimized E7, TriMix and luciferase mRNAs were prepared by eTheRNA by in vitro transcription (IVT) from eTheRNA plasmids. No nucleotide modifications were used. The E7 mRNA used in the DoE was ARCA capped. All later experiments were performed using CleanCapped mRNAs. After IVT, dsRNA was removed by cellulose purification. mRNA quality was monitored by capillary gel electrophoresis (Agilent, Belgium).
For biodistribution and cellular uptake studies, LNPs were loaded with a mixture of Firefly luciferase (Fluc) encoding mRNA (eTheRNA immunotherapies NV) and Cleancap® Cy5-labelled Fluc mRNA (TriLink Biotechnologies) in a 1:1 ratio. For the DoE immunogenicity study, LNPs were loaded with E7 mRNA. All other studies were performed with a mixture of E7, CD40L, CD70 and TLR4 mRNA in a 3:1:1:1 ratio. The mRNA was diluted in 100 mM sodium acetate buffer (pH 4) and lipids were dissolved and diluted in ethanol. The mRNA and lipid solutions were mixed using a NanoAssemblr Benchtop microfluidic mixing system (Precision Nanosystems) followed by dialysis overnight against Tris-buffered saline (TBS, 20 mM Tris, 0.9% NaCl, pH 7.4). Amicon Ultra Centrifugal Filters (10 kD) were used for concentration of LNPs. Size, polydispersity index and zeta potential was measured with a Zetasizer Nano (Malvern). mRNA encapsulation efficiency was determined via ribogreen assay (ThermoFisher). Composition of all LNPs are summarized in table 3 of example 3.
Mice were injected intravenously via the tail vein with 10 μg of mRNA in selected LNP formulations. After 4 hours, mice were anesthetized with 250 μL of pentobarbital (6 mg/mL). Blood samples were collected in tubes with gel clotting factor (Sarstedt). Subsequently, the chest cavity was opened, the portal vein was cut, and mice were perfused with 7 mL of PBS through the right ventricle. Organs were removed and snap-frozen in liquid nitrogen. For liver and spleen tissues, a part of the organ was kept in ice-cold PBS for flow cytometry analysis.
Liver and spleen tissues were placed in petri dishes with RPMI 1640 medium containing 1 mg/mL Collagenase A (Roche) or 20 μg/mL Liberase™ (Roche), respectively, and 10 μg/mL DNAse I, grade II (Roche). Tissues were minced using surgical blades and incubated for 30 min at 37° C. Subsequently, tissue suspensions were passed through 100 μm nylon cell strainers. Liver suspensions were centrifuged for 3 min at 70×g to remove parenchymal cells. Supernatants and spleen suspensions were centrifuged 7 min at 500×g to pellet cells. Red blood cells were lysed in ACK buffer (Gibco) for 5 min, inactivated with PBS, and subsequently passed through a 100 μm cell strainer. Cells were washed with RPMI 1640 containing 1% fetal bovine serum (FBS), mixed with trypan blue and counted using a Luna-II Automated Cell Counter (Logos Biosystems). 3×105 (liver) or 6×105 (spleen) live cells were seeded in 96-well plates, pelleted for 5 min at 500×g and resuspended in 2% BSA in PBS (2% PBSA) containing 50% Brilliant Stain Buffer (BD Biosciences) and 2 μg/mL TruStain FcX (BioLegend). Cells were incubated for 10 min on ice and mixed 1:1 with 2% PBSA containing applicable antibody cocktails (three in total) in duplicate. Cells were incubated for 15 min at room temperature on a shaker, washed two times with 2% PBSA and were resuspended in 2% PBSA containing 0.25 μg/mL 7-AAD Viability Stain (BioLegend). Samples were acquired on a 4-laser BD LSRFortessa flow cytometer. Analysis was sone using FlowJo software.
Approximately 50-100 mg of each tissue was dissected, weighed and placed in 2 mL microtubes with a layer of approximately 5 mm of 1.4 mm ceramic beads (Qiagen). For each mg of tissue, 3 μL of cold Cell Culture Lysis Reagent (Promega) was added, and tissues were homogenized using a Mini-BeadBeater-8 (BioSpec) at full speed for 60 s at 4° C. Homogenates were stored at −80° C., thawed, centrifuged at 10.000×g for 10 min at 4° C. to remove beads and debris, and supernatants were stored again at −80° C. Ten microliters of each lysate was aliquoted in duplicate a white 96-well plate. Using a SpectraMax iD3 platereader equipped with injector, 50 μL of Luciferase Assay Reagent (Promega) was dispensed in each well while mixing, followed by a delay of 2 seconds and luciferase emission recording for 10 s. Luciferase activity was normalized for background signal obtained from organ lysates of mice injected with TBS.
Mice were immunized intravenously via the tail vein with 10 μg of mRNA in selected LNPs in a weekly interval. Blood for flow cytometry stainings was collected 5 to 7 days after immunizations. After lysing of red blood cells, the cells were incubated with FcR block and viability dye. After incubation and washing, APC labelled E7(RAHYNIVTF)-tetramer was added and incubated at RT for 30 minutes. Excess tetramer was washed away and an antibody mixture for surface molecules CD3, CD8, was added to the cells and incubated for 30 minutes at 4° C. Samples were acquired on a 3-laser AtuneNxt flow cytometer or a 4-laser BD LSRFortessa flow cytometer.
Intracellular cytokine production was determined in spleen 7 days after the third immunization. Single cell suspensions of splenocytes were prepared by crushing the spleens, lysing the red blood cells and filtering the samples over a 40 μM cell strainer. 200.000 cells/well/sample were plated in duplicate in a 96 well plate. 4 ug of E7 peptide (Genscript) was added for stimulation before cells were incubated at 37° C. After 1 hour of peptide stimulation, GolgiPlug (BD Cytofix/Cytoperm kit (BD Biosciences)) was added. Cells were incubated for another 4 hours. Hereafter, cells were incubated with FcR block and viability dye. After incubation and washing, APC labelled E7(RAHYNIVTF)-textramer was added and incubated at RT for 30 minutes. Excess dextramer was washed away and an antibody mixture for surface molecules CD3 and CD8 was added to the cells and incubated for 30 minutes at 4° C. Further steps were according to the manufacturer's instructions of the BD Cytofix/Cytoperm kit (BD Biosciences). After permeabilization, cells were stained for IFN-γ and TNF-α. Samples were acquired on a 4-laser BD LSRFortessa flow cytometer. Analysis was done using FlowJo software.
Mice were injected intravenously via the tail vein with 5 μg of mRNA in selected LNPs. Spleens were harvested 4 hours later for flow cytometry staining. Single cell suspensions of splenocytes were prepared and incubated with digestion buffer (DMEM with DNAse-1 and collagenase-III) for 20 minutes with regular shaking. Hereafter, samples were incubated with Fc block and viability dye. After incubation and washing, cells were stained with cell lineage markers and activation markers. Samples were acquired on a 3-laser AtuneNxt flow cytometer. Analysis was done using FlowJo software.
TC-1 cells were obtained from Leiden University Medical Center. 0.5 million TC-1 cells in 50 μL PBS were injected subcutaneously on the right flank of the mice. Tumor measurements were performed using a caliper. Tumor volume was calculated as (smallest diameter2×largest diameter)/2. Ant-PD-1 and isotype control antibodies were freshly diluted in PBS to a concentration of 200 μg in 200 μL per mouse and injected intraperitoneally. Mice received either antiPD-1 antibody (monotherapy or combined with mRNA LNP immunization) or isotype control (combined with LNP immunization). Antibodies were injected every 3 to 4 days starting 3 days after the first mRNA LNP immunization and ending 2 weeks after the last LNP injection. For analysis of tumor infiltrating lymphocytes, tumors were isolated 3 days after the second mRNA LNP immunization and placed in a 24-well plate filled with MACS tissue storage buffer (Miltenyi Biotec). Tumors were minced and incubated in digestion buffer for 1 hour with regular shaking. Hereafter, red blood cells were lysed and all samples were filtered over a 70 μM cell strainer. Lymphocytes were enriched by ficoll-paque density gradient purification before proceeding with staining. First, the cells were incubated with FcR block and viability dye. After incubation and washing, APC labelled E7(RAHYNIVTF)-tetramer was added and incubated at RT for 30 minutes. Excess tetramer was washed away and an antibody mixture for surface molecules CD45 and CD8 was added to the cells and incubated for 30 minutes at 4° C. Samples were acquired on a 3-laser AtuneNxt flow cytometer. Analysis was done using FlowJo software.
Blood samples were collected in tubes with gel clotting factor (Sarstedt). Clotted blood samples were centrifuged for 5 min at 10.000g to obtain serum. Serum samples were stored at −80° C. until analysis. ProcartaPlex multiplex assay (ThermoFisher) was used to determine concentration of inflammatory cytokines, such as IFN-γ, TNF-α, IP-10. Serum samples were diluted 3 times in assay buffer and incubated with fluorescently labelled beads for 120 minutes. Further steps were performed according to protocol. Samples were acquired on a MagPix instrument (Luminex). Data was analysed using ProcartaPlex Analyst software.
LNP-libraries were created by combining the commercially available ionizable lipid Coatsome SS-EC with cholesterol, DOPE and a PEGylated lipid. DOPE is already part of several approved liposomal products and mRNA-vaccines under investigation. For the current experiment, different LNP compositions comprising DSG-PEG2000 lipids were explored. The differential behavior of PEG-lipids was described to have strong influence on the pharmacokinetics and pharmacodynamics of siRNA LNPs upon i.v. administration.
A first LNP-library was designed to address whether lipid molar ratios and PEG-lipid chemistry indeed impact the T-cell response elicited by i.v. mRNA-LNP-vaccination and hence represent variables that can be optimized to improve vaccine potency. The molar percentages of SS-EC, DOPE and PEG-lipid were considered as independent variables, whereas cholesterol was considered a filler lipid to balance the molar percentage to 100%. By using DOE-methodology, an experimental design involving 11 LNPs was created (see composition in table 3).
The 11 lipid ratios were uniformly distributed in the experimental domain (data not shown). For immunogenicity screening, the percentage of E7-specific CD8 T cells in blood after three i.v. immunizations was considered the response variable to be maximized. To this end, all LNPs packaged mRNA encoding the Human Papillomavirus 16 (HPV16) oncoprotein E7 as an antigen. Results confirm our assumption that the magnitude of the CD8 T-cell response is strongly dependent on the LNP-composition. Several mRNA-LNP-vaccines gave rise to over 50% of E7-specific CD8 T cell responses, whereas other mRNA-LNP-vaccines induced hardly any response (
Bayesian regression modelling was applied to the data to create response surface models (data not shown) that can predict the immunogenicity of a certain LNP-composition. The quality of the response surface models for each of the PEG-lipid chemistries is reflected by the coefficient of determination R2, which indicated the capacity of the model to explain variability in T-cell responses based on the input variables (% SS-EC, DOPE and PEG-lipid). For DSG-PEG2000 LNPs, mean R2 values of 0.74 were obtained To validate the predictive value of the models, 2 new LNP-compositions (table 4) were assessed.
Mice immunized with LNP36 (DSG-PEG2000) had an over 90% probability to elicit>30% E7-specific CD8 T cells (optimal LNPs), whereas LNP37 (DSG-PEG2000) was predicted to yield poor T-cell responses (non-optimal LNPs) (
The success of cancer immunotherapy is impacted by a multitude of factors, including the T cell phenotype, functionality and tumor infiltration. We first assessed the quality and boostability of the T-cell response evoked by the optimal LNPs. To this aim, mice received three prime immunizations at days 0, 7 and 14 followed by a final immunization at day 50. E7 mRNA was supplemented with TriMix, a mix of 3 immunostimulatory mRNAs Bonehill et al., 2008), which increases the strength of the T-cell response.
Following 3 immunizations with E7-TriMix, over 70% of E7-specific T cells were present in blood (
To assess T-cell functionality, we performed an intracellular cytokine staining after three immunizations with LNP36. Polyfunctional CD8 T cells, who produce more than one cytokine simultaneously, are associated with better control of infectious diseases and tumors and accounted for approximately 30% of E7-specific CD8 T cells (
Therapeutic antitumor efficacy was assessed in syngeneic mouse tumor model TC-1, generated by retroviral transduction with HPV16 E6/E7 antigens. Treatment with 5 μg E7-TriMix delivered by LNP36 was initiated when tumors reached a mean diameter of 55 mm3. In addition, mice were treated with anti-PD-1 (or isotype control antibody). PD-1 is expressed on activated T cells and upon interaction with PD-L1 inhibits T cell function and induces tolerance. PD-1 checkpoint blockade sustains T-cell reactivity and is approved for the first line treatment of patients with metastatic or unresectable recurrent HNSCC. LNP36 vaccination resulted in profound regression of TC-1 tumors (
Finally, we assessed the capacity of the vaccine elicited T cells to reach the tumorbed. Two vaccinations with the respective mRNA-LNP-vaccines led to a strong infiltration of CD8+ tumor-infiltrating T cells into the tumor (
To address whether correlations exist between the magnitude of the evoked T-cell response and the biodistribution of mRNA uptake and expression at the organ and cell type level, we encapsulated Cy5-labeled Firefly Luciferase mRNA in the DSG-PEG2000 LNPs that were previously screened for immunogenicity. Luciferase activity was measured in isolated liver, spleen, lungs, heart and kidneys four hours after LNP-injection. As anticipated, LNP-composition had strong impact on the intensity and organ specificity of mRNA-expression. Liver was the primary target organ, followed by the spleen, but the ratio liver to spleen differed strongly between LNPs (
We next assessed whether immunogenicity is linked to early mRNA-uptake and activation of specific immune cell types in the spleen. LNPs accumulated mainly in macrophages and monocytes (
To further validate the importance of mRNA-uptake and expression in the spleen we compared the biodistribution and cellular uptake profiles of the optimal, highly immunogenic LNPs (LNP36) with the non-optimal, poorly immunogenic LNPs (LNP37). Relative to non-optimal LNPs, LNP36 dramatically increased mRNA-expression in the spleen (
Compared to LNP37, formulated with 1,5% DSGPEG2000, the optimal mRNA LNP composition LNP36 triggered higher levels of inflammatory cytokines in blood and elevated expression of CD86 on splenic DC subsets (
Recently a pilot study in non-human primates (NHP) was executed to evaluate the translational value of the optimal LNPs (LNP36) In NHP, spleen showed the highest accumulation of E7 mRNA per g tissue, followed by liver and bone marrow. (
LNP composition is a critical determinant of the T cell response evoked upon systemic administration of mRNA vaccines. LNPs having DSG-PEG2000 as the LNP stabilizing PEG-lipid elicited increased T cell responses compared to DPG-PEG2000 and DMG-PEG2000 containing LNPs. Furthermore, reducing the molar percentage of DSG-PEG2000 to 0,5-0,9% strongly increased the T cell response. mRNA vaccines delivered by such optimized LNP compositions induce high magnitude/high quality T cell responses that can be boosted by repeated administration and confer antitumor efficacy in murine syngeneic tumor models. Mechanistically, optimal LNP compositions are characterized by increased mRNA expression in the spleen, involving increased mRNA uptake by various antigen presenting cell types.
Optimal LNP formulations trigger increased activation of splenic dendritic cells and result in enhanced release of IFN-a and IP-10 in the blood.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20152938.5 | Jan 2020 | EP | regional |
| 20152995.5 | Jan 2020 | EP | regional |
| 20179434.4 | Jun 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/051290 | 1/21/2021 | WO |
