Patent Application
20250235532
The present application claims the right of priority of European patent application 22158324 filed with the European Patent Office on 23 Feb. 2022, the entire content of which is incorporated herein for all purposes.
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
The present invention relates to a polymer-lipid hybrid nanoparticles comprising a lipid and a block copolymer, wherein the amount of said lipid expressed in mole percentage (mole %) present in the polymer-lipid hybrid nanoparticle is greater than the amount of said block copolymer expressed in mole percentage present in the polymer-lipid hybrid nanoparticle. The invention also relates to such a polymer-lipid hybrid nanoparticle/s further comprising a soluble encapsulated antigen/s, wherein said soluble encapsulated antigen/s is a protein/s and/or polynucleotide/s. The invention further relates to a method of encapsulating such an antigen/s in such a polymer-lipid hybrid nanoparticles as well as to a composition/s comprising such a polymer-lipid hybrid nanoparticle/s and uses of such a polymer-lipid hybrid nanoparticle/s and/or composition/s as a vaccine, a pharmaceutical, means of targeting cells, tissues and/or organs and/or non-viral delivery system capable of delivering nucleotides, e.g., to inside a cell. In particular, in the course of the present invention, synthetic polymer-lipid hybrid nanoparticle comprising block copolymer PBD-PEO (non-degradable) or Poly(ε-caprolactone)-poly (ethylene glycol) (PCL-PEO) (biodegradable) have been explored as novel platform for polynucleotide (e.g., mRNA) delivery.
Although immunization is a well-established process, there are differences in the response level elicited between different immunogens or antigens. For example, membrane proteins form a class of antigens that produce a low response level, which in turn means that large amounts of membrane proteins are required to generate or elicit an immune response to the desired level. Membrane proteins are notoriously difficult to synthesize and are insoluble in water without the presence of a detergent. This makes it expensive and difficult to obtain membrane proteins in sufficient quantity for immunization. Furthermore, membrane proteins require proper folding to function correctly. The immunogenicity of correctly folded native membrane proteins is typically much better than that of their solubilized forms, which may not be folded in a physiologically relevant manner. Thus, even though adjuvants may be used to boost the immunogenicity of such solubilized antigens, it is an inefficient method that does not provide too much of an advantage (e.g., WO2014/077781A1).
Although transfected cells and lipid-based systems have been used to present membrane protein antigens to increase the chances of isolating antibodies that may be efficient in vivo, these systems are often unstable (e.g., oxidation sensitive), tedious and costly. Moreover, the current state of the art for such membrane protein antigens is to use inactive virus-like particles for immunization.
On the other hand, vaccines are the most efficient way to prevent diseases, mainly infectious diseases [e.g., Liu et al., 2016]. As of today, most of the licensed vaccines are made of either live or killed viruses. Despite their effectiveness in generating a humoral response (an antibody mediated response) to prevent viral propagation and entry into cells, safety of such vaccines remains a concern. In the past few decades, scientific advances have helped to overcome such issues by engineering vaccine vectors that are non-replicating recombinant viruses. In parallel, protein based antigens or sub-unit antigens have been explored as safer alternatives. However, such protein based vaccines typically illicit poor immune (both humoral and cellular response). To improve immunogenic properties of antigens, several approaches have been used. For example, microencapsulation of antigens into polymers has been investigated extensively, although it did enhance the immunogenicity, aggregation and denaturing of antigens remain unsolved [e.g., Hilbert et al., 1999]. Furthermore, adjuvants (e.g., oil in water emulsions or polymer emulsions) [e.g., U.S. Pat. No. 9,636,397B2, US2015/0044242 A1] are used together with antigens to elicit a more pronounced humoral and cellular response. Despite these advances, they are less efficient in uptake and cross-presentation. To promote cross-presentation, based on the available information of the immune system during infection by viruses, viral like particles that mimic such properties have been exploited. Synthetic architectures such as liposomes with encapsulated antigens are particularly attractive. Liposomes are unilamellar self-assembling structures made of lipids and, cationic liposomes are more attractive and promising as delivery vehicles because of their efficient uptake by Antigen Presenting Cells (APCs) [e.g., Maji et al., 2016]. Furthermore, it allows integrating immunomodulators such as Monophosphoryl Lipid A (MPL), CpG oligodeoxynucleotide, that are toll-like receptor (TLR) agonists which stimulate immune cells through receptors. Despite these opportunities of such delivery vehicles, one of the limiting factors is stability of liposomes in the presence of serum components. By PEGylations, loading with high melting temperature lipids, stability issues of liposomes are somewhat reduced with and one such well characterized example being inter bilayered-crosslinked multilamellar vesicles (ICMVs), formed by stabilizing multilamellar vesicles with short covalent crosslinks linking lipids [e.g., Moon et al., 2011]. Other nanoparticle architectures have led to successful immunisations using nanodiscs [e.g., Kuai et al., 2017] or pH sensitive particles [e.g., Luo et al., 2017]. But such strategies either still requires adjuvants or are not as efficient outside the prototypical Ovalbumin (OVA) models.
In addition, polymersomes, offer as a stable alternative for liposomes and they have been used to integrate membrane proteins to elicit immune response [e.g., Quer et al., 2011, WO2014/077781A1]. Protein antigens were also encapsulated in a chemically altered membrane of the polymersome (however oxidation-sensitive membranes) to release antigens and the adjuvants to dendritic cells [e.g., Stano et al., 2013].
On the other hand messenger RNA (mRNA) has arisen as a promising strategy for prevention and treatment of various diseases including infections, cancer and gene disorders. However, the clinical translation of mRNA therapeutic is impeded by its instability and inefficient in vivo delivery. Recently, advanced lipid nanoparticles (LNP) systems have demonstrated potency in preclinical trials and successfully entered the clinics. For instance, LNP-Onpattro (LNP-ON) containing DMG-PEG, DSPC, MC3 and Chol (at a ratio 1.5:10.0:50: 38.5) has been approved by the FDA in 2018, where a therapeutic siRNA (pastisiran) was encapsulated for treatment of hereditary mediated amyloidosis. To date, many other LNPs have been developed for mRNA delivery. Notably, mRNA-1273 and BNT162b have been used in clinics globally for the prevention of coronavirus disease 2019 (COVID-19). Despite some positive results, it remains a challenge to maintain the long-term stability and potency of mRNA loaded LNP. For instance, mRNA-1273 and BNT162b are recommended to be stored at −80° C. and −20° C., respectively. As cold chain transportation and storage are not available in many areas, there is an urgent need to develop therapeutics with enhanced long-term stability.
Accordingly, despite this progress made by the use of polymers, there remains a need to provide for efficient and stable uptake, delivery and/or stable cross-presentation delivery vehicles/systems and methods based thereon that overcome, or at least alleviate, the above problems as well as possess an improved functionality inter alia in that they are also capable of eliciting a CD8(+) T cell-mediated immune response, which is particularly important in treatment and/or prevention of infectious diseases, cancers and autoimmune diseases.
The present invention relates to a polymer-lipid hybrid nanoparticle comprising a lipid and a block copolymer, wherein the amount of said lipid, expressed in mole percentage (i.e., a mole %) present in the polymer-lipid hybrid nanoparticle, wherein the mole percentage refers to the total amount of all components that form the polymer-lipid nanoparticle, is greater than the amount of said block copolymer, expressed in mole percentage, present in the polymer-lipid hybrid nanoparticle.
The present invention further relates to such a polymer-lipid hybrid nanoparticle, wherein the lipid (e.g., ionizable lipid) is selected from a group consisting of: an ionizable lipid DLin-MC3-DMA (also referred to as MC3) and an ionizable lipid C12-200. The present invention further relates to such a polymer-lipid hybrid nanoparticle, wherein the block copolymer is selected from a group consisting of: poly(butadiene)-b-poly(ethylene glycol) (PBD-PEO) block copolymer, poly caprolactone (PCL)-PEO block copolymer, poly(Lactide-co-glycolide) (PLGA)-PEO (e.g., with various LA to GA ratios) and DMG-PEG block copolymer. The present invention further relates to such a polymer-lipid hybrid nanoparticle, wherein a mole % ratio of the lipid to the block copolymer is between 31.8 to 12 and about 35 to 2.5. The present invention further relates to such a polymer-lipid hybrid nanoparticle, further comprising a stabilizer, e.g., comprising or consisting of cholesterol (also referred to as CHOL). The present invention further relates to such a polymer-lipid hybrid nanoparticle, further comprising another lipid, wherein said another lipid is selected from a group consisting of: DMPC, DSPC, DOPE, DOTAP, DODAP, DOTMA, DODMA, DDA, 18:1 PA (1,2-dioleoyl-sn-glycero-3-phosphate), 14:0 PA (1,2-dimyristoyl-sn-glycero-3-phosphate), 18:1 BMP (bis(monooleoylglycero)phosphate). The present invention further relates to such a polymer-lipid hybrid nanoparticle, consisting of: (i) PBD-PEO, MC3, CHOL; (ii) PBD-PEO, C12-200, CHOL; (iii) PBD-PEO, DOPE, C12-200, CHOL; (iv) PBD-PEO, DOPE, C12-200, CHOL; (v) PBD-PEO, DOPE, C12-200, CHOL; (vi) PBD-PEO, DOPE, C12-200, CHOL; (vii) DMG-PEG, DSPC, MC3, CHOL; (viii) PCL-PEO, DMPC, MC3, CHOL; (ix) PCL-PEO, DMPC, MC3, CHOL; (x) PCL-PEO, DMPC, MC3, CHOL; or (xi) PCL-PEO, DMPC, MC3, CHOL. The present invention further relates to such a polymer-lipid hybrid nanoparticle, further comprising a soluble encapsulated antigen, wherein said soluble encapsulated antigen is a protein and/or polynucleotide.
The present invention further relates to a composition comprising such a polymer-lipid hybrid nanoparticle.
The present invention further relates to a method of delivering nucleotide/s to inside a cell without using viral vector/s as delivery means, said method comprising: (i) providing the polymer-lipid hybrid nanoparticle and/or composition of the present invention; and (ii) contacting said polymer-lipid hybrid nanoparticle and/or composition with a cell.
Illustrative polymer-lipid hybrid nanoparticles of the present invention exhibit favorable physicochemical properties and/or superior encapsulation efficiency (˜100%). In comparison to benchmark LNP-ON (i.e., LNP-Onpattro or LNP-ONP, which can be used interchangeably herein), the polymer-lipid hybrid nanoparticles of the present invention outperform and enhance in vitro transfection efficacy and/or long term thermostability of polynucleotides (e.g., mRNA), Moreover, polymer-lipid hybrid nanoparticles formulations of the present invention display less cytotoxicity as compared to benchmark LNP-ON. Furthermore, illustrative polymer-lipid hybrid nanoparticles formulations of the present invention can strongly activate cDC1 and cDC2 in the lymph nodes to promote antigen surface presentation. Taken together, the present invention provides a novel class of polymer lipid hybrid nanoparticles with efficient protein and antigen expression as well as enhanced thermostability, which makes them suitable for delivery of therapeutic mRNA over a wide range of diseases.
Therefore, the present invention satisfies this demand by provision of stable polymer-lipid hybrid nanoparticles comprising a lipid and a block copolymer as described herein, methods based thereon as well as methods for their production and compositions comprising such a polymer-lipid hybrid nanoparticle, described herein, characterized in the claims and illustrated by the appended Examples and Figures.
The following detailed description refers to the accompanying Examples and Figures that show, by way of illustration, specific details and embodiments, in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized such that structural, logical, and eclectic changes may be made without departing from the scope of the invention. Various aspects of the present invention described herein are not necessarily mutually exclusive, as aspects of the present invention can be combined with one or more other aspects to form new embodiments of the present invention.
A messenger RNA (mRNA) has arisen as a promising strategy for prevention and treatment of various diseases including infections, cancer and gene disorders.
However, the clinical translation of mRNA therapeutic is impeded by its instability and inefficient in vivo delivery. Recently, advanced lipid nanoparticles (LNP) systems have demonstrated potency in preclinical trials and successfully entered the clinics. For instance, LNP-Onpattro (LNP-ON or LNP-ONP) containing DMG-PEG, DSPC, MC3 and Chol (1.5:10.0:50: 38.5) has been approved by the FDA in 2018, where therapeutic siRNA (pastisiran) was encapsulated for treatment of hereditary mediated amyloidosis. To date, many other LNPs have been developed for mRNA delivery. Notably, mRNA-1273 and BNT162b have been used in clinics globally for the prevention of coronavirus disease 2019 (COVID-19).
However, despite the positive results, it remains a challenge to maintain the long-term stability and potency of mRNA loaded LNP. For instance, mRNA-1273 and BNT162b are recommended to be stored at −80° C. and−20° C., respectively. As cold chain transportation and storage are not available in many areas, there is an urgent need to develop therapeutics with enhanced long-term stability.
In the course of the present invention, synthetic polymer-lipid hybrid nanoparticles comprising PBD-PEO (non-degradable) or Poly(ε-caprolactone)-poly(ethylene glycol) (PCL-PEO) (biodegradable) block copolymer, PLGA-PEO have been explored as novel platform for polynucleotide (e.g., mRNA) delivery.
PBD-PEO, PCL-PEO, PLGA-PEO polymer-lipid hybrid nanoparticle were synthesized with well-defined molecular weight and narrow polydispersity. For example, such synthetic polymers can be integrated with helper lipid and ionized lipid and formulated to create a new class of polymer-lipid hybrid nanoparticles (e.g., PBD-PEO polymer lipid hybrid nanoparticles can be interchangeably referred to as “BNPs” herein and PCL-PEO polymer lipid hybrid nanoparticles can be interchangeably referred to as “PCLs” herein) for e.g., mRNA delivery. The effects of compositions and N/P ratios (N in the ionized cationic lipid and P in mRNA) on the performance of the BNPs (prepared by solvent dispersion method) was systematically evaluated in terms of particle size, polydispersity, surface charge, morphology, encapsulation efficiency, loading level and in vitro transfection. The optimal formulation was further produced by Precision Nanosystem Incorporation NanoasembirPatform (PNI). The in vivo delivery efficacy of BNPs and PCLs was further evaluated using Luciferase protein expression model in mice. Importantly, the optimum formulation demonstrated potent mRNA delivery both in vitro and in vivo yet with enhanced storage stability as compared to benchmark LNP-ON. Overall, BNPs demonstrated great potential for delivery of therapeutic mRNA.
In the present context, the term “polynucleotide” (also “nucleic acid”, which can be used interchangeably with the term “polynucleotide”) refers to macromolecules made up of nucleotide units which e.g., can be hydrolysable into certain pyrimidine or purine bases (usually adenine, cytosine, guanine, thymine, uracil), d-ribose or 2-deoxy-d-ribose and phosphoric acid. Non-limiting examples of “polynucleotide” include DNA molecules (e.g. cDNA or genomic DNA), RNA (e.g., siRNA, an mRNA, guide RNA or self-amplifying mRNA (saRNA)), oligonucleotide (e.g., antisense oligonucleotide), combinations thereof or hybrid molecules comprised of DNA and RNA. The nucleic acids can be double- or single-stranded and may contain double- and single-stranded fragments at the same time. Most preferred are double stranded DNA molecules and mRNA molecules.
In the present context, the term “antisense oligonucleotide” refers to a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid which is present in a normal cell or in an affected cell. Exemplary “antisense oligonucleotide” include antisense RNA, SiRNA, RNAi.
In the present context, polymersomes are vesicles with a polymeric membrane, which are typically, but not necessarily, formed from the self-assembly of dilute solutions of one or more amphiphilic block copolymers, which can be of different types such as diblock and triblock (A-B-A or A-B-C). Polymersomes may also be formed of tetra-block or penta-block copolymers. For tri-block copolymers, the central block is often shielded from the environment by its flanking blocks, while di-block copolymers self-assemble into bilayers, placing two hydrophobic blocks tail-to-tail, much to the same effect. In most cases, the vesicular membrane has an insoluble middle layer and soluble outer layers. The driving force for polymersome formation by self-assembly is considered to be the microphase separation of the insoluble blocks, which tend to associate in order to shield themselves from contact with water. Polymersomes possess such properties due to the large molecular weight of the constituent copolymers. Vesicle formation is favored upon an increase in total molecular weight of the block copolymers. As a consequence, diffusion of the (polymeric) amphiphiles in these vesicles is very low compared to vesicles formed by lipids and surfactants. Owing to this less mobility of polymer chains aggregated in vesicle structure, it is possible to obtain stable polymersome morphologies. Unless expressly stated otherwise, the term “polymersome” and “vesicle”, as used herein, are taken to be analogous and may be used interchangeably. Importantly, a polymersome can be formed from either one kind of block copolymers or from two or more kinds of block copolymers, meaning a polymersome can also be formed from mixtures of polymersomes and thus can contain two or more block copolymers.
In the present context, polymer-lipid hybrid nanoparticles of the present invention comprising a lipid and a block copolymer, wherein the amount of said lipid, expressed in mole percentage (mole %) present in the polymer-lipid hybrid nanoparticle, wherein the mole percentage refers to the total amount of all components that form the polymer-lipid nanoparticle is greater than the amount of said block copolymer, expressed in mole percentage, present in the polymer-lipid hybrid nanoparticle. Such polymer-lipid hybrid nanoparticles are not polymersomes. They may have electro-lucent amorphous internal structure surrounded by a peripheral bilayer. Exemplary polymer-lipid hybrid nanoparticles of the present invention having one or more of the following characteristics: (i) a diameter greater than 75 nm, e.g., said diameter ranging from about 80 nm to about 450 nm or said diameter ranging from about 80 nm to about 140 nm, or said diameter ranging from about 100 nm to about 140 nm (The diameter can, for example, be determined by a dynamic light scattering (DLS) instrument using Z-average (d, nm), a preferred DLS parameter. Z-average size is the intensity weighted harmonic mean particle diameter (cf.
The polymer-lipid hybrid nanoparticle of the present invention, may comprise a soluble encapsulated antigen, wherein said soluble encapsulated antigen is a protein and/or polynucleotide, preferably said protein is a nuclease involved in gene- or RNA-editing, polynucleotide is selected from a RNA (e.g., siRNA, an mRNA, guide RNA or self-amplifying mRNA (saRNA)) molecule or a DNA molecule.
In the present context, the term “encapsulated” means enclosed by a membrane (e.g., membrane of the polymer-lipid hybrid nanoparticle of the present invention, e.g., embodied inside the lumen of said polymer-lipid hybrid nanoparticle). With reference to an antigen the term “encapsulated” further means that said antigen is neither integrated into-nor covalently bound to-nor conjugated to said membrane (e.g., of a polymer-lipid hybrid nanoparticle of the present invention).
In the present context, the term “antigen” means any substance that may be specifically bound by components of the immune system. Only antigens that are capable of eliciting (or evoking or inducing) an immune response are considered immunogenic and are called “immunogens”. Exemplary non-limiting antigens are proteins and polynucleotides. Exemplary non-limiting protein antigen is a nuclease involved in gene- or RNA-editing. Exemplary non-limiting polynucleotide is selected from a RNA (e.g., siRNA, an mRNA (e.g., as set forth in SEQ ID NOs: 1, 2 or 3), guide RNA or self-amplifying mRNA (saRNA) molecule or a DNA molecule. The antigen may originate from within the body (“self-antigen”) or from the external environment (“non-self”).
The term “polypeptide” is equally used herein with the term “protein”. Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term “polypeptide” as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms “polypeptide” and “protein” also refer to naturally modified polypeptides/proteins wherein the modification is effected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
In the present context, the term “CD8 (+) T cell-mediated immune response” refers to the immune response mediated by cytotoxic T cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cells, cytolytic T cells, CD8 (+) T-cells or killer T cells). Example of cytotoxic T cells include, but are not limited to antigen-specific effector CD8 (+) T cells. In order for the T-cell receptors (TCR) to bind to the class I MHC molecule, the former must be accompanied by a glycoprotein called CD8, which binds to the constant portion of the class I MHC molecule.
Therefore, these T cells are called CD8 (+) T cells. Once activated, the TC cell undergoes “clonal expansion” with the help of the cytokine Interleukin-2 (IL-2), which is a growth and differentiation factor for T cells. This increases the number of cells specific for the target antigen that can then travel throughout the body in search of antigen-positive somatic cells.
In the present context, the term “cellular immune response” refers to an immune response that does not involve antibodies, but rather involves the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.
In the present context, the term “humoral immune response” refers to an immune response mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Its aspects involving antibodies are often called antibody-mediated immunity.
In the present context, the term “stabilizer” may refer to a substance that renders or maintains a solution, mixture (e.g., polymer-lipid hybrid nanoparticle), suspension or state resistant to chemical change. Exemplary non-limiting stabilizers of the present invention comprise or consist of cholesterol, substituted or unsubstituted cholesterol moiety, or cholesterol derivative, preferably said cholesterol derivative is a hydroxylated cholesterol derivative (e.g., a hydroxycholesterol).
In the present context, the term “B cells”, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system by secreting antibodies.
An “antibody” when used herein is a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term “immunoglobulin” (lg) is used interchangeably with “antibody” herein. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. In particular, an “antibody” when used herein, is typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, IgA1, and IgA2, with IgG being preferred in the context of the present invention. An antibody relating to the present invention is also envisaged which has an IgE constant domain or portion thereof that is bound by the Fc epsilon receptor I. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The constant domains are not involved directly in binding an antibody to an antigen, but can exhibit various effector functions, such as participation of the antibody dependent cellular cytotoxicity (ADCC). If an antibody should exert ADCC, it is preferably of the IgG1 subtype, while the lgG4 subtype would not have the capability to exert ADCC.
The term “antibody” also includes, but is not limited to, but encompasses monoclonal, monospecific, poly- or multi-specific antibodies such as bispecific antibodies, humanized, camelized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies, with chimeric or humanized antibodies being preferred. The term “humanized antibody” is commonly defined for an antibody in which the specificity encoding CDRs of HC and LC have been transferred to an appropriate human variable frameworks (“CDR grafting”). The term “antibody” also includes scFvs, single chain antibodies, diabodies or tetrabodies, domain antibodies (dAbs) and nanobodies. In terms of the present invention, the term “antibody” shall also comprise bi-, tri- or multimeric or bi-, tri- or multifunctional antibodies having several antigen binding sites.
Furthermore, the term “antibody” as employed in the invention also relates to derivatives of the antibodies (including fragments) described herein. A “derivative” of an antibody comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions or additions. Additionally, a derivative encompasses antibodies which have been modified by a covalent attachment of a molecule of any type to the antibody or protein. Examples of such molecules include sugars, PEG, hydroxyl-, ethoxy-, carboxy- or amine-groups but are not limited to these. In effect the covalent modifications of the antibodies lead to the glycosylation, pegylation, acetylation, phosphorylation, amidation, without being limited to these.
The antibody relating to the present invention is preferably an “isolated” antibody. “Isolated” when used to describe antibodies disclosed herein, means an antibody that has been identified, separated and/or recovered from a component of its production environment. Preferably, the isolated antibody is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.
The term “amino acid” or “amino acid residue” typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).
“Polyclonal antibodies” or “polyclonal antisera” refer to immune serum containing a mixture of antibodies specific for one (monovalent or specific antisera) or more (polyvalent antisera) antigens which may be prepared from the blood of animals immunized with the antigen or antigens.
The term “immunizing” refers to the step or steps of administering one or more antigens to a non-human animal so that antibodies can be raised in the animal.
Specifically, the non-human animal is preferably immunized at least two, more preferably three times with said polypeptide (antigen), optionally in admixture with an adjuvant. An “adjuvant” is a nonspecific stimulant of the immune response. The adjuvant may be in the form of a composition comprising either or both of the following components: (a) a substance designed to form a deposit protecting the antigen(s) from rapid catabolisme.g. mineral oil, alum, aluminium hydroxide, liposome or surfactant (e.g. pluronic polyol) and (b) a substance that nonspecifically stimulates the immune response of the immunized host animal (e.g. by increasing lymphokine levels therein).
Exemplary molecules for increasing lymphokine levels include lipopolysaccaride (LPS) or a Lipid A portion thereof; Bordetalla pertussis; pertussis toxin; Mycobacterium tuberculosis; and muramyl dipeptide (MDP). Examples of adjuvants include Freund's adjuvant (optionally comprising killed M. tuberculosis; complete Freund's adjuvant); aluminium hydroxide adjuvant; and monophosphoryl Lipid A-synthetic trehalose dicorynomylcolate (MPL-TDM).
The “non-human animal” to be immunized herein is preferably a rodent. A “rodent” is an animal belonging to the rodentia order of placental mammals. Exemplary rodents include mice, rats, guinea pigs, squirrels, hamsters, ferrets etc, with mice being the preferred rodent for immunizing according to the method herein. Other non-human animals which can be immunized herein include non-human primates such as Old World monkey (e.g. baboon or macaque, including Rhesus monkey and cynomolgus monkey; see U.S. Pat. No. 5,658,570); birds (e.g. chickens); rabbits; goats; sheep; cows; horses; pigs; donkeys; dogs etc.
By “screening” is meant subjecting one or more monoclonal antibodies (e.g., purified antibody and/or hybridoma culture supernatant comprising the antibody) to one or more assays which determine qualitatively and/or quantitatively the ability of an antibody to bind to an antigen of interest.
By “immuno-assay” is meant an assay that determines binding of an antibody to an antigen, wherein either the antibody or antigen, or both, are optionally adsorbed on a solid phase (i.e., an “immunoadsorbent” assay) at some stage of the assay. Exemplary such assays include ELISAs, radioimmunoassays (RIAs), and FACS assays. Given the above, the present invention provides thus a monoclonal or polyclonal antibody obtainable by the aforedescribed methods for the generation of an antibody, i.e., by immunizing a non-human animal as described before.
As used herein, “cancer” refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumors or cells that invade neighboring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.
Non-limiting examples of cancers include squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g. clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer (or carcinoma), gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, melanoma (e.g., metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally-induced cancers including those induced by asbestos, virus-related cancers (e.g., human papilloma virus (HPV)-related tumor), and hematologic malignancies derived from either of the two major blood cell lineages, i.e., the myeloid cell line (which produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells), such as all types of luekemias, lymphomas, and myelomas, e.g., acute, chronic, lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML), undifferentiated AML (MO), myeloblastic leukemia (MI), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplantation, lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome), and lymphoplasmacytoid lymphoma (LPL) with Waldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary, plasmocytoma, and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, hematopoietic tumors of lymphoid lineage, for example T-cell and B-cell tumors, including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including of the small cell and cerebriform cell type; large granular lymphocyte leukemia (LGL) preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head or neck, renal cancer, rectal cancer, cancer of the thyroid gland; acute myeloid lymphoma, as well as any combinations of said cancers. The methods described herein may also be used for treatment of metastatic cancers, refractory cancers (e.g., cancers refractory to previous immunotherapy, e.g., with a blocking CTLA-4 or PD-1 or PD-L1 antibody), and recurrent cancers.
The term “subject” is intended to include living organisms. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. The subject (animal) can however be a non-mammalian animal such as a bird or a fish. In some preferred embodiments of the invention, the subject is a human, while in other some other preferred embodiments, the subject might be a farm animal, wherein the farm animal can be either a mammal or a non-mammalian animal. Examples of such non-mammalian animals are birds (e.g. poultry such as chicken, duck, goose or turkey), fishes (for example, fishes cultivated in aquaculture such as salmon, trout, or tilapia) or crustacean (such as shrimps or prawns). Examples of mammalian (life stock) animals includes goats; sheep; cows; horses; pigs; or donkeys. Other mammals include cats, dogs, mice and rabbits, for example. In illustrative embodiments the polymer-lipid hybrid nanoparticles of the present invention are used for the vaccination or immunization of the above-mentioned farm animals, both mammalian farm animals and non-mammalian farm animals (a bird, a fish, a crustacean) against virus infections (cf. the Example section in this regard). Accordingly, in such cases, polymer-lipid hybrid nanoparticle of the invention may have encapsulated therein soluble viral full length proteins or soluble fragments of viral full-length proteins.
When used for vaccinations of both humans and non-humans animals, polymer-lipid hybrid nanoparticle or compositions comprising polymer-lipid hybrid nanoparticle of the invention may be administered orally to the respective subject (cf. also the Example Section) dissolved only in a suitable (pharmaceutically acceptable) buffer such as phosphate-buffered saline (PBS) or 0.9% saline solution (an isotonic solution of 0.90% w/v of NaCl, with an osmolality of 308 mOsm/L).
As used herein, the term “LNP-Onpattro”, which can be used interchangeably with the terms “LNP-ON” or “LNP-ONP” may refer to lipid nanoparticles containing DMG-PEG, DSPC, MC3 and Chol, e.g., at a mole ratio 1.5:10.0:50: 38.5.
In illustrative embodiments of these polymer-lipid hybrid nanoparticles and oral formulations, the polymer-lipid hybrid nanoparticles that are used for vaccination have encapsulated therein a viral antigen that comprises a soluble portion of Influenza hemagglutinin, Swine Influenza hemagglutinin, Foot and Mouth Disease (FMD) virus protein such as the VP1, VP2 or VP3 coat protein (the VP1 coat protein contains the main antigenic determinants of the FMD virion, and hence changes in its sequence should be responsible for the high antigenic variability of the virus), Ovalbumin (OVA) or of the Porcine epidemic diarrhea (PED) virus SPIKE protein.
The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the infection and the general state of the subject's own immune system. The term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.
The appropriate dosage, or therapeutically effective amount, of the antibody or antigen binding portion thereof will depend on the condition to be treated, the severity of the condition, prior therapy, and the patient's clinical history and response to the therapeutic agent. The proper dose can be adjusted according to the judgment of the attending physician such that it can be administered to the patient one time or over a series of administrations. The pharmaceutical composition can be administered as a sole therapeutic or in combination with additional therapies as needed.
If the pharmaceutical composition has been lyophilized, the lyophilized material is first reconstituted in an appropriate liquid prior to administration. The lyophilized material may be reconstituted in, e.g., bacteriostatic water for injection (BWFI), physiological saline, phosphate buffered saline (PBS), or the same formulation the protein had been in prior to lyophilization.
Pharmaceutical compositions for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In addition, a number of recent drug delivery approaches have been developed and the pharmaceutical compositions of the present invention are suitable for administration using these new methods, e.g., Inject-ease, Genject, injector pens such as Genen, and needleless devices such as MediJector and BioJector. The present pharmaceutical composition can also be adapted for yet to be discovered administration methods. See also Langer, 1990, Science, 249:1527-1533.
The pharmaceutical composition can also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously, into the ligament or tendon, subsynovially or intramuscularly), by subsynovial injection or by intramuscular injection. Thus, for example, the formulations may be modified with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions may also be in a variety of conventional depot forms employed for administration to provide reactive compositions. These include, for example, solid, semi-solid and liquid dosage forms, such as liquid solutions or suspensions, slurries, gels, creams, balms, emulsions, lotions, powders, sprays, foams, pastes, ointments, salves, balms and drops.
The pharmaceutical compositions may, if desired, be presented in a vial, pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. In one embodiment, the dispenser device can comprise a syringe having a single dose of the liquid formulation ready for injection. The syringe can be accompanied by instructions for administration.
The formulations described herein are useful as pharmaceutical compositions in the treatment and/or prevention of the pathological medical condition as described herein in a patient in need thereof. The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of the formulation to the body, an isolated tissue, or cell from a patient who has a disease/disorder, a symptom of a disease/disorder, or a predisposition toward a disease/disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease.
As used herein, the term “treating” and “treatment” refers to administering to a subject a therapeutically effective amount of a pharmaceutical composition according to the invention. A “therapeutically effective amount” refers to an amount of the pharmaceutical composition or the antibody which is sufficient to treat or ameliorate a disease or disorder, to delay the onset of a disease or to provide any therapeutic benefit in the treatment or management of a disease.
As used herein, the term “prophylaxis” refers to the use of an agent for the prevention of the onset of a disease or disorder. A “prophylactically effective amount” defines an amount of the active component or pharmaceutical agent sufficient to prevent the onset or recurrence of a disease.
As used herein, the terms “disorder” and “disease” are used interchangeably to refer to a condition in a subject. In particular, the term “cancer” is used interchangeably with the term “tumor”.
The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
In the present context, the term “soluble antigen” as used herein means an antigen capable of being dissolved or liquefied. The term “soluble antigen” includes antigens that were “solubilized”, i.e., rendered soluble or more soluble, especially in water, by the action of a detergent or other agent. Exemplary non-limiting soluble antigens of the present invention include: polypeptides derived from a non-soluble portion of proteins, hydrophobic polypeptides rendered soluble for encapsulation as well as aggregated polypeptides that are soluble as aggregates. In some aspects, the antigens (e.g., membrane proteins) of the present invention are solubilized with the aid of detergents, surfactants, temperature change or pH change.
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle comprising a lipid and a block copolymer, wherein the amount of said lipid, expressed in mole percentage (i.e., a mole %) present in the polymer-lipid hybrid nanoparticle, wherein the mole percentage refers to the total amount of all components that form the polymer-lipid nanoparticle, is greater than the amount of said block copolymer, expressed in mole percentage, present in the polymer-lipid hybrid nanoparticle.
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, wherein the lipid (e.g., ionizable lipid) is selected from a group consisting of: an ionizable lipid DLin-MC3-DMA (also referred to as MC3) and an ionizable lipid C12-200.
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, wherein the block copolymer is selected from a group consisting of: PBD-PEO block copolymer, PCL-PEO block copolymer and DMG-PEGblock copolymer (e.g., Table 1).
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, wherein a mole % ratio of the lipid to the block copolymer is between 31.8 to 12 and about 35 to 2.5.
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, further comprising a stabilizer, e.g., comprising or consisting of cholesterol (also referred to as CHOL).
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, further comprising another lipid, wherein said another lipid is selected from a group consisting of: DMPC, DSPC, DOPE, DOTAP, DODAP, DOTMA, DODMA, DDA, 18:1 PA (1,2-dioleoyl-sn-glycero-3-phosphate), 14:0 PA (1,2-dimyristoyl-sn-glycero-3-phosphate), 18:1 BMP (bis(monooleoylglycero)phosphate) (e.g., Table 1).
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, consisting of: (i) PBD-PEO, MC3, CHOL; (ii) PBD-PEO, C12-200, CHOL; (iii) PBD-PEO, DOPE, C12-200, CHOL; (iv) PBD-PEO, DOPE, C12-200, CHOL; (v) PBD-PEO, DOPE, C12-200, CHOL; (vi) PBD-PEO, DOPE, C12-200, CHOL; (vii) DMG-PEG, DSPC, MC3, CHOL; (viii) PCL-PEO, DMPC, MC3, CHOL; (ix) PCL-PEO, DMPC, MC3, CHOL; (x) PCL-PEO, DMPC, MC3, CHOL; or (xi) PCL-PEO, DMPC, MC3, CHOL; (xii) PLGA-PEO, DMPC, MC3, CHOL.
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, further comprising a soluble encapsulated antigen, wherein said soluble encapsulated antigen is a protein and/or polynucleotide.
In some aspects, the invention provides a polymer-lipid hybrid nanoparticle as described herein, capable of maintaining long-term stability and/or potency of said polynucleotide (e.g., mRNA, e.g., as set forth in SEQ ID NOs: 1, 2 or 3).
In some aspects, the invention provides a composition comprising a polymer-lipid hybrid nanoparticle as described herein.
In some aspects, the invention provides a method of delivering nucleotide/s to inside a cell without using viral vector/s as delivery means, said method comprising: (i) providing the polymer-lipid hybrid nanoparticle and/or composition of the present invention; and (ii) contacting said polymer-lipid hybrid nanoparticle and/or composition with a cell.
In some aspects of the present invention, a polymer-lipid hybrid nanoparticle of the present invention is selected from the group consisting of: (a) BNP-012 having 10 mM (Molar %) of DOTAP: Cholesterol: DSPC: PBD-b-PEO (40:48:10:2) and/or BNP-025 having 10 mM (Molar %) of DOTMA: Cholesterol: DSPC: PBD-b-PEO (40:48:10:2); (b) BNP-002 having 5 mM (Molar %) of DLin-MC3-DMA: Cholesterol: PBD-b-PEO (49:39:12); or (c) BNP-002.2 having 5 mM (Molar %) of DLin-MC3-DMA: Cholesterol: DSPC: PBD-b-PEO (49.3:39.0:10.1:1.6).
In some aspects of the present invention, a polymer-lipid hybrid nanoparticle of the present invention is capable of targeting (e.g., predominantly targeting) a tissue/s and/or cell/s of an organ selected from the group consisting of: liver, spleen, lung/s, preferably said targeting is carried out without using a functional ligand/s; further preferably wherein: (a) the following polymer-lipid hybrid nanoparticles are suitable (e.g., is used) for said lung targeting: BNP-012 having 10 mM (Molar %) of DOTAP: Cholesterol: DSPC: PBD-b-PEO (40:48:10:2) and/or BNP-025 having 10 mM (Molar %) of DOTMA: Cholesterol: DSPC: PBD-b-PEO (40:48:10:2); (b) the following polymer-lipid hybrid nanoparticle/s are suitable (e.g., is used) for said liver targeting: BNP-002 having 5 mM (Molar %) of DLin-MC3-DMA: Cholesterol: PBD-b-PEO (49:39:12); (c) the following polymer-lipid hybrid nanoparticle/s are suitable (e.g., is used) for said spleen targeting: BNP-002.2 having 5 mM (Molar %) of DLin-MC3-DMA: Cholesterol: DSPC: PBD-b-PEO (49.3:39.0:10.1:1.6).
Based on the above, a new class of lipid hybrid nanoparticles has been developed in the course of the present invention, which is particularly suitable for mRNA delivery. Illustrative optimal polymer-lipid hybrid nanoparticles of the present invention exhibite favorable physicochemical properties and/or superior encapsulation efficiency (˜100%). In comparison to benchmark LNP-ON, the optimal formulation of the polymer-lipid hybrid nanoparticle of the present invention out perform with enhance in vitro transfection efficacy and/or long term thermostability, as can be evidenced by high levels of Luc protein expression and OVA protein expression (e.g., see below in the Experimental Section). Moreover, the ACM polymer-lipid hybrid nanoparticle formulations display less cytotoxicity as compared to benchmark LNP-ON (e.g., see below in the Experimental Section). Importantly, the optimal formulation of the polymer-lipid hybrid nanoparticle of the present invention demonstrate potent in vivo mRNA delivery efficacy, which is comparable to that of benchmark LNP-ON. Furthermore, OVA mRNA formulation can strongly activate cDC1 and cDC2 in the lymph nodes to promote antigen surface presentation. Taken together, the present invention provides a novel class of polymer lipid hybrid nanoparticles with efficient protein and antigen expression as well as enhanced thermostability, which hold great potential for delivery of therapeutic mRNA over a wide range of diseases.
The invention is also characterized by the following items:
wherein n=22; b denotes block, m=12; *=OCH3;
wherein n=22; m=29; *=H;
wherein x=23; y=4; m=26; n=29
In order that the invention may be readily understood and put into practical effect, some aspects of the invention are described by way of the following illustrative non-limiting examples.
All solvents and chemicals were purchased from Merck and used as received unless otherwise mentioned. Block copolymers including PBD1.2k-b-PEO0.6k (PBD-PEO) and PCL3.3k-b-PEO 1K(PCL-PEO), PLGA (LA: GA=85:15) 1.9K-PEO1K were synthesized in the lab (e.g., Table 1). 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC, Avanti), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti), DLin-MC3-DMA (MC3, Avanti), DMG-PEG-2K (DMG-PEG) and Cholesterol (Chol) were bought from Merck. 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) was purchased from Corden pharm. EZ Cap™ Firefly Luciferase mRNA (Luc mRNA-SEQ ID NO: 1) was purchased from APExBIO. CD19 mRNA (SEQ ID NO: 3) and OVA mRNA (SEQ ID NO: 2) were bought from Trilink Biotech and stored at −80° C. Quant-iTRiboGreen RNA assay, Lipofectamine™ MessengerMAX™, MultiTox-Fluor™ Multiplex Cytotoxicity Assay, ONE-Glo™ Luciferase Assay were bought from Thermofisher. Human embryonic kidney (HEK293T) cell lines (CRL-11268™) were obtained from ATCC, U.S.A., and cultured according to ATCC's recommendation. The cells were cultured in Roswell Park Memorial Institute (RPMI 1640) medium enriched with 10% fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, U.S.A.) at 37° C. with 5% CO2.
Fabrication of mRNA Loaded Polymer Lipid Hybrid Nanoparticles
Polymer lipid hybrid nanoparticles encapsulation mRNA was prepared by the solvent dispersion method, which was followed by dialysis. Briefly, polymers, lipid, ionized lipid and cholesterol were dissolved in ethanol at predetermined molar ratios with a total concentration of 5 mM (Table 2).
The aqueous solution was prepared in 20 mM acetic acid buffer (pH 5.0) with mRNA (Luc mRNA or OVA mRNA or CD19 mRNA). Ethanol phase was slowly injected into aqueous phase at a 3:1 ratio with vortex using a vortex mixer. The nanoparticles formed while vortexing and then dialysed against buffer (20 mM Trisbuffer, 4.5 mM Acetate, 5% Sucrose, pH 7.4) overnight at 4° C. overnight using dialysis membrane (300 kDa molecular weight cut-off (MWCO) cellulose ester membrane, Spectrum Laboratories Inc., cat. no. 131450) to remove organic solvents and unencapsulated mRNA. The dialyzed solution was sterile filtered using 0.22 μm sterile filter (Sartorius) and stored at 4° C. Lipid nanoparticles (LNP) encapsulating mRNA was prepared using a similar molar composition as reported in the literature and used as control, where the molar ratio among ionized lipid/cholesterol/DSPC/DMG-PEG-2Kis set 49:39: 10.5:1. 5. The N/P ratio ((N in the ionized cationic lipid and P in mRNA) was ranged from 4-40 in this method.
Polymer Lipid Hybrid Nanoparticles Encapsulation mRNA Produced by PNI
Polymer lipid hybrid nanoparticles encapsulation mRNA was produced in a similar molar composition as presented above by Precision Nanosystem Incorporation Nanoasemblr system (Ignite™, PNI, Canada)). Briefly, ethanol phase containing mixture of polymers and lipids with predetermined molar ratios (see e.g., Tables 2 and 3) and mRNA containing aqueous phase (20 mM acetic acid buffer, pH 5.0) injected simultaneously in a Y-shaped staggered herringbone micromixer of 300 μm width and 130 μm height). Nanoparticles were produced at 5 mM polymer/lipid concentration, 3:1 aqueous: organic flow rate ratio (FRR), 12 mL/min total flow rate (TFR). The nanoparticles were dialyzed against buffer (20 mM TRIS, 4.5 mM Acetate, 5% sucrose, pH 7.4) overnight at 4° C. using 300 kDa molecular weight cut-off (MWCO) cellulose ester membrane, Spectrum Laboratories Inc., cat. no. 131450) with magnetic stirring. The dialyzed solution was sterile filtered using 0.22 μm sterile filter (Sartorius) and stored at 4° C. for further usage.
EnGen@ Lba Cas12a (Cpf1) NED (Cas12a) and gRNA with ASF p52 were mixed at 250 nM concentration. The solution was incubated at room temperature (RT) for 10-15 mins for Cas12a to bind to gRNA. This was further encapsulated in the BNP-002 (Table 2) using the PNI system with TFF 12 ml/min and FRR of 3:1 at 1 ml scale (
The particle size, polydispersity and zeta potential of the nanoparticles was measured by ZetasizerNano ZS system (Malvern Instrument Ltd., Malvern, UK) equipped with a He-Ne laser beam at 658 nm (scattering angle: 90°). A 50 μL of sample was diluted 10 times using dialyzed buffer and an average of 3 measurements (10 runs per measurement) was collected and the data were presented as average.
4 μL of the mRNA encapsulated nanoparticles (5 mM) was adsorbed onto a lacey holey carbon-coated Cu grid, 200 mesh size (Electron Microscopy Sciences). The grid was surface treated for 20 s via glow discharge before use. After surface treatment, a 4 μL sample was added and the grid was blotted with Whatman filter paper (GE Healthcare Bio-Sciences) for 2 s with blot force 1 and then plunged into liquid ethane at −178° C. using a Vitrobot (FEI Company). The cryo-grids were imaged using a FEG 200 keV transmission electron microscope (Arctica; FEI Company) equipped with a direct electron detector (Falcon III; Fei Company).
Quantification of mRNA Encapsulated in the Nanoparticles
mRNA encapsulated in the nanoparticles was quantified using a modified Quant-iTRiboGreen RNA assay. A 20 μL portion of mRNA encapsulated nanoparticles or free mRNA at known concentrations was added to a 384-well black plate. A 10 μL amount of TE buffer or TE buffer with 10% Triton-X 100 was added into each well, and the plate was incubated for 15 min at 37° C. to lyse ACM vesicles before adding 20 μL of 1×Quant-iT RiboGreen (Invitrogen, Thermo Fisher Scientific). Each sample and standard were prepared in triplicate. The plate was incubated for 10 min at 25° C., and fluorescence was measured (excitation, 500 nm; emission, 525 nm) using a plate reader (Biotek). The concentration of mRNA was calculated according to the standard curve. Encapsulation efficiency (EE) was calculated as (FI-Fo)/F*100, where Ft and Fo are the amount of mRNA quantified in presence and absence of 1% triton X-100, F; was the initial amount of mRNA used for preparing nanoparticles.
Gel electrophoresis was used to analyse the integrity of mRNA in various formulations. Agarose was dissolved in 1 xTris-acetate-EDTA (TAE) buffer to form a 1% w/v solution upon heating, followed by the addition of SybrSafe Dye (5 μl per 50 mL). The solution was mixed well and pour on to the casting tray with comb placed on it. The gel was allowed to solidify. The comb was removed, and the gel was placed on buffer tank with 0.5× TAE buffer. 20 UL of mRNA encapsulated nanoparticles (mRNA equal amount 500 ng) were mixed with 2 μL of 20% Triton X for 30 min, followed by the addition of 20 μl of 2× RNA loading dye. The samples were heated at 70° C. for 10 mins on heat block. The samples were cool down and then loaded on the gel (20 μ per well). It is noted that RNA Ladder was used as molecular weight standard. A power supply was connected to the chamber and a voltage of 80 V applied for 40-45 mins. The gel was then visualized using an ImageQuant LAS 500 system.
Tested samples (20 μL) were challenged with RNase (1 μl of 10 μg/ml stock), followed by incubation at 37 degrees for 20 mins. Subsequently, 0.2% SDS (4 μl of 1% SDS stock) was added to each sample. It is noted that 0.2% SDS works as RNAse inhibitor. Positive control samples were incubated with 1% of triton X at room temperature for 30 min. For negative control groups, samples (20 μL) were challenged with RNase (1 μl of 10 μg/ml stock), followed by incubation at 37° C. for 20 mins. Subsequently, 2% Triton X (2 μl of 20% Triton X) was added to each sample. The mixture was incubated at room temperature for 30 min. addition of 20 μl of 2× RNA loading dye. The samples were heated at 70° C. for 10 mins on heat block. The samples were cool down and then loaded on the gel (20 μl per well). It is noted that RNA Ladder was used as molecular weight standard. A power supply was connected to the chamber and a voltage of 80 V applied for 40-45 mins. The gel was then visualized using an ImageQuant LAS 500 system.
mRNA Transfection
HEK293T cells (CRL-11268™) were seeded in 96 well plates at a density of 25000 cells per well. After overnight incubation at 37° C., the cells were transfected with Luc mRNA encapsulated nanoparticles or control Luc mRNA using Lipofectamine™ MessengerMAX™ (MM, Thermo Fisher). The cytotoxicity of free mRNA and mRNA-loaded nanoparticles against HEK293 cells was determined after 24-h incubation through MultiTox-Fluor™ Multiplex Cytotoxicity Assay. Luciferase activity was measured with the ONE-Glo™ Luciferase Assay according to the manufacturer's instructions (Promega).
HEK293T cells were seeded in 24 well plates at 250,000 cells per well. After overnight incubation, the cells were transfected with OVA mRNA nanoparticles (OVA mRNA equal amount: 1 μg) or control OVA mRNA (100 ng and 200 ng) using Lipofectamine™ Messenger MAX™ (Thermo Fisher). The cells were collected after 24 h transfection. The cells were lysed, and protein was quantified using BCA assay (Thermo Fisher) according to manufacturer's protocol. 50 μL of sample (containing 150 ng of protein) was mixed with 50 μL of loading buffer and the mixture was heated at 95° C. for 10 min. The samples (20 μL) were loaded for SDS-PAGE. The OVA protein was then detected by western blotting with a monoclonal antibody against the OVA protein. The gel was then visualized using an ImageQuant LAS 500 system.
In Vivo Luc mRNA Delivery
The animal studies were approved by Institutional Animal Care and Use Committee, A-star, Singapore. 6-8-week-old female C57BL/6 mice were randomly grouped. C57BL/6 mice were injected with Luc mRNA with dose of 0.35 mg/kg by IM (thigh muscle), SC (flank) and IV (tail vein), respectively. There were 6 groups for each administration route3 mice per group, total 18 mice per administration route). In the case of intramuscularly (IM) administration route, mice were intramuscularly (IM) injected at the inner thigh with Luc mRNA encapsulated within LNP-ON, BNP-002, BNP-008, PCL-008 and PCL-012 at a dosage of 0.35 mg/kg, where PBS was used as negative control and LNP-ON was used as positive control for comparison. At 6 h post-injection, mice were anesthetized with 2% isofluorane in oxygen and imaged 10 min after intraperitoneal injection of D-Luciferin (150 mg/Kg). Bioluminescence imaging was performed using an IVIS Spectrum imaging system. Organs collected for ex vivo imaging. Mice were imaged at 10 minutes post administration of D-luciferin. Bioluminescence values were quantified by measuring photon flux in the region of interest using the Living IMAGE Software provided by Caliper.
Mice Injected with ACM-OVA mRNA Polymer-Lipid Hybrid Nanoparticles Expressed OVA Peptide on Surfaces of Dendritic Cells
The animal studies were approved by Institutional Animal Care and Use Committee, A-star, Singapore. 6-8-week-old female C57BL/6 mice were intramuscularly (IM) injected at the inner thigh with 3-4 μg OVA mRNA encapsulated within LNP or ACM carrier. Two days after, animals were sacrificed and inguinal lymph nodes that drain the site of injection were harvested. To release DCs for analysis, lymph nodes were cut into tiny pieces and digested with 0.2 mg/ml collagenase and 0.05 mg/ml DNAse I in complete RPMI medium for 30 min at 37° C. Cells were passed through 70 μm cell strainers. To prepare for flow cytometry, cells were stained using the following antibody panel: BUV395-CD45, FITC-CD3, FITC-CD19, FITC-CD49b, BV510-MHC-II, BV650-CD64, PE-CD594-CD11c, PerCP-Cy5.5-XCR1, APC-CD172a, APC-Cy7-CD86, and PE-SIINFEKL-H2kb. Live/dead discrimination was done using fixable viability dye eFluor 455 UV. Non-specific staining was reduced using mouse FcR block reagent. Cells were analysed using LSR Fortessa (BD) and data was analysed using FlowJo V10.
OVA mRNA Vaccine Adaptive Immunity Study
Mouse vaccination. The animal studies were approved by Institutional Animal Care and Use Committee, A-star, Singapore. 6-8 weeks old, female C57BL/6 (n=5 per group) were intramuscularly (IM) injected at each thigh muscle with total of 5 μg OVA mRNA on Days 0 and 14. Blood was collected by retro-orbital puncture on Day 7 and 21 for assessment of circulating T cells and Days 14 and 24 for serum IgG titres.
T cell analysis. Blood was collected in 0.1% EDTA. Cells were pelleted at 500 g, 40 C, 5 min and erythrocytes were lysed using RBD lysis buffer (Thermo Fisher). White blood cells were surface stained with antibodies and pentamer for analysis by flow cytometry (Table 7). Cells were acquired on LSR II cytometer (BD) and data analysed using FlowJo V10 software.
OVA IgG titre. 96-well Corning EIA/RIA plate was coated with 2 μg/ml OVA protein overnight at 40 C. The next morning, the plate was washed thrice with PBS+0.1% v/v Tween-20 before blocking with 2% w/v BSA in wash buffer for 1.5 h at 370 C. Serum was serially diluted with Assay Diluent (PBS+0.5% w/v BSA+0.1% v/v Tween-20) and applied to corresponding wells of the ELIA plate. Samples were incubated 1 h at 370 C before the plate was washed thrice. HRP-conjugated goat anti-mouse IgG (H+L) (BioRad) was diluted 1:10,000 and applied to the ELISA plate. The plate was incubated 1 min at 370 C before washing thrice. To visualize antibody binding, TMB substrate (Sigma Aldrich) was added and incubated 30 min at room temperature. The reaction was stopped with Stop Solution (Thermo Fisher) and absorbance at 450 nm measured. Data was analyzed using 5-parameter non-linear regression (GraphPad Prism version 9.1.2). Antibody titer, defined as the reciprocal of the highest dilution giving an OD value 3X background, was interpolated from the titration curve.
Statistical significance was evaluated via an independent two-tailed Student's t test or two-way ANOVA. P-values less than 0.05 were considered statistically significant. Data were analyzed using GraphPad Prism 7 software.
Physicochemical Characterization of mRNA Loaded Polymer-Lipid Hybrid Nanoparticles Prepared by Solvent Dispersion Method (Co-Solvent Method/Nano-Preciptation Method)
BNPs composed of PBD-PEO, MC3 and Chol encapsulating mRNA were first prepared by solvent dispersion method. LNP-onpattro (LNP-ON or LNP-ONP) containing DMG-PEG, DSPC, MC3 and Chol (1.6:10.1:49.3:39.0) with mRNA was prepared via the same method and used as control. The physicochemical properties of nanoparticles including particle size, polydispersity, zeta potential, mRNA encapsulation efficiency, loading concentration were summarized in Table 2. BNP-002 has an average particle size of 138 nm with a relative lower polydispersity (PDI: 0.176). It is noted that the hydrodynamic diameter of LNP-ON was 158 nm and the PDI of LNP-ON was 0.17. The results indicated that the particle size and PDI of BNP-002 are comparable to those of LNP-ON. The surface potential of BNP-002 was 27.8 mV, which is significanlty higher than that of LNP-ON (11.1 mV). This was in line with the fact that the N/P value of BNP-002 was significanlty higher than that of LNP-ON (27). To explore the potential of mRNA loaded nanoparticles with tumor antigens for cancer immunotherapy, OVA mRNA was loaded into BNPs via solvent dispersion method. Interestingly, it was found that OVA mRNA BNPs showed an diameter value of 107 nm with a low PDI value of 0.137. The morphology of Luc mRNA loaded BNPs and OVA mRNA loaded BNPs and were analyzed by cryo-TEM, as illustrated in
The ionized lipid has been reported to play a critical role on the performance of mRNA nanoparticles. Numerous studies have shown that ionized lipids including DLin-KC2-DMA and DLin-MC3-DMA achieved maximum activity on mRNA delivery. It was recently reported by Kauffmann et al. that lipid like material (lipidoid) C12-200 nanoparticles incorporated with DOPE, DMG-PEG and Chol (35:16: 2.5:46.5) (LLNPs) remarkably increased EPO expression seven-fold in serum as compared to the benchmark formulation LNP-ON. Inspired by Kauffmann's design, we integrated PBD-PEO with LLNPs and investigated the functionality of resulting Luc mRNA BNPs. BNP based on C12-200 were prepared with solvent dispersion method as listed in Table 2, BNP-008 had an average particle size ranging from 121 to 200 nm, lower PDI values less than 0.22, surface charge ranging from 25 to 30 mV. BNP-008 had the smallest particle size and highest in vitro transfection efficiency (data not shown). It was therefore selected for further studies.
mRNA integrity in BNPs were analyzed by gel electrophoresis. As illustrated in
It should be mentioned that the BNPs produced by solvent dispersion method had lower encapsulation efficiency and lower loading concentration. To improve the encapsulation efficiency and loading levels of BNPs, mRNA loaded nanoparticles were further prepared by Precision Nanosystem Incorporation (PNI Nanoasemblr Patform.
Physicochemical Properties of mRNA Loaded Nanoparticles Prepared by Precision NanoSystem Incorporation (PNI) Nanoasemblr Patform
Physicochemical Properties of mRNA Loaded Nanoparticles Prepared by the PNI Method
BNPs and PCLs were formulated as specified compositions (e.g., Table 2, Table 3, Table 4 and Table 5) at N/P molar ratio of 10 using the PNI method.
Briefly, mRNA diluted in acetic acid buffer (20 mM, pH5: 0) was rapidly mixed with polymer and/or lipids in ethanol at 3:1 aqueous: ethanol volume ratio. The aqueous to organic. flow rate ratio (FRR) was set to be 3:1 and the total follow rate (TFR) was set to be 12 mL/min. Interestingly, BNP-002, BNP-008, PCL-008 and PCL-012 with N/P ratio at: 10 showed 80-130 nm in z-average diameter with lower polydispersity (Tables 3 and 6A). It is noteworthy that all formulations exhibited superior encapsulation efficiency (˜100%) (Table 6B), Benchmark LNP-ON with N/P at 4 prepared by PNI yielded particles with 73 nm, low polydispersity (0.11) and strikingly high encapsulation efficiency (˜100%) (Table 6A and 6B), which is consistent with previous studies. Importantly, all formulations demonstrated high load concentrations (more than 75 μg/mL) (Table 6B), which is significantly increased as compared to those produced by solvent dispersion method.
1 This is to measure any free unencapsulated mRNA or mRNA that binds to the surface of the vesicles.
2 This is to measure the total mRNA present in the sample as Triton was added to break open the vesicle.
3 mRNA loading is calculated by using the concentration with Triton minus the concentration without Triton.
Among these, BNP-008 with: N/P at 10 exhibited strikingly higher loading concentrations, which is comparable to that of benchmark LNP-ON with N/P at 4 (Table 6B). The surface charge of BNP-002 and BNP-008 were ˜ 22 mV, while the surface charge of PCL-008 and PCL-012 were ˜ 5-8 mV (Table 6A). The morphology of BNPs and PCLs were analyzed by cryo-TEM. All formulations formed spherical particles with 50-200 nm in diameter, which agrees with DLS analysis. The nanoparticles had an electro-lucent amorphous internal structure surrounded by a peripheral bilayer (
OVA mRNA was also encapsulated into BNPs and PCLs using PNI. All formulations exhibited small particle size, low polydispersity, strikingly higher encapsulation efficiency (˜100%) and high mRNA loading level (data not shown). The integrity of OVA mRNA encapsulated in BNPs and PCLs was assessed by gel electrophoresis. As seen in
Cas12a, CRISP effector protein, has shown great promise in the treatment of genetic diseases. CRISPR technology based on Cas12a and gRNA has been reported to be powerful for RNA-based gene regulation. To explore the potential of polymer lipid hybrid nanoparticles as carriers for Cas12a and gRNA, we developed BNP-002 nanoparticles for the encapsulation of Cas12a and gRNA. Cas12a and gRNA with ASF p52 were first mixed and incubate for 10-15 min. This was further encapsulated in the BNP-002 using the PNI system. The resulting nanoparticles were 285.2 nm by dynamic light scattering (
In Vitro Transfection Efficiency of mRNA Loaded Nanoparticles Prepared by Solvent Dispersion Method
The in vitro delivery efficacy and cytotoxicity of BNPs was investigated by normalized luminescence intensity after transfection of Luc mRNA into HEK293T cells. As shown in
Next, BNPs was validated as nano vaccine to deliver OVA mRNA encoding antigen in HEK293 cells. As illustrated
In Vitro Transfection Efficiency of mRNA Loaded Nanoparticles Prepared by Precision NanoSystem Incorporation (PNI) Nanoasemblr Patform
As stated earlier, all formulations produced by PNI demonstrated remarkably higher encapsulation efficiency (˜100%) at N/P ratio of 10. In vitro delivery efficacy of these formulations was evaluated for delivery of Luc mRNA in HEK293T cells (
HEK293T cells were treated with OVA mRNA formulations and the OVA protein expression was assessed by western blot assay. As illustrated in
In Vivo Delivery Efficacy of Luc mRNA
In vivo delivery efficacy of BNPs and PCLs was evaluated in C57BL/6 mice via different administration routes (IM, SC and IV). For IM administration routes, mice were randomly assigned into six different groups (3 mice in each group): control group (1×PBS), BNP-002, BNP-008, PCL-008 and PCL-012. All formulations were adminstrated via intramuscle (IM) route at the thigh muscle region at dosage of 0.35 mg/kg. Strong expression of luciferase protein was observed at the injection site and upper abdomen in the mice 6 h after IM injection (
Being encouraged by comparable Luciferase protein expression between Luc mRNA BNPs, PCLs and benchmark LNP-ONPs administrated via IM, we further investigated the effects of administration routes (subcutaneous (SC) and intravenous (IV)) of BNPs and PCLs for delivering of mRNA. SC administration of Luc mRNA resulted in protein expression mainly at the site of injection (data not shown). Strong bioluminescent signal was also observed at 6 hours post SC administration at upper abdomen for LNP-ONP LNP-ON (data not shown). Organs/tissues were then excised, and bioluminescence was further analyzed. Luciferase protein generated by LNP-ONP LNP-ON was expressed mainly in the liver (74%), a less extent was seen in injection site (16%) and lymph nodes. In contrast, Luciferase protein generated by ACM formulations of the present invention was expressed mainly in the injection site (55-83%). It is noteworthy that Luc protein expression level was similar in inguinal lymph nodes among LNP-ONP, BNP-008 and PCL-008 after 6 h SC administration.
When Luc mRNA nanoparticles were injected intravenously, strong bioluminescent signal was detected at liver for LNP-ONP, PCL-008 and PCL-012 (
Classical dendritic cells consist of two subsets (cDC1 and cDC2) which are crucial for initiating the adaptive immune response. Two days after injection of LNP-mRNA or BNP-mRNA vaccine, strong upregulation of CD86 activation marker was seen on both cDC subsets (
ACM-OVA mRNA Vaccine Adaptive Immunity Study
Mice were IM injected twice with LNP, BNP or PCL formulation encapsulating 5 μg per mouse of OVA mRNA (
In conclusion, a new class of polymer and lipid hybrid nanoparticles have been developed for mRNA delivery. The optimal formulation exhibited favourable physicochemical properties and superior encapsulation efficiency (˜100%). In comparison to benchmark LNP-ON, the optimal formulation outperformed with enhanced in vitro transfection efficacy and long term thermostability, as evidenced by high levels of Luc protein expression and OVA protein expression. Moreover, the ACM formulations displayed less cytotoxicity as compared to benchmark LNP-ON. Importantly, the optimal formulation demonstrated potent in vivo mRNA delivery efficacy, which is comparable to that of benchmark LNP-ON. Furthermore, OVA mRNA formulation strongly activated cDC1 and cDC2 in the lymph nodes to promote antigen surface presentation. BNP-008 consistently generated comparable CD8+ T cell and IgG response as LNP controls. The findings strongly suggested that ACM-OVA mRNA would likely induce OVA-specific adaptive immunity. Taken together, our work reports a novel class of polymer lipid hybrid nanoparticles with efficient protein and antigen expression as well as enhanced thermostability, which hold great potential for delivery of therapeutic mRNA over a wide range of diseases.
1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA, Avanti) was bought from Merck, all the other chemicals and reagents as well as methods have been described above. Accordingly, if not specified otherwise materials and methods were as described above for example 1. In this example Luc mRNA loaded nanoparticles were prepared by microfluidizer according to Table 8, wherein for mRNA loaded polymer lipid hybrid nanoparticles mRNA was produced in a molar composition as presented in Table 8 by microfluidizer, where N/P molar ratio is 10:
After that, the polymer-lipid hybrid nanoparticles were characterized as shown in Table 9:
Accordingly, BNP-002.2 showed 93 nm in z-average diameter with lower polydispersity. BNP-012 had a particle size of 51 nm with a polydispersity of 0.188. BNP-025 exhibited a particle size of 52 nm and a polydispersity of 0.198. BNP-012 and BNP-025 displays zeta potential of ˜ 45 mV. Finally, all formulations demonstrated high Luc-mRNA encapsulation efficiency (>90%).
After that, exemplary Cryo-TEM images were produced for BNP-002.2, BNP-012 and BNP-025 (all loaded with Luciferase mRNA) (
After that, Luc mRNA encapsulation efficiency was measured using Ribogreen Assay as shown in Table 10:
1 This is to measure any free un-encapsulated mRNA or mRNA that binds to the surface of the vesicles.
2 This is to measure the total mRNA present in the sample as Triton was added to break open the vesicle.
3 mRNA loading is calculated by using the concentration with Triton minus the concentration without Triton.
4% EE is calculated by using Loading divided by concentration with Triton into percentage.
Accordingly, Ribogreen assay indicated that all formulations exhibited high encapsulation efficiency (>90%).
After that, an agarose gel image for Luc mRNA loaded nanoparticles prepared by microfluidizer were carried out indicating that all samples contain intact mRNA as no degradation observed in the gel (
This was followed by an in vitro Luciferase mRNA nanoparticles transfection efficiency profiles in HEK293T cells indicating that all formulations showed high expression of luciferase protein, which was comparable to that of LNP-ON and that BNP-002.2 demonstrated remarkably high in vitro transfection potency as compared to LNP-ON (p<0.05).
After that, an endotoxin (Lonza LAL Assay) analysis of Luc mRNA nanoparticles was carried out as shown in Table 11, wherein the endotoxin levels of and Luc mRNA nanoparticles were investigated using Lonza Kinetic Chromogenic LAL Assay according to the manufacturer instructions. It was shown that Spike recovery of all nanoparticles met the acceptable range and that the endotoxin level of all nanoparticles were around ˜ 2 EU/mL, which also met the requirements (<10 EU/mL).
After that, Luciferase Protein expression Biodistribution Percentage Profile via IV Administration was carried out (
This was followed by Tissue Expression Profiles (Raw value of Flux) of the Luc mRNA-encoded Protein in Mice 6 h Post Administration via IV (
Finally, Luciferase Protein expression Biodistribution Percentage Profile (Flux) via IV Administration was performed (
Conclusion: Organ Specific Delivery of mRNA to Liver, Spleen and Lung has been Achieved Via Engineered Block Copolymer Lipid Hybrid Nanoparticles of the Present Invention
One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of certain embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing “, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. All documents, including patent applications and scientific publications, referred to herein are incorporated herein by reference for all purposes.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22158324.8 | Feb 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/054613 | 2/23/2023 | WO |
