KR20210052924A - Small lipid nanoparticle and cancer vaccine comprising the same - Google Patents

Abstract

The present invention relates to a combination therapy of small lipid nanoparticles, a small lipid nanoparticle (SLNP)-based nanovaccine platform comprising the same, and an immune checkpoint inhibitor. The lipid nanoparticles of the present invention can easily deliver antigens and anionic drugs into cells, and exhibit strong antitumor effects when loaded with tumor-associated antigens. In particular, by using a cancer vaccine kit of the present invention comprising the lipid nanoparticles of the present invention as a primary vaccine composition and the lipid nanoparticles and an immune checkpoint inhibitor as a secondary vaccine composition, it is possible to effectively inhibit regrowth and recurrence of tumors caused by immunosuppression against cancer nano vaccines.

Description

Small lipid nanoparticle and cancer vaccine comprising the same

The present invention relates to a small lipid nanoparticle, a small lipid nanoparticle (SLNP)-based nanovaccine platform comprising the same, and a combination treatment regimen with an immune checkpoint inhibitor.

Cancer nano-vaccines based on nanomaterials carrying tumor-associated antigens or tumor-specific neoantigens have shown promising therapeutic efficacy in preclinical animal models, but the clinical results of these nano vaccines Enemy use has been limited. Despite the ability of cancer nano-vaccines to expand the pool of tumor-specific T cells, the occurrence of immune evasion and immune suppression in the tumor microenvironment (TME) has been shown to cause poor therapeutic responses in nano-vaccine trials. It is believed. In particular, increased expression at the immune checkpoint of programmed death ligand 1 (PD-L1), which binds to the programmed cell death-1 receptor in T cells, is a vaccine-mediated immune response. responses), thereby limiting the therapeutic efficacy of these vaccines. This immune suppression could be overcome by the combination of cancer nano vaccines and immune checkpoint blockers (ICBs) such as anti-PD-1 and anti-PD-L1 antibodies, which could improve the therapeutic effect. However, to date, few studies have evaluated the optimal timing and sequence of cancer nano vaccines and ICBs. For example, it is unclear whether simultaneous treatment results in better treatment results or sequential treatment results in better treatment results. Moreover, the timing of the optimal combination of the two modalities has not been determined. A rational approach is needed to determine the timing and sequence of administration of cancer nano vaccines and ICBs. The present invention relates to a novel combination treatment regimen that can effectively inhibit tumor growth and recurrence by improving nano vaccine-induced adaptive immune resistance. In addition, the present invention is a neutral-charged small antigen that acts as both antigen and adjuvant-carrying nano-vaccines to investigate whether rationally designed antigen-carrying nanomaterials are suitable candidates for cancer nano-vaccines. Small lipid nanoparticles (SLNP) were prepared. The present inventors have confirmed that sequential administration and combination of nano vaccine and anti-PD1 antibody can exhibit an effective anti-tumor effect and suppression of tumor recurrence, and have completed the present invention.

Shirota et al., Vaccines 2015, 3, 390-407.

An object of the present invention is to provide a lipid nanoparticle comprising an antigen, a phospholipid, a cationic lipid, and an adjuvant.

Another object of the present invention is to provide a vaccine composition comprising the above-described lipid nanoparticles as an active ingredient.

Another object of the present invention is to include lipid nanoparticles comprising a tumor-associated antigen, a phospholipid, a cationic lipid, and an anionic drug as a first vaccine composition; It is to provide a cancer vaccine kit comprising the lipid nanoparticles and an immune checkpoint inhibitor as a second vaccine composition.

According to one aspect of the present invention, the present invention provides a lipid nanoparticle comprising an antigen, a phospholipid, a cationic lipid, and an adjuvant.

In one embodiment of the present invention, the antigen is a tumor-associated antigen.

More specifically, the tumor-associated antigens are MAGE-1, MAGE-2, MAGE-3, MAGE-12, BAGE, GAGE, NY-ESO-1, tyrosinase, TRP-1, TRP-2, gp100, MART-1 , MCIR, Ig idotype, CDK4, Caspase-B, beta-catenin, CLA, BCR/ABL, mutated p21/ras, mutated p53, proteinase 3, WT1, MUC-1, Her2/neu, PAP, PSA, PSMA, G250 , HPV E6/E7, EBV LMP2a, CEA, alpha-Fetoprotein, 5T4, onco-trophoblast glycoprotein, etc. are selected from the group consisting of, but are not limited to, and those skilled in the art may apply various antigens that can be applied in cancer vaccines It will be easy to understand that you can.

In one embodiment of the present invention, the tumor (or cancer) includes breast cancer, head and neck cancer, bladder cancer, gastric cancer, rectal/colon cancer, pancreatic cancer, lung cancer, melanoma, prostate cancer, kidney cancer, liver cancer, cervical cancer, etc. , But is not limited thereto.

In one embodiment of the present invention, the phospholipid is a phospholipid having 14 to 22 aliphatic carbon atoms.

In a specific embodiment of the present invention, the phospholipid is 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol )-1000] DSPE-PEG derivatives containing (DSPE-PEG ₁₀₀₀ ), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000] (DSPE-PEG ₂₀₀₀ -PDP ) And 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG ₂₀₀₀ -Maleimide), etc., 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DPPE-Rhodamine) , 1,2-Didecanoyl- sn -glycero-3-phosphocholine ( DDPC ), 1,2-Dierucoyl- sn -glycero-3-phosphate (DEPA), 1,2-Dierucoyl- sn -glycero-3-phosphocholine (DEPC ), 1,2-Dierucoyl- sn -glycero- 3-phosphoethanolamine (DEPE), 2-Dierucoyl- sn -glycero-3-Phospho-rac- (1-glycerol) (DEPG), 1,2-Dilinoleoyl- sn - glycero-3-phosphocholine (DLOPC), 1,2-Dilauroyl- sn -glycero- 3-phosphate (DLPA), 1,2-Dilauroyl- sn -glycero-3-phosphocholine (DLPE), 1,2-Dilauroyl- sn -glycero-3-Phospho-rac-(1-glycerol) (DLPG), 1 ,2-Dilauroyl- sn -glycero-3-phosphoserine (DLPS), 1,2-Dimyristoyl- sn -glycero-3-phosphate (DMPA), 1,2-Dimyristoyl- sn -glycero-3-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero- 3-phosphoethanolamine (DMPE), 1,2-Dimyristoyl- sn -glycero-3-Phospho-rac- (1-glycerol) (DMPG), 1,2-Dimyristoyl- sn - glycero-3-phosphoserine (DMPS), 1,2-Dioleoyl- sn -glycero-3-phosphate (DOPA), 1,2-Dioleoyl- sn -glycero-3-phosphocholine (DOPC), 1,2-Dioleoyl- sn -glycero-3-Phospho-rac-(1-glycerol) (DOPG), 1,2-Dioleoyl- sn -glycero-3-phosphoserine (DOPS), 1,2-Dipalmitoyl- sn -glycero-3-phosphate (DPPA ), 1,2-Dipalmitoyl- sn -glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl- sn -glycero-3-phosphoethanolamine (DPPE), 1,2-Dipalmitoyl- sn -glycero-3-Phospho- rac-(1-glycerol) (DPPG), 1,2-Dipalmitoyl- sn -glycero-3-phosphoserine (DPPS), 1,2-Distearoyl- sn -glycero-3-phosphate (DSPA), 1,2 -Distearoyl- sn -glycero-3-phosphocholine (DSPC), 1,2-Distearoyl- sn -glycero-3-Phospho-rac-(1-glycerol) (DSPG), 1,2-Distearoyl- sn -glycero-3 -phosphoserine (DSPS), Egg-PC (EPC), Hydrogenated Egg PC (HEPC), Hydrogenated Soy PC (HSPC), 1-Myristoyl-sn-glycero-3-phosphocholine (LYSOPC MYRISTIC), 1-Palmitoyl- sn -glycero -3-phosphocholine (LYSOPC PALMITIC), 1-Stearoyl-sn-glycero-3-phosphocholine (LYSOPC STEARIC), 1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (Milk Sphingomyelin MPPC), 1-Myristoyl-2 -stearoyl- sn -glycero-3-phosphocholine (MSPC), 1-Palmitoyl-2-myristoyl- sn -glycero-3-phosphocholine (PMPC), 1-Palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC ), 1-Palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine (POPE), 1-Palmitoyl-2-oleoyl- sn -glycero-3-Phospho-rac-(1-glycerol) (POPG), 1- Palmitoyl-2-stearoyl- sn -glycero-3-phosphocholine (PSPC), 1-Stearoyl-2-myristoyl- sn -glycero-3-phosphocholine (SMPC), 1-Stearoyl-2-oleoyl- sn -glycero-3- phosphocholine (SOPC) and 1-Stearoyl-2- One or more phospholipids selected from the group consisting of palmitoyl- sn -glycero-3-phosphocholine (SPPC), but are not limited thereto.

In one embodiment of the present invention, the cationic lipid is Dimethyldioctadecyl-ammoniumbromide (DDAB), Dimethyldioctadecylammonium (DDAB), (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis( dioleyloxy)-1-propaniminium pentahydrochloride) (DOSPA), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium) (DOTMA), (N-[1-(2,3 -dioleoyloxy)propyl]-N,N,N-trimethylammonium) (DOTAP), 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N4-Cholesteryl-Spermine (GL67) , 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC) containing O-alkyl phosphatidylcholines derivatives, N-(4-carboxybenzyl)- Selected from the group consisting of DAP derivatives including N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ) and 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP) One or more cationic lipids, but are not limited thereto.

In one embodiment of the present invention, the cationic lipid is a cationic cholesterol derivative. Specifically, the cationic cholesterol derivative is Monoarginine-cholesterol (MA-Chol).

In one embodiment of the present invention, the lipid nanoparticle may contain an anionic drug in addition to the adjuvant. In a specific embodiment of the present invention, the anionic drug is an oligonucleotide, an aptamer, mRNA, siRNA, miRNA, or a combination thereof.

In one embodiment of the present invention, the adjuvant is an immunostimulatory single-chain or double-chain oligonucleotide, an immunostimulatory low-molecular compound, or a combination thereof.

In one embodiment of the present invention, the immune-stimulating single-chain or double-chain oligonucleotide is known as a useful adjuvant (immune adjuvant). They often contain a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked to guanosine). Oligonucleotides, including a TpG motif, a palindrome arrangement, a plurality of contiguous thymidine nucleotides (e.g. TTTT), a plurality of contiguous cytosine nucleotides (e.g. CCCC) or poly(dG) arrangements, are also also available. Like double-stranded RNA, it is a known adjuvant. Any of these various immunostimulatory oligonucleotides can be used without limitation with the present invention.

The oligonucleotide typically has 10 to 100 nucleotides, for example 15 to 50 nucleotides, 20 to 30 nucleotides, or 25 to 28 nucleotides. It is typically a single chain.

The oligonucleotide may include only natural nucleotides, only non-natural nucleotides, or a mixture of both. For example, the oligonucleotide contains one or more phosphorothioate linkages, and/or may be one or more 2'-O-methyl alterations.

In a specific embodiment of the present invention, the single-chain or double-chain oligonucleotide is a CpG oligonucleotide, a STING active oligonucleotide, or a combination thereof.

As used herein, the term "Stimulator of Interferon Genes (STING)" refers to a stimulating factor (STING) of the interferon gene, which is a molecule that plays a major role in the innate immune response. STING mainly contains five putative transmembrane (TM) regions present in the endoplasmic reticulum (ER), induces type I IFG, and NF-kB and IRF3 transcription pathways that exert a strong antiviral state after expression. Can be activated (see U.S. Patent Application Serial No. 13/057,662 and PCT/US2009/052767). Loss of STING reduces the ability of polyIC to activate type I IFN, lacks STING generated by targeted homologous recombination, and is susceptible to vesicular stomatitis virus (VSV) infection. (-/- MEF) is provided. In the absence of STING, the DNA-mediated type I IFN response is inhibited, indicating that STING can play an important role in recognizing DNA from viruses, bacteria and other pathogens that can infect cells. Yeast double hybridization and coimmunoprecipitation studies show that STING interacts with RIG-I and Ssr2/TRAPβ (a member of the translocon-associated protein (TRAP) complex required for protein transfer across the ER membrane after translation). Indicated that. RNAi removal of TRAPβ inhibited STING function and prevented the production of type I IFN in response to polyIC. Further experiments have shown that STING itself binds nucleic acids, including single and double-stranded DNA, for example from pathogens or apoptotic DNA, and modulates pro-inflammatory gene expression in inflammatory conditions such as DNA-mediated arthritis and cancer. Has shown that it plays a central role. Various novel methods of up-regulating STING expression or function and various novel compositions for up-regulating STING expression or function are described herein along with further characterization of other cellular molecules that interact with STING. These findings allow the design of new adjuvants, vaccines and therapies to modulate the immune system and other systems.

The STING active oligonucleotide may be a nucleic acid molecule that binds the STING function to STING. The STING-binding nucleic acid molecule may be single-stranded DNA of 40 to 150 base pairs in length or double-stranded DNA of 40 to 150, 60 to 120, 80 to 100, or 85 to 95 base pairs or more in length. The STING binding nucleic acid molecule may be nuclease resistant, for example made of nuclease resistant nucleotides. STING-binding nucleic acid molecules can also bind molecules that promote transmembrane transport. In this way, the disease or disorder may be a DNA-dependent inflammatory disease. Also described herein are methods of treating cancer in a subject having a cancerous tumor infiltrated with inflammatory immune cells. Such methods may include administering to the subject any amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent that down-regulates the function or expression of STING.

More specifically, the oligonucleotide is an antisense oligonucleotide, a CpG oligonucleotide, or a combination thereof.

In the present specification, the term CpG oligonucleotide (CpG oligodeoxynucleotide, or CpG oligodeoxynucleotide, CpG ODN) is a short term including unmethylated cytosine triphosphate deoxynucleotide ("C") and guanine triphosphate deoxynucleotide ("G"). As a single-stranded synthetic DNA molecule, it is known as an immunostimulant. When the CpG is included as a component of the nano vaccine of the present invention, it serves as an adjuvant that enhances the immune response of dendritic cells.

The immunostimulatory low-molecular compound is also called a small molecule adjuvant, and there are synthetic low-molecular adjuvants and natural small-molecular adjuvants. Examples of the immunostimulatory low-molecular compound or low-molecular adjuvant include monophosphoryl lipid A, Muramyl dipeptide, Bryostatin-1, Mannide monooleate (Montanide ISA 720), Squalene, QS21, Bis-(3',5')-cyclic dimeric guanosine monophosphate, PAM2CSK4, PAM3CSK4, Imiquimod, Resiquimod, Gardiquimod, cl075, cl097, Levamisole, 48/80, Bupivacaine, Isatoribine, Bestatin, Sm360320, and Loxoribine, but are not limited thereto. For small molecule adjuvants, Flower DR et al . (Expert Opin Drug Discov. 2012 Sep;7(9):807-17.).

In another aspect of the present invention, the present invention provides a vaccine composition comprising the above-described lipid nanoparticles as an active ingredient.

The vaccine composition is a pharmaceutical composition, and includes a pharmaceutically acceptable excipient or carrier in addition to the above-described lipid nanoparticles.

As used herein, the term "pharmaceutically acceptable" refers to properties that are acceptable to patients from a pharmacological/toxicological point of view and acceptable to a pharmaceutical pharmacist from a physical/chemical point of view for composition, formulation, safety, patient acceptability and bioavailability. And/or a substance. “Pharmaceutically acceptable carrier” refers to a medium that does not interfere with the effect of the biological activity of the active ingredient(s) and is non-toxic to the host upon administration.

The subject to which the present vaccine composition is applied may be any animal, specifically mammals, such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, pigs, and the like. Most preferred subjects are humans.

The vaccine composition may be freeze-dried or formulated as a liquid formulation according to any means suitable in the art. Non-limiting examples of formulations in liquid form include solutions, suspensions, syrups, slurries and emulsions. Suitable liquid carriers include all suitable organic or inorganic solvents, such as water, alcohol, saline, buffered saline, physiological saline, dextrose solution, propylene glycol solution, and the like, preferably in sterile form.

The vaccine composition may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the active polypeptide), which are inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Formed together. In addition, salts formed from free carboxyl groups include inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine. , Procaine, etc.

The vaccine composition is preferably formulated for inoculation or injection into a subject. For injection, the vaccine composition of the present invention may be formulated in an aqueous solution, such as water or alcohol, or in a physiologically suitable buffer, such as Hanks' solution, Ringer's solution or physiological saline buffer. I can. Solutions may include formulating agents, such as suspending, preserving, stabilizing and/or dispersing agents. In addition, injection formulations can be prepared as solid form formulations that are converted to liquid form formulations suitable for injection immediately prior to use, for example by reconstitution with a suitable vehicle, such as sterile water, saline or alcohol, prior to use.

In addition, the vaccine composition can be formulated as a delayed release vehicle or depot formulation. Such long acting formulations can be administered by inoculation or implantation (eg subcutaneous or intramuscular) or by injection. Thus, for example, the vaccine composition can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a poorly soluble derivative, e.g., a sparingly soluble salt. have. Liposomes and emulsions are well known examples of delivery vehicles suitable for use as carriers.

The vaccine composition may include an agent, such as an adjuvant, that enhances the protective efficacy of the vaccine. Adjuvants include any compound or compounds that act to enhance a protective immune response against the peptide antigen, thereby reducing the amount of antigen required for the vaccine and/or the number of administrations required to generate a protective immune response. Adjuvants are, for example, emulsifiers, muramyl dipeptide, abridin, aqueous adjuvants, such as aluminum hydroxide, chitosan-based adjuvants, and any of a variety of saponins, oils and known in the art. Other substances, such as Amphigen, LPS, bacterial cell wall extract, bacterial DNA, CpG sequences, synthetic oligonucleotides and combinations thereof [Schijins et al. (2000) Curr. Opin. Immunol. 12:456], mycobacterial play (M. phlei) cell wall extract (MCWE) (US Pat. No. 4,744,984), M. play DNA (M-DNA) and M-DNA-M. play cell wall complex (MCC). Can include. Compounds that can be used as emulsifiers include natural and synthetic emulsifiers and anionic, cationic and nonionic compounds. Among synthetic compounds, anionic emulsifiers include, for example, calcium, sodium and aluminum salts of lauric acid and oleic acid, calcium, magnesium and aluminum salts of fatty acids, and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltriethylammonium bromide, and synthetic nonionic agents include glyceryl esters (eg glyceryl monostearate), polyethylene glycol esters and ethers, and sorbitan fatty acid esters. (E.g. sorbitan monopalmitate) and polyoxyethylene derivatives thereof (e.g. polyoxyethylene sorbitan monopalmitate). Natural emulsifiers include acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants may be formed from oil components, for example single oils, oil mixtures, water-in-oil emulsions or oil-in-water emulsions. The oil can be mineral oil, vegetable oil or animal oil. Mineral oil is a liquid hydrocarbon obtained from petrolatum through distillation techniques, and is also referred to in the art as liquid paraffin, liquid petrolatum or white mineral oil. Suitable animal oils include, for example, cod liver oil, flounder oil, large herring oil, orange roughy oil and shark liver oil, all of which are commercially available. Suitable vegetable oils include, for example, canola oil, almond oil, cottonseed oil, corn milk, olive oil, peanut oil, safflower oil, sesame oil, soybean oil and the like. Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA) are two common adjuvants commonly used in vaccine formulations, and are also suitable for use in the present invention. Both FCA and FIA are water-in-mineral oil emulsions; However, FCA also includes killed Mycobacterium sp.

In addition, immunomodulatory cytokines can also be used in vaccine compositions to enhance vaccine efficacy, for example as adjuvants. Non-limiting examples of such cytokines include interferon alpha (IFN-α), interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF) or combinations thereof. GM-CSF is highly preferred.

Vaccine compositions comprising an antigen and further comprising an adjuvant can be prepared using techniques well known to those of skill in the art, including, but not limited to, mixing, sonication and microfluidation. The adjuvant is from about 10% to about 50% (v/v) of the vaccine composition, more preferably from about 20% to about 40% (v/v), more preferably from about 20% to about 30% (v /v), or any integer within these ranges. About 25% (v/v) is very preferred.

The vaccine composition may be administered by infusion or injection (eg, intravenous, intramuscular, intradermal, subcutaneous, intrathecal, intraduodenal, intraperitoneal, etc.). In addition, the vaccine composition may be administered intranasally, vaginal, rectal, oral, or transdermally. Additionally, the vaccine composition can be administered by a “needle-free” delivery system. Preferably, the composition is administered by intradermal injection. Administration may be as directed by a physician or medical assistant.

The injection can be divided into several injections, and these divided inoculations are preferably administered substantially simultaneously. When administered in divided inoculations, the dose of the immunogen is preferably, although not necessarily, allocated equally at each separate injection. When an adjuvant is present in the vaccine composition, the dose of the adjuvant is preferably, but not necessarily, allocated equally at each separate injection. Separate injections for split inoculations are administered substantially adjacent to each other in the patient's body in some aspects. In some aspects, the injections are administered at least about 1 cm apart from each other in the body. In some aspects, the injections are administered at least about 2.5 cm apart from each other in the body. In some aspects, the injections are administered at least about 5 cm apart from each other in the body. In some aspects, the injections are administered at least about 10 cm apart from each other in the body. In some aspects, the injections are administered more than 10 cm from each other in the body, such as at least about 12.5, 15, 17.5, 20 cm or more apart. Primary vaccination injections and booster injections can be administered in divided doses as described and exemplified herein.

A variety of alternative pharmaceutical delivery systems can be used. Non-limiting examples of such systems include liposomes and emulsions. Certain organic solvents such as dimethylsulfoxide can also be used. Additionally, the vaccine composition can be delivered using a delayed release system, for example a semipermeable matrix of solid polymer comprising the therapeutic agent. A variety of delayed release materials available are well known to those of skill in the art. Delayed release capsules can release the vaccine composition over a range of days to weeks to months, depending on their chemical properties.

In order to prevent recurrence of cancer in a patient in a cancer remission state, a therapeutically effective amount of a vaccine composition is administered to the subject. A therapeutically effective amount, as determined by any means suitable in the art, is a clinically significant increase in the number of ^{tumor-associated antigen-specific cytotoxic T-lymphocytes (CD8 +) in the patient and the cytotoxic T to the antigen.} -Will provide a clinically significant increase in lymphocyte response. In general, in a patient, a therapeutically effective amount of a vaccine composition will destroy the remaining microscopic disease and significantly reduce or eliminate the risk of recurrence of cancer in the patient.

An effective amount of the vaccine composition significantly increases the likelihood that, without limitation, race, breed, pulp, height, weight, age, overall health condition of the patient, type of formulation, mode or method of administration, or cancer recurrence in the patient. It can be dependent on a number of variables, including the presence or absence of risk factors that cause it. These risk factors include, but are not limited to, the type of surgery, the condition and number of benign lymph nodes, the size of the tumor, the histological grade of the tumor, the presence/absence of hormone receptors (estrogen and progesterone receptors), HER2/neu expression, lymph. Vascular invasion and genetic predisposition (BRCA 1 and 2, etc.). In some preferred embodiments, the effective amount depends on whether the patient is lymph node positive or lymph node negative, and if the patient is lymph node positive, the number and extent of positive lymph nodes. In all cases, a suitable effective amount can generally be determined by a person skilled in the art using conventional optimization techniques and skilled and knowledgeable judgment of experts and other factors apparent to those skilled in the art. Preferably, a therapeutically effective amount of the vaccine composition described herein will provide a therapeutic prophylactic benefit without causing substantial toxicity to the subject.

The toxicity and therapeutic efficiency of the vaccine composition can be determined in cell cultures or experimental animals, for example, to determine _{LD 50} (a dose lethal to 50% of the population) and ED _{50 (a therapeutically effective dose to 50% of the population).} It can be measured by standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as a ratio of _{LD 50} /ED _50. Vaccine compositions exhibiting a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in patients. The dose of such a vaccine composition preferably falls within a range of circulating concentrations including the _{ED 50 with very little or no toxicity.} The dosage may vary within this range depending on the dosage form used and the route of administration used.

Toxicity information can be used to more accurately determine useful doses in a particular subject, such as a human. The treating physician may end, discontinue or control administration due to toxicity or organ dysfunction, and may adjust treatment as necessary if the clinical response is not suitable to improve the response. In the prevention of recurrent cancer, the size of the dose administered will vary depending on the severity of the patient's condition, the relative risk for recurrence, or the route of administration, among other factors. The severity of the patient's condition can be assessed, for example, in part by standard prognostic evaluation methods.

The vaccine composition may be administered to a patient on any suitable schedule to induce and/or support protective immunity against recurrence of cancer, to induce and/or support a cytotoxic T lymphocyte response. For example, after administering to a patient a vaccine composition as described and exemplified herein as a primary vaccination, a booster may be administered to support and/or maintain protective immunity. In some aspects, the vaccine composition may be administered to a patient once, twice or more times per month.

Vaccine dosing schedules, including primary vaccinations and booster vaccinations, can be continued over the course of, for example, several years to extend the patient's lifespan, as long as the patient needs it. In some aspects, the vaccine schedule includes more frequent dosing at the beginning of the vaccine regimen and less frequent dosing (eg booster) during the time to maintain protective immunity.

The vaccine composition is administered at lower doses at the beginning of the vaccine regimen, and higher doses may be administered over time. In addition, the vaccine is administered in higher doses at the beginning of the vaccine regimen, and lower doses may be administered over time.

The number of primary vaccine and booster administrations and the dose of antigen administered can be tailored and/or adjusted to meet the specific needs of the individual patient, as determined by the attending physician according to any means suitable in the art. have.

The vaccine composition according to an aspect of the present invention is a composition including the above-described lipid nanoparticles in common, and the description thereof will be omitted within the overlapping range in order to avoid the complexity of the specification.

In one embodiment of the present invention, the vaccine composition is for cancer prevention. When the vaccine composition is for cancer prevention, the antigen of the nano-lipid particle as an active ingredient is a tumor-associated antigen.

As used herein, the term "prevention" is used to prevent recurrence/relapse of cancer in patients in clinical remission as measured by any objective or subjective variable including the results of radiological or physical irradiation. Mention any success or signs of success.

In another aspect of the present invention, the present invention comprises a lipid nanoparticle comprising a tumor-associated antigen, a phospholipid, a cationic lipid, and an anionic drug as a first vaccine composition; It provides a cancer vaccine kit comprising the lipid nanoparticles and an immune checkpoint inhibitor as a second vaccine composition.

As used herein, "immune checkpoint" refers to a modulator of the immune system. Immune checkpoint molecules include stimulating immune checkpoint molecules and inhibitory immune checkpoint molecules. Inhibitory immune checkpoint molecules are targets for cancer immunotherapy. As the inhibitory immune checkpoint molecules, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TMI-3, VISTA, SIGLEC7, etc. are known, but limited thereto. It does not become. Immune checkpoint inhibitors approved to date target CTLA4, PD-1, and PD-L1.

In one embodiment of the present invention, the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

The present invention provides a lipid nanoparticle comprising an antigen, a phospholipid, a cationic lipid, and an anionic drug, a vaccine composition comprising the same, and a cancer vaccine kit. When using the lipid nanoparticles of the present invention, antigens and anionic drugs can be easily delivered into cells. In particular, the lipid nanoparticles of the present invention are included as a first vaccine composition, and lipid nanoparticles and an immune checkpoint inhibitor are prepared. Using the cancer vaccine kit of the present invention, which is included as a secondary vaccine composition, can effectively suppress regrowth and recurrence of tumors due to the occurrence of immunosuppression against the cancer nano vaccine.

1A is a diagram showing a method for synthesizing monoarginine-cholesterol (MA-Chol) used in the present invention.
1B is a diagram showing a ¹ H-NMR spectrum of MA-Chol in DMSO-d ^{6 -.}
1C is a diagram showing the MALDI-TOF mass spectrum of MA-Chol.
Figure 2a is a DSPE-PEG ₂₀₀₀ -OVA _PEP used in the present invention. It is a diagram showing the conjugation method of.
Figure 2b is DSPE-PEG ₂₀₀₀ -OVA _PEP . It is a diagram showing the purification results using HPLC.
Figure 2c is a diagram showing the MALDI-TOF mass spectrum _{of DSPE-PEG 2000} -OVA _PEP.
Figure 3a is a diagram showing the expected structure _{of the OVA PEP-SLNP@CpG nanoparticles of the present invention.}
Figure 3b is a diagram showing the expected principle of action _{of the OVA PEP-SLNP@CpG nanoparticles of the present invention.}
4 is a diagram evaluating the loading efficiency of CPG-ODN. Specifically, the CpG ODN loading efficiency was evaluated through Sepharose CL-4B size exclusion column. When 1.65 nmol of CpG ODN was loaded in 8 μmol of OVAPEP-SLNP, the loading efficiency of CpG ODN was nearly 100%.
5 is a diagram showing an electron micrograph (TEM) _{of the OVA PEP-SLNP@CpG nanoparticles of the present invention and their diameters.}
6 is a diagram showing a result of measuring the hydrodynamic diameter and zeta potential of the nanoparticles of the present invention by dynamic light scattering (DLS).
_{7 is a diagram evaluating the cytotoxicity of the OVA PEP-} SLNP@CpG nanoparticles of the present invention to dendritic cells (DC2.4) by WST-1 analysis.
_{Figure 8 is a diagram evaluating the intracellular uptake of the OVA PEP-} SLNP@CpG nanoparticles of the present invention in dendritic cells and bone marrow-derived DCs using flow cytometry. Rhodamine dye-labeled OVA _PEP- SLNP@CpG was used for flow cytometry.
9 is a view observed with a confocal laser scanning microscope to confirm the intracellular absorption _{of OVA PEP-} SLNP@CpG nanoparticles of the present invention.
10 is a diagram showing the frequency of mature dendritic cells by treatment with _{OVA PEP-SLNP@CpG nanoparticles of the present invention.}
11 is a diagram showing the expression level of CD80, a co-stimulatory molecule, by treatment with _{OVA PEP-SLNP@CpG nanoparticles of the present invention.}
12 is a diagram showing the expression level of CD86, a co-stimulatory molecule, by treatment with _{OVA PEP-SLNP@CpG nanoparticles of the present invention.}
13 is a diagram showing the B3Z reaction by the treatment _{of OVA PEP-SLNP@CpG nanoparticles of the present invention.}
14 is a result of measuring the secretion level of IL-2 by the treatment _{of OVA PEP-} SLNP@CpG nanoparticles of the present invention through ELISA.
15 is a diagram showing the lymphatic discharge of _{the OVA PEP-SLNP@CpG nanoparticles of the present invention.} Near-infrared dye-loaded OVA _PEP- SLNP@CpG nanoparticles were measured using IVIS.
Figure 16 is a diagram showing the lymphatic discharge of _{the OVA PEP-SLNP@CpG nanoparticles of the present invention.} The intensity of fluorescence over time was shown after subcutaneous injection.
17 is a diagram showing the in vivo distribution _{of OVA PEP-} SLNP@CpG nanoparticles of the present invention in lymph nodes.
18 is a diagram showing a flow cytometric gating strategy for confirming the distribution of specific cells in a lymph node. FSC x SSC gating was used to obtain singlets and lymphocytes based on size and granulation, and CD45 was used as a leukocyte marker. CD3 ^- CD19 ^- 7-AAD ^- cells were gated to exclude T cells, B cells and dead cells.
19 is a diagram showing the absorption of the nanoparticles of the present invention by antigen-presenting cells in lymph nodes through flow cytometry. Rhodamine-labeled OVA _PEP- SLNP@CpG was injected into the paws of mice, and the popliteal lymph nodes were excised.
Fig. 20 is a diagram showing a strategy for evaluating dendritic cell maturity in lymph nodes by gating ^{CD11c +} MHCII ⁺ cells, which are considered mature dendritic cells.
21 is a result of evaluating the maturity of dendritic cells in vivo by treatment of the nanoparticles of the present invention. Maturity markers CD40 and CD86 were measured by flow cytometry.
22 is a diagram showing an immunization schedule for evaluating the in vivo antigen-specific T cell response enhancing effect _{of the OVA PEP-SLNP@CpG nano vaccine of the present invention.}
Fig. 23 is a diagram showing the level of interferon-gamma secreted from splenocytes after restimulation of splenocytes from mice immunized with _{OVA PEP-SLNP@CpG nanovaccines measured by ELISA.}
FIG. 24 is a diagram showing the number of IFN-γ spot forming cells (SFCs) secreting interferon-gamma from splenocytes after collecting splenocytes from mice immunized with _{OVA PEP-SLNP@CpG nanovaccine and restimulated.} .
25 is a diagram showing the proportion of CD8+ T cells that produce interferon gamma.
26 is a diagram showing the proportion of CD8+ T cells producing interferon gamma and granzyme B.
27 is a diagram showing the immunization and experimental schedule of mice used for in vivo CTL analysis to evaluate the antigen-specific killing ability _{of OVA PEP-SLNP@CpG of the present invention.}
28 is a _PEP -SLNP OVA OVA @ CpG of _PEP of the invention is also a cell by measuring CFSE ^high and CFSE ^low cells quantitatively comparing CTL killing ability to analyze the apoptosis-specific splenocytes.
29 is a diagram showing the immunization and tumor inoculation schedule for evaluating the tumor antigen-specific tumor prophylactic effect _{of OVA PEP-SLNP@CpG of the present invention.}
30 and 31 are diagrams showing the average tumor size and tumor size in individual mice after EL tumor cell inoculation after immunization with _{OVA PEP-SLNP@CpG of the present invention.}
32 is a diagram showing the average tumor weight after inoculation of EL4 tumor cells after immunization with _{OVA PEP-SLNP@CpG of the present invention.}
33-34 are diagrams showing the average tumor size (FIG. 33) and tumor size in individual mice (FIG. 34) after inoculation of E.G7-OVA tumor cells after immunization with _{OVA PEP-} SLNP@CpG of the present invention.
35 is a diagram showing the average tumor weight after inoculation of E.G7-OVA tumor cells after immunization with _{OVA PEP-SLNP@CpG of the present invention.}
Fig. 36 is a tumor photograph of individual mice after inoculation of E.G7-OVA tumor cells after immunization with _{OVA PEP-SLNP@CpG.}
37 is a diagram showing an experimental schedule for evaluating the therapeutic efficacy _{of OVA PEP-SLNP@CpG.}
38-39 are diagrams showing the mean tumor size (FIG. 38) and tumor size in individual mice (FIG. 39) after E.G7-OVA tumor cell inoculation.
40 is a diagram showing the average tumor weight after inoculation of E.G7-OVA tumor cells.
Fig. 41 is a photograph of tumors of individual mice after inoculation of E.G7-OVA tumor cells.
42 is a _{diagram showing the number of TUNEL-positive cells in order to remove tumor tissue from mice immunized with OVA PEP-} SLNP@CpG of the present invention and to evaluate the antitumor efficacy of the nano vaccine of the present invention at the cellular level.
_{43 shows damaged cells in tumor tissues of mice immunized with OVA PEP-} SLNP@CpG of the present invention, and is a tissue stained with H&E.
Fig. 44 shows cells subjected to apoptosis in tumor tissues of mice immunized with _{OVA PEP-SLNP@CpG of the present invention, and brown cells show TUNEL-positive cells.}
45 is a diagram showing the number of TUNEL-positive cells counted in three random fields for each group in tumor tissues of mice immunized with _{OVA PEP-SLNP@CpG of the present invention.}
46 shows PD-L1 expression in tumor tissues through immunohistochemical (IHC) analysis. PD-L1 ⁺ cells were stained green and cell nuclei identified by Hoechst staining were indicated in blue.
47 is ^{a diagram evaluating CD8 +} T cell infiltration into tumor tissue through IHC analysis. CD8 ⁺ T cells were stained in red, and cell nuclei identified by Hoechst staining were indicated in blue.
48 is a diagram showing the expression level of PD-L1 in E.G7-OVA tumor cells with or without interferon-gamma treatment.
49 is a diagram showing an experimental schedule for evaluating the efficacy of inhibiting tumor recurrence by sequential combination treatment of _{the OVA PEP-SLNP@CpG nano vaccine of the present invention and an ICB antibody.}
Fig. 50 shows the gating strategy of mouse PBMCs for tetramer analysis, in which singlelets and lymphocytes are obtained according to the size and degree of granulation by FSC x SSC gating. CD45 was used as a leukocyte marker, and CD3 and CD8 were used as T cell markers. 7-AAD ^- cells were gated to exclude dead cells, and CD3 ⁺ CD8 ⁺ T cells were gated for tetrameric staining analysis.
51 is a diagram showing representative flow cytometric results of peripheral blood CD8 ⁺ _{T cells positive for OVA PEP} tetramer 20 days after tumor inoculation.
_{Figures 52 and 53 show OVA PEP} -specific CD8 ⁺ T cells (Figure 52) and PD- in peripheral blood of mice immunized with the nano-vaccine of the present invention at day 20 after tumor inoculation determined by flow cytometry (n = 6). It ^{is a diagram showing the percentage of 1 +} CD8 ⁺ T cells (FIG. 53).
54 is a diagram showing the size of tumors after inoculation of E.G7-OVA cells in mice. After the first vaccination cycle, 40 good responders were divided into 4 groups. When the tumor volume ^{reached ~ 2000 mm 3} poor responders were sacrificed.
55 is a diagram showing the weight of tumors at the time of sacrifice of mice for each group. Data are expressed as mean±SEM.
56 is a diagram visually showing the sequence and time of using _{the OVA PEP-SLNP@CpG nano vaccine of the present invention together with an immune checkpoint treatment.}

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol) %.

Experimental materials and test methods

Materials

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000] (DSPE-PEG ₁₀₀₀ ), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP (polyethylene glycol)-2000] (DSPE-PEG ₂₀₀₀ -PDP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-( lissamine rhodamine B sulfonyl) (DPPE-Rhodamine) was purchased from Abanti Polar Lipid (Alabaster, AL, USA). Boc-Arg(Pbf)-OH and cholesterol were purchased from Sigma Aldrich (St. Louis, MO, USA). CpG oligodeoxynucleotide (CpG ODN; 5'-TCC ATG ACG TTC CTG ACG TT-3') and control ODN (control ODN; 5'-TCC ATG AGC TTC CTG AGC TT-3') were from Genotech (Daejeon, Korea). It was synthesized using a phosphorothioate backbone. OVA _{257 -264} SIINFEKL (OVA _PEP) and SIINFEKL (OVA _PEP-C) peptide with a N- terminal cysteine was synthesized by Cosmo Tec Geneva (Seoul, Korea). All other reagents were purchased from Sigma Aldrich unless otherwise indicated.

Animals and cells

Female C57BL/6 mice were obtained from Orient Bio (Korea) and housed under pathogen-free conditions. Animal care and laboratory procedures were approved by the Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST). The DC2.4 murine dendritic cell line was provided by Dr. KL Rock (University of Massachusetts Medical School, Worcester, MA, USA). The B3Z murine CD8 ⁺ T hybridoma cell line was provided by Professor Lim Yong-taek (Sungkyunkwan University). DC2.4 and B3Z cells were 10% heat-inactivated fetal bovine serum (FBS; Welgene), 1% penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1X non-essential It was maintained using RPMI-1640 medium (Welgene, Gyeongsan, Korea) supplemented with amino acids and 50 μM 2-mercaptoethanol. The EL4 murine lymphoma cell line and the E.G7-OVA murine EL4 lymphoma cell line transfected with Ovalbumin were purchased from ATCC (American Type Culture Collection; Manassas, VA, USA). EL4 cells were in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol. Grew. E.G7-OVA cells were treated with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 0.5 mg/ It was grown in RPMI-1640 medium supplemented with mL G418 (Gibco, Grand Island, NY, USA). All cells were maintained at 37 °C in a humidified atmosphere containing _{5% CO 2.}

Flow cytometry Analysis (Flow flow cytometry )

All antibodies were purchased from BioLegend (San Diego, CA, USA), eBiosciences (San Diego, CA, USA) and Tonbo Biosciences (San Diego, CA, USA). Anti-CD16/CD32 (clone 2.4G2), anti-CD45 (clone 30-F11), anti-CD3 (clone 145-2C11), anti-CD8 (clone 53-6.7), anti-CD19 (clone 1D3), anti-CD169 (clone 3D6.112), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-MHCⅡ (clone M5/114.15.2), anti-CD40 (clone 3/ 23), anti-CD80 (clone 16-10A1), anti-CD86 (clone GL-1), anti-IFN-γ (clone XMG1.2), anti-Granzyme B (clone 16G6), anti-PD-1 ( clone 29F.1A12), and anti-PD-L1 (clone 10F.9G2). Cells were blocked with anti-CD16/CD32 antibody at 4° C. for 10 minutes and immunostained with other antibodies at 20-30 min at 4° C. for 20-30 minutes. Dead cells were excluded by staining with 7-AAD viability staining solution (Biolegend) or Ghost Dye ^TM Violet 450 (Tonbo Biosciences). Flow cytometry was performed using an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and data was analyzed using FlowJo software (TreeStar).

OVA _PEP -Phospholipid Conjugated Synthesis of OVA _PEP - phospholipid conjugate)

The C-OVA _PEP peptide was conjugated to _{DSPE-PEG 2000} -PDP by disulfide exchange reaction. Briefly, 2 mg of C-OVA _PEP and 7.8 mg of DSPE-PEG ₂₀₀₀ -PDP were dissolved in 200 μl DMSO and the solution was gently vortexed overnight at room temperature.

The mixture was quenched by addition of 200 μl of acetonitrile and purified by high performance liquid chromatography (HPLC, Agilent) using a C4 column (Nomura Chemical). The product-containing fraction was lyophilized and the conjugate (DSPE-PEG ₂₀₀₀ -OVA _PEP ) was obtained as a white solid. The conjugate (conjugate) was further analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectroscopy (Bruker).

OVA _PEP -SLNP@CpG Nano vaccine Preparation and characterization of OVA _PEP -SLNP@CpG nanovaccine )

Monoarginine-cholesterol (MA-Chol) was synthesized as described above (Lee, J. et al. Theranostics 6, 192-203, 2016). _{A nano vaccine (OVA PEP-} SLNP@CpG) based on small lipid nanoparticles (SLNP) was prepared by film formation and rehydration. Briefly, MA-Chol (3.89 μmol), DOPE (3.89 μmol), DSPE-PEG ₁₀₀₀ (0.2 μmol) and DSPE-PEG ₂₀₀₀ -OVA _PEP (0.02 μmol) were added to a glass vial and dried under vacuum overnight to remain. The solvent was completely removed. The resulting lipid film was rehydrated with 1 ml of HEPES-buffered glucose (HBG) containing 1.65 nmol of CpG ODN. After the solution was sonicated for 10 minutes, it was stirred for at least 4 hours at room temperature with a magnetic bar, and extruded at least 11 times using a mini extruder (Avanti Polar Lipids). The morphology and size of OVA _PEP- SLNP@CpG were evaluated by transmission electron microscopy (TEM) with 1% uranyl acetate solution for negative staining. The average size of the nanoparticles was measured using ImageJ software (National Institutes of Health), and its hydrodynamic size and zeta potential were measured at ambient temperature using a Zetasizer Nano range system (Malvern, Worcestershire, UK) using DLS (dynamic light scattering). ). The efficiency of CpG ODN loading was evaluated using a Sepharose CL-4B size exclusion column (Sigma Aldrich).

OVA _PEP- SLNP@CpG was loaded onto the column washed with HEPES-buffered saline (HBS); Fifteen eluted fractions were collected, and each CpG ODN was measured using Quant-iT OliGreen ssDNA reagent (Thermo Fisher). The loading efficiency of CpG ODN was determined by mixing 100 μl of each fraction with 20 μl of 5% Triton-X 100 and 100 μl of OliGreen, and measuring fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Fractions 2-4 contained nanovaccines, but fractions 6-10 contained free CpG ODN due to their size difference. The loading efficiency of 1.65 nmol of CpG ODN in 8 μmol of SLNP was nearly 100%.

cell Survivability Analysis (Cell viability assays)

The cytotoxicity of the nano vaccine was evaluated by analysis of water-soluble tetrazolium salt (WST-1) using the EZ-Cytox Cell Viability Assay kit (DoGenBio, Seoul, Korea) according to the manufacturer's instructions. Briefly, DC2.4 cells were seeded in 96-well plates at a density of ^{1 x 10 4} cells per well in 100 μl medium and incubated overnight at 37°C. Cells were treated with OVA _PEP- SLNP @ CpG and incubated at 37° C. for 24 hours. A 10% volume of WST-1 reagent was added to each well and the plate was incubated at 37° C. for 4 hours. Absorbance was measured at 450 nm in a micro plate reader (VERASmax ^{™, Molecular Devices).}

Dendritic Absorption in vitro ( In vitro uptake of DCs ).

The intracellular uptake of the OVA _PEP- SLNP@CpG nano vaccine was evaluated by flow cytometry and confocal laser scanning microscopy. Briefly, DC2.4 cells were seeded in 6-well plates at a density of ^{5 x 10 5} cells per well in 2 ml medium and allowed to attach overnight. To detect the intracellular uptake of the nano vaccine, 0.5% by weight of DPPE-rhodamine dye was added to the lipid nanoparticle formulation. Cells were incubated with 200 μM of rhodamine-labeled nano vaccine for 4 hours and washed with PBS, and cell uptake was assessed by flow cytometry. To confirm cell uptake by confocal microscopy, DC2.4 cells were seeded and grown at a density of ^{4 x 10 4} cells per well in 0.5 ml medium on a cover slip of a 24-well plate to attach overnight. Cells were incubated with 200 μM rhodamine-labeled nano vaccine for 4 hours, washed with PBS, fixed with 10% formalin solution and their nuclei stained with DRAQ5 (Thermo Fisher). All samples were imaged by confocal laser scanning microscope (LSM 780; Carl Zeiss).

marrow Of dendritic cells (BMDC) Generation, uptake, maturation and T cell cross-priming of BMDCs .)

BMDC is described in Kang, S. et al. It was created as described in (J Control Release 256 , 56-67, 2017). To evaluate the intracellular uptake of the nano vaccine, BMDC was seeded into 12-well plates at a density of ^{3 x 10 5 cells per well in 0.5 ml medium and attached overnight.} After incubation with 200 μM rhodamine-labeled nano vaccine for 4 hours, cells were washed, harvested, and stained with anti-CD11c-PE/Cy7 and anti-MHCII-APC antibodies.

Cell uptake was assessed by flow cytometry. To evaluate the ability of nano vaccines to enhance DC maturation, immature BMDCs were cultured in 12-well plates at a density of ^{5 x 10 5 cells per well in 0.5 ml medium and attached overnight.} BMDC for 24 hours in HBG buffer, soluble CpG, soluble OVA _PEP , soluble OVA _PEP + CpG, OVA _PEP -SLNP@ODN or OVA _PEP -SLNP@CpG (CpG: 0.1 μM; OVA _PEP : 1.2 μM; SLNP: 0.48 mM ) And incubated for 24 hours. BMDCs were then washed, harvested, stained with anti-CD11c-PE/Cy7, anti-MHC±anti-CD80-FITC and anti-CD86-PE antibodies and analyzed by flow cytometry.

To evaluate the cross-priming of T cells, BMDCs were seeded into 12-well plates at a density of ^{1×10 6 cells per well in 1 ml medium and adhered overnight.} BMDC with HBG buffer, soluble CpG, soluble OVA _PEP , soluble OVA _PEP + CpG, OVA _PEP -SLNP @ ODN or OVA _PEP -SLNP @ CpG (CpG: 0.1 μM; OVA _PEP : 1.2 μM; SLNP: 0.48 mM) Incubated for 18 hours, harvested and washed with citrate-phosphate buffer (pH 3.2) for 3 minutes on ice to remove the peptide/MHC class I complex from the surface.

These BMDCs were then ^{co-cultured with B3Z CD8 +} T hybridoma cells for 24 hours. Briefly, BMDC is seeded in a 96-well U bottom plate at a density of ^{2 x 10 4} cells per well in 0.1 ml buffer, then B3Z cells are added to each well at a density of ^{4 x 10 4 cells per well in 0.1 ml medium.} And incubated for 24 hours. The suspension was centrifuged to separate the cell pellet and the supernatant.

β-galactosidase activity was assayed in cell pellets. Briefly, the harvested cell pellet was washed and CPRG assay buffer (0.1% Triton X-100, 100 μM 2-mercaptoethanol, 10 mM MgCl ₂ and chlorophenol red-β-D-galactopyranoside (chlorophenol) red-β-D-galactopyranoside, CPRG). Each resuspended pellet was transferred to a well of a 96-well plate, and the plate was incubated for 3 hours at 37° C. in the dark. The absorbance of each well at 570 nm was measured using a microplate reader. The concentration of IL-2 in the collected supernatant was evaluated using an IL-2 ELISA kit (R & D Systems, Minneapolis, USA) according to the manufacturer's instructions.

Lymphatic drainage, absorption and Dendritic cells Maturity( In vivo lymphatic drainage, uptake and DCs maturation.)

To evaluate the lymphatic drainage of the OVA _PEP- SLNP @ CpG nano vaccine, 0.37% by weight of PEGylated cypate dye was added to the nanoparticle formulation. Pegylated cypate-loaded nano vaccines were injected subcutaneously into the soles of C57BL/6 mice. After 2, 4, 8 and 12 hours, the fluorescence signal was evaluated using an in vivo imaging system (IVIS). Lymphatic drainage was also evaluated by confocal microscopy. The rhodamine dye-labeled nano vaccine was injected subcutaneously into the sole of the mouse, and the popliteal LN was removed 8 hours later. The removed LN was embedded in an OCT compound (Leica, Germany), frozen, and split into 15 μm slices using a freezing microtome (CM1850; Leica), and mounted on a glass slide. LN sections were fixed in 10% formalin solution and blocked with PBS containing 2% bovine serum albumin (BSA) for 1 hour at room temperature. Slides were ^{mounted with VectaMount™} AQ mounting medium (Vector Laboratories, Burlingame, CA, USA) and imaged by confocal laser scanning microscope. In order to confirm the absorption by APC in LN, rhodamine-labeled nano vaccine was injected subcutaneously into the sole of the mouse, and the popliteal lymph node LN was removed 8 hours later. The removed LN was washed, excised and digested in collagenase type IV solution (1 mg/ml; Sigma Aldrich) for 30 minutes at 37°C. The cells were washed again and a single cell suspension was recovered by passing through a 70 μm cell filter (Falcon). LN cells were treated with anti-CD45-Pacific Blue, anti-CD3-PerCP/Cy5.5, anti-CD19-PerCP/Cy5.5, anti-CD169-FITC, anti-CD11b-APC, anti-CD11c-PE/Cy7 antibodies. And incubated at 4° C. for 20 minutes with 7-AAD. These cells were washed and analyzed by flow cytometry.

Immunization

6 week old female C57BL/6 mice were immunized using homologous prime-boost therapy. Mice were divided into 4 groups, which were then divided into HBG buffer vehicle, soluble OVA _PEP + CpG, OVA _PEP -SLNP @ ODN, or OVA _PEP -SLNP @ CpG (CpG: 0.4 nmol per mouse; OVA _PEP : 5 nmol per mouse; SLNP) : 2 μmol per mouse) were immunized by subcutaneous injection into the soles of both feet.

Evaluation of antigen-specific T cell responses

Mice divided into four groups as described above were immunized three times at 10-day intervals and sacrificed 3 weeks after the last immunization. To assess antigen-specific T cell responses, splenocytes were _{restimulated ex vivo with OVA PEP} (SIINFEKL peptide; 10 μg/ml). The amount of secreted IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) and the number of INF-γ producing cells was assessed by enzyme-linked immune spot (ELISpot) assay. INF-γ and granzyme B produced by CD8 ⁺ T cells were quantified by intracellular cytokine staining (ICS). To measure INF-γ levels by ELISA, splenocytes were seeded in 96-well U bottom plates at a density of ^{3 x 10 5} _{cells per well and restimulated with OVA PEP} for 72 hours. Culture supernatant was collected and IFN-γ concentration was measured using an IFN-γ ELISA kit (R&D Systems). To measure INF-γ-producing cells by ELISpot, splenocytes were seeded into 96-well microplates coated with a monoclonal antibody specific for mouse IFN-γ at a density of ^{3 x 10 5 cells per well, and cells were seeded at 30 cells.} Restimulated with OVAPEP for hours. The INF-γ production point was developed using the mouse IFN-γ ELISpot kit (R&D Systems) according to the manufacturer's protocol. After development, an automatic ELISpot reader (AID GmbH, Strassberg, Germany) was used to count the blue-black spots at the site of cytokine localization. For ICS analysis, splenocytes (3 x 10 ⁶ _{cells per round bottom test tube) were restimulated} with OVA PEP for 1 hour. To inhibit intracellular transport of cytokines, GolgiStop ^™ or GolgiPlug ^™ (BD Biosciences) was added to each tube. ^{Cells were incubated for 5 hours, stained with Ghost Dye TM} Violet 450 at 4° C. for 30 minutes to differentiate dead cells, and then anti-CD3-PerCP/Cy5.5 and anti-CD8-APC at 4° C. for 20 minutes. /Cy7 antibody stained. For intracellular cytokine staining, cells were permeabilized using Cytofix/Cytoperm ^™ solution (BD Biosciences) and incubated with PE-conjugated anti-IFN-γ and Alexa Fluor 647-conjugated anti-Granzyme B antibody. . Samples were washed and analyzed by flow cytometry.

In vivo cytotoxic T lymphocyte analysis (In vivo cytotoxic T lymphocyte assay)

Mice were divided into 4 treatment groups as described above and immunized 3 times at 7 day intervals. Seven days after the last immunization, mice were injected with a mixture of cells prepared from splenocytes of non-immune C57BL/6 mice. _{Half of the splenocytes were pulsed with OVA PEP} (1 μg/ml) at 37° C. for 1 hour and the other half was not. Non-pulsed cells were labeled with 0.5 μM CFSE (carboxyfluorescein succinimidyl ester), and OVA _PEP pulsed cells were labeled with 5 μM CFSE for 10 min. A 1:1 mixture of pulsed (CFSE ^high ) and non-pulsed (CFSE ^low ) cells was injected intravenously into immunized mice. Eighteen hours after injection, splenocytes from recipient mice were harvested and analyzed by flow cytometry to determine the relative numbers of ^{CFSE high} and CFSE ^{low cells.} Antigen-specific target cell death was calculated using the following equation:

expression

Specific target cell death percentage = 100-[100 x {(%CFSE ^high immunized mice / %CFSE ^low immunized mice) / (%CFSE ^high non-immunized mice/%CFSE ^low non-immunized mice)}]

Anti-tumor effect test ( Antitumor efficacy test)

To evaluate the therapeutic effect in tumor prevention, mice were immunized three times at 10 day intervals with each of the vaccine modalities described above. Three weeks after the last immunization, 2 ^{×10 5} EL4 cells were inoculated subcutaneously ^{on one flank of each mouse, and 2×10 5} E.G7-OVA cells were inoculated on the other side. Tumor growth was monitored every two days using a digital caliper and the tumor volume was calculated ^{as 0.5 x length x width 2.} Mice were euthanized when the average tumor volume reached the ethical endpoint (~2000 mm ^{3 ).}

To analyze the effect of treatment on tumor volume reduction, 2 ^{×10 5} E.G7-OVA cells were inoculated subcutaneously in the right flank of each mouse. When the average tumor volume ^{reached ∼50 mm 3} , mice were randomly divided into 4 treatment groups and immunized 3 times at 4 day intervals. To evaluate the efficacy of combination immunotherapy, mice were subjected to two immunization cycles with or without antibodies to mouse-PD-1 (alpha PD-1; BioXcell; clone: RMP1-14). Mice were inoculated by subcutaneous injection of 2 x 10 ⁵ E.G7-OVA cancer cells into the right flank. The first immunization cycle, consisting of three subcutaneous injections _{of the OVA PEP-} SLNP@CpG nanovaccine at 4-day intervals, began after 6 days. On day 20, mice were divided into mice with small-tumor (good responder) and mice with large-tumor (poor responder). When the tumor volume ^{reached ~ 2000 mm 3} , poor responders were sacrificed. Good responders began the second immunization cycle starting on day 26. The second immunization cycle consisted of two subcutaneous injections at 6-day intervals. In addition, alpha PD-1 (200 μg per injection) was administered intraperitoneally on days 1, 3 and 5 after each vaccination. Upon reaching the endpoint, the tumor tissue was removed, weighed and photographed.

In vitro induction of PD-L1 ( In vitro induction of PD-L1)

E.G7-OVA cells were seeded in 24-well plates at a density of ^{1 x 10 5} cells per well in 0.5 ml medium. Cells were treated with recombinant murine IFN-γ (100 ng/ml; Peprotech, Rocky Hill, NJ, USA) for 48 hours, washed, harvested and stained with PE-conjugated anti-PD-L1 antibody. In vitro PD-L1 induction was confirmed by flow cytometry.

Histopathology analysis of tumor tissue ( Histopathological analysis of tumor tissues)

The excised tumor tissue was embedded in the OCT solution, immediately frozen, and sectioned into 20 μm slices using a frozen slide, and mounted on a glass slide. Tissue sections were fixed in 10% formalin solution for 10 minutes and blocked with PBS containing 2% BSA for 1 hour at room temperature. ^{CD8 +} T cell infiltration into tumor tissue was assessed by incubating tissue sections at 4° C. overnight with biotin-conjugated anti-mouse CD8a antibody (1:100 dilution; Tonbo Biosciences).

Tissue sections were then washed and incubated for 1 hour at room temperature with PE-conjugated anti-streptavidin antibody (1:200 dilution; BD Biosciences). PD-L1 induction in tumor tissue was evaluated by incubating tissue sections at 4° C. overnight with rat monoclonal anti-PD-L1 antibody (1:100 dilution; Abcam). These sections were then washed and incubated with Alexa Fluor 488-conjugated goat anti-rat IgG antibody (1:100 dilution; Abcam) for 1 hour at room temperature.

Nuclei were stained with Hoechst 33342 (1:5000 dilution) and slides were mounted with VectaMount AQ mounting medium. All sections were imaged by confocal laser scanning microscope. The resected tumor tissue was fixed in 10% formalin solution, embedded in paraffin, and cut into 4 μm slices. These sections were stained with H&E, and apoptotic cells were measured using the Dead End Colorimetric TUNEL system (Promega, Madison, WI, USA) according to the manufacturer's instructions. All slides were analyzed using a Nikon upright fluorescence microscope.

Tetramer analysis( Tetramer analysis.)

Mouse peripheral blood obtained from the posterior orbital blood was collected in a serum separator tube (BD). Red blood cells (RBCs) were removed by incubating with 1 ml of RBC lysis buffer (Biolegend) at room temperature with gentle shaking for 2 minutes. For tetramer staining, blood cells were incubated with iTAg H-2Kb OVA tetramer-PE (MBL, Japan) at 4° C. for 30 minutes. Cells were then washed and 4° C. with Pacific Blue-conjugated anti-CD45, PE/Cy7 conjugated anti-CD3, Alexa Fluor 488 conjugated anti-CD8, and APC conjugated anti-PD-1 antibody and 7-AAD. Incubated for 20 minutes. Cells were washed again and analyzed by flow cytometry.

Statistical analysis.

Data are expressed as mean±SEM. Groups were compared by one-way analysis of variance (ANOVA) with a post hoc Tukey test using GraphPad Prism 5 (GraphPad Software). Statistical significance was defined as P <0.05.

Example 1: antigen of the present invention- and Adjuvant -Transport nano vaccine (OVA _PEP -SLNP@CpG) synthesis and characterization

Small lipid nanoparticles (SLNP) used as antigen- and adjuvant-carrying nano vaccines in the present invention are two phospholipids i) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and ii) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol) ₁₀₀₀ ] (DSPE-PEG ₁₀₀₀ ); And monoarginine-cholesterol (MA-Chol), a cholesterol derivative.

The DOPE is a neutral lipid involved in endosome escape of lipid nanoparticles. Thus, incorporation of DOPE into SLNP can promote antigen expression in cell membranes by enhancing antigen migration from endosomes to cytoplasm. DSPE-PEG ₁₀₀₀ is a PEGylated phospholipid (PEGylated phospholipid) that promotes lymphatic drainage of SLNP by increasing the colloidal stability of SLNP under physiological conditions.

The MA-Chol is a cationic molecule composed of arginine, cholesterol, and major components of SLNP, and enables the formation of a complex between SLNP and oligonucleotide (FIGS. 1A-1C ).

The adjuvant used in the present invention is a TLR9 functional CpG oligodeoxynucleotide (Toll-like receptor 9 agonistic CpG oligodeoxynucleotide, CpG ODN). The combination of CpG ODN and ICB immunotherapy has been reported to have potent synergistic anti-tumor efficacy, and several clinical trials of this combination are currently in progress. The model tumor antigen was the MHC class I-restricted epitope of ovalbumin (SIINFEKL; _{named OVA PEP} ), which was shown to stimulate ^{CD8 + T cell responses.} The OVA _PEP was chemically attached to the end of the PEGylated DSPE through a disulfide bond (Figs. 2a-2c).

When internalized into cells, this molecule was cleaved by glutathione in the cytoplasm, and the released free OVA _PEP (free OVA _PEP ) was presented to the cell membrane by MHC class I or II (Figs. 3a-3b).

An _{antigen-labeled, CpG adjuvant-containing SLNP, designated OVA PEP-} SLNP@CpG, was prepared by a one-pot sequential process of film formation and rehydration (FIG. 1A). Size exclusion chromatography showed almost complete loading of CpG ODN on _{OVA PEP-SLNP (FIG. 4 ).}

Transmission electron microscopy (TEM) showed that OVA _{PEP- SLNP@CpG prepared using 0.25 mol% of OVA PEP} antigen has a spherical shape, and has an average diameter of ~ 72 nm by analyzing 155 particles. It was confirmed (Fig. 5).

These OVA _PEP- SLNP@CpG particles had a hydrodynamic size of ˜104.5 nm and a zeta potential of +0.23 mV indicating a neutral surface charge. OVAPEP-SLNP@CpG and a control nano-vaccine with similar size and zeta potential, a non-immunostimulant control ODN-complex SLNP (OVA _PEP- SLNP@ODN) was prepared using the same procedure (FIG. 6).

Example 2: OVA of the present invention _PEP -SLNP@CpG of nano vaccine in vitro DC maturation and T cell cross-over in Priming Enhancement effect

Because in vivo nano vaccines are expected to be uptaken by dendritic cells (DC) or macrophages, the _{cytotoxicity of OVA PEP-} SLNP@CpG to DC was evaluated using the WST-1 assay. . The nano vaccine of the present invention did not affect the viability of DC2.4 murine DC even at a high CpG concentration of 500 nM (FIG. 7).

Intracellular uptake of nano vaccines by DC was _{evaluated using OVA PEP-} SLNP@CpG labeled with rhodamine dye, flow cytometry revealed a new band of rhodamine-positive cells, and the original DC2 It was different from the band of .4 cell and bone marrow-derived DC (BMDC) (FIG. 8).

Confocal laser scanning microscopy further confirmed the intracellular location of the nano vaccine (FIG. 9).

The maturation of DC and membrane presentation of the delivered antigen via MHC class I molecules are essential to induce an ^{effective CD8 + T cell response.} Flow cytometry was performed to evaluate whether OVA _PEP- SLNP@CpG can enhance DC maturation. The frequency of mature DCs expressing the marker CD11c ⁺ MHCII ^high and the expression of co-stimulatory molecules (CD80 and CD86) were significantly higher than that of other cell groups (FIGS. 10-12 ).

The mixture of soluble antigen and adjuvant (OVA _PEP + CpG ODN) or control OVA _PEP -SLNP@ODN did not induce DC maturation, indicating the importance of the CpG adjuvant. However, soluble CpG alone could not induce DC maturation, suggesting that a suitable delivery system for DC is required. These findings indicate that the OVA _PEP- SLNP@CpG nano vaccine can be taken up by DC and induce maturation.

Cross-priming ability of nano vaccines ^{in BMDC and CD8 +} T cell hybridoma B3Z cells engineered to secrete β-galactosidase when T cell receptors _{specifically recognize OVA PEP (SIINFEKL)-MHC complexes} Was evaluated. BMDC was treated with each vaccine modality for 18 hours, and rigorously washed to remove any antigenic peptides present on the MHC molecule extracellularly so that there was no intracellular processing and cross-presentation, and 24 hours While co-cultured with B3Z cells. β-galactosidase assay and IL-2 enzyme-linked immunosorbent assays (ELISA) showed that OVA _PEP- SLNP@CpG secreted significantly higher levels of β-galactosidase and IL-2 than other therapies. Was shown to induce (Fig. 2d). Both OVA _PEP -SLNP@ODN and OVA _PEP + CpG _{showed higher activity than soluble OVA PEP} or CpG alone (Figs. 13 and 14), but much lower than _{OVA PEP -SLNP@CpG.}

Taken together, these in vitro studies show that the OVA _PEP- SLNP@CpG nano vaccine is easily absorbed by DC to induce maturation, and effectively crosses ^{CD8 + T cells against the antigen by presenting the antigen released to the surface through MHC.} -Demonstrated that it can be primed.

Example 3: OVA _PEP -SLNP@CpG Induction of maturation of DC in lymph nodes by local injection (Local injection of OVA) _PEP -SLNP@CpG results in maturation of DCs in lymph nodes)

Depending on the size and function of the surface, topically injected nano-vaccines have been shown to flow into local lymph nodes (LNs), where nano-vaccines are produced by antigen presenting cells (APCs) such as DCs and macrophages. Is absorbed. To evaluate the lymphatic drainage of the nano vaccine, the present inventors _{injected OVA PEP-} SLNP@CpG labeled with a near-infrared dye subcutaneously into the soles of C57BL 6 mice. The in vivo imaging system (IVIS) showed a clear fluorescence signal intensity around the discharged LN, and the fluorescence signal started after 2 hours and lasted for 12 hours (FIGS. 15-16).

To investigate the distribution of the nano-vaccine in the lymph nodes, OVA _PEP- SLNP@CpG labeled with rhodamine dye was injected subcutaneously and draining popliteal LN was excised 8 hours later. Confocal microscopy showed that most of the nano-vaccines are localized in the subcapsular sinus region of lymph nodes (FIG. 17 ).

When OVA _PEP- SLNP@CpG reaches the nearest lymph node within 2 hours, the nano-vaccine is more likely to be excreted directly into the LN through the lymphatic vessel, rather than being absorbed by the DC at the injection site and delivered to the lymph nodes, and this process is ~ It takes 24 hours. The ability of APC to absorb dye-labeled nano-vaccines in popliteal lymph nodes was assessed by flow cytometry-based gating (Figure 18).

Approximately 19.7% of macrophages (CD169 ⁺ CD11b ^{+ ) and 25% of DC (CD169-} CD11c ⁺ ) were rhodamine fluorescence-positive, suggesting high levels of nano-vaccine uptake by APC in lymph nodes (Figure 19). .

After confirming the ability of the OVA _PEP- SLNP@CpG of the present invention to activate and promote DC maturation in vitro, the present inventors demonstrated DC maturation in popliteal lymph nodes including nano vaccines by a gating strategy using maturation markers CD40 and CD86. Was evaluated (FIG. 20).

OVA _PEP- SLNP@CpG significantly increased the expression of CD40 and CD86, but _{a mixture of soluble OVA PEP} and CpG, or a control OVA _PEP- SLNP@ODN slightly increased the expression of these markers (Fig. 21).

Taken together, these in vivo results show that the OVA _PEP- SLNP@CpG nano vaccine of the present invention can be delivered directly to local lymph nodes with high efficiency, is absorbed by DCs and macrophages residing in the lymph nodes, and effectively induces DC maturation. Proved that it can.

Example 4: In vivo Of antigen-specific cytotoxic T cell responses in cytotoxic T cell responses in vivo )

i) vehicle, ii) soluble OVA _PEP + CpG, iii) OVA _PEP -SLNP@ODN or iv) OVA _PEP -SLNP@CpG To evaluate antigen-specific CD8 ⁺ T cell responses, each substance was immunized to mice 3 times at 10-day intervals (day 0). , 10 days, 20 days), and sacrificed at 3 weeks (41 days) after the third immunization (FIG. 22).

Splenocytes were isolated from immunized mice, _{restimulated with OVA PEP} (SIINFEKL peptide), and the secretion of interferon-gamma (IFN-γ), a representative cytokine secreted by ^{activated CD8 + T cells, was performed by ELISA and} Measured by ELISpot analysis.

OVA _PEP- SLNP@CpG immunity induced greater secretion of INF-γ in ELISA (Figure 3b), and production of INF-γ spot forming cells (SFC) in ELISpot assay was much higher than that of other immunogens (Fig. Fig. 24).

Intracellular cytokine staining (ICS) was performed ^{to test the functionality of activated CD8 +} T cells, and INF-γ and granzyme B were measured using the gating strategy shown in Supplementary Figure 6. Soluble OVA _PEP +CpG and OVA _PEP -SLNP@ODN were not effective in inducing antigen-specific T cell responses, but ^{CD8 +} _{T cells isolated from OVA PEP} -SLNP@CpG immunized mice of the present invention were much more effective. High levels of INF-γ and granzyme B were produced (FIGS. 25-26).

The in vivo antigen-specific killing activity of these CD8 ⁺ T cells was determined by bilateral delivery of a mixture of halves of splenocytes _{obtained from unpulsed mice and halves of splenocytes pulsed with OVA PEP to recipient mice immunized with each vaccine therapy.} It was evaluated by doing. After 18 hours of adoptive transfer, ^{the antigen-specific killing ability of CD8 +} T cells was evaluated by flow cytometry (FIG. 27 ).

_{OVA PEP} -specific _{of metastatic splenocytes in mice immunized with OVA PEP-} SLNP@CpG (89%) than in mice immunized with soluble OVA _PEP +CpG (60.7%) or OVA _PEP- SLNP@ODN (82.9%). The percentage of enemy death was higher (Figure 28).

Collectively, these results suggest that OVA _PEP- SLNP@CpG can induce much higher antigen-specific killing activity in ^{CD8 +} T cells than the physical mixture of soluble antigen + adjuvant.

Example 5: preventive effect: OVAPEP - SLNP @ CpG Prophylactic effect: tumor prevention by OVA _PEP -SLNP@CpG nanovaccine )

The present inventors investigated the in vivo effect of each vaccine therapy to prevent tumor growth using two mouse lymphoma cell lines, EL4 and E.G7-OVA. E.G7-OVA is derived from EL4 cells by transfection of the OVA gene.

First, mice were immunized three times at 10 day intervals each with four different vaccine modalities; Three weeks after the third immunization, EL4 and E.G7-OVA cells were injected into the opposite flanks of these mice, respectively (Fig. 29).

OVA _PEP- SLNP@CpG did not inhibit the growth of EL4-derived tumors, but completely prevented the growth of E.G7-OVA-derived tumors in the opposite flank (FIGS. 30-31).

Soluble OVA _PEP + CpG had no effect in preventing tumor growth in both cell lines, whereas OVA _PEP -SLNP@ODN was moderately effective against both, but _{compared to OVA PEP} -SLNP @ CpG against E.G7-OVA-derived tumors. It was not effective (Figures 32-36).

These findings mean that the nano vaccines of the present invention prevented tumor growth in an antigen-specific manner.

Example 6: OVA in established tumor model _PEP -Therapeutic efficacy of SLNP@CpG

_{The present inventors next evaluated the therapeutic efficacy of the OVA PEP-} SLNP@ CpG nano vaccine of the present invention in E.G7-OVA tumor bearing mice. When the average tumor volume ^{reached -50 mm 3} , mice were randomly divided into 4 groups and immunized 3 times at 4 day intervals with each vaccine modality (FIG. 37 ).

Immunization induced with soluble OVA _PEP + CpG or OVA _PEP -SLNP @ ODN showed only moderate tumor growth inhibition compared to vehicle control, but immunization induced with _{OVA PEP -SLNP@CpG of the present invention inhibited tumor growth.} Significantly inhibited, and 2 out of 7 mice became tumor-free (Figures 38-41).

Histopathological analysis of tumor tissue was performed to better understand the effects of nano vaccines at the cellular level. Immunohistochemistry (IHC) showed that CD8 ⁺ T cell infiltration was significantly higher in tumor tissues from mice immunized with _{the OVA PEP-} SLNP @ CpG of the present invention than in the other groups (Figure 42).

Tumor tissue was analyzed during the late rejection phase of the CTL response, but the difference in the degree of T cell invasion between groups was significant.

Hematoxylin and eosin (H & E) staining of tumor tissues results in massive cellular damage such as modified nuclei, enucleated necrotic cells and dead cell-derived debris in _{the OVA PEP-SLNP@CpG-immunized group of the present invention.} Yes, but not in the other groups (Fig. 43).

Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) analysis confirmed large amounts of apoptosis in tumor tissues from _{the OVA PEP-SLNP@CpG-immunized group of the present invention.} (Figures 44-45).

Collectively, this histopathological analysis indicates _{that the therapeutic efficacy due to OVA PEP-} SLNP@CpG immunization is due to mass death of cancer cells following increased T cell invasion into the tumor.

Example 7: Immune checkpoint blocking and OVA _PEP - SLNP @ CpG nanovaccine Inhibitory effect of sequential combination of tumor regrowth

The OVA _PEP- SLNP @ CpG nano vaccine was very effective in preventing and inhibiting tumor growth, but the treatment response varied in individual mice. To evaluate the change in anti-tumor effect, tumors _{obtained from OVA PEP-} SLNP@CpG-immunized mice were arbitrarily divided into 2 groups based on their relative size: large tumor group (> ~ 60 mm ³ , 7 mice. 2 of mice) and small tumor group (<~ 60 mm ³ , 3 of 7 mice).

IHC showed that PD-L1 expression was significantly higher in the larger tumor group (classified as'poor responder') or in the smaller tumor group (classified as'good responder') than the unvaccinated control group (Figure 46). . In addition, CD8 ⁺ T cell infiltration was much better in good responders than in poor responders or unvaccinated mice (FIG. 47 ).

The present inventors also found that PD-L1 expression in E.G7-OVA cancer cells was significantly induced by treatment with IFN-γ, a representative anti-tumor cytokine secreted by ^{activated CD8 + T cells (Fig. 48).} ).

Because PD-L1 expression in TME is increased by IFN-γ secreted from T cells, this finding suggests that greater anti-tumor efficacy in good responders results from higher CD8 ⁺ T cell infiltration and cancer cell death by IFN-γ. Suggests that it can be. However, since PD-L1 expression and co-localization of tumor infiltrating T cells in tumor tissues are closely related to adaptive immune suppression and resistance, good responders with high PD-L1 induction in tumors are adaptive immunity through T cell depletion. It can develop resistance and lead to tumor recurrence. These findings suggest that there is a need to investigate new combinatorial strategies including the order and timing of treatment of cancer nano vaccines and ICBs.

To investigate the feasibility of a therapy in which the nano vaccine was sequentially combined with ICB therapy, 50 mice were vaccinated twice with or without an antibody against mouse PD-1 (αPD-1). Specifically, the first immunization was performed by subcutaneous injection of _{OVA PEP-} SLNP@CpG nano vaccine three times 6 days after inoculation to the side with E.G7-OVA cancer cells (FIG. 49). On the 20th day after inoculation of E.G7-OVA cancer cells, mice were divided into two groups based on the treatment response to the nano vaccine. 10 (20%) were poor responders and 40 (80%) were good responders. CD8 ⁺ T cells were isolated from both poor and good responders, and their phenotype was analyzed by flow cytometry using a gating strategy (FIG. 50 ).

The tetramer assay _{showed that the percentage of OVA PEP} (SIINFEKL)-specific CD8 ⁺ T cells was about 2 times higher in poor responders and in good responders than in non-immunized controls (FIGS. 51-52 ). In addition, ^{the expression of PD-1 by CD8 +} T cells was much higher in poor responders and in good responders than in unvaccinated controls (FIG. 53 ).

This finding is in good agreement with reports showing that PD-1 expression is upregulated in antigen-specific CD8 ^{+ T cells induced by vaccination and can be considered to reflect T cell depletion and activation.} The number of PD-1 ⁺ CD8 ⁺ T cells in TME was shown to be positively correlated with the number of these cells in peripheral blood. In addition, ^{invasion of PD-1 +} CD8 ⁺ T cells into the tumor has been reported to be a positive marker for response to ICB therapy. Since the number of PD-1 ⁺ CD8 ⁺ T cells in peripheral blood was high in good responders (Fig. 53) and PD-L1 was expressed in tumor tissues (Fig. 46), it seemed reasonable to treat only good responders with αPD-1. .

The 40 excellent responders were randomly divided into 4 dogs of 10 each (Figures 49 and 54).

i) One group was vehicle control, ii) the other was treated with αPD-1 alone, iii) the other was _{immunized again with OVA PEP} -SLNP @ CpG, iv) the other with OVA _PEP -SLNP@CpG Reimmunized and treated with αPD-1.

Starting on the 26th day, mice of the last two groups were _{immunized twice with OVA PEP-} SLNP @ CpG of the present invention at intervals of 6 days, and mice of the 2nd and 4th groups were immunized with 6 αPD-1 at intervals of 2 days. I had an intraperitoneal injection.

Only the vehicle control showed rapid regrowth of the tumor within a few days (FIG. 54 ), which is presumed to be due to exhaustion of antigen-specific T cells in the tumor and consequently unable to control tumor recurrence.

Contrary to our initial expectations, αPD-1 alone, which was expected to re-energize ^{exhausted PD-1 +} CD8 ^{+ T cells, could hardly inhibit tumor regrowth.} Because antigen-specific T cells only have short-term activity, an additional nano vaccine was needed to boost the CTL response again. The second cycle of OVA _PEP- SLNP@CpG alone immunization of the present invention partially inhibited tumor regrowth, but its efficacy was not significantly different compared to the vehicle control group or αPD-1 group, which is the tumor after the first immunization cycle. This suggests that if PD-L1 expression is induced at a high level in, the treatment result of the second cycle immunization using the nano vaccine may be poor. In contrast, tumor regrowth was effectively inhibited by the combination of the second cycle _{of OVA PEP-} SLNP@CpG nano vaccine of the present invention + αPD-1 (Figs. 54-55).

These results suggest that the effectiveness of the combination therapy differs depending on the order and timing of administration of the nano vaccine and ICB therapy.

That is, initial immunization with nano vaccines can lead to high tumor growth inhibition, but at the same time induce tumor expression of PD-L1, resulting in antigen-specific T cell depletion. Treatment of a good responder to cycle 1 immunization with a combination of nano vaccine immunization + ICB of cycle 2 can lead to a potent therapeutic response (FIG. 56 ).

Discussion

Cancer nano vaccines using nanomaterials as antigen and/or adjuvant-delivery carriers can induce tumor antigen-specific T cell immunity and have shown potential as a therapeutic method in an in vivo animal model. In addition, the combination of ICB immunotherapy and cancer nano vaccines can further enhance the anti-tumor efficacy of cancer nano vaccines.

However, the lack of an optimal vaccination regimen capable of solving the problems associated with the antigen-transmitting nanomaterial itself, such as toxicity and manufacturability, and the adaptive resistance of tumors to cancer vaccines hindered the clinical application of cancer nano-vaccines. To solve this problem, the present inventors have developed a novel antigen/adjuvant-carrying nanoparticle made of a biocompatible lipid component. These nanoparticles, in combination with ICB immunotherapy, showed very strong anti-tumor efficacy in both prophylactic and therapeutic tumor models, and the validity of a new treatment regimen based on the sequence and timing of modalities that effectively inhibit tumor recurrence. Proved.

The lack of toxicity associated with antigen-transmitting nanomaterials and the antitumor efficacy of nanovaccines are key factors for successful clinical application. The present invention discloses the construction of a cancer nano vaccine using biocompatible and non-toxic naturally occurring or synthetic components. Two biocompatible and non-toxic neutral-charged phospholipids, DOPE and DSPE-PEG _1000, have been widely used in clinically available liposome-based treatments. In previous studies, the present inventors have shown that MA-Chol, a cationic cholesterol derivative, can form a stable complex with oligonucleotides such as siRNA and CpG ODN. MA-Chol is biodegradable and non-toxic because it is synthesized from endogenous arginine and cholesterol through ester linkages. In fact, all three biocompatible and non-toxic components were able to successfully form SLNPs with CpG ODN, and antigen/adjuvant-carrying nano vaccines (OVA _PEP -SLNP @ CpG) are sufficiently stable in physiological media, so local injections It was excreted directly into local LN according to. These results indicate that SLNP is clinically suitable for use in cancer nano vaccines. In addition, the model tumor antigen (OVA _PEP ) was linked to the SLNP surface through disulfide bonds, so that the intact form of the antigen was released in the cytoplasm and effectively displayed on the MHC of APC. In fact, the OVA _PEP- SLNP@CpG nano vaccine presented antigens that were efficiently absorbed by DCs in vitro and in vivo and released on the DC surface via MHC, thereby effectively crossing ^{CD8 + T cells against the antigen-} I was able to prime. Experimental conditions used in this example may be different from those of other nano vaccine systems, but the _{antitumor efficacy of OVA PEP-} SLNP@CpG was 4 out of 6 mice in the prophylactic tumor model and 7 mice in the therapeutic tumor model. It was impressive because there were no tumors in 2 of the mice. The efficacy of these nano vaccines may be due to their ability to induce strong CTL responses against antigen-expressing E.G7 tumors. OVA _PEP- SLNP@CpG is made of a biocompatible, non-toxic substance and exhibits strong anti-tumor activity, so it has clinical potential for use as a therapeutic cancer nano vaccine.

The generation of adaptive resistance to cancer vaccines in tumors has disrupted the clinical application of cancer vaccines. PD-L1 expression and ^{colocalization of tumor infiltrating CD8 +} T cells are closely related to adaptive immune resistance. This study focused on the differences in treatment response to nanovaccines in individual E.G7 tumor-bearing mice. Tumor expression of PD-L1, ^{infiltration of CD8 +} T cells, and _{proportion of circulating OVA PEP} -specific/PD-1 + CD8 ⁺ T cells in peripheral blood were significant in poor responders and better responders than in the unvaccinated controls. Was higher. This suggests that good responders can be regarded as'hot tumors' that respond better to ICB therapy than'cold tumors'. On the other hand, these findings also indicated that good responders develop adaptive resistance to nano vaccines, leading to tumor recurrence if not treated properly. Thus, only good responders were treated with the combination of nano vaccine and ICB therapy. Efforts have been made to maximize treatment outcomes by varying the order and timing of each modality. The first cycle of vaccination was performed to systemically increase antigen-specific T cell immunity and induce PD-L1 expression in tumors. Subsequently, good responders sensitive to ICB therapy were treated with a second vaccination in combination with ICB antibodies. However, treatment of good responders with αPD-1 alone was entirely ineffective in inhibiting tumor regrowth, suggesting that booster vaccination is necessary to reactivate antigen-specific memory T cells derived from the first immunization cycle. do. _{In addition, the method of treating good responders only with OVA PEP-} SLNP@CpG vaccination of the second cycle was ineffective, indicating that T cell depletion by high PD-L1 induction in tumors is possible. The present inventors _{found that only the combination of αPD-1 with the second cycle of OVA PEP-} SLNP@CpG vaccination significantly improved the treatment outcome, resulting in effective inhibition of tumor regrowth or recurrence. This may be due to the re-boosting of the antigen-specific T cell response by the second vaccination, along with the reversal of immune suppression by ICB antibodies. Taken together, these findings clearly demonstrate the importance of the order and timing of treatment of each modality in combination therapy involving nano vaccines and ICB antibodies.

Despite the high therapeutic potential, cancer vaccines can stimulate cancer cells to produce immunosuppressive molecules and recruit immune regulatory cells to TME. For example, vaccine-derived CD8 ⁺ T cells upregulate PD-L1 and indoleamine-2,3-dioxygenase (IDO) expression and regulate in a model of metastatic melanoma. It has been shown to mobilize T cells (Treg) to induce immune suppression. In addition, cancer vaccines have been shown to upregulate the expression of NKG2A inhibitory receptors ^{on tumor infiltrating CD8 + T cells.} Despite the presence of various inhibitory and immunosuppressive molecules, this study investigated the effect of nano vaccines on the expression of only one inhibitory molecule, PD-L1. Therefore, there is a need to investigate other immunosuppressive molecules induced by nano vaccines and their mechanisms of activity. In addition, the reason for the difference in treatment response to the nano-vaccines in individual mice with the same genetic background is unclear. Nevertheless, the results of the present invention indicate the importance of the order and timing of each modality in designing a combination immunotherapy comprising a nano vaccine. Although tumor size may not be a good marker for distinguishing between good and poor responders, the sequential combination strategy proposed in this study requires additional clinical evaluation of personalized therapy. Imaging modalities, such as computed tomography to monitor tumor size and positron emission tomography to monitor tumor activity, can be a criterion for distinguishing between good and bad responders.

In conclusion, the present inventors have developed a new type of antigen/adjuvant-carrying nano vaccine composed of biocompatible and non-toxic lipid components. These nano vaccines showed very potent anti-tumor efficacy in both prophylactic and therapeutic tumor models. In addition, according to the treatment sequence and timing, a new combination treatment regimen consisting of cancer nano vaccine and ICB immunotherapy was proposed. These protocols can improve the persistence of anti-tumor immunity, including effective inhibition of tumor growth and recurrence. These findings further suggest the need to evaluate these new combination treatment regimens in other immunotherapy modalities.

Claims (12)

Lipid nanoparticles comprising antigens, phospholipids, cationic lipids, and adjuvants. The lipid nanoparticle of claim 1, wherein the antigen is a tumor-associated antigen. The lipid nanoparticle of claim 1, wherein the phospholipid is a phospholipid having 14 to 22 aliphatic carbon atoms. The method of claim 1, wherein the cationic lipid is Dimethyldioctadecyl-ammoniumbromide (DDAB), Dimethyldioctadecylammonium (DDAB), (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)- 1-propaniminium pentahydrochloride) (DOSPA), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium) (DOTMA), (N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium) (DOTAP), 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N4-Cholesteryl-Spermine (GL67), 1, Lipid nanoparticles, which are at least one cationic lipid selected from the group consisting of 2-dioleyloxy-3-dimethylaminopropane (DODMA), O-alkyl phosphatidylcholines derivatives, and dimethylammonium-propane (DAP) derivatives. The lipid nanoparticle of claim 1, wherein the cationic lipid is a cationic cholesterol derivative. The lipid nanoparticle of claim 6, wherein the cationic cholesterol derivative is Monoarginine-cholesterol (MA-Chol). The lipid nanoparticle according to claim 1, wherein the adjuvant is an immunostimulatory single-chain or double-chain oligonucleotide, an immunostimulatory low-molecular compound, or a combination thereof. The lipid nanoparticle according to claim 8, wherein the single-chain or double-chain oligonucleotide is a CpG oligonucleotide, a STING active oligonucleotide, or a combination thereof. A vaccine composition comprising the lipid nanoparticles of any one of claims 1 to 8 as an active ingredient. The vaccine composition of claim 9, wherein the vaccine composition is for preventing or treating cancer. A lipid nanoparticle comprising a tumor-associated antigen, a phospholipid, a cationic lipid, and an anionic drug as a first vaccine composition;
Cancer vaccine kit comprising the lipid nanoparticles and an immune checkpoint inhibitor as a second vaccine composition. The cancer vaccine kit according to claim 11, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

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