HK1228756B - Vaccine delivery systems using yeast cell wall particles - Google Patents

Description

Vaccine delivery system using yeast cell wall particles

Technical Field

The present invention generally relates to compositions comprising yeast cell wall particles (YCWP) and methods for delivering vaccines. The compositions and methods disclosed herein are particularly useful for preparing prophylactic and therapeutic vaccines.

Background Art

Vaccines are biological materials or preparations that, when administered to a subject, induce immune-mediated resistance to a disease. Vaccines have been widely used over the past 200 years to combat both infectious and non-infectious diseases.

Vaccine comprises immunogen, and it is the antigen that can induce experimenter to produce humoral and/or cell-mediated immune response.Antigen presenting cells comprise macrophage and the cell of other mononuclear phagocyte system, and they actively engulf antigen particles and play a key role in immune response.Macrophage is the cell that is derived from monocyte in tissue.These monocyte/macrophage phagocytosis microorganism, is then digested into smaller antigen part in lysosome/phagosome.The antigen that produces circulates back to the surface and is used for being presented to the armed (cellular arms) of the cell composition of humoral and immune system.Therefore, monocyte/macrophage is particularly useful, and this is because it all plays an important role in the nonspecific and specific defense of host antagonism antipathogen.

Dendritic cells are also antigen-presenting cells that express both MHC class I and class II molecules. In addition to conventional dendritic cells, dermal dendritic cells are important members of the skin's immune system. This is because dermal dendritic cells carry a high number of MHC class II molecules, allowing them to function as highly potent antigen-presenting cells.

An ideal vaccine would mimic the rapid uptake and translocation of pathogen structures without actually establishing infection and without causing inhibition of the MHC class I pathway.

Recently, many studies have focused on the targeted delivery of biomaterials to monocyte-derived cells to improve the therapeutic effects of biomaterials. It has been reported that many media including microspheres/microparticles, liposomes, nanoparticles, dendrimers, vesicles and carbon nanotubes can be used for this purpose. It is ideal to achieve sustained release, extended duration of action, reduced dosage and adverse side effects, and to improve patient compliance with this new delivery method. Jain et al., Expert Opin. Drug Deliv. 10(3): 353-367 (2013).

Yeast cell wall particles have become one of the preferred delivery vehicles because they form a hollow, porous microsphere structure through a glucan shell derived from natural yeast. Soto et al., Journal of Drug Delivery 2012 (2011). Yeast cell wall particles are used to deliver a variety of substances, such as nucleic acids, proteins, and imaging reporters. See, for example, Bioconjug. Chem. 19(4):840-848 (2008); and Figueiredo et al., Chemical Communications 47:10635-10637 (2011).

There remains a need in the art for improved immunogenicity by effectively delivering vaccines comprising exogenous proteins, antigenic determinants, antigens, peptides, and/or nucleic acids using only minimal amounts of exogenous material for MHC presentation. Furthermore, there remains a need in the art for yeast cell wall particles for delivering biomaterials to improve delivery efficiency, reducing the amount of biomaterial while achieving the same or increased levels of targeting efficacy to monocyte-derived cells. The present invention addresses this need.

Summary of the Invention

In one aspect, the present invention relates to a composition for delivering a vaccine comprising (i) particles and (ii) an exogenous biological material such as a protein or fragment thereof, a nucleic acid, a carbohydrate, a tumor cell lysate, or a combination thereof, loaded within the particles. In a specific embodiment, the vaccine comprises an antigen that induces an immune response when administered to a subject. Preferably, the antigen or fragment thereof is ultimately presented on a class I MHC molecule or a class II MHC molecule.

In some embodiments, the protein or fragment thereof, or nucleic acid is selected from the group consisting of a protein, a peptide, an antigenic determinant, an antigen, DNA, RNA, cDNA, and an immunogenic fragment or subunit thereof. In other embodiments, the vaccine is a live vaccine, an inactivated vaccine, or an attenuated vaccine. In other embodiments, the vaccine is a recombinant vaccine.

In some embodiments, the particles are digestible or biodegradable particles. Particles suitable for the present invention are synthetic or derived from natural sources and have a hollow interior or porous structure. Exemplary particles include yeast cell wall particles.

In some embodiments, the loaded particles are incubated with the isolated dendritic cells for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 1 hour prior to administration. Preferably, the dendritic cells are immature cells that have been isolated for no more than 8 days. In other embodiments, the loaded particles are administered without prior incubation with the dendritic cell population.

In some embodiments, the compositions of the present invention further comprise one or more adjuvants, excipients and/or preservatives. It is within the skill of those skilled in the art to select appropriate adjuvants, excipients and/or preservatives for a particular vaccine.

In a preferred embodiment, a small amount of one or more immune response enhancing adjuvants are added to the composition. The addition of one or more adjuvants increases the immunogenicity effect of the vaccine. Commonly used adjuvants include but are not limited to proteins, peptides, nucleic acids and carbohydrates. Exemplary adjuvants include but are not limited to monophosphoryl lipid A, CpG oligonucleotides (such as CpG DNA), polyinosinic acid cytidylic acid (Poly I: C), Poly ICLC, potent MHC II antigen epitope peptides, beta glucan and cytokines that stimulate dendritic cells such as IL-12 and IFN-γ, and cytokines that make DC mature such as IL-4 and GM-CSF. Suitable adjuvants are those molecules known to have the following effects: it makes DC mature and interacts with receptors on dendritic cells to activate dendritic cells and further stimulates the stronger generation of T cells such as CD4+T cells and CD8+T cells.

In one embodiment, the amount of one or more immune response enhancing adjuvants is at least about 10 ng, at least about 50 ng, at least about 100 ng, at least about 200 ng, at least about 300 ng, at least about 400 ng, at least about 500 ng, at least about 600 ng, at least about 700 ng, at least about 800 ng, at least about 900 ng, at least about 1 μg, at least about 5 μg, at least about 10 μg, at least about 15 μg, at least about 20 μg, at least about 25 μg, at least about 30 μg, at least about 35 μg, at least about 40 μg, at least about 45 μg, at least about 50 μg, at least about 60 μg, at least about 70 μg, at least about 80 μg, at least about 80 μg, at least about 90 μg, or at least about 100 μg. In one embodiment, the amount of the adjuvant accounts for 1-10% of the composition. The amount of the adjuvant is enough to stimulate receptors such as toll-like receptors on dendritic cells.

In a related aspect, the present invention relates to a method for effectively delivering a vaccine to a subject, comprising administering a composition comprising (i) particles and (ii) an exogenous protein, fragment thereof, nucleic acid, or combination thereof, as disclosed above, loaded into the dermis of the subject. Dermal dendritic cells phagocytose the loaded particles, thereby triggering an immune response to the vaccine.

In another related aspect, the present invention relates to a method for preparing incubated dendritic cells containing biomaterial-loaded particles, the method comprising: (i) loading the particles with biomaterial to produce loaded particles; (ii) lyophilizing the biomaterial-loaded particles; and (iii) incubating the biomaterial-loaded particles with dendritic cells, wherein the biomaterial comprises a protein or fragment thereof, a nucleic acid, or a combination thereof, and wherein incubating the loaded particles with the dendritic cells causes phagocytosis of the loaded particles by the dendritic cells.

In a specific embodiment, the method further comprises (a) resuspending the particles loaded with biological material in a solution and (b) freeze-drying the resuspended solution before step (iii). The biological material comprises a protein or a fragment thereof, a nucleic acid or a combination thereof.

In a specific embodiment, step (iii) comprises: (a) adding a biological material to the yeast cell wall particles, (b) incubating the yeast cell wall particles, (c) lyophilizing the yeast cell wall particles, and (d) washing the yeast cell walls, wherein the biological material comprises a protein or a fragment thereof, a nucleic acid or a combination thereof, and wherein steps (b) to (c) are repeated at least once, and the step of adding water to the yeast cell wall particles before step (b) is also repeated.

In specific embodiments, step (iii) comprises: (a) contacting the vaccine-loaded particles with dendritic cells at a ratio of about 1:1 to about 100:1, including about 1:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and about 100:1; (b) incubating the vaccine-loaded particles with the dendritic cells for 1-2 hours and (c) collecting the dendritic cells and washing the cells.

Furthermore, the present invention relates to a method for preventing and treating infectious and non-infectious diseases, comprising administering a composition comprising (i) particles and (ii) exogenous biological material loaded within the particles, wherein the biological material comprises a protein or a fragment thereof, a nucleic acid or a combination thereof, as disclosed above.

In specific embodiments, infectious diseases include, but are not limited to, viral, bacterial, or parasitic diseases that are currently susceptible to vaccine-stimulated protective immune responses, or those infectious diseases that are slightly susceptible to existing vaccine technologies that can be improved by the present invention. In other embodiments, non-infectious diseases include, but are not limited to, cancers that are targeted by generating similar protective immune responses against known or unknown immunogenic tumor-associated antigens.

On the other hand, the present invention relates to a composition comprising yeast cell wall particles and a silicate, wherein the yeast cell wall particles are modified by "capping" with a silicate. In some embodiments, the composition further comprises an exogenous biological material loaded into the yeast cell wall particles. In other embodiments, the composition encompassed by the present invention comprises yeast cell wall particles loaded with biological material and optionally one or more adjuvants. Preferably, one or more adjuvants are loaded into the yeast cell wall particles. In addition, suitable excipients and/or preservatives may be included in the composition of the present invention. Selecting suitable adjuvants, excipients and/or preservatives is within the skill of those skilled in the art. Preferably, the silicate is an organic moiety connected to each of the four oxygen-containing compounds of the orthosilicate, such as tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, or tetrabutyl orthosilicate.

In some embodiments, the biological material includes, but is not limited to, a specific protein or fragment thereof, a nucleic acid, a carbohydrate, a tumor cell lysate, or a combination thereof. The protein or fragment thereof or the nucleic acid is selected from the group consisting of a protein, a peptide, an antigenic determinant, an antigen, DNA, RNA, cDNA, and an immunogenic fragment or subunit thereof.

In some embodiments, a small amount of one or more immune response enhancing adjuvants can be loaded into the yeast cell wall particles or co-administered with the loaded yeast cell wall particles. Adding one or more adjuvants to the interior of the yeast cell wall particles can increase the immunogenicity of the composition. Commonly used adjuvants include but are not limited to small molecule compounds, proteins, peptides, nucleic acids and carbohydrates. Suitable adjuvants are those molecules known to have the following effects: they mature dendritic cells and interact with receptors on dendritic cells to activate dendritic cells and further stimulate the stronger generation of T cells such as CD4+ T cells and CD8+ T cells. Exemplary adjuvants include but are not limited to monophosphoryl lipid A, CpG oligonucleotides (such as CpG DNA), polyinosinic acid cytidylic acid (Poly I: C), Poly ICLC, potent MHC II antigen epitope peptides, beta glucan and cytokines that stimulate dendritic cells such as IL-12, IL-15 and IFN-γ, imiquimod, and cytokines that mature DC such as IL-4 and GM-CSF.

In one embodiment, the amount of one or more immune response enhancing adjuvants is at least about 10 ng, at least about 50 ng, at least about 100 ng, at least about 200 ng, at least about 300 ng, at least about 400 ng, at least about 500 ng, at least about 600 ng, at least about 700 ng, at least about 800 ng, at least about 900 ng, at least about 1 μg, at least about 5 μg, at least about 10 μg, at least about 15 μg, at least about 20 μg, at least about 25 μg, at least about 30 μg, at least about 35 μg, at least about 40 μg, at least about 45 μg, at least about 50 μg, at least about 60 μg, at least about 70 μg, at least about 80 μg, at least about 80 μg, at least about 90 μg, or at least about 100 μg. In one embodiment, the amount of the adjuvant accounts for 1-10% (w/w) of the composition. The amount of adjuvant is sufficient to stimulate receptors on dendritic cells, such as toll-like receptors.

In a related aspect, the present invention relates to a method for preparing a composition comprising yeast cell wall particles and a silicate. The method comprises contacting the yeast cell wall particles with the silicate in the presence of ammonia, such that the yeast cell wall particles are "capped" by the silicate. Preferably, the silicate is tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, or tetrabutyl orthosilicate.

In another related aspect, the present invention relates to a method for effectively delivering a biomaterial to a subject, comprising administering to the subject a composition comprising: (i) silicate-capped yeast cell wall particles; and (ii) a biomaterial loaded within the particles. Preferably, the composition is administered directly to the subject's dermis, causing dermal dendritic cells to engulf the loaded particles, thereby triggering an immune response to the biomaterial. Monocyte-derived cells engulf the composition comprising silicate-capped yeast cell wall particles loaded with the biomaterial, thereby promoting the differentiation of mature dendritic cells for appropriate antigen presentation.

In another related aspect, the present invention relates to a method for producing a cell mixture comprising yeast cell wall particles loaded with a biological material and capped with silicate, the method comprising: (i) loading the biological material into yeast cell wall particles to produce loaded particles; (ii) capping the loaded particles with silicate, (iii) lyophilizing the capped loaded particles; and (iv) incubating the capped loaded particles with monocyte-derived cells, such as pre-dendritic cells, dendritic cells, or partially differentiated dendritic cells, wherein the incubation causes the monocyte-derived cells to phagocytose the capped loaded particles. In some embodiments, the monocyte-derived cells are dendritic cells, and the biological material includes, but is not limited to, a specific protein or fragment thereof, a nucleic acid, a carbohydrate, a tumor cell lysate, or a combination thereof.

In a specific embodiment, the loading step of the above method for preparing capped loaded yeast cell wall particles comprises: (a) suspending the yeast cell wall particles and the biological material in a diluent and incubating for a period of time, such as about 2 hours, to allow the biological material to be absorbed by the yeast cell wall particles; and (b) lyophilizing the suspension to load the biological material into the yeast cell wall particles. If necessary, steps (a) and (b) are repeated at least once to improve the loading efficiency.

In a specific embodiment, the incubation step of the above method for preparing capped loaded yeast cell wall particles further comprises: (a) contacting the capped loaded particles with dendritic cells at a ratio of about 1:1 to about 100:1, including about 1:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, and about 100:1; (b) incubating the capped loaded particles with dendritic cells for 1-2 hours and (c) collecting the dendritic cells and washing the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 depicts a method for preparing dendritic cells.

Figure 2 depicts the method used to prepare tumor cell lysates.

FIG3 depicts a method for preparing yeast cell wall particles.

FIG4 depicts a method for loading biological material into yeast cell wall particles.

FIG5 depicts a method for preparing dendritic cells loaded with vaccine particles.

Figure 6A depicts the lungs of unvaccinated mice after 21 days of IV incubation with one million B16 tumor cells (dark spots are melanoma metastases). Figure 6B depicts the lungs of vaccinated mice after 21 days of IV incubation with one million B16 tumor cells, where the mice were vaccinated with a simple mixture of tumor cell lysate and yeast cell wall particles 3 days before tumor challenge. Figure 6C depicts the lungs of vaccinated mice after 21 days of IV incubation with one million B16 tumor cells, where the mice were vaccinated with yeast cell wall particles loaded with tumor cell lysate 3 days before tumor challenge.

Figure 7 depicts the structure of silicate-capped yeast cell wall particles. Silicon is represented in a darker gray, while a lighter gray represents carbon. Figure 8A shows the structure of tetraethyl orthosilicate (TEOS). Figure 8B shows the partial hydrolysis product of TEOS (ethyl orthosilicate) initiated by ammonia in the reaction mixture, forming H-bonds between its OH groups. Figure 8C shows the silanol condensation product, which results from the loss of water from the H-bonded ethyl orthosilicate molecules. This reaction continues, gradually forming polymeric silicates. The structural group in Figure 8D shows H-bonding similar to that between the polymeric silicate structure and the primary hydroxyl groups of β-glucan, between the polymeric silicate structure and the primary hydroxyl groups of β-glucan in the yeast cell wall particle. Figure 8E shows the resulting covalent bond formed between the polymeric silicate and the yeast cell wall particle, which originates from this silanol condensation and causes the capping of the loaded yeast cell wall particle.

Figure 8 depicts the survival percentage of each group of mice: the “control” group, which received intravenous injection of B16 melanoma tumor cells only; the “conventional yeast cell wall particles” group, which received intravenous injection of B16 melanoma tumor cells and intradermal injection of uncapped yeast cell wall particles loaded with B16 tumor cell lysate; the “Si-capped yeast cell wall particles” group, which received intravenous injection of B16 melanoma tumor cells and intradermal injection of silicate-capped yeast cell wall particles loaded with B16 tumor cell lysate; and the “conventional yeast The "cell wall particles + AD" group received intravenous injection of B16 melanoma tumor cells and intradermal injection of uncapped yeast cell wall particles loaded with B16 tumor cell lysate and adjuvants (including CpG oligonucleotides and monophosphoryl lipid A); and the "Si-capped yeast cell wall particles + AD" group received intravenous injection of B16 melanoma tumor cells and intradermal injection of silicate-capped yeast cell wall particles loaded with B16 tumor cell lysate and adjuvants (including CpG oligonucleotides and monophosphoryl lipid A).

DETAILED DESCRIPTION

Various methodologies known to those skilled in the art are cited herein. The cited publications and other materials describing these known methodologies are incorporated herein by reference in their entirety, as if set forth in their entirety.

definition

The term "about" in conjunction with numerical values and ranges indicates that the numbers are not to be considered limited to the exact numbers set forth herein, but rather are intended to refer to numerical ranges that are substantially within the ranges mentioned without departing from the scope of the present invention. As used herein, "about" is understood by those skilled in the art and will vary to a certain degree in the context in which it is used. For example, "about" means +/- 10% of the specific numerical value following the term.

As used herein, "subject" or "patient" refers to any animal in need of treatment with a vaccine. For example, a subject may have or be at risk of developing a condition that can be treated or prevented with a vaccine. As used herein, "subject" or "patient" includes humans.

As used herein, the phrases "therapeutically effective amount" and "therapeutic amount" refer to a vaccine dose or plasma concentration, respectively, of a composition described herein in a subject that provides for administration of a biological material or vaccine to a subject in need of such treatment to produce a specific response. For convenience only, exemplary dosages, delivered amounts, therapeutically effective amounts, and therapeutic amounts are provided below with reference to adult subjects. Such amounts may be adjusted as needed by one skilled in the art in accordance with standard practice to treat a particular subject and/or condition/disease.

As used herein, the term "capping" or "capping" means that a polymeric structure such as a "net-like net" covers or encapsulates the yeast cell wall particles, such that the biological material loaded into the yeast cell wall particles is retained or trapped therein. The polymeric structure can be formed by silicates such as tetraalkyl orthosilicates.

vaccine

The term "immunization" refers to a process by which a subject is protected against a particular condition, disease, or diseases, usually by receiving a vaccine.

The term "vaccine" is a biological material or product that, when administered (e.g., by injection, oral administration, or by aerosol administration), induces an immune response in a subject. Vaccines include at least one active ingredient, such as an antigen that induces an immune response, and other ingredients, such as adjuvants, conjugates, preservatives, and other excipients (including diluents, stabilizers), etc.

Exemplary antigens for vaccine applications include allergens, viral antigens, bacterial antigens, and antigens derived from parasites. For the prevention and treatment of infectious diseases, preferred bacterial antigens and viral antigens. Suitable viral antigens include HIV, EBV, HBV, HCV, CMV, and herpes viruses. In addition, toxins (usually produced by bacteria) can be used as antigens. For non-infectious diseases such as cancer, preferred antigens include tumor-associated antigens, which are familiar to those skilled in the art (for example, carcinoembryonic antigen, prostate-specific membrane antigen, melanoma antigen, adenoma antigen, leukemia antigen, lymphoma antigen, sarcoma antigen, MAGE-1, MAGE-2, MART-1, Melan-A, p53, gp 100, antigens associated with colon cancer, antigens associated with breast cancer such as HER2 and mammaglobulin A, Muc1, Trp-2, telomerase, PSA, and antigens associated with renal cancer), which may include a combination of antigens or antigenic fragments. In one embodiment, the particles are loaded with tumor cell lysates.

Biomaterials

The biological materials encompassed by the present invention include, but are not limited to, specific proteins or fragments thereof, nucleic acids, carbohydrates, tumor cell lysates, or combinations thereof. It will be appreciated by those skilled in the art that protein fragments, such as peptides of any length, antigenic determinants, or subunits of proteins, which, when administered, produce an immunogenic response in a subject.

In recent years, nucleic acids such as DNA, RNA, cDNA or fragments thereof have also been used as vaccines. Generally speaking, DNA is extracted from the DNA of an infectious source and then modified/enhanced by genetic engineering before being delivered to a subject by electroporation, gene gun, etc.

The biomaterials of the present invention can be live, wild-type pathogens. Preferably, the antigen is in an inactive or attenuated form, such as inactivated viruses, bacterial fragments, and subunits of proteins or immunogenic functional fragments, polypeptides, or nucleic acids. More preferably, the biomaterial does not cause disease but is effective in eliciting an immune response in a subject and protecting the subject against future infection with the specific disease.

It is understood that yeast cell wall particles have a pore size of at least about 30 nm, and therefore any molecule/object with a radius of gyration of 30 nm or less can be loaded into yeast cell wall particles. For example, some viruses or viral particles having a size less than 30 nm (e.g., tobacco mosaic virus), as well as other antigens (including tumor cell lysates), can be loaded into yeast cell wall particles.

adjuvant

A variety of immune response enhancers can be added to the composition as adjuvants to enhance the immune response, such that when the composition is administered to a subject (e.g., directly to the subject's dermis), the immune response is enhanced by the adjuvant compared to administration of a composition without any adjuvant. Alternatively, when a composition comprising particles loaded with biomaterials is incubated with dendritic cells, the adjuvant exhibits an increased effect on dendritic cells while significantly reducing any systemic effects from the adjuvant. Biomaterials include proteins or fragments thereof, nucleic acids, carbohydrates, tumor cell lysates, or combinations thereof.

A variety of immune response enhancers can be added to the composition for loading into the yeast cell wall particles, because the adjuvant enhances the immune response, so that when the composition is administered to a subject (e.g., directly to the subject's dermis), the immune response is enhanced by the adjuvant compared to administration of the composition without any additional adjuvant. For example, when yeast cell wall particles are loaded with a biomaterial and the loaded yeast cell wall particles are also incubated with dendritic cells, the adjuvant exhibits an increased effect on the dendritic cells while significantly reducing any local or systemic effects from the adjuvant.

It is within the skill of those skilled in the art to select one or more adjuvants suitable for use in the present invention. For example, monophosphoryl lipid A, CpG oligonucleotides, polyinosinic acid-cytidylic acid (Poly I:C), Poly ICLC, potent MHC II epitope peptides, and cytokines that stimulate dendritic cells such as IL-12, IL-2, and GM-CSF are good adjuvant candidates for the present invention.

Suitable adjuvants are molecules known to have the following effects: they mature dendritic cells and interact with receptors on dendritic cells to activate dendritic cells and further stimulate the stronger generation of T cells such as CD4+ T cells and CD8+ T cells. For example, monophosphoryl lipid A, CpG oligonucleotides, polyinosinic acid cytidylic acid (Poly I: C), Poly ICLC, potent MHC II epitope peptides and cytokines that stimulate dendritic cells such as IL-12, IL-2 and GM-CSF, small molecules such as imiquimod are good adjuvant candidates for the present invention.

particles

As used herein, "particle" refers to any hollow and porous structure that can contain a vaccine and also allow the vaccine to leave the structure. In some embodiments, the size of the particle is about 0.5 to about 5 μm, which is close to the size of bacteria to allow the particle to be digested by monocytes such as dendritic cells. In a specific embodiment, the size of the particle is about 0.5 to about 1 μm. In a specific embodiment, the size of the particle is about 0.5 to about 2.5 μm. In some embodiments, the particle can be any particle having a glycan network, as long as the particle size is about 0.5 to about 5 μm.

Preferably, the particles are digestible or biodegradable particles. In some embodiments, the particles are not limited to a specific shape or material, but can be any shape, size or material with a hollow or porous structure that allows the particles to be phagocytosed by monocytes (including dendritic cells).

Yeast cell wall granules

In another embodiment, the particles are yeast cell wall particles, yeast cell wall particles, which are prepared from yeast cell walls so that the particles have a hollow or porous structure to encapsulate biomaterials therein. The biomaterial comprises a protein or a fragment thereof, a nucleic acid or a combination thereof. In one embodiment, the yeast cell wall particles are prepared from Saccharomyces cerevisiae. In another embodiment, the yeast cell wall particles are approximately the size of microbial structures that are commonly ingested by cells of the mononuclear phagocyte system and other phagocytes. In a specific embodiment, the yeast cell wall particles are about 1-25 μm, preferably 1-5 μm, 5-10 μm, 10-15 μm, 15-20 μm, 15-25 μm, or 20-25 μm. For example, the yeast cell wall particles are about 20 μm.

In one embodiment, yeast cell wall particles are prepared by: (a) suspending yeast to produce a suspension, (b) incubating the suspension, (c) centrifuging the suspension and removing the supernatant, and (d) recovering the resulting yeast cell wall particles. In another embodiment, steps (a) to (d) are repeated at least once, twice, three times, or four times.

In another embodiment, yeast cell wall particles are prepared by (a) suspending yeast in a solution to produce a first suspension, (b) incubating the first suspension, (c) centrifuging the first suspension and removing the supernatant, (d) suspending the resulting pellet to produce a second suspension, (e) incubating the second suspension, (f) centrifuging the second suspension and removing the supernatant, and (g) washing the resulting pellet to recover the yeast cell wall particles. In another embodiment, the yeast cell wall particles are sterilized.

In a specific embodiment, the yeast is suspended in NaOH comprising 1M NaOH. In a specific embodiment, the first suspension is incubated at about 80°C for about 1 hour or 1 hour. In a specific embodiment, the centrifugation is performed at about 2000 times gravity for about 10 minutes, or at 2000 times gravity for about 10 minutes. In a specific embodiment, the pellet is suspended in water comprising water at about pH 4.5 or at pH 4.5. In a specific embodiment, the second suspension is incubated at about 55°C for about 1 hour or at 55°C for 1 hour. In a specific embodiment, the pellet is washed in water at least once, twice, three times, or four times. In a specific embodiment, the pellet is washed once.

In another embodiment, the yeast cell wall particles are sterilized using isopropyl alcohol and/or acetone, and then the precipitate is washed. In a specific embodiment, other known alcohols are suitable. In a specific embodiment, the yeast cell wall particles are allowed to dry completely after sterilization. In another embodiment, the yeast cell wall particles are resuspended after drying. In a specific embodiment, the yeast cell wall particles are resuspended in PBS, such as 1X PBS.

In another embodiment, the yeast cell wall particles are allowed to dry and then frozen before loading the biological material into the yeast cell wall particles and/or before capping with silicate, and the yeast cell wall particles are then stored before use. In a specific embodiment, the yeast cell wall particles are lyophilized and stored at about 4°C or lower. In a specific embodiment, the yeast cell wall particles are lyophilized and stored at 4°C. The biological material comprises a specific protein or fragment thereof, a nucleic acid, a carbohydrate, a tumor cell lysate, or a combination thereof.

Particles loaded with biomaterials

Particles, such as yeast cell wall particles, are loaded with biological material, such as a specific protein or fragment thereof, a nucleic acid, a carbohydrate, a tumor cell lysate, or a combination thereof. In one embodiment, the biological material is loaded into the particles by incubating the biological material with a suspension of particles, such as yeast cell wall particles, to allow the biological material to penetrate the hollow interior of the particles.

In another embodiment, after granules or yeast cell wall particles are incubated with biomaterial or loaded with biomaterial, the combination is freeze-dried to generate anhydrous vaccine in the granules. By freeze-drying, the biomaterial is trapped in the granules and is prepared to be engulfed by mononuclear cells such as dendritic cells. In a specific embodiment, freeze-drying is the only mechanism for retaining the biomaterial in the granules. In a specific embodiment, retention is not caused by blocking the biomaterial from leaving the granules (for example, by physical retention, hydrophobic binding, any other combination) by a separate component. In a specific embodiment, capture is not caused by crosslinking or otherwise connecting the biomaterial to any connection that can occur after freeze-drying outside the granules. In a specific embodiment, the compositions of the present invention do not include any other components that specifically assist in avoiding lysosomes. The biomaterial includes, for example, specific proteins or fragments thereof, nucleic acids, tumor cell lysates or their combinations.

In another embodiment, the biological material is incorporated into yeast cell wall particles. In a specific embodiment, the number of yeast cell wall particles is about 1 x 10 ⁹ and the volume of the biological material is about 50 μL. In a specific embodiment, the incubation is at about 4°C for about 1 hour or less. In some embodiments, the combination of yeast cell wall particles and biological material is lyophilized over a period of less than or about 2 hours.

In another embodiment, the loaded yeast cell wall particles are capped with a silicate. Specifically, in some embodiments, the loaded yeast cell wall particles are capped by contacting the yeast cell wall particles with a silicate, such as a tetraalkyl orthosilicate, in the presence of ammonia, such that the loaded yeast cell wall particles are capped with the silicate. In preferred embodiments, the loaded yeast cell wall particles are capped with the silicate within about 60 minutes, about 45 minutes, about 30 minutes, about 15 minutes, about 10 minutes, about 5 minutes, or about 2 minutes. The reactivity of the tetraalkyl orthosilicate allows it to react with the primary hydroxyl groups of the β-glucan structure of the yeast cell wall particles under ammonia-mediated hydrolysis. The tetraalkyl orthosilicate also self-reacts with the termini of these cell wall silicates to form "bridges" such as -O-Si(OH) ₂ -O- or three-dimensional bridges such as -O-Si(-O-Si-O-)(OH)-O- or -Si(-O-Si-O-) ₂ -O-. These bridges can occur across the pores in the yeast cell wall particles, resulting in increased retention of the drug or biological material loaded therein. The structure of the capped yeast cell wall particle is illustrated in Figure 7E. Such capped loaded yeast cell wall particles can be lyophilized.

The inventors of the present invention have unexpectedly discovered that loaded yeast cell wall particles capped with silicate are an effective vaccine delivery system. More specifically, the capped yeast cell wall particles retain more of the loaded material than uncapped yeast cell wall particles. Even more surprisingly, as detailed in the working examples, the capped yeast cell wall particles not only deliver significantly more of the released biological material to the cytoplasm of phagocytes, but also deliver significantly more loaded particles to the phagocytes, compared to uncapped yeast cell wall particles.

In another embodiment, after lyophilization, the loaded particles are resuspended in a diluent or solution. In a specific embodiment, the diluent or solution is water. In a specific embodiment, the loaded particles are resuspended and/or incubated with other biological materials, such as vaccines, to penetrate the particles, and then the combination is lyophilized again. In other embodiments, the combination is subjected to multiple lyophilization and resuspension. In other embodiments, after lyophilization and before use, the particles loaded with biological materials are sterilized in ethanol. The biological materials include, for example, proteins or fragments thereof, nucleic acids, carbohydrates, tumor cell lysates, or combinations thereof.

In a specific embodiment, the biological material is loaded into the particles by (a) incubating the biological material with a suspension of the particles, allowing the biological particles to penetrate the hollow interior of the particles, and lyophilizing the suspension of loaded particles, and (b) optionally resuspending the particles, incubating the resuspended particles, and lyophilizing the resuspended particles and any vaccine not in the particles.

In a specific embodiment using yeast cell wall particles, the number of yeast cell wall particles is about 1x10 ⁹ and the volume of the biological material is about 50 μL. In a specific embodiment, the number of yeast cell wall particles is 1x10 ⁹ and the volume of the biological material is 50 μL. In a specific embodiment, the incubation in step (a) lasts for less than 1 hour at about 4°C. In a specific embodiment, the incubation in step (a) lasts for about 1 hour at 4°C. In some embodiments, the suspension is lyophilized in step (a) for a period of less than 2 hours or about 2 hours. In some embodiments, the yeast cell wall particles in step (b) are resuspended in water, the water comprising about 50 μL of water or 50 μL of water. In some embodiments, the resuspended yeast cell wall particles are incubated in step (b) at about 4°C for less than 1 hour or about 1 hour, or at 4°C for less than 2 hours or about 2 hours. The biological material comprises a specific protein or fragment thereof, a nucleic acid, a carbohydrate, a tumor cell lysate, or a combination thereof.

Prior to administration, the capped yeast cell wall-loaded particles are resuspended in a pharmaceutically acceptable excipient such as PBS or saline solution.

dendritic cells

As used herein, "dendritic cells" refer to cells generated from peripheral blood mononuclear cells ("PBMCs"). In one embodiment, dendritic cells are prepared by: (a) collecting blood, (b) diluting the blood, (c) performing density gradient separation of PBMCs, (d) lysing red blood cells and washing PBMCs, (e) incubating PBMCs, (f) removing non-adherent cells, and (g) incubating adherent cells in medium.

In some embodiments, the dendritic cells are immature dendritic cells that have been incubated for no more than 5 days. In other embodiments, the dendritic cells have been incubated for 6-8 days.

In a specific embodiment, blood is heparinized. In a specific embodiment, the density gradient separation in step (c) includes placing blood in a lymphocyte separation medium (Lymphocyte Separation Medium) and then centrifuging the blood. In a specific embodiment, centrifugation is carried out for about 20 minutes or 20 minutes at 1000 times of gravity. In a specific embodiment, a second centrifugation is carried out before step (d) and is carried out for about 5 minutes at about 500g or 5 minutes at 500g. In a specific embodiment, a third centrifugation is carried out before step (d) and is carried out for about 10 minutes at about 500g or 10 minutes at 500g. In a specific embodiment, centrifugation is carried out for about 10 minutes at about 1200 times of gravity or is carried out for about 15 minutes at 1200 times of gravity. In a specific embodiment, a second centrifugation is carried out before step (d) and is carried out for about 5 minutes at about 500g or 5 minutes at 500g. In a specific embodiment, ACK lysis solution is used to carry out lysis, then incubated, preferably incubated at room temperature for about 5 minutes, then subsequent centrifugation. In a specific embodiment, the PBMCs are washed in RPMI medium. In a specific embodiment, the PBMCs are incubated in a flask at about 37° C. for about 1-2 hours or at 37° C. for 1-2 hours in step (e). In a specific embodiment, serum-free DC medium is added to the flask.

In some embodiments, one or more cytokines are present in the incubation medium, including but not limited to granulocyte-macrophage stimulating factor (eg, 800 units/ml) and IL-4 (eg, 500 units/ml).

Vaccine composition

In some embodiments, the particles loaded with biomaterials are injected directly into the dermis of the subject so that the loaded particles are engulfed by dermal dendritic cells. In some embodiments, optionally, the particles loaded with biomaterials are engulfed in monocytes (preferably dendritic cells) in vitro. In some embodiments, yeast cell wall particles loaded with biomaterials and capped with silicate are injected directly into the dermis of the subject so that the particles are engulfed by dermal dendritic cells. In one embodiment, the particles loaded with biomaterials are incubated with dendritic cells so that the cells engulf the particles loaded with biomaterials. Biomaterials include, for example, specific proteins or fragments thereof, nucleic acids, carbohydrates, tumor cell lysates or combinations thereof. In other embodiments, the capped loaded particles are engulfed by monocytes in vitro, wherein the monocytes are preferably dendritic cells.

In specific embodiments, the particles are incubated with dendritic cells at a ratio of about 1 : 1 to about 100: 1, optionally prior to administration to a human. The incubation can be for about 1 hour, 1 hour, or preferably less than 1 hour.

In specific embodiments, the dendritic cells containing the capped loaded particles are collected and washed, for example, at least once, twice, three times, or four times. In other embodiments, the dendritic cells are incubated after washing, resuspended in a freezing medium, and cryopreserved prior to use. In specific embodiments, the resuspension has a concentration of approximately 10 x ¹⁰ cells/ml or 10 x ¹⁰ cells/ml. In specific embodiments, the resuspension is cryopreserved prior to use.

preparation

The compositions of the present invention can be formulated for mucosal administration (e.g., intranasal and inhalation administration) or for transdermal administration. The compositions of the present invention can also be formulated for parenteral administration (e.g., intramuscular injection, intravenous injection, or subcutaneous injection), and are directly injected into the patient and monocyte-derived target cells, such as macrophages and dendritic cells. In a specific embodiment, the particles of the capped loaded biomaterial that were not previously incubated with dendritic cells are directly injected into the dermis of the experimenter. Therefore, the compositions of the present invention can be used like conventional vaccines. This also significantly reduces costs because only a lower level of skill is required. In other embodiments, before being administered to the experimenter, first the capped loaded particles and monocyte-derived cells such as dendritic cells are incubated.

Preparations for injection may be present in, for example, ampules or multidose containers in unit dosage form, optionally with the addition of a preservative. The composition may be in the form of a suspension, solution, or emulsion, for example, in an oily or aqueous vehicle, and may include preparatants such as suspending agents, stabilizers, and/or dispersants. The composition of the present invention may also be formulated with pharmaceutically acceptable excipients. These excipients are well known in the art, but will generally be physiologically tolerated aqueous solutions. Physiologically tolerated solutions are those that are essentially non-toxic. Preferred excipients will be inert or synergistic, but inhibitory compounds may also be used to achieve a tolerance response. Alternatively, the composition is not administered with any other immunosuppressive treatments such as steroids or chemotherapy.

Treatment

The compositions of the present invention attract phagocytes, such as cells of the mononuclear phagocyte system, including monocytes, macrophages, dendritic cells, or immature dendritic cells, and therefore can be used as vaccines. In the field of immunology, cells of the mononuclear phagocyte system are considered "professional" antigen-presenting cells and are therefore ideal targets for vaccine delivery. It is well known that presentation of antigens in APCs is far more effective in generating a strong cellular immune response than expression of the same antigen in any other cell type. Therefore, the ability of the compositions of the present invention to present antigens on the surface of antigen-presenting cells via class I and class II MHC molecules significantly enhances the effectiveness of the vaccine.

The present invention contemplates the use of the compositions disclosed herein for the prophylactic and therapeutic treatment of infectious and non-infectious diseases (including cancer), which are viral-mediated diseases, bacterial-mediated diseases, and parasitic diseases that are currently targeted by vaccine strategies, or those that are slightly susceptible due to limitations in existing vaccine technologies. The disease to be treated is not particularly limited, but depends on the biomaterial loaded into the particle. Exemplary biomaterials include tumor cell lysates, proteins or fragments thereof, carbohydrates, or combinations thereof.

The compositions of the present invention are contacted with phagocytes in vivo or in vitro. Thus, both in vivo and in vitro methods can be used. For in vivo methods, the compositions of the present invention are typically administered parenterally, often intravenously, intramuscularly, subcutaneously, interdermally, or intradermally. They can be administered, for example, by bolus injection or continuous infusion. In in vitro methods, monocytes are contacted outside the body and the contacted cells are then administered parenterally to the patient.

dose

In some embodiments, approximately 200 μL of dendritic cells containing biomaterial-loaded particles or capped biomaterial-loaded yeast cell wall particles at a concentration of 10x10 ⁶ forms a therapeutic dose. In another embodiment, the dose is administered as follows: before administering the dose to a subject, a 200 μL aliquot is diluted to a final volume of 1 ml. In a specific embodiment, the aliquot is diluted with sterile saline containing 5% human serum albumin. In a specific embodiment, the 200 μL aliquot will need to be thawed before dilution. In such a case, the time between thawing and administering the dose to the subject will not exceed 2 hours. In some embodiments, the diluted aliquot is administered in a 3cc syringe. In some embodiments, a syringe needle of no less than 23 gauge is used.

In another embodiment, at least 1, 2, 3 or 4 doses of the composition of the present invention are administered to the subject. In a specific embodiment, the subject is re-vaccinated once every 4 weeks. In some embodiments, the composition comprising particles loaded with biomaterials is administered to the subject without first fusing with dendritic cells. In a specific embodiment, the composition is re-administered to the subject once every 4 weeks. In a specific embodiment, at each vaccination, approximately 1-2 million dendritic cells comprising particles loaded with biomaterials or comprising capped loaded particles are optionally administered by injection. In a specific embodiment, the particles loaded with biomaterials or capped loaded particles are injected into the subject at or near the following locations: (1) infection or disease site, or (2) lymph node. The biomaterial includes, for example, a protein or fragment thereof, a nucleic acid or a combination thereof.

Vaccine compositions may also include biological adjuvants, including but not limited to nucleic acids such as CpG oligonucleotides, proteins, or peptide epitopes such as tetanus toxoid MHC class II-binding p30 peptide.

The present invention is further illustrated by the following working examples, which are for illustrative purposes only and do not limit the scope of protection of the present invention in any way.

Example

Example 1: Preparation of dendritic cells

Dendritic cells are generated from the patient's PBMC. PBMCs are collected by drawing 200ml of blood from the patient according to standard operating procedures. The blood is then transferred to a 250ml centrifuge tube and diluted 1:1 with 1X PBS. 35ml of diluted blood is then overlaid on a 15ml room temperature lymphocyte separation medium (LSM; Mediatech) layer in a 50ml tube and centrifuged at 1000g for 20 minutes at room temperature. The PBMC layer is removed by pipetting from the LSM gradient and placed in a clean 50ml centrifuge tube. Four volumes of 1XPBS are added and the tube is inverted to mix the contents. The PBMCs are then centrifuged at room temperature at 500g for 5 minutes. 10ml of 1X PBS is added to each tube, and the cells are resuspended and collected in 1 tube. The PBMCs are centrifuged again at room temperature at 500g for 10 minutes, resuspended in 20-40ml of ACK lysis solution (Cambrex), and incubated at room temperature for 5 minutes. The cells are then centrifuged again at 1500rpm for 5 minutes. PBMCs were resuspended in 30ml of RPMI-1640 medium (Mediatech). The cells were then transferred to 2-4 T75 flasks. The flasks were incubated at 37°C for 1-2 hours. Non-adherent cells were then removed by rinsing. Afterwards, 10ml of 1X PBS was added to each flask, the flasks were swirled, and the PBS was removed. Afterwards, 10ml of complete DC medium (serum-free DC medium + 800U/ml GM-CSF + 1000U/ml IL-4) was added to each flask. The flasks were then incubated at 37°C, 5% _CO2 for 2 days. On the 3rd day, 10ml of complete DC medium was added to each flask. The cells were then incubated for 2 more days. On the 6th or 7th day, the resulting immature dendritic cells were ready.

Figure 1 provides an overview of the generation of dendritic cells.

Example 2: Preparation of antigens

Synthetic antigens such as peptides can be readily produced commercially and provided in a lyophilized state. These peptides can be reconstituted and incubated with prepared yeast cell wall particles for loading. Similarly, recombinant proteins and/or isolated proteins can be suspended in solution and incubated with yeast cell wall particles for loading, as discussed below.

Example 3: Preparation of tumor cell lysates

Tumor samples are obtained from patients. After separating fat and necrotic tissue from the tumor tissue, the tissue is weighed and 1XPBS is added (50 μL of PBS per 200 μg of tissue), and the tumor is thoroughly minced with a scalpel in 1X PBS. The tumor cells are then subjected to 4 cycles of freezing and thawing. Freezing is performed in liquid nitrogen for 20 minutes and thawing is performed at room temperature. The prepared tumor cell lysate is quantified by spectrometer. An aliquot is taken for quality control testing. The remainder is stored in the formulation at ≤-135°C for vaccine preparation. After the freeze-thaw cycle, a small amount of adjuvant can be optionally added.

Figure 2 provides an overview of tumor cell lysate processing.

Example 4: Preparation of yeast cell wall particles

Yeast cell wall particles are prepared from Fleishmans Baker yeast or equivalent. In short, 10g of Fleishmans Baker yeast is suspended in 100ml of 1M NaOH, heated to 80°C and kept for 1 hour. Undissolved yeast cell walls are recovered by centrifugation at 2000x g for 10 minutes. The recovered yeast cell walls are then resuspended in 100ml of water (wherein the pH is adjusted to 4.5 by HCl) and incubated at 55°C for another 1 hour, then recovered by centrifugation. The recovered yeast cell wall particles are then washed once with water, 4 times with isopropyl alcohol, and finally 2 times with acetone. Once the yeast cell wall particles are completely dry, they are resuspended in PBS, counted, aliquoted into groups of ^1X10 particles and lyophilized for the manufacture of vaccines.

Figure 3 provides an overview of yeast cell wall particle processing.

Example 5: Preparation of yeast cell wall particles

Wash 3 grams of active dry yeast (Fleischmann or equivalent) three times in water as follows: suspend the yeast in 30 mL of sterile water, vortex, and centrifuge at 800-1000 x g for 5 minutes at room temperature. After decanting the supernatant, resuspend the yeast pellet in 50 mL of 1 M NaOH and heat in a 90°C water bath for 1 hour.

The yeast suspension was then centrifuged at 800-1000 x g for 5 minutes, and the pellet was resuspended in 25-30 mL of acidic water (pH adjusted to 4.5 with HCl). The acidic water wash step was repeated until the pH of the suspension was <7.0. The pellet was then resuspended in 30 mL of acidic water and incubated in a 75°C water bath for 1 hour. The yeast pellet was recovered by centrifugation at 1000 x g for 5 minutes, then washed three times with 10 mL of sterile water, four times with 10 mL of isopropanol, and finally twice with 10 mL of acetone. The acetone was carefully removed, and the pellet was evenly spread on the glass surface of a beaker and allowed to air dry overnight.

The dried YWCP was collected and stored in a vacuum jar at 4°C and then washed three times in 10-15 mL of filtered 70% ethanol. The YWCP was quickly sonicated on the final wash and repeated as needed to disperse clumps. Once the ethanol was removed, the YWCP was washed in sterile water. A 100 μl aliquot of YWCP was dispensed into 2.0 mL round-bottomed, buckled-top centrifuge tubes, refrigerated for 1 hour, lyophilized, and stored in a vacuum jar at 4°C for future use.

Example 6: Preparation of yeast cell wall particles

Yeast cell wall particles (YCWP) are prepared as follows: Saccharomyces cerevisiae (100 g of Fleishmans Bakers yeast, AB Mauri Food Inc., Chesterfield, MO) are suspended in 1 L of 1 M NaOH, heated to 80 ° C and kept for 1 hour. The insoluble material containing the yeast cell walls is collected by centrifugation at 2000 x g for 10 minutes. The insoluble material is then suspended in 1 L of water, the pH is adjusted to 4-5 with HCl, and then incubated at 55 ° C for 1 hour. The insoluble residue is collected again by centrifugation, washed once with 1 L of water, washed 4 times with 200 mL of isopropyl alcohol, and washed 2 times with 200 mL of acetone. The resulting slurry is dried at room temperature in a sterile hood to produce 12.4 g of a fine, slightly off-white powder. A powder consisting of dried yeast cell wall particles was carefully weighed and suspended in sterile distilled water at a concentration of 10 mgs/ml. 1 ml aliquots were placed in sterile Eppendorf tubes, frozen at -60°C, and lyophilized at 0.012 mBar. Since the boiling points of isopropanol and acetone are much lower than those of water, any possible contamination from these solvents was removed under these high vacuum conditions.

Example 7: Preparation of tumor cell lysate and loading of yeast cell wall particles

Tumor protein antigens are released from tumor tissue as follows: three freeze (-60°C)/thaw cycles are performed, followed by centrifugation at 21,000g to remove all insoluble material. Soluble tumor antigen material and optionally included adjuvant material are loaded into the interior of hollow yeast cell wall particles by incubation at 4°C for 2 hours to allow a small volume of soluble tumor cell lysate to completely penetrate the hollow interior of the yeast cell wall particles. The volume of soluble tumor cell lysate used is carefully calculated to be very close to the internal volume of the yeast cell wall particles so that a large amount of soluble tumor cell lysate remains in the hollow interior of the yeast cell wall particles after incubation. After incubation, the fully solvated yeast cell wall particles are frozen at -60°C and all water is removed by vacuum freeze drying at 0.012mBar for 8 hours, so that most of the anhydrous tumor cell lysate antigen material remains inside the hollow yeast cell wall particles. To force any remaining tumor cell lysate into the interior of the yeast cell wall particles, sterile water equal to the carefully calculated volume of the yeast cell wall particle interior was added to the partially dried loaded yeast cell wall particles, incubated again at 4°C for 2 hours, and then lyophilized again at 0.012 mBar vacuum for 8 hours.

Example 8: Loading of biological materials into yeast cell wall particles

A suspension of completely anhydrous yeast cell wall particles (1×10 ⁹ ) was contacted with 50 μL of peptide in PBS at 4° C. for a period of 2 hours to allow the peptide to penetrate into the hollow interior of the yeast cell wall particles to produce loaded yeast cell wall particles. The suspension was then lyophilized for 2 hours. After lyophilization, 50 μL of water was added to the loaded yeast cell wall particles, incubated at 4° C. for another 2 hours, and lyophilized again to obtain yeast cell wall particles with dried biomaterial in their hollow interiors. The loaded yeast cell wall particles were then sterilized by washing in ethanol and stored in ethanol.

Figure 4 provides an overview of the yeast cell wall particle loading protocol.

Example 9: Loading Yeast Cell Wall Particles with Tumor Cell Lysate

Patient tumor biopsy samples were carefully mixed with 50-100 μl of lysis buffer (PBS) (depending on the amount of tumor sample), avoiding the generation of bubbles during mixing, and then incubated at 4°C for 30 minutes. The mixture was subjected to three freeze-thaw cycles in an acetone-dry ice bath and a 37°C water bath, and centrifuged at maximum speed at 4°C for 10 minutes. 50 μl of the prepared tumor cell lysate was added to a sterile 2 mL centrifuge tube containing 10 mg of dry yeast cell wall pellets so that the liquid tumor cell lysate covered the yeast cell wall pellets. The mixture was incubated at 4°C for 2 hours until the liquid tumor cell lysate penetrated the yeast cell wall pellets.

The tubes were then placed in a -85°C freezer for 30 minutes to quickly freeze the pellet. The tubes were then placed in a freeze dryer overnight. 50 μl of sterile water was added to the dried yeast pellet and incubated at 4°C for 2 hours to allow the liquid to penetrate the pellet.

The tube was placed in a -85°C freezer for 30 minutes to quickly freeze the pellet. The tube was then placed in a freeze dryer overnight. The dried pellet was then resuspended in 1 mL of 70% ethanol and stored at 4°C for future use.

Example 10: Administration of loaded yeast cell wall particles to a subject

Under sterile conditions, the yeast cell wall-loaded pellet prepared according to the above example is resuspended in 1 mL of a solution suitable for injection, such as sterile water for injection or sterile saline for injection, optionally containing 5% human serum albumin. Once the yeast cell wall-loaded pellet is carefully resuspended, the entire volume is withdrawn and injected into the patient's dermis using a syringe.

Example 11: Preparation of dendritic cells containing loaded particles

Centrifuge a suspension of the loaded yeast cell wall particles prepared according to the above example in 70% ethanol. Carefully remove the ethanol and replace it with 1 mL of PBS. Sonicate the loaded yeast cell wall particles. Wash the loaded yeast cell wall particles with sterile 1X PBS. After the final wash, resuspend the loaded yeast cell wall particles in PBS to approximately 1 x ¹⁰ particles/100 μl PBS.

The loaded yeast cell wall particles were added to the dendritic cell culture at a ratio of 1:100, and the culture was returned to the 37°C incubator. The following factors were then added to the culture: 50 μg/mL of TNF-α in sterile water was added to the culture at a volume ratio of 1:5000 (2 μL per 10 mL culture); 10 μg/mL of IL-1β in sterile water was added to the culture at a volume ratio of 1:1000; 10 μg/mL of IL-6 in sterile water was added to the culture at a volume ratio of 1:1000; and 1 mg/mL of PGE2 in 100% ethanol was added to the culture at a volume ratio of 1:1000. After all factors were added and mixed into the culture, the culture was incubated overnight.

Example 12: Harvesting dendritic cells, preparing and cryopreserving vaccine compositions

Remove the dendritic cell culture prepared according to Example 11 from the incubator. Perform the following steps under sterile conditions in a hood: Remove 10 mL of medium from the culture flask. Rinse the culture flask with 4.0-4.5 mL of 1X PBS and add this to the medium.

Add 1.5-2.0 mL of CellStripper ^™ to the culture flask. Place the culture flask in a 37°C incubator for 10-20 minutes. Add approximately 4 mL of culture medium from the tube back to the flask to wash and remove the cells. Wash the flask to harvest as many cells as possible. Count the cells on a hemocytometer or Cellometer ^™ . Centrifuge and remove the supernatant.

The cells were then resuspended at 5 x ¹⁰⁶ cells/mL in a CryoStor ^™ 10 and aliquoted into cryovials appropriately labeled with the patient ID number, date, and a cell concentration of 1.25 x ¹⁰⁶ cells/mL/vial (approximately 250 μL). A 250-500 μL portion was retained in the cryovial for sterility testing, and the remaining vials were stored in Styrofoam containers and placed at -86°C for gradual freezing.

Example 13: Preparation of a solid dose of vaccine for patient administration

Remove one cryovial of patient cells from cryopreservation and carefully thaw in a 37°C water bath. Under aseptic conditions, gently add 1 mL of sterile saline containing 5% human serum albumin for injection (or 1 mL of sterile 1X PBS) to the cryovial containing the cells. After carefully resuspending the cells, withdraw the entire volume from the cryovial and use the syringe containing the vaccine for administration to the patient.

Example 14: Immunization Protocol

To vaccinate the subjects, a dose of 1.25 million dendritic cells containing vaccine-loaded particles was cryopreserved in 0.2 mL of serum-free 10% dimethyl sulfoxide freezing medium (CryoStor ^™ CS-10, BioLife Solutinos, Inc.). Prior to injection, the dendritic cells were thawed and diluted to 1 mL with sterile saline for injection containing 5% human serum albumin (Albuminar-25, AventisBehring). The dilution was then transferred to a 3.0 cc syringe for injection using a needle of no less than 23 gauge and administered within 2 hours of thawing. The injection solution can be administered subcutaneously to the lymph node region or intradermally.

Example 15: Isolation of mononuclear cells from whole peripheral blood using the SepMate-50 system

Sepmate-50 tubes with spigots allow for rapid layering of diluted blood onto density gradient medium (LSM) and prevent mixing of the layers. Following centrifugation with braking, enriched peripheral blood mononuclear cells (PBMCs) are poured into fresh tubes and processed for future culture as described below.

Procedure 1:

Procedure 2:

Example 16: Generation of dendritic cells in combination with loaded yeast cell wall particles

Following the procedure in Example 15, the following method was performed:

I. Adding Yeast Cell Wall Particles

II. Preparation and Addition of Cytokines

step Procedures/Work Instructions 1 Add TNF-α, 1β, IL-6 and PGE2 to each culture flask.

Example 17: Harvesting cells, preparing and cryopreserving vaccine compositions

Proceed with the following method:

Harvesting cells:

II. Preparation of vaccine composition and cryopreservation:

step Procedures/Work Instructions 1

Example 18: Experiments in mouse models

Yeast cell wall particles loaded with B16 murine tumor cell lysate were used as a mouse vaccine. Unimmunized mice vaccinated IV with 1 million B16 tumor cells served as controls. Three days prior to IV challenge with the same tumor, mice were vaccinated with: (i) a simple mixture of tumor cell lysate and yeast cell wall particles; or (ii) yeast cell wall particles loaded with tumor cell lysate. The total protein content and number of yeast cell walls in the tumor cell lysate were identical in both groups of vaccinated mice. Twenty-one days after IV inoculation with 1 million B16 tumor cells, the lungs of each group of mice were examined.

Figures 6A, 6B, and 6C show the results for the lungs of each group of mice 21 days after tumor challenge.

Example 19: Preparation of Silicate-Capped Yeast Cell Wall Particles

Yeast cell wall particles (YCWP) were prepared and loaded with peptide as described in the previous examples. 1 mg of yeast cell wall particles was loaded with 500 μg of peptide. The particles were then lyophilized, suspended in 1 ml of absolute ethanol, and a suspension of 100 μl of tetraethyl orthosilicate and 100 μl of a 10% aqueous ammonia solution was added. The mixture was gently shaken at room temperature for 15 minutes. The yeast cell wall particles were then washed thoroughly with absolute ethanol and stored in ethanol at 4°C until use.

Example 20: In vitro leakage test

Yeast cell wall particles were loaded with fluorescently labeled albumin, and then a portion of the loaded yeast cell wall particles was capped with silicate according to the above example, while the other portion remained uncapped.

Both uncapped and silicate-capped yeast cell wall particles loaded with fluorescently labeled albumin were first read on the reader to obtain an initial reading of total fluorescence counts, and then both uncapped and silicate-capped yeast cell wall particles were vigorously shaken. Supernatants were taken from both uncapped and silicate-capped yeast cell wall particles at 1 hour and 2 hours to obtain fluorescence readings, as detailed below.

As shown in the table above, after 1 hour of shaking, uncapped yeast cell wall particles leaked 24.73% of the total fluorescence, while silicate-capped yeast cell wall particles leaked 15.81%. After 2 hours of shaking, uncapped yeast cell wall particles leaked 16.6% of the total fluorescence, while silicate-capped yeast cell wall particles leaked 6.65%. Overall, after 2 hours, uncapped yeast cell wall particles lost 41.33% of the fluorescently labeled albumin, while silicate-capped yeast cell wall particles only lost 22.46% of the loaded albumin.

Example 21: In vivo loading and release test

Mouse macrophage primary cells, a phagocytic monocytic cell line, were plated in 6-well plates and cultured overnight. The following morning, both uncapped and silicate-capped yeast cell wall particles loaded with fluorescently labeled albumin were added to the cells. After overnight incubation, the cells were washed several times with PBS to lyse them. The lysate was centrifuged to collect the supernatant for fluorescence readings. The supernatant and pellet of the uncapped yeast cell wall particles had fluorescence readings of 1042 and 1094, respectively; while the supernatant and pellet of the silicate-capped yeast cell wall particles had fluorescence readings of 1945 and 878, respectively. Thus, the silicate-capped yeast cell wall particles delivered 86.6% more released albumin to the cytoplasm of the phagocytes than the uncapped yeast cell wall particles.

Example 22: In vitro phagocytosis assay

Mouse macrophage primary cells were plated in 24-well plates and cultured overnight. The following morning, uncapped yeast cell wall particles and silicate-capped yeast cell wall particles, both loaded with fluorescently labeled albumin, were added to the cells. Cell fluorescence readings were measured at 20 minutes, 1 hour, and 2 hours.

As shown in the table above, silicate-capped yeast cell wall particles delivered 82% more loaded albumin to phagocytes than uncapped yeast cell wall particles.

Example 23: Mouse survival study

Survival studies were conducted on five groups of mice, with five mice in each group in Groups I-IV and ten mice in Group V. The control group (Group I) received an intravenous injection of 0.5×10 ⁶ B16 melanoma tumor cells. The "conventional yeast cell wall particles" group (Group II) received an intravenous injection of 0.5×10 ⁶ B16 melanoma tumor cells and was vaccinated intradermally with uncapped yeast cell wall particles loaded with B16 tumor cell lysate one week before and weekly thereafter until week 6. The "Si-capped yeast cell wall particles" group (Group III) received an intravenous injection of 0.5×10 ⁶ B16 melanoma tumor cells and was vaccinated intradermally with silicate-capped yeast cell wall particles loaded with B16 tumor cell lysate one week before and weekly thereafter until week 6. The "conventional yeast cell wall particles + AD" group (Group IV) received an intravenous injection of 0.5×10 ⁶ B16 melanoma tumor cells and was vaccinated with uncapped yeast cell wall particles loaded with B16 tumor cell lysate together with GpC oligonucleotides and monophosphoryl lipid A adjuvants by intradermal injection one week before and weekly thereafter until week 6. The "Si-capped yeast cell wall particles + AD group" (Group V) received an intravenous injection of 0.5×10 ⁶ B16 melanoma tumor cells and was vaccinated with silicate-capped yeast cell wall particles loaded with B16 tumor cell lysate together with GpC oligonucleotides and monophosphoryl lipid A adjuvants by intradermal injection one week before and weekly thereafter until week 6.

As shown in Figure 8, all mice in the control group died within approximately 22 days. All mice in the conventional yeast cell wall particle group died within approximately 25 days, all mice in the silicate-capped yeast cell wall particle group died within approximately 35 days, and all mice in the conventional yeast cell wall particle and adjuvant group died within approximately 45 days. In contrast, approximately 40% of the mice in the silicate-capped yeast cell wall particle and adjuvant group were still alive after 100 days.

Claims (19)

1. A composition for delivering a vaccine comprising (i) yeast cell wall particles, (ii) tetraalkyl orthosilicate and (iii) tumor cell lysate loaded within the yeast cell wall particles, wherein the yeast cell wall particles are modified by capping with tetraalkyl orthosilicate. 2. The composition of claim 1, further comprising one or more adjuvants. 3. The composition of claim 2, wherein one or more adjuvants are loaded within the yeast cell wall particles. 4. The composition of claim 2, wherein the adjuvant is monophosphoryl lipid A or CpG oligonucleotide. 5. The composition of any one of claims 1-4, wherein the tetraalkyl orthosilicate is tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, or tetrabutyl orthosilicate. 6. The composition of any one of claims 1-4, further comprising an excipient or a preservative. 7. A method for preparing a cell mixture comprising yeast cell wall particles loaded with tumor cell lysates and capped with tetraalkyl orthosilicate, comprising: (i) Loading tumor cell lysates into yeast cell wall particles to produce loaded particles; (ii) The yeast cell wall particles are contacted with the tetraalkyl orthosilicate in the presence of ammonia, such that the yeast cell wall particles are capped by the tetraalkyl orthosilicate to prepare capped loaded particles. (iii) freeze-drying the capped loaded particles; and (iv) Incubate the capped loaded particles with mononuclear cell-derived cells. The incubation process causes mononuclear cell-derived cells to phagocytose the capped loaded particles. 8. The method of claim 7, wherein the mononuclear cell-derived cell is a precursor dendritic cell, a dendritic cell, or a partially differentiated dendritic cell. 9. The method of any one of claims 7-8, wherein step (i) comprises: (a) Suspending yeast cell wall particles and tumor cell lysates in a diluent and incubating for a period of time to allow the tumor cell lysates to be absorbed by the yeast cell wall particles; and (b) Freeze-dry the suspension obtained in step (a) to load the tumor cell lysate into the yeast cell wall particles. 10. The method of claim 9, wherein steps (a) and (b) are repeated at least once. 11. The method of any one of claims 7-8, wherein the tetraalkyl orthosilicate is tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, or tetrabutyl orthosilicate. 12. Use of the composition of any one of claims 1-4 in the preparation of a medicament for treating cancer. 13. The use of claim 12, wherein the cancer is selected from the group consisting of: breast cancer, small cell lung cancer, non-small cell lung cancer, glioma, medulloblastoma, neuroblastoma, nephroblastoma, rhabdomyosarcoma, osteosarcoma, liver cancer, pancreatic cancer, melanoma, and prostate cancer. 14. The use of claim 12, wherein the cancer is ocular melanoma. 15. A method for preparing yeast cell wall particles loaded with tumor cell lysates and capped with tetraalkyl orthosilicate, comprising: (i) Loading tumor cell lysates into yeast cell wall particles to prepare loaded particles; (ii) Contacting the yeast cell wall particles with the tetraalkyl orthosilicate in the presence of ammonia, thereby capping the yeast cell wall particles with the tetraalkyl orthosilicate to prepare capped loaded particles; and (iii) Freeze-dry the capped loaded particles. 16. The method of claim 15, wherein step (i) comprises: (a) Suspending yeast cell wall particles and tumor cell lysates in a diluent and incubating for a period of time to allow the tumor cell lysates to be absorbed by the yeast cell wall particles; and (b) Freeze-dry the suspension obtained in step (a) to load the tumor cell lysate into the yeast cell wall particles. 17. The method of claim 16, wherein steps (a) and (b) are repeated at least once. 18. The method of any one of claims 15-17, wherein the tetraalkyl orthosilicate is tetraethyl orthosilicate, tetramethyl orthosilicate, tetrapropyl orthosilicate, or tetrabutyl orthosilicate. 19. The composition of any one of claims 1-4, wherein the composition is produced by the following steps: (i) Loading tumor cell lysates into yeast cell wall particles to prepare loaded particles; (ii) Contacting the yeast cell wall particles with the tetraalkyl orthosilicate in the presence of ammonia, thereby capping the yeast cell wall particles with the tetraalkyl orthosilicate to prepare capped loaded particles; and (iii) Freeze-dry the capped loaded particles.