Autologous dendritic cell vaccine kit and use

Abstract

We provide a kit for producing personalized vaccines based on autologous dendritic cells. [Solution] A kit for producing personalized vaccines based on autologous dendritic cells is disclosed herein. The kit contains all the materials, reagents, and information necessary to produce a specific dose of a live dendritic cell vaccine against a pathogen, part of a pathogen, toxin, venom, or structure obtained by recombinant or chemical synthesis.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Application No. 63 / 033,678, filed on 2 June 2020, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Personalized vaccines have attracted significant research interest, but their transition to clinical practice has been limited. The production of personalized vaccines typically requires equipment and trained individuals at laboratories or dedicated manufacturing facilities, which limits availability and contributes to their high cost, hindering their adoption. Personalized vaccines are primarily pursued for cancer treatment, where their high cost is more bearable. [Overview of the Initiative]

[0003] A simple vaccine kit containing all the necessary components for producing dendritic cell-based autoimmune vaccines enables rapid vaccine production even in emergency situations. Ex vivo-prepared dendritic cell-based vaccines avoid the potential for antigenic toxicity as well as the possibility of inducing immune tolerance. The personal autoimmune vaccine kit disclosed herein enables vaccine production in facilities such as local community hospitals that have only basic research equipment.

[0004] The complete composition or kit of the components is disclosed herein, and these are required for each step in the process of producing an autoimmune vaccine.

[0005] Furthermore, a method for using a vaccine manufacturing kit to produce an individual vaccine against an antigen is disclosed herein.

[0006] One embodiment is a kit for producing a personalized dendritic cell (DC) vaccine for an individual. The kit includes a kit container for containing the other components of the kit. The components of the kit include blood collection supplies, monocyte isolation medium or inertial isolation device, DC differentiation medium components, cell culture vessels, unique identification marks, and antigens.

[0007] In some embodiments, the monocyte isolation medium is contained in a blood collection vacuum tube. In some embodiments, the monocyte isolation medium is FICOLL®, a neutral, highly branched, high-mass, hydrophilic polysaccharide.

[0008] The DC differentiation medium components include basal cell culture media such as RPMI-1640, PRIME-XV Dendritic Cell Maturation known composition medium, or AIM-V medium. In some embodiments, a bicarbonate-free CO2-isolated medium is used. In some embodiments, HEPES is used as a CO2-isolated buffer. In some embodiments, the medium contains interleukin-4 (IL-4). In some embodiments, the medium contains IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the medium does not contain GM-CSF. In some embodiments, the medium contains interferon-alpha (IFNα), and in some embodiments, the medium contains interferon-gamma (IFNγ). In some embodiments, the medium contains interleukin-2 (IL-2). In some embodiments, the medium contains up to 30% autologous plasma.

[0009] In some embodiments, the cell culture vessel is a closed system with at least one access port. In some embodiments, the cell culture vessel is a bag, while in other embodiments, it is a rigid container with a flat inner surface. In some embodiments, the inner surface is hydrophobic.

[0010] In various embodiments, the unique identifier may be a series of alphanumeric characters, a barcode, or a QR code.

[0011] In some embodiments, the antigen is the entire pathogen or a fragment derived from a pathogen such as bacteria, fungi, viruses, rickettsiae, mycoplasma, or parasites.

[0012] In some embodiments, the antigen is a toxin or poison. Examples of non-limiting examples include toxins and poisons derived from bacteria, insects, and plants, or synthetically produced compounds.

[0013] In some embodiments, the antigen is a purified molecule such as a protein or peptide, or a fragment thereof. In some embodiments, the antigen is produced by recombinant technology.

[0014] In some embodiments, the antigen is produced by chemical synthesis.

[0015] In some embodiments, the antigen is the full-length spike protein of SARS-CoV-2.

[0016] In some embodiments, the kit container can function as an incubator. In some embodiments, the kit container has insulating walls. In some embodiments, the kit container has a rechargeable power source, such as a lithium battery. In some embodiments, the power source is a lithium polymer battery that can be formed to fit into the kit container. In some embodiments, the kit container includes a thermostat rather than a temperature controller. In some embodiments, the thermostat includes a phase exchange material and a positive temperature coefficient material.

[0017] One embodiment is a method for producing a personalized autologous DC vaccine using a kit disclosed herein. In some embodiments, the method includes collecting blood from an individual, isolating peripheral blood mononuclear cells (PBMCs), differentiating the PBMCs by adding the DCs to a cell culture vessel and incubating the cells for 2–5 days to generate immature DCs, then adding antigens to the cell culture vessel and incubating for a further 1–2 days to load the immature DCs with antigens (the antigens function as immunogens to induce an immune response to a pathogen, toxin, or component of a venom that is the target of the vaccine), and collecting the antigen-loaded immature DCs.

[0018] Some embodiments of a method for producing a personalized autologous DC vaccine further include storing autologous plasma obtained from the isolation step. Some embodiments of a method for producing a personalized autologous DC vaccine further include marking the containers containing cells or plasma obtained from the individual with an identifying mark. Some embodiments of a method for producing a personalized autologous DC vaccine further include resuspending the collected antigen-loaded immature DCs in autologous plasma.

[0019] Some embodiments of methods for producing personalized autologous DC vaccines further include storing collected, resuspended, antigen-loaded immature DCs prior to administration to an individual. Some embodiments include storing collected, resuspended, antigen-loaded immature DCs at room temperature for up to 5 hours. Some embodiments include storing collected, resuspended, antigen-loaded immature DCs at 4°C for up to 48 hours. Some embodiments include storing collected, resuspended, antigen-loaded immature DCs at -80°C for up to 21 days.

[0020] One embodiment is a personalized autologous dendritic cell (DC) vaccine prepared by any of the vaccine preparation methods disclosed herein.

[0021] One aspect is a method of immunizing an individual, comprising administering to the individual the personalized autologous DC vaccine disclosed herein. In some embodiments, the personalized autologous DC vaccine is administered by subcutaneous injection. In some embodiments, the personalized autologous DC vaccine is administered by intradermal injection. In some embodiments, immunization is achieved with a single dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] It is a diagram showing the principle mechanism of an autologous dendritic cell vaccine. A blood sample is obtained from a subject, monocytes are isolated from the blood sample, differentiated into dendritic cells loaded with antigen in vitro, and reinfused into the same individual from whom the sample was obtained. [Figure 2] It is a diagram showing the reactivity of endogenously produced IgG antibodies against recombinant viral spike protein in patients infected with SARS-CoV-2 virus. [Figure 3] It is a diagram showing the reactivity of endogenously produced IgG antibodies against recombinant viral nucleocapsid protein in 11 different patients infected with SARS-CoV-2 virus. [Figure 4] It is a diagram showing the reactivity of endogenously produced IgA antibodies against recombinant viral spike protein in 11 different patients infected with SARS-CoV-2 virus. [Figure 5] It is a diagram showing the reactivity of endogenously produced IgA antibodies against recombinant viral nucleocapsid protein in 11 different patients infected with SARS-CoV-2 virus. [Figure 6] It is a diagram showing flow cytometry data demonstrating that monocytes differentiate into CD11c+ and CD14-negative dendritic cells at a high percentage. [Figure 7] It is a diagram comparing cytokine secretion versus a control during co-culture of autologous mixed lymphocytes with antigen-loaded dendritic cells. [Figure 8] It is a diagram showing the percent increase in cytokine secretion during co-culture of autologous mixed lymphocytes with dendritic cells loaded with antigen at various antigen loading amounts. [Figure 9] This figure shows flow cytometry data demonstrating an increase in Tbet transcription factor-positive lymphocytes after exposure to antigen-loaded dendritic cells. [Figure 10] This diagram illustrates a typical manufacturing process for the autologous DC vaccine disclosed herein. [Figure 11] This figure shows the percentage of SARS-CoV-2 spike protein-specific IFNγ-secreting lymphocytes in the PBMC population before (Figure 11A) and 2 weeks after (Figure 11B) immunization using an autologous DC vaccine specific to the SARS-CoV-2 spike protein, as determined by the ELISpot assay. [Figure 12] This figure shows the mean number of SARS-CoV-2 spike protein-specific IFNγ-secreting lymphocyte spots in a PBMC population before and 2 weeks after immunization with a SARS-CoV-2 spike protein-specific autologous DC vaccine, as determined by the ELISpot assay with or without antigen stimulation in the assay. [Figure 13] This figure illustrates the evaluation of the memory response. Figure 13A shows the percentage of subjects showing a memory response to the SARS-CoV-2 spike protein before and 2 weeks after immunization with an auto-DC vaccine specific to the SARS-CoV-2 spike protein, as determined by the ELISpot assay for SARS-CoV-2 spike protein-specific IFNγ-secreting lymphocytes, which uses antigen stimulation in the assay. Figure 13B shows the number of subjects 2 weeks after immunization, categorized by memory status: converted, boosted, or unchanged. [Modes for carrying out the invention]

[0023] While proven safe, effective, and superior to other vaccine approaches in research settings, personalized dendritic cell (DC) immunotherapy presents an unresolved challenge in scaling up to industrial production scale. The compositions and methods disclosed herein address the scaling problem and provide a viable solution for industry.

[0024] Dendritic cell (DC) treatment and viral antigen presentation are well-established with demonstrated immunological effects in vitro and in animal models. Vaccines using dendritic cells have demonstrated protection against leishmaniasis, herpes simplex virus, influenza virus, Candida albicans, and human immunodeficiency virus (HIV). In a model of APC-specific immunosuppression, DC-based influenza vaccines rapidly induced significant antigen-specific antibody titers that could not be achieved with protein-based vaccination.

[0025] The application of DC immunotherapy as a preventive measure against infectious diseases is both scientifically rational and cost-effective, particularly for certain niche populations at risk of increased morbidity and those that do not respond well to conventional vaccinations.

[0026] DCs spontaneously phagocytose and digest soluble antigens for presentation to other immune cells. In this process, particles are endocytized after recognition by cell surface receptors, or by microphagocytosis, or by non-selective endocytosis of solutes. Antigen uptake results in activation signals leading to DC maturation, which promotes antigen presentation and maximal stimulation by cells for adaptive immune responses. Human monocyte-derived DCs and monocyte-derived macrophages can be generated from monocytes in vitro. Culture using either macrophage colony-stimulating factor (M-CSF) or GM-CSF alone produces macrophages, while culture of monocytes using GM-CSF and IL-4 produces DCs. Monocyte-derived DCs are superior in antigen presentation and exhibit antigen specificity of CD4 + and CD8 +T cells are induced, which express multiple pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), to recognize pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or modified glycosylated autoantigens such as tumor antigens. TLR recognition induces intracellular signaling and the expression of antigen-presenting molecules (MHC II molecules), costimulatory molecules (CD80 / 86, CD40), inflammatory and / or antiviral cytokines (TNF-α, IL-12, IL-23, IFNα / β, etc.), and chemokines (i.e., IL-8, RANTES).

[0027] Vaccines using non-viable or attenuated pathogens require large amounts of antigen and extensive testing. DNA / RNA vaccines are a reasonable alternative because the antigens provide immediate MHC presentation. However, an effective delivery system remains a major problem. Genomic delivery systems can be toxic, immunogenic, and could prevent any future use of the same carrier (e.g., adenovirus or adeno-associated virus vectors).

[0028] Pattern recognition receptor expression is regulated by the differentiation and maturation of mononuclear phagocytes. We have established culture conditions that produce immature DCs with high capacity for antigen uptake and cross-presentation. Ex vivo maturation is promoted by exposure to TLR4 ligand (LPS, poly(I:C)). Following peripheral injection (subcutaneous or intradermal), further maturation is achieved in vivo during migration to regional lymph nodes.

[0029] Ex vivo loading of immature dendritic cells (DCs) to facilitate the uptake, processing, and presentation of viral antigens can overcome inhibition caused by several pathogens, including, but not limited to, coronaviruses, HIV, influenza, Ebola, HSV-1, measles, hepatitis C, and dengue virus. By circumventing inhibitory pathways, ex vivo antigen treatment enhances the induction of humoral and cellular immune responses to viral antigens.

[0030] The autovaccine presented herein, which contains ex vivo antigen-loaded immature DCs, is an effector CD8 + This provides an essential mechanism that highlights the advantages of a Th1-biased immune response mediated by cytotoxic cells, which is superior to the Th2 response in combating viral infections.

[0031] Effector CD8 + Lymphocyte-mediated cell-mediated responses do not need to target specific antigenic epitopes; responses to any immunogenic epitopes in any expressed antigen can also be advantageous. In contrast, antibody-mediated immunity must be directed to specific epitopes of viral surface antigens. In many cases, neutralization can be involved in preventing lysis for uncoated or enveloped viruses, but neutralizing antibodies are directed to the receptor binding site of the viral receptor. If the receptor region mutates and neutralizing antibodies are ineffective, as in the recent coronavirus (SARS-CoV-2) pandemic, there is a risk of disease-enhancing antibodies increasing due to the accumulation of non-neutralizing antibodies.

[0032] Regardless of the recognized epitope, cell-mediated immunity is always neutralizing it, targeting infected host cells and thereby further disrupting viral replication.

[0033] The disclosed DC vaccine protozoa are far more adaptable to mutations than conventional vaccines, which aim to become antireceptor-binding domain antibodies optimized by their adjuvant system to maximize humoral response.

[0034] Certain viruses, such as SARS-CoV-2, circulate CD8 +These viruses are known to inhibit cell-mediated immunity through a rapid decrease in cells and suppression of interferon production. Many of the immune responses generated by these viruses are humoral, with antibodies initially produced against non-mutated epitopes, followed by antibodies against mutated portions of the virus 7–14 days later. If the mutant is located in the receptor-binding domain (RBD), there is a 7–14-day period of viral replication and non-neutralizing antibody production. This phenomenon particularly triggers congenital immune cell activation and general inflammation, explaining the pathogenesis of acute respiratory distress syndrome (ARDS) in some patients with other comorbidities that lead to Th2 bias (such as age, diabetes, and obesity).

[0035] Manufacturing process The manufacturing process disclosed herein is based on individual, single-use kits assigned to a single organism. The kit contains all the materials and reagents necessary for the production and quality control of the final product.

[0036] The kit typically includes blood collection supplies, monocyte isolation medium, DC differentiation medium, plasticware, dosage containers, QC sampling containers, QC reagent labels, and documentation. Nevertheless, other configurations are also possible.

[0037] Each kit component is uniquely identified, recorded in the manufacturing documentation, and traceable according to current regulatory requirements. In various embodiments, the distinctive mark for the unique identification of individual kits may be a series of alphanumeric characters, a barcode, a QR code, or the like. In some embodiments, the mark is printed on a sticker that can be attached to the kit components when used, and in a hard copy of the patient record.

[0038] In some embodiments, the kit container can also function as an incubator in addition to containing various components during storage and transport.

[0039] Blood collection is performed using standard heparinized vacuum tubes and venotomy kits. Collection tube sizes vary between 5 mL and 50 mL, and each tube optionally contains a Ficol isolation layer. One collection method involves using vacuum tubes pre-loaded with isolation medium.

[0040] Another method of collection involves using inertial microfluidic devices for blood separation. Such devices utilize non-equilibrium inertial separation arrays.

[0041] Peripheral blood mononuclear cells (PBMCs) are concentrated by centrifugation or tangential flow filtration in the same closed-loop system. A small amount of plasma is collected for the final product composition and stored separately. In some embodiments, heparin is used as an anticoagulant.

[0042] Subsequently, the isolated PBMCs are exposed to dendritic cell differentiation medium for 2–5 days.

[0043] Exemplary media for DC differentiation are CO2-independent media containing non-bicarbonate buffers. Examples of CO2-independent media include RPMI-1640, bicarbonate-free, and AIM V medium (ThermoFisher). One example of a non-bicarbonate buffer suitable for tissue culture is HEPES.

[0044] In some embodiments, the DC differentiation medium contains antioxidants and free radical scavengers. Examples of free radical scavengers include N-acetyl-cysteine, carboxy-PTIO, flavonoids, and L-NG-methylarginine.

[0045] In some embodiments, the DC differentiation medium contains GM-CSF and IL-4.

[0046] In some embodiments, the DC differentiation medium does not contain GM-CSF.

[0047] In some embodiments, the DC differentiation medium contains IFNγ.

[0048] In some embodiments, the DC differentiation medium contains IFNα.

[0049] In some embodiments, the DC differentiation medium contains IL-2.

[0050] In some embodiments, the DC differentiation medium may contain up to 30% of the autologous plasma preserved during the blood collection and PBMC isolation process. In some embodiments, the medium also contains 5% to 30% or any integer value within that range of autologous plasma, including both ends.

[0051] Subsequently, the PBMC cell suspension is transferred to a closed cell culture vessel for DC differentiation and antigen exposure.

[0052] An example of a closed cell culture vessel is approximately 50 cm². 2 It is a flexible bag with an internal cell culture surface and at least one access port.

[0053] An alternative closed-system cell culture vessel is approximately 50 cm². 2 It is a rigid container equipped with a flat internal cell culture surface and an access port.

[0054] The inner surface of a closed cell culture vessel is hydrophobic to prevent cell adhesion.

[0055] Exemplary materials for closed cell culture vessels are gas-permeable materials such as fluorinated polyethylene and its copolymers.

[0056] An alternative material for closed cell culture vessels is chemically modified cellulose for hydrophobicity. In various embodiments, the chemically modified cellulose is cellulose acetate that has been acylated or esterified with fatty acids (e.g., palmitic acid, stearic acid, etc.).

[0057] Alternatively, hydrophobic properties can be achieved by coating the inner surface with a hydrophobic material. Such a material could be, for example, a hydrophobic silane.

[0058] The containers containing the PBMC and culture medium are incubated at 37°C for 2 to 5 days.

[0059] An exemplary incubator is a kit container containing a rechargeable power supply and a thermostat. The kit container walls provide insulation that allows for minimal energy dissipation to maintain the contents at approximately 37°C for 5 to 7 days. In some embodiments, the power supply consists of a lithium polymer battery that can be formed to fit into the box.

[0060] Exemplary thermostats include positive temperature coefficient (PTC) materials that eliminate the need for phase exchange materials and / or temperature controllers. As used herein, a temperature controller is a mechanical or electronic system having sensors and feedback loops to enable or disable the injection of energy (heat), while a thermostat is any system that maintains a constant temperature (within an acceptable range). As used herein, a phase exchange material is a substance that provides heating or cooling by releasing or absorbing sufficient energy during a phase transition. For example, if heating raises the temperature of a solid phase exchange material to its melting point, heat is absorbed with virtually no change in temperature until the material is completely melted. Similarly, if heat is dissipated and the temperature of the liquid phase decreases toward its melting point, the material solidifies, and heat is released with virtually no change in temperature until the material is completely solidified. In some embodiments, the phase exchange material is Paraffin 20-Carbons (melting point 36.7°C). In some embodiments, the phase exchange material is Camphenilon (melting point 39°C). In some embodiments, the phase exchange material is referred to as a means for releasing or absorbing energy or a means for storing latent heat.

[0061] PTC materials exhibit increased electrical resistance with increasing temperature. Because they receive greater electrical resistance with further temperature increases at a certain point, PTC materials can be designed to reach the maximum temperature for a given input voltage. Unlike linear resistance heating or negative temperature materials, PTC materials are inherently self-limiting. In some embodiments, the PTC material is a silicone rubber that conducts electricity by exponentially increasing resistance with increasing temperature up to a temperature where the resistivity increases infinitely. At this temperature, the PTC rubber is an electrical insulator. In particular, the PTC rubber can be made from polydimethylsiloxane (PDMS) loaded with carbon nanoparticles. In some embodiments, the PTC material is a carbon-based PTC ink. The PTC ink is placed on the outer surface of a cell culture vessel. In some embodiments, the PTC material is referred to as a means for PTC limiting heating.

[0062] In various embodiments, the thermostat includes a phase exchange material, a PTC material, or both. In some embodiments, the thermostat is referred to as a means for maintaining a constant temperature. In some embodiments, the constant temperature is approximately 37°C.

[0063] Subsequently, immature dendritic cells are exposed to the antigen for one or two days. The antigen may be added to a closed cell culture vessel via a corresponding port, such as a self-sealing swabbing valve. In some embodiments, the culture medium is not changed during the 3–7 day culture period.

[0064] Appropriate antigens for DC loading include water-soluble or insoluble antigens derived from pathogens, toxins, and venom, or insoluble antigens containing the whole pathogen, attenuated organisms, non-developing organisms, fragments, or whole protein complexes. Various synthetic or recombinant structures designed from the DNA / RNA sequence of a pathogen can be used as antigen sources. Furthermore, antigens can be fused with terminal peptide sequences that enhance antigenicity or stimulate dendritic cells. Fusion peptide sequences may include, for example, fragments of human immunoglobulins and chemical structures that activate Toll-like receptors (TLRs) on dendritic cells.

[0065] As published elsewhere, recombinant methods can be used to produce antigens. Recombination methods use sequences and chemical structures derived from DNA / RNA analysis of pathogens. Antigenicity predictions can also be based on matching human HLA on major and non-major subclasses.

[0066] Antigen target validation may be performed by antibody binding studies in survivors of infection with the pathogen or in convalescent patients who have tested positive for the targeted pathogen.

[0067] Antigen combinations could potentially be used to produce vaccines that offer broader protection.

[0068] For dose preparation, the cell-containing material in the container is collected in a centrifuge tube, and the supernatant is removed by centrifugation and replaced with the patient's own plasma.

[0069] If it is anticipated that the dose will be frozen before administration, a cryopreservation solution may be added at this step. In some embodiments, the cryopreservation agent is mixed with an equal amount of autologous plasma to resuspend the cells. Exemplary cryopreservations contain trehalose and glycerin. The use of trehalose and glycerin as cryopreservations allows for direct injection of the thawed progenitor. These are permitted for direct injection as indicated in the USP as vaccine adjuvants. Other cryopreservations, such as DMSO, must be removed before the thawed progenitor can be injected.

[0070] A small number of cells are subjected to quality control at the discretion of the organism. In some embodiments, the cell population contains 5–30% dendritic cells and 70–95% lymphocytes, with an undifferentiated monocyte content of less than 1%. The absence of macrophages indicates the effectiveness of DC differentiation and the elimination of the need to remove adherent cells before DC differentiation.

[0071] In some embodiments, the dose does not contain residual antigen or cell culture medium.

[0072] The dose is then transferred to a final container and stored at room temperature for immediate use, at 4-8°C for use within 2 days, at -65--85°C for use up to 21 days, and in liquid nitrogen (<-165°C) for long-term storage.

[0073] Quality control may include identification tests, e.g., testing for CD14-CD11+ cells; safety tests for microbial contamination (mycoplasma, endotoxins, and sterility); and efficacy tests, e.g., testing for the presence of IL-12 in the supernatant.

[0074] Examples of efficacy tests include rapid evaluation methods such as lateral flow immunoassays.

[0075] An exemplary rapid sterility test is based on solid-phase laser scanning cytometry to rapidly count viable microorganisms in an aqueous sample. This method is included in commercially available devices such as ScanRDI® (bioMerieux Inc.).

[0076] In some embodiments, the dose is verified by patient identity and administered by subcutaneous injection.

[0077] Process and product distribution The production and distribution of vaccine products may be coordinated from a central location. In some embodiments, personalized DC vaccines are produced in low-technology laboratories using general bioengineering or clinical laboratory environments and techniques. In some embodiments, the production and distribution of vaccine products take place in the same location.

[0078] In the initial stage, the laboratory is evaluated for space, equipment, and personnel qualifications. A typical laboratory includes a biosafety level 2 space, including an incubator, centrifuge, refrigerator, freezer, microscope, and common laboratory equipment.

[0079] In some embodiments, specific software modules are implemented within the facility's existing quality control and oversight system (QMS). The software provides instructions, manufacturing records, controls, and release procedures in accordance with the U.S. Food and Drug Administration or equivalent regulatory bodies in other countries. This module communicates with a centralized QMS located in a coordinating center to ensure the effectiveness of manufacturing kits, training, and troubleshooting.

[0080] Production capacity at each location is assessed, and manufacturing kits are planned or stored accordingly. The supply chain for each component is secured by contract.

[0081] The kit may be manufactured according to the bill of materials by a professional third-party assembly and warehousing organization, or it may be distributed according to local production.

[0082] The exemplary kit configuration includes all reagents as dry materials already present in the cell culture vessel, and the liquid format is reconstituted by adding cells suspended in a 0.8% sodium chloride solution after PBMC isolation. In embodiments where the DC differentiation medium contains autologous plasma, the autologous plasma is also added to the cell culture at this point.

[0083] Immature DCs loaded with antigen can be used immediately after resuspending, but a certain storage period is expected to be necessary; for example, quality control assays can typically be completed in about 3 hours. Furthermore, patients do not necessarily need to be present at the facility where the vaccine is prepared. To account for the time required for patient travel and / or vaccine transport, the vaccine may be stored at room temperature for at least 5 hours, at 4°C for at least 48 hours, or at -80°C for at least 21 days. [Examples]

[0084] Example 1. Generation of dendritic cells from individual blood samples and loading using recombinant antigens derived from the SARS-CoV-2 virus genome. In the initial step, the reactivity of the target antigen was confirmed using antibodies spontaneously produced in known COVID-19 patients. For this purpose, serum was collected from consenting donors who had previously been diagnosed with SARS-CoV-2 infection by PCR testing, and exposed to ELISA plates coated with recombinant peptides obtained from the DNA sequences of the SARS-CoV-2 virus spike protein and nucleocapsid protein.

[0085] The reactivity with recombinant antigens was confirmed by analyzing the ELISA plates shown in Figures 2 to 5.

[0086] Following antigen validation, 50 mL of whole blood was collected from four volunteer donors in heparinized vacutainers to produce autologous DC vaccines.

[0087] Complete blood count (CBC) was performed to evaluate the starting mononuclear cell population. All volunteers had CBC values ​​within the normal range.

[0088] Within 6 hours of collection, whole blood was subjected to Ficol separation. A portion of the mononuclear cells was extracted in 75 cm³. 2 The cells were transferred to a cell culture dish, and a portion of the cells were dispensed into a 12-well plate.

[0089] The floating cells, which consisted mainly of lymphocytes, were removed and saved for later use.

[0090] The adherent population was incubated for 5 days in AIM-V medium containing GM-CSF and IL-4. The monocytes differentiated into dendritic cells, with 90% of the population being CD11c-positive / CD14-cells (Figure 6).

[0091] The culture medium was then removed and replaced with a new medium containing SARS-CoV-2 antigen, obtained from each patient culture in 3 μg portions, extracted by recombinant DNA sequencing of spike proteins (S1, S2) and nucleocapsids.

[0092] Two days later, the loaded dendritic cells were sampled for phenotyping and remixed with the corresponding autologous lymphocytes at a ratio of 1:3.

[0093] DCs and autologous lymphocytes were co-cultured for 72 hours and analyzed for markers suggestive of lymphocyte activation.

[0094] The results demonstrate successful differentiation of monocytes into dendritic cells, lack of antigen toxicity, and activation of cytotoxicity in lymphocytes co-cultured with loaded dendritic cells (Table 1, Figure 7, and Figure 8).

[0095]

Table 1

[0096] Compared with the control in which differentiated DCs were not exposed to the antigen, CD8 + The population increased to 41% after exposure to the antigen.

[0097] The CD4 helper population showed a significant activation, observed as an average 17% increase relative to the Tbet transcription factor-positive non-antigen control (Figure 9), and lack of immune tolerance due to the absence of FoxP3-positive cells (average 0.06%). Both Th1 and Th2 activation of the adaptive immune system is attributable to Tbet-positive cells.

[0098] These experiments demonstrate that recombinant antigens are effective targets of naturally produced antibodies, and therefore immunization against these antigens makes it possible to produce antibodies that react to natural viral antigens.

[0099] The experiments further demonstrate the lack of direct antigen toxicity on dendritic cells and the activation of lymphocytes in a cytotoxic mode.

[0100] Furthermore, clinical-grade manufacturing (Figure 10) was confirmed using patient samples from six more individuals. All pre-packaged materials and reagents from individual patient-specific kits were used in the manufacturing process. Logistics for controlling the blood collection processing and distribution process, as well as maintaining the final dose, were integrated into the component labeling system.

[0101] In terms of practicality, each kit is divided into four parts: parts A and B are used in the manufacturing plant depending on storage conditions, while parts C and D are used in the clinical setting for blood collection and dose administration.

[0102] Each kit includes GMP-compliant documentation, and the collected data is expected to be centralized in the company's database for traceability. This disclosure is also intended as training material for on-site use.

[0103] The electronic GMP documentation and data collection software are installed locally and have central reporting capabilities. The software is capable of being updated remotely, thereby avoiding the need for local document change control systems.

[0104] In a business model that ensures material coverage and avoids supply chain depletion during peak demand, manufacturing sites can secure assembled kits ahead of the vaccination schedule.

[0105] Example 2. Safety and efficacy of autologous DC vaccine loaded with recombinant full-length SARS-CoV-2 spike protein. Clinical trials were conducted using autovaccine products prepared from 40 mL of peripheral blood lymphocytes differentiated from monocytes and loaded with 0.1, 0.33, or 1 μg of full-length recombinant SARS-CoV-2 spike protein. Broad inclusion criteria were used in the trials, excluding only patients with unstable medical conditions and individuals belonging to certain protective categories (i.e., children, pregnant women, and individuals who were physically, socially, or mentally incapacitated). The primary endpoints evaluated were safety as assessed by clinical laboratory values ​​and efficacy as measured by surrogate markers.

[0106] Vectors encoding the SARS-CoV-2 spike protein may contain a signal sequence and a His tag or other sequences to facilitate purification, but these portions are typically absent in the mature recombinant protein. In some embodiments, the mature full-length recombinant SARS-CoV-2 spike protein has the following amino acid sequence:

[0107] The preparations were carried out according to the methods described herein. Each subject was assigned a specific identified kit, and the same certificate was given to the subject. PBMCs were separated using a 1.073 density gradient Ficol reagent.

[0108] PBMCs were differentiated for 5 days in 25 mL of PRIME-XV Dendritic Cell Maturation known composition medium (FujiFilm Irvine Scientific) or AIM-V medium (Thermo Fisher) in the presence of 250 μg / L GM-CSF and 100 μg / L IL4 in VueLife bioprocess bags (Saint Gobain). On day 5, a total amount of 0.1, 0.33, or 1 μg of antigen was introduced into the bag. After 2 days, cells were collected and doses were prepared by resuspending cells in autologous plasma. For collection, cells were precipitated by centrifugation, the supernatant was aspirated, and the cells were washed by resuspending them in physiological saline, followed by centrifugation and aspiration of the supernatant. Washing removed culture medium components and any free antigens. The doses were stored at 4°C and administered by subcutaneous injection the following day.

[0109] Subjects were observed 3 hours after injection, daily for the following 3 days, and weekly for 4 weeks. Blood samples were taken for laboratory safety and surrogate efficacy.

[0110] Surrogate efficacy testing was performed using the ELISPOT assay for interferon-γ under non-stimulated versus antigen-stimulated conditions. This assay detects antigen-specific activated IFN-gamma secreting cells. 8 mL of blood was collected in a CPT vacutainer (Becton Dickinson), PBMCs were separated by centrifugation, and the assay was performed by plating at standardized concentrations in 24-well plates with or without spike protein antigen and IL-2. After 10 days without further antigen stimulation, the cells were transferred to 96-well ELISPOT plates (three wells per plate) under each condition at the specified concentrations. Spots were stained and counted according to the manufacturer's (Becton Dickinson) standard procedure.

[0111] A total of 138 subjects accepted the treatment, of whom 216 underwent screening. 61 subjects experienced at least one adverse event (AE). The total number of adverse events was 100. All events were considered mild (94%) to moderate (6%).

[0112] No severe or serious adverse events were recorded. No participants discontinued the study due to adverse events.

[0113] None of the subjects developed symptomatic COVID-19 symptoms after vaccination.

[0114] The most common adverse event (AE) was localized injection site reactions, common to all vaccines (Table 2).

[0115] [Table 2]

[0116] Baseline ELISPOT data demonstrate that 30% of the subjects had a history of SARS-CoV-2 exposure (natural infection or undisclosed vaccine), but none of the subjects showed antibodies during screening (Figure 11A). Because recombinant SARS-CoV-2 spike protein was used as a stimulant, positive responses are expected to reflect actual exposure to SARS-CoV-2 rather than cross-reactivity with other coronaviruses to which the subjects may have been exposed.

[0117] Within two weeks post-vaccination, ELISPOT responsiveness increased to 92.9% of tested subjects (Figure 11B). The mean number of spots increased significantly in both stimulated and unstimulated states (p << 0.001) (Figure 12, Table 3), and 43% of subjects demonstrated cytotoxic memory cells specific to the SARS-CoV-2 spike protein (IFNγ secretory lymphocytes; Figure 13A). The majority of subjects with cytotoxic memory cells two weeks post-vaccination were attributable to transformation. Some subjects who were previously responsive showed a booster effect after vaccination, while a small number remained unchanged (Figure 13B).

[0118] [Table 3]

[0119] The ELISPOT data demonstrates high reactivity two weeks after vaccination, suggesting that the majority of subjects experience a primary immune response.

[0120] Data from a safety pilot study of 28 patients showed that cytotoxic memory responses last for at least four months after vaccination, but are expected to persist even longer.

[0121] In conclusion, these data indicate the induction of cellular immunity.

[0122] Unless otherwise indicated, all numbers used in the specification and claims to represent properties such as the amount of a component, molecular weight, reaction conditions, etc., should be understood in all examples to be modified by the term “about.” As used herein, the terms “about” and “approximately” mean within 10 to 15%, preferably within 5 to 10%. Therefore, unless otherwise indicated, the numerical parameters described in the specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the invention. At a minimum, each numerical parameter should be interpreted, in light of the number of significant figures reported, by the application of ordinary rounding techniques, without any attempt to limit the application of the doctrine of equivalents to the scope of the claims. Despite the numerical ranges and parameters describing the broad scope of the invention being approximations, the numerical values ​​stated in specific examples are reported as accurately as possible. However, each numerical value inherently contains a certain error, which is necessarily due to the standard deviation observed in the respective test measurements.

[0123] The terms “a,” “an,” and “the,” and similar references used in reference to the present invention (in particular in reference to the following claims), should be interpreted as including both singular and plural forms unless otherwise specified herein or expressly contradicted by the context. Enumerations of value ranges herein are intended merely as abbreviations to refer individually to each distinct value contained within that range. Unless otherwise specified herein, each individual value is incorporated herein as if it were individually enumerated herein. Unless otherwise specified herein or expressly contradicted by the context, all methods described herein may be performed in any suitable order. The use of all examples or illustrative words (e.g., “such as”) provided herein is intended merely to better illustrate the present invention and does not limit the scope of the invention as otherwise required. No word herein should be interpreted as indicating an element essential to the carrying out of the invention that is not described in the claims.

[0124] The grouping of alternative elements or embodiments of the Invention disclosed herein should not be construed as limiting. Members of each group may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or excluded from a group for convenience and / or patentability. In the event of such inclusion or exclusion, this Specification shall be deemed to include the group to be qualified and thus satisfy the written specification of all Markush groups used in the appended claims.

[0125] Specific embodiments of the invention are described herein and include the best modes known to the inventor for carrying out the invention. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the foregoing description. The inventor anticipates that those skilled in the art will utilize such variations appropriately and intends that the invention may be carried out in ways other than those specifically described herein. Accordingly, the present invention includes all modifiers and equivalents to the subject matter described in the appended claims, as permitted by applicable law. Furthermore, unless otherwise specified herein or expressly rejected by context, all combinations of the above elements in all possible variations are incorporated into the present invention.

[0126] The specific embodiments disclosed herein may be further limited in the claims, which consist of or are essentially derived from the language. When used in the claims, the transitional term "consisting of" excludes any elements, processes, or components not embodied in the claims, whether in the filing or amended form. The transitional term "essentially derived from" limits the claims to the specified materials or processes and those that do not substantially affect the fundamental and novel features. Thus, embodiments of the claimed inventions are essentially or expressly described and made available herein.

[0127] Furthermore, numerous references to patents and publications are made throughout this specification. Each of these references and publications is incorporated herein by reference in its entirety.

[0128] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be used are within the scope of the invention. Thus, as non-limiting examples, alternative configurations of the invention may be used in accordance with the teachings herein. Accordingly, the invention is not limited to those precisely described.

Claims

1. a. Kit container, b. blood collection supplies, c. Monocyte isolation medium or inertial microfluidic device, d. Components of dendritic cell (DC) differentiation medium, e. A cell culture vessel equipped with at least one access port, f. Unique identifiers, and g. antigen Personalized vaccine kits, including

2. The personalized vaccine kit according to claim 1, wherein the kit container includes an insulating wall and can function as an incubator.

3. The personalized vaccine kit according to claim 2, wherein the incubator includes a power supply and a thermostat system.

4. The personalized vaccine kit according to claim 3, wherein the thermostat system includes a phase exchange material and a positive temperature coefficient (PTC) material, but does not include a temperature controller.

5. The individualized vaccine kit according to any one of claims 1 to 4, wherein the DC differentiation medium component is provided in a dry state in the cell culture vessel.

6. The personalized vaccine kit according to any one of claims 1 to 5, wherein the DC differentiation medium component comprises IL-4.

7. The personalized vaccine kit according to claim 6, wherein the DC differentiation medium component includes GM-CSF.

8. The personalized vaccine kit according to any one of claims 1 to 7, wherein the DC differentiation medium component comprises IFNγ, IFNα, IL-2, or any combination thereof.

9. The personalized vaccine kit according to any one of claims 1 to 8, wherein the antigen is a full-length recombinant SARS-CoV-2 spike protein.

10. A method for producing a personalized autologous dendritic cell (DC) vaccine using a kit according to any one of claims 1 to 9, a) A step of collecting 5 to 50 mL of blood from the individual, b) A step of isolating peripheral blood mononuclear cells (PBMCs) from the blood, c) A step of differentiating the PBMC by adding the DCs to the cell culture vessel and incubating the cells for 2 to 5 days in order to generate immature DCs, and thereafter d) Adding the antigen to the cell culture vessel and incubating it for a further 1 to 2 days in order to load the immature DCs with the antigen, e) A step of collecting the antigen-loaded immature DCs. Methods that include...

11. The method according to claim 10, further comprising the step of storing the autologous plasma obtained from the isolation step.

12. The method according to claim 10 or 11, further comprising the step of collecting the antigen-loaded immature DC in the autologous plasma and resuspending it.

13. The method according to any one of claims 10 to 12, further comprising the step of marking a container containing cells or plasma obtained from the individual with the distinctive identification mark.

14. The method according to any one of claims 10 to 13, further comprising the step of collecting and resuspending the antigen-loaded immature DCs and storing them before administration to the organism.

15. The method according to claim 14, comprising the step of storing the collected and resuspended antigen-loaded immature DCs at room temperature for up to 6 hours.

16. The method according to claim 14, comprising the step of storing the collected and resuspended antigen-loaded immature DCs at 4°C for a maximum of 48 hours.

17. The method according to claim 10 or 11, further comprising the steps of resuspending the collected and antigen-loaded immature DCs in a mixture of autologous plasma and a cryopreservative, and storing the collected, resuspended and antigen-loaded immature DCs at -80°C for up to 21 days.

18. A personalized autologous dendritic cell (DC) vaccine prepared by the method described in any one of claims 10 to 17.

19. A method for immunizing an individual, comprising the step of administering the individualized autologous dendritic cell (DC) vaccine to the individual by subcutaneous or intradermal injection.

Images