JP2022036961A - Lipids as synthetic vectors to enhance antigen processing and presentation ex-vivo in dendritic cell therapy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for activating dendritic cells ex vivo.

SOLUTION: The present invention discloses a composition for producing vaccine, containing a population of isolated dendritic cells, at least one cationic lipid, and at least one peptide antigen.

SELECTED DRAWING: None

COPYRIGHT: (C)2022,JPO&INPIT

Description

Cross-reference to related applications

This international application is prioritized by US Patent Provisional Application Nos. 62 / 254,794 filed on November 13, 2015 and US Patent Provisional Application Nos. 62 / 404,504 filed on October 5, 2016. It asserts the interests of the right and is incorporated herein by reference in its entirety.

The present invention relates to dendritic cell therapy in cancer. For more than 10 years, adoptive transfer of ex vivo autologous hematopoietic progenitor cells or monocyte-derived dendritic cells (DCs) has been tested as a cancer vaccine. These studies suggest that dendritic cell vaccines are safe and can induce the proliferation of tumor antigen-specific and circulating CD4 ⁺ T cells and CD8 ⁺ T cells. Some patients had an objective clinical response. Although it takes time for a clinical response to be established, it has been established that remission may continue for a very long period of time. Preclinical antitumor studies have shown that DCs isolated from animals, loaded with tumor antigens ex vivo and administered as cell therapy induce both prophylactic and therapeutic effects.

Studies of the antigen-presenting properties of DC have shown promise in recent years in the development of effective cancer immunotherapies, with promising results in recent clinical trials. Treatment of metastatic prostate cancer with sipuleucel-T resulted in a median survival of approximately 4 months in Phase III trials. Sipuleucel-T (Provenge®) is a dendritic cell-based cell taken from a patient and cultured with a fusion protein of prostatic acid phosphatase (PAP) and GM-CSF from concentrated blood. It is a product of. Dendritic cells are then returned to the patient by intravenous injection (intravenous injection). This process is repeated twice more every two weeks to allow the patient to receive 3 doses of cells. With the US Food and Drug Administration (FDA) approval for the treatment of metastatic prostate cancer, sipuleucel-T paved the way for clinical development and regulation of next-generation cellular immunotherapeutic products. Among metastatic melanoma patients who received a vaccine of activated DC loaded with a tumor antigen peptide, there were patients who showed antigen-specific CD4 ⁺ T cell response and CD8 ⁺ T cell response. Despite this promising view, the clinical response was not as good as expected, and the traditional objective antitumor response rate was barely over 15%.

Dendritic cell therapy is a promising method for treating various debilitating diseases such as cancer. This technique allows the development of personalized and personalized therapies for the patient. For example, instead of using a protein antigen specific for a particular cancer, the antigen can be obtained from the patient's tumor for treatment and loaded onto the patient's own dendritic cells. The technique of using dendritic cell therapy in ex vivo is an approved product that has shown a sustained immune response in patients 2 years after treatment and has been shown to show significant prospects. However, treatment results have not been excellent, and several methods for improving these treatments have been studied.

One of the important issues is the selection of tumor antigens for loading DC. Candidate antigens include specific (mutant) antigens and non-specific non-mutant self-antigens. Non-mutant self-antigens are most preferred for producing widely applicable therapies, even though the negative selection eliminates a population of high-affinity clones. Mutant antigens could be used to avoid this shortcoming. The development of RNA sequencing technology has helped determine the range of mutated antigens from primary tumors and metastatic tumors, allowing treatment to be tailored to specific patients. It has become.

For peptide or protein antigens, and tumor-derived self-antigens, a key limiting factor in the successful application of DC vaccines is very often the amount of antigen available. One of the technical barriers to overcome is the effective presentation of these antigens to dendritic cells, allowing for more effective presentation by MHC class I (CD8 + T cells) and MHC class II (CD4 + T cells). Is to. This also directly affects the efficacy and stability of the resulting T cell response, which is still not the best.

The DC can also cross-present the antigen. Cross-presentation is the pathway by which an exogenous soluble protein or peptide is taken up by antigen-presenting cells, where the peptide or protein is MHC class I rather than degraded and enters the pathway presented with MHC class II. Refers to the route that enters the processing route of. This can occur in two ways: the cytoplasmic pathway or the endosome pathway. In either pathway, proteins are first incorporated into endosomes / phagosomes. In the cytoplasmic pathway, some of the partially degraded endosome proteins eventually enter the cytoplasm. Although the mechanism is poorly understood, endosome proteins are processed in the proteasome, and the resulting peptides are transported by TAP into either the endoplasmic reticulum or other endosomes for binding to MHC class I. Alternatively, the protein may be degraded in the endosome and the peptide may bind to MHC class I present in the endosome. This latter pathway is inefficient because it is proteasome-independent and depends on the accidental production of the correct peptide by endosome proteases. Invasion of proteins into early endosomes with limited proteolytic activity favors cross-presentation, whereas late endosomes with high levels of proteolytic activity may inhibit cross-presentation.

From the above considerations, it is clear that proteins entering the endosomal pathway, especially the early endosomal pathway, can be cross-presented on MHC class I. Also, the degree of cross-presentation will depend on the amount of protein / peptide taken up by the early endosomes and the amount of antigen fragment subsequently carried to the cytoplasm. Soluble proteins that bind to the DC scavenger receptor can be cross-presented. A classic example is ovalbumin, which binds to the mannose receptor. However, there are various approaches to target receptors that have already been introduced into proteins / peptides, and not all bind to DC scavenger receptors. These pathways include targeting Fc receptors, various C-type lectin receptors such as CD205, CD207, CLEC9a, integrins or glycolipids. These methods have some drawbacks. The antigen needs to be bound to a specific receptor-binding protein, usually a monoclonal antibody, which is a very cumbersome technique. The amount of protein uptake is limited to the amount of possible receptor uptake into the cell, and once taken up, it is released into the cytoplasm, albeit in a limited manner. Some DC receptors target anaphase endosomes, resulting in inefficient cross-presentation. Finally, the distribution of cross-presentation DC receptors on human DC subsets is poorly understood, which makes the design of such techniques difficult and makes studies in mice into humans. It can explain why it is not replaced successfully. Another method with a little lower specificity is to attach a soluble antigen to nanoparticles and convert them into fine particles. This technique suffers from the difficulty of carrying sufficient antigen and the fact that it loses its phagocytic capacity as DC matures and is delivered to the lymph nodes.

This application demonstrates that the uptake of antigen by dendritic cells is highly dependent on the amount of antigen in the development of peptide-based DC vaccines and autologous DC vaccines in which the antigen is derived from tumors. Cationic lipids can be safely used to facilitate uptake by this dendritic cell and promote cross-presentation of important antigens.

Therefore, this application focuses on the development of DNA / RNA-free dendritic cell vaccines, and develops vaccines that improve the uptake and processing of antigens by DC while limiting toxicity to DC. Shows the means to facilitate.

Yet another significant impediment to the success of immunotherapy is the tumor suppressive microenvironment, which is responsible for promoting various immunosuppressive mechanisms that impede antitumor cell-lytic T cell responses. This effect has been called "antigenic escape" or "immune tolerance". Clinical trials were conducted in adjuvant-administered patients with minimal residual lesions and a high risk of recurrence to avoid the inhibitory effects present in end-stage tumors [Sears AK, Perez SA, Clifeton]
GT, et al. , AE37: a novel T-cell-eliciting vaccine for breast cancer. Expert Opin Biol Ther. 2011; 11 (11): 1543-1550]. A reasonable support for vaccination in this clinical setting is to ensure that patients with minimal tumor burden still have a sufficiently competent immune system to provide a reliable antitumor response. In addition, vaccination in early stage cancer or adjuvant administration has the advantage of minimizing T cell accumulation in an immunosuppressive tumor environment in which T cells can be inactivated.

An exemplary embodiment of the invention is shown below. It should be understood that these embodiments are presented solely to provide the reader with a brief overview of the forms in which the invention may be taken and are not intended to limit the scope of the invention. In fact, the invention can embrace a wide variety of aspects not explicitly described below.

In one embodiment described herein, a composition for dendritic cell activation in ex vivo is provided. The composition comprises one or more lipids having at least one cationic lipid and at least one antigen. The composition may contain other lipids as well as growth factors for enhancing the viability and proliferation of dendritic cells.

The composition for DC activation in ex vivo comprises one or more lipids having at least one cationic lipid and at least one antigen, where the antigen is a protein or polypeptide found in tumors or cancers. be. Certain cationic lipids are unique in their ability to rapidly bind to dendritic cells in a receptor-independent manner and are rapidly incorporated into early endosomes. Importantly, once entering the early endosomes, cationic lipids facilitate endosome destabilization and release of the contents into the cytoplasm to enter the class I processing pathway. This allows the release of significantly more endosomal contents into the cytoplasm than would result from the uptake of targeted receptors. Appropriate cationic lipids also provide an immunological danger signal (Danger Signal) that induces the production of certain cytokines and chemokines that activate and proliferate T cells and cause T cell migration to lymph nodes. You can also do it. Also, suitable cationic lipids can reduce the population of Treg cells in the tumor microenvironment, preferably when used in combination with tumor antigens.

In another embodiment, an ex vivo method of treating a subject's dendritic cells, wherein the subject is a mammal, is provided. The method comprises treating dendritic cells with one or more lipids comprising an effective amount of at least one cationic lipid and at least one antigen. The composition also contains growth factors such as GM-CSF and cytokines to promote cell maintenance and proliferation in vitro. At least one antigen is a cancer antigen.

A method for producing a cancer DC vaccine is provided. The method comprises treating dendritic cells with one or more lipids comprising an effective amount of at least one cationic lipid and at least one antigen. The composition also contains growth factors such as GM-CSF and cytokines to promote cell maintenance and proliferation in vitro.

A method for producing a cancer DC vaccine capable of inducing high levels of tumor infiltrating T cells while inducing a significant reduction in the Treg population within the tumor microenvironment is provided. The method comprises activating dendritic cells with one or more lipids comprising an effective amount of at least one cationic lipid and at least one antigen. The method also comprises further activating DC with growth factors such as GM-CSF and cytokines to promote cell maintenance and proliferation in vitro. When a cationic lipid is used in vitro to activate DC in vitro and then injected into a subject with cancer, DC increases the amount of tumor-specific CD8 + T in the tumor microenvironment. Thereby, the tumor microenvironment can be changed and the Treg population can be significantly reduced. As a result, the ratio of Treg to CD8 + T cells is significantly reduced.

Immunizing a subject with a DC vaccine, comprising administering to the patient DC pretreated with one or more lipids comprising an effective amount of at least one cationic lipid and at least one antigen, wherein the antigen is a cancer antigen. A way to do it is provided. This method involves immunizing the subject more than once. The method comprises ascertaining cancer-specific CD8 + T cell levels and Treg cell levels in post-immune subjects.

Yet another embodiment provides a method of enhancing a prophylactic or therapeutic immune response in a mammal. This method comprises activating dendritic cells with one or more lipids, optionally with growth factors such as GM-CSF and cytokines, as well as effective amounts of at least one cationic lipid and an antigen. Includes the step of administering dendritic cells to a mammal. In various embodiments, the mammal is a human.

One embodiment of the invention relates to the use of tumor-derived antigens and cationic lipids to facilitate the uptake and presentation of protein and peptide antigens when administered ex vivo in combination with dendritic cells. This practice has limited toxicity to dendritic cells and should be used in the presence of growth factors and cytokines intended to maintain and promote dendritic cell viability and growth. Can be done.

Cationic liposomes are widely used in vivo in RNA and DNA transfection and delivery for use in gene therapy, as well as in DNA-based dendritic cell vaccines. Recently, cationic lipids have also been reported to be potent adjuvants capable of stimulating a strong immune response through activation of MAP kinase.

Cationic lipids are unique in their ability to rapidly bind to dendritic cells in a receptor-independent manner and are rapidly incorporated into early endosomes. Importantly, once entering the early endosomes, cationic lipids appear to facilitate endosome destabilization and release of the contents into the cytoplasm to enter the class I processing pathway. This allows higher endosome content to be released into the cytoplasm than would result from target receptor uptake. This allows the release of significantly more endosomal contents into the cytoplasm than would result from the uptake of targeted receptors.

In one embodiment of the invention, the disclosure discloses MHC class I and MHC class II with orders of magnitude more protein than current approaches such as cationic lipids using an adjuvant to induce dendritic cell maturation. Show that it can help you enter the route.

In one embodiment, the invention provides a composition comprising a cationic lipid and a particular tumor antigen necessary for preparing a DC vaccine against a tumor ex vivo.

In one embodiment, the composition required to prepare a DC vaccine against a tumor comprises a non-nucleic acid antigen, protein, polypeptide, peptide, lipoprotein or lipopeptide.

In one embodiment, the antigen is a tumor antigen or a mutant tumor antigen. An antigen is a protein product of a patient's gene, and the gene is a cancer gene, a tumor suppressor gene, a gene having a mutation, a gene having a rearrangement peculiar to a tumor cell, a reactivated embryo gene product, a cancer fetal antigen, It is selected from the group consisting of tissue-specific differentiation genes, growth factor receptors, and cell surface protein genes.

In one embodiment, the antigen is a microbial antigen, a viral antigen, a bacterial antigen, a fungal antigen or a natural isolate of a microbial antigen, a fragment and a derivative thereof.

In one embodiment, the antigen is a viral antigen.

In one embodiment, the composition for stimulating or activating DC for producing a DC vaccine comprises a plurality of peptide antigens.

In one embodiment, the composition that stimulates or activates DC for producing a DC vaccine comprises a peptide antigen that is a self-associating complex.

In one embodiment, the cationic lipid and the antigen are not structurally bound by a chemical bond.

In one embodiment, the cationic lipid and the antigen are bound by a chemical bond.

In one embodiment, the cationic lipid and the antigen are bound by a linker.

In one embodiment, the cationic lipid encapsulates the antigen.

In one embodiment, the cationic lipid forms liposomes.

In one embodiment, cationic lipids and antigens form an emulsion.

In one embodiment, cationic lipids promote the uptake of protein or peptide antigens.

In one embodiment, the peptide or protein antigen of the composition for uptake and stimulation of DC during the development of the DC vaccine is further modified to reduce the hydrophobicity of the antigen. The hydrophobicity of an antigen can be enhanced while preserving the antigenic properties of the molecule, for example by binding to a lipid chain or hydrophobic amino acid to improve the solubility of the antigen in the hydrophobic acyl chain of a cationic lipid. can.

The modified antigen is a modified lipoprotein with enhanced hydrophobicity in the amino acid sequence.
It may be a lipopeptide, a protein or a peptide, or a combination thereof.

The modified antigen can bind a linker between the lipid and the antigen, for example, the N-terminal α or ε-palmitoyl lysine via a serine-serine dipeptide linker.

In one embodiment, the cationic lipid comprises R-DOTAP, S-DOTAP, DOEPC, DDA, and DOTMA. Cationic lipids are non-toxic and can promote antigen internalization and DC maturation. More specifically, cationic lipids promote not only the internalization of the antigen by dendritic cells, but also its processing, cytoplasmic invasion, and subsequent enhanced presentation by the MHC class I and II pathways in vivo. be able to. This improves the antigen-specific immune response.

In another embodiment, the cationic lipid is DOTAP.

In yet another embodiment, the cationic lipid is DOTMA.

In another embodiment, the cationic lipid is DOEPC.

In some embodiments, the cationic lipid has been purified.

In some embodiments, the cationic lipid is an enantiomer.

In some embodiments, the enantiomer is purified.

In some embodiments, the composition comprising a cationic lipid and a protein antigen forms a particulate composition for DC uptake in the production of a DC cancer vaccine.

In some embodiments, the composition comprising a cationic lipid and a protein antigen forms a nanoparticle composition for DC uptake in the production of a DC cancer vaccine.

In some embodiments, the nanoparticles are less than about 10,000 nm in diameter.

In some embodiments, the invention treats isolated dendritic cells ex vivo with one or more lipids, including an effective amount of at least one cationic lipid and at least one cancer antigen. Provided is a method for producing a cancer dendritic cell vaccine, including the above. antigen.

Thus, the present disclosure provides isolated dendritic cells with (a) an effective amount of at least one cationic lipid, (b) at least one cancer antigen, and (c) at least a growth factor or at least a cytokine. By activating isolated dendritic cells with a composition comprising, or a combination thereof, activated dendritic cells are produced and an appropriate number of activated dendritic cells is administered to the patient. Provided are methods for treating cancer patients, including treating cancer patients by administering an appropriate number of activated dendritic cells.

Also disclosed is a method for assessing the success of treatment of a cancer patient by the method described above, comprising periodically checking the level of CD8 + T cells specific for at least one antigen in the composition. The evaluation step comprises periodically checking the level of CD8 + T cells specific for at least one antigen in the composition and checking the level of Treg cells in the patient after administering the dendritic cells to the patient. High levels of specific CD8 + T cells and low levels of Treg cells provide a method to show that patients are effectively treating cancer.

Geometric mean fluorescence intensity emitted by cells incorporating fluorescent ovalbumin. When statistical significance was evaluated using binary ANOVA, the * values were significantly different between treatments. Average fluorescence intensity of green fluorescence in gated CD11c positive cells, representing the amount of DQ-OVA taken up and processed by DC. Average fluorescence intensity indicating uptake of DQ-OVA by DC and TC1 cells in the presence of DOTAP at the indicated concentrations. Flow cytometric analysis of green vs. red fluorescence showing uptake and processing of DQ-OVA by mouse DC. Green vs. red fluorescence indicating uptake and processing of DQ-OVA by mouse DC in the presence of DOTAP or MPL. Laser scanning confocal microscopy and flow cytometry showing uptake and processing of DQ-OVA. A beta-galactosidase assay showing a measured value (arbitrary unit) of relative absorbance (570 nm). When statistical significance was evaluated using binary ANOVA, the * values were significantly different between treatments. Average CPM of ^3H -thymidine uptake by OT1 cells in the presence of OVA-stimulated BDMC with or without DOTAP. Average CPM of ^3H -thymidine uptake by DO11.10 splenocytes and OVAp323-339. Flow cytometric analysis of the CFSE dilution profile of T cells from the influx region lymph nodes of mice injected with OVA alone or OVA and DOTAP. OT1 T cell proliferation in the presence of BMDCs "pulsed" with OVA and three cationic lipids. Average CPM of ^3H -thymidine uptake in the last 18 hours of culture. IFN-ELISPOT assay of T cells from mice vaccinated with palmitoylation-KSSIINFEKL peptide or isotonic sucrose (280 mM) pulsed with palmitoylation mixed with cationic lipids (RDOTAP, DOTMA) or neutral lipids (DOPC). Vaccinated with tumor-related peptide (a) mixed with cationic lipid (RDOTAP, DOTMA) or neutral lipid (DOPC), HPV-related antigen and (b) BMDC pulsed with mucin 1 or isotonic sucrose (280 mM). IFN-ELISPOT assay of T cells from mice. A: Effect of ELISpot on HPV16-specific CD8 + T cell induction by HPV16-E7, R-DOTAP / HPV16-E7, S-DOTAP / HPV16-E7 and Alum / MPL / HPV16-E7 vaccination. B. Effect of R-DOTAP, R-DOTAP / HPV16-E7, S-DOTAP / HPV16-E7 vaccination on the regression of established HPV16-positive TC-1 tumors. Quantification of tumor infiltration HPV16-specific CD8 + T by RF9-specific dextramers analyzed using flow cytometry. The data represent the mean + SEM of 4-5 mice in each group. Quantification of regulatory T cells in tumors after treating tumor-bearing mice with various vaccines. The data represent the mean + SEM of 4-5 mice in each group. * Statistically significant R-DOTAP + antigen compared to all other groups (except R-DOTAP only). P <0.01. The ratio of T-regulatory cells (Tregs) to HPV16 E7-specific CD8 + T cells in CD45 + cells. The data represent the mean + SEM of 4-5 mice in each group. The effect of vaccination on the established regression of TC-1 tumors. * Statistically significant R-DOTAP + antigen tumor regression compared to all other groups. P <0.01. Quantification of CD8 + T cell induction by IFN-γ ELISPOT. The data represent the mean + SEM of 4-5 mice in each group. * Statistically significant R-DOTAP + antigen compared to all other groups. P <0.01. Dunnett's multiple comparison test. Quantification of T lymphocytes and total lymphocytes in inflow area lymph nodes after R-DOTAP cationic lipid vaccination. Quantification of MCP-1 and IP-10 levels in response to vaccination. The figure shows the levels of MCP-1 and IP-10 12 hours, 24 hours and 48 hours after vaccination.

Cationic lipids are rarely used in pharmaceuticals envisioned as gene transfection agents, especially since they have been reported to be toxic to cells in vitro. On the other hand, we have succeeded in using cationic lipids as transfection agents for complexing and delivering DNA and RNA to cells including dendritic cells. This technique has also been used to deliver RNA / DNA agents as antigens in DC vaccines. In such cases, the positive charge is neutralized and toxicity is minimized.

The methods described herein can improve protein and peptide based dendritic cell therapy. When treated ex vivo, classes of cationic lipids including R-DOATP, S-DOTAP, DOEPC, DDA, DOTMA can be administered to dendritic cells under conditions that limit toxicity and are dendritic. It can promote not only the internalization of the antigen by cells, but also its processing, cytosol entry, and subsequent presentation by the MHC class I and II pathways in vivo. This improves the antigen-specific immune response. This effect was not common to other lipids, but rather unique to cationic lipids.

Also, this effect is independent of the recently reported effect of cationic lipids as an immune adjuvant. Because both strong (R-DOTAP, DOTMA) cationic lipid adjuvants and weak (S-DOTAP, DDA) cationic lipid adjuvants, as well as neutral lipids that have been shown to have no action as immunoadjudicants in vivo. This is because the same effect was brought about in. Also, neither S-DOTAP nor DDA induced an effective antigen-specific immune response in vivo in previously reported studies, but now ex vivo promotes antigen processing and antigen presentation. It is shown that. This effect in facilitating antigen uptake, processing and presentation is in
When setting conditions for in vitro / ex vivo, it may be facilitated by bringing dendritic cells and cationic lipids close to each other, but this does not always occur in vivo. This important discovery has led to new uses for cationic lipids in the development of more effective ex vivo dendritic cell therapies. To date, this ability of cationic lipids has not been known, and therefore it has not been reported that cationic lipids are used to promote protein and peptide uptake and presentation in ex vivo dendritic cell therapy.

Hereinafter, various embodiments of the present invention will be described. In one embodiment described herein, ex vivo dendritic cell therapy compositions are provided. The composition comprises one or more lipids having at least one cationic lipid and at least one antigen. The composition may contain other lipids as well as growth factors for enhancing the viability and proliferation of dendritic cells.

If the subject is a mammal, a method of treating the subject's dendritic cells with ex vivo is provided. This method comprises an effective amount of at least one cationic lipid and antigen, optionally with growth factors such as GM-CSF and cytokines, to facilitate cell maintenance and growth in vitro. The step of treating dendritic cells with the above lipids is included.

Methods are provided for enhancing a prophylactic or therapeutic immune response in mammals. This method matures with the step of treating dendritic cells with one or more lipids containing at least one cationic lipid and antigen in effective amounts, optionally with growth factors such as GM-CSF and cytokines. Includes a step of administering a dendritic cell to a subject. In various embodiments, the composition comprises at least one cationic lipid and one or more lipids having at least one antigen.

This discovery will lead to the development of dendritic cell vaccines based on self-tumor-derived antigens capable of presenting antigens in very limited amounts, and peptide-based proteins to enable administration of very low doses of antigens. It can provide great advantages in dendritic cell vaccines. Although the dendritic cell vaccine approach offers significant potential, its lack of robust T-cell response limits its effectiveness and use as a viable cancer therapy. The present disclosure demonstrates that the use of cationic lipids can be used with limited toxicity to enhance the efficacy of such dendritic cell vaccines utilizing their own tumor-derived antigens or protein and peptide-based antigens. do.

Hereinafter, various embodiments of the present invention will be described as follows. In one embodiment described herein, an ex vivo dendritic cell therapy composition is provided. The composition comprises one or more lipids having at least one cationic lipid and at least one antigen. The composition may contain other lipids as well as growth factors to improve the viability and proliferation of dendritic cells.

The present invention shows that in the context of MHC class I and class II, cationic lipids can be used as vaccines to safely promote antigen presentation to dendritic cells and presentation to CD4 + and CD8 + T cells. Cationic lipids promote the induction of T cells that infiltrate high levels of tumors, while also being effective in inducing a significant reduction in the Treg population within the tumor microenvironment. These effects significantly alter the tumor microenvironment by reducing the Treg to CD8 + T cell ratio, which leads to the highly effective killing of tumor cells. Therapeutic
Cancer Vaccines J Clin Invest. In a recent review of 2015; 125 (9): 3401-3421, Melief et al. Stated: "Unoptimal vaccine design and immunosuppressive cancer microenvironments are the root causes of cancer eradication. Drugs and physical treatments can relieve immunosuppressive cancer microenvironments. It can include chemotherapy, radiation, indolamine 2,3-dioxygenase (IDO) inhibitors, T cell checkpoint inhibitors, selected TNF receptor family member agonists, and unwanted cytokine inhibitors. The specificity of therapeutic vaccination combined with such immunosuppression provides an attractive tool for the development of future cancer therapies.
antigen

In one embodiment, the cationic lipid is administered with an autoantigen such as an antigen derived from the patient's own tumor. In another embodiment, the cationic lipid is administered in combination with a non-self antigen such as a synthetic peptide, recombinant protein or DNA. In each case, the purpose is to elicit an antigen-specific immune response, along with which dendritic cells are treated with cationic lipids. The in vivo response that occurs upon injection of ex vivo-treated dendritic cells may include the production of certain cytotoxic T cells, memory T cells or B cells, and certain specific antigens associated with these antigens. It leads to prevention of the disease or therapeutic response to the disease. The antigen may be any tumor-related antigen or microbial antigen, and may be any other antigen known to those of skill in the art.

As used herein, a "tumor-related antigen" is associated with a tumor or cancer cell and, when expressed on the surface of an antigen-presenting cell in the context of an MHC molecule, provides an immune response (humoral and / or cellular). A molecule or compound that can be triggered (eg, protein, peptide, polypeptide, lipoprotein, lipopeptide, glycoprotein, glycopeptide, lipid, glycolipid, carbohydrate, RNA and / or DNA). Tumor-related antigens include self-antigens and other antigens that may not be specifically associated with cancer but nevertheless enhance and / or reduce the immune response of the tumor or cancer cells when administered to animals. included. More specific embodiments are provided herein.

As used herein, "microbial antigen" is an antigen of a microorganism, including, but not limited to, infectious viruses, infectious bacteria, infectious parasites and infectious fungi. Microbial antigens are intact microorganisms, natural isolates, fragments or derivatives thereof, synthetic compounds identical to or similar to naturally occurring microbial antigens, preferably (origin from which naturally occurring microbial antigens were obtained). ) Induces an immune response specific to the corresponding microorganism. In a preferred embodiment, the compound is equivalent to a naturally occurring microbial antigen as long as it induces an immune response (humoral and / or cellular) similar to that of a naturally occurring microbial antigen. Compounds or antigens that are equivalent to naturally occurring microbial antigens include, for example, proteins, peptides, polypeptides, lipoproteins, lipopeptides, glycoproteins, glycopeptides, lipids, glycolipids, carbohydrates, RNA, and / or DNA. Etc., which are well known to those in the art. Another non-limiting example of a compound that is similar to a naturally occurring microbial antigen is a peptide mimetic of a polysaccharide antigen.

The term "antigen" is further intended to include peptide or protein analogs of known or wild-type antigens, such as those described herein. Analogs are more soluble or stable than wild-type antigens and may also contain mutations or modifications that further immunologically activate the antigen. The antigen can be modified in any way, including addition of lipid or sugar moieties, mutations in peptide or protein amino acid sequences, mutations in DNA or RNA sequences, or any other modification known to those of skill in the art. The antigen can be modified using standard methods known to those of skill in the art.

Also useful in the compositions and methods of the invention are peptides or proteins having an amino acid sequence that is homologous to the amino acid sequence of the desired antigen, in which case the antigen having that homology is the respective tumor, microorganism or infected cell. Induces an immune response to.

In one embodiment, the antigen of the cationic lipid complex comprises a tumor or cancer-related antigen, i.e., a tumor-related antigen, for producing a vaccine for preventing or treating a tumor. Thus, in one embodiment, the tumor vaccine or cancer vaccine of the invention further comprises at least one epitope of at least one tumor-related antigen. In another preferred embodiment, the tumor vaccine or cancer vaccine of the invention further comprises a plurality of epitopes derived from one or more tumor-related antigens. The tumor-related antigens used in the cationic lipid complexes and methods of the invention can be immunogenic, non-immunogenic, or slightly immunogenic in nature. As evidenced herein, even tumor-related self-antigens can be used in favor of the vaccine to obtain therapeutic effects. This is because the composition can disrupt immune tolerance to such antigens. Exemplary antigens include, but are not limited to, synthetic antigens, recombinant antigens, foreign antigens or antigens with homology, and the materials for the antigens include proteins, peptides, polypeptides and lipos. Examples include, but are not limited to, proteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA and DNA. Examples of such treatments are breast cancer, head and neck cancer, melanoma, cervical cancer, lung cancer, prostate cancer, intestinal cancer, or the treatment or prevention of other cancers known in the art that are sensitive to immunotherapy. However, it is not limited to these. In such ex vivo treatments, it is also possible to combine the antigen with a cationic lipid without encapsulation.

Tumor-related antigens suitable for use in the present invention can be indicators of a single tumor type, are shared by several types of tumors, and / or are exclusively expressed or normal in tumor cells. These include both naturally occurring and modified molecules that are overexpressed compared to normal cells. In addition to proteins, glycoproteins, lipoproteins, peptides and lipopeptides, tumor-specific patterns of expression of carbohydrates, gangliosides, glycolipids and mutin have also been shown. Exemplary tumor-related antigens used in cancer vaccines include tumor genes, tumor suppressor genes, protein products of other genes with mutations or rearrangements specific to tumor cells, reactivated embryo gene products, and cancer fetal. These include antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, exogenous viral proteins, and numerous other autoproteins.

Specific embodiments of tumor-related antigens include, for example, mutant or modified antigens such as Ras p21 proto-oncogene, tumor suppressor p53, HER-2 / neu and protein products of BCR-abl cancer genes, and CDK4, MUM1. , Caspase 8, beta-catenin; overexpressing antigens such as galectin 4, galectin 9, carbonate dehydratase, aldolase A, PRAME, Her2 / neu, ErbB-2 and KSA; alphafetoprotein (AFP), human villous gonadotropin (hCG). Cancer fetal antigens such as; self-antigens such as cancer embryonic antigens (CEA) and melanosite differentiation antigens such as Mart1 / MelanA, gp100, gp75, tyrosinase, TRP1 and TRP2; PSA, PAP, PSMA, PSM-P1 and PSM- Prostate-related antigens such as P2; reactivated embryonic gene products such as MAGE1, MAGE3, MAGE4, GAGE1, GAGE2, BAGE, RAGE, and other cancer testicular antigens such as NY-ESO1, SSX2 and CSP1; Muc-1 and Muc Mutin such as -2; gangliosides such as GM2, GD2, GD3, neutral glycolipids and glycoproteins such as Lewis (y) and globo-H; Be done. Tumor-related antigens herein include whole cells and tumor cell lysates and their immunogenic portions, as well as immunoglobulin idiotypes expressed by monoclonal proliferation of B lymphocytes used for B cell lymphoma. Can be given.

Tumor-related antigens and their respective tumor cell targets include, for example, cytokeratin, particularly cytokeratin 8, 18, 19 as an antigen for malignant cancer. Capsular antigen (EMA), human embryo antigen (HEA-125), human milk fat globules, MBr1, MBr8, Ber-EP4, 17-1A, C26, T16 are also known carcinoma antigens. Desmin and muscle-specific actin are antigens of myogenic sarcoma. Placental alkaline phosphatase, β-human chorionic gonadotropin, and α-fetoprotein are antigens for trophoblasts and germ cell tumors. Prostate-specific antigens are prostate cancer antigens and carcinoembryonic antigens of colon adenocarcinoma. HMB-45 is an antigen for melanoma. In cervical cancer, useful antigens may be encoded by the human papillomavirus. Chromagranin-A and synaptophysin are antigens for neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid tumor masses with necrotic areas. Dissolution of such necrotic cells is a rich source of antigen for antigen-presenting cells, and therefore this treatment finds advantageous uses in combination with conventional chemotherapy and / or radiation therapy. Can be done.

Tumor-related antigens can be prepared by methods well known in the art. For example, these antigens prepare a crude extract of cancer cells (eg, as described in Cohen et al., Cancer Res., 54: 1055 (1994)), partially purifying the antigens. It can be prepared from cancer cells by methods such as by recombinant techniques or by de novo synthesis of known antigens. The antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in the subject and presentation to the immune system of the subject to be immunized. Further, the antigen may be a complete antigen or may be a fragment of a complete antigen containing at least one epitope.

Antigens derived from pathogens known to predispose to certain types of cancer can also be advantageously included in the cancer vaccines of the invention. It can be estimated that nearly 16% of global cancer incidence is due to infectious pathogens, and some common malignancies are characterized by the expression of specific viral gene products. Thus, inclusion of one or more antigens from pathogens involved in the development of cancer can help expand the immune response of the host and enhance the prophylactic or therapeutic effect of the cancer vaccine. Of particular interest as pathogens used in the cancer vaccines provided herein are hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (hepatic tumor), Epstein-Barr virus (EBV) (Berkit lymphoma, nasopharyngeal cancer). , PTLD in immunosuppressed individuals), HTLVL (adult T-cell leukemia), carcinogenic human papillomavirus types 16, 18, 33, 45 (adult cervical cancer), Helicobacter pylori (B-cell gastric lymphoma). Other medically related microorganisms that can act as antigens in mammals, especially humans, include C.I. G. A It is described in documents such as Thomas, Medical Microbiology, Balliere Tindall, Great Britain 1983, and the entire contents thereof are incorporated herein by reference.

In another embodiment, the antigen comprises an antigen derived from or associated with a pathogen, i.e. a microbial antigen. Thus, in one embodiment, the pathogen vaccine of the invention further comprises at least one epitope of at least one microbial antigen. Pathogens that can be targeted by this vaccine include, but are not limited to, viruses, bacteria, parasites and fungi. In another embodiment, the pathogen vaccine of the invention further comprises a plurality of epitopes derived from one or more microbial antigens.

Microbial antigens useful in cationic lipid complexes and methods may have intrinsic immunogenicity, may be non-immunogenic, or may be slightly immunogenic. Exemplary antigens include, but are not limited to, synthetic antigens, recombinant antigens, foreign antigens or antigens with homology, and the materials for the antigens include proteins, peptides, polypeptides and lipos. Examples include, but are not limited to, proteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA and DNA.

Exemplary viral pathogens include, but are not limited to, viruses that infect mammals, especially humans. Examples of viruses include human immunodeficiency viruses such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III / LAV or HIV-III; and other isolates such as HIV-LP). Picornavirus family (eg, poliovirus, hepatitis A virus; enterovirus, human coxsackie virus, rhinovirus, echovirus, etc.), calicivirus family (eg, strains that cause gastroenteritis), togavirus family (eg, etc.) , Horse encephalitis virus, ruin virus, etc.), Flavivirus family (eg, dengue virus, encephalitis virus, yellow fever virus, etc.), Coronavirus family (eg, coronavirus, etc.), Rabdovirus family (eg, bullous stomatitis virus, mad dog disease, etc.) Family of phylloviruses (eg, Ebola virus), Paramyxoviruses (eg, parainfluenza virus, epidemic parotitis, measles virus, respiratory follicles virus, etc.), Orthomyxoviruses (eg, respiratory follicles virus, etc.) , Influenza virus, etc.), Bungavirus family (eg, Hantan virus, Bungavirus, Pravovirus, Nyrovirus, etc.), Arenavirus family (hemorrhagic fever virus), Leovirus family (eg, Leovirus, Arbovirus, Rotavirus) , Birnavirus family, Hepadnavirus family (hepatitis B virus), Parvovirus family (parvovirus), papovavirus family (papillomavirus, polyomavirus), adenovirus family (most adenovirus), herpesvirus Family (simple herpesvirus (HSV) 1 and 2, varicella herpes virus, cytomegalovirus (CMV), herpesvirus), Poxylidae (pox virus, vaccinia virus, poxvirus), iridvirus family (eg , African pig cholera virus, etc.), unclassified virus (eg, causal encephalopathy pathogen, delta hepatitis factor (believed to be a hepatitis B virus deficient satellite), non-A non-B hepatitis Factors (class 1 = endogenous transmission, class 2 = parenteral transmission (ie, hepatitis C), nowalk virus and related viruses, astrovirus) are, but are not limited to.

Also, in vertebrates, Gram-negative and Gram-positive bacteria can be targeted by the compositions and methods. Examples of such Gram-positive bacteria include, but are not limited to, Pasteurella species, Staphylococcus species, and Streptococcus species. Examples of Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, and Salmonella. Specific examples of infectious bacteria, Helicobacter pylori, Borella burgdorferiLegionella pneumophiliaii, Mycobacteria sps (e.g., M.tuberculosis, M.avium, M.intracellulare, M.kansaii, etc. M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis , Listeria monocytogenes, Streptococcus pyogenes (A group Streptococcus), Streptococcus agalactiae (B group Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sps. , Enterococcus sp. , Haemophilus influenza, Bacillus anthracis, corynebacterium diphtheria, corynebacterium sp. , Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pastelella muscle. , Fusobacterium nucleatumi, Streptobacillus moniliformis, Treponema pallidium, Treponema pallidium, Leptospira, Rickettsia, Actinomyces, but not limited to Actinomyces.

Bacterial pathogen polypeptides that may be useful as sources of microbial antigens in the composition include iron-regulated outer membrane proteins (“IOLP”), outer membrane proteins (“OMP”), and Aeromonis salmonicida, which causes flunegrisis. Protein, p57 protein of Renibacterium salmoninarum that causes bacterial kidney disease (“BKD”), major surface-related antigen (“msa”), surface-expressed cytotoxicity (“mpr”), surface-expressed hemolytic element (“ish”), Yersinosis Foal antigens, extracellular proteins (“ECP”), iron-regulated outer membrane proteins (“IROP”), structural proteins of pasturella disease, OMP and Vibrosis angillum and V.I. Ordalii's flagellar proteins, Edwardiellosis ictaluri and E. coli. Examples include, but are not limited to, tarda flagellar proteins, OMP proteins, aroA, purA, surface antigens of Ichthyophthirius, structural and regulatory proteins of Cytophaga colimunari, and structural and regulatory proteins of Rickettsia. Such antigens can be isolated or prepared by recombination or other means known in the art.

Examples of pathogens include, but are not limited to, fungi that infect mammals, especially humans. Examples of fungi include Cryptococcus neoformansi, Histoplasma capsulatum, Coccidioides imitis, Chlamydia trachomatis, Candida albicans, and not limited to Candida albicans. Examples of infectious parasites include Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and other plasmodium protozoa. Other infectious organisms (ie, protists) include Toxoplasma gondii. Polypeptides of parasite pathogens include, but are not limited to, surface antigens of Ichthyophythirius.

Other medically relevant microorganisms that act as antigens in mammals, especially humans, are widely described in the literature. For example, C.I. G. A. Thomas, Medical Microbiology, Balliere Tindall, Great Britain 1983 (the entire content of which is incorporated herein by reference). In addition to treating infectious human diseases and human pathogens, the compositions and methods of the invention are also useful in treating infections in non-human mammals. Many vaccines for treating non-human mammals are available in Bennett, K. et al. Compendium of Veterinary Products, 3rd ed. North American Compendium, Inc. , 1995, also see WO02 / 069369 (the disclosure of which is expressly incorporated herein).

Exemplary non-human pathogens include mouse breast tumor virus (“MMTV”), laus sarcoma virus (“RSV”), trileukemia virus (“ALV”), trimyelyoblastosis virus (“AMV”), and mice. Leukemia virus (“MLV”), feline leukemia virus (“FeLV”), mouse sarcoma virus (“MSV”), tenagazal leukemia virus (“GALV”), splenic necrosis virus (“SNV”), reticular endothelosis virus (“SNV”) "RSV"), monkey sarcoma virus ("SSV"), Masson Faiser monkey virus ("MPMV"), monkey retrovirus type 1 ("SRV-1"), as well as HIV-1, HIV-2, SIV, Visna virus, lentivirus such as feline immunodeficiency virus ("FIV"), horse infectious anemia virus ("EIAV"), HTLV-1, HTLV-II, monkey T-cell leukemia virus ("STLV"), bovine leukemia virus Examples include, but are limited to, T-cell leukemia virus such as (“BLV”), human foam virus (“HFV”), monkey foam virus (“SFV”), and bovine foam virus (“BFV”). It is not something that will be done.

In some embodiments, as used herein with respect to an infectious agent, "treating,""treating," and "treating" increase the subject's resistance to infection by the pathogen, or the subject is the pathogen. Prophylactic treatment and / or treatment to combat infection after the subject has been infected, for example, reducing or eliminating the infection or preventing the infection from getting worse.

Microbial antigens can be prepared by methods well known in the art. For example, these antigens can be prepared directly from viral and bacterial cells by preparing crude extracts, partially purifying the antigens, by recombinant techniques, or by de novo synthesis of known antigens. The antigen may also be in the form of a nucleic acid encoding an antigenic peptide in a form suitable for expression in the subject and presentation to the immune system of the subject to be immunized. Further, the antigen may be a complete antigen or may be a fragment of a complete antigen containing at least one epitope.
Lipids

In order to improve the uptake of the antigen into the cationic lipid vesicles and also to improve the delivery of the immune system to cells, the antigen may be modified to increase its hydrophobicity or the negative charge on the surface of the antigen. For example, in order to improve the solubility of an antigen in the hydrophobic acyl chain of a cationic lipid while maintaining the antigenic properties of the molecule, the hydrophobicity of the antigen may be enhanced by binding to the lipid chain or a hydrophobic amino acid. The modified antigen can be a modified lipoprotein, lipopeptide, protein or peptide, or a combination thereof, with enhanced hydrophobicity in the amino acid sequence. The modified antigen may have a linker bound between the lipid and the antigen, for example, N-terminal α or ε-palmitoyl lysine bound to the antigen via a serine-serine dipeptide linker. You may. As discussed in more detail below, the DOTAP / E7-lipopeptide complex had an enhanced in vivo functional antigen-specific CD8 T lymphocyte response compared to the DOTAP / E7 preparation. In addition, the negative charge may be increased by changing the buffer of the preparation in which the antigen is encapsulated in a cationic lipid complex, or by covalently attaching an anionic moiety, such as an anionic amino acid, to the antigen. The antigen may be manipulated.

As shown in Example 1 described herein, the immunogenicity of the E7 antigen was increased by covalently modifying the antigen. It was possible to covalently bind an amino acid sequence to the antigen so that the obtained antigen amino acid sequence would not be found in the parent protein that originally obtained the antigen. Studies were conducted to show that modified antigens have better MHC class I binding affinity than native antigens. The excellent binding affinity as demonstrated in this study resulted in an excellent in vivo antitumor immune response to HPV-positive TC-1 tumors. The present invention will be further understood in the light of the following examples.

In some embodiments described herein, the cationic lipid may be in the form of a nanoparticle assembly. As used herein, the term "nanoparticles" refers to particles whose measured size is in the nanometer range. For example, "nanoparticles" can refer to particles having a structure less than about 10,000 nanometers in size. In some embodiments, the nanoparticles are liposomes.

As used herein, the term "cationic lipid" is a large number that retains a net positive charge at physiological pH or has a protonatable group that is positively charged at a pH lower than pKa. Refers to all of the lipid species of. Suitable cationic lipids according to the present disclosure include 3-β [ ⁴ N- ( ¹ N, ⁸ N ^- diguanidinospermidine) -carbamoyl] cholesterol (BGSC), 3-β [N, N-diguanidinoethyl-aminoethane. )-Carbamoylcholesterol ( ^BGTC ), N, N1, ^N2 , N3 Tetramethyltetramilspermin (selffectin), Nt-butyl-N'-tetradecyl- ³ -tetradecylaminopropionamidin (CLONfectin), dimethyl Dioctadecylammonium bromide (DDAB), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N- [2 (spermine-carboxamide) ethyl]- N, N-dimethyl-1-propaneammonium trifluorosate) (DOSPA), 1,3-dioreyloxy-2- (6-carboxyspermil) -propylamide (DOSPER), 4- (2,3-) Bis-palmitoyloxy-propyl) -1-methyl-1H-imidazole (DPIM) Ν, Ν, Ν', Ν'-tetramethyl-N, N'-bis (2-hydroxyethyl) -2,3-diore Oiloxy-1,4-butane-diammonium iodide) (Tfx-50), N-1- (2,3-dioleoyloxy) propyl-Ν, Ν, Ν-trimethylammonium chloride (DOTMA) or others N- (N, N-1-dialcooxy) -alkyl-N, N, N-trisubstituted ammonium surfactant, 1,2 dioleoil-3- (4'-trimethylammonio) butanol-sn-glycerol (DOBT) ) Or cholesteryl (4'trimethylammonium) butanoate (ChOTB) (where the trimethyl-ammonium group is connected to a double chain (in the case of DOTB) or a cholesteryl group (in the case of ChOTB) via a butanol spacer arm. ), DORI (DL-1,2-diore oil-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-diore oil-3-dimethylaminopropyl-β-hydroxyethylammonium) ( DORIE) or an analog thereof (such as those disclosed in WO93 / 03709), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), co. Lipopolyamines such as resteryl hemiscusinate ester (ChOSC), dioctadecylamide glycyl spermin (DOGS) and dipalmitoyl phosphatidyl ethanol amyl spermin (DPPES), cholesteryl-3β-carboxyl-amide-ethylenetrimethylammonium iodide, 1- Dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3-O-carboxyamide ethyleneamine, cholesteryl-3-β-oxysuccinamide-ethylenetrimethylammonium iodide, 1 -Dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide, 2- (2-trimethylammonio) -ethylmethylaminoethyl-cholesteryl-3-β- Oxysuccinate iodide, 3-β-N- (N', N'-dimethylaminoethane) carbamoyl cholesterol (DC-chol), 3-β-N- (polyethyleneimine) -carbamoyl cholesterol, Ο, Ο' -Dimyristyl-N-Resil Aspartate (DMKE), Ο, Ο'-Dimyristyl-N-Lisyl-Glutamate (DMKD), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 1 , 2-Jirauroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1,2-dimiristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dioleoyl-sn-glycero- 3-Ethylphosphocholine (DOEPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC), 1 , 2-diore oil-3-trimethylammonium propane (DOTAP), diore oil dimethylaminopropane (DODAP), 1,2-palmitoyle-3-trimethylammonium propane (DPTAP), 1,2-distearoyl-3-trimethylammonium Examples include, but are not limited to, propane (DSTAP), 1,2-myristoyl-3-trimethylammonium propane (DMTAP), and sodium dodecyl sulfate (SDS). In addition, structural variants and derivatives of any of the cationic lipids described are contemplated.

In certain embodiments, the cationic lipid is selected from the group consisting of DOTAP, DOTMA, DOEPC, and combinations thereof. In another embodiment, the cationic lipid is DOTAP. In yet another embodiment, the cationic lipid is DOTMA. In another embodiment, the cationic lipid is DOEPC. In some embodiments, the cationic lipid is purified.

In some embodiments, the cationic lipid is an enantiomer of the cationic lipid. The term "enantiomer" refers to a pair of stereoisomers, such as R enantiomers and S enantiomers, which are mirror images of cationic lipids that cannot be superimposed on each other. In various examples, the enantiomer is R-DOTAP or S-DOTAP. In one example, the enantiomer is R-DOTAP. In another example, the enantiomer is S-DOTAP. In some embodiments, the enantiomer is purified. In various examples, the enantiomer is R-DOTMA or S-DOTMA. In one example, the enantiomer is R-DOTMA. In another example, the enantiomer is S-DOTMA. In some embodiments, the enantiomer is purified. In various examples, the enantiomer is R-DOPEC or S-DOPEC. In one example, the enantiomer is R-DOPEC. In another example, the enantiomer is S-DOPEC. In some embodiments, the enantiomer is purified.

Note that for illustrative purposes, all examples are well studied and performed with ovalbumin, a model protein available with dual fluorescent labels. Using model proteins provides excellent examples of how cationic lipids enhance antigen uptake processing and presentation. In addition, the availability of TCR transgenic T cells specific for class I and class II restrictive OVA peptides allows for detailed studies and confirmation of antigen presentation via both pathways.

All in vitro studies reported in the Examples were performed using the model protein ovalbumin as a representative antigen. Fluorescent OVA conjugates (DQ-OVA conjugates, Alexa Fluor® 647 OVA) that can be easily traced using a flow cytometer to assess the effect of cationic lipids on antigen uptake and processing by antigen presenting cells. Conjugate) was used. In addition, by using the ovalbumin protein as an antigen, ovalbumin-specific T-cell hybriddoma cells and TCR transgenic mice (OT-1 and DO11.10) with ovalbumin-specific CD4 and CD8 T cell receptors are used. MHC
Confirmation of antigen presentation by I and MHC II became easier. The results shown in this study are generally applicable to all protein and peptide antigens.
<Example 1: Effect of cationic lipid on antigen uptake by dendritic cells>

To determine the effect of cationic lipids on protein antigen uptake, mouse bone marrow-derived dendritic cells (BMDCs) are pulsed with Alexa Fluor® 647-OVA conjugate and ovo by BMDCs using flow cytometry. Albumin uptake was quantified. Briefly, serum-free RPMI 1640 containing 20 μg / ml ovalbumin (Ovalbumin AlexaFluor® 647 conjugate; Life Technologies, Catalog No. 0347884) and 50 μM cationic lipid (RDOTAP) or 280 mM sucrose diluent. In cell culture medium, 2 × 10 ⁶ cells / ml BMDCs were incubated at 37 ° C. for 10-60 minutes. The cells were washed after pulse to remove ovalbumin not bound to the cells and fixed with 1% formaldehyde for analysis on a flow cytometer. Ovalbumin uptake was quantified using a BD LSR II flow cytometer. As shown in FIG. 1, cationic lipids significantly increased protein uptake by BMDCs at all measured time points. In addition, protein uptake occurred so rapidly in the presence of cationic nanoparticles that cationic lipids are beneficial in significantly reducing antigen pulse time during the preparation of dendritic cell vaccines. It suggests.
<Example 2: Effect of cationic lipid on antigen processing by dendritic cells and epithelial cells>

A fluorescent ovalbumin protein called DQ-OVA was used to determine the effect of cationic lipids on ex vivo antigen uptake and processing by dendritic cells. DQ-OVA is not fluorescent in the intact state, but emits both red and green fluorescence when the protein is degraded. BMDCs were incubated with DQ-OVA alone or mixed with different concentrations of cationic lipid DOTAP at 37 ° C or 4 ° C for 1 hour. The cells were then washed, fixed and stained with a fluorescent antibody against CD11c, a marker for dendritic cells. The cells were then analyzed with an LSRII flow cytometer for both red and green fluorescent channels.

The results in FIG. 2 show that BMDCs incubated with fluorescent DQ-OVA in medium alone increased fluorescence at 37 ° C. for uptake and processing. This represents DC-mediated uptake of the well-known mannose receptor for OVAs. DOTAP enhances the uptake and processing of antigens by DC. Graph display of fluorescence uptake of DQ-OVA to BMDC measured by flow cytometry. The plot shows the average fluorescence intensity of the green fluorescence of the gated CD11c positive cells, which represents the amount of DQ-OVA taken up and processed by DC.

This uptake and processing was inhibited at 4 ° C, confirming that this type of uptake requires active cytoskeleton reconstruction. BMDCs incubated with DQ-OVA in the presence of pure R-enantiomer (R-DOTAP) of DOTAP showed doubling of fluorescence and the cationic lipid R-DOTAP with protein uptake at DC. It has been shown to significantly enhance processing. Significant uptake was still observed at 4 ° C., indicating that R-DOTAP can facilitate protein uptake in the absence of active cell metabolism. The action by R-DOTAP was concentration-dependent, and the maximum action was observed at 50 μM. Mouse epithelial cell lines were incubated with DQ-OVA under the same conditions as BMDCs to determine if enhanced protein uptake by R-DOTAP was cell-dependent. The results in FIG. 3 show that this OVA uptake and processing is observed only in DC, not in TC1 epithelial cells. These data indicate that DOTAP can significantly enhance complete protein uptake and processing into dendritic cells ex vivo. Furthermore, it can be seen that this enhancement is selective for dendritic cells and not for other non-antigen presenting cell types.
<Example 3: Comparison of the effects of cationic lipids and known adjuvants on antigen processing and endosome invasion>

BMDCs were incubated with DQ-OVA using medium alone or R-DOTAP to determine if the lipid adjuvant could intervene in the same action as R-DOTAP, as described in FIG. .. The BMDC was also incubated under the same conditions with lipopolysaccharide (LPS), a potent lipid adjuvant. Incubate mouse bone marrow DC for 1 hour in the presence of fluorescent DQ-OVA at either 37 ° C or 4 ° C / azide in the presence of 25 μM DOTAP nanoparticles, 10 μg / ml LPS or medium alone, and flow cytometry. Analyzed in. As shown in FIG. 1, DQ-OVA was actively taken up and processed by DC in the absence of R-DOTAP, apparently from the strong increase in red fluorescence in the presence of R-DOTAP. As such, uptake was significantly enhanced. In contrast, as shown in FIG. 4, no such increase was observed with treatment with LPS. Monophosphoryl lipid A (MPL) is a derivative of LPS with low toxicity and is currently an FDA-approved adjuvant for several vaccines. Similar to the LPS results in FIG. 5, MPL did not show the ability to enhance protein uptake in BMDCs.
<Example 4: Effect of cationic lipid on antigen processing in human monocyte cell line>

To determine the action of cationic lipids on human cells, the human monocyte cell line THP-1 was used to evaluate the uptake of DQ-OVA ex vivo. THP-1 is representative of monocyte cells derived from human blood and is the same cells used to make DCs from patients in ex vivo DC therapy techniques. THP-1 cells with DQ-OVA (10 μg / ml) at 37 ° C. for 1 hour in the presence (A, B) or non-existence (C, D) of R-DOTAP (25 μg / ml). Incubated. The same cells were imaged using a laser scanning confocal microscope and quantified by measuring green vs. red fluorescence by flow cytometry. The results in FIG. 6 show that R-DOTAP dramatically increased the uptake of DQ-OVA in THP-1 cells. Unlike mouse BMDCs, no uptake of DQ-OVA was observed in the absence of R-DOTAP. This is thought to be because blood-derived monocytes are precursors of dendritic cells but do not yet have the receptors required for protein uptake. This result is important. This is because R-DOTAP has been shown to enhance uptake and processing in cells that would normally not be uptake by the receptor. Another prominent observation from FIGS. 6A and B is the accumulation of processed OVA in the endocytic vesicles. Endocytotic vesicle proteins can be efficiently incorporated into MHC class II molecules to stimulate CD4 T cells, or cross-presentation pathways for presentation from MHC class I to CD8 T cells. Well known. Therefore, cationic lipids promote protein uptake in a manner that maximizes the presentation of antigen to both MHC class I and class II molecules, maximally stimulating CD8 and CD4 T cells, respectively. ..
<Example 5: Action of DOTAP and DOTMA on antigen processing and cross-presentation on MHC class I restrictive T cells>

To evaluate cross-presentation in the presence of cationic lipids to verify that facilitating antigen uptake in cationic lipids actually leads to enhanced antigen presentation in MHC class I (cross-presentation). Two different methods were used. In the first method, a T cell hybridoma cell line (B3Z cell) was used. This cell line can respond to antigen-presenting cells that cross-present the SIINFEKL peptide via MHC I by inducing β-galactosidase enzyme, which can be quantified using the β-gal assay. It is possible. Only BMDC (OVA241-270; SMLVLLPDEVSGLEQLESIINFEKLTEWTS) or isotonic sucrose (280 mM) (Suc) with an ovalbumin peptide mixed with 50 μM RDOTAP. Peptide-pulsed BMDCs were washed and co-cultured overnight at 37 ° C. with an antigen-specific T-cell hybriddoma cell line (B3Z cells) capable of recognizing SIINEKL epitopes presented by antigen-presenting cells via MHCI (H2Kb). B3Z cells responded to the SIINFFEKL epitope by producing a β-galactosidase enzyme, which was quantified using a colorimetric β-galactosidase assay as an indicator of peptide cross-presentation by dendritic cells. The data represent relative absorbance (570 nm) in the test well (arbitrary unit). Statistical significance was evaluated using a binary ANOVA, and the * values were significantly different for each treatment (shown in FIG. 7).

As shown in FIG. 7, peptide pulses with cationic nanoparticles were observed to significantly reduce the concentration of peptide required for efficient pulses. This method utilizing cationic lipids is particularly advantageous under conditions where cross-presentation of dendritic cells is restricted due to the amount of peptide antigen available or the concentration of peptide (eg, for autologous tumor vaccines with limited epitopes). Provides significant advantages in peptide loading in.

In the second method, T cells derived from TCR transgenic mice (OT-1), in which all T cells are specific for the internal peptide of OVA, were used. These T cells are treated with OVA and proliferate only when presented at the DC presenting the OVA peptide to the MHC class I molecule.

Therefore, this is a rigorous assay for cross-presentation. BMDCs were incubated with different concentrations of OVA complete protein for 1 hour at 37 ° C. in the presence or absence of either of the two cationic lipids DOTAP or DOTMA. The DCs were then washed, immobilized and added to OVA peptide-specific T cells. The results in FIG. 8 show that DCs incubated with OVA in the presence of DOTAP or DOTMA cross-presented the antigen to CD8 + T cells much more strongly than DCs incubated with OVA without cationic lipids. ing. This response was dose-dependent with respect to OVA concentration and was also evident when DC was incubated with OVA at 4 ° C.

These results show that cationic lipids enhance antigen uptake, resulting in CD8.
It has been shown to actually lead to efficient antigen processing and peptide uptake into the MHC class I pathway, which are absolute prerequisites for effective activation of T cells.
<Example 6: Effect of DOTMA and DOTAP on antigen processing and cross-presentation on MHC class II restrictive T cells>

Derived from DO11.10 transgenic mice with T cells specific for the OVA peptide presented on MHC class II molecules to determine if cationic lipids actually increase antigen presentation to CD4 T cells. Cells were used. The results of FIG. 9 show that the same trends as those observed in the class I presentation of FIG. 8 were observed in the class II presentation. Incorporation of OVA in the presence of cationic lipids enhanced antigen presentation to CD4 T cells.

These results show that cationic lipids enhance antigen uptake, resulting in CD4.
It has been shown to actually lead to efficient antigen processing and peptide uptake into the MHC class II pathway, which are absolute prerequisites for effective activation of T cells.
<Example 7: Effect of DOTAP on antigen presentation in actual vaccine setting in vivo>

A T cell receptor adoption system was used to model the effects of DOTAP on actual vaccine settings. This system uses the same TCR transgenic mouse-derived T cells as described in FIGS. 8 and 9, but analyzes how these cells respond in vivo after vaccination. First, OT-1 (OVA-specific CD8 +) or DO11.10 (OVA-specific CD4 +) T cells were labeled with the follow-up fluorescent dye CFSE. Twenty-four hours later, mice were injected with OVA alone or in the presence of DOTAP. If these T cells recognize the antigen presented by DC in the influx region lymph node after immunization, the T cells will proliferate and the CFSE dye will be diluted in all daughter cells. Next, mice were injected with OVA with and without DOTAP. Three days later, the inflow region lymph nodes at the vaccination site were removed, T cells were stained with anti-CD8 and anti-CD4 antibodies, and CFSE levels were visualized by flow cytometry. The results in FIG. 10 show CFSE-labeled OT1 (CD8 T cells) or DO11.10 (CD4 T cells) when either complete OVA or complete OVA mixed with DOTAP is further injected (immunized). Significant T cell division occurred only when mice were vaccinated with OVA + DOTAP. These results indicate that the antigen delivery properties of DOTAP lead to an increased T cell response in the influx region lymph nodes after vaccination.
<Example 8: Action of various lipids on antigen processing and cross-presentation on MHC class I restrictive T cells>

To confirm the effect of other cationic and triglyceride on antigen uptake and cross-presentation, BMDCs at 37 ° C. or in the presence of complete OVA protein and various concentrations of DOTAP, DOTMA, DOPC, DOEPC or DDA. Incubated at 4 ° C. for 30 minutes. Then, the DCs were washed, added to OT1 splenocytes (TCR transgenic T cells specific for class I-restricted OVA peptide SIINFFEKL) on a microtiter plate, and cultured at 37 ° C. for 3 days. The plot shows the average CPM of ^3H thymidine uptake during the last 18 hours of culture. Control cultures contain only OT1 splenocytes and SIINFFEKL, which avoids the need for antigen processing.

The results in FIG. 11 show that the cationic lipids DOTAP, DOTMA, DOEPC, and DDA all promote OVA uptake and cross-presentation enhancement by BMDCs. The cationic lipid S-DOTAP also facilitated uptake and presentation. The triglyceride DOPC also facilitated uptake and presentation. These results indicate that a class of cationic lipids is effective in mediating the effective uptake of antigens and the delivery of protein antigens by dendritic cells.
<Example 9: Cationic lipid improves the efficacy of dendritic cell vaccine>

The effect of cationic lipids on CTL induction by dendritic cell vaccines was evaluated to show evidence of the concept that cationic lipids improve the efficacy of dendritic cell vaccines in vivo. B6 mice were immunized with only peptide-pulsed BMDC or isotonic sucrose with peptide alone (palmitoylated-KSSSINFEKL) or a peptide mixed with cationic (50 μMR-DOTAP, DOTMA) nanoparticles or neutral lipid nanoparticles (DOPC). .. On days 0 and 7, a group of C57BL6 / J mice (n = 5) was subcutaneously immunized with peptide-pulsed BMDCs, and on day 14 an antigen-specific IFN-γ response was obtained using the ELISPOT assay. Vaccine response was assessed by measurement. The data represent spot-forming cells in each mouse in a representative study.

As shown in FIG. 12, pulsed dendritic cells with peptide-loaded cationic nanoparticles significantly increases the vaccine-induced antigen-specific T cell response, and thus the cations seen in the in vitro assay. It is directly shown that the beneficial effects of sex nanoparticles can affect the efficacy of dendritic cell vaccines. In the following studies, the effectiveness of cationic nanoparticles in inducing CTL was investigated using tumor-related antigens derived from HPV-related tumors and mucin 1-related tumors. As expected, the cationic nanoparticles loaded with tumor-related antigens improved the antigen-specific T cell immune response mounted in vaccinated mice (FIG. 13). In this experiment, a mouse BMDC with a peptide mixture or isotonic sucrose (280 nM) containing a tumor-related antigen (HPV tumor-related (a) or mucin 1-related (b)) mixed with 50 μM cationic lipid (RDOTAP). Was pulsed for 10 minutes. On days 0 and 7, a group of C57BL6 / J mice (n = 5) was subcutaneously immunized with peptide-pulsed BMDCs or pulse-free BMDCs, and on day 14 antigen-specific IFN- using the ELISPOT assay. Vaccine response was evaluated by measuring γ response. The data represent spot-forming cells in each mouse in a representative study. Some enhancement was observed with DOTAP, DOTMA, and DOPC, whereas antigen presentation was significantly enhanced with DOEPC and DDA. Note: DDA formed a precipitate when diluted with OVA. All of the cationic lipids are found to be enhanced, but the overall size varies depending on the experiment. DOPC, which is a triglyceride, was also found to be somewhat enhanced in this experiment.

These results indicate that a class of cationic lipids is effective in mediating the effective uptake of antigens and the delivery of protein antigens by dendritic cells. In addition, dendritic cells pulsed with antigen-loaded cationic lipids can significantly improve the efficacy of dendritic cell vaccines.
<Example 10: Effect of R-DOTAP on a population of T cells and regulatory T cells in the tumor microenvironment>

Antigen-specific CD8 + T cells were induced by R-DOTAP and S-DOTAP.

C57BL mice were vaccinated with various preparations.
Group 1: KF18 HPV peptide (GQAEPDRAHYNIVTF)
Group 2: KF18 HPV peptide + R-DOTAP liposome Group 3: KF18 HPV peptide + S-DOTAP liposome Group 4: KF18 peptide + MPL / Alum adjuvant

Five mice per group were injected with various preparations. Mice were vaccinated on days 0 and 7 and sacrificed on day 14. Spleen cells were removed and ELISPOT studies were performed. Spleen cells were stimulated with the peptide RAHYNIVTF (RF9) and the HPV16 CD8 + T cell epitope peptide recognized by C57 mice was recognized by C57 mice. This study shows that R-DOTAP is effective in inducing a strong HPV-specific CD8 + T cell response. However, S-DOTAP, which showed the same ability to promote antigen uptake, internalization, processing, and dendritic cell maturation, did not lead to an increased CD8 + T cell response as seen with peptides alone. (FIG. 14A). Since MPL was not effective in promoting antigen uptake compared to either R-DOTAP or S-DOTAP, it was assumed that the CD8 + T cell response would be significantly lower than R-DOTAP. Another example of this effect is also observed with the cationic lipid DDA. FIG. 14B shows the ability of DDA to facilitate antigen uptake and presentation. However, it has been reported that DDA is used in combination with a potent adjuvant to induce a strong antigen-specific T cell response (Brandt L. et al, ESAT-6 Subunit Vaccination aggregation Mycobacterium tuberculosis, Infect Immuno. 2000 Feb; 68 (2): 791-795).

Directly using R-DOTAP and GM-CSF-based immunotherapy with tumor antigens, as enhanced and presented antigen uptake by R-DOTAP and excellent CD8 + T cell induction in vivo are observed. A comparative (head-to-head) study was conducted to study the effect of these two vaccines on the infiltration of T cells into the tumor microenvironment and the ability to downregulate the immunosuppressive tumor microenvironment.

Eight C57 mice per group were divided into R-DOTAP + HPV16 E7 peptide KF18 (GQAEPDRAHYNIVTF), GM-CSF + HPV16 E7 peptide KF18, R-DOTAP, GM-CSF, HPV16 E7 peptide KF18, and untreated groups. .. On day 1, 1 × 10 ⁵ TC-1 tumor cells were injected into the flank of the mouse. Various preparations were administered 12 and 19 days after tumor transplantation. On day 19, 4-5 mice per group were sacrificed and multiple assessments were performed to assess the immunology of the tumor microenvironment.

RF9-specific dextramer staining and flow cytometry were used to quantify the number of HPV-specific CD8 + T cells infiltrating the tumor microenvironment. In this study, the number of CD8 + T cells specific for mouse epitope RF9 was quantified. These CD8 + T cells were measured as a percentage of all immune cells (CD45, CD3, CD8) present in the tumor. Antigen-specific T cells infiltrating the tumor were measured by flow cytometry using RF9-specific dextramers. FIG. 15 shows the results of the study, showing a statistically significant increase in HPV-specific T cells compared to R-DOTAP / HPV compared to all other groups. The data represent the mean + SEM of 4-5 mice in each group.

On day 19, flow cytometry was used to study immunosuppressive tumor microenvironments, especially regulatory T cell populations. On the 14th and 19th days, T cells (CD45 + CD3 + CD4 + CD25 + Foxp3 +) cells invaded the tumor. The results are shown in FIG. This study shows that a statistically significant reduction of about 40% of the Treg population within the tumor is observed with R-DOTAP + antigen alone within 1 week after vaccination (P <0.01). Statistical significance was not achieved in this group, but none of the groups other than the R-DOTAP group showed the ability to reduce the Treg population.

Very important to the clinical efficacy of any immunotherapy is the ratio of immunosuppressive cells to tumors that target CD8 + T cells in the tumor microenvironment. The lower the ratio of immunosuppressive cells to CD8 + T cells, the better the prognosis towards antitumor effect. In this study, the Treg / CD8 + T cell ratio dropped dramatically to less than 0.13 for R-DOTAP + antigen, compared to a ratio of about 1 for GM-CSF + antigen or for antigen alone. It shows that it becomes. The ratio was about 32 in the tumor antigen-free group (shown in FIG. 17). Cationic lipids appear to promote preferential expansion of the correct phenotype of effector T cells over Tregs. This leads to significant alteration of the tumor microenvironment, which makes immunotherapy highly effective because the attacker produces a "force shift" that favors CD8 + T cells over immunosuppressive Tregs "advocates".

The same study evaluated the effects of various preparations on established TC-1 tumors. FIG. 18 shows that the tumors had completely disappeared by day 26 in all animals treated with R-DOTAP + antigen (Treg / CD8 + ratio <0.13). Tumor volume was measured using a caliper. The naive mouse group is mice that remain untreated and have tumors. The HPV16 E7 peptide used in the vaccine is KF18. Neither the GM-CSF + antigen nor the antigen alone (Treg / CD8 + ratio of about 1.0) induced tumor regression, but tumor growth was inhibited and the tumor volume was about 200 mm ³ on day 26. became. In the third group (Treg / CD8 + ratio> 30) consisting of animals treated with R-DOTAP or GM-CSF without antigen or untreated animals, the tumor volume was 300-700 mm ³ .

In this study, IFN-γ ELISPOT studies were performed to quantify and understand the “quality” of tumor-specific T cells produced. In this study, animals were sacrificed on day 26 and splenocytes were used. The results are shown in FIG. This study shows that when cells were stimulated with HPV16 CD8 + mouse epitope RF9, the R-DOTAP + antigen preparation produced about 4-5 times more IFN-γ than the GM-CSF + antigen. This is because, in FIG. 11, cationic lipids are "higher quality" than GM-CSF due to the fact that the amount of CD8 + cells infiltrating the tumor microenvironment is less than twice the result with GM-CSF. It is suggested that T cells can be generated. The reason for the superior priming of T cells was evaluated in another study.
<Example 11: Evaluation of R-DOTAP vaccination against infiltration of T cells and B cells into lymph nodes>

12 mM R-DOTAP or sucrose as a control was injected into the toes of the right and left foot of the mice, respectively, and the influx of T cells and total lymphocytes into the inflow region lymph nodes was quantified by flow cytometry. In this experiment, popliteal lymph nodes were removed and analyzed 15 hours after vaccination. FIG. 17 shows that R-DOTAP induced significant infiltration of T cells into the lymph nodes. In the second experiment, analysis was performed at 5 hours, 16 hours, 3rd day, and 4th day, and it was found that the infiltration of lymphocytes into the lymph nodes increased over 4 days (Fig. 20). Five mice were used in one study.
Example 12: Evaluation of the role of chemokines in lymphocyte infiltration into lymph nodes

The main purpose of the current experiment was to perform the study described in Example 7 using 5 mice to visualize the homing of adopted CFSE-labeled cells. This study included a population of cells in vitro treated with pertussis toxin to inactivate chemokine receptors. These cells were labeled with two concentrations of CFSE so that pertussis toxin and untreated cells could be distinguished by flow cytometry. Lymphocytes should be guided back to the lymph nodes. However, if the DOTAP-enhanced homing is due to chemokines, the pertussis toxin population should not be present in the DLN, or it should only be present at significantly reduced levels.

Spleen cells were prepared from one B6 mouse and split in half. Half of the cells were treated with 100 ng / ml pertussis toxin at 37 ° C. for 1 hour and washed. The two cell populations were then labeled with two concentrations of CFSE and mixed together so that the two cell populations could be distinguished by flow cytometry. The mixture ( ¹⁰⁷ cells) was intravenously injected into the tail vein of 5 B6 mice. Mice were then anesthetized and either sucrose (right toe) or R-DOTAP (left toe, 50 μl, 600 nanomoles) was injected into the toe.

After 16 hours, the mice were sacrificed and the knee fossa LN and spleen were harvested. The total number of cells collected from the left and right nodes of each mouse was counted. The migrated CFSE-labeled lymphocytes also infiltrated the lymph nodes during R-DOTAP vaccination. However, this did not occur in cells treated with whooping cough, indicating that cationic lipids induce the influx of lymphocytes into the lymph nodes, probably mediated by chemokines.

Previous studies (Yan et al.) Suggested that cationic lipids induce chemokines CCL2, 3, and 4. However, these chemokines are not involved in lymph node homing. Therefore, this study suggests that cationic lipids such as R-DOTAP also induce other lymph node homing chemokines such as CCL21 or CXCL12.
<Example 13: Induction of cytokines and chemokines in lymph nodes>

One of the important side effects of an adjuvant is to induce cytokines and increase such cytokines in the circulating blood. The presence of cytokines in the blood often results in a significant inflammatory response, which results in fever, nausea, vomiting, headache, and in extreme cases poisonous shock and even death. Cytokine storms are often associated with the administration of various adjuvants.

Therefore, this study focused on assessing the presence of cytokines systemically after subcutaneous administration of a cationic lipid vaccine. High-dose and low-dose R-DOTAP + antigens were administered to human HLA-A2 mice capable of recognizing human HPV antigens.
Group 1:
Mice vaccinated with 100 μL of a 1: 1 mixture of high dose R-DOTAP (3.4 mg / mL) and sucrose solution Group 2:
Vaccinated with 50 μg LPS as a positive control for cytokine induction Group 3:
High dose R-DOTAP + HPV antigen (RDOTAP 3.4 mg / mL and HPVMi
Mice vaccinated with 100 μL (1: 1 mixture with x of 0.14 mg / mL) Group 4:
Mice are vaccinated with 100 μL of low dose R-DOTAP + HPV antigen (1: 1 mixture of RDOTAP 3.4 mg / mL and HPV Mix 0.14 mg / mL).

After one vaccination, blood was collected from all mice as follows.
1. 1. Before blood sampling (before vaccination)
2.12 hours 3.24 hours 4.48 hours

Approximately 200 μL of blood was collected from each mouse at the time point above.

Cytokine analysis was performed in the Luminex assay according to the manufacturer's instructions.

As a positive control, mice were vaccinated with a well-studied Toll-like receptor (TLR) agonist lipopolysaccharide (LPS).

Study Results: Mouse sera were analyzed using the Luminex Mouse cytokine 20-plex panel (cytokines are listed below). Luminex software was used to quantify cytokine intensity levels by comparison with cytokine standards performed on the same plate. The positive control group (LPS) showed increased systemic levels of IL-12, IP-10, KC, MCP-1, and MIG at the time of vaccination. As shown in FIG. 21, neither systemic induction of the cytokines or chemokines studied was induced beyond the pre-vaccination baseline with high and low doses of PDS0101.

Mouse Cytokine 20-plex Panel:
FGF, IL-1b, IL-10, IL-13, IL-6, IL-12 (P40 / P70), IL-17, MIP-1a, GM-CSF, MCP-1, IL-5, VEGF, IL -1a, IFN-γ, TNFa, IL-2, IP-10, MIG, KC, IL-4.

The study results for MCP-1 (CCL2) and IP-10, which are typical of the results found in all the cytokines tested, are shown in FIG. This study shows that in the case of cationic lipids, cytokine and chemokine induction appears to be predominantly restricted to lymph nodes. For LPS, a typical TLR agonist, cytokine induction is not limited to lymph nodes and systemic spikes at cytokine levels are observed within 12 hours of vaccination. The absence of cytokines in the blood circulation suggests that cationic lipids provide a unique and safe means of immunotherapy for altering the tumor microenvironment.
Equivalent

One of ordinary skill in the art will be able to recognize or confirm many equivalents to the specific embodiments of the invention described herein using only idiomatic experiments. .. Such equivalents are intended to be included in the appended claims.

Claims (41)

With a population of isolated dendritic cells,
With at least one cationic lipid,
A composition for producing a vaccine comprising at least one peptide antigen. The composition of claim 1, wherein the at least one peptide antigen is a self-associating complex. The composition according to claim 1, wherein the at least one antigen is a modified protein, lipoprotein, lipopeptide, peptide or a combination thereof, which has an amino acid with enhanced hydrophobicity. The composition according to any one of claims 1 to 3, wherein the at least one antigen comprises a cancer or tumor-related antigen. The at least one antigen comprises a protein product of a gene in a patient, wherein the gene is a cancer gene, a tumor suppressor gene, a gene having a mutation, a gene having a rearrangement peculiar to a tumor cell, a reactivated embryo gene product, and the like. The composition according to any one of claims 1 to 4, selected from the group consisting of a cancer fetal antigen, a tissue-specific differentiation gene, a growth factor receptor, and a cell surface protein gene. The composition according to any one of claims 1 to 5, wherein the at least one antigen contains a microbial antigen. The composition according to claim 6, wherein the microbial antigen is selected from the group consisting of a viral antigen, a bacterial antigen, a fungal antigen, and a natural isolate, fragment, and derivative thereof of the microbial antigen. The composition according to any one of claims 1 to 7, which comprises a plurality of antigens. The composition according to any one of claims 1 to 8, wherein the at least one antigen comprises a non-immunogenic peptide or a peptide having poor immunogenicity. The composition according to claim 1, wherein the at least one cationic lipid is structurally bound to the at least one antigen by a linker. The composition according to claim 10, wherein the linker is a serin-serine dipeptide linker. The composition according to claim 1, wherein the cationic lipid encapsulates the antigen. The composition according to any one of claims 1 to 12, wherein the cationic lipid contains liposomes. The composition according to any one of claims 1 to 12, wherein the cationic lipid and the antigen are emulsions. The composition according to claim 1-14, wherein the at least one cationic lipid is selected from the group consisting of R-DOTAP, S-DOTAP, DOEPC, DDA, and DOTMA. The composition according to claim 1 to 15, wherein the cationic lipid is purified. The composition according to claim 1 to 16, wherein the cationic lipid is an enantiomer. The composition according to claim 1 to 17, wherein the cationic lipid forms nanoparticles. 18. The composition of claim 18, wherein the nanoparticles have a diameter of less than about 10,000 nm. A method of activating dendritic cells ex vivo,
The process of obtaining isolated dendritic cells and
A step of treating isolated dendritic cells with a composition comprising an effective amount of at least one cationic lipid and at least one antigen.
Including
A method in which the isolated dendritic cells are activated by treating the isolated dendritic cells. The at least one cationic lipid is selected from the group consisting of R-DOTAP, S-DOTAP, DOEPC, DDA, and DOTMA.
The method of claim 20, wherein the at least one antigen is a cancer antigen. 20. The method of claim 20, further comprising treating the dendritic cells with at least a growth factor or at least a cytokine or a combination thereof. The method according to any one of claims 20 to 22, wherein the isolated dendritic cells are human. The method according to any one of claims 20 to 23, wherein the at least one cytokine is GMCSF. A method of ex vivo activation of dendritic cells for use in the production of cancer vaccines.
The process of obtaining isolated dendritic cells and
The isolated dendritic cells
With an effective amount of at least one cationic lipid,
With at least one cancer antigen,
With at least growth factors or at least cytokines or combinations thereof,
And the process of treating with a composition containing
Including
A method in which the dendritic cells are activated by treating the dendritic cells. 25. The method of claim 25, wherein the dendritic cell is a human dendritic cell. 25. The method of claim 25 or 26, wherein the at least one cytokine is GMCSF. A method for producing a cancer dendritic cell vaccine,
The process of obtaining isolated dendritic cells and
The isolated dendritic cells
With an effective amount of at least one cationic lipid,
With at least one cancer antigen,
With at least growth factors or at least cytokines or combinations thereof,
And the process of treating with a composition containing
Including
A method in which the dendritic cells are activated by treating the dendritic cells. 28. The method of claim 28, further comprising treating the dendritic cells with growth factors and GM-CSF. A method for enhancing the therapeutic immune response in cancer patients
The process of obtaining isolated dendritic cells and
The isolated dendritic cells
With an effective amount of at least one cationic lipid,
With at least one cancer antigen,
With at least growth factors or at least cytokines or combinations thereof,
And the process of treating with a composition containing
Including
A method in which the dendritic cells are activated by treating the dendritic cells. A method for treating cancer patients
The process of obtaining isolated dendritic cells and
The isolated dendritic cells
With an effective amount of at least one cationic lipid,
With at least one cancer antigen,
With at least growth factors or at least cytokines or combinations thereof,
And the step of activating with a composition containing
The process of producing activated dendritic cells and
The step of administering an appropriate number of activated dendritic cells to the patient, and
Including
A method of treating said patient with said cancer by administering the appropriate number of activated dendritic cells. A step of periodically confirming the level of CD8 + T cells specific to the at least one antigen in the composition.
Following the administration of the dendritic cells to the patient, a step of confirming the level of Treg cells in the patient, and
Including
31. The method of claim 31, wherein high levels of antigen-specific CD8 + T cells and low levels of Treg cells indicate to the patient that the cancer is being effectively treated. The method according to any one of claims 28, 30, 31 or 32, wherein the cationic lipid is selected from the group consisting of R-DOTAP, S-DOTAP, DOEPC, DDA, and DOTMA. Use of cationic lipids in combination with tumor antigens to alter the tumor microenvironment. 34. The use according to claim 34, wherein altering the tumor microenvironment is to reduce the population of immunosuppressive regulatory T cells. 34. The method of claim 34, wherein the population of immunosuppressive regulatory T cells within the tumor is reduced and the population of tumor-specific CD8 + T cells and / or CD4 + T cells is increased. Three important functions for effective immunotherapy: i) presentation of the antigen to CD8 + T cells by MHC class I and CD4 + T cells by MHC class II, ii) proliferation of T cells and T cells. Cationic for inducing the cytokines and chemokines that promote activation of the tumor, iii) inducing T cells that infiltrate the tumor, and iv) reducing the immunosuppressive cell population in the tumor microenvironment. Use of lipid-based vaccines. 37. The use according to claim 37, wherein the immunosuppressive cell population is the Treg population. Use of cationic lipids in protein and peptide-based dendritic cell vaccines to enhance antigen-specific T cell responses in vivo. The use according to claims 34 to 39, wherein the cationic lipid is selected from the group consisting of R-DOTAP, S-DOTAP, DOEPC, DDA, and DOTMA. The use according to claims 34 to 39, wherein the cationic lipid is a combination with at least one cancer peptide antigen.

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