JP2019500021A - Lipids as synthetic vectors to enhance ex vivo antigen processing and presentation in dendritic cell therapy - Google Patents

Abstract

  The present invention encompasses the use of a class of lipids including cationic lipids in dendritic cell therapy ex vivo. Cationic lipids enhance the presentation of processed antigen to CD8 + and CD4 + T cells, respectively, by dendritic cells via antigen uptake, processing, and MHC class I and II presentation pathways, respectively. When antigens are taken up by dendritic cells via cationic lipids, the population of immunosuppressive regulatory T cells in the tumor is greatly reduced and the number of cytotoxic T cells targeting the tumor is greatly increased. The loss of regulatory T cells and the increase of tumor-specific cytotoxic cells will contribute to the effective removal of the tumor.

Description

Cross-reference to related applications

This international application is the priority of US Provisional Application No. 62 / 254,794 filed on November 13, 2015 and US Provisional Application No. 62 / 404,504 filed on October 5, 2016. Allegedly to the benefit of the right, the entire contents of which are incorporated herein by reference.

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

Studies of the antigen presentation properties of DC have recently shown promise in the development of effective cancer immunotherapy and have shown promising results in recent clinical trials. Treatment of metastatic prostate cancer with sipuleucel-T increased median survival for about 4 months in the Phase III trial. sipuleucel-T (Provenge®) is a dendritic cell-based cell obtained from concentrated blood that was collected from a patient and cultured with a fusion protein of prostatic acid phosphatase (PAP) and GM-CSF It is a product. Dendritic cells are then returned to the patient by intravenous infusion (intravenous injection). This process is repeated twice more every two weeks, so that the patient is given 3 doses of cells. Sipuleucel-T has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic prostate cancer, paving the way for clinical development and regulation of next generation cellular immunotherapy products. Among patients with metastatic melanoma who received a vaccine of activated DC loaded with tumor antigen peptides, there were patients who showed antigen-specific CD4 ⁺ T cell responses and CD8 ⁺ T cell responses. Despite this promise, 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 technique for treating various debilitating diseases including cancer. This approach enables the development of treatments that are customized and personalized for the patient. For example, rather than using a specific protein antigen for a particular cancer, the antigen can be obtained from the patient's tumor and loaded into the patient's own dendritic cells for treatment. The ex vivo approach using dendritic cell therapy has been shown to be a significant advance with an approved product that has demonstrated a sustained immune response in patients 2 years after treatment. However, treatment results are not optimal and several approaches to improve these treatments have been studied.

  One important issue is the selection of tumor antigens to load DC. Candidate antigens include specific (mutant) antigens and non-specific non-mutated autoantigens. Non-mutated autoantigens are most preferred, even if negative selection removes a population of high affinity clones to produce a widely applicable therapy. Using mutant antigens would avoid this drawback. The development of RNA sequencing technology has helped to determine the full range of primary and metastatic variant antigens, so that treatment can be tailored to specific patients. It has become.

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

  DCs can also cross-present antigens. Cross-presentation is the pathway by which exogenous soluble protein or peptide is taken up by antigen presenting cells and does not enter the pathway in which the protein is degraded and presented with MHC class II, but the peptide or protein is MHC class I The path that enters the processing path. This can occur in two ways: a cytoplasmic pathway or an endosomal pathway. In both pathways, the protein is first taken up by the endosome / phagosome. In the cytoplasmic pathway, a portion of the partially degraded endosomal protein eventually enters the cytoplasm. Although the mechanism is hardly understood, endosomal proteins are processed in the proteasome and the resulting peptides are transported by TAP and enter 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 proteasome-independent and is inefficient because it relies on the accidental production of the correct peptide by endosomal proteases. Protein entry into early endosomes with limited proteolytic activity favors cross-presentation, while late endosomes with high levels of proteolytic activity may inhibit cross-presentation.

  From the above discussion, it is clear that proteins that enter the endosomal pathway, particularly 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 that is taken up by early endosomes and the amount of antigen fragments that are subsequently delivered to the cytoplasm. Soluble proteins that bind to DC scavenger receptors can be cross-presented. A classic example is ovalbumin that binds to the mannose receptor. However, the approaches to target receptors already introduced in proteins / peptides vary 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 approaches have several disadvantages. It is necessary to bind the antigen to a specific receptor binding protein, usually a monoclonal antibody, which is a very tedious technique. The amount of protein uptake is limited to the amount of possible receptor uptake into the cell, and once incorporated, is released to the cytoplasm, albeit limitedly. Some DC receptors target late endosomes, which results in inefficient cross-presentation. Finally, the distribution of cross-presented DC receptors on human DC subsets is poorly understood, which makes it difficult to design such techniques and makes mouse studies more human. It can be an explanation of why the replacement is not successful. As another technique with a little lower specificity, there is a technique in which soluble antigen is attached to nanoparticles and converted into fine particle form. This approach suffers from the fact that it is difficult to carry enough antigen and that it loses phagocytic ability as the DC matures and is delivered to the lymph nodes.

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

  Therefore, this application focuses on the development of dendritic cell vaccines that are not based on DNA / RNA, and the development of vaccines that improve the uptake and processing of antigens by DC while limiting the toxicity to DC. Means to facilitate.

  Yet another significant obstacle to successful immunotherapy is the tumor's inhibitory microenvironment, which plays a key role in advancing various immunosuppressive mechanisms that interfere with anti-tumor cytolytic T cell responses. This effect has been called "immune evasion" or "immune tolerance". To avoid the inhibitory effects present in end-stage tumors, clinical trials were conducted in patients with minimal residual disease and high risk of recurrence and given adjuvant [Sears AK, Perez SA, Clifton GT, et al. , AE37: a novel T-cell-eliminating vaccine for breast cancer. Expert Opin Biol Ther. 2011; 11 (11): 1543-1550]. The rationale supporting vaccination in this clinical setting is to still have a sufficiently capable immune system that can provide a reliable anti-tumor response in patients with minimal tumor burden. Furthermore, vaccination in early cancer or adjuvant administration has the advantage of minimizing T cell accumulation in an immunosuppressive tumor environment where T cells can be inactivated.

  Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented only to provide a brief summary of the forms that the invention can take, and do not limit the scope of the invention. Indeed, the invention may encompass a wide variety of aspects not explicitly described below.

  In one embodiment described herein, a composition for ex vivo dendritic cell activation is provided. The composition includes one or more lipids having at least one cationic lipid and at least one antigen. The composition may include other lipids and growth factors to enhance dendritic cell viability and proliferation.

  A composition for DC activation ex vivo comprises one or more lipids having at least one cationic lipid and at least one antigen, wherein the antigen is a protein or polypeptide found in a tumor or cancer is there. Certain cationic lipids are unique in their ability to rapidly bind to dendritic cells in a receptor-independent manner and are rapidly taken up by early endosomes. Importantly, once in the early endosome, the cationic lipids help destabilize the endosome to enter the class I processing pathway and release the contents into the cytoplasm. This allows release of much more endosomal content into the cytoplasm than would occur with target receptor uptake. 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 cells to migrate to lymph nodes. You can also. Also, suitable cationic lipids can reduce the population of Treg cells within the tumor microenvironment, preferably when combined with a tumor antigen.

  In another embodiment, there is provided a method of treating a subject's dendritic cells ex vivo, wherein the subject is a mammal. The method includes 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 includes growth factors such as GM-CSF and cytokines to facilitate 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 includes 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 includes growth factors such as GM-CSF and cytokines to facilitate cell maintenance and proliferation in vitro.

  Provided is a method of producing a cancer DC vaccine that can induce high levels of tumor-infiltrating T cells while inducing a significant decrease in the Treg population within the tumor microenvironment. The method includes 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 includes further activating DCs with growth factors such as GM-CSF and cytokines to facilitate cell maintenance and proliferation in vitro. When cationic lipids are used in combination with tumor antigens to activate DCs in vitro and then injected into a subject with cancer, DCs increase the amount of tumor-specific CD8 + T within the tumor microenvironment This can change the tumor microenvironment and greatly reduce the Treg population. As a result, the ratio of Treg to CD8 + T cells is significantly reduced.

  Immunizing a subject with a DC vaccine, wherein the DC is pre-treated 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 method is provided. The method includes immunizing the subject more than once. The method includes ascertaining the level of cancer-specific CD8 + T cells and the level of Treg cells in the post-immunization subject.

  In yet another embodiment, a method for enhancing a prophylactic or therapeutic immune response in a mammal is provided. The method comprises activating dendritic cells with one or more lipids comprising an effective amount of at least one cationic lipid and an antigen, optionally with growth factors such as GM-CSF and cytokines, and activated Administering dendritic cells to the mammal. In various embodiments, the mammal is a human.

  One embodiment of the invention relates to the use of cationic lipids to facilitate uptake and presentation of tumor-derived antigens and 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 that are intended to maintain and promote the proliferation and proliferation of dendritic cells. Can do.

  Cationic liposomes are widely used in vivo in RNA and DNA transfection and delivery for use in gene therapy and in DNA-based dendritic cell vaccines. Recently, cationic lipids have also been reported to be potent adjuvants that can stimulate 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 taken up by early endosomes. Importantly, once in the early endosome, the cationic lipid appears to help destabilize the endosome to enter the class I processing pathway and release the contents into the cytoplasm. This allows more of the endosome content to be released into the cytoplasm than occurs upon target receptor uptake. This allows release of much more endosomal content into the cytoplasm than would occur with target receptor uptake.

  In one embodiment of the present invention, the present disclosure provides that MHC class I and MHC class II proteins are several orders of magnitude more than current approaches where cationic lipids use adjuvants to induce dendritic cell maturation. Indicates that you can help enter the path.

  In one embodiment, the present invention provides a composition comprising a cationic lipid and a specific tumor antigen necessary to prepare a DC vaccine against a tumor ex vivo.

  In one embodiment, the composition necessary 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. The antigen is a protein product of a patient gene, and the gene is an oncogene, a tumor suppressor gene, a gene having a mutation, a gene having a rearrangement specific to tumor cells, a reactivated embryo gene product, an oncofetal antigen, Selected from the group consisting of a tissue-specific differentiation gene, a growth factor receptor, and a cell surface protein gene.

  In one embodiment, the antigen is a microbial antigen, viral antigen, bacterial antigen, fungal antigen or natural isolate of microbial antigens, fragments and derivatives thereof.

  In one embodiment, the antigen is a viral antigen.

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

  In one embodiment, a composition for stimulating or activating DC to produce 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 linked by chemical bonds.

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

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

  In one embodiment, the cationic lipid encapsulates the antigen.

  In one embodiment, the cationic lipid forms a liposome.

  In one embodiment, the cationic lipid and antigen form an emulsion.

  In one embodiment, the cationic lipid facilitates protein or peptide antigen uptake.

  In one embodiment, the peptide or protein antigen of the composition to further incorporate and stimulate DC in the course of DC vaccine development is further modified to reduce the hydrophobicity of the antigen. The hydrophobicity of an antigen can be increased while maintaining the antigenic properties of the molecule, for example by conjugating to a lipid chain or a hydrophobic amino acid to improve the solubility of the antigen in the hydrophobic acyl chain of the cationic lipid. it can.

  The modified antigen may be a modified lipoprotein, lipopeptide, protein or peptide, or combinations thereof with increased amino acid sequence hydrophobicity.

  The modified antigen can attach a linker between the lipid and the antigen, for example, N-terminal α or ε-palmitoyl lysine can be attached to the antigen 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 antigen internalization by dendritic cells but also their processing, entry into the cytoplasm, 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 is 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 manufacture 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 manufacture of a DC cancer vaccine.

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

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

  Thus, the present disclosure provides isolated dendritic cells, wherein (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. Or a combination thereof, to produce an activated dendritic cell by activating the isolated dendritic cell with a composition comprising, and subjecting an appropriate number of activated dendritic cells to the patient A method for treating a cancer patient is provided, comprising administering an appropriate number of activated dendritic cells.

  The present disclosure is also a method for assessing the success of treating a cancer patient by the above method comprising periodically checking the level of CD8 + T cells specific for at least one antigen in the composition comprising: The evaluating 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, A method is provided wherein high levels of specific CD8 + T cells and low levels of Treg cells indicate that the patient is being effectively treated for cancer.

Geometric mean fluorescence intensity emitted by cells taking up fluorescent ovalbumin. Statistical significance was assessed using two-way ANOVA and * values were significantly different between treatments. Average fluorescence intensity of green fluorescence of gated CD11c positive cells, representing the amount of DQ-OVA taken up and processed by DC. Mean fluorescence intensity showing DQ-OVA uptake by DC and TC1 cells in the presence of DOTAP at the indicated concentrations. Flow cytometric analysis of green vs. red fluorescence showing DQ-OVA uptake and processing by mouse DCs. Green vs. red fluorescence showing DQ-OVA uptake and processing by mouse DC in the presence of DOTAP or MPL. Laser scanning confocal microscopy and flow cytometry showing DQ-OVA uptake and processing. Beta galactosidase assay showing relative absorbance (570 nm) measurements (arbitrary units). Statistical significance was assessed using two-way ANOVA and * values were significantly different between treatments. Average CPM of ³ H-thymidine uptake by OT1 cells in the presence of OVA-stimulated BDMC with or without DOTAP. Mean CPM of ³ H-thymidine incorporation by DO11.10 splenocytes and OVAp323-339. Flow cytometric analysis of CFSE dilution profiles of T cells from draining lymph nodes of mice injected with OVA alone or OVA plus DOTAP. OT1 T cell proliferation in the presence of OVA and BMDC “pulsed” with three cationic lipids. Average CPM of ³ H-thymidine incorporation during the last 18 hours of culture. IFN-ELISPOT assay of T cells from mice vaccinated with BMDC pulsed with palmitoylated-KSSSIINFEKL peptide or isotonic sucrose (280 mM) mixed with cationic lipids (RDOTAP, DOTMA) or neutral lipids (DOPC). Vaccinated with tumor-associated peptide (a), HPV-associated antigen and (b) mucin 1 or isotonic sucrose (280 mM) pulsed with cationic lipids (RDOTAP, DOTMA) or neutral lipids (DOPC) IFN-ELISPOT assay of T cells from mice. A: Effect of vaccination with HPV16-E7, R-DOTAP / HPV16-E7, S-DOTAP / HPV16-E7 and Alum / MPL / HPV16-E7 on induction of HPV16-specific CD8 + T cells by ELISpot. B. Effect of R-DOTAP, R-DOTAP / HPV16-E7, S-DOTAP / HPV16-E7 vaccination on regression of established HPV16 positive TC-1 tumors. Quantification of tumor-infiltrating HPV16-specific CD8 + T by RF9-specific dextramers, analyzed using flow cytometry. Data represent the mean + SEM of 4-5 mice in each group. Quantification of regulatory T cells in tumors after treatment of tumor-bearing mice with various vaccines. 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. Ratio of T regulatory cells (Treg) to HPV16 E7 specific CD8 + T cells in CD45 + cells. Data represent the mean + SEM of 4-5 mice in each group. Effect of vaccination on the regression of established 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. 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. Dunnet's multiple comparison test. Quantification of T lymphocytes and total lymphocytes in draining lymph nodes after R-DOTAP cationic lipid-based vaccination. Quantification of MCP-1 and IP-10 levels in response to vaccination. The figure shows the levels of MCP-1 and IP-10 at 12, 24 and 48 hours after vaccination.

  Cationic lipids are rarely used in pharmaceuticals that are supposed as gene transfection agents, since toxicity to cells has been reported, particularly in vitro. On the other hand, cationic lipids have been successfully used as transfection agents for complexing and delivering DNA and RNA to cells including dendritic cells. This approach 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. It can facilitate not only the internalization of antigens 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.

  This effect is also independent of the recently reported effect of cationic lipids as immune adjuvants. Because both strong (R-DOTAP, DOTMA) and weak (S-DOTAP, DDA) cationic lipid adjuvants, as well as neutral lipids that have been shown not to act as immune adjuvants in vivo This is because the same effect was brought about. In addition, neither S-DOTAP nor DDA induced effective antigen-specific immune responses in vivo in previously reported studies, but now promotes antigen processing and presentation of antigens ex vivo. It has been shown. This effect in facilitating antigen uptake, processing and presentation may be facilitated by bringing dendritic cells and cationic lipids in close proximity when setting in vitro / ex vivo conditions, but in vivo Then it does not necessarily happen. This important discovery has led to new uses for cationic lipids in the development of more effective ex vivo dendritic cell therapy. To date, this ability of cationic lipids has not been known, so it has not been reported that ex vivo dendritic cell therapy uses cationic lipids to promote protein and peptide uptake and presentation.

  Hereinafter, various embodiments of the present invention will be described. In one embodiment described herein, an ex vivo dendritic cell therapeutic composition is provided. The composition includes one or more lipids having at least one cationic lipid and at least one antigen. The composition may include other lipids and growth factors to enhance dendritic cell viability and proliferation.

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

  Methods are provided for enhancing a prophylactic or therapeutic immune response in a mammal. The method comprises treating dendritic cells with one or more lipids comprising an effective amount of at least one cationic lipid and antigen, optionally together with growth factors such as GM-CSF and cytokines; Administering dendritic cells to the subject. In various embodiments, the composition comprises one or more lipids having at least one cationic lipid and at least one antigen.

  This discovery has led to the development of dendritic cell vaccines based on autologous tumor-derived antigens that can present antigens in very limited quantities, and peptide systems to enable administration of very low doses of antigens for proteins. In dendritic cell vaccines, it can provide significant advantages. While the dendritic cell vaccine approach has shown significant promise, its lack of a robust T cell response has limited 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 antigens or proteins and peptide-based antigens derived from autologous tumors. To do.

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

The present invention shows that in the context of MHC class I and class II, cationic lipids can be used as vaccine agents that safely promote antigen presentation to dendritic cells and presentation to CD4 + and CD8 + T cells. Cationic lipids are effective in promoting the induction of T cells that infiltrate high levels of tumors while also inducing a significant decrease in the Treg population within the tumor microenvironment. These effects drastically change the tumor microenvironment by reducing the Treg to CD8 + T cell ratio that leads to very effective killing of tumor cells. Therapeutic Cancer Vaccines J Clin Invest. In a recent review 2015; 125 (9): 3401-3412, Mellief et al. “The suboptimal vaccine design and immunosuppressive cancer microenvironment are the root causes that do not lead to the eradication of cancer. Drugs and physical treatment can relieve the immunosuppressive cancer microenvironment. And include chemotherapy, radiation, indoleamine 2,3-dioxygenase (IDO) inhibitors, inhibitors of T cell checkpoints, agonists of selected TNF receptor family members, inhibitors of unwanted cytokines. The specificity of therapeutic vaccination combined with such immunomodulation provides an attractive means for the development of future cancer therapies.
antigen

  In one embodiment, the cationic lipid is administered with a self-antigen, such as an antigen 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 objective is to elicit an immune response specific for the antigen, and dendritic cells are treated with cationic lipids along with the antigen. The in vivo response that occurs upon infusion of ex vivo treated dendritic cells may involve the production of specific cytotoxic T cells, memory T cells, or B cells, and specific antigens associated with these antigens. Leading to disease prevention or therapeutic response to the disease. The antigen can be any tumor-associated antigen or microbial antigen, and any other antigen known to those skilled in the art.

  As used herein, a “tumor associated antigen” is associated with a tumor or cancer cell and produces an immune response (humoral and / or cellular) when expressed on the surface of antigen presenting cells in the context of MHC molecules. A molecule or compound that can be triggered (eg, protein, peptide, polypeptide, lipoprotein, lipopeptide, glycoprotein, glycopeptide, lipid, glycolipid, carbohydrate, RNA and / or DNA). Tumor-associated 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 an animal. included. More specific embodiments are provided herein.

  As used herein, a “microbial antigen” is an antigen of a microorganism, including but not limited to infectious viruses, infectious bacteria, infectious parasites and infectious fungi. The microbial antigen is an intact microorganism, a natural isolate, a fragment or a derivative thereof, a synthetic compound that is the same or similar to a naturally occurring microbial antigen, preferably (the origin from which the naturally occurring microbial antigen was obtained) Induces an immune response specific to the corresponding microorganism. In a preferred embodiment, a compound is equivalent to a naturally occurring microbial antigen provided that it induces an immune response (humoral and / or cellular) similar to a naturally occurring microbial antigen. A compound or antigen that is equivalent to a naturally occurring microbial antigen is, for example, a protein, peptide, polypeptide, lipoprotein, lipopeptide, glycoprotein, glycopeptide, lipid, glycolipid, carbohydrate, RNA, and / or DNA. Etc. are well known to those skilled in the art. Another non-limiting example of a compound that is similar to a naturally occurring microbial antigen is a peptidomimetic of a polysaccharide antigen.

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

  Also useful in the compositions and methods of the present invention are peptides or proteins having an amino acid sequence 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 an antigen associated with a tumor or cancer, ie, a tumor-associated antigen, for producing a vaccine for preventing or treating the tumor. Thus, in one embodiment, the tumor vaccine or cancer vaccine of the present invention further comprises at least one epitope of at least one tumor associated antigen. In another preferred embodiment, the tumor vaccine or cancer vaccine of the present invention further comprises a plurality of epitopes derived from one or more tumor associated antigens. Tumor-associated antigens used in the cationic lipid complexes and methods of the present invention can be essentially immunogenic or non-immunogenic, or slightly immunogenic. As demonstrated herein, even tumor-associated autoantigens can be advantageously used in the vaccine to obtain a therapeutic effect. This is because the composition can destroy immune tolerance to such antigens. Exemplary antigens include, but are not limited to, synthetic antigens, recombinant antigens, foreign antigens or homologous antigens, and antigen materials include proteins, peptides, polypeptides, Examples include, but are not limited to proteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA, and DNA. Examples of such treatments include the treatment or prevention of breast cancer, head and neck cancer, melanoma, cervical cancer, lung cancer, prostate cancer, intestinal cancer, or other cancers known in the prior art that are sensitive to immunotherapy. However, it is not limited to these. In such ex vivo treatment methods, it is also possible to combine the antigen with a cationic lipid without encapsulation.

  Tumor-associated antigens suitable for use in the present invention can be indicative of a single tumor type, are shared by several types of tumors, and / or are expressed exclusively in tumor cells or normal Both naturally occurring and modified molecules that are overexpressed compared to normal cells. In addition to proteins, glycoproteins, lipoproteins, peptides, lipopeptides, tumor-specific patterns of carbohydrate, ganglioside, glycolipid, mucin expression are also shown. Exemplary tumor-associated antigens used in cancer vaccines include oncogenes, tumor suppressor genes, protein products of other genes with mutations or rearrangements unique to tumor cells, reactivated embryo gene products, carcinoembryonicity Antigens, tissue specific (but not tumor specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, foreign viral proteins, and many other self-proteins.

  Specific embodiments of tumor-associated antigens include, for example, mutant or modified antigens such as Ras p21 proto-oncogene, tumor suppressor p53, HER-2 / neu and BCR-abl oncogene protein products, and CDK4, MUM1 , Caspase 8, beta catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, aldolase A, PRAME, Her2 / neu, ErbB-2 and KSA; alpha fetoprotein (AFP), human chorionic gonadotropin (hCG) Carcinoembryonic antigens such as: carcinoembryonic antigen (CEA) and self antigens and melanocyte differentiation antigens such as Mart1 / MelanA, gp100, gp75, tyrosinase, TRP1 and TRP2; PSA, PAP, PSMA, PSM-P1 and P Reactivated embryonic gene products such as prostate-related antigens such as M-P2; MAGE1, MAGE3, MAGE4, GAGE1, GAGE2, BAGE, RAGE, and other cancer testis antigens such as NY-ESO1, SSX2 and SCP1; Muc-1 And mucins such as Muc-2; gangliosides such as GM2, GD2, GD3, neutral glycolipids and glycoproteins such as Lewis (y) and globo-H; glycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn Can be given. In addition, the tumor-related antigens used herein include whole cell and tumor cell lysates and immunogenic portions thereof, as well as immunoglobulin idiotypes expressed by monoclonal proliferation of B lymphocytes used against B cell lymphomas. Can be given.

  Examples of tumor-associated antigens and their respective tumor cell targets include cytokeratins, particularly cytokeratins 8, 18, and 19, as antigens for malignant cancers. Epithelial membrane antigen (EMA), human embryonic antigen (HEA-125), human milk fat globule, MBr1, MBr8, Ber-EP4, 17-1A, C26, T16 are also known carcinoma antigens. Desmin and muscle-specific actin are myogenic sarcoma antigens. Placental alkaline phosphatase, β-human chorionic gonadotropin, α-fetoprotein are antigens of trophoblast and germ cell tumors. The prostate specific antigen is an antigen of prostate cancer, an oncofetal antigen of colon adenocarcinoma. HMB-45 is a melanoma antigen. In cervical cancer, useful antigens may be encoded by human papillomavirus. Chromagranin-A and synaptophysin are antigens of neuroendocrine and neuroectodermal tumors. Of particular interest are aggressive tumors that form solid tumor masses with necrotic areas. Such lysis of necrotic cells is an abundant source of antigen for antigen-presenting cells, and therefore the present therapy finds advantageous applications in combination with conventional chemotherapy and / or radiation therapy. Can do.

  Tumor associated antigens can be prepared by methods well known in the art. For example, these antigens can be used to prepare a crude extract of cancer cells (eg, as described in Cohen et al., Cancer Res., 54: 1055 (1994)), partially purify 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 that encodes an antigenic peptide in a form suitable for expression in the subject and presentation to the immune system of the subject to be immunized. Furthermore, the antigen may be a complete antigen or a fragment of a complete antigen containing at least one epitope.

  Antigens from pathogens known to predispose to certain types of cancer can also be advantageously included in the cancer vaccines of the present invention. It can be estimated that nearly 16% of cancer incidence worldwide is due to infectious agents, 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 to expand the immune response of the host and increase the preventive or therapeutic effect of the cancer vaccine. Particularly interesting pathogens used in the cancer vaccines provided herein include hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (hepatoma), Epstein-Barr virus (EBV) (Burkitt lymphoma, nasopharyngeal carcinoma) , PTLD in immunosuppressed individuals), HTLVL (adult T cell leukemia), oncogenic human papillomavirus type 16, 18, 33, 45 (adult cervical cancer), Helicobacter pylori (B cell gastric lymphoma). Other medically relevant microorganisms that can act as antigens in mammals, particularly humans, are C.I. G. A Thomas, Medical Microbiology, Bailier Tindall, Great Britain 1983, etc., the entire contents of which are incorporated herein by reference.

  In another embodiment, the antigen comprises an antigen derived from or associated with a pathogen, ie, a microbial antigen. Thus, in one embodiment, the pathogen vaccine of the present 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 present invention further comprises a plurality of epitopes derived from one or more microbial antigens.

  Microbial antigens useful in the cationic lipid complexes and methods may have intrinsic immunogenicity, or may be non-immunogenic or slightly immunogenic. Exemplary antigens include, but are not limited to, synthetic antigens, recombinant antigens, foreign antigens or homologous antigens, and antigen materials include proteins, peptides, polypeptides, 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, particularly humans. Examples of viruses include retroviridae (eg, human immunodeficiency viruses such as HIV-1 (also called HTLV-III, LAV or HTLV-III / LAV or HIV-III; and other isolates such as HIV-LP) Picornaviridae (eg poliovirus, hepatitis A virus; enterovirus, human coxsackie virus, rhinovirus, echovirus etc.), caliciviridae (eg strain causing gastroenteritis), togaviridae (eg , Equine encephalitis virus, rubella virus, etc.), flaviviridae (eg, dengue virus, encephalitis virus, yellow fever virus), coronaviridae (eg, coronavirus), rhabdoviridae (eg, vesicular stomatitis virus) Rabies virus), filoviridae (eg Ebola virus), paramyxoviridae (eg parainfluenza virus, mumps, measles virus, respiratory syncytial virus), orthomyxoviridae (eg For example, influenza virus, etc., Bungaviridae (for example, Hantan virus, Bungavirus, Pravovirus, Nairovirus), Arenaviridae (haemorrhagic fever virus), Reoviridae (for example, reovirus, arbovirus, rota) Virus, etc.), birnaviridae, hepadnaviridae (hepatitis B virus), parvoviridae (parvovirus), papovaviridae (papillomavirus, polyomavirus), adenoviridae (most adenoviruses), f Pesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus), Poxiridae (decubitus virus, vaccinia virus, poxvirus), Iridoviridae (E.g., African swine fever virus), unclassified viruses (e.g., causative factor of spongiform encephalopathy, hepatitis delta factor (considered to be a hepatitis B virus deficient satellite), non-A non-B type Factors of hepatitis (class 1 = intrinsically transmitted, class 2 = parenterally transmitted (ie, hepatitis C), Norwalk virus and related viruses, astrovirus) include, but are not limited to Absent.

  Also, in vertebrates, Gram negative and Gram positive bacteria can be targeted by the present compositions and methods. Examples of such Gram-positive bacteria include, but are not limited to, Pasteurella species, Staphylococcus species, and Streptococcus species. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. 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 (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), treptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sps. Enterococcus sp. Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp. , Erysiperothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multerocid. , Fusobacterium nucleatumii, Streptobacillus moniliformis, Treponema pallidium, Treponema parttenue, Leptospira, Rickettsia, Actinomyce.

  Bacterial pathogen polypeptides that may be useful as a source of microbial antigens in the present compositions include iron-regulated outer membrane protein (“IRROM”), outer membrane protein (“OMP”), and Aeromonis salmonida A Protein, p57 protein of Renibacterium salmoninarum causing bacterial kidney disease (“BKD”), major surface associated antigen (“msa”), surface expressed cytotoxin (“mpr”), surface expressed hemolysin (“ish”), Yersinosis Flagellar antigen, extracellular protein ("ECP"), iron-regulated outer membrane protein ("IRROM"), structural protein of Pasteurellosis, OMP and Vibrosis angulararum and V. ordali flagellar protein, Edwardsiellosis icitaluri and E. coli. These include, but are not limited to, tarda flagellar proteins, OMP proteins, aroA, purA, Ichthyophthirius surface antigens, Cytophaga columnari structural and regulatory proteins, and Rickettsia structural and regulatory proteins. Such antigens can be isolated or prepared by recombinant or other means known in the art.

  Examples of pathogens include, but are not limited to, fungi that infect mammals, particularly humans. Examples of fungi include but are not limited to Cryptococcus neoformansi, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitis, Chlamydia trachomatis, Candida albicans. Examples of infectious parasites include Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax and other malaria parasites. Another infectious organism (ie, protist) is Toxoplasma gondii. Parasite pathogen polypeptides include, but are not limited to, Ichthyophthirius surface antigens.

  Other medically relevant microorganisms that act as antigens in mammals, particularly humans, are widely described in the literature. For example, C.I. G. See A Thomas, Medical Microbiology, Bailier Tindall, Great Britain 1983, the entire contents of which are incorporated herein by reference. In addition to treating infectious human diseases and human pathogens, the compositions and methods of the present invention are also useful for treating infections in non-human mammals. Many vaccines for treating non-human mammals are described in Bennett, K. et al. Compendium of Veterinary Products, 3rd ed. North American Compensium, Inc. , 1995, and also refer to WO 02/069369 (the disclosure of which is expressly incorporated herein).

  Exemplary non-human pathogens include mouse mammary tumor virus (“MMTV”), Rous sarcoma virus (“RSV”), avian leukemia virus (“ALV”), avian myeloblastosis virus (“AMV”), mouse Leukemia virus (“MLV”), feline leukemia virus (“FeLV”), mouse sarcoma virus (“MSV”), gibbon leukemia virus (“GALV”), spleen necrosis virus (“SNV”), reticuloendotheliosis virus ( “RSV”), simian sarcoma virus (“SSV”), Mason Pfizer monkey virus (“MPMV”), simian retrovirus type 1 (“SRV-1”), and HIV-1, HIV-2, SIV, Lentiviruses such as Visna virus, feline immunodeficiency virus (“FIV”), equine infectious anemia virus (“EIAV”) T cell leukemia viruses such as HTLV-1, HTLV-II, simian T cell leukemia virus (“STLV”), bovine leukemia virus (“BLV”), human foam virus (“HFV”), simian foam virus (“SFV”) ), And foam viruses such as bovine foam virus (“BFV”), but are not limited thereto.

  In some embodiments, as used herein with respect to infectious pathogens, “treatment”, “treating”, “treating” increases the resistance of a subject to infection by a pathogen, or the subject Prophylactic treatment that reduces the likelihood of becoming infected and / or treatment to fight infection after the subject is infected, such as 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 antigen, by recombinant techniques, or by de novo synthesis of known antigens. The antigen may also be in the form of a nucleic acid that encodes an antigenic peptide in a form suitable for expression in the subject and presentation to the immune system of the subject to be immunized. Furthermore, the antigen may be a complete antigen or a fragment of a complete antigen containing at least one epitope.
Lipid

  In order to improve antigen uptake into cationic lipid vesicles and improve delivery to cells of the immune system, the antigen may be modified to increase its hydrophobicity or negative charge on the antigen surface. For example, in order to improve the solubility of the antigen in the hydrophobic acyl chain of the cationic lipid while maintaining the antigenic properties of the molecule, the hydrophobicity of the antigen may be increased by binding to a lipid chain or a hydrophobic amino acid. The modified antigen can be a modified lipoprotein, lipopeptide, protein or peptide, or combinations thereof with increased amino acid sequence hydrophobicity. The modified antigen may have a linker attached between the lipid and the antigen, eg, N-terminal α or ε-palmitoyl lysine bound to the antigen via a serine-serine dipeptide linker. May be. As discussed in more detail below, the DOTAP / E7-lipopeptide complex had an enhanced antigen-specific CD8 T lymphocyte response in vivo compared to the DOTAP / E7 preparation. In addition, the negative charge can 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. Antigens 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 to the antigen an amino acid sequence that resulted in an antigenic amino acid sequence that was not found in the parent protein from which the antigen was originally obtained. Studies were conducted to show that the modified antigen has better MHC class I binding affinity than the native antigen. The excellent binding affinity as demonstrated in this study resulted in a superior anti-tumor immune response in vivo against HPV positive TC-1 tumors. The invention will be further understood in 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 “nanoparticle” refers to a particle whose measured size is in the nanometer range. For example, “nanoparticles” can refer to particles having a structure that is less than about 10,000 nanometers in size. In some embodiments, the nanoparticle is a liposome.

As used herein, the term “cationic lipid” refers to a number that retains a net positive charge at physiological pH or has a protonatable group and is positively charged at a pH lower than pKa. All of the lipid species. Suitable cationic lipids according to the present ^{disclosure, 3-β [4 N- (} 1 N, 8 N - di guanidino spermidine) - carbamoyl] cholesterol (BGSC), 3-β [ N, N- di guanidinoethyl - aminoethane ) -Carbamoylcholesterol (BGTC), N, N ¹ , N ² , N ³ tetramethyltetramilspermine (cellfectin), Nt-butyl-N′-tetradecyl-3-tetradecylaminopropionamidine (CLONfectin), dimethyl Dioctadecyl ammonium bromide (DDAB), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N- [2 (spermine-carboxamido) ethyl]- N, N-dimethyl- -Propanaminium trifluorocetate) (DOSPA), 1,3-dioleyloxy-2- (6-carboxyspermyl) -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-dioleoyloxy-1,4- Butane-diammonium iodide) (Tfx-50), N-1- (2,3-dioleoyloxy) propyl-Ν, Ν, Ν-trimethylammonium chloride (DOTMA) or other N- (N, N -1-dialkoxy) -alkyl-N, N, N-trisubstituted ammonium surfactants, 1,2 dioleoyl-3- (4′-trimethylammonio) butanol-sn -Glycerol (DOBT) or cholesteryl (4'trimethylammonia) butanoate (ChOTB), where the trimethyl-ammonium group is double-stranded (in the case of DOTB) or cholesteryl group (in the case of ChOTB) via a butanol spacer arm Connected), DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxy) Ethyl ammonium) (DORIE) or analogs thereof (such as those disclosed in WO 93/03709), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl hemisuccinate ester (ChOSC), dioctadecylamidoglycylspermine (DOGS), and lipopolyamines such as dipalmitoylphosphatidylethanolamylspermine (DPPES), cholesteryl-3β-carboxyl-amide-ethylenetrimethylammonium iodide, 1-dimethylamino-3- Trimethylammonio-DL-2-propyl-cholesteryl carboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamide-ethylenetrimethylammonium iodide, 1-dimethylamino-3 -Trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide, 2- (2-trimethylammonio) -ethylmethylaminoethyl-core Steryl-3-β-oxysuccinate iodide, 3-β-N- (N ′, N′-dimethylaminoethane) carbamoylcholesterol (DC-chol), 3-β-N- (polyethyleneimine) -carbamoyl Cholesterol, Ο, Ο'-Dimyristyl-N-lysyl aspartate (DMKE), Ο, Ο'-Dimyristyl-N-lysyl-glutamate (DMKD), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium Bromide (DMRIE), 1,2-jirouroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2- Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dipalmi Yl-sn-glycero-3-ethylphosphocholine (DPEPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), Dioleoyldimethylaminopropane (DODAP), 1,2-palmitoyl-3-trimethylammoniumpropane (DPTAP), 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), 1,2-myristoyl-3-trimethyl Examples thereof include, but are not limited to, ammonium propane (DMTAP) and sodium dodecyl sulfate (SDS). Furthermore, any structural variants and derivatives of the described cationic lipids are contemplated.

  In certain embodiments, the cationic lipid is selected from the group consisting of DOTAP, DOTMA, DOEPC, and combinations thereof. In other embodiments, the cationic lipid is DOTAP. In still other embodiments, the cationic lipid is DOTMA. In other embodiments, 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 stereoisomer of a cationic lipid that is a non-superimposable mirror image of each other, such as its paired stereoisomers, eg, the R and S enantiomers. 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.

  It should be noted that for purposes of illustration, all examples are performed using ovalbumin, a model protein that has been thoroughly studied and is available with dual fluorescent labels. Using model proteins provides an excellent example 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 restricted 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 ovalbumin, a model protein, as a representative antigen. Fluorescent OVA conjugate (DQ-OVA conjugate, Alexa Fluor® 647 OVA) that can be easily followed using a flow cytometer to assess the effect of cationic lipids on antigen uptake and processing by antigen presenting cells Conjugate) was used. Furthermore, by using ovalbumin protein as an antigen, ovalbumin-specific T cell hybridoma cells and OCR albumin-specific T cell hybridoma cells (OT-1 and DO11.10) having CD4 and CD8 T cell receptors are used. Confirmation of antigen presentation by MHC I and MHC II became easier. The results shown in this study are generally applicable to any protein and peptide antigen.
<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 (BMDC) were pulsed with Alexa Fluor® 647-OVA conjugate and ovo-induced by BMDC using flow cytometry. Albumin uptake was quantified. Briefly, serum-free RPMI 1640 containing 20 μg / ml ovalbumin (Ovalbumin AlexaFluor® 647 conjugate; Life Technologies, catalog number 034884) and 50 μM cationic lipid (RDOTAP) or 280 mM sucrose diluent. In cell culture medium, 2 × 10 ⁶ cells / ml BMDC were incubated at 37 ° C. for 10-60 minutes. Cells were washed after the pulse to remove unbound ovalbumin and fixed with 1% formaldehyde for analysis on a flow cytometer. Ovalbumin incorporation was quantified using a BD LSR II flow cytometer. As shown in FIG. 1, cationic lipids significantly increased protein uptake by BMDC at all time points measured. In addition, since protein uptake occurred very rapidly in the presence of cationic nanoparticles, cationic lipids would be beneficial in significantly reducing the time of antigen pulses when preparing dendritic cell vaccines. It suggests.
<Example 2: Effect of cationic lipid on antigen processing by dendritic cells and epithelial cells>

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

  The results in FIG. 2 show that BMDC incubated with fluorescent DQ-OVA in medium alone increased fluorescence at 37 ° C. and was taken up and processed. This represents uptake by DCs mediated by the well-known mannose receptor of OVA. DOTAP enhances antigen uptake and processing by DCs. Graph display of fluorescence uptake of DQ-OVA into BMDC measured by flow cytometry. The plot shows the mean fluorescence intensity of the green fluorescence of gated CD11c positive cells representing 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 reconstitution of the active cytoskeleton. In BMDC incubated with DQ-OVA in the presence of pure R-enantiomer of DOTAP (R-DOTAP), a doubling of fluorescence is observed, and the cationic lipid R-DOTAP is associated with protein uptake in DC. It has been shown to greatly enhance processing. Significant uptake was still observed at 4 ° C., indicating that R-DOTAP can facilitate protein uptake without active cellular metabolism. The effect of R-DOTAP was concentration-dependent, and the maximum effect was observed at 50 μM. To determine whether the enhancement of protein uptake by R-DOTAP is cell dependent, a mouse epithelial cell line was incubated with DQ-OVA under the same conditions as BMDC. The results in FIG. 3 indicate that this OVA uptake and processing is observed only in DC, but not in TC1 epithelial cells. These data show that DOTAP can greatly enhance uptake and processing of complete proteins into dendritic cells ex vivo. It can also 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 endosomal entry

To determine whether lipid adjuvants can mediate the same effect as R-DOTAP, BMDC were incubated with DQ-OVA using medium alone or R-DOTAP as described in FIG. . BMDCs were also incubated under the same conditions using lipopolysaccharide (LPS), which is a potent lipid adjuvant. Mouse bone marrow DCs were incubated for 1 hour in the presence of fluorescent DQ-OVA either in the presence of 25 μM DOTAP nanoparticles, 10 μg / ml LPS or media alone, either at 37 ° C. or 4 ° C./azide, and flow cytometry Analyzed with As shown in FIG. 1, DQ-OVA was actively taken up and processed by DCs in the absence of R-DOTAP, but it is evident from the strong increase in red fluorescence in the presence of R-DOTAP. As such, uptake was greatly enhanced. In contrast, as shown in FIG. 4, such an increase was not observed upon treatment with LPS. Monophosphoryl lipid A (MPL) is a derivative of LPS with low toxicity and is currently an FDA approved adjuvant in several vaccines. Similar to the results with LPS in FIG. 5, MPL did not show the ability to enhance protein uptake in BMDC.
<Example 4: Effect of cationic lipid on antigen processing in human monocyte cell line>

To determine the effect of cationic lipids on human cells, the uptake of DQ-OVA ex vivo was evaluated using the human monocyte cell line THP-1. THP-1 is representative of human blood-derived monocytic cells and is the same cell used to make DCs from patients with ex vivo DC therapy techniques. THP-1 cells with DQ-OVA (10 μg / ml) for 1 hour at 37 ° C. in the presence (A, B) or absence (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 versus red fluorescence by flow cytometry. The results in FIG. 6 show that R-DOTAP dramatically increased DQ-OVA uptake in THP-1 cells. Unlike mouse BMDC, DQ-OVA uptake was not 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 receptors necessary for protein uptake. This result is important. This is because R-DOTAP has been shown to enhance uptake and processing in cells that would otherwise not be uptaken by the receptor. Another remarkable observation from FIGS. 6A and B is the accumulation of processed OVA in endocytic vesicles. Endocytotic vesicle proteins are either efficiently taken up by MHC class II molecules to stimulate CD4 T cells or cross the cross-presentation pathway for presentation from MHC class I to CD8 T cells. Well known. Thus, cationic lipids facilitate protein uptake in a manner that maximizes the presentation of antigen to both MHC class I and class II molecules and maximizes stimulation of each of CD8 and CD4 T cells. .
<Example 5: Effects of DOTAP and DOTMA on antigen processing and cross-presentation on MHC class I-restricted T cells>

  To evaluate that cross-presentation in the presence of cationic lipids in order to verify that the promotion of antigen uptake by 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 the β-galactosidase enzyme, which can be quantified using a β-gal assay. Is possible. Only BMDC (OVA241-270; SMLVLPLPDEVSGLEQLESIINFEKLTEWTS) or isotonic sucrose (280 mM) (Suc) with ovalbumin peptide mixed with 50 μM RDOTAP. The peptide-pulsed BMDCs were washed and co-cultured overnight at 37 ° C. with an antigen-specific T cell hybridoma cell line (B3Z cells) capable of recognizing the SINEKL epitope presented by antigen-presenting cells via MHCI (H2Kb). B3Z cells responded to the SIINFEKL epitope by producing β-galactosidase enzyme, which was quantified using a colorimetric β-galactosidase assay as an indicator of peptide cross-presentation by dendritic cells. Data represent relative absorbance (570 nm) in test wells (arbitrary units). Statistical significance was assessed using two-way ANOVA and * values were significantly different from treatment to treatment (shown in FIG. 7).

  As shown in FIG. 7, it was observed that peptide pulses with cationic nanoparticles significantly reduced the concentration of peptide required for efficient pulses. This method, which utilizes cationic lipids, is particularly under conditions where cross-presentation of dendritic cells is limited due to the amount of peptide antigen available or the concentration of the peptide (eg in the case of self-tumor vaccines with limited epitopes). Provides significant advantages in peptide loading at

  The second method used T cells derived from TCR transgenic mice (OT-1), where all T cells are specific for the internal peptide of OVA. These T cells proliferate only when presented with DCs that have processed OVA and presented OVA peptides to MHC class I molecules.

  This is therefore a rigorous assay for cross-presentation. BMDC were incubated for 1 hour at 37 ° C. with different concentrations of OVA complete protein in the presence or absence of either of two cationic lipids, DOTAP or DOTMA. Thereafter, DCs were washed, fixed 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 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 apparent when DC were incubated with OVA at 4 ° C.

These results show that the uptake of antigens by cationic lipids is an absolute prerequisite for effective activation of CD8 T cells, efficient antigen processing and peptides into the MHC class I pathway. It shows that it actually leads to the incorporation of.
Example 6 Effect of DOTMA and DOTAP on Antigen Processing and Cross-presentation on MHC Class II Restricted T Cells>

  To ascertain whether cationic lipids actually increase antigen presentation to CD4 T cells, derived from DO11.10 transgenic mice with T cells specific for OVA peptides presented on MHC class II molecules Cells were used. The results in FIG. 9 show that the same trend was observed in the class II presentation as observed in the class I presentation in FIG. Incorporation of OVA in the presence of cationic lipid enhanced antigen presentation to CD4 T cells.

These results show that enhanced antigen uptake by cationic lipids is an absolute prerequisite for effective activation of CD4 T cells, efficient antigen processing and peptides into the MHC class II pathway It shows that it actually leads to the incorporation of.
<Example 7: Effect of DOTAP on antigen presentation in actual vaccine setting in vivo>

A T cell receptor adoptive transfer system was used to model the effects of DOTAP in actual vaccine settings. This system uses T cells from the same TCR transgenic mice 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 tracking fluorescent dye CFSE. After 24 hours, mice were injected with OVA alone or OVA in the presence of DOTAP. If these T cells recognize the antigen presented by DCs in the draining lymph nodes after immunization, the T cells proliferate and the CFSE dye is diluted in all daughter cells. Next, mice were injected with OVA with and without DOTAP. Three days later, draining lymph nodes at the vaccination site were removed, T cells were stained with anti-CD8 and anti-CD4 antibodies, and the level of CFSE was visualized by flow cytometry. The results in FIG. 10 show CFSE-labeled OT1 (CD8 T cells) or DO11.10 (CD4 T cells) when further injected (immunized) with either complete OVA or complete OVA mixed with DOTAP. 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 increased T cell responses in draining lymph nodes after vaccination.
<Example 8: Effects of various lipids on antigen processing and cross-presentation on MHC class I-restricted T cells>

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

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

  To demonstrate as a proof of concept that cationic lipids improve the efficacy of dendritic cell-based vaccines in vivo, the effect of cationic lipids on CTL induction by dendritic cell vaccines was evaluated. B6 mice were immunized with only BMDC or isotonic sucrose pulsed with peptides alone (palmitoylated-KSSSIINFEKL) or peptides 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 immunized subcutaneously with peptide-pulsed BMDC, and on day 14 an antigen-specific IFN-γ response was obtained using an ELISPOT assay. By measuring the vaccine response was evaluated. Data represent spot forming cells for each mouse in a representative study.

  As shown in FIG. 12, pulsing dendritic cells with cationic nanoparticles loaded with peptides significantly increases the antigen-specific T cell response induced by the vaccine, so that the cation seen in in vitro assays It is directly shown that the beneficial effects of sex nanoparticles can affect the effectiveness of dendritic cell-based vaccines. In the following study, the effectiveness of cationic nanoparticles in CTL induction was examined using tumor-associated antigens derived from HPV-related tumors and mucin 1-related tumors. As expected, cationic nanoparticles loaded with tumor-associated antigens improved the antigen-specific T cell immune response mounted in vaccinated mice (FIG. 13). In this experiment, mouse BMDCs were prepared with a peptide mixture or isotonic sucrose (280 nM) containing a tumor associated antigen (HPV tumor associated (a) or mucin 1 associated (b)) mixed with 50 μM cationic lipid (RDOTAP). For 10 minutes. On days 0 and 7, groups of C57BL6 / J mice (n = 5) were immunized subcutaneously with peptide-pulsed or non-pulsed BMDC, and on day 14 antigen-specific IFN- using an ELISPOT assay. Vaccine response was assessed by measuring gamma response. Data represent spot forming cells for each mouse in a representative study. Some enhancement was observed in DOTAP, DOTMA, and DOPC, whereas antigen presentation was greatly enhanced in DOEPC and DDA. Note: DDA formed a precipitate when diluted with OVA. All cationic lipids show enhancement, but the overall size varies from experiment to experiment. Some enhancement of DOPC, a neutral lipid, was also observed in this experiment.

These results indicate that a class of cationic lipids is effective in mediating effective antigen uptake and delivery of protein antigens by dendritic cells. Furthermore, dendritic cells pulsed with cationic lipids loaded with antigen can greatly improve the effectiveness of dendritic cell vaccines.
<Example 10: Effect of R-DOTAP on populations of T cells and regulatory T cells in 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 liposomes Group 3: KF18 HPV peptide + S-DOTAP liposomes Group 4: KF18 peptide + MPL / Alum adjuvant

  Five mice per group were injected with various preparations. On days 0 and 7, mice were vaccinated and sacrificed on day 14. Splenocytes 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 peptide alone. (FIG. 14A). Since MPL was not effective at promoting antigen uptake compared to either R-DOTAP or S-DOTAP, it was assumed that the CD8 + T cell response was significantly lower than R-DOTAP. Another example of this effect is also observed with DDA, which is a cationic lipid. 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 powerful adjuvant to induce strong antigen-specific T cell responses (Brandt L. et al, ESAT-6 Subunit Vaccination amybacterium tuberculosis, Infect Immun. 2000 Feb; 68 (2): 791-795).

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

C57 mice were divided into 8 groups per group, R-DOTAP + HPV16 E7 peptide KF18 (GQAEPDRAHYNIVTF), GM-CSF + HPV16 E7 peptide KF18, R-DOTAP, GM-CSF, HPV16 E7 peptide KF18, untreated group . On day 1, 1 × 10 ⁵ TC-1 tumor cells were injected into the flank of the mice. Various preparations were administered on days 12 and 19 after tumor implantation. 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 that infiltrated the tumor microenvironment. In this study, the number of CD8 + T cells specific for the 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 and shows a statistically significant increase in HPV specific T cells compared to R-DOTAP / HPV compared to all other groups. Data represent the mean + SEM of 4-5 mice in each group.

  On day 19, flow cytometry was used to study the immunosuppressive tumor microenvironment, particularly the population of regulatory T cells. On days 14 and 19, T cells (CD45 + CD3 + CD4 + CD25 + Foxp3 +) cells infiltrated the tumor. The results are shown in FIG. This study shows that a statistically significant decrease 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). Although no statistical significance was achieved in this group, none of the groups other than the R-DOTAP group showed the ability to reduce the Treg population.

  Of great importance for 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 prognostic improvement towards antitumor effects. This study shows that the ratio of Treg / CD8 + T cells is dramatically reduced to less than 0.13 for R-DOTAP + antigen, compared to a ratio of about 1 for GM-CSF + antigen and antigen alone. It shows that it becomes. The ratio in the group without tumor antigen was about 32 (shown in FIG. 17). Cationic lipids appear to promote preferential expansion of the correct phenotype of effector T cells over Tregs. This leads to a significant modification of the tumor microenvironment, where the immunotherapy is very effective because the attacker produces a “power shift” that favors CD8 + T cells over the immunosuppressive Tregs “advocate”.

The same study evaluated the effect of various preparations on established TC-1 tumors. FIG. 18 shows that all animals treated with R-DOTAP + antigen (Treg / CD8 + ratio <0.13) had their tumors completely disappeared by day 26. Tumor volume was measured using calipers. The naïve mouse group is the untreated mouse with tumor. The HPV16 E7 peptide used in the vaccine is KF18. Both GM-CSF + antigen and antigen alone (Treg / CD8 + ratio was about 1.0) did not induce tumor regression, but tumor growth was inhibited and on day 26 the tumor volume was about 200 mm ³ . became. In the third group of animals treated with R-DOTAP or GM-CSF without antigen or untreated animals (Treg / CD8 + ratio> 30), the tumor volume was 300-700 mm ³ .

In this study, IFN-γ ELISPOT studies were performed to quantify and understand the “quality” of the tumor-specific T cells that are generated. 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, R-DOTAP + antigen preparation produced approximately 4-5 times more IFN-γ than GM-CSF + antigen. This is because in FIG. 11 the cationic lipid is “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. This suggests that T cells can be generated. The reason for the superiority of T cell priming was evaluated in another study.
<Example 11: Evaluation of R-DOTAP vaccination against T cell and B cell infiltration into lymph nodes>

12 mM R-DOTAP or sucrose as a control were injected into the right and left footpads of mice, respectively, and the influx of T cells and total lymphocytes into the draining 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, 3 days, 4 days, and it was found that lymphocyte infiltration into lymph nodes increased over 4 days (FIG. 20). Five mice were used in one study.
Example 12: Evaluation of the role of chemokines on lymphocyte infiltration into lymph nodes

  The main purpose of the current experiment was to conduct the study described in Example 7 using 5 mice to visualize the homing of adoptively transferred CFSE-labeled cells. This study included a population of cells that were treated with pertussis toxin in vitro 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 induced to return to the lymph nodes. However, if homing enhanced by DOTAP is due to chemokines, there should be no pertussis toxin population in DLN or only at significantly reduced levels.

Spleen cells were prepared from one B6 mouse and divided in half. Half of the cells were treated with 100 ng / ml pertussis toxin for 1 hour at 37 ° C. and washed. The 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 was injected intravenously (10 ⁷ cells) into the tail vein of five B6 mice. Next, the mouse was anesthetized and either sucrose (right footpad) or R-DOTAP (left footpad, 50 μl, 600 nmol) was injected into the meatpad.

  After 16 hours, the mice were sacrificed and popliteal LN and spleen were collected. The total number of cells collected from the left and right nodes of each mouse was counted. Migrated CFSE-labeled lymphocytes also infiltrated the lymph nodes at the time of R-DOTAP vaccination. However, since this did not occur in cells treated with pertussis, cationic lipids induced lymphocyte influx into lymph nodes, indicating that this phenomenon is probably mediated by chemokines.

Previous studies (Yan et al.) Suggested that cationic lipids induce chemokines CCL2,3,4. 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 important side effect of adjuvants is to induce cytokines and increase them in the circulating blood. The presence of cytokines in the blood often results in a serious inflammatory response, which results in fever, nausea, vomiting, headache, and, in extreme cases, toxicity that can even lead to toxic shock and death. Cytokine storms are often associated with the administration of various adjuvants.

This study therefore focused on assessing the presence of cytokines throughout the body after subcutaneous administration of cationic lipid vaccines. Human HLA-A2 mice capable of recognizing human HPV antigen were administered high and low doses of R-DOTAP + antigen.
Group 1:
Vaccinate mice with 100 μL of a 1: 1 mixture of high dose R-DOTAP (3.4 mg / mL) and sucrose solution Group 2:
Vaccination with 50 μg LPS as a positive control for cytokine induction Group 3:
Vaccinate mice with 100 μL of high dose of R-DOTAP + HPV antigen (1: 1 mixture of RDOTAP 3.4 mg / mL and HPVMix 0.14 mg / mL) Group 4:
Vaccinate mice with 100 μL of low dose R-DOTAP + HPV antigen (1: 1 mixture of RDOTAP 3.4 mg / mL and HPVMix 0.14 mg / mL)

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

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

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

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

  Study Results: Mouse serum was analyzed using the Luminex Mouse cytokin 20-plex panel (cytokines are listed below). Luminex software was used to quantify the intensity level of cytokines by comparison with cytokine standards run on the same plate. The positive control group (LPS) showed increased systemic levels of IL-12, IP-10, KC, MCP-1, MIG at the time of vaccination. As shown in FIG. 21, no systemic induction of the cytokines and 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 typical of the results seen with all cytokines tested are shown in FIG. This study shows that in the case of cationic lipids, cytokine and chemokine induction appears to be mainly restricted to lymph nodes. In the case of LPS, which is a typical TLR agonist, cytokine induction is not limited to lymph nodes and a cytokine level systemic spike is 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

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. . Such equivalents are intended to be encompassed by the following claims.

Claims (41)

A population of isolated dendritic cells;
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 of claim 1, wherein the at least one antigen is a modified protein, lipoprotein, lipopeptide, peptide, or combinations thereof having amino acids with increased hydrophobicity.   4. The composition of any one of claims 1-3, wherein the at least one antigen comprises a cancer or tumor associated antigen.   The at least one antigen comprises a protein product of a gene in a patient, the gene comprising an oncogene, a tumor suppressor gene, a gene having a mutation, a gene having a rearrangement specific to tumor cells, a reactivated embryo gene product, The composition according to any one of claims 1 to 4, which is selected from the group consisting of an oncofetal antigen, a tissue-specific differentiation gene, a growth factor receptor, and a cell surface protein gene.   6. The composition of any one of claims 1-5, wherein the at least one antigen comprises a microbial antigen.   7. The composition of claim 6, wherein the microbial antigen is selected from the group consisting of viral antigens, bacterial antigens, fungal antigens, and natural isolates, fragments, and derivatives of microbial antigens.   The composition according to any one of claims 1 to 7, comprising a plurality of antigens.   9. The composition of any one of claims 1 to 8, wherein the at least one antigen comprises a non-immunogenic peptide or a poorly immunogenic peptide.   The composition of claim 1, wherein the at least one cationic lipid is structurally linked to the at least one antigen by a linker.   11. The composition of claim 10, wherein the linker is a serine-serine dipeptide linker.   The composition of claim 1, wherein the cationic lipid encapsulates the antigen.   The composition according to claim 1, wherein the cationic lipid includes a liposome.   The composition according to claim 1, wherein the cationic lipid and the antigen are emulsions.   15. The composition of claims 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, wherein the cationic lipid is purified.   The composition according to claim 1, wherein the cationic lipid is an enantiomer.   18. A composition according to claims 1-17, wherein the cationic lipid forms nanoparticles.   19. The composition of claim 18, wherein the nanoparticles are less than about 10,000 nm in diameter. A method of activating dendritic cells ex vivo comprising:
Obtaining an isolated dendritic cell;
Treating the isolated dendritic cells with a composition comprising an effective amount of at least one cationic lipid and at least one antigen;
Including
A method wherein 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;
21. The method of claim 20, wherein the at least one antigen is a cancer antigen.   21. The method of claim 20, further comprising the step of treating the dendritic cell with at least a growth factor or at least a cytokine or a combination thereof.   23. A method according to any one of claims 20 to 22, wherein the isolated dendritic cells are human.   24. A method according to any one of claims 20 to 23, wherein the at least one cytokine is GMCSF. A method of activating dendritic cells ex vivo for use in the manufacture of a cancer vaccine comprising:
Obtaining an isolated dendritic cell;
The isolated dendritic cells,
An effective amount of at least one cationic lipid;
At least one cancer antigen;
At least growth factors or at least cytokines or combinations thereof;
Treating with a composition comprising:
Including
A method wherein the dendritic cells are activated by treating the dendritic cells.   26. The method of claim 25, wherein the dendritic cell is a human dendritic cell.   27. The method of claim 25 or 26, wherein the at least one cytokine is GMCSF. A method for producing a cancer dendritic cell vaccine comprising:
Obtaining an isolated dendritic cell;
The isolated dendritic cells,
An effective amount of at least one cationic lipid;
At least one cancer antigen;
At least growth factors or at least cytokines or combinations thereof;
Treating with a composition comprising:
Including
A method wherein the dendritic cells are activated by treating the dendritic cells.   30. The method of claim 28, further comprising treating the dendritic cell with a growth factor and GM-CSF. A method for enhancing a therapeutic immune response in a cancer patient comprising:
Obtaining an isolated dendritic cell;
The isolated dendritic cells,
An effective amount of at least one cationic lipid;
At least one cancer antigen;
At least growth factors or at least cytokines or combinations thereof;
Treating with a composition comprising:
Including
A method wherein the dendritic cells are activated by treating the dendritic cells. A method for treating a cancer patient, comprising:
Obtaining an isolated dendritic cell;
The isolated dendritic cells,
An effective amount of at least one cationic lipid;
At least one cancer antigen;
At least growth factors or at least cytokines or combinations thereof;
Activating with a composition comprising:
Creating a dendritic cell activated thereby,
Administering an appropriate number of activated dendritic cells to the patient;
Including
A method wherein the patient with the cancer is treated by administering the appropriate number of activated dendritic cells. Periodically checking the level of CD8 + T cells specific for the at least one antigen in the composition;
Confirming the level of Treg cells in the patient subsequent to administering the dendritic cells to the patient;
Further including
32. The method of claim 31, wherein a high level of antigen-specific CD8 + T cells and a low level of Treg cells indicates that the patient is effectively treating the cancer.   33. The method of 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.   35. Use according to claim 34, wherein altering the tumor microenvironment is reducing the population of immunosuppressive regulatory T cells.   35. The method of claim 34, wherein the population of immunosuppressive regulatory T cells within the tumor is decreased and the population of tumor specific CD8 + T cells or CD4 + T cells or both is increased.   Three important functions for effective immunotherapy: i) presentation of the antigen to MHC class I CD8 + T cells and MHC class II CD4 + T cells, ii) T cell proliferation and T cells Cationic for each of the following: induction of cytokines and chemokines that promote activation, iii) induction of T cells infiltrating the tumor, iv) reduction of the immunosuppressive cell population within the tumor microenvironment Use of lipid-based vaccines.   38. 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.   40. Use according to claims 34-39, wherein the cationic lipid is selected from the group consisting of R-DOTAP, S-DOTAP, DOEPC, DDA, and DOTMA. 40. Use according to claims 34-39, wherein the cationic lipid is in combination with at least one cancer peptide antigen.