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BioNTech and Pfizer’s BNT162 Vaccine Patent Landscape
By Mario Gaviria and Burcu Kilic November 16, 2020
BNT162 Vx patent landscape
Safe and effective vaccines are key to combating the Covid-19 pandemic; however, patents and other intellectual property claims directed at vaccine technologies create legal barriers for equitable access and fair allocation. No corporation produces at scale to supply the world. Providing timely global access will depend in significant part on increasing supply, including by transferring technology to qualified manufacturers. Much of this technology is claimed as patented, proprietary, or confidential in nature.

German company BioNTech and its U.S. partner Pfizer’s[1] vaccine candidate, BNT162 SARS-CoV-2, employs the use of lipid nanoparticle (NP) technology to deliver mRNA to cells. Once the lipid nanoparticle is injected into a patient, it travels into the cells and instructs them to produce the SARS-CoV-2 spike protein. The presence of this corona virus protein is thought to trigger an immune response leading to the production of antibodies.[2] If the patient is infected with coronavirus, the antibodies will identify and bind to the virus, which triggers a series of events resulting in the elimination of the virus.
BNT162 is in Phase 3 clinical trials. Pfizer announced promising but preliminary trial results on November 9th.[3]
We identified several patents claimed by BioNTech relating to the pertinent vaccine technologies.[4] We placed them in three groups based on their description and their primary independent claim:

·   Patents directed at RNABelow is our non-exhaustive list.
·   Patents directed at Lipids/NP + mRNA
·   Patents specifically directed at pharmaceutical compositions involving lipid NP + mRNA
Below is our non-exhaustive list. In a recent financial statement, BioNTech suggested that its patent claims extend to mRNA structure, formulations, and manufacturing, and relies on trade secrets and confidential know-how to protect aspects of mRNA manufacturing technologies.[5]

Patent/Published Application   Applicant/Assignee   Filing Date   Status   Invention Type   
US 10,576,146BioNTechMarch 15, 2018ActiveLipids/NP + mRNAUS 10,485,884BioNTechMarch 5, 2013ActiveLipids/NP + mRNAUS 9,950,065BioNTechSeptember 26, 2013ActiveLipids/NP + mRNAUS2020/0155671BioNTechJanuary 22, 2020PendingLipids/NP + mRNAUS2020/0197508BioNTechMarch 21, 2018PendingRNA immune responseUS2019/0153428BioNTechAugust 24, 2016PendingRNA immunogenicityUS2019/0321458BioNTechJuly 14, 2017PendingPC: Lipids/NP + mRNAUS2018/0263907BioNTechMarch 30,2016PendingLipids/NP + mRNAUS2017/0273907BioNTechSeptember 17, 2015PendingLipids/NP + mRNAUS2014/0030808BioNTechDecember 2, 2011PendingRNA expressionWO2016/156398BioNTechMarch 30,2016PublishedLipids/NP + mRNAWO2015/043613BioNTechSeptember 26, 2013PublishedLipids/NP + mRNAWO2013/087083BioNTechDecember 15, 2011PublishedLipids/NP + mRNA[1] All patents and patent applications identified in this study were claimed by BioNTech indicating that they are the inventor of the relevant vaccine technology, while Pfizer is acting as the innovator and leading the large-scale manufacturing, development, and regulatory approval process.
[2] https://www.nejm.org/doi/10.1056/NEJMoa2027906
[3] https://www.pfizer.com/news/press-release/press-release-detail/pfizer-and-biontech-announce-vaccine-candidate-against
[4] Pharmaceutical companies are not the only claimants of key technology. The U.S. government claims a patent on a key technology which may be relevant for BioNTech and Pfizer to stabilize the spike protein. See Public Citizen, Leading COVID-19 Vaccine Candidates Depend on NIH Technology (Nov. 10, 2020), https://www.citizen.org/article/leading-covid-19-vaccines-depend-on-nih-technology/.
[5] “Certain of our technologies, including in particular certain proprietary manufacturing processes or technologies and/or neoantigen prediction technologies, are protected as trade secrets”,.BioNTech SE, SEC Filing (July 21 2020), https://www.sec.gov/Archives/edgar/data/1776985/000119312520195911/d939702df1.htm.
Public Citizen

Moderna mRNA Patent 2013
Rna formulation for immunotherapy
Abstract
The present invention is in the field of immunotherapy, in particular tumor immunotherapy. The present invention provides pharmaceutical formulations for delivering RNA to antigen presenting cells such as dendrite cells (DCs) in the spleen after systemic administration. In particular, the formulations described herein enable to induce an immune response after systemic administration of antigen-coding RNA.
Classifications
A61K48/0033 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the ‘non-active’ part of the composition delivered, e.g. wherein such ‘non-active’ part is not delivered simultaneously with the ‘active’ part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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WO2013143555A1
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Inventor
Ugur Sahin
Heinrich Haas
Sebastian Kreiter
Mustafa DIKEN
Daniel Fritz
Martin MENG
Lena Mareen KRANZ

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2012 WO 2013 JP TR ES CA BR US PT AU HU CN LT CA RS CN SI PT HU NZ LT MX DK ES WO 2015 HK 2017 JP AU 2018 JP 2019 US 2020 AU JP HR

Application PCT/EP2012/001319 events
2012-03-26
Application filed by Biontech Ag, TRON – Translationale Onkologie an der Johannes Gutenberg-Universität Mainz gemeinnützige GmbH, Universitätsmedizin Der Johannes Gutenberg-Universität Mainz
2012-03-26
Priority to PCT/EP2012/001319
2013-10-03
Publication of WO2013143555A1
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Description
RNA FORMULATION FOR IMMUNOTHERAPY
TECHNICAL FIELD OF THE INVENTION
The present invention is in the field of immunotherapy, in particular tumor immunotherapy. The present invention relates to the provision of pharmaceutical formulations for delivering RNA with high selectivity to antigen presenting cells such as dendrite cells (DCs) in the spleen after systemic administration. In particular, the formulations described herein enable to induce an immune response after systemic administration of antigen-coding RNA.
BACKGROUND OF THE INVENTION
Nucleic acids like DNA, siRNA or RNA are of interest for various therapeutic interventions in patients. A relatively new immunological approach in tumor therapy is based on tumor antigen expression by coding RNA in antigen presenting cells (APCs) in order to induce a T- cell response to the tumor (Weide, B. et al. (2008) Journal of Immunotherapy 31(2): 180-188; Weide, B. et al. (2009) Journal of Immunotherapy 32(5): 498-507; Kreiter, S. et al. (2010) Cancer Res 70(22): 9031-9040; Kuhn, A. N. et al. (2010) Gene Ther 17(8): 961-971). Target cells for such intervention are dendritic cells (DCs) which reside, for example, in the lymph nodes (LNs) or in the spleen.
In order to provide sufficient uptake of the RNA by DCs, local administration of RNA to lymph nodes has proven to be successful. However, such local administration requires specific skills by the physician. Therefore, there is a need for RNA formulations which can be administered systemically, for example intravenously (i.v.), subcutaneously (s.c), intradermally (i.d.) or by inhalation. From the literature, various approaches for systemic administration of nucleic acids are known. In non-viral gene transfer, cationic liposomes are used to induce DNA/RNA condensation and to facilitate cellular uptake. The cationic liposomes usually consist of a cationic lipid, like DOTAP, and one or more helper lipids, like DOPE. So-called ‘lipoplexes’ can be formed from the cationic (positively charged) liposomes and the anionic (negatively charged) nucleic acid. In the simplest case, the lipoplexes form spontaneously by mixing the nucleic acid with the liposomes with a certain mixing protocol, however various other protocols may be applied. Electrostatic interactions between the positively charged liposomes and the negatively charged nucleic acid are the driving force for the lipoplex formation. Besides the lipid composition, the charge ratio between cationic and anionic moieties plays a key role for efficient condensation and transfection. Generally, an excess positive charge of the lipoplexes is considered necessary for efficient transfection (Templeton, N. S. et al. (1997) Nature Biotechnology 15(7): 647-652; Zhdanov, R. I. et al. (2002) Bioelectrochemistry 58(1): 53-64; Templeton, N. S. (2003) Current Medicinal Chemistry 10(14): 1279-1287). Most natural membranes are negatively charged, and therefore the attractive electrostatic interaction between the positively charged lipoplexes and the negatively charged biomembrane may play a role in cell binding and uptake of the lipoplexes. Typical ranges of +/- ratios which are considered optimal for transfection are between 2 and 4. With lower excess positive charge, the transfection efficacy goes drastically down to virtually zero. Unfortunately, for positively charged liposomes and lipoplexes elevated toxicity has been reported, which can be a problem for the application of such preparations as pharmaceutical products.
The above described lipolexes have proven to enable transfection in various organs. The detailed organ distribution of expression depends on the formulation and administration parameters (lipid composition, size, administration route) in a complex manner. So far, selective expression in a given target organ or cellular moiety, avoiding expression in off- target organs, could not be realized sufficiently. Using luciferase DNA or RNA as a reporter, for example, transfection in lung, liver, spleen, kidneys, and heart has been reported. Avoiding targeting of lung and liver has proven to be particularly difficult, because, in many cases, lung and liver targeting are predominant. Lung has a very large surface and it is the first organ which the i.v. injected compounds pass after administration. Liver is a typical target organ for liposomes and formulations with lipophilic compounds like the lipids present in the lipoplexes.
For RNA based immunotherapy, lung or liver targeting can be detrimental, because of the risk of an immune response against these organs. Therefore, for such therapy, a formulation with high selectivity only for the DCs, such as in the spleen is required. Certain ligands have been proposed to improve targeting selectivity. For example, liposomes which comprise mannose functionalized lipids are considered to improve macrophage targeting. However, such components make the formulations more complex, which makes practical pharmaceutical development more complicated. Furthermore, the selectivity is limited and a certain fraction of the liposomes is still taken up by other organs. Another problem is serum interactions and RNA degradation in serum, which is favored by positively charged lipoplexes. Also, for therapeutic applicability, requirements for pharmaceutical products such as chemical and physical stability, need to be fulfilled. In addition, products for intraperitoneal application need to be sterile and have to fulfill certain requirements regarding particle characteristics. Additionally, the products have to be suitable for manufacturing.
Summarizing, the problem of development of an injectable RNA formulation with high spleen selectivity, which fulfills the criteria for products for application to patients, still needs to be solved.
The present invention provides a solution to the above described problem. According to the invention, nanoparticulate RNA formulations with defined particle size are provided wherein the net charge of the particles is close to zero or negative. In one particularly preferred embodiment, said RNA nanoparticles are RNA lipoplexes. Surprisingly it was found that electro-neutral or negatively charged lipoplexes from RNA and liposomes lead to substantial RNA expression in spleen DCs after systemic administration. A strong expression of reporter gene in the target cells (spleen) was determined while the expression in other organs was low. Furthermore, a strong immune response against a model antigen could be induced. This was unexpected, because usually, excess positive charge is considered a prerequisite for successful uptake and expression. Here we have found that, although the absolute amount of expression decreases with decreasing excess of positive charge, the expression is still sufficiently high to provide therapeutic efficacy of the lipoplexes after systemic administration.
According to the invention it was possible to form the lipoplexes with a well-defined particle size distribution profile as measured by dynamic light scattering and with low fraction of subvisible particles, which is required for intravenous administration to patients. If formed by incubation of liposomes with RNA by self-assembly, the particle size of the original liposomes is only little affected, and no undesired moieties of large aggregates are found. Different sizes can be obtained by selecting the size of the precursor liposomes and the mixing conditions. This was surprising because usually formation of large aggregates on incubation of RNA with cationic liposomes is observed. This aggregate formation is one major obstacle for developing lipoplex formulations which are acceptable for intravenous or subcutaneous administration. The particles were stable for at least 24 hours and did not tend to aggregate over time. The particles could be frozen and thawed without formation of aggregates, while maintaining the original particle size profile, and maintaining the biological activity. The particles could be lyophilized and reconstituted with water without formation of aggregates, while maintaining the original particle size profile and maintaining the biological activity. The particles can be manufactured by different protocols which are scalable and which can be performed under controlled conditions. With such properties the lipoplex formulations of the invention fulfill important requirements for pharmaceutical formulations for application to patients, in terms of particle size distribution profile and stability. Furthermore, compared to positively charged lipopexes, the RNA nanoparticles described herein are expected to be less toxic and to display less undesired serum interactions. In particular, the formulations are suitable for parenteral administration, including intravenous and subcutaneous administration.
DESCRIPTION OF INVENTION
Summary of the invention
Immunotherapeutic strategies are promising options for the treatment of e.g. infectious diseases and cancer diseases. The identification of a growing number of pathogen- and tumor- associated antigens (also termed tumor antigens herein) led to a broad collection of suitable targets for immunotherapy.
The present invention generally embraces the immunotherapeutic treatment of diseases by targeting diseased cells. The invention provides for the selective eradication of cells that express an antigen thereby minimizing adverse effects to normal cells not expressing said antigens. Thus, preferred diseases for a therapy are those in which one or more antigens are expressed such as cancer diseases or infectious diseases.
The present invention aims at specifically targeting antigen-expressing cells by active immunization inducing and expanding T cells in the patient, which are able to specifically recognize and kill diseased cells. Specifically, the present invention enables selective incorporation of an antigen represented as RNA into antigen-presenting cells such as dendritic cells in vivo. The antigen may be processed to produce a peptide partner for the MHC molecule or may be presented without the need for further processing, if it can bind to MHC molecules. Preference is given to administration forms in which the complete antigen is 5 EP2012/001319 processed in vivo by antigen-presenting cells, since this may also produce T helper cell responses which are needed for an effective immune response. Thus, the compositions provided according to the invention when administered to a patent provide one or more MHC presented epitopes for stimulating, priming and/or expanding T cells directed against cells expressing antigens from which the MHC presented epitopes are derived. Accordingly, the compositions described herein are preferably capable of inducing or promoting a cellular response, preferably cytotoxic T cell activity, against a disease characterized by presentation of antigens with class I MHC.
In particular, the present invention relates to a pharmaceutical composition comprising nanoparticles which comprise RNA encoding at least one antigen, wherein:
(i) the number of positive charges in the nanoparticles does not exceed the number of negative charges in the nanoparticles and/or
(ii) the nanoparticles have a neutral or net negative charge and/or
(iii) the charge ratio of positive charges to negative charges in the nanoparticles is 1.4: 1 or less and/or
(iv) the zeta potential of the nanoparticles is 0 or less.
Preferably, the nanoparticles described herein are colloidally stable for at least 2 hours in the sense that no aggregation, precipitation or increase of size and polydispersity index by more than 30% as measured by dynamic light scattering takes place
In one embodiment, the charge ratio of positive charges to negative charges in the nanoparticles is between 1.4: 1 and 1 :8, preferably between 1.2: 1 and 1 :4, e.g. between 1 : 1 and 1 :3 such as between 1 : 1.2 and 1 : 1.8.
In one embodiment, the zeta potential of the nanoparticles is -5 or less, -10 or less, -15 or less, -20 or less or -25 or less. In various embodiments, the zeta potential of the nanoparticles is -35 or higher, -30 or higher or -25 or higher. In one embodiment, the nanoparticles have a zeta potential from 0 mV to -50 mV, preferably 0 mV to -40 mV or -10 mV to -30 mV.
In one embodiment, the nanoparticles comprise at least one lipid. In one embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphophilic molecule, eg, a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning of the present invention. In one embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the RNA. In one embodiment, the nanoparticles comprises at least one helper lipid. The helper lipid may be a neutral or an anionic lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In one embodiment, the cationic lipid and/or the helper lipid is a bilayer forming lipid.
In one embodiment, the at least one cationic lipid comprises l ,2-di-0-octadecenyl-3- trimethylammonium propane (DOTMA) or analogs or derivatives thereof and/or 1,2-dioleoyl- 3-trimethylammonium-propane (DOTAP) or analogs or derivatives thereof.
In one embodiment, the at least one helper lipid comprises l ,2-di-(9Z-octadecenoyl)-sn- glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and/or l ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or analogs or derivatives thereof.
In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9: 1 to 3:7, 4: 1 to 1 :2, 4: 1 to 2:3, 7:3 to 1 : 1 , or 2: 1 to 1 : 1 , preferably about 1 : 1. In one embodiment, in this ratio, the molar amount of the cationic lipid results from the molar amount of the cationic lipid multiplied by the number of positive charges in the cationic lipid.
In various embodiments, the lipids are not functionalized such as functionalized by mannose, histidine and/or imidazole, the nanoparticles do not comprise a targeting ligand such as mannose functionalized lipids and/or the nanoparticles do not comprise one or more of the following: pH dependent compounds, cationic polymers such as polymers containing histidine and/or polylysine, wherein the polymers may optionally be PEGylated and/or histidylated, or
2+
divalent ions such as Ca .
In various embodiments, the RNA nanoparticles may comprise peptides, preferentially with a molecular weight of up to 2500 Da. EP2012/001319
In the nanoparticles described herein the lipid may form a complex with and/or may encapsulate the RNA. In one embodiment, the nanoparticles comprise a lipoplex or liposome. In one embodiment, the lipid is comprised in a vesicle encapsulating said RNA. The vesicle may be a multilamellar vesicle, an unilamellar vesicle, or a mixture thereof. The vesicle may be a liposome.
In one embodiment, the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 10:0 to 1 :9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1 :2, even more preferably 1.4:2 to 1.1 :2 and even more preferably about 1.2:2.
In one embodiment, the nanoparticles are lipoplexes comprising DOTMA and Cholesterol in a molar ratio of 10:0 to 1 :9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1 :2, even more preferably 1.4:2 to 1.1 :2 and even more preferably about 1 .2:2.
In one embodiment, the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 10:0 to 1 :9, preferably 8:2 to 3:7, and more preferably of 7:3 to 5:5 and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.8:2 to 0.8:2, more preferably 1.6:2 to 1 :2, even more preferably 1.4:2 to 1.1 :2 and even more preferably about 1.2:2.
In one embodiment, the nanoparticles are lipoplexes comprising DOTMA and DOPE in a molar ratio of 2: 1 to 1 :2, preferably 2: 1 to 1 : 1 , and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4: 1 or less.
In one embodiment, the nanoparticles are lipoplexes comprising DOTMA and cholesterol in a molar ratio of 2: 1 to 1 :2, preferably 2:1 to 1 : 1 , and wherein the charge ratio of positive charges in DOTMA to negative charges in the RNA is 1.4: 1 or less. In one embodiment, the nanoparticles are lipoplexes comprising DOTAP and DOPE in a molar ratio of 2: 1 to 1 :2, preferably 2: 1 to 1 : 1 , and wherein the charge ratio of positive charges in DOTAP to negative charges in the RNA is 1 .4: 1 or less.
In one embodiment, the nanoparticles have an avarage diameter in the range of from about 50 nm to about 1000 nm, preferably from about 50 nm to about 400 nm, preferably about 100 nm to about 300 nm such as about 150 nm to about 200 nm. In one embodiment, the nanoparticles have a diameter in the range of about 200 to about 400 nm.
In one embodiment, the polydispersity index of the nanoparticles described herein as measured by dynamic light scattering is 0.5 or less, preferably 0.4 or less or even more preferably 0.3 or less.
In one embodiment, the nanoparticles described herein are obtainable by one or more of the following: (i) incubation of liposomes in an aqueous phase with the RNA in an aqueous phase, (ii) incubation of the lipid dissolved in an organic, water miscible solvent, such as ethanol, with the RNA in aqueous solution, (iii) reverse phase evaporation technique, (iv) freezing and thawing of the product, (v) dehydration and rehydration of the product, (vi) lyophilization and rehydration of the of the product, or (vii) spray drying and rehydration of the product.
In one embodiment, the nanoparticles are produced by a process comprising a step of incubating the RNA with bivalent cations preferably at a concentration of between 0.1 mM to 5 mM such as 0.1 mM to 4 mM or 0.3 mM to 1 mM prior to incorporation into said nanoparticles and/or by incubating the RNA with monovalent ions preferably at a concentration of between 1 mM to 500 mM such as 100 mM to 200 mM or 130 mM to 170 mM prior to incorporation into said nanoparticles and/or by incubating the RNA with buffers prior to incorporation into said nanoparticles.
In one embodiment, after incubation of the bivalent cations to RNA a step of dilution by adding liposomes and/or other aqueous phases by at least a factor of more than 1.5, preferably by a factor of more than 2, or by a factor of more than 5 is involved. In one embodiment, the bivalent cations are calcium ions, where the final concentration of said calcium ions is less than 4 mM, preferably less than 3 mM and even more preferably 2.2 mM or less.
In one embodiment, the nanoparticles described herein are produced by a process comprising a step of extruding and/or a step of filtration and/or a step of lyophilizing the nanoparticles.
In one embodiment, after systemic administration of the nanoparticles, RNA expression in the spleen occurs. In one embodiment, after systemic administration of the nanoparticles, no or essentially no RNA expression in the lung and/or liver occurs. In one embodiment, after systemic administration of the nanoparticles, RNA expression in the spleen is at least 5-fold, preferably at least 8-fold, preferably at least 10-fold, preferably at least 20-fold, preferably at least 50-fold, preferably at least 100-fold, preferably at least 1000-fold or even more the amount of RNA expression in the lung. In one embodiment, after systemic administration of the nanoparticles, RNA expression in antigen presenting cells, preferably professional antigen presenting cells in the spleen occurs.
In one embodiment, the nanoparticles when administered systemically target or accumulate in the spleen. Preferably, the nanoparticles when administered systemically deliver the RNA to antigen presenting cells, preferably professional antigen presenting cells such as dendritic cells and/or macrophages in the spleen. Preferably the nanoparticles release the RNA at the target organ or tissue and/or enter cells at the target organ or tissue. Preferably, the target organ or tissue is spleen and the cells at the target organ or tissue are antigen presenting cells such as dendritic cells. In one embodiment, the nanoparticles when administered systemically do not or do not essentially target or accumulate in the lung and/or liver. In one embodiment, the amount of the nanoparticles targeting or accumulating in the spleen is at least 5-fold, preferably at least 8-fold, preferably at least 10-fold, preferably at least 20-fold, preferably at least 50-fold, preferably at least 100-fold, preferably at least 1000-fold or even more the amount targeting or accumulating in the lung.
According to the invention, systemic administration is preferably by parenteral administration, preferably by intravenous administration, subcutaneous administration, intradermal administration or intraarterial administration. The antigen encoded by the RNA comprised in the nanoparticles described herein preferably is a disease-associated antigen or elicts an immune response against a disease-associated antigen or cells expressing a disease-associated antigen.
The pharmaceutical composition of the invention may further comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical composition of the invention may further comprise at least one adjuvant.
The pharmaceutical composition of the invention may be formulated for systemic administration.
The pharmaceutical composition of the invention may be used for inducing an immune response, in particular an immune response against a disease-associated antigen or cells expressing a disease-associated antigen, such as an immune response against cancer. Accordingly, the pharmaceutical composition may be used for prophylactic and/or therapeutic treatment of a disease involving a disease-associated antigen or cells expressing a disease- associated antigen, such as cancer. Preferably said immune response is a T cell response. In one embodiment, the disease-associated antigen is a tumor antigen.
In one embodiment, the RNA comprised in the nanoparticles described herein does not comprise pseudouridine residues and preferably does not comprise modified nucleosides.
The present invention also relates to a method for delivering an antigen to antigen presenting cells, preferably professional antigen presenting cells such as dendritic cells and/or macrophages in the spleen or expressing an antigen in antigen presenting cells, preferably professional antigen presenting cells such as dendritic cells and/or macrophages in the spleen comprising administering to a subject a pharmaceutical composition of the invention.
The present invention also relates to a method for inducing an immune response, preferably an immune response against cancer, in a subject comprising administering to the subject a pharmaceutical composition of the invention. The present invention also relates to a method for stimulating, priming and/or expanding T cells in a subject comprising administering to the subject a pharmaceutical composition of the invention.
The present invention also relates to a method of treating or preventing a disease involving an antigen, preferably a cancer disease, in a subject comprising administering to the subject a pharmaceutical composition of the invention.
In the above aspects, the disease may be tumor growth and/or tumor metastasis. Accordingly, the present invention also relates to a method of treating or preventing tumor growth and/or tumor metastasis in a subject that has or is at risk of developing tumors and/or tumor metastases comprising administering to the subject a pharmaceutical composition of the invention.
In one aspect, the invention also provides the agents and compositions described herein for use in the methods of treatment described herein.
The present invention also relates to particles as set forth herein.
Other features and advantages of the instant invention will be apparent from the following detailed description and claims.
Detailed description of the invention
Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject-matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
The present invention describes agents and compositions that upon administration induce an immune response, in particular a cellular immune response, directed against a disease- associated antigen or cells expressing a disease-associated antigen such as cancer cells. In particular, the present invention envisions the use of RNA encoding antigenic proteins or peptides (also termed “antigen” herein) inducing an immune response, in particular a T cell response, against the disease-associated antigen or cells expressing the disease-associated antigen. These antigenic proteins or peptides may comprise a sequence essentially corresponding to or being identical to the sequence of the disease-associated antigen or one or more fragments thereof. In one embodiment, the antigenic protein or peptide comprises the sequence of an MHC presented peptide derived from the disease-associated antigen. Immunisation with RNA encoding intact or substantially intact disease-associated antigen or fragments thereof such as MHC class I and class II peptides makes it possible to elicit a MHC class I and/or a class II type response and thus, stimulate T cells such as CD8+ cytotoxic T lymphocytes which are capable of lysing diseased cells and/or CD4+ T cells. Such immunization may also elicit a humoral immune response (B cell response) resulting in the production of antibodies against the antigen. Accordingly, the pharmaceutical composition of the present invention may be used in genetic vaccination, wherein an immune response is stimulated by introduction into a subject a suitable RNA molecule which codes for an antigenic protein or peptide. The agents and compositions disclosed herein may be used as a therapeutic or prophylactic vaccine for the treatment or prevention of a disease such as a disease as disclosed herein. In one embodiment, a disease-associated antigen is a tumor antigen. In this embodiment, the agents and compositions described herein may be useful in treating cancer or cancer metastasis. Preferably, the diseased organ or tissue is characterized by diseased cells such as cancer cells expressing a disease-associated antigen and preferably presenting the disease-associated antigen in the context of MHC molecules.
The term “immune response” refers to an integrated bodily response to an antigen or a cell expressing an antigen and preferably refers to a cellular immune response or a cellular as well as a humoral immune response. The immune response may be protective/preventive/prophylactic and/or therapeutic.
“Inducing an immune response” may mean that there was no immune response against a particular antigen or a cell expressing an antigen before induction, but it may also mean that there was a certain level of immune response against a particular antigen or a cell expressing an antigen before induction and after induction said immune response is enhanced. Thus, “inducing an immune response” also includes “enhancing an immune response”. Preferably, after inducing an immune response in a subject, said subject is protected from developing a disease such as an infectious disease or a cancer disease or the disease condition is ameliorated by inducing an immune response. For example, an immune response against a viral antigen may be induced in a patient having a viral disease or in a subject being at risk of developing a viral disease. For example, an immune response against a tumor antigen may be induced in a patient having a cancer disease or in a subject being at risk of developing a cancer disease. Inducing an immune response in this case may mean that the disease condition of the subject is ameliorated, that the subject does not develop metastases, or that the subject being at risk of developing a cancer disease does not develop a cancer disease.
A “cellular immune response”, a “cellular response”, a “cellular response against an antigen” or a similar term is meant to include a cellular response directed to cells expressing an antigen and being characterized by presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes which act as either “helpers” or “killers”. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as infected cells or cancer cells, preventing the production of more diseased cells. In preferred embodiments, the present invention involves the stimulation of an anti-tumor CTL response against cancer cells expressing one or more tumor antigens and preferably presenting such tumor antigens with class I MHC.
According to the present invention, the term “antigen” comprises any molecule, preferably a peptide or protein, which comprises at least one epitope that will elicit an immune response and/or against which an immune response is directed. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune response, which is preferably specific for the antigen or cells expressing the antigen. In particular, an “antigen” relates to a molecule which, optionally after processing, is presented by MHC molecules and reacts specifically with T lymphocytes (T cells).
Thus, an antigen or fragments thereof should be recognizable by a T cell receptor. Preferably, the antigen or fragment if recognized by a T cell receptor is able to induce in the presence of appropriate co-stimulatory signals, clonal expansion of the T cell carrying the T cell receptor specifically recognizing the antigen or fragment. In the context of the embodiments of the present invention, the antigen or fragment is preferably presented by a cell, preferably by an antigen presenting cell and/or a diseased cell, in the context of MHC molecules, which results in an immune response against the antigen or cells expressing the antigen.
According to the present invention, any suitable antigen is envisioned which is a candidate for an immune response, wherein the immune response is preferably a cellular immune response.
An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen. According to the present invention, an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.
The term “pathogen” relates to pathogenic microorganisms and comprises viruses, bacteria, fungi, unicellular organisms, and parasites. Examples for pathogenic viruses are human immunodeficiency virus (HIV), cytomegalovirus (CMV), herpes virus (HSV), hepatitis A- virus (HAV), HBV, HCV, papilloma virus, and human T-lymphotrophic virus (HTLV). Unicellular organisms comprise plasmodia, trypanosomes, amoeba, etc. The term “disease-associated antigen” refers to all antigens that are of pathological significance and includes “tumor antigens”. According to the invention it is desired to induce an immune response to a disease-associated antigen or cells expressing a disease-associated antigen and preferably presenting a disease-associated antigen in the context of MHC molecules. Preferably, a disease-associated antigen is a naturally occurring antigen. In one embodiment, a disease-associated antigen is expressed in a diseased cell and preferably presented by MHC molecules of the cell.
An antigen encoded by the RNA comprised in the nanoparticles described herein should induce an immune response which is directed against the disease-associated antigen to be targeted or cells expressing the disease-associated antigen to be targeted. Thus, an antigen encoded by the RNA comprised in the nanoparticles described herein may correspond to or may comprise a disease-associated antigen or one or more immunogenic fragments thereof such as one or more MHC binding peptides of the disease-associated antigen. Thus, the antigen encoded by the RNA comprised in the nanoparticles described herein may be a recombinant antigen.
The term “recombinant” in the context of the present invention means “made through genetic engineering”. Preferably, a “recombinant object” such as a recombinant nucleic acid in the context of the present invention is not occurring naturally.
The term “naturally occurring” as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
In a preferred embodiment, an antigen may be a tumor antigen, i.e., a constituent of cancer cells such as a protein or peptide expressed in a cancer cell which may be derived from the cytoplasm, the cell surface or the cell nucleus, in particular those which primarily occur intracellularly or as surface antigens on cancer cells. For example, tumor antigens include the carcinoembryonal antigen, a 1 -fetoprotein, isoferritin, and fetal sulphoglycoprotein, cc2-H- ferroprotein and γ-fetoprotein. According to the present invention, a tumor antigen preferably comprises any antigen which is expressed in and optionally characteristic with respect to type and/or expression level for tumors or cancers as well as for tumor or cancer cells. In the 1 7 T EP2012/001319 context of the present invention, the term “tumor antigen” or “tumor-associated antigen” preferably relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and/or organs or in specific developmental stages, for example, the tumor antigen may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are expressed or aberrantly expressed in one or more tumor or cancer tissues. In this context, “a limited number” preferably means not more than 3, more preferably not more than 2 or 1 . The tumor antigens in the context of the present invention include, for example, differentiation antigens, preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, cancer/testis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta, and germ line specific antigens. In the context of the present invention, the tumor antigen is preferably not or only rarely expressed in normal tissues. Preferably, the tumor antigen or the aberrant expression of the tumor antigen identifies cancer cells. In the context of the present invention, the tumor antigen that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer disease, is preferably a self-protein in said subject. In preferred embodiments, the tumor antigen in the context of the present invention is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e., tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen which is expressed in normal tissues and the tumor antigen which is expressed in cancer tissues. Preferably, a tumor antigen is presented by a cancer cell in which it is expressed.
Examples for tumor antigens that may be useful in the present invention are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1 , CASP-8, CDC27/m, CD 4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1 , G250, GAGE, GnT-V, Gapl OO, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE- A, preferably MAGE-A1 , MAGE-A2, MAGE- A3, MAGE-A4, MAGE- A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A1 1 , or MAGE- A12, MAGE-B, MAGE-C, MART- 1 /Melan-A, MC1 R, Myosin/m, MUC1 , MUM-1 , -2, -3, NA88-A, NF1 , NY-ESO-1 , NY-BR-1 , pl 90 minor BCR-abL, Pm l/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RUl or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP 1 , SCP2, SCP3, SSX, SURVrVIN, TEL/AMLl , TPI/m, TRP-1 , TRP-2, TRP-2/1NT2, TPTE and WT, preferably WT-1.
The term “epitope” refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by a T cell, in particular when presented in the context of MHC molecules. An epitope of a protein such as a tumor antigen preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present invention is a T cell epitope.
According to the invention an epitope may bind to MHC molecules such as MHC molecules on the surface of a cell and thus, may be a “MHC binding peptide”. The term “MHC binding peptide” relates to a peptide which binds to an MHC class I and/or an MHC class II molecule. In the case of class I MHC/peptide complexes, the binding peptides are typically 8-10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC/peptide complexes, the binding peptides are typically 10-25 amino acids long and are in particular 13-18 amino acids long, whereas longer and shorter peptides may be effective.
According to the invention, an antigen encoded by the RNA comprised in the nanoparticles described herein may comprise an immunogenic fragment of a disease-associated antigen such as a peptide fragment of a disease-associated antigen (also termed antigen peptide herein) which preferably is a MHC binding peptide.
An “immunogenic fragment of an antigen” according to the invention preferably relates to a portion or fragment of an antigen which is capable of stimulating an immune response, preferably a cellular response against the antigen or cells expressing the antigen and preferably presenting the antigen such as diseased cells, in particular cancer cells. Preferably, an immunogenic fragment of an antigen is capable of stimulating a cellular response against a cell characterized by presentation of an antigen with class I MHC and preferably is capable of 19 T EP2012/001319 stimulating an antigen-responsive CTL. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules. Certain preferred immunogenic fragments bind to an MHC class 1 or class II molecule. As used herein, an immunogenic fragment is said to “bind to” an MHC class I or class II molecule if such binding is detectable using any assay known in the art.
Preferably, an immunogenic fragment of an antigen according to the invention is an MHC class I and/or class II presented peptide or can be processed to produce a MHC class I and/or class II presented peptide. Preferably, an immunogenic fragment of an antigen comprises an amino acid sequence substantially corresponding and preferably being identical to the amino acid sequence of a fragment of the antigen. Preferably, said fragment of an antigen is an MHC class I and/or class II presented peptide.
If a peptide is to be presented directly, i.e., without processing, in particular without cleavage, it has a length which is suitable for binding to an MHC molecule, in particular a class I MHC molecule, and preferably is 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-1 1 amino acids in length, in particular 9 or 10 amino acids in length.
If a peptide is part of a larger entity comprising additional sequences, e.g. of a polypeptide, and is to be presented following processing, in particular following cleavage, the peptide produced by processing has a length which is suitable for binding to an MHC molecule, in particular a class I MHC molecule, and preferably is 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-1 1 amino acids in length, in particular 9 or 10 amino acids in length. Preferably, the sequence of the peptide which is to be presented following processing is derived from the amino acid sequence of an antigen, i.e., its sequence substantially corresponds and is preferably completely identical to a fragment of an antigen.
Thus, an antigen encoded by the RNA comprised in the nanoparticles described herein may comprise a sequence of 7-20 amino acids in length, more preferably 7-12 amino acids in length, more preferably 8-1 1 amino acids in length, in particular 9 or 10 amino acids in length which substantially corresponds and is preferably completely identical to a MHC presented fragment of a disease-associated antigen and following processing makes up a presented peptide.
Peptides having amino acid sequences substantially corresponding to a sequence of a peptide which is presented by the class I MHC may differ at one or more residues that are not essential for TCR recognition of the peptide as presented by the class I MHC, or for peptide binding to MHC. Such substantially corresponding peptides are also capable of stimulating CTL having the desired specificity and may be considered immunologically equivalent.
A peptide when presented by MHC should be recognizable by a T cell receptor. Preferably, the presented peptide if recognized by a T cell receptor is able to induce in the presence of appropriate co-stimulatory signals, clonal expansion of the T cell carrying the T cell receptor specifically recognizing the presented peptide. Preferably, antigen peptides, in particular if presented in the context of MHC molecules, are capable of stimulating an immune response, preferably a cellular response against the antigen from which they are derived or cells expressing the antigen and preferably presenting the antigen. Preferably, an antigen peptide is capable of stimulating a cellular response against a cell presenting the antigen with class I MHC and preferably is capable of stimulating an antigen-responsive CTL. Such cell preferably is a target cell for the purposes of the invention.
“Target cell” shall mean a cell which is a target for an immune response such as a cellular immune response. Target cells include cells that express an antigen such as a disease- associated antigen and preferably present said antigen (which, in particular, means that the antigen is processed in the cells and one or more fragments of the antigen are presented in the context of MHC molecules on the cells). Target cells include any undesirable cell such as an infected cell or cancer cell. In preferred embodiments, the target cell is a cell expressing an antigen as described herein and preferably presenting said antigen with class I MHC.
“Antigen processing” refers to the degradation of an antigen into procession products, which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, preferably antigen presenting cells to specific T cells.
An antigen-presenting cell (APC) is a cell that presents, i.e. displays, antigen in the context of major histocompatibility complex (MHC) on its surface. This, includes the situation where only one or more fragments of an antigen are presented. T cells may recognize this complex using their T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells.
Professional antigen-presenting cells are very efficient at internalizing antigen, either by phagocytosis or by receptor-mediated endocytosis, and then displaying a fragment of the antigen, bound to a class II MHC molecule, on their membrane. The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen- presenting cell. An additional co-stimulatory signal is then produced by the antigen- presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules is a defining feature of professional antigen-presenting cells.
The main types of professional antigen-presenting cells are dendritic cells, which have the broadest range of antigen presentation, and are probably the most important antigen- presenting cells, macrophages, B-cells, and certain activated epithelial cells.
Dendritic cells (DCs) are leukocyte populations that present antigens captured in peripheral tissues to T cells via both MHC class II and I antigen presentation pathways. It is well known that dendritic cells are potent inducers of immune responses and the activation of these cells is a critical step for the induction of antitumoral immunity.
Dendritic cells are conveniently categorized as “immature” and “mature” cells, which can be used as a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation.
Immature dendritic cells are characterized as antigen presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e. g. CD54 and CD1 1 ) and costimulatory molecules (e. g., CD40, CD80, CD86 and 4-1 BB). Dendritic cell maturation is referred to as the status of dendritic cell activation at which such antigen-presenting dendritic cells lead to T cell priming, while presentation by immature dendritic cells results in tolerance. Dendritic cell maturation is chiefly caused by biomolecules with microbial features detected by innate receptors (bacterial DNA, viral RNA, endotoxin, etc.), pro-inflammatory cytokines (TNF, IL- 1, IFNs), ligation of CD40 on the dendritic cell surface by CD40L, and substances released from cells undergoing stressful cell death. The dendritic cells can be derived by culturing bone marrow cells in vitro with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.
Non-professional antigen-presenting cells do not constitutively express the MHC class II proteins required for interaction with naive T cells; these are expressed only upon stimulation of the non-professional antigen-presenting cells by certain cytokines such as IFNy.
Antigen presenting cells can be loaded with MHC presented peptides by transducing the cells with nucleic acid, such as RNA, encoding a peptide or protein comprising the peptide to be presented, e.g. a nucleic acid encoding the antigen. Transfection of dendritic cells with mRNA is a promising antigen-loading technique of stimulating strong antitumor immunity.
The term “immunogenicity” relates to the relative efficiency of an antigen to induce an immune reaction.
The terms “T cell” and “T lymphocyte” are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells.
T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors (TCR). The thymus is the principal organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with a distinct function.
T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
Cytotoxic T cells destroy diseased cells, e.g. infected cells such as virally infected cells and cancer cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.
A majority of T cells have a T cell receptor (TCR) existing as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCRa and TCRP) genes and are called a- and β-TCR chains, γδ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is made up of one γ-chain and one δ-chain. This group of T cells is much less common (2% of total T cells) than the αβ T cells.
All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors derived from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classed as double-negative (CD4-CD8-) cells. As they progress through their development they become double-positive thymocytes (CD4+CD8+), and finally mature to single-positive (CD.4+CD8- or CD4-CD8+) thymocytes that are then released from the thymus to peripheral tissues.
The first signal in activation of T cells is provided by binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually a professional antigen presenting cell (APC), usually a dendritic cell in the case of naive responses, although B cells and macrophages can be important APCs. The peptides presented to CD8+ T cells by MHC class I molecules are 8-10 amino acids in length; the peptides presented to CD4+ T cells by MHC class II molecules are longer, as the ends of the binding cleft of the MHC class II molecule are open.
The term “clonal expansion” refers to a process wherein a specific entity is multiplied. In the context of the present invention, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the specific lymphocyte recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the lymphocytes.
According to the invention, cytotoxic T lymphocytes may be generated in vivo by incorporation of an antigen or an antigen peptide into antigen-presenting cells in vivo. The antigen or antigen peptide is represented as RNA. The antigen may be processed to produce a peptide partner for the MHC molecule, while a fragment thereof may be presented without the need for further processing. The latter is the case in particular, if these can bind to MHC molecules. The resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes which then propagate.
Specific activation of CD4+ or CD8+ T cells may be detected in a variety of ways. Methods for detecting specific T cell activation include detecting the proliferation of T cells, the production of cytokines (e.g., lymphokines), or the generation of cytolytic activity. For CD4+ T cells, a preferred method for detecting specific T cell activation is the detection of the proliferation of T cells. For CD8+ T cells, a preferred method for detecting specific T cell activation is the detection of the generation of cytolytic activity.
The term “major histocompatibility complex” and the abbreviation “MHC” include MHC class I and MHC class II molecules and relate to a complex of genes which occurs in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptides and present them for recognition by T cell receptors. The proteins encoded by the MHC are expressed on the surface of cells, and display both self antigens (peptide fragments from the cell itself) and nonself antigens (e.g., fragments of invading microorganisms) to a T cell.
The MHC region is divided into three subgroups, class I, class II, and class III. MHC class I proteins contain an a-chain and 2-microglobulin (not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. On most immune system cells, specifically on antigen-presenting cells, MHC class II proteins contain a- and β- chains and they present antigen fragments to T-helper cells. MHC class III region encodes for other immune components, such as complement components and some that encode cytokines.
In humans, genes in the MHC region that encode antigen-presenting proteins on the cell surface are referred to as human leukocyte antigen (HLA) genes. However the abbreviation MHC is often used to refer to HLA gene products. HLA genes include the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1 , HLA-DPB 1 , HLA-DQA1 , HLA-DQB1 , HLA-DRA, and HLA-DRB 1.
In one preferred embodiment of all aspects of the invention an MHC molecule is an HLA molecule.
By “cell characterized by presentation of an antigen”, “cell presenting an antigen”, “antigen presented by a cell”, “antigen presented” or similar expressions is meant a cell, in particular a diseased cell or target cell such as an infected cell or a cancer cell, or an antigen presenting cell presenting the antigen it expresses or a fragment derived from said antigen, e.g. by processing of the antigen, in the context of MHC molecules, in particular MHC Class I molecules. Similarly, the terms “disease characterized by presentation of an antigen” denotes a disease involving cells characterized by presentation of an antigen, in particular with class I MHC. Presentation of an antigen by a cell may be effected by transfecting the cell with a nucleic acid such as RNA encoding the antigen.
The term “immunologically equivalent” means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect such as induction of a humoral and/or cellular immune response, the strength and/or duration of the induced immune reaction, or the specificity of the induced immune reaction.
The term “immune effector functions” in the context of the present invention includes any functions mediated by components of the immune system that result, for example, in the killing of infected cells or cancer cells, or in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis. Preferably, the immune effector functions in the context of the present invention are T cell mediated effector functions. Such functions comprise in the case of a helper T cell (CD4+ T cell) the recognition of an antigen or an antigen peptide in the context of MHC class II molecules by T cell receptors, the release of cytokines and/or the activation of CD8+ lymphocytes (CTLs) and/or B-cells, and in the case of CTL the recognition of an antigen or an antigen peptide in the context of MHC class I molecules by T cell receptors, the elimination of cells presented in the context of MHC class I molecules, i.e., cells characterized by presentation of an antigen with class I MHC, for example, via apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-γ and TNF-a, and specific cytolytic killing of antigen expressing target cells.
A nucleic acid is according to the invention preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. Nucleic acids include according to the invention genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may according to the invention be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle. A nucleic can be employed for introduction into, i.e. transfection of, cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.
Nucleic acids may be comprised in a vector. The term “vector” as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expre

Patents Assigned to modeRNA Therapeutics
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Publication number: 20180256750

Abstract: The invention features polynucleotides encoding a polypeptide including a 3?-stabilizing region and having increased stability compared to wild-type polynucleotides.

Type: Application

Filed: September 19, 2016

Publication date: September 13, 2018

Applicant: MODERNA THERAPEUTICS, INC.

Inventors: Gabor BUTORA, Andrew W. FRALEY, Edward John MIRACCO, Jennifer NELSON, Amy RHODEN-SMITH, Matthew STANTON
·   CHIMERIC POLYNUCLEOTIDES

Publication number: 20170204152

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of chimeric polynucleotide molecules, which allow for customized placement, position and percent load of chemical modifications, which improve, alter or optimize certain physicochemical and pharmaceutical properties of the polynucleotides. In one non-limiting embodiment, such chimeric polynucleotides take the form or function as modified mRNA molecules which encode a polypeptide of interest. In one embodiment, such chimeric polynucleotides are substantially noncoding.

Type: Application

Filed: July 16, 2015

Publication date: July 20, 2017

Applicant: Moderna Therapeutics, Inc.

Inventors: Jennifer NELSON, Andrew W. FRALEY, Amy RHODEN-SMITH
·   Modified polynucleotides encoding hepatitis A virus cellular receptor 2

Patent number: 9675668

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

Type: Grant

Filed: December 13, 2013

Date of Patent: June 13, 2017

Assignee: Moderna Therapeutics, Inc.

Inventors: Stephane Bancel, Tirtha Chakraborty, Antonin de Fougerolles, Susan Whoriskey, Ron Weiss
·   ALTERNATIVE NUCLEIC ACID MOLECULES AND USES THEREOF

Publication number: 20170136132

Abstract: The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them.

Type: Application

Filed: June 19, 2015

Publication date: May 18, 2017

Applicant: Moderna Therapeutics, Inc.

Inventors: Atanu ROY, Christopher R. CONLEE, Antonin DE FOUGEROLLES, Andrew W. FRALEY
·   ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF

Publication number: 20170065675

Abstract: Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to kill or reduce the growth of microorganisms. Such compositions and methods include the use of modified messenger RNAs, and are useful to treat or prevent microbial infection, or to improve a subject’s heath or wellbeing.

Type: Application

Filed: September 15, 2016

Publication date: March 9, 2017

Applicant: Moderna Therapeutics, Inc.

Inventors: Stephane Bancel, Jason P. Schrum, Alexander Aristarkhov
·   NUCLEIC ACID VACCINES

Publication number: 20160331828

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use ribonucleic acid vaccines (NAVs) comprising polynucleotide molecules encoding one or more antigens.

Type: Application

Filed: April 5, 2016

Publication date: November 17, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Giuseppe Ciaramella, Axel Bouchon, Eric Yi-Chun Huang
·   NUCLEIC ACID VACCINES

Publication number: 20160317647

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use ribonucleic acid vaccines (NAVs) comprising polynucleotide molecules encoding one or more antigens.

Type: Application

Filed: April 1, 2016

Publication date: November 3, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Giuseppe Ciaramella, Axel Bouchon, Eric Yi-Chun Huang
·   Engineered nucleic acids and methods of use thereof

Patent number: 9464124

Abstract: Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to kill or reduce the growth of microorganisms. Such compositions and methods include the use of modified messenger RNAs, and are useful to treat or prevent microbial infection, or to improve a subject’s heath or wellbeing.

Type: Grant

Filed: November 5, 2014

Date of Patent: October 11, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Stephane Bancel, Jason P. Schrum, Alexander Aristarkhov
·   ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF

Publication number: 20160271272

Abstract: Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to kill or reduce the growth of viruses. Such compositions and methods include the use of modified messenger RNAs, and are useful to treat or prevent viral infection, or to improve a subject’s heath or wellbeing.

Type: Application

Filed: June 6, 2016

Publication date: September 22, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Stephane Bancel, Jason P. Schrum, Alexander Aristarkhov
·   Engineered nucleic acids and methods of use thereof

Patent number: 9447164

Abstract: Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the use of modified messenger RNAs, and are useful to treat or prevent diseases, disorders or conditions, or to improve a subject’s heath or wellbeing.

Type: Grant

Filed: October 8, 2015

Date of Patent: September 20, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Jason P. Schrum, Stephane Bancel, Noubar B. Afeyan, Kenechi Ejebe
·   POLYNUCLEOTIDE MOLECULES AND USES THEREOF

Publication number: 20160264614

Abstract: The present disclosure provides alternative nucleosides, nucleotides, and polynucleotides, and methods of use thereof.

Type: Application

Filed: October 2, 2014

Publication date: September 15, 2016

Applicant: MODERNA THERAPEUTICS, INC.

Inventors: Christopher R. CONLEE, Andrew W. FRALEY, Atanu ROY
·   Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

Patent number: 9428535

Abstract: The present disclosure provides methods of increasing the level of a polypeptide of interest in a mammalian subject by administering a polynucleotide having one or more chemical modifications and a Protein:Cytokine ratio of greater than 100.

Type: Grant

Filed: October 3, 2012

Date of Patent: August 30, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Antonin de Fougerolles, Atanu Roy, Jason P. Schrum, Suhaib Siddiqi, Paul Hatala, Stephane Bancel
·   Polynucleotides Encoding Low Density Lipoprotein Receptor

Publication number: 20160244501

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotide molecules encoding low density lipoprotein receptor comprising at least one mutation (e.g., an LDLR signally enhancing mutation).

Type: Application

Filed: October 3, 2014

Publication date: August 25, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Jeff Lynn ELLSWORTH, Joseph Beene BOLEN, Francine M. GREGOIRE, Justin GUILD
·   COMPOSITIONS AND METHODS FOR TOLERIZING CELLULAR SYSTEMS

Publication number: 20160243221

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of molecules for tolerizing cellular systems.

Type: Application

Filed: October 17, 2014

Publication date: August 25, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Stephen G. Hoge, Eric Yi-Chun Huang
·   CIRCULAR POLYNUCLEOTIDES

Publication number: 20160194368

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of circular polynucleotides.

Type: Application

Filed: September 3, 2014

Publication date: July 7, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Stephen G. HOGE, Antonin DE FOUGEROLLES
·   Compositions and Methods of Altering Cholesterol Levels

Publication number: 20160136236

Abstract: The present invention relates to compositions, methods and kits using polynucleotides, primary transcripts and mmRNA molecules. The present invention also relates to compositions and methods for altering cholesterol levels using polynucleotides, primary transcripts and mmRNA molecules.

Type: Application

Filed: March 14, 2014

Publication date: May 19, 2016

Applicant: Moderna Therapeutics, Inc.

Inventors: Stephen G. HOGE, Eric Yi-Chun HUANG, Joseph Beene BOLEN, Justin GUILD, Antonin DE FOUGEROLLES, Jeff Lynn ELLSWORTH
·   Modified nucleosides, nucleotides, and nucleic acids, and uses thereof

Patent number: 9334328

Abstract: The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using thereof.

Type: Grant

Filed: January 11, 2013

Date of Patent: May 10, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Jason P. Schrum, Suhaib Siddiqi, Kenechi Ejebe
·   Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins

Patent number: 9303079

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

Type: Grant

Filed: March 9, 2013

Date of Patent: April 5, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Tirtha Chakraborty, Antonin de Fougerolles
·   Modified polynucleotides encoding apoptosis inducing factor 1

Patent number: 9301993

Abstract: The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of oncology-related polynucleotides, oncology-related primary transcripts and oncology-related mmRNA molecules.

Type: Grant

Filed: December 16, 2013

Date of Patent: April 5, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Tirtha Chakraborty, Antonin de Fougerolles
·   Formulation and delivery of PLGA microspheres

Patent number: 9295689

Abstract: The present disclosure provides, inter alia, formulation compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor. The formulation composition may further include a modified nucleic acid molecule and a delivery agent. The present invention further provides nucleic acids useful for encoding polypeptides capable of modulating a cell’s function and/or activity.

Type: Grant

Filed: May 18, 2013

Date of Patent: March 29, 2016

Assignee: Moderna Therapeutics, Inc.

Inventors: Antonin de Fougerolles, Kristy M. Wood, Pedro Valencia

https://patents.justia.com/assignee/moderna-therapeutics

I have put this together from a number of different resources with my free salt water cure in the middle of it, of course. I hope it is not too disjointed and flows consistently to arrive at the final outcome which I have established – please pass it on if you think it has merit.
If people are able to view the situation objectively, and ask themselves why a ruling class with a longstanding history of psychopathy, war-mongering, and merciless killing, would be incentivised to keep around billions of people they no longer “need”, the stark – yet really very simple – truth becomes apparent.
The ultra-wealthy intergenerational dynasties who possess control of all the world’s land and resources, believe that they have inalienable ownership rights to the earth, and that we “tenants” are only permitted to reside in “their” world so long as we perform some sort of useful function for them. Once we no longer do, since AI can take our place, then they – the self-proclaimed owners of the earth – are incentivised to do nothing more than accelerate our demise. That’s why they staged “the pandemic” when they did – when AI had finally become advanced enough to replace millions of people – and that’s why ChatGPT was launched in November 2022 – just as excess deaths from the depopulating injection are really starting to spike and grab the headlines.
That AI is imminently to make millions, if not billions of people, surplus to commercial requirements, is certainly no “conspiracy theory”, and many thinkers and scholars have been warning for years that this is inevitable – the question for them has been how will human beings practically and financially support themselves, without the option to earn a wage through work, and how will they find wider meaning to their lives, when work is so fundamental to so many people’s identities and sense of purpose? by MiriAF

It’s just plainly obvious common sense, extrapolated from the way the ruling classes have made it clear they see everyone else, that when the masses are no longer seen to have a useful function, then – just like farm animals that can no longer fulfil the purpose for which they were initially maintained – they will be destroyed. You may think, “but I am a human being! I am not defined by my “usefulness”, I have an intrinsic value of my own.” – and that’s true, objectively.

The point is that the ultra-wealthy and well-resourced ruling classes who control the world don’t see you like that, and no amount of appealing to their conscience or humanity will ever change that. They see themselves as a completely different species, a breed apart (that’s why they are obsessive about interbreeding and always marrying their cousins etc. – they believe their bloodlines are ‘special’ and cannot be tainted by the blood of lowly lifeforms like ordinary human beings). Consider how differently you see yourself to a cow or a chicken – well, that’s the same difference as to how they see us. They even refer to us as ‘human cattle’. Not incidentally, the word ‘vaccine’ comes from the Latin, ‘vacca’ – cow. MiriAF
RIO DE JANEIRO, BRAZIL – Scientists have discovered genetic material in the spike protein of SARS-CoV-2, which was patented by vaccine manufacturer Moderna in 2016. The researchers’ discovery again supports the hypothesis that the Coronavirus originated in the laboratory.
The identified genetic code snippet was found at the furin cleavage site of the virus: The part of SARS-CoV-2 in which it differs significantly from other coronaviruses and thanks to which the virus can so successfully infect humans.
Even before that, because of this structure, it had been repeatedly argued that the virus must have originated in the laboratory: Some scientists doubted that it could have evolved naturally in this way.
The critical physicist Roland Wiesendanger also sees the unique furin cleavage site of SARS-CoV-2 as an important indication that the virus was produced artificially.
Moderna filed the patent as part of its cancer research. According to the researchers’ analysis, the virus shares a sequence of 19 nucleotides in total with the said Moderna genome, twelve of which form the structure of the furin cleavage site. The sequence is part of a gene called MSH3, which affects how damaged cells in the body repair themselves. Link here: https://twitter.com/CuriousCanuckle/status/1496189358621458433
Moderna filed a patent application in February 2016. Researchers say the discovery cannot be a coincidence – “the chance that it arose this way through natural evolution is one in three trillion”.
https://www.riotimesonline.com/brazil-news/modern-day-censorship/moderna-patented-component-of-coronavirus-three-years-before-pandemic/

In a new hour-long presentation – watch it here: https://sensereceptornews.com/?p=15980 – Latypova lays out the copious evidence she has compiled – including “receipts” – to show that covid injections are nothing more than a bioweapon that was unleashed on the world by the United States Department of Defense (DoD) via the corrupt U.S. Food and Drug Administration (FDA).

Moderna is suing Pfizer for patent infringement is that Moderna helped to create the Covid-19 virus as early as 2013 during gain of function research and then patented parts of the virus. This means Moderna essentially owns SARS-CoV-2.
Moderna has filed patent infringement lawsuits in the U.S. and Germany accusing Pfizer and its partner BioNTech of stepping on patents that Moderna says it filed between 2010 and 2016.
But official documents and evidence suggest the patent infringement may be due to Moderna helping to create the Covid-19 virus in a lab during gain of function research.
Moderna then patented parts of the SARS-CoV-2 virus as early as 2013. And this may explain why further official documents prove Moderna had a Covid-19 vaccine candidate months before Covid-19 was known to exist officially. The 2013 Patent here: https://patents.google.com/patent/WO2013143555A1/en
Let’s start by looking at the confidential agreement proving Moderna had a Coronavirus vaccine candidate at least nineteen days before the alleged emergence of SARS-CoV-2 in Wuhan, China.
The confidential agreement which can be viewed here states that providers ‘Moderna’ alongside the ‘National Institute of Allergy and Infectious Diseases’ (NIAID) agreed to transfer ‘mRNA coronavirus vaccine candidates’ developed and jointly-owned by NIAID and Moderna to recipients ‘The University of North Carolina at Chapel Hill’ on the 12th December 2019.
https://expose-news.com/2022/09/04/real-reason-moderna-suing-pfizer/

There were no COVID-19 vaccines close to approval on August 27, 2020. In fact, the Pfizer/BioNTech vaccine trial phase 2/3 had only started a month earlier on July 27.The first contract, with General Dynamics, is dated August 27, 2020. It outlines a series of services the company was to provide to the CDC pursuant to the “anticipated increase” in VAERS reports due to the COVID-19 vaccines.

It certainly appears that by August, 2020, the impending emergency use authorization of at least one COVID-19 vaccine was a foregone conclusion.

BioNTech CEO Ugur Sahin says that his mRNA vaccines rolled out in January this year (2021): Link here: https://www.ibtimes.sg/fact-check-biontech-ceo-ugur-sahin-refuses-take-pfizer-covid-19-vaccine-due-safety-concerns-61652 (If he dies from his own vaccines, he won’t be able to make any more, to save all of you?)

In 2020, BioNTech CEO Ugur Sahin proudly announced, “…we chose lipid nanoparticles that promote migration from the muscle cells into the lymph nodes.” – https://groups.google.com/g/town-square-news/c/6XrY4yTa8JQ/m/qFqqyvFJBQAJ

But by August 27, 2020 – The first contract, with General Dynamics, is dated August 27, 2020 had been let and the contract states that they were expecting up to 1,000 VAERS reports to be filed per day, with up to 40% of the reports being serious in nature and the CDC was already anticipating that the COVID vaccines might generate nearly seven times as many reports as all other vaccines combined (a 600% increase),

The amounts paid out under the contracts with General Dynamics were redacted. But according to this site, the initial amount paid was $9.45 million, with $4.4 million added in late February, and then an additional $16.3 million tacked on in early March. In March of 2022 there was an additional $5.2 million added (increases in deaths and injuries beyond the initial contracts)

The Contracts 23 00099 General Dynamics Information Te.. https://substack.com/redirect/dac78dc8-9d2d-4280-a390-218d47bb331d?j=eyJ1IjoibjFlaXcifQ.OkComRnvTz45cW2ospKdwvhGbhkMepFwvepUF91fYF023 00099

Eagle Health Analytics, Ll
https://substack.com/redirect/bcba05ff-1dce-4b15-a6a7-0f59f3cb115a j=eyJ1IjoibjFlaXcifQ.OkComRnvTz45cW2ospKdwvhGbhkMepFwvepUF91fYF0

Grand total? $35,425,642 The Vaccine Adverse Event Reporting System (VAERS) records 1% of all reports: VAERS updated its numbers showing a staggering 1,481,226 reports of adverse events (x41 to arrive at December 2022 numbers) following COVID-19 vaccines were submitted between Dec. 14, 2020, and Dec. 9, 2022. …The above information posted by Josh Guetzkow

FDA assigned Pfizer’s COVID vaccine a license number months prior to actually licensing it | 6 Jan 2023 | Back in July 2020, when the U.S. Government committed to purchasing millions of doses of Pfizer’s Covid vaccines prior to the vaccines actually being authorized for use in the American public, some wondered whether the FDA’s “review” process for granting Emergency Use Authorization (EUA) was just a mere formality…

Based on an FDA document that ICAN’s (Informed Consent Action Network) attorneys recently obtained, it appears we can now say that the FDA’s post-EUA procedure for determining whether to formally license Pfizer’s Covid vaccine was indeed just that – a mere formality.

On June 17, 2021, the FDA drafted a memo announcing that it was issuing a license number for Pfizer’s Covid vaccine. But the FDA didn’t actually license the Pfizer’s Covid vaccine until several months later on August 23, 2021. In the document, the FDA makes the stunning admission that, while granting a BLA license number prior to actual licensing was a “deviation from [the FDA’s] normal practice,” it was doing so to “facilitate product labeling and distribution” because it had reviewed “much” of the information in Pfizer’s licensing application.

So Moderna and Pfizer (etc) have reinvented the wheel with their Scientific Frankenstein Monstrosity and that is called progress – the original wheel being this:

Millions saved from viruses and bacteria – are you one of those? 30 years never had a virus or bacteria infection = never ill:

Mix one heaped teaspoon of salt in a mug of clean warm water – cup a hand and in stages, sniff or snort the mugful up your nose spitting out anything which comes down into your mouth. If burning sensation, you have a virus and the salt solution is disinfecting it, so wait 2-3 minutes until burning sensation goes away, then blow out your nose on toilet paper and flush away, washing your hands afterwards.

Do my free salt water cure morning, noon, night or more often if you want, until it feels like you are flushing with water only – job done. 3 minutes idea to job done – simple.

No virus, no Covid or Long Covid in your head possible.

You cannot catch Covid, you have to catch a Coronavirus first and let it become Covid in the nasal passages of your head, later transported down into your body in the one liter of snot, or mucus, we each produce daily – the engine oil of the body.

Vaccines – what for – I never have any. My method is like using a fire hose to put out a fire. It takes 3 minutes to prepare and do with salt and clean water and over the 30 years I and others have been doing it, it has NOT killed or injured ONE PERSON, unlike those synthetic mRNA vaccines.

ALSO verified by Alberta Medical University – Canada.

Do it – you will be amazed at how quickly it destroys colds and flu in the nasal passages of your head. No infection, no Covid possible.

Spread the word to everyone please. Neti pots are like using a garden sprinkler to put out a house fire. your life, your choice!! AND free!!

So what if the “virus and vaccines” were never about what everyone had been nudged to believe, all smoke and mirrors to confuse and misdirect all:

See Electron Microscope studies of blood at cellular level and the nanotechnology to be found there: https://www.drrobertyoung.com/post/the-mindset-of-dr-robert-young-on-blood-clots-pleomorphism-the-jibby-jab-t-cruzi-h-vulgaris?postId=3704e561-ae34-4a1b-98f5-ded468531d93&utm_campaign=5003d1c8-c42a-4ec7-837d-42a0d74c29a2&utm_source=so&utm_medium=mail&utm_content=ca364f20-ac6e-41bf-9814-33ce0b02ff77&cid=8bec1d4c-3773-415a-83c2-1694daa095a4

The obvious question then is, for the vaccinated – what to do about the installed nanotechnology which is far ahead of our technical ability – how to explain that.
So let’s go with aircraft engines:

Up to the end of World War 2 (1939-1945) all aircraft were powered by piston engine technology and it was only towards the end of World War 2 (1944) that the jet engine was invented, in its infancy and because it delivered more thrust, provided faster speeds, took up less space, than piston engine aircraft – it eventually became our main mode of aircraft propulsion.

So with the nanotechnology in 2023 – we are at the beginning of World War 2 (1939) as above now and the vaccines are jet engines, way beyond our understanding or mechanical ability to create now, because we do not have the specialized equipment, or the engineering capability to create them and won’t reach that level of capability for at least 10 years forwards from today, let’s see 2033 and here we have them being injected with the Pfizer’s (and other vaccines) from January 2021

The virus drives the vaccines which create a once in a lifetime opportunity to get everyone vaccinated, but “controlled” by 5G and if so, then all of the above was “smoke and mirrors” to hide what has “really” been going on here from the outset – to take each body away from the spirit or soul which inhabited it, on call and by 5G and by its 10 digit call sign which can only be seen with an Android iPhone – Not Apple iPhone’s they don’t work:  

Dr. Madej is also featured in a viral video warning people about experiment mRNA and viral vector shots. She said in a recent interview with The New American:
“The Supreme Court ruled that if there is anything synthetic, not from nature, inside of our genome, then whoever owns the patent on those synthetic parts now owns part or all of you as a human. That means Bill and Melinda Gates, The Department of Defense, [and others] can literally own a human being. If this synthetic code is taken up into your genome, by law, you could be owned overnight.”
Living, man-made micro-organism is patentable subject matter as a “manufacture” or “composition of matter” within the meaning of the Patent Act of 1952. The fact that the organism sought to be patented is alive is no bar to patentability.
The case that provides the blueprint for pharmaceutical companies claiming ownership of your genes is Association for Molecular Pathology v. Myriad Genetics, Inc., 569 U.S. 576 (2013). This case originated from a Utah-based company called Myriad Genetics.
The company isolated the location and sequence of naturally-occurring genes called BRCA1 and BRCA2. Mutations in these genes positively correlate with predispositions to breast and ovarian cancers. Myriad filed patents on these genes in 1994 and 1995, respectively. The patents gave Myriad exclusive rights to cancer genetic testing that isolated natural DNA strands and created synthetic complementary DNA (cDNA) that resembled the original isolated strands.
The USPTO granted both patents in 1998. At least 2,000 other human genes had been patented through 2010, according to the New York State Bar. But the Myriad patents hindered other scientists from doing research on naturally-occurring BRCA1 and BRCA2, and thus hindered breast and ovarian cancer testing by other companies.

Notice how all these so-called doctors and scientists avoid pointing fingers at Pfizer, Moderna, Johnson & Johnson et al. when someone dies within hours, days or weeks of receiving their shots. It’s almost as if said doctors and scientists are carefully navigating the trademark and patent landscape. They don’t want to trespass on someone else’s property, if you will. Moderna owns several mRNA patents. Doctors and hospitals wanting a piece of the mRNA pie cannot bite the hand that feeds them.
The synthetic mRNA of Pfizer and Moderna, along with the viral vector DNA delivery systems of Johnson & Johnson and AstraZeneca, change your genetic code, making you “genetically-modified.” Granted mainstream media say the foregoing is “conspiracy theory.” But Moderna Chief Medical Officer Tal Zaks tells you straight up that 1) the shots change your genetic code and 2) the shots do not stop the spread of COVID-19. He says the Moderna shot is “hacking the software of life” (at the 0:43 second mark, but the whole video is…disturbing). Viral vectors do the same thing.
The Covid Blog
So do these companies “own you” once you get the shots? Well, they own mice and bacteria created with their inventions. Once you get these shots, you are no longer a “naturally-occurring” human being. Prosthetic limbs, breast implants, etc. are not “natural” per se. But they are removable and not part of what fundamentally makes you human. Gene therapy is irreversible. Do the math yourself.

Which begs the question: Why then the money spent on these vaccines, the nudging by press of the obsolete humans, or the ulterior motive behind them, which is what – destroy humanity down to 40,000 tops or less – conveniently blaming a virus or the vaccines, as the cause for that happening, while repurposing obsolete humans by taking control of the mind and bodies of those who survive the virus and the vaccines, for what purpose: a bio/robot, which in turn osoletes all military technology immediately, so win/win and little financial expenditure necessary, from the legally vaccinated, non-human multiple resources, to hand?

However, the installed nanotechnology runs on the bodies electricity and therein is its vulerability – touch a 12 volt DC cattle fence with both hands to short out the nanotechnology and it takes one second to do. in the same way as killing Lyme Disease, possibly onset Cancer and other blood borne diseases and no more contact from the computer to its human clone possible, or for any other reason, like a self destruct order

An alternative way: Touching the 12 volt DC car spark plug leads with both hands – turn over a cold car engine once, the jolt will do the job and both ways take one second to do – a good defense from an incoming bio/robot too – short the fucking thing out.

There is risk in everything and this too, but what is your alternative in the short or longer term, when you do nothing?

Just think – so many tries to successfully exterminate us, at the costs of billions of tax payers dollars and political nudging and it all comes down to, probably, is my simple free salt water cure for all viruses and bacteria, so you can’t get Covid ever and my one second 12 volt DC electric shock to kill off their injected nanotechnology, which would make 5G robots of all the vaccinated, that “they” did not kill first by 5G command (Android looking for Bluetooth to discover your vaccine injected nanotechnology number) FAIL Moderna, Pfizer, all other vaccine makers, Politicians, The Elite and all. Much Laughter!!

Tomorrow’s World Today?

FDA assigned Pfizer’s COVID vaccine a license number months prior to actually licensing it | 6 Jan 2023 | Back in July 2020, when the U.S. Government committed to purchasing millions of doses of Pfizer’s Covid vaccines prior to the vaccines actually being authorized for use in the American public, some wondered whether the FDA’s “review” process for granting Emergency Use Authorization (EUA) was just a mere formality… Based on an FDA document that ICAN’s (Informed Consent Action Network) attorneys recently obtained, it appears we can now say that the FDA’s post-EUA procedure for determining whether to formally license Pfizer’s Covid vaccine was indeed just that – a mere formality. On June 17, 2021, the FDA drafted a memo announcing that it was issuing a license number for Pfizer’s Covid vaccine. But the FDA didn’t actually license the Pfizer’s Covid vaccine until several months later on August 23, 2021. In the document, the FDA makes the stunning admission that, while granting a BLA license number prior to actual licensing was a “deviation from [the FDA’s] normal practice,” it was doing so to “facilitate product labeling and distribution” because it had reviewed “much” of the information in Pfizer’s licensing application.
There were no COVID-19 vaccines close to approval on August 27, 2020. In fact, the Pfizer/BioNTech vaccine trial phase 2/3 had only started a month earlier on July 27.The first contract, with General Dynamics, is dated August 27, 2020. It outlines a series of services the company was to provide to the CDC pursuant to the “anticipated increase” in VAERS reports due to the COVID-19 vaccines. It certainly appears that by August, 2020, the impending emergency use authorization of at least one COVID-19 vaccine was a foregone conclusion. BioNTech CEO Ugur Sahin says that his mRNA vaccines rolled out in January this year (2021): Link here: https://www.ibtimes.sg/fact-check-biontech-ceo-ugur-sahin-refuses-take-pfizer-covid-19-vaccine-due-safety-concerns-61652
In 2020, BioNTech CEO Ugur Sahin proudly announced, “…we chose lipid nanoparticles that promote migration from the muscle cells into the lymph nodes.” – https://groups.google.com/g/town-square-news/c/6XrY4yTa8JQ/m/qFqqyvFJBQAJ
But by August 27, 2020 – The first contract, with General Dynamics, is dated August 27, 2020 had been let and the contract states that they were expecting up to 1,000 VAERS reports to be filed per day, with up to 40% of the reports being serious in nature and the CDC was already anticipating that the COVID vaccines might generate nearly seven times as many reports as all other vaccines combined (a 600% increase), The amounts paid out under the contracts with General Dynamics were redacted. But according to this site, the initial amount paid was $9.45 million, with $4.4 million added in late February, and then an additional $16.3 million tacked on in early March. In March of 2022 there was an additional $5.2 million added (increases in deaths and injuries beyond the initial contracts)
The Contracts 23 00099 General Dynamics Information Te..https://substack.com/redirect/dac78dc8-9d2d-4280-a390-218d47bb331d?j=eyJ1IjoibjFlaXcifQ.OkComRnvTz45cW2ospKdwvhGbhkMepFwvepUF91fYF023 00099 Eagle Health Analytics, Llhttps://substack.com/redirect/bcba05ff-1dce-4b15-a6a7-0f59f3cb115a j=eyJ1IjoibjFlaXcifQ.OkComRnvTz45cW2ospKdwvhGbhkMepFwvepUF91fYF0 Grand total? $35,425,642 The Vaccine Adverse Event Reporting System (VAERS) records 1% of all reports:
VAERS updated its numbers showing a staggering 1,481,226 reports of adverse events (x41 to arrive at December 2022 numbers) following COVID-19 vaccines were submitted between Dec. 14, 2020, and Dec. 9, 2022. …The above information posted by Josh Guetzkow
BioNTech CEO Ugur Sahin says that his mRNA vaccines rolled out in January this year (2021): Link here: https://www.ibtimes.sg/fact-check-biontech-ceo-ugur-sahin-refuses-take-pfizer-covid-19-vaccine-due-safety-concerns-61652 but by August 27, 2020 – The first contract, with General Dynamics, is dated August 27, 2020 had been let and the contract states that they were expecting up to 1,000 VAERS reports to be filed per day, with up to 40% of the reports being serious in nature and the CDC was already anticipating that the COVID vaccines might generate nearly seven times as many reports as all other vaccines combined (a 600% increase), with a rate of serious adverse events that could be up to 8 times higher, (bearing in mind that VAERS represents only 1% of all injuries and deaths recorded and multiplying the numbers given by 41 (x41) gives a much better appreciation of just how many American’s were being injured or had died, after vaccinations began in early 2021), on a daily basis: The Vaccine Adverse Event Reporting System (VAERS) Friday updated its numbers showing a staggering 1,481,226 reports of adverse events (x41) following COVID-19 vaccines were submitted between Dec. 14, 2020, and Dec. 9, 2022, so for 100% x41 = 60,730,266 dead or injured Americans so far and it does not include the numbers your governments are inflicting on you, if you don’t live in America.
See Electron Microscope studies of blood at cellular level and the nanotechnology to be found there: https://www.drrobertyoung.com/post/the-mindset-of-dr-robert-young-on-blood-clots-pleomorphism-the-jibby-jab-t-cruzi-h-vulgaris?postId=3704e561-ae34-4a1b-98f5-ded468531d93&utm_campaign=5003d1c8-c42a-4ec7-837d-42a0d74c29a2&utm_source=so&utm_medium=mail&utm_content=ca364f20-ac6e-41bf-9814-33ce0b02ff77&cid=8bec1d4c-3773-415a-83c2-1694daa095a4
The US Department of Defense (US DoD) has had a dominant role in the response to the SARS-CoV-2 virus and the US DoD took charge of the Covid vaccine funding, development and testing from the very start of the perceived threat in early 2020 and in the development, and distribution of the Covid 19 vaccines, a fact hidden from the general public. In those processes many standard steps and procedures, otherwise required for pharmaceutical products, were omitted or circumvented. 
The US FDA’s website (FDA, 2020) reveals that the United States Department of Defence (DoD) has been in full control of the Covid Vaccine development program since its beginning. The DoD has been responsible for development, manufacturing, clinical trials, quality assurance, distribution and administration, since that time (FDA, 2020; Rees and Latypova, 2022; KEI, 2022; Medical Defense Consortium, 2022; Rees, 2022). The major pharmaceutical companies have been involved as “Project Coordination Teams” effectively performing as subcontractors to the DoD. The Chief Operating Officer for (Trump’s) Warp Speed vaccine program is the US Department of Defence, and the Chief Science Advisor is the US Department of Health and Human Services (HHS).
Definition of these vaccines as “countermeasures” rather than therapeutic agents has permitted their expedited progression to emergency use authorisation and widespread rollouts. Many adverse consequences have been the outcome of this secret military response to a public health matter. Excepts From: Phillip Altman’s Essay he is Pharmacologist and Clinical trial and drug regulatory affairs consultant in Melbourne, Australia and Brownstone Institute