[go: up one dir, main page]

WO2023209619A1 - Protéines miniace2 solubles qui interagissent avec le sars cov 2 et utilisations de ces dernieres - Google Patents

Protéines miniace2 solubles qui interagissent avec le sars cov 2 et utilisations de ces dernieres Download PDF

Info

Publication number
WO2023209619A1
WO2023209619A1 PCT/IB2023/054335 IB2023054335W WO2023209619A1 WO 2023209619 A1 WO2023209619 A1 WO 2023209619A1 IB 2023054335 W IB2023054335 W IB 2023054335W WO 2023209619 A1 WO2023209619 A1 WO 2023209619A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
ace2
cells
cov
sars
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2023/054335
Other languages
English (en)
Spanish (es)
Inventor
Paulino GOMEZ PUERTAS
Cesar Augusto RAMÍREZ SEGURA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Instituto Distrital De Ciencia Biotecnologia E Innovacion En Salud Idcbis
Consejo Superior de Investigaciones Cientificas CSIC
Original Assignee
Instituto Distrital De Ciencia Biotecnologia E Innovacion En Salud Idcbis
Consejo Superior de Investigaciones Cientificas CSIC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Instituto Distrital De Ciencia Biotecnologia E Innovacion En Salud Idcbis, Consejo Superior de Investigaciones Cientificas CSIC filed Critical Instituto Distrital De Ciencia Biotecnologia E Innovacion En Salud Idcbis
Publication of WO2023209619A1 publication Critical patent/WO2023209619A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention provides miniature versions of the angiotensin-converting enzyme 2 (min!ACE2) with improved binding to the SARS-CoV-2 spike protein that, upon binding to it, allow the interaction of the SARS-CoV virus to be blocked. -2 with the natural receptor on the human host cell.
  • the invention also contemplates the use of min ⁇ ACE2 to treat or prevent coronavirus infection with min ⁇ ACE2 in humans or animals and to be used as a substrate for capturing viral particles.
  • the invention also comprises chimeric antigen receptors (CAR) designed from min ⁇ ACE2 to specifically recognize SARS-CoV-2 infected cells that can be incorporated into NK cells.
  • min ⁇ ACE2 or min ⁇ ACE2-based CAR NK cells can be administered to people with COVID-19 to induce a cellular immune response against cells infected with SARS-CoV-2.
  • the invention belongs to the technical field of cellular and molecular engineering. More specifically, the invention relates to the design and construction of miniature versions of the human angiotensin 2 converting enzyme that specifically bind to the spike protein of the SARS-CoV-2 virus and prevent or neutralize the infection caused by it. Likewise, the invention describes the design and production of chimeric antigen receptors related to said miniature versions. BACKGROUND
  • the first step that the virus that causes severe acute respiratory syndrome type 2 (SARS-CoV-2) must take to infect cells is to bind to the angiotensin-converting enzyme 2 (ACE2, by its English name “Angiotensin - Converting Enzyme 2”), present on the surface of the cells of the lungs, arteries, heart, kidneys and intestines.
  • ACE2 angiotensin-converting enzyme 2
  • RBD receptor binding domain
  • S protein spike protein
  • ACE2 serves as an entry receptor for SARS-CoV-2 and protects the lung from injury. ACE2 is primarily expressed on the surface of type II alveolar epithelial cells and in multiple extrapulmonary tissues, including the heart, kidney, blood vessels, and intestine, so SARSCoV-2 can also infect these tissues.
  • This protein contains an N-terminal domain with M2 metallo-peptidase activity (amino acids 19-615) and a C-terminal domain of renal amino acid transporter collectrin (amino acids 616-768) that ends with a single helix or transmembrane anchor. and an intracellular segment.
  • the C-terminal domain is a membrane anchoring domain that exposes the peptidase domain to the cell surface.
  • the N-terminal peptidase domain consists of two subdomains or lobes that together form the active site of the enzyme and are joined by an alpha helix.
  • Hydrolysis of the extracellular domain of ACE2 results in a soluble protein that is released into the blood circulation that lowers blood pressure by hydrolyzing angiotensin I (a vasoconstrictor) to heptapeptide angiotensin 1-7 (a vasodilator).
  • angiotensin I a vasoconstrictor
  • heptapeptide angiotensin 1-7 a vasodilator
  • Protein S is a trimeric glycoprotein that protrudes from the surface of the SARS-COV-2 virion and is responsible for mediating ACE2 receptor recognition and fusion to the membrane of human cells. Each monomer has two subunits (S1 and S2) separated by a cleavage site that is recognized by host cell proteases. During viral infection, the S protein is cleaved into S1 and S2 subunits. The S1 subunit contains the receptor binding domain (RBD), which binds directly to the peptidase domain of ACE2, while the S2 subunit is responsible for membrane fusion.
  • RBD receptor binding domain
  • the RBD domain is composed of a 5-stranded antiparallel beta sheet between strands (34-7 and is an extended insertion of short strands [35-6.
  • the extension carries the receptor binding motif (RBM) that binds to the receptor ACE2 to enter cells.
  • Some of the strategies to combat SARS-CoV-2 have focused on preventing the virus from entering cells by blocking the interaction of the viral S protein with its cellular receptor ACE2.
  • neutralizing antibodies have been studied (including plasma from convalescent patients and monoclonal antibodies directed at RBD) and soluble versions of ACE2 that can act as a decoy to bind to the S protein of SARS-CoV-2. thus preventing the entry of the virus.
  • the virus shows a rapid accumulation of escape mutations when under selective pressure.
  • the virus appears to have limited potential to escape soluble ACE2-mediated neutralization without simultaneously decreasing affinity for the native ACE2 receptor, so administration of soluble ACE2 is predicted to prevent the occurrence of escape variants. of the virus.
  • the administration of the recombinant ACE2 protein in people infected with SARS-CoV-2 may also help mitigate lung, heart, and kidney damage caused by COVID-19 given the role of soluble ACE2 in regulating blood pressure.
  • Soluble fragments of ACE2 have been used, for example, in mouth and nose washing solutions that inactivate the virus and allow it to be eliminated. These washing solutions can prevent infection in people who have been exposed to infected people for a short period of time, or reduce the viral load in the mouth and nose of highly infectious people to reduce the risk of transfer.
  • patent document WO 2021/228854 discloses the neutralization of the SARS-COV-2 virus by means of a washing solution that comprises a soluble fragment of the ACE2 receptor, which comprises the peptidase domain or a fragment and/or derivative. of the same. Said soluble ACE2 receptor fragments are modified by introducing mutations in at least 13 positions of the PD domain to inactivate the catalytic activity of ACE2, while the amino acids of the S protein binding region remain unchanged.
  • FIG. 2020 Another example can be found in the document by Romano et al. (2020) describes the creation of a modified miniprotein expressed recombinantly in E. coli (termed Spikeplug), which acts as a soluble and stable cap of protein S.
  • the miniprotein herein was designed using the Helix 1-Helix region. 2 of ACE2 as a basic scaffold and a series of mutations were included to increase its stability and solubility and to compensate for missing interactions mediated by residues outside helices H1 and H2. Also An extra helix was added to guarantee helical folding of the miniprotein in solution.
  • the soluble plug can interact with the RBD of the SARS-CoV-2 S protein with nanomolar affinity of 14.7 nM with a dissociation constant of 40 nM.
  • ACE2 spike protein binding peptides
  • WO2021188966A1 describes peptides of 6-100, 6-70 or 6-25 amino acids that specifically bind to a receptor binding domain (RBD).
  • RBD receptor binding domain
  • WO2021216845A1 provides antiviral peptides between 20 and 100 amino acids in length that inhibit the interaction between helix 1 of ACE2 and the receptor binding domain of SARS-CoV-2 type viruses.
  • W02021203098A2 provides binding proteins that may include the extracellular portion of ACE2, excluding the collectrin domain, and a flexible flexible polypeptide linker that couples the ACE2 portion to an Fe domain.
  • ACE2 Since administration of ACE2 can potentially excessively reduce blood pressure and affect kidney function, there is a need to produce high affinity soluble versions that can be used at low doses with few adverse effects. Thus, developing versions of the ACE2 protein with improved binding to RDB may help decrease the amount of protein that needs to be administered and reduce the occurrence of adverse effects. Additionally, it has been observed that the use of short versions of ACE2 may be particularly useful to prevent and/or treat kidney disease associated with COVID1 -9.
  • CAR T cells chimeric antigen receptor T cells that combine, in a single chimeric protein, a specific antigen recognition domain (e.g., derived from an antibody or a receptor) with an intracellular domain. T cell receptor activator. This strategy allows a patient's T cells to be given the ability to recognize and kill infected cells directly. Furthermore, CAR T cells (CAR-T cells) have the ability to multiply in vivo, which is why they are considered a living active ingredient.
  • NK-CAR cells which describes the genetic engineering of primary human CD8 T cells to express specific CARs of the S protein with the extracellular domain of ACE2 (ACE2 CAR) from the sequence obtained in Ensembl Gene Browser, which selectively kills the cells that expressed the S protein.
  • ACE2 CAR ACE2 CAR
  • NK-CAR cells Therapy with natural killer cells (NK cells) that express a CAR may serve as an alternative to CAR T-cell therapy because NK-CAR cells have considerably fewer safety concerns, such as off-target effects.
  • CAR-NK cells the extracellular, transmembrane, and intracellular signaling domains are present as they are in CAR-T cells.
  • NK-CAR cells often have CD3 as an initial signaling domain and CD28 or CD137 (4-1 BB) as a costimulatory domain to form an intracellular signaling motif, both of which increase the cytotoxic capacity of NK cells and the production of cytokines is increased through two more costimulatory molecules, namely, NKG2D and CD244.
  • CAR-T cell immunotherapy may have side effects, such as cytokine release syndrome and T cell exhaustion.
  • Another potential disadvantage could be that ACE2 in the ACE2 CAR may interact with its physiological ligands and interfere with the regulation of blood pressure. Consequently, the development of ACE2 CAR NK cells that have improved affinity for protein S may contribute to decreasing such side effects.
  • the present technology refers to the design and construction of miniature versions of the human angiotensin-converting enzyme 2 (hACE2), called min ⁇ ACE2, from the molecular surface regions of ACE2 that mediate the interaction of ACE2 with the RBD domain of the SARS-CoV-2 spike protein.
  • hACE2 human angiotensin-converting enzyme 2
  • min!ACE2 neutralize the contact reaction of the S protein of SARS-CoV-2 by interacting with its amino acids that bind to the natural receptor of the cells (hACE2), so it is predicted that the mutations that potentially allow the virus to evade interaction with min!ACE2, negatively impact the binding capacity to the natural ACE2 receptor and therefore affect its ability to infect human cells. Therefore, an additional effect of the use of min ⁇ ACE2 is to prevent the occurrence of escape variants of the SARS-CoV-2 virus.
  • the min ⁇ ACE2 of the invention can also be used to selectively retain the SARS-CoV-2 virus, through affinity binding to the S protein of SARS-CoV-2, this can be exploited in the production of tests diagnostics or methods to concentrate the virus from biological or environmental samples.
  • the invention also relates to the salts, solvates, hydrates, isomers and tautomers of the miniACEs of the invention.
  • compositions comprising the min ⁇ ACE2 of the invention, or a salt, solvate, hydrate, isomer or tautomer thereof.
  • These compositions are specially formulated to allow min ⁇ ACE2 to be infused into the circulation of patients infected with SARS-CoV-2 to neutralize the infectivity of the virus, reduce the viral load and prevent the severe effects caused by COVID19.
  • the soluble miniACE2 disclosed here by interacting strongly with the RBD of the S protein on the viral surface, coat the virus in a way that blocks its interaction with the natural ACE2 receptor on the surface of human cells and therefore neutralizes the ability infectivity of the SARS-CoV-2 virus (see Figure 1).
  • the present invention also contemplates the design and development of chimeric antigen receptors (CAR by its name in English "Chimeric antigen receptor"), which comprise surface domains based on minIACE2. These CARs can be incorporated into Natural Killer cells (NK cells) to give them the ability to recognize infected cells that present the S protein on their cell surface and eliminate them specifically, thus reducing the reservoirs of the SARS-CoV-2 virus and the side effects on cells other than the target cells. Likewise, the methods for producing the CARs of the invention and the compositions that comprise them are also part of the present application.
  • CAR by its name in English "Chimeric antigen receptor”
  • NK cells Natural Killer cells
  • the methods for producing the CARs of the invention and the compositions that comprise them are also part of the present application.
  • the present invention also contemplates genetically modifying mesenchymal cells to induce the secretion of miniACE2 in them in order to reduce the severe pulmonary effects in severe cases of COVID19.
  • genetic modification of mesenchymal cells can be beneficial since that these have mainly pulmonary tropism, which would allow min ⁇ ACE2 to be concentrated in one of the organs most affected by COVID19.
  • the present invention also provides NK cells and mesenchymal cells modified to express the CARs or miniACEs of the invention.
  • Another aspect of the present application relates to the use of min ⁇ ACE2 and CARs according to embodiments of the present invention in methods for the treatment of the human or animal body by surgery or therapy, as a vaccine or in diagnostic methods practiced on the human body or animal body.
  • Still another aspect of the present invention refers to methodologies for diagnosing SARS-CoV-2 from different types of samples based on the retention of the virus by affinity using the min ⁇ ACE2 disclosed here.
  • Figure 1 shows a scheme of the neutralization of SARS-CoV-2 by the min ⁇ ACE2 of the invention.
  • the soluble miniACE2s of the present invention strongly interact with the spike protein of SARS-CoV-2 by blocking the interaction of the virus with the ACE2 receptor expressed on the surface of human cells.
  • Figure 2 shows a multiple alignment of the partial sequence of the N-terminal domain of the native ACE2 protein (corresponding to residues 1 to 420) and the sequences of the three min ⁇ ACE2 of the invention BP2 (SEQ ID NO : 9), BP2 (SEQ ID NO: 13) and BP12 (SEQ ID NO: 17), where the 3 discontinuous conformational regions (R1, R2 and R3) of the native ACE2 and the regions R1, R2 and R3 are observed of the min!ACE2 of the invention, each enclosed within the respective black boxes, with their corresponding connector sequences (SC1 -2, SC2-3, and SC1 -3).
  • the alignment also shows the position of R1, R2 and R3 with respect to the structure of the native ACE2 receptor, and indicates the residues that participate directly in the interaction (underlined residues), as well as amino acids that have been substituted or deleted with respect to the native ACE2 sequence (residues in bold).
  • R1 encompasses helices H2, H3, H4 and H5 of ACE2
  • R2 encompasses helix H19
  • R3 encompasses two folded sheets (C), linked by a disulfide bridge and a beta hairpin, and helix H20.
  • Figure 3 shows constructs for the expression of the CARs of the invention in NK cells.
  • Figure 4 shows constructs for modification of mesenchymal cells.
  • Figure 5 shows constructs for inducible protein expression in CHO cells.
  • Figure 6 shows constructs for inducible protein expression in yeast of the genus Pichia spp.
  • Figure 7 shows constructs for inducible expression of RFP-tagged protein in yeast of the genus Pichia spp.
  • Figure 8 shows the three-dimensional structure of the miniACEs of the invention A) BP2, B) BP9 and C) BP1 1.
  • Figure 9 shows the three-dimensional structure of the CARs of the invention A) BP2-CAR1, B) BP9-CAR1, C) BP11-CAR1, D) BP2-CAR2, E) BP9-CAR2, F) BP11 - CAR2, G) BP2-CAR3, H) BP9-CAR3, I) BP11-CAR3, J) BP2-CAR4, K) BP9-CAR4, L) BP11 -CAR4, M) BP2-CAR5, N) BP9-CAR5, and O) BP11 -CAR5.
  • SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2, which is the virus that causes a respiratory illness known as coronavirus disease 2019 (COVID-19). 19). This virus belongs to the Coronaviridae family, a family of lipid-enveloped positive-sense RNA viruses.
  • SARS-CoV-2 preferably refers to all variants of SARS-CoV-2, but also to all variants that show single mutations or multiple mutations or combinations of mutations, but are still They name it as SARS-CoV-2.
  • ACE2 refers to the soluble or membrane-bound angiotensin-converting enzyme 2 (ACE2).
  • ACE2 comprises a sequence of 805 amino acids reported in UniProtKB with the ID Q9BYF1.
  • ACE2 It comprises an N-terminal M2 metallopeptidase activity domain (amino acids 19-615), a C-terminal collection renal amino acid transporter domain (amino acids 616-768), a neck domain (amino acids 616-726), a transmembrane anchor and an intracellular segment.
  • ACE2 peptidase domain or “peptidase domain” refer to the peptidase domain of ACE2 and consists of two subdomains or lobes that together form the active site of the enzyme and are linked by an alpha helix.
  • the peptidase domain of ACE2 lacks the neck domain of ACE2.
  • S protein refers to the trimeric glycoprotein protruding from the surface of the SARS-CoV-2 virion, in which each monomer has two subunits (S1 and S2) separated by a cleavage site.
  • RBD domain of protein S refers to the receptor binding domain (RBD) contained in subunit 1 (S1) of protein S that binds directly to the peptidase domain of protein S. ACE2.
  • the RBD domain is composed of a 5-stranded antiparallel beta sheet between strands (34-7 and is an extended insertion of short strands [35-6.
  • the extension carries the receptor binding motif (RBM) that binds to the receptor ACE2 to enter cells.
  • contact region refers to the particular region(s) of the peptidase domain of ACE2 and the RBD domain of protein S where the proteins interact/associate with each other.
  • the contact interface comprises one or more amino acid residues that are capable of interacting with the corresponding amino acid residues that come into contact or association when the protein-receptor interaction occurs. Generally, the interaction is mediated by the formation of hydrogen bonds.
  • ACE2 discontinuous conformational regions refers to the regions of native ACE2 that form three-dimensional structures that participate in the contact interface with the RBD domain of the S protein.
  • the three conformational regions are located discontinuously in the linear sequence of ACE2 from one another, separated by connecting segments of amino acids that do not directly participate in the contact interface.
  • miniACE2 discontinuous conformational regions refers to the discontinuous conformational regions of the miniACE2 of the invention that may comprise substitutions, insertions or deletions of amino acids with respect to to the native ACE2 sequence that improve the affinity of min!ACE2 with the RBD domain of the S protein.
  • miniACE2 refers to chimeric proteins that block the interaction of SARS-CoV-2 with the native ACE2 receptor. It is a miniature version of the ACE2.
  • the min!ACE2 may comprise two or three of the discontinuous conformational regions R1, R2 and R3 of ACE2, where one or more amino acids have been modified or eliminated with respect to the sequence of the native ACE2 with the aim of improving the interaction of the min ⁇ ACE2 with RBD.
  • R1, R2 and R3 are linked by two or three amino acid connecting segments.
  • min ⁇ ACE2 comprises R1, R2 and R3, they are connected by two connector segments.
  • min!ACE2 comprises R1 and R3, they are connected by a connector segment.
  • the min!ACE2 can be cloned and expressed recombinantly from genes that encode the respective amino acid sequence by techniques widely known in the state of the art, for example, by expression systems in mammalian cells, such as cells CHO, HEK293 or mesenchymal cells, and in yeasts, such as yeasts of the genus Pichia.
  • amino acid linker segments refers to at least three amino acid segments that connect each of the discontinuous conformational regions of min ⁇ ACE2 and that allow min ⁇ ACE2 to maintain the three-dimensional structure of the contact matrix. with the RBD domain.
  • the amino acid linker segments are selected in such a way as to ensure correct folding of the min!ACE2s of the invention and to maximize their interaction with RBD.
  • the amino acid connecting segments can be of variable length. For example, amino acid linker segment segments may comprise between 1 to 15 amino acids.
  • the min ⁇ ACE2 comprises one or two linker segments depending on whether the min ⁇ ACE2 comprises two or three conformation regions of ACE2.
  • the connector segment that connects R1 with R2 is called SC1 -2.
  • the connector segment that connects R2 to R3 is called SC2-3 and the connector segment that connects R1 to R3 is called SC1 -3.
  • chimeric antigen receptor (CAR) miniACE2 or “CAR-miniACE2” refers to a chimeric protein that comprises a miniACE2 of the invention as an extracellular antigen recognition domain, linked through a region connector or hinge to a transmembrane region, followed by an intracellular signaling domain that activates the functioning of the immune cell.
  • CAR-miniACE2 NK cells refers to natural killer cells (NK cells) genetically modified to express a CAR-miniACE2 as an extracellular antigen recognition domain linked to an intracellular signaling domain through a region connector or hinge from the protein CD8, CD28, CD16, ICOS, CD137 or lgG1 and a transmembrane region selected from CD8, CD28, CD16, 0X40 or ICOS. Additionally, NK cells with CAR-miniACE2 also have a costimulatory domain of CD16, CD28 CD137, 0X40 or ICOS and a CD3z domain as an intracellular signaling domain. NK cells with CAR-miniACE2 are generally obtained from autologous NK cells of the patient to be treated or from umbilical cord blood. Because NK cells do not require HLA compatibility, they can be used as allogeneic effector cells.
  • 'CAR-miniACE2 mesenchymal cells refers to mesenchymal cells genetically modified to express a CAR-miniACE2 as an extracellular antigen recognition domain linked to a signaling domain.
  • substitution refers to the presence of an amino acid residue at a certain position in the derived sequence that is different from the amino acid residue that is present or absent at the corresponding position in the reference sequence.
  • a derivative of the present invention may exhibit 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of such substitutions.
  • percent sequence identity refers to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences and amino acid sequences) in a comparison window, in which the Part of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result times 100 to produce the percent sequence identity.
  • the term "therapeutically acceptable” refers to compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.) that are suitable for use in contact with the tissues of a patient without excessive toxicity, irritation or allergic response, with a reasonable benefit/harm, and that are effective for their intended use.
  • the invention relates to miniature versions or soluble fragments of human angiotensin-converting enzyme 2 (hACE2), called min ⁇ ACE2, which specifically bind to the S protein of SARS-CoV-2. More specifically, the min!ACE2 of the invention specifically bind to the RBD domain of the S protein.
  • the min!ACE2 of the invention show a higher affinity for the RBD domain compared to native ACE2.
  • the binding strength of the native ACE2 receptor and the RBD in terms of binding affinity (AG) is: -1 1.9 and the dissociation constant (Kd) is 3.8x10' 9 .
  • the min ⁇ ACE2 of the invention comprise at least three discontinuous conformational regions of ACE2, called R1, R2 and R3 that may or may not be modified with respect to the regions R1, R2 and R3 of the native ACE2 (which correspond to the SEQ ID Nos: 1, 2 and 3).
  • the discontinuous conformational regions R1, R2 and R3 of the min!ACE2 comprise amino acid sequences that form three-dimensional structures that participate in the formation of contact with the RBD, but the amino acid sequence is not consecutive from one to the other.
  • R1, R2 and R3 of the min ⁇ ACE2 comprise substitutions, insertions or deletions of amino acids with respect to the native ACE2 that improve the affinity of the min ⁇ ACE2 for the native receptor, and that facilitate its correct folding.
  • the discontinuous conformational regions of minIACE2 comprise the amino acids at positions R1a: 24-45, R1b: 62-96; R2: 324 - 334; R3a: 341 - 359, and R3b: 363 - 385 of native ACE2 ( Figure 2).
  • the modifications introduced eliminate N-glycosylation sites.
  • minIACE2 contains a region R1: 20 - 96, without alterations in the sequence with respect to native ACE2, a region R2: 324 - 334 with the substitution of thptophan for serine at position 328 ( W328S), and a region R3: 341 - 362 without alterations in the sequence with respect to native ACE2.
  • min ⁇ ACE2 contains R1: 21 - 96, with a substitution of asparagine for glutamine at positions 53 and 90 (N53Q and N90Q), thus eliminating N-glycosylation sites, and R3: 341 - 385 with a substitution of lysine for glycine at position 341 (K341 G) and methionine for serine at position 360 (M360S).
  • the min ⁇ ACE2 contains R1: 21 - 96, with substitution of asparagine for glutamine at position 90 (N90Q), thus eliminating an N-glycosylation site, and R3: 341 - 385 with a substitution of lysine for glycine at position 341 (K341 G) and of methionine for serine in the 360 position (M360S).
  • R1 has the amino acid sequence set forth in SEQ ID NO: 4
  • R2 has the amino acid sequence set forth in SEQ ID NO: 6
  • R3 has the amino acid sequence set forth in SEQ ID NO: 8.
  • the min ⁇ ACE2 of the invention comprise discontinuous conformational regions R1, R2 and R3 linked together by amino acid linker segments, with lengths between 1 and 15 amino acids, more preferably lengths between 1 to 10 amino acids. More preferably, the R1, R2 and R3 regions are linked by one or two amino acid linker segments. Preferably, the amino acid linker segments are selected so that the correct folding of the min!ACE2s is favored and the interaction with RBD is maximized. More preferably, the linker segments comprise amino acid sequences that enhance their affinity to the RBD domain of the S protein while maintaining the conformational structure of the ACE2 binding base. In preferred embodiments the linkers comprise between 1 and 10 amino acid residues, preferably selected from serine and glycine. In some embodiments the linker has a single amino acid.
  • min ⁇ ACE2 comprises a first connector segment connecting R1 and R2 and a second connector segment connecting R2 to R3.
  • the miniACE2 comprises a connector segment connecting R1 and R3.
  • the first connector segment may have the sequence NGTIYSTG (SEQ ID NO: 5) and the second connector segment the sequence S (SEQ ID NO: 7).
  • the connector segment may have the sequence LQALQQNSGSG (SEQ ID NO: 1 1 or 15).
  • the min ⁇ ACE2 of the invention comprises an amino acid sequence that has at least 90% identity with the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 1 1 and SEQ ID NO: 14.
  • the min!ACE2 of the invention comprises an amino acid sequence selected from the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 1 1 and SEQ ID NO: 14.
  • the invention in a second embodiment, relates to a chimeric antigen receptor (CAR) that recognizes protein S. More specifically, the CAR of the invention specifically binds to the RBD domain of protein S. Such CARs may be used in NK cells to give them the ability to recognize the S protein in infected cells and specifically eliminate them to reduce the cellular reservoirs of the SARS-CoV-2 virus.
  • CAR chimeric antigen receptor
  • the CAR of the invention comprises a specific antigen recognition domain that has at least 90% identity with one of the min!ACE2 of the amino acid sequences set forth in SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 14.
  • the CAR-miniACE2 of the invention comprises a specific antigen recognition domain with an amino acid sequence selected from the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 1 1 and SEQ ID NO: 14.
  • the CAR of the invention comprises a linker or hinge region derived from the protein CD8 (SEQ ID NO: 35), CD28 (SEQ ID NO: 36), CD16 (SEQ ID NO: 37), ICOS (SEQ ID NO: 38), CD137 or lgG1.
  • the CAR of the invention also comprises a transmembrane region derived from CD8 (SEQ ID NO: 39), CD28 (SEQ ID NO: 40), CD16 (SEQ ID NO: 41), 0X40 (SEQ ID NO: 42) and ICOS ( SEQ ID NO: 43).
  • the CAR of the invention also comprises a costimulatory domain derived from the proteins CD16 (SEQ ID NO: 44), CD28 (SEQ ID NO: 45), CD137 (SEQ ID NO: 47), 0X40 (SEQ ID NO: 46), CD137 (SEQ ID NO: 47) or ICOS (SEQ ID NO: 48).
  • the CAR of the invention also comprises the CD3z domain for signal transduction (SEQ ID NO: 49), and a guide peptide derived from CD8 (SEQ ID NO: 33) or CD16 (SEQ ID NO: 34).
  • the CAR-miniACE2 of the invention is expressed in NK cells through transduction with lentivirus or retrovirus vectors.
  • NK cells with CAR-miniACE2 comprise the chimeric gene encoding the CAR protein followed downstream by the complete interleukin 15 (IL-15) gene, including a recognition target for fuhna upstream of the sequence encoding the guide peptide with in order for the protein to be cleaved correctly so that it is secreted by NK cells with CAR-miniACE2.
  • IL-15 interleukin 15
  • min!ACE2 blocking proteins comprised one, two or all of the R1, R2 and R3 regions of the native ACE2 receptor modified or without modification and joined by amino acid linker segments derived from the peptidase domain of the native ACE2 to put the modified and unmodified regions R1, R2 and R3 together.
  • modifications could comprise amino acid substitutions or deletions. For example, substitutions to remove N-glycosylation sites.
  • the resulting min ⁇ ACE2 sequences were used to model the 3D structure using the Robetta protein structure prediction service (https://robetta.bakerlab.orq/) and the Phyre2 program.
  • a structural alignment was carried out on the native ACE2 models available in the Protein Data Bank (PDB) database (6VXX, 6LZG, 6VSB, 6M0J), with the help of the Pymol software.
  • PDB Protein Data Bank
  • Synthetic genes were constructed to express the amino acid sequences of the min ⁇ ACE2 proteins according to the amino acid sequences of SEQ ID NO: 7, 11 and 14 in a CHO or HEK293 cell system (see Figure 5 ), in yeasts of the genus Pichia (See Figure 6) or mesenchymal cells (see Figure 4).
  • the amino acid sequence of the min ⁇ ACE2 candidates were used to construct the corresponding genes, including restriction targets to facilitate their cloning and the nucleotide sequence of these was optimized for expression in Pichia pastoris,
  • the secretion signal of the factor a of Saccharomyces cerevisiae is also included, which promotes the efficient translocation and secretion of the recombinant protein to the culture medium.
  • a tail of 6 histidines is added that facilitates the process of purification.
  • Gene expression is under the control of the methanol-inducible AOX1 gene promoter.
  • the genes were subcloned into the pPICZaA expression vector.
  • the plasmid DNAs were constructed: pZ-H-Xa-BP2 (SEQ ID NO: 49), pZ-H-RFP-Xa-BP2 (SEQ ID NO: 50), pZ-H-Xa-BP9 (SEQ ID NO: 51), pZ-H-RFP-Xa-BP9 (SEQ ID NO: 52), pZ-H-Xa-BP1 1 (SEQ ID NO: 53) and pZ-H-RFP-Xa-BP1 1 ( SEQ ID NO: 54) (see Figures 6 and 7), using the pPICZaA expression vector as a skeleton, sequences to facilitate the detection and purification process of the recombinant protein were included in the plasmid DNA constructs as the inclusion of the sequence which encodes the red fluorescent protein (RFP), a 6-histidine tail and a cleavage site with the Xa protease.
  • RFP red fluorescent protein
  • the recombinant expression vector for each of the proteins of interest was linearized by digestion with the restriction enzyme Pmel to transform competent Pichia pastor ⁇ s GS1 15 cells by electroporation.
  • Recombinant P. pastor ⁇ s clones were selected on YPDS plate (YPD medium with 1 M sorbitol) with a zeocin concentration of 100 pg/mL, and the cells were incubated for 3 days, at 30° C. Successful integration of the plasmid Recombinant was confirmed by POR and sequencing of the amplified products.
  • the expression of the gene is under the control of a TET-ON promoter, to increase the stability and expression of the mRNA, Combinations of the 5' UTR region of the human cytochrome P450 gene (NCBI RefSeq: NM_000773.4) and the 3' UTR regions of the genes for apolipoprotein A2 (NCBI RefSeq: NM_001643.2) and the alpha subunit will be added to the gene.
  • eF1 a elongation factor 1 a
  • Example 3 Obtaining genetically modified NK cells with the expression of CAR-miniACE2 to eliminate infected cells that act as a reservoir of the virus.
  • NK cells recognize cells infected with SARS-Cov-2 virus due to the presence of the S protein on their surface.
  • the surface domain of the CAR that confers recognition was designed so that it corresponded to each of the min!ACE2 of SEQ ID NO: 9, 13 and 17, followed by a linker or hinge region from the CD8 protein (SEQ ID NO: 35), CD28 (SEQ ID NO: 36), CD16 (SEQ ID NO: 37), ICOS (SEQ ID NO: 38), CD137 or lgG1.
  • the transmembrane region was selected from the transmembrane regions of CD8 (SEQ ID NO: 39), CD28 (SEQ ID NO: 40), CD16 (SEQ ID NO: 41), 0X40 (SEQ ID NO: 42) or ICOS (SEQ ID NO: 43).
  • the costimulatory domain was selected from the costimulatory domains of CD16 (SEQ ID NO: 44), CD28 (SEQ ID NO: 45), 0X40 (SEQ ID NO: 46), CD137 (SEQ ID NO: 47) or ICOS (SEQ ID NO: 48).
  • the guide peptide was derived from CD8 (SEQ ID NO: 33) or CD16 (SEQ ID NO: 34).
  • the CD3z domain (SEQ ID NO: 39) was included as a recognition domain for function upstream of the sequence that encodes the guide peptide so that the protein is correctly cleaved to be secreted by NK cells.
  • the complete amino acid sequence of the CARs designed here is shown in Table 4.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Mycology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des versions en miniature de l'enzyme de conversion de l'angiotensine 2 (miniACE2) avec une liaison améliorée à la protéine spike du SARS-CoV-2 qui, avec sa liaison à celle-ci, permettent de bloquer l'interaction du virus du SARS- CoV-2 avec le récepteur naturel dans la cellule hôte humaine. L'invention concerne également l'utilisation des miniACE2 pour traiter ou prévenir l'infection par le coronavirus avec miniACE2 chez l'homme et l'animal et pour servir de substrat de capture de particules virales. L'invention comprend également des récepteurs d'antigène chimériques (CAR) conçus à partir des miniACE2 pour reconnaître spécifiquement les cellules infectées par le SARS-CoV-2 qui peuvent être incorporés dans des cellules NK. Les miniACE2 ou les cellules NK avec CAR basées sur les miniACE2 peuvent être administrées à des personnes atteintes de la COVID-19 pour induire une réponse immune cellulaire contre les cellules infectées avec le SARS-CoV-2.
PCT/IB2023/054335 2022-04-27 2023-04-27 Protéines miniace2 solubles qui interagissent avec le sars cov 2 et utilisations de ces dernieres Ceased WO2023209619A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CONC2022/0005322A CO2022005322A1 (es) 2022-04-27 2022-04-27 Proteínas miniace 2 solubles que interaccionan con sars cov 2 y usos de las mismas
CONC2022/0005322 2022-04-27

Publications (1)

Publication Number Publication Date
WO2023209619A1 true WO2023209619A1 (fr) 2023-11-02

Family

ID=86059789

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2023/054335 Ceased WO2023209619A1 (fr) 2022-04-27 2023-04-27 Protéines miniace2 solubles qui interagissent avec le sars cov 2 et utilisations de ces dernieres

Country Status (2)

Country Link
CO (1) CO2022005322A1 (fr)
WO (1) WO2023209619A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210388031A1 (en) * 2020-06-16 2021-12-16 New Jersey Institute Of Technology Designer Peptide Opsonins
WO2022035998A1 (fr) * 2020-08-11 2022-02-17 City Of Hope Compositions et utilisations de cellules nk modifiées d'un récepteur antigénique chimérique ciblant le sars-cov-2

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210388031A1 (en) * 2020-06-16 2021-12-16 New Jersey Institute Of Technology Designer Peptide Opsonins
WO2022035998A1 (fr) * 2020-08-11 2022-02-17 City Of Hope Compositions et utilisations de cellules nk modifiées d'un récepteur antigénique chimérique ciblant le sars-cov-2

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "A Phase I/II Study of Universal Off-the-shelf NKG2D-ACE2 CAR-NK Cells for Therapy of COVID-19", CLINICALTRIALS.GOV, 27 March 2020 (2020-03-27), XP093107570, Retrieved from the Internet <URL:https://classic.clinicaltrials.gov/ct2/show/NCT04324996?term=CAR&cond=COVID-19&draw=2&rank=1> [retrieved on 20231130] *
MARIA ROMANO, RUGGIERO ALESSIA, SQUEGLIA FLAVIA, BERISIO RITA: "An engineered stable mini-protein to plug SARS-Cov-2 Spikes", BIORXIV, 29 April 2020 (2020-04-29), XP055737733, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.04.29.067728v1.full.pdf> [retrieved on 20201007], DOI: 10.1101/2020.04.29.067728 *
YANXIAO HAN, PETR KRáL: "Computational Design of ACE2-Based Peptide Inhibitors of SARS-CoV-2", ACS NANO, AMERICAN CHEMICAL SOCIETY, US, vol. 14, no. 4, 28 April 2020 (2020-04-28), US , pages 5143 - 5147, XP055732485, ISSN: 1936-0851, DOI: 10.1021/acsnano.0c02857 *

Also Published As

Publication number Publication date
CO2022005322A1 (es) 2023-04-27

Similar Documents

Publication Publication Date Title
CN111560074B (zh) 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白单区域亚单位纳米疫苗
CN111607002B (zh) 一种基于幽门螺旋杆菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111217919B (zh) 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
ES2753854T3 (es) Antígenos del virus de la inmunodeficiencia humana, vectores, composiciones y métodos de uso de los mismos
US20230190917A1 (en) Viral vaccine vector for immunization against a betacoronavirus
CN111217918A (zh) 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗
JP2003517006A (ja) ５−ヘリックスタンパク質
US6497873B1 (en) Recombinant Rhabdovirus containing a heterologous fusion protein
WO2022096899A1 (fr) Protéines de spicule virale et fusion de celles-ci
CN113264990B (zh) 抑制新型冠状病毒(sars-cov-2)的多肽和其应用
KR20130063497A (ko) 하나 이상의 ｃｘｘｃ 모티프를 포함하는 폴리펩티드 및 이종 항원을 포함하는 약학 조성물 및 이의 용도
KR20230084478A (ko) 면역원성 코로나 바이러스 융합 단백질 및 관련 방법
CN112226413A (zh) 基于SARS-CoV-2 S蛋白RBD区域的靶向性外泌体及其制备方法
CN101007168B (zh) 一种sars疫苗及其制备方法
US20230090422A1 (en) Novel coronavirus s protein double-region subunit nano-vaccine based on bacterial complex
JP5823296B2 (ja) Ｐｉｖ−５及びｐｉｖ−２のｆタンパク質の突然変異タンパク質
ES2326217T3 (es) Glicoproteinas quimericas mejoradas y vectores lentivirales pseudotipados.
WO2023209619A1 (fr) Protéines miniace2 solubles qui interagissent avec le sars cov 2 et utilisations de ces dernieres
JP5813509B2 (ja) Ｐｉｖ−５およびｐｉｖ−２のｆタンパク質の突然変異タンパク質
JP2018052953A (ja) インフルエンザウイルスワクチンおよびその使用
KR20140046994A (ko) 인간 ＮＬＢＰ 유래의 ＮＰ2 폴리펩티드 또는 ｄＮＰ2 폴리펩티드를 포함하는 세포 투과 펩티드 및 이를 이용한 카고 전달 시스템
CN119161495B (zh) 呼吸道合胞病毒重构多肽及其应用
AU2022317127B2 (en) Modified t cell receptors for the prevention and treatment of viral infections and cancer
KR20230136628A (ko) Ace2-수용체 엑토도메인 융합 분자 및 이의 용도
WO2025102071A1 (fr) Polypeptides d&#39;hémagglutinine et leurs utilisations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23795755

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 23795755

Country of ref document: EP

Kind code of ref document: A1