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WO2021201679A1 - Compositions et procédés ciblant des coronavirus - Google Patents

Compositions et procédés ciblant des coronavirus Download PDF

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Publication number
WO2021201679A1
WO2021201679A1 PCT/NL2021/050207 NL2021050207W WO2021201679A1 WO 2021201679 A1 WO2021201679 A1 WO 2021201679A1 NL 2021050207 W NL2021050207 W NL 2021050207W WO 2021201679 A1 WO2021201679 A1 WO 2021201679A1
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Prior art keywords
antibodies
cell
cov
cells
sars
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Robert Heinz Edward Friesen
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Kiadis Pharma Intellectual Property BV
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Kiadis Pharma Intellectual Property BV
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention relates to pharmaceutical compositions for treating or preventing infection by coronaviruses as well as coronavirus mediated disease, e.g., respiratory disorders.
  • the disclosure provides treatments comprising immune cells, such as natural killer cells, and antibodies or vaccines that target coronaviruses. Compositions and kit-of-parts comprising said immune cells and antibodies or vaccines are also provided.
  • the disclosure also provides engineered immune cells expressing a chimeric antigen receptor (CAR). Such CAR expressing cells are useful for treatment.
  • Exemplary viral targets of the present invention include the coronavirus spike protein, in particular evolutionary conserved epitopes thereof.
  • Coronaviruses are a significant threat to human health. Coronaviruses are a group of RNA viruses that derive its name from the characteristic appearance of its virions, which carry a “corona” of spike protein peplomers. The original genus of coronavirus has been subdivided into four genera; Alphacoronavirus, Betacoronavirus,
  • Gammacoronavirus and Deltacoronavirus Of these genera, Alphacoronavirus and Betacoronavirus infect mammals, whereas Gamma- and Deltacoronavirus primarily infect birds. As of 2020, at least 39 types of coronavirus have been officially recognized. Several of these types are coronaviruses that readily infect humans (i.e., human coronaviruses) and produce mild symptoms like the common cold (e.g., 229E, NL63, OC43, and HKU1).
  • MERS Middle East respiratory-syndrome-related coronavirus
  • SARS-CoV-1 and SARS-CoV-2 severe acute respiratory syndrome-related coronaviruses
  • SARS-CoV-1 and SARS-CoV-2 severe acute respiratory syndrome-related coronaviruses
  • SARS-CoV-2 severe acute respiratory syndrome-related coronaviruses
  • the symptoms from these viruses are much more severe and have higher fatality rates that the “common cold” coronaviruses.
  • Vaccination strategies against coronavirus are primarily directed to the spike protein, which is in most coronaviruses responsible for interaction with the host cell. On average, each virion has 74 spikes, and each spike is composed of 3 spike proteins.
  • the spike protein itself is composed of an SI and an S2 subunit.
  • the spike protein interacts with the host cell via the receptor binding domain (RBD), which is present on the SI subunit.
  • RBD receptor binding domain
  • the SI subunit exists of four core domains: S1 A through Si D .
  • SI proteins are highly variable among different species, and can bind different receptors on the host cell, depending on the N-terminal domain (Sl-NTD) and C- terminal domain (Sl-CTD).
  • Sl-NTD N-terminal domain
  • Sl-CTD C- terminal domain
  • the host-receptor for SARS-CoV-1 and SARS-CoV-2 is Angiotensin-converting enzyme 2 (ACE2), which is bound by the S1 B domain, while dipeptidyl peptidase 4 (DPP4) is the host receptor for MERS-CoV.
  • ACE2 Angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • the spike protein shows variability between coronavirus species and mutates over time. See, e.g., table 1 of US8, 106,170 which describes a number of different SARS- CoV isolates and their respective spike protein sequences. This results in the necessity to generate new vaccines with every outbreak. While a number of therapies and vaccines against SARS-CoV-2 are under investigation, effective treatments are still required.
  • One object of the invention is to provide effective treatments for SARS-CoV-1, SARS- CoV-2, and/or MERS-CoV.
  • a further object is to provide a broad-spectrum treatment for coronavirus infections.
  • a further object of the invention is to provide treatments for immunocompromised and at-risk individuals individuals.
  • the disclosure provides an engineered immune cell expressing a chimeric antigen receptor (CAR), wherein the CAR binds to a coronavirus spike (S) protein.
  • CAR chimeric antigen receptor
  • S coronavirus spike
  • the disclosure further provides engineered immune cells as disclosed herein for use in the treatment and/or prevention of coronavirus infection and/or coronavirus mediated disease in an individual.
  • the disclosure further provides a method of treating or preventing coronavirus infection and/or coronavirus mediated disease in an individual, said method comprising administering to an individual in need thereof an effective amount of engineered immune cells as described herein.
  • the individual is immunocompromised or at high-risk.
  • the CAR binds to the spike protein from SARS-CoV-1, MERS-CoV, and/or SARS-CoV-2.
  • the CAR binds to the spike protein from SARS-CoV-1 and SARS-CoV-2.
  • the CAR binds to the spike protein from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.
  • the CAR binds to a coronavirus spike (S) protein from at least two different coronaviruses.
  • the immune cell or engineered immune cell expresses an NK cell activating receptor such as NKp46, NKp44, NKp30, NKG2C and/or NKG2D.
  • the immune cell or engineered immune cell is a natural killer cell.
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of NK cell stimulating feeder cells, NK cell stimulating molecules, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • CAR chimeric antigen receptor
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of an NK cell stimulating feeder cell that is a K562 cell that expresses one or more of IL-15, IL-21 and/or 4-1BBL; and optionally wherein said cell culture further comprising IL-2.
  • the initial population of NK cells is derived or isolated from at least one of peripheral blood, placental blood, lymphatic fluid, iPSCs, ESCs, MSCs, or an CD56+CD3- cell-containing source.
  • the disclosure further provides pharmaceutical composition comprising a collection of the engineered immune cells as disclosed herein.
  • the disclosure further provides a pharmaceutical composition comprising a collection of immune cells and one or more antibodies that bind to a coronavirus spike (S) protein.
  • the disclosure further provides immune cells (including engineered immune cells) as disclosed herein and one or more antibodies that bind to a coronavirus spike (S) protein for use in the treatment or prevention of coronavirus infection and/or coronavirus mediated disease in an individual.
  • the antibodies and immune cells may be provided in a single composition, such as the pharmaceutical composition described herein.
  • the treatment also comprises the separate administration of antibodies and immune cells as described further herein.
  • the disclosure further provides a method of treating or preventing coronavirus infection and/or coronavirus mediated disease in an individual, said method comprising administering to an individual in need thereof an effective amount of immune cells or engineered immune cells as described herein and one or more antibodies that bind to a coronavirus spike (S) protein.
  • the individual is immunocompromised or at high-risk.
  • the immune cells express a chimeric antigen receptor (CAR) (i.e., is an engineered immune cell, preferably the CAR binds to a coronavirus spike (S) protein).
  • CAR chimeric antigen receptor
  • the immune cell or engineered immune cell expresses NKp46, NKp44, NKp30, NKG2C and/or NKG2D.
  • the immune cell or engineered immune cell is a natural killer cell.
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of NK cell stimulating feeder cells, NK cell stimulating molecules, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • CAR chimeric antigen receptor
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of an NK cell stimulating feeder cell that is a K562 cell that expresses one or more of IL-15, IL-21 and/or 4-1BBL; and optionally wherein said cell culture further comprising IL-2.
  • the initial population of NK cells is derived or isolated from at least one of peripheral blood, placental blood, lymphatic fluid, iPSCs, ESCs, MSCs, or an CD56+CD3- cell-containing source.
  • one or more antibodies individually or collectively, binds to a coronavirus spike (S) protein from at least two different coronaviruses one or more antibodies, individually or collectively, binds to the spike protein from SARS-CoV-1, MERS-CoV, and/or SARS-CoV-2, one or more antibodies, individually or collectively, binds to the spike protein from SARS-CoV-1 and SARS-CoV-2, and/or one or more antibodies, individually or collectively, binds to the spike protein from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.
  • S coronavirus spike
  • the disclosure provides a multi-specific antibody comprising: i) at least one antigen binding domain that binds a coronavirus spike (S) protein as described herein and ii) at least one antigen binding domain that binds an NK cell activating receptor, preferably NKp46.
  • the disclosure provides a multi-specific antibody comprising: i) at least one antigen binding domain that binds a coronavirus spike (S) protein as described herein and ii) at least one antigen binding domain that binds CD 16.
  • the disclosure provides a multi-specific antibody comprising: i) at least one antigen binding domain that binds a coronavirus spike (S) protein as described herein, ii) at least one antigen binding domain that binds an NK cell activating receptor, preferably NKp46, and ii) at least one antigen binding domain that binds CD 16.
  • the multi-specific antibody binds to a coronavirus spike (S) protein from at least two different coronaviruses binds to the spike protein from SARS-CoV-1, MERS-CoV, and/or SARS-CoV-2, binds to the spike protein from SARS-CoV-1 and SARS-CoV-2, and/or binds to the spike protein from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.
  • S coronavirus spike
  • MERS-CoV coronavirus spike
  • the immune cells express a chimeric antigen receptor (CAR) (i.e., is an engineered immune cell, preferably the CAR binds to a coronavirus spike (S) protein).
  • CAR chimeric antigen receptor
  • the immune cell or engineered immune cell expresses NKp46, NKp44, NKp30, NKG2C and/or NKG2D.
  • the immune cell or engineered immune cell is a natural killer cell.
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of NK cell stimulating feeder cells, NK cell stimulating molecules, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • CAR chimeric antigen receptor
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of an NK cell stimulating feeder cell that is a K562 cell that expresses one or more of IL-15, IL-21 and/or 4-1BBL; and optionally wherein said cell culture further comprising IL-2.
  • the initial population of NK cells is derived or isolated from at least one of peripheral blood, placental blood, lymphatic fluid, iPSCs, ESCs, MSCs, or an CD56+CD3- cell-containing source.
  • the multi-specific antibodies and immune cells may be provided in a single composition, such as the pharmaceutical composition.
  • the treatment also encompasses the separate administration of antibodies and immune cells as described further herein.
  • the individual is immunocompromised or at high-risk.
  • the disclosure provides an immune cell as described herein (including an engineered immune cell) and an immunogenic composition that elicits a humoral immune response against coronavirus virus, for use in the treatment and/or prevention of coronavirus infection and/or coronavirus mediated disease in an individual.
  • the disclosure provides a method of treating or preventing coronavirus infection and/or coronavirus mediated disease in an individual, said method comprising administering to an individual in need thereof an effective amount of an immune cell as described herein (including an engineered immune cell) and an immunogenic composition that elicits a humoral immune response against coronavirus.
  • the individual is immunocompromised or at high-risk.
  • the immunogenic composition and immune cells may be provided in a single composition, such as the pharmaceutical composition.
  • the treatment also encompasses the separate administration of an immunogenic composition and immune cells as described further herein.
  • the immune cells express a chimeric antigen receptor (CAR) (i.e., is an engineered immune cell, preferably the CAR binds to a coronavirus spike (S) protein).
  • CAR chimeric antigen receptor
  • the immune cell or engineered immune cell expresses NKp46, NKp44, NKp30, NKG2C and/or NKG2D.
  • the immune cell or engineered immune cell is a natural killer cell.
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of NK cell stimulating feeder cells, NK cell stimulating molecules, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • CAR chimeric antigen receptor
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells, preferably an initial population of engineered NK cells that express a chimeric antigen receptor (CAR) as defined herein, in a cell culture in the presence of an NK cell stimulating feeder cell that is a K562 cell that expresses one or more of IL-15, IL-21 and/or 4-1BBL; and optionally wherein said cell culture further comprising IL-2.
  • the initial population of NK cells is derived or isolated from at least one of peripheral blood, placental blood, lymphatic fluid, iPSCs, ESCs, MSCs, or an CD56+CD3- cell-containing source.
  • the immunogenic composition comprises a coronavirus spike protein or an antigenic fragment thereof.
  • the immunogenic composition elicits a humoral immune response against a coronavirus spike protein.
  • the at spike protein antigen comprises an epitope that is evolutionary conserved between different coronaviruses.
  • the humoral immune response elicits one or more antibodies which, individually or collectively, bind to SARS-CoV-1, MERS-CoV, and/or SARS-CoV-2.
  • the humoral immune response elicits one or more antibodies which, individually or collectively, bind to SARS-CoV-1 and SARS-CoV-2.
  • the immunogenic composition is selected from an inactivated coronavirus vaccine; a live attenuated coronavirus virus vaccine; a coronavirus recombinant protein vaccine comprising a recombinant protein of at least one antigen of spike protein; an nucleic acid vaccine comprising a nucleic acid encoding at least one antigen of spike protein; an vaccine comprising a vector, preferably a viral vector, that expresses at least one spike protein antigen; and an vaccine comprising a VLP that displays on its outer surface at least one spike protein antigen.
  • the disclosure also provides direct viral killing/neutralization (e.g.
  • NK cells by freshly isolated donor-derived NK cells/K-NK cells/Fc-imprinted K-NK cells.
  • the disclosure also provided the combination of such NK cells with polyclonal or monoclonal antibodies, in particular antibodies that bind the spike protein of the SARS-CoV-2 virus.
  • the Fc domains of these antibodies can subsequently bind to the CD 16 activating receptor on NK cells resulting in NK cell activation leading to viral killing/neutralization.
  • antivirals may include chloroquine and hydrochloroquine, nucleotide analogs such as but not limited to Remdesivir (Gilead GS-5734), RNA polymerase inhibitors such as but limited to favipiravir (Toyama), and neuraminidase inhibitors such as oseltamivir (Tamiflu), interferons, nucleoside analogs, protease inhibitors, Reverse transcriptase inhibitors, and neuraminidase inhibitors.
  • nucleotide analogs such as but not limited to Remdesivir (Gilead GS-5734
  • RNA polymerase inhibitors such as but limited to favipiravir (Toyama)
  • neuraminidase inhibitors such as oseltamivir (Tamiflu)
  • interferons nucleoside analogs
  • protease inhibitors Reverse transcriptase inhibitors
  • neuraminidase inhibitors osel
  • antivirals which may be used include Foscavir, Acyclovir, Cymevene, Ribavirin, Lamivudine, Zidovudine, Fortovase, Viracept, Crixivan, Relenza, Interferon a-2a (Roferon) Roche 100,000 IU/mL, Interferon a-2b (Intron A) Schering-Plough 500,000 IU/mL Interferon a-nl (Wellferon) GlaxoSmithKline 500,000 IU/mL, Interferon a-n3 (Alferon) Hemispheryx 10,000 IU/mL, Interferon a- la (Rebif) Serono 500,000 IU/mL, Interferon a- lb (Betaferon) Sobering AG 100,000 IU/mL, Acyclovir Faulding 1,000 ig/mL u.
  • Ganciclovir Roche 50,000 ig/mL, Ribavirin ICN Pharma 10,000 ig/mL, Indinavir (Crixivan) Merck 100 imol/L, Nelfinavir (Viracept) Roche 10,000 nmol/L, Saquinavir (Fortovase) Roche 10,000 nmol/L, Lamivudine (Epivir) GlaxoSmithKline 1,000 imol/L, Zidovudine (Retrovir) GlaxoSmithKline 1,000 ig/mL, Oseltamivir (Tamiflu) Roche 10,000 imol/L, Zanamivir (Relenza) GlaxoSmithKline 1,000 imol/L dd.
  • NK cells may include those that are collected from peripheral blood, collected through apheresis, differentiated from iPSC/ESC, etc.
  • NK cells may be activated and/or expanded, ii. activation and expansion may be through contact with cytokines (IL-1)
  • cytokines may be present on feeder cells, plasma membrane (PM) particles or exosome platforms.
  • PM plasma membrane
  • K562, 721.22 cells, other tumor cell lines, EBV-LCLs, lymphocytes, etc can be used as a base for feeder cells or as sources for PM particles or exosomes
  • other signaling molecules such as 4-lbbl may be in combination.
  • NK cells may include those that are collected from peripheral blood, collected through apheresis, differentiated from iPSC/ESC, etc.
  • NK cells may be activated and/or expanded, ii. activation and expansion may be through contact with cytokines (IL-1)
  • cytokines may be present on feeder cells, plasma membrane (PM) particles or exosome platforms.
  • PM plasma membrane
  • K562, 721.22 cells, other tumor cell lines, EBV-LCLs, lymphocytes, etc can be used as a base for feeder cells or as sources for PM particles or exosomes
  • other signaling molecules such as 4-lbbl may be in combination
  • NK cells for in vitro diagnostic for recognition and detection of virally compromised cells in human samples: a. for triggering production and/or release of induced NK cell products and detection of. i. for detection through IFN ⁇ production. ii. for detection of CD 107a production iii. for detection of degranulation. b. for adding viral targeted or viral recognizing or virus neutralizing antibodies to stimulate NK cells through CD 16 engagement. c. for adding convalescent serum obtained from recovering patients
  • source NK cells may be as from l.a.
  • NK cells activated with PM21 result in the upregulation of CD 16, activating KIR and NCR receptors, and upregulated secretion of IFN-gamma (Denman et al., PloS one, 2012; 7(l):e30264; Oyer et al. Oncolmmunology 2018;7:11)).
  • Activating receptors such as NKp46, KIRXDSX, and NKG2D and inhibitory receptors such as KIRXDLX are depicted as well as antibody binding via CD 16.
  • Figure 2 Two embodiments of NK cell-based therapy.
  • Anti- viral antibodies as disclosed herein can be monospecific or multi-specific, and can be monovalent or multivalent.
  • Type I antibodies bind to epitopes on the most immunogenic parts of the viral protein (typically the head region). These Abs neutralize the virus by interfering with the binding to the target cells (typically epithelial cells of the lung). Typically, these epitopes are less conserved and prone to viral escape.
  • Type II Abs bind to the receptor binding region which are more conserved, less prone to viral escape, and neutralize as described for type I Abs.
  • Type III Abs bind also to epitopes on less conserved regions but are non-neutralizing.
  • Type IV Abs bind to conserved epitopes and are neutralizing, e.g. by interfering with the viral and target cell membrane fusion, or the viral and endosomal membrane fusion.
  • NK-cells (CD56+, CD3-) are innate lymphoid immune cells that are widely recognized as first responders to viral infections. They recognize target cells through multiple different stress-related and viral proteins on the surface of virus-infected cells, rather than a single protein or antigen. NK-cells trigger direct cytotoxic effector mechanisms as well as cytokine production associated with cross-talk to the adaptive immune system. Early NK-cell responses are associated with more rapid adaptive responses, clearance of virus, and recovery. Individuals with NK-cell deficiencies are particularly susceptible to infections with certain viruses.
  • NK cells are known to play an active role in the immune response against a number of viruses. NK-cells are actively recruited to the lungs and airways during infection of the respiratory system. Infected respiratory epithelial cells release chemokines that attract NK-cells. Migration of NK-cells is dictated by the severity of respiratory infection, and partially dependent on CXCR3 and CCR5 receptors on NK-cells and their ligands.
  • the disclosure provides, in some embodiments, pharmaceutical compositions and methods of treatment using one or more antibodies and an immune cell, such as an NK-cell.
  • the disclosure provides that the Fc domain of an antibody targeting a virally infected cell can subsequently bind to the CD 16 activating receptor on immune cells, such as NK cells, mast cells, and macrophages, leading to immune cell activation, resulting in viral killing/neutralization.
  • immune cells such as NK cells, mast cells, and macrophages
  • the disclosure also provides engineered CAR-expressing immune cells, which can be combined with antibody treatment.
  • virus infected individuals have low NK cell counts with an exhausted phenotype.
  • the pharmaceutical compositions and treatments described herein provide a source of activated NK cells to kill or neutralize virally infected target cells, e.g., through cytotoxic mechanism such as perforin and granzyme release.
  • NK cells can produce and secrete IFN ⁇ which has a direct anti-viral effect and additionally stimulates humoral responses, specifically the production of IgG2a and IgG3.
  • IFN ⁇ can also activate macrophages that then can clear opsonized cells and particles.
  • the adoptive administration of NK cells that recognize virally compromised cells can induce IFN ⁇ release. While not wishing to be bound by theory, the induced IFN ⁇ can then enhance overall humoral immunity as well as clearance of opsonized cellular targets or opsonized viral particles.
  • cross-reactive antibodies targeting e.g., evolutionarily conserved domains are preferred.
  • the combination of such antibodies with immune cells, or the presentation of the antigen binding region of such low potent cross-reactive antibodies in a CAR format in immune cells increases potency, while harnessing the breadth of the specificity.
  • Suitable immune cells include lymphocytes, such as natural killer cells (NK cells) and T-cells; monocytes, macrophages, basophils, neutrophils, and eosinophils.
  • NK cells natural killer cells
  • Preferred immune cells include cytotoxic T-cells and NK cells.
  • the immune cell expresses NKp46 (CD335). NKp46 is considered the major lysis receptor for NK cells.
  • NKp46 In addition to NK cells, NKp46 (CD335) is also expressed by T-cell neoplasms such as T- cell large granular lymphocytic leukemia, mycosis fungoides, and ALK+ anaplastic large cell lymphoma (Freud et al., American Journal of Clinical Pathology, 2013 140:853-866).
  • the immune cell is modified to express an NK activating receptor, such as NKp46.
  • the immune cell may be transfected with a nucleic acid expressing NKp46.
  • NKp46 amino acid sequences are known and an exemplary human sequence has accession number NP_004820.2.
  • the expanded and/or activated NK cells described further herein demonstrate increased NKp46 expression.
  • the skilled person is aware how to increase NKp46 expression on NK cells, for instance by selecting specific NK cell donors and/or imprinting with feeder cells or with membrane particles as disclosed herein.
  • compositions comprising an immune cell and one or more antibodies as described herein that bind a coronaviral spike (S) protein.
  • the disclosure also provides a combined treatment comprising an immune cell and one or more antibodies as described herein that bind a coronaviral spike (S) protein. See e.g., Figure 2A.
  • the immune cell may also be an engineered immune cell expressing a chimeric antigen receptor (CAR-cell) as described herein.
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair of chain consist of one “heavy” chain with one “fight” chain.
  • the human fight chains are classified as kappa and lambda.
  • the heavy chains comprise different classes namely: mu, delta, gamma, alpha or epsilon. These classes define the isotype of the antibody, such as IgM, IgD, IgG, IgA and IgE, respectively. These classes are important for the function of the antibody and help to regulate the immune response. Both the heavy chain and the light chain consist of a variable and a constant region.
  • Each heavy chain variable region (VH) and fight chain variable region (VL) comprises complementary determining regions (CDR) interspersed by framework regions (FR).
  • the variable region consists in total four FRs and three CDRs. These are arranged from the amino - to the carboxyl-terminus as follows: FR1. CDR1, FR2, CDR2, FR3, CDRS, FR4.
  • the variable regions of the fight and heavy chain together form the antibody binding site and defines the specificity for the epitope.
  • the antibodies of the present disclosure preferably comprise an Fc region.
  • the antibody is an IgG, more preferably IgG 1 or IgG 3 .
  • Suitable antibodies comprise one or more antigen-binding domains or fragments.
  • antibody antigen-binding domains or fragments include Fab, F(ab'), F(ab') 2 , complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single domain antibodies, and other antigen recognizing immunoglobulin fragments. These antigen binding domains or fragments can be finked to suitable Fc regions.
  • single domain antibodies refer to a single variable antibody domain.
  • Exemplary single domain antibodies include V H H fragments, such as those found in camelids, V NAR fragments (such as those derived from sharks), and preferable human single-domain antibodies.
  • Suitable antibodies may have an antigen binding region that cross-react with multiple coronaviruses.
  • the antigen binding regions recognize epitopes that are evolutionarily conserved across multiple coronaviruses.
  • compositions and methods of treatments described herein may also comprise several antibodies, wherein the antibodies recognize different and/or overlapping coronaviruses.
  • the composition or method of treatment may comprise a first antibody that recognizes the SARS-CoV-1 spike protein and a second antibody that recognizes the SARS-CoV-2 spike protein.
  • Antibodies suitable for use in the present disclosure include those having at least two different antigen binding domains, wherein the antigen binding domains recognize different coronaviruses.
  • the antibody may be a multi-specific antibody, e.g, a bi- or tri-specific antibody, that comprises multiple antigen binding domains. See Runcie et al. for a review of various formats of multi-specific antibodies
  • the multidomain antibodies of Laursen are an example of a multi-specific (against multiple epitopes) and multivalent (repeats of the same binding region).
  • the one or more antibodies, individually or collectively bind to a coronaviral spike (S) protein.
  • the one or more antibodies, individually or collectively bind to the spike protein from SARS-CoV-1, MERS-CoV, and/or SARS-CoV-2.
  • the one or more antibodies, individually or collectively bind to the spike protein from SARS-CoV-1 and SARS-CoV-2.
  • the one or more antibodies, individually or collectively bind to the spike protein from SARS- CoV-1 and MERS-CoV.
  • the one or more antibodies, individually or collectively bind to the spike protein from MERS-CoV and SARS-CoV- 2.
  • the one or more antibodies, individually or collectively bind to the spike protein from SARS-CoV-1, MERS-CoV, and SARS-CoV-2.
  • antibody binding “individually or collectively” to at least two different targets encompasses an antibody that binds to two different targets as well as the combination of a first antibody that binds a first target and a second antibody that binds a second target.
  • the disclosure encompasses the use of a first antibody that binds at least two different coronavirus spike proteins, either because, e.g., the antigen binding domain is cross-reactive for two different spike proteins or the first antibody comprises two different antigen binding domains, wherein the first antigen binding domain binds, e.g., SARS-CoV-1, and the second antigen binding domain binds, e.g., SARS-CoV-2.
  • the disclosure also encompasses the use of a first antibody that binds a first coronavirus spike protein (e.g., SARS-CoV-1) and a second antibody that binds a second coronavirus spike protein (e.g., SARS-CoV-1) and a first antibody that binds a first coronavirus spike protein (e.g., SARS-CoV-1) and a second antibody that binds a second coronavirus spike protein (e.g., SARS-CoV-1)
  • a second antibody that binds a second coronavirus spike protein e.g., SARS-CoV-
  • antibodies suitable for use in the present disclosure may also be hi- or tri-specific antibodies that comprise multiple antigen binding domains or fragments.
  • an antibody comprises, in addition to one or more viral specific antigen-binding domains as disclosed herein, one or more antigen-binding domains or fragments that specifically binds to an NK cell activating receptor, preferably NKp46.
  • This antigen-binding domain or fragment stimulates or activates signalling through said NK cell activating receptor, preferably stimulates or activates signalling through NKp46.
  • the additional antigen-binding domain or fragment binds said NK cell activating receptor, preferably NKp46, agonistically.
  • NK cell activating receptors such as NKp46.
  • Exemplary bi- or tri-specific antibodies are described in Gauthier et al. Cell 2019 177:1701-1713.el6, which is hereby incorporated by reference.
  • Preferred multi-specific antibodies comprise at least one viral specific antigen-binding domain as disclosed herein and the heavy chain CDRs from at least one of the antibodies described in Gauthier et al.
  • the multi-specific antibody may further comprise the light chain CDRs and in some embodiments, the heavy and/or light chain variable domains of the NKp46 antibodies described in Gauthier et al.
  • NK cell activity and cytokine release are increased by NKp46 activation. While not wishing to be bound by theory, such multi-specific antibodies are particularly useful to increase NK cell activity. Such antibodies allow for both NK cell targeting to the virally infected cell, as well as activating the NK cells.
  • the antibody may comprise an Fc-region or Fc domain, preferably an IgG Fc-region or Fc domain that specifically binds to CD 16, preferably Fc ⁇ RIIIa (CD 16a).
  • Fc ⁇ RIIIa is expressed on NK cells and such binding further activates NK cells.
  • the conformation of the bispecific antibodies is preferably such to allow binding to both the virally infected cell and an NK cell.
  • the conformation of the bispecific antibodies allows binding to both a virion and an NK cell.
  • the viral specific antigen-binding domain(s) and the NKp46 binding domain(s) are separated by linkers, for example glycine-serine linkers. Other linkers are also known to a skilled person and the optimal length of such hnkers can also be determined.
  • an antibody as disclosed herein comprises, in addition to one or more viral specific antigen-binding domains as disclosed herein, one or more antigen- binding domains or fragments that specifically binds to CD 16, preferably Fc ⁇ RIIIa (CD 16a).
  • This antigen-binding domain or fragment stimulates or activates signalling through CD 16.
  • the additional antigen-binding domain or fragment binds CD 16 agonistically.
  • Agonistic anti-CD 16 antibodies are known in the art and are described for example in Behar et al. (Protein Engineering, Design & Selection vol.21:l, pp. 1-10 (2008)). Debar et al.
  • sdAbs anti-Fc ⁇ RIII single domain antibodies
  • sdAbs anti-Fc ⁇ RIII single domain antibodies
  • the amino acid sequences of isolated anti-Fc ⁇ RIII sdAbs C13, C21, C28 and C72 are displayed in Figure 1 of Debar et al, which are explicitly incorporated by reference herein. Debar et al. discloses that these sdAbs bind Fc ⁇ RIIIA+ NK cells.
  • CDR1, CDR2 and/or 3 of each one of sdAbs C13, C21, C28 and C72 are incorporated herein by reference.
  • Preferred multi-specific antibodies comprise at least one viral specific antigen-binding domain as disclosed herein and the heavy chain CDRs from at least one of the antibodies C13, C21, C28 and C72.
  • the antibody may further comprise the light chain CDRs. Since such multi-specific antibodies recognize CD 16 via the antigen-binding domain, an Fc region is not necessary, though may also be included.
  • antibodies comprising one or more viral specific antigen-binding domains as disclosed herein, one or more NK cell activating receptor binding domains as disclosed herein, and one or more CD 16 binding domains as disclosed herein.
  • a tri-specific antibody has the advantages that it activates NK cells by binding to an NK cell activating receptor such as NKp46 as well as CD 16 and brings the activated NK cells in the proximity of coronavirus and/or coronavirus- infected cells by virtue of its binding specificity towards both a coronavirus epitope. Since such multi-specific antibodies recognize CD 16 via the antigen-binding domain, an Fc region is not necessary, though may also be included.
  • NK cells results in NK cell activation, resulting in viral killing/neutralization by for instance antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • a further aspect of the present disclosure provides engineered immune cells expressing a chimeric antigen receptor (CAR-cell).
  • the engineered immune cells disclosed herein are modified to express chimeric antigen receptors.
  • CARs comprise an extracellular domain (or ectodomain) that comprises one or more antigen recognition domains, and optionally a signal peptide to direct the CAR to the endoplasmic reticulum for processing; a transmembrane domain; and one or more intracellular signalling domains.
  • the stimulatory domain(s) produce an activation signal that induces the NK cell to kill the targeted cell. See for an exemplary embodiment Figure 2B and 2C.
  • the ectodomain domain is linked to a transmembrane domain.
  • CARs may also comprise a spacer or hinge region linking the antigen-recognition domain to the transmembrane domain.
  • Such hinge regions are known in the art and include IgG sequences (such as CH3 of IgG 4 ), IgD domains, and CD8a hinge region.
  • Suitable transmembrane domains include a transmembrane region of a natural cytotoxicity receptor expressed in NK cells such as, for example, CD 16, NKp44, NKp46, NKp30, NKp80, DNAM-1 or NKG2D.
  • Other suitable transmembrane domains include those from CD4, CD8a, and CD28.
  • Suitable intracellular signaling domains also referred to as stimulatory domains, include a signaling domain of an NK cell membrane-bound signaling adaptor protein. Exemplary signalling domains are those present in, for example, 2B4, NKG2D,
  • the CAR comprises a portion of CD3 ⁇ and a second or third signalling domain (e.g., 2B4, NKG2D, DAP10, DAP 12, IL21R, CD 134 (0X40), and/or CD137 (4- 1BB).
  • the antigen recognition domain comprises a polypeptide sequence that recognizes an antigen. Suitable antigen recognition domains include one or more antibody antigen binding domains or fragments as described herein (e.g., scFv and single- domain antibodies). Multiple antigen binding domains can also be linked together to provide CARs with multiple antigen recognition domains.
  • the CARs described herein may have an antigen recognition region that cross-reacts with multiple coronaviruses.
  • the CARs described herein may also comprise several antigen recognitions regions, wherein the antigen recognitions regions recognize different and/or overlapping coronaviruses.
  • the CAR may comprise a first antigen recognitions region that recognizes SARS-CoV-1 and a second antigen recognitions region that recognizes SARS-CoV-2.
  • the CAR may further comprise, e.g., a third antigen recognition region that recognizes MERS-CoV. Further antigen recognition regions with various specificities may be used.
  • the present disclosure provides CARs and antibodies that target coronavirus.
  • Suitable antigen binding sequences may be identified by any method known in the art, including in vitro animal mouse immunization protocols to obtain antibodies, screening antibody or CDR3 libraries (e.g., phage libraries), and from the plasma of coronavirus infected convalescent donors, e.g., those recovering from COVID-19.
  • in vitro animal mouse immunization protocols to obtain antibodies, screening antibody or CDR3 libraries (e.g., phage libraries), and from the plasma of coronavirus infected convalescent donors, e.g., those recovering from COVID-19.
  • the CAR or one or more antibodies recognizes at least two different coronaviruses. In some embodiments, the CAR or one or more antibodies binds to spike protein from at least two different coronaviruses or at least three different coronaviruses. In some embodiments, the CAR or one or more antibodies binds to the spike protein from SARS-CoV-1, MERS-CoV, and/or SARS-CoV-2. In some embodiments, the CAR or one or more antibodies binds to the spike protein from SARS-CoV-1 and SARS-CoV-2. In some embodiments, the CAR or one or more antibodies binds to the spike protein from SARS-CoV-1 and MERS-CoV.
  • the CAR or one or more antibodies binds to the spike protein from MERS-CoV and SARS-CoV-2. In some embodiments, the CAR or one or more antibodies binds to the spike protein from SARS-CoV- 1, MERS-CoV, and SARS-CoV-2.
  • the antigen binding regions or CARs bind to epitopes depicted as Type I in Figure 3. In some embodiments the antigen binding regions or CARs bind to epitopes depicted as Type III in Figure 3. In some embodiments the antigen binding regions or CARs bind to epitopes depicted as Type III in Figure 3. In some embodiments the antigen binding regions or CARs bind to epitopes depicted as Type IV in Figure 3.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein preferably bind to an evolutionarily conserved epitope of the spike protein. In some embodiments, the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein bind to the SI domain of the spike protein. In some embodiments, the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein bind to the S2 domain of the spike protein. It is within the skill of one in the art to identify antibodies that bind to the spike protein. Such antibodies may target the SI or S2 subunit. For example, Wrapp et al.
  • VHH-Fc fusion is also generated and can be produced at high yields in a CHO cell system.
  • a number of antibodies are known to a skilled person which target, for example, the N-terminal domain or receptor-binding domain of the SI spike protein subunit or the C-terminal S2 subunit.
  • the CARs or antibodies recognize SARS-CoV-2.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated COV2-2050, COV2-2082, COV2-2094, COV2-2096, COV2-2165, COV2-2479, COV2-2499, COV2-2677 and/or COV2-2832 as described in Zost et al (Zost et al. 2020, Nature, 584:443-449).
  • said CARs and antibodies may further comprise the light chain CDRs.
  • said CARs and antibodies may comprise the heavy and/or light chain variable domains of the antibodies.
  • Hansen et al. describes antibodies targeting the SARS-CoV-2 receptor binding domain of the spike protein (Hansen et al. 2020 Science 369, 1010-1014).
  • REGN10933, REGN10934, REGN10977, REGN10964, REGN10954, REGN10984, and REGN 10986 are depicted in Data SI.
  • REGN10987 and REGN 10933 are described as an interesting therapeutic antibody cocktail since they target non-overlapping epitopes on the spike protein (Baiun et al. 2020 bioRxiv 2020.08.02.233320; doi: https://doi.org/10.1101/2020.08.02.233320). This cocktail is referred to as REGN-
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated REGN 10989, REGN 10987, REGN10933,
  • CARs and antibodies may comprise the heavy chain CDRs from both REGN 10987 and REGN 10933.
  • said CARs and antibodies may further comprise the light chain CDRs.
  • said CARs and antibodies may comprise the heavy and/or light chain variable domains of the antibodies.
  • Nanobodies Sb#14, Sb#15, Sb#16, Sb#42, Sb#45 and Sb#68 inhibit the RBD-ACE2.
  • the sequences of these antibodies are depicted in table 2 of Walter et al. Of these antibodies, Sb#68 is the strongest binder. Sb#68 can be combined with Sb#15, as both nanobodies bind different, non-overlapping epitopes.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more nanobodies designated Sb#14, Sb#15, Sb#16, Sb#42, Sb#45 and Sb#68 as depicted in table 2 of Walter et al.
  • CARs and antibodies comprise the heavy chain CDRs of both Sb#15 and Sb#68.
  • said CARs and antibodies may comprise the heavy chain variable domains of the antibodies.
  • Huo et al. 2020 describe nanobodies binding the RBD of the SARS-CoV-2 spike protein (Nature Struct & Mol. Biol., 27:846-854). The nanobodies were fused to the Fc domain of IgGl.
  • Nanobodies H11-D4 and H11-H4 bind the RBD with high affinity and block binding of the RBD with ACE-2.
  • the CDR3 sequences of these antibodies are depicted in figure 2a.
  • Huo et al. also describes the combination of nanobody H11-H4 and CR3022, which is cross reactive to both SARS-CoV-1 and SARS-CoV-2.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs in particular CDR3 from one or more nanobodies designated H11-D4 or H11-H4 as depicted in figure 2a of Huo et al.
  • CARs and antibodies comprise the heavy chain CDRs of H11-D4 or H11-H4 and the heavy chain of VHH-72 or CR3022.
  • CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • the antibodies are a combination of nanobody H11- H4 and CR3022.
  • Wu et al. 2020 describe human single domain antibodies binding the RBD of the SARS-CoV-2 spike protein (Cell Host & Microbe 27:891-898). These single domain antibodies include n3001, n3002, n3003, n3004, n3008, n3009, n3011, n3014, n3020, n3021, n3025, n3026, n3047, n3051, n3055, n3065, n3063, n3010, n3063, n3010, n3021, n3077, n3068, n3130, n3086, n3113.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated n3001, n3002, n3003, n3004, n3008, n3009, n3011, n3014, n3020, n3021, n3025, n3026, n3047, n3051, n3055, n3065, n3063, n3010, n3063, n3010, n3021, n3077, n3068, n3130, n3086, n3113.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated n3021, n3063, n3010, n3088, n3130, n3086, n3113.
  • CARs and antibodies comprise the heavy chain variable domains of the antibodies, in particular n3088 and n3113 or the heavy chain of n3130 and n3113.
  • Valenzuela Nieto et al. 2020 describe alpaca nanobodies binding the RBD of the SARS-CoV-2 spike protein
  • CARs and antibodies include W25 and W23 of which the sequences are depicted in figure 2c of Nieto et al.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated W23 and W25.
  • CARs and antibodies comprise the heavy chain CDRs from the antibody designated W25.
  • CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • VHH antibodies binding the RBD of SARS-CoV-2 spike protein https://www.biorxiv.org/content/10.1101/2020.07.24.219857v3, accessed on 16 September 2020.
  • These antibodies include NIH-CoVnd-101, NIH-CoVnd-102, NIH-CoVnd- 103, NIH-CoVnd-104, NIH-CoVnd-105, NIH-CoVnd-106, NIH-CoVnd- 107, NIH-CoVnd- 108, NIH-CoVnd- 109, NIH-CoVnd- 110, NIH-CoVnd- 1011, NIH- CoVnd-112, NIH-CoVnd- 113.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated NIH-CoVnd- 101, NIH- CoVnd-102, NIH-CoVnd- 103, NIH-CoVnd- 104, NIH-CoVnd- 105, NIH-CoVnd- 106, NIH-CoVnd- 107, NIH-CoVnd- 108, NIH-CoVnd- 109, NIH-CoVnd- 110, NIH-CoVnd- 1011, NIH-CoVnd- 112, NIH-CoVnd- 113.
  • CARs and antibodies comprise the heavy chain CDRs from NIH-CoVnd- 112.
  • CARs and antibodies comprise the heavy chain variable domains of the antibodies, in particular of NIH-CoVnd- 112.
  • VHH binding the RBD of SARS-CoV-2 spike protein (https://www.biorxiv.org/content/10.1101/2020.06.02.130161v2, accessed on 16 September 2020).
  • This VHH is designated Tyl, of which the amino acid sequence is depicted in figure lc.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from the antibody designated Ty1. In some embodiments CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • Chi et al 2020 describe humanized single domain antibodies binding RBD of SARS- CoV-2 spike protein (Nature communications, 11:4528). These single domain antibodies are designated 1E2, 2F2, 3F11, 4D8, 5F8. Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more of the humanized single domain antibodies designated 1E2, 2F2, 3F11, 4D8, 5F8. In some embodiments CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • V H ab8 a human V H domain binding the RBD of SARS-CoV-2 spike protein (Cell 183:1-13). This V H domain is designated V H ab8.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from the V H antibody designated V H ab8. In some embodiments CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • Gai et al. 2020 disclose nanobodies based on camelid VHH’s binding the RBD of SARS-CoV-2 spike protein
  • These antibodies include Nb3-85, Nb4-14, Nb4-43, Nb7-17, Nb8-65, Nb8-87, Nb9-22, Nb9-56, Mb 10-46, Nb 10-51, Mb 11-25, Nb 11-59, Mb 13-8, Nb13-10,
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated Nb4-43, Nb 11-59, Nb15-61, Nb 16-52, Nb 14-33, Nb 16-68, Nb 16-75 of which the CDR sequences are depicted in figure 3E of Gai et al.
  • CARs and antibodies comprise the heavy chain CDRs from the antibody designated Nb 11-59.
  • CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • V H domains binding the RBD of SARS-CoV-2 spike protein (mAbs 12:1). These V H domains are designated Vh-Fc ab6 and m397.
  • CARs and antibodies comprise the heavy chain CDRs from the antibodies designated Vh-Fc ab6 and m397.
  • CARs and antibodies comprise the heavy chain variable domains of the antibodies.
  • CARS and antibodies comprise the heavy chain CDRs from one or more antibodies 44B3, 45E10, 46F11, 39F9, 41A7, 28E3, 34C10, 16C10, 14B1, 30B1, 28G10, 28F6, 40H10, 39A4, 37G1, 44E11, 19C1, 58D2, 14C1, 45H1, 24F5, 52D9, 45E6,
  • the antibodies were obtained from hybridomas generated by sequential immunization with trimeric spike protein of three human coronaviruses.
  • antibodies 47D11 and 49F1 which both bind to the SI domain.
  • Antibody 47D11 more specifically binds the S1 B domain, but does not interfere with receptor binding.
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and hght chain variable domains of the antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from antibody CR03-022, which was subsequently described to be cross-reactive to both SARS-CoV-1 and SARS-CoV-2 (Tian et al 2020 Microbes & Infections, 9:1, 382-385, and Greany et. al 2020 biorxiv.org/content/10.1101/2020.09.10.292078vl).
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • the CARs or antibodies recognize SARS-CoV-1.
  • US8,106,170 hereby incorporated by reference, describes binding molecules that recognize the SARS-CoV-1 spike protein.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from antibodies CR03-014 and CR03-022.
  • US7,728,110 hereby incorporated by reference, describes binding molecules that recognize the SARS-CoV- 1 spike protein.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated 1B5, 1G3, 2E8.1, 2E8.2, 2B10.1,
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and hght chain variable domains of the antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from the 80R antibody.
  • said CARs and antibodies may further comprise the hght chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated F26G1, F26G2, F26G4, F26G5, F26G6, F26G8, F26G12, F26G13, F26G14, F26G16, F26G17, F26G3, F26G7, F26G9, F26G10, G26G18 and F26G19.
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and hght chain variable domains of the antibodies. Wrapp et al.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated SARS VHH-6, SARS VHH-72, and SARS VHH-44.
  • said CARs and antibodies may further comprise the hght chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated S230.15 and m396 described in Zhu, Z. et al. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12123-12128.
  • said CAEs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated S 109.8 and S227.14 described in Rockx.
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more mouse antibodies designated 33G4, 35B5, and 30F9 described in He, Y. et al. (2006) J. Immunol. 176, 6085-6092.
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • the CARs or antibodies recognize MERS-CoV.
  • US 9,718,872 describes binding molecules that recognize the MERS-CoV spike protein.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies depicted in Tables 2-7 of US 9,718,872.
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated JC57-13, JC57-14, JC57-11, C2, C5, A2, A10, FIB_B2, FIB_H1, G2, G4, D12, and Fll, described therein.
  • said CARs and antibodies may further comprise the light chain CDRs or said CARs and antibodies may comprise the heavy and light chain variable domains of the antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated MERS VHH-2, MERS VHH-20, MERS VHH-15, MERS VHH-55, MERS VHH-12, MERS VHH-34, and MERS VHH-40 described therein.
  • said CARs and antibodies may further comprise the heavy chain variable region.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated VHH-01, VHH-06, VHH-08, VHH-20, VHH-21, VHH-28, VHH-38, VHH-46, VHH-50, VHH-70, VHH-83, VHH-84, VHH-184, VHH-59, VHH-101, VHH-111, VHH-152, VHH-203, VHH-04, VHH-18, VHH-02, VHH-07, VHH-10, VHH-12, VHH-26, VHH-44, VHH-44, VHH-58, VHH-62, VHH-91, VHH-93, VHH-129, VHH-136, and VHH-174 described therein.
  • said CAR comprises the heavy chain CDRs from one or more antibodies designated VHH-01, VHH-06, VHH-08, VHH-20, VHH-21, VHH-28, VHH-38, VHH-46, VHH-50, VHH-70, VHH-83, V
  • Wan et al. 2020 describe hitman antibodies binding the RBD of SARS-CoV-2 spike protein (Cell Rep., 32(3):107918). These antibodies include 515-1, 505-5, 553-49, 505- 3, 553-15, 553-63, 414-1 and 553-60. Particularly useful CARs and antibodies comprise an antigen binding domain of antibodies 414-1 or 533-15. In some embodiments CARs and antibodies as disclosed herein comprise a heavy chain CDR3 and/or hght chain CDR3 of antibody 414-1 or 533-15. These CDR3 sequences are disclosed in Figure 3A of Wan et al. 2020, the contents of which are incorporated herein by reference. Brouwer et al.
  • CARs and antibodies as disclosed herein comprise an antigen binding domain of COVA1-16, COVA1-18, COVA2-02 or COVA1-15.
  • the contents of Brouwer et al. 2020 are incorporated herein by reference.
  • Seydoux et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Immunity, 53(l):98-105.e5). These antibodies include CVS, CV30 and CV43. Particularly useful CARs and antibodies as disclosed herein comprises an antigen binding domain of CV30. In some embodiments CARs and antibodies comprise domains of the antibodies, in particular of CV30.
  • the contents of Seydoux et al. 2020 are incorporated herein by reference.
  • Rogers et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Science, 369(6506):956-963). These antibodies include CC12.1 and CC6.33. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of CC12.1 or CC6.33. The contents of Rogers et al. 2020 are incorporated herein by reference. Lv et al. 2020 describe a humanized antibody binding the RBD of SARS-CoV-2 spike protein (Science, eabc5881). This antibody is H014. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of H014. The contents of Lv et al. 2020 are incorporated herein by reference. Lui et al.
  • CARs and antibodies as disclosed herein comprise an antigen binding domains of one of antibodies 2-15, 2-7, 1-57 and 1-20.
  • the contents of Lui et al. 2020 are incorporated herein by reference.
  • Robbiani et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Nature, 584:437-442). These antibodies include C121, C135 and C144. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of one of antibodies C121, C135 and C144. The contents of Robbiani et al. 2020 are incorporated herein by reference.
  • Zhou et al. 2020 describe a murine antibody binding the RBD of SARS-CoV-2 spike protein (Nature Structural & Molecular Biology, 27:950-958). This antibody is EY6A.
  • CARs and antibodies as disclosed herein comprise an antigen binding domain of antibody EY6A.
  • the contents of Zhou et al. 2020 are incorporated herein by reference.
  • Cao et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Cell, 182(1):73-84.e16). These antibodies include BD-361, BD-368.
  • CARs and antibodies as disclosed herein comprise an antigen binding domain of one of the aforementioned antibodies, for instance BD-368-2, BD-218 and BD-23.
  • the contents of Cao et al. 2020 are incorporated herein by reference.
  • Shi et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Nature, 584:120-124). These antibodies include CA1 and CB6. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of CA1 or CB6. The contents of Cao et al. 2020 are incorporated herein by reference.
  • Wu et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Science, 368(6496): 1274- 1278). These antibodies include B38 and H4. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of one of antibodies B38 or H4. The contents of Wu et al. 2020 are incorporated herein by reference. Chen et al. 2020 describe human antibodies binding the RBD of SARS-CoV-2 spike protein (Cell Mol Immunol., 17(6):647-649). These antibodies include 311mab31B5 and 311mab32D4. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of one of antibodies 311mab31B5 or 311mab32D4. The contents of Chen et al. 2020 are incorporated herein by reference.
  • Particularly useful CARs and antibodies as disclosed herein comprise an antigen binding domain of P2C-1F11, P2B- 2F6, P2A-1A10, P2A-1B3, P2C-1A3 or P2C-1C10.
  • Wee et al. 2020 describe hitman antibodies binding the RBD of SARS-CoV-2 spike protein (Science, 369(6504):731-736). These antibodies include ADI55689 and ADI56046. In some embodiments CARs and antibodies as disclosed herein comprise an antigen binding domain of the one of ADI55689 or ADI56046. The contents of Wee et al. 2020 are incorporated herein by reference.
  • Pinto et al. 2020 describe a human antibody binding the RBD of SARS-CoV-2 spike protein (Nature, 583:290-295). This antibody is S309.
  • CARs and antibodies as disclosed herein comprise an antigen binding domain of antibody S309. The contents of Pinto et al. 2020 are incorporated herein by reference.
  • VIR-7831 GSK4182136
  • VIR-7832 GSK4182137
  • CARs and antibodies as disclosed herein comprise an antigen binding domain of antibody VIR-7831 or VIR-7832.
  • CARs and antibodies as disclosed herein comprise an antigen binding domain of antibody casirivimab or imdevimab.
  • antigen recognition domains and antigen binding domains recognize multiple different coronaviruses.
  • antigen recognition domains or antigen binding domains recognize both SARS-CoV- 1 and SARS-CoV-2.
  • the spike proteins of SARS-CoV-2 and SARS-CoV are 77.5% identical by primary amino acid sequence, structurally very similar, and bind ACE2.
  • a number of antibodies have been identified which recognize both SARS-CoV- 1 and SARS-CoV-2 including 47D11, 52D9, 49F1 and 65H9 (Wang et al.
  • SARS VHH-72 also recognizes a bat coronavirus WIV-1 CoV.
  • antigen binding molecules e.g., antibodies
  • SARS-CoV- 1 binders will also bind other coronaviruses, in particular SARS-CoV-2.
  • the disclosure provides a number of suitable antigen binding domains that can be used in the CARs or antibodies disclosed herein.
  • the antigen recognition domain of a CAR or antigen binding fragment of an antibody will preferably comprise the heavy chain CDR3 of the exemplary antibodies disclosed herein.
  • the antigen recognition domain of a CAR or antigen binding fragment of an antibody will preferably comprise the heavy chain CDR1, CDR2, and CDR3 of the exemplary antibodies.
  • the antigen recognition domain of a CAR or antigen binding fragment of an antibody will preferably comprise the heavy chain variable region of the exemplary antibodies.
  • the antigen recognition domain of a CAR or antigen binding fragment of an antibody will preferably comprise the light chain CDR1, CDR2, and CDR3 of the exemplary antibodies. In some embodiments, the antigen recognition domain of a CAR or antigen binding fragment of an antibody will preferably comprise the light chain variable region of the exemplary antibodies.
  • single domain antibodies e.g., VHH do not have fight chains.
  • the disclosure provides antibodies and CARs that bind and recognize particular antigens and epitopes.
  • a skilled person recognizes that such antibodies and CARs exhibit appreciable binding affinity for an antigen or a particular epitope and, preferably, do not exhibit significant cross-reactivity to unrelated proteins.
  • Target binding can be determined according to any art-recognized means for determining such binding (e.g., ELISA, immunoblotting, flow cytometry and surface plasma resonance).
  • Natarajan et al. also describes a luciferase-based assay to detect antigen binding to antibodies and CARs (Scientific Reports 2020 10, Article number: 2318).
  • the disclosure provides nucleic acid molecules encoding said CARs and antibodies.
  • codon usage bias in different organisms can affect gene expression level.
  • Various computational tools are available to the skilled person in order to optimize codon usage depending on which organisms the desired nucleic acid will be expressed.
  • the nucleic acid sequences are optimized for human codon usage for expression in human cells.
  • the disclosure provides a vector comprising the nucleic acid molecule(s) as taught herein, which is capable of expressing the CARs and antibodies.
  • vector is well-known in the art and is understood to refer to a nucleic add molecule capable of artificially carrying or transporting foreign genetic material (i.e. nucleic acid molecule) to which it has been linked, into another cell, where it can be replicated and/or expressed.
  • certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • the CARs are genomically integrated into the immune cells.
  • vectors may comprise promoters that are capable of directing the expression of genes to which they are operatively linked.
  • Vectors may be “expression vectors”. Methods and standard protocols for the preparation of suitable vectors are also well known to the skilled person.
  • the immune cells of the present disclosure are NK cells.
  • NK cell includes reference to large granular lymphocytes involved in the innate immune response. Functionally, NK cells exhibit cytolytic activity against a variety of targets via exocytosis of cytoplasmic granules containing a variety of proteins, including perforin, and granzyme proteases. Killing is triggered in a contact-dependent, non-phagocytotic process.
  • Human NK cells are characterized by the presence of the cell-surface markers CD 16 and CD56, and the absence of the T cell receptor (CD3).
  • Human bone marrow-derived NK cells may further be characterized by the CD2+CD 16+CD56+CD3- phenotype, further containing the T-cell receptor zeta-chain [zeta(Q-TCR], and can be characterized by NKp46, NKp30 and/or NKp44 expression.
  • the NK cell is preferably a human NK cell such as an NK cell that is obtained from a human donor subject prior to genetic engineering.
  • An engineered immune cell of the disclosure preferably an engineered NK cell, expresses a chimeric antigen receptor (CAR) as disclosed herein.
  • CAR chimeric antigen receptor
  • a suitable source of NK cells that can be engineered with a CAR as disclosed herein are primary NK cells (e.g., NK cells isolated directly from a human or animal tissue).
  • primary NK cells are harvested from blood, preferably peripheral blood or cord blood, of a (donor) subject, preferably a human (donor) subject.
  • the donor NK cells are harvested from a living donor, preferably a living human subject.
  • Other sources of NK cells, that can make up the initial population of NK cells include NK cells derived from iPSCs, ESCs, MSCs, or any other CD56+CD3- cell-containing source.
  • NK cells purified from peripheral blood of a human subject are a preferred source of NK cells in the present disclosure
  • cell lines derived from NK cells that can also be used in the present disclosure, including NK-92, NKL, KYHG-1, YT, NK-YS, HANK-1, YTS and NKG cells. These well- characterized, clonal cell populations can be used to produce CAR-expressing NK cells.
  • NK cell prior to engineering an NK cell to express a CAR as disclosed herein, said NK cell may already have been engineered to express a different protein such as a different CAR.
  • the skilled person is aware of various standard methods for isolating NK cells, for instance from peripheral blood.
  • PBMCs are separated into lymphocytes and monocytes, and the lymphocytes are further divided into T cells, B cells, and natural killer cells for isolation.
  • the NK cell can be an expanded and/or an activated NK cell.
  • An NK cell useful in the present disclosure can also be not expanded and/or not activated such as for instance a naive or unprimed NK cell.
  • NK cell refers to NK cells having a phenotype that is more characteristic of a quiescent NK cell, for example, lower (or decreased) expression levels of GDI la, NKG2D, and/or NKp46.
  • an NK cell is an expanded and/or activated NK cell.
  • NK cell expansion and/or activation may occur by in vitro or ex vivo cell culture, preferably ex vivo cell culture.
  • expanded or “expansion”, as used herein, includes reference to an increase in number of NK cells by any suitable NK cell expansion method. Expansion generally occurs ex vivo in a cell culture medium. Suitable cell culture mediums are known to those skilled in the art.
  • NK cell activating markers include NKG2D, signaling lymphocytic activation molecule (SLAM) family molecule 2B4 (CD244), the DNAX accessory molecule (DNAM-1, CD226) and the NK cell receptors (NKR) NKp30, NKp44, NKp46, NKp80 and/or NKG2D.
  • SLAM signaling lymphocytic activation molecule
  • DNAM-1 DNAX accessory molecule
  • NNKR NK cell receptors
  • This change in biological state can be produced by primary stimulatory signals such as co-culturing NK cells with NK cell feeder cells and/or co-culturing with NK cell-stimulation compositions.
  • Co-stimulatory signals may amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity.
  • Established methods to activate and expand NK cells include for instance culturing of NK cells in the presence of a myeloid leukemia cell line such as a K562 myeloid cell line, which may be engineered to express membrane bound growth factors, cytokines and/or ligands to further increase expansion and activation of NK cells.
  • a preferred method both activates and expands NK cells.
  • the skilled person has standard assays at his disposal to measure expressing, or increased expression, of NK cell receptors on NK cells.
  • NK cell expression of NKp30, NKp46, NKp44, NKG2C and/or NKG2D can be determined by routine assays available in the art, including performing a FACS analysis on NK cells incubated with antibodies specifically binding to NKp30, NKp46, NKp44, NKG2C and/or NKG2D.
  • anti-NKG2C-Pe, anti-NKG2D-Pe antibodies are commercially available through R&D (R&D Systems, Wiesbaden, Germany), and anti- NKp30-Pe, anti-NKp44-Pe and anti-NKp46-Pe antibodies are commercially available through Beckman Coulter (Heidelberg, Germany).
  • An exemplary method of performing such a FACS analysis involves incubating NK cells with an amount of antibody and an incubation time that is sufficient for the antibody to bind the NK cell receptor, and washing said NK cell bound to said antibody, for instance in phosphate buffered saline, and analyzing said samples on FACS apparatus such as a FACScalibur using appropriate FACS software such as CellQuest software.
  • the term “increase”, as used herein, includes reference to any change that results in a greater amount of a protein expression, for instance expression of an NK cell receptor, a symptom, disease, composition, condition or activity.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% or more increase.
  • the term “decrease”, as used herein, includes reference to any change that results in a smaller amount of a protein expression, symptom, disease, composition, condition, or activity.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease.
  • the NK cells of the present disclosure are obtained in a method comprising expanding an initial population of NK cells or NK precursor cells (such as CD34+ stem cells).
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells in a cell culture in the presence of NK cell stimulating feeder cells, NK cell stimulating molecules, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • NK cell stimulating feeder cells NK cell stimulating molecules, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • NK cell stimulating molecules are known in the art. Cytokines, growth factors and/or ligands have been employed to expand and/or activate NK cells. For instance, a number of cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, type I IFNs, and TGF-B) and/or ligands (4-1BBL) have been shown to be effective in expanding and/or activating NK cells, for instance primary NK cells that are naive NK cells, ex vivo. Combinations of cytokines, growth factors and/or ligands typically lead to several dozen fold expansion of NK cells (Wagner et al., Frontiers in immunology.
  • CD34+ cord blood stem cells can be cultured in the presence of a cocktail of cytokines to provide CD34+ cells-derived NK cells (Spanholtz et al., PloS one. 2010; 5(2):e9221). Additionally, anti-CD3 antibodies (such as OKT-3) have been reported in NK cell expansion protocols, in particular when combined with IL-2.
  • NK cells as disclosed herein can be IL-21 expanded NK cells, optionally wherein said IL-21 is combined with at least one other cytokine and/or ligand selected from IL-2, IL-12, IL-15, IL-18, IL-21, type I IFNs, TGF-B, OX40L, and/or 4-1BBL, preferably wherein said NK cell is expanded and/or activated with IL-21 and/or 4-1BBL and optionally another cytokine or ligand.
  • cytokine and/or ligand selected from IL-2, IL-12, IL-15, IL-18, IL-21, type I IFNs, TGF-B, OX40L, and/or 4-1BBL
  • NK cell feeder cell lines are another example of an NK cell expansion and/or activation platform.
  • Preferred feeder cells include EBV-LCL cells, RPMI8866 cells, Wilnis tumor cell line HFWT cells (see Harada et al., Jpn J Cancer Res. 2002 Mar; 93(3): 313-319) and K562 cells.
  • growth factors and/or cytokines can be included in the NK cell culture, such as for instance IL-2.
  • An EBV-LCL feeder cell line is for instance an example of an NK cell feeder cell line expansion and/or activation platform. This platform involves contacting an EBV infected feeder line with NK cells during NK cell culture (Berg et al., Cytotherapy. 2009; 11(3):341-55).
  • Another, and preferred, NK cell expansion platform employs a K562 NK cell feeder cell line.
  • K562 feeder cells may be engineered to express NK stimulating molecules, in particular one or more membrane bound cytokines, preferably IL-15 and/or IL-21. Particular useful feeder cells include K562 cells expressing 4- IBB and mIL-15 (membrane bound IL-15).
  • Suitable feeder cells also include K562 engineered to express the OX40L (see, Kweon et al. Front Immunol. 2019; 10: 879).
  • K562-OX40L feeder cells may be used in combination with various cytokines such as IL-2, IL-15, and/or IL-21.
  • Preferred K562 feeder cells are engineered to express mIL-21 (membrane bound IL- 21).
  • Such a feeder cell fine can very beneficially be used to expand and activate NK cells (Denman et al., PloS one, 2012; 7(l):e30264; and EP2866834 Bl).
  • NK cells expanded and/or activated by co-culturing with an K562 NK cell feeder cell line express, or have an increased expression, of activating NK cell receptors such as NKp46, NKp44 NKp30, NKG2C and/or NKG2D.
  • the NK cells of the disclosure are obtained in a method comprising expanding an initial population of NK cells in the presence of NK cell stimulating particles.
  • NK cell stimulating particles are plasma membrane vesicles purified from NK cell feeder cells, such as those disclosed herein.
  • the plasma membrane vesicles comprise one or more NK stimulating agents such as IL-15, IL-21, IL-2, 4-1BBL, IL-12, IL-18, MICA, 2B4, LFA-1, or BCM1/SLAMF2.
  • Exemplary particles are disclosed in US9623082, which is hereby incorporated by reference.
  • the NK cell stimulating particles are plasma membrane vesicles comprising a membrane bound cytokine (in particular IL15 and/or IL21) and optionally 4-1BBL.
  • the K562 NK cell feeder cell line that is engineered to express mIL-21 (membrane bound IL-21) and optionally 4-1BBL, such as for instance described in US9623082 can be provided in the form of plasma membrane vesicles purified from said NK cell feeder cell. These vesicles are also referred to as “PM21 particles”, see e.g., Oyer et al. Cytotherapy 2016 May;18(5):653-63. A depiction of NK cells activated with PM21 is shown in Figure 1. Vesicle membrane vesicles can be provided by different standard techniques such as nitrogen cavitation.
  • Plasma membrane (PM) particles are vesicles made from the plasma membrane of a cell or artificially made (i.e., liposomes).
  • a PM particle can contain a lipid bilayer or simply a single layer of lipids.
  • a PM particle can be prepared in single lamellar, multi-lamellar, or inverted form.
  • PM particles can be prepared from feeder cells using known plasma membrane preparation protocols or protocols for preparing liposomes such as those described in U.S. Pat. No. 9,623,082, the entire disclosure of which is herein incorporated by reference.
  • the natural killer cell is obtained in a method comprising expanding an initial population of NK cells in the presence of NK cell-stimulating exosomes.
  • Exosomes are cell-derived vesicles that are present in many and perhaps all eukaryotic fluids. Exosomes contain RNA proteins, lipids and metabolites that is reflective of the cell type of origin.
  • the exosomes are secreted from feeder cells, in particular from feeder cells as disclosed herein. Suitable exosomes and their method of preparation are disclosed in US2017333479, hereby incorporated by reference.
  • the NK cell-stimulating exosomes are obtained from K562 cells engineered to express mbIL21 and optionally 4-1BBL.
  • the IL-21 and/or 4-1BBL and optionally further cytokines and/or ligands are provided on the surface of NK cell feeder cells, plasma membrane vesicles (for instance plasma membrane vesicles purified from said NK cell feeder cells), liposomes, and/or exosomes.
  • the IL-21 and/or 4-1BBL and optionally further cytokines and/or ligands are provided on the surface of engineered feeder cells, engineered plasma membrane vesicles, engineered liposomes, and/or engineered exosomes.
  • the NK cells disclosed herein are expanded NK cells, wherein the NK cells are expanded in vivo or ex vivo by contacting an NK cell (preferably a non- activated and non-exp anded NK cell such as a primary NK cell that is a naive NK cell) with a plasma membrane vesicle, liposome, exosome, or feeder cell that was engineered to express membrane bound IL-21.
  • an NK cell preferably a non- activated and non-exp anded NK cell such as a primary NK cell that is a naive NK cell
  • a plasma membrane vesicle, liposome, exosome, or feeder cell that was engineered to express membrane bound IL-21.
  • NK cells feeder cell line that can be co-cultured is NK-92, which is an NK cell leukemia cell line and is used as an NK cell feeder cell.
  • an NK cell can be obtained in a method comprising expanding and/or activating an initial population of NK cells in the presence of NK cell (stimulating) feeder cells, NK cell stimulating cytokines, NK cell stimulating particles, NK cell stimulating exosome, or any combination thereof.
  • ex-vivo refers to a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube).
  • Engineering of an immune cell to express a CAR as disclosed herein may occur before, during, or after expansion and/or activation of NK cells as disclosed herein.
  • primary NK cells such as (naive) primary NK cells isolated from blood of a human donor
  • nucleic acid molecules, including vectors, that encode the CAR as disclosed herein can be transduced with nucleic acid molecules, including vectors, that encode the CAR as disclosed herein.
  • the following methods and means for transducing an immune cell with nucleic acid molecules, including vectors, that encode the CAR as disclosed herein are considered standard and routine.
  • transduction methods for introducing a CAR into an immune cell such as an NK cell employ viral vectors or non-viral vectors that encode a CAR as disclosed herein.
  • retrovirus- or lentivirus-based vectors may be employed to genetically engineer NK cells, preferably primary NK cells, due to their stable integration into the genome.
  • Preferred vectors are AAV vectors.
  • Non-viral methods, such as the use of naked DNA, are advantageous in that they have a low immunogenicity and are inexpensive, but do not integrate into the genome.
  • Transposon systems can also be employed as non-viral means to transduce an NK cell with a CAR.
  • the Sleeping Beauty (SB) transposon system is an example of a non-viral NK cell gene transfer system that combines the advantages of viral and non-viral vectors since they mediate stable transgene expression.
  • NK cells Another non-viral transduction system for NK cells includes electroporation of a nucleic acid (e.g. mRNA or DNA) that encodes a CAR as disclosed herein in an NK cell as disclosed herein.
  • a nucleic acid e.g. mRNA or DNA
  • An example of a transfection method that involves NK cell electroporation with plasmid DNA encoding a CAR is for instance reported in Igegnere et al., Front. Immunol. 10:957 (2019).
  • This NK cell transfection method can either be performed on expanded and/or activated NK cells and on nonexpanded and/or nonactivatived NK cells.
  • the nucleic acid molecule and/or vector as disclosed herein is genomically integrated into an NK cell as disclosed herein.
  • the present disclosure provides engineered immune cells, coronavirus antibodies, and pharmaceutical compositions for the treatment and prevention of coronavirus infection and/or coronavirus mediated disease in an individual.
  • Immune cells in particular NK cells, are provided for the treatment and prevention of coronavirus infection and/or coronavirus mediated disease.
  • the immune cells for treatment are engineered immune cells expressing the CARs described herein (immune cell-CAR).
  • the treatment further comprises the administration of one or more coronavirus antibodies as disclosed herein, preferably in combination with an engineered NK cell as disclosed herein or an NK cell that is not engineered with a CAR as disclosed herein.
  • the antibodies and immune cells may be provided in the same pharmaceutical composition.
  • the antibodies When used as a combined treatment, the antibodies may be administered simultaneously or separately from the immune cells.
  • the immune cells may be administered as a single dose and the antibodies may be provided one daily or once weekly.
  • the present disclosure also provides (i) immune cells, such as immune cells engineered with a CAR as disclosed herein or immune cells that are not engineered with a CAR as disclosed herein and (ii) an immunogenic composition, preferably a vaccine composition, that elicits, raises or induces an immune response, preferably a humoral immune response, against coronavirus.
  • said immunogenic composition comprises at least one coronavirus antigen, more preferably at least one spike protein antigen, against which a humoral immune response is to be raised.
  • the immune response product (preferably an antibody) of the immune response that is to be raised binds an epitope that is evolutionary conserved between different coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, and MERS-CoV).
  • an evolutionary conserved epitope is bound by an antibody and/or CAR as disclosed herein.
  • said immunogenic composition comprises an antigen, more preferably a spike protein antigen that comprises at least part, or all, of the spike protein.
  • antigen can be used interchangeably with the term “antigenic protein” herein.
  • the (i) immune cells and (ii) immunogenic composition can be provided in the same pharmaceutical composition. Preferably, they are provided in separate pharmaceutical compositions, e.g., as a kit of parts.
  • the immune cells preferably NK cells engineered or not engineered with a CAR as disclosed herein, may be administered simultaneously or separately from the immunogenic composition.
  • the immune cells may be administered as a single dose or as a multiple dose.
  • the immune cells are administered daily, weekly, monthly or quarterly. For instance, during a coronavirus epidemic, the immune cells can be administered once monthly.
  • dosage levels of NK cells range from 1x10 6 cells/dose to 1x10 12 cells/dose, in particular from 1x10 7 cells/dose to 2x10 9 cells/dose, preferably administered intravenously.
  • the immune cells are administered to a subject separately from the immunogenic composition. More preferably, the immune cells are administered after the immunogenic composition has been administered.
  • NK cells are administered when a humoral immune response has been raised against an immunogenic composition as disclosed herein. Without being bound by theory, this allows for the NK cells to mediate ADCC through Fc-binding with the antibodies raised. Additionally, and depending on the time of dosing, NK cells can stimulate the adoptive immune system and therefore improve the immune response of a vaccine.
  • Common vaccines against viruses contain inactivated or live attenuated viruses.
  • Inactivated vaccines are based on subunit viral antigens or split viruses.
  • Split virus vaccines are prepared by disrupting viral particles by treatment with detergents or other chemicals.
  • Live- attenuated viral vaccines are typically produced from viruses that do not replicate well at body temperature for instance because they are cold- adapted viruses.
  • Subunit vaccine types are vaccines that comprise recomb inantly viral proteins, such as the spike protein.
  • Vaccine platforms include nucleic acid-based vaccines such as vaccines that comprise DNA or mRNA encoding a coronavirus spike protein, or part thereof.
  • Another example of a vaccine platform is a vector-based, preferably viral vector-based, coronavirus vaccine such as a vaccine that comprises an alphavirus vector that expresses a coronavirus spike protein, or part thereof or an adenovirus vector that expresses a coronavirus spike protein, or part thereof.
  • Chimpanzee adenovirus and modified vaccinia virus Ankara are other examples of suitable vectors for expression of viral antigens.
  • VLP virus-like particles
  • coronavirus vaccines that can be employed as an immunogenic composition as disclosed herein are the following:
  • the immunogenic composition is directed to SARS-CoV-2.
  • Follegatti and colleagues describe a vaccine called ChAdOx1 nCoV-19 directed against SARS-CoV-2.
  • This virus is a viral vector vaccine that expresses the spike protein of SARS-CoV-2.
  • the ChAdOx1 nCoV-19 vaccine consists of the replication- deficient simian adenovirus vector ChAdOx1, containing the full-length structural surface glycoprotein (spike protein) of SARS-CoV-2., with a tissue plasminogen activator leader sequence.
  • the ChAdOx1 nCoV-19 vaccine expresses a codon-optimized coding sequence for the spike protein.
  • the vaccine mRNA-1273 is a lipid nanoparticle— encapsulated, nucleoside-modified messenger RNA (mRNA)-based vaccine that encodes the SARS-CoV-2 spike glycoprotein stabilized in its prefusion conformation.
  • mRNA messenger RNA
  • This new type of vaccine involves making a synthetic version of the coronavirus spike proteins’ RNA. It is believed that the vaccinated individual will make the spike protein itself and thereby induce an immune response.
  • McKay and colleagues describe a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle (LNP) as a vaccine.
  • LNP lipid nanoparticle
  • Mulligan and colleagues describe a phase 1/2 study of the COVID-9 RNA vaccine BNT162b1 (BioNTech's mRNA COVID-19 vaccine).
  • BNT162b1 is a lipid nanoparticle- formulated, nucleoside-modified mRNA vaccine that encodes trimerized SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD).
  • RBD trimerized SARS-CoV-2 spike glycoprotein receptor-binding domain
  • the vaccine RNA is formulated in lipid nanoparticles (LNPs) for more efficient delivery into cells after intramuscular injection.
  • LNPs lipid nanoparticles
  • the BNT162b2 vaccine encodes the SARSCoV-2 full-length spike, modified by 2 proline mutations (P2 S) to lock it in the profusion conformation to increase its potential to elicit virus-neutralizing antibodies (Walsh, E.E. et al. RNA-Based COVID-19 Vaccine BNT162b2 Selected for a Pivotal Efficacy Study, concurrently undergoing scientific peer review for potential publication. medRxiv preprint doi: https://doi.org/10.1101/2020.08.17.20176651.this version posted August 28, 2020) Inovio has developed a DNA vaccine called INO-4800.
  • Gao and colleagues describe a purified inactivated SARS-CoV-2 virus for a traditional method to develop a vaccine.
  • the virus strain CN2 was chosen to develop a purified inactivated SARS-CoV-2 virus vaccine, PiCoVacc (Gao Q, Bao L, Mao H, et al. Development of an inactivated vaccine candidate for SARS-CoV-2. Science 2020;369:77-81).
  • the vaccine is an inactivated SARS-CoV-2 whole virion vaccine with aluminium hydroxide as adjuvant developed by Sinovac Life Sciences Co. These traditional techniques have been used for decades to make vaccines to protect against diseases including influenza and polio.
  • the vaccine comprises virus particles that have been killer or inactivated and do no longer cause infection.
  • the immune system will recognize the virus provoking an immune response.
  • the immunogenic composition is directed to MERS-CoV.
  • MERS-CoV Muthumani and colleagues reported on the development of a synthetic DNA vaccine against MERS-CoV.
  • the DNA vaccine encoded the MERS spike protein and triggered an immune response in nonhuman primates (Muthumani, D. et al., A synthetic consensus anti-spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates. Sci. Transl. Med.7, 301ra132 (2015)).
  • rRBD recombinant receptor-binding domain
  • the vaccine comprises the MERS- CoV rRBD protein containing an 240 amino acid fragment spanning residues 367-606 (Lan J., et al., Recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against Middle East respiratory syndrome coronavirus challenge. EBioMedicine 2, 1438-1446 (2015)).
  • VLPs virus-like particle Virus-like particles by co- expressing the S, envelope (E) and membrane (M) genes of the MERS CoV.
  • E S, envelope
  • M membrane
  • VLPs are protein-only subunit vaccines that emulate the morphology of the native virus. Compared with inactivated or live-attenuated virus vaccines, VLPs are able to induce robust humour al and cellular immune responses without the risk of reversion to virulence (Wang C et al., MERS-CoV virus-like particles produced in insect cells induce specific humour al and cellular imminity in rhesus macaques. Oncotarget 8, 12686-12694 (2017)).
  • ChAdOx1 MERS a replication- deficient simian adenovirus vector
  • ChAdOx1 MERS vaccine was shown to protect rhesus macaques from infection (Van Doremalen N, et al. A single dose of ChAdOx1 MERS provides protective immunity in rhesus macaques. Sci Adv 2020; 6: eaba8399).
  • GLS-5300 is a DNA vaccine that expresses the full-length MERS coronavirus S-glycoprotein antigen.
  • Inovio Pharmaceuticals (Modjarrad K., et al. Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial. Lancet Infect Dis 2019; 19: 1013-22. http://dx.doi.org/10.1016/S 1473-3099(19)30266-X)
  • the immunogenic composition is directed to SARS-CoV-1.
  • SARS-CoV- 1 inactivated SARS coronavirus
  • ICV inactivated SARS CoV-1 vaccine
  • VRC- SRSDNA015-00-VP The VRC-SRSDNA015-00-VP vaccine is based upon cDNA expression of SARS Spike glycoprotein (Urbani strain) with codon-modification to optimize expression in human cells. It expresses the full sequence except for deletion of the last 13 COOH-terminal amino acids (J.E. Martin, M.K. Louder, L.A. Holman, I.J. Gordon, M.E. Enama, B.D. Larkin, C.A. Andrews, L. Vogel, R.A. Koup, M. Roederer, et al., VRC 301 Study Team.
  • a SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial. Vaccine, 26 (2008), pp. 6338-6343).
  • the immunogenic composition is an inactivated coronavirus vaccine such as a subunit vaccine comprising coronavirus antigens or a split virus vaccine; a live attenuated virus vaccine; an recombinant protein vaccine comprising a recombinant protein of at least one of the spike antigens disclosed herein; a vaccine comprising a nucleic acid, such as a DNA or mRNA, encoding a coronavirus spike protein or antigenic part thereof; a vaccine comprising a vector, preferably a viral vector, that expresses an antigen as disclosed herein; or a vaccine comprising a VLP that displays on its outer surface at least one antigen disclosed herein.
  • a coronavirus vaccine such as a subunit vaccine comprising coronavirus antigens or a split virus vaccine; a live attenuated virus vaccine; an recombinant protein vaccine comprising a recombinant protein of at least one of the spike antigens disclosed herein; a vaccine comprising a nucle
  • the live attenuated virus is a coronavirus selected from SARS- CoV-1, MERS-CoV, or SARS-CoV-2.
  • the antigen is a coronavirus spike protein or an antigenic fragment thereof.
  • the antigen is a coronavirus spike protein or an antigenic fragment thereof from SARS-CoV-1, MERS-CoV, or SARS-CoV-2.
  • the immunogenic composition as disclosed herein elicits, raises or induces antibodies that bind to an evolutionary conserved epitope of the spike protein, preferably an epitope that is universal (or shared) between SARS-CoV-1 and SARS- CoV-2; or between SARS-CoV-1, MERS-CoV, and SARS-CoV-2.
  • the antibodies elicited by an immunogenic composition as disclosed herein bind to an evolutionary conserved epitope of spike protein that is bound by an antibody and/or CAR as disclosed herein.
  • the antibodies that are raised by administration of an immunogenic compositions may have an antigen binding region that cross-re acts with coronaviruses.
  • the antigen binding regions recognize epitopes that are evolutionarily conserved across multiple coronaviruses.
  • An immunogenic composition as disclosed herein can be a multivalent vaccine, i.e. a vaccine that comprises more than one antigen, for instance antigens of both SARS- CoV- 1 and SARS-CoV-2.
  • the antigen(s) as disclosed herein is (are) an immunogen(s).
  • an immunogenic composition as disclosed herein can be either adjuvanted or not adjuvanted.
  • the immunogenic composition comprises an adjuvant, for example an oil-in-water emulsion.
  • the oil and emulsifier are in an aqueous carrier such as an aqueous solution.
  • the aqueous carrier may, for example, be a phosphate buffered saline or a citrate buffer.
  • An example of an adjuvant is MF59, which is an oil-in-water emulsion of squalene oil.
  • An immunogenic composition as disclosed herein is preferably administered by parental administration, preferably by intramuscular, subcutaneous or intradermal administration.
  • Said immunogenic composition can be administered in the form of a single dose or in the form of a multiple dose such as a prime-boost regimen.
  • the immunogenic composition can be provided in the form of an off-the-shelf, pre- filled parenteral injection device, such as a syringe, or said immunogenic composition can be formulated, and filled in a parenteral injection device, moments prior to administration.
  • the immune cells preferably NK cells
  • Anti-viral antibodies may induce antibody-dependent enhancement (ADE) of viral infection.
  • Antibody-dependent enhancement of infection refers to the promotion of viral infection of host cells as a result of an interaction between virus-bound antibodies and Fc receptors on host cells and subsequent phagocytosis of the virion (Van Erp et al. Front Immunol. 2019; 10:548). More specifically, in antibody-dependent enhancement, the number of virus- infected host cells is increased in the presence of antibodies or antibody concentration that are suboptimal. This may result in more severe symptoms.
  • NK cell CARs are different from antibodies in that they do not contain an Fc region, they have the advantage of not contributing to antibody- dependent enhancement of viral infection to the same extent as anti-viral antibodies and vaccines. Therefore, in method of treating and/or preventing viral-mediated disease, preferably NK cell CARs as disclosed herein are employed so as to counteract, inhibit, prevent, or minimize or reduce the risk of, antibody- dependent enhancement (ADE) of infection.
  • NK cells as described herein can be CD 16 knockout NK cells.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed.
  • treatment may be administered in the absence of symptoms, e.g., after viral infection has been suspected or confirmed or after potential exposure to the virus.
  • prevent refers to the reduction in viral infection, reduction in the severity of viral infection, and/or reduction of resulting symptoms from infection.
  • the compositions disclosed herein may not prevent viral infection, but rather prevent the severity of disease progression.
  • the compositions may be provided prophylactically.
  • compositions may be provided at the beginning of the seasonal flu season or during a coronavirus epidemic.
  • prevention and/or treatment may not be 100% effective in 100% of individuals administered.
  • the pharmaceutical compositions may comprise one or more antibodies as disclosed herein and an immune cell, such as an NK-cell. While not wishing to be bound by theory, the disclosure provides that the Fc domain (or an antigen binding domain specific for CD 16) of an antibody targeting a virally infected cell can subsequently bind to the CD 16 activating receptor on NK cells resulting in NK cell activation, resulting in viral killing/neutralization.
  • the pharmaceutical compositions may comprise engineered CAR-expressing immune cells.
  • the pharmaceutical compositions provide a source of activated NK cells to kill or neutralize virally infected target cells, e.g., through cytotoxic mechanism such as perforin and granzyme release.
  • NK cells can produce and secrete IFN ⁇ which can then stimulate humoral responses, specifically the production of IgG2a and IgG3.
  • IFN ⁇ can also activate macrophages that then can clear opsonized cells and particles.
  • the adoptive administration of NK cells that recognize virally compromised cells can induce IFN ⁇ release. While not wishing to be bound by theory, the induced IFN ⁇ can then enhance overall humoral immunity as well as clearance of opsonized cellular targets or opsonized viral particles.
  • the immune cells of the present disclosure may be autologous or allogeneic immune cells.
  • Autologous immune cells are cells derived from the subject that is to be treated.
  • Allogeneic immune cells are immune cells derived from a subject different from the subject that is to be treated, said subjects having a non-identical gene at one or more loci. If the immune cells are derived from an identical twin, the v cells can be referred to as “syngeneic”.
  • the term “individual”, as used herein, can be used interchangeably with the term “patient”, and includes reference to a mammal, preferably a human, who is in need of treatment or prevention of a viral infection, preferably coronavirus infection and/or coronavirus mediated disease.
  • the subject can be at least 30 or at least 40 years old. More preferably, the individual is at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or at least 65 years old and/or is an individual that is immunocompromised (also referred to as immunosuppressed). Even more preferably, a subject is at least 66, 67, 68, 69 or at least 70 or at least 80 years old. In some embodiments the individual is a neonate, i.e., less than 12, 6, or 3 months old.
  • the individual is infected with human immunodeficiency virus (HIV), is afflicted with cancer, is or has undergone cancer treatment, such as chemotherapy or radiation, is a transplant recipient, uses immunosuppressant agents (e.g., glucocorticoids), suffers from an auto-immune disease (such as rheumatoid arthritis, Crohn’s disease or multiple sclerosis), suffers from a respiratory condition (such as COPD or asthma), is afflicted with an immune disorder (such as SCID or primary immunodeficiency), seriously ill patients such as those in intensive care, is a neonate, is pregnant, is diabetic, is obese, is older than 65 years, or is a nursing home resident.
  • immunosuppressant agents e.g., glucocorticoids
  • Suitable dosage levels of NK cells range from 1x10 6 cells/dose to 1x10 12 cells/dose, in particular from 1x10 7 cells/dose to 2x10 9 cells/dose, preferably administered intravenously.
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb “to consist” may be replaced by “to consist essentially of' meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.

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Abstract

L'invention concerne des compositions pharmaceutiques pour le traitement ou la prévention d'une infection par des coronavirus ainsi que des maladies médiées par le coronavirus, par exemple des troubles respiratoires. La divulgation concerne des traitements comprenant des cellules immunitaires, telles que des cellules tueuses naturelles, et des anticorps ou des vaccins ciblant des coronavirus. Des compositions et des kits de composants comprenant lesdites cellules immunitaires et lesdits anticorps ou lesdits vaccins sont également prévus. La divulgation concerne également des cellules immunitaires modifiées exprimant un récepteur antigénique chimérique (CAR). Ces cellules exprimant le CAR sont utiles pour le traitement. Des exemples de cibles virales selon la présente invention comprennent la protéine spike du coronavirus, en particulier des épitopes conservés évolutifs de celle-ci.
PCT/NL2021/050207 2020-04-01 2021-03-31 Compositions et procédés ciblant des coronavirus Ceased WO2021201679A1 (fr)

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WO2023086494A1 (fr) * 2021-11-10 2023-05-19 Regents Of The University Of Minnesota Procédés, compositions et kits pour la multiplication de cellules tueuses naturelles
CN116135881A (zh) * 2021-11-17 2023-05-19 中国科学院微生物研究所 一株羊驼源纳米抗体n36及其应用
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US20240058380A1 (en) * 2020-10-22 2024-02-22 Albert-Ludwigs-Universität Freiburg Corona virus-specific t cell receptor fusion constructs, vectors encoding the same, t cells comprising the same and uses thereof
WO2023086494A1 (fr) * 2021-11-10 2023-05-19 Regents Of The University Of Minnesota Procédés, compositions et kits pour la multiplication de cellules tueuses naturelles
CN116135881A (zh) * 2021-11-17 2023-05-19 中国科学院微生物研究所 一株羊驼源纳米抗体n36及其应用
CN116135881B (zh) * 2021-11-17 2025-11-28 中国科学院微生物研究所 一株羊驼源纳米抗体n36及其应用

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