WO2021201677A1 - Compositions et procédés ciblant la grippe - Google Patents

Compositions et procédés ciblant la grippe Download PDF

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Publication number
WO2021201677A1
WO2021201677A1 PCT/NL2021/050205 NL2021050205W WO2021201677A1 WO 2021201677 A1 WO2021201677 A1 WO 2021201677A1 NL 2021050205 W NL2021050205 W NL 2021050205W WO 2021201677 A1 WO2021201677 A1 WO 2021201677A1
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influenza
protein
antibodies
cell
cells
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PCT/NL2021/050205
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English (en)
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Robert Heinz Edward Friesen
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Kiadis Pharma Intellectual Property B.V.
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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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—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 influenza.
  • the disclosure provides treatments comprising immune cells, such as natural killer cells, and antibodies or vaccines that target influenza.
  • 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).
  • CAR chimeric antigen receptor
  • Exemplary influenza targets of the present invention include the influenza viral hemagglutinin (HA) protein, the extracellular domain of an influenza viral M2 protein (M2e), and the influenza viral neuraminidase (NA) protein, in particular evolutionary conserved epitopes thereof.
  • HA influenza viral hemagglutinin
  • M2e extracellular domain of an influenza viral M2 protein
  • NA influenza viral neuraminidase
  • Influenza is an infectious disease that can lead to mild to severe symptoms such as fever and cough and can lead to severe illness including death.
  • Complications associated with influenza include pneumonia, sinus infections, and even heart failure.
  • Immunocompromised individuals are an example of a patient group that is particularly susceptible to severe illness and complications from influenza.
  • Influenza is caused by infection from the influenza virus.
  • Influenza viruses have been grouped into four genus; Influenza type A, Influenza type B, Influenza type C, and Influenza type D.
  • Influenza A viruses can also be divided into different subtypes based on the type of HA protein and NA proteins. These proteins are expressed on the surface of the viral envelope. At least sixteen subtypes of HA (H1-H16) and eleven NA (Nl-Nll) antigenic variants are known in influenza A virus.
  • Type A influenza viruses are further divided into Group 1 and Group 2. These Groups are further divided into subtypes, which refers to classification of a virus based on the sequence of its HA protein.
  • NA is a type II transmembrane glycoprotein. There are nine NA antigenic variants known for Influenza A virus, divided in group I and group II. Group I contains Nl,
  • Group II contains N2, N3, N6, N7 and N9 serotypes.
  • Bat NAdike proteins N10 and Nil are Bat NAdike proteins.
  • NA forms a tetramer of four NA polypeptides on the surface of the virion. Each polypeptide is composed of a highly conserved cytoplasmic tail, a transmembrane region, a stalk and the catalytic head. As opposed to the cytoplasmic tail, the stalk region varies in number and sequence of amino acids (McAuley et al. 2019).
  • the four head domains of the tetramer together form a functional catalytic site capable of cleaving sialic acid, thus enabling viral escape.
  • the catalytic site encompasses an active site and an outer shell. The active site is responsible for directly interacting with sialic acid and is composed of eight highly conserved amino acid residues.
  • the M2 ion channel is a highly conserved viral protein essential for viral budding and assembly of the viral core.
  • the M2 protein is composed of a cytoplasmic C-terminus and an N-terminal ectodomain (M2e), which is exposed on surface of the virion (Wei et al. 2020).
  • HA hemagglutinin
  • HA is a glycoprotein on the surface of the virus that is responsible for interaction of the virus with host cell receptors that contain sialic acid molecules. Typical host cells are epithelial cells of the upper and/or lower airways. NKp46 receptors are expressed on NK cells and contain sialic acid molecules which allow NK cells to engage and bind influenza virions and virally infected cells. In this way, virally infected cells are killed by NK cells.
  • HA proteins are enzymatically cleaved to yield amino-terminal HA1 and carboxy-terminal HA2 polypeptides.
  • the globular head comprises the receptor binding domain (RBD) and is formed from the HA1 polypeptide, whereas the stem that anchors the hemagglutinin protein into the viral lipid envelope comprises a small part of HA1 and HA2.
  • One object of the invention is to provide a broad-spectrum treatment for influenza.
  • a further object of the invention is to provide influenza treatments for immunocompromised and at-risk individuals.
  • the disclosure provides an engineered immune cell expressing a chimeric antigen receptor (CAR), wherein the CAR binds to an influenza viral hemagglutinin (HA) protein, the extracellular domain of an influenza viral M2 protein (M2e), and/or an influenza viral neuraminidase (NA) protein.
  • CAR chimeric antigen receptor
  • the disclosure further provides engineered immune cells as disclosed herein for use in the treatment or prevention of influenza in an individual.
  • the disclosure further provides a method of treating or preventing influenza 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 an influenza viral HA protein an influenza viral M2e protein, and/or an influenza viral NA protein from at least two different influenza virus type A strains.
  • the CAR binds to a viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein from an influenza virus type A strain and from an influenza virus type B strain.
  • the CAR binds to the HA protein from at least two different influenza virus type A HA subtypes.
  • the CAR binds to the HA2 region of viral HA protein.
  • 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
  • a pharmaceutical composition comprising a collection of immune cells and one or more antibodies that bind to an influenza viral HA protein, an influenza viral M2e protein and/or an influenza viral NA protein.
  • the disclosure further provides immune cells (including engineered immune cells and immune cells not engineered with a CAR) as disclosed herein and one or more antibodies that bind to an influenza viral HA protein, an influenza viral M2e protein and/or an influenza viral NA protein for use in the treatment or prevention of influenza 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 influenza 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 an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA 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 an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein.)).
  • CAR chimeric antigen receptor
  • 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.
  • one or more antibodies individually or collectively, binds to a viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein from at least two different influenza virus type A strains
  • one or more antibodies individually or collectively, binds to a viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein from an influenza virus type A strain and from an influenza virus type B strain
  • one or more antibodies individually or collectively, binds to the HA protein from at least two different influenza virus type A HA subtypes
  • one or more antibodies binds to the HA2 region of viral HA protein.
  • the disclosure also provides a multi-specific antibody comprising: i) at least one antigen binding domain that binds an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA 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 an influenza viral HA protein, an influenza viral M2e protein,) and/or an influenza viral NA 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 an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA 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 is HAI+ and/or binds the head region of viral HA protein.
  • the disclosure provides a multi-specific antibody that binds: i) an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein and ii) an NK cell activating receptor, preferably NKp46 and/or CD 16.
  • the antibody is HAI+ and/or binds the head region of viral HA protein.
  • the multi- specific antibody is HAI+ and/or binds the head region of viral HA protein.
  • the disclosure provides the multi-specific antibodies described herein for the treatment and or prevention of influenza.
  • a method is provided comprising administering to an individual in need thereof an effective amount of the multi specific antibody as described herein.
  • the treatments may further comprise the use or administration of immune cells (including engineered immune cells) as describe herein.
  • the immune cells express a chimeric antigen receptor (CAR) (i.e., is an engineered immune cell, preferably the CAR binds to an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein.)).
  • CAR chimeric antigen receptor
  • the immune cell or engineered immune cell expresses NKp46, NKp44, NKp30,
  • 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 influenza virus, for use in the treatment and/or prevention of influenza in an individual.
  • the disclosure provides a method of treating or preventing influenza 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 influenza virus.
  • 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 an influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein.)).
  • CAR chimeric antigen receptor
  • the immune cell or engineered immune cell expresses NKp46, NKp44, NKp30,
  • 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 at least one HA, NA and/or Me2 antigen, preferably at least one HA antigen.
  • the immunogenic composition elicits a humoral immune response against influenza viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein.
  • the humoral immune response elicits one or more antibodies which, individually or collectively, bind to a viral HA protein, an influenza viral M2e protein, and/or an influenza viral NA protein from at least two different influenza virus types, influenza subtypes and/or influenza strains A strains.
  • the immunogenic composition is selected from an inactivated influenza virus vaccine; a live attenuated influenza virus vaccine; an influenza virus recombinant protein vaccine comprising a recombinant protein of at least one antigen of HA, NA and/or M2e; an influenza nucleic acid vaccine encoding at least one influenza antigen of HA, NA and/or M2e; an influenza vaccine comprising a vector, preferably a viral vector, that expresses at least one HA, NA and/or M2e antigen; and an influenza vaccine comprising a VLP that displays on its outer surface at least one HA, NA and/or M2e antigen.
  • the disclosure also provides direct viral killing/neutralization (e.g. SARS-CoV-2 and influenza) by freshly isolated donor-derived NK cells/K-NK cells/Fc-imprinted K-NK cells.
  • direct viral killing/neutralization e.g. SARS-CoV-2 and influenza
  • the disclosure also provided the combination of such NK cells with polyclonal or monoclonal antibodies, in particular antibodies that bind Hemagglutinin of the influenza 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) Schering 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 activated with PM21 result in the upregulation of CD16, 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 for viruses like influenza.
  • Figure 4 Schematic depiction of influenza HA protein indicating the head region comprising the receptor binding site and the stem region.
  • 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.
  • Influenza infection causes potentially lethal pneumonia. Shortly after influenza infection, NK-cell become hyperresponsive with an increased killing of influenza- infected cells, facilitated by the activating NKp46 receptor on NK-cells, which recognize viral HA on the surface of infected cells. NK-cells are actively recruited to the lungs and airways during influenza infection. Infected respiratory epithelial cells release chemokines that attract NK-cells. Migration of NK-cells is dictated by the severity of influenza 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. While not wishing to be bound by theory, 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.
  • the disclosure also provides engineered CAR-expressing immune cells, which can be combined with antibody treatment. Typically, virus infected individuals have low NK cell counts with an exhausted phenotype.
  • NK cells can produce and secrete IFNy which has a direct anti-viral effect and additionally stimulates humoral responses, specifically the production of IgG2a and IgG3.
  • IFNy 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 IFNy release. While not wishing to be bound by theory, the induced IFNy can then enhance overall humoral immunity as well as clearance of opsonized cellular targets or opsonized viral particles.
  • cross-reactive antibodies e.g., HA stem binding antibodies
  • HA stem binding antibodies lack potency and clinical trials with such stem-binding antibodies have been unsuccessful.
  • the combination of such antibodies with immune cells, or the presentation of the antigen binding region of such low potent cross -re active 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 a , 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 an influenza viral HA protein, an influenza viral M2e protein, and/or a viral NA protein.
  • the disclosure also provides a combined treatment comprising an immune cell and one or more antibodies as described herein that bind an influenza viral HA protein and/or a viral NA 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 “light” chain.
  • the human light 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 light 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, CDR3, FR4.
  • the variable regions of the light 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 IgGi or IgG3.
  • 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 linked to suitable Fc regions.
  • single domain antibodies refer to a single variable antibody domain.
  • Exemplary single domain antibodies include VHH fragments, such as those found in camelids, VNAR 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 influenza strains or subtypes.
  • the antigen binding regions recognize epitopes that are evolution arily conserved across multiple influenza strains or subtypes.
  • epitopes are generally present on the stem region of HA, but also occur on the head region and may interfere with receptor binding or HA trimer integrity.
  • compositions and methods of treatments described herein may also comprise several antibodies, wherein the antibodies recognize different and/or overlapping influenza strains or subtypes.
  • the composition or method of treatment may comprise a first antibody that recognizes HA1 subtype influenza strains and a second antibody that recognizes HA2 subtype influenza strains.
  • the composition or method of treatment may comprise an antibody that binds an HA1 subtype influenza strain and an HA2 subtype influenza strain.
  • 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 and/or overlapping influenza strains or subtypes.
  • the antibody maybe a multi-specific antibody, e.g, abi- or tri-specific antibody, that comprises multiple antigen binding domains.
  • abi- or tri-specific antibody e.g. abi- or tri-specific antibody.
  • 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 viral HA protein from at least two different influenza viruses (e.g., different type A strains or subgroups).
  • the one or more antibodies individually or collectively, bind to a viral NA protein from at least two different influenza viruses (e.g., different type A strains or subgroups).
  • the one or more antibodies individually or collectively, bind to a viral NA protein from at least two different influenza viruses and a viral HA protein from at least two different influenza viruses.
  • the one or more antibodies, individually or collectively bind to a Me2 from at least two different influenza viruses (e.g., different type A strains or subgroups).
  • 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 HA proteins, either because, e.g., the antigen binding domain is cross-reactive for two different HA proteins or the first antibody comprises two different antigen binding domains, wherein the first antigen binding domain binds, e.g., HI, H2, H5, H6 and H9, and the second antigen binding domain binds, e.g., HI, H3, H5, H7, and H9; together with a second antibody that binds at least two different NA proteins, either because, e.g., the antigen binding domain is cross -re active for two different NA proteins or the second antibody comprises two different antigen binding domains, wherein the first antigen binding domain binds, e.g., NA1, and the second antigen binding domain binds, e.g., NA2.
  • antibodies suitable for use in the present disclosure may also be bi- 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.
  • NK cells are generally activated by the NK-46 receptor which engages viral HA on the surface of influenza infected cells through a sialic acid molecule that is on the NK-46 receptor and which binds the receptor -binding domain (RBD) of HA.
  • the viral specific antigen binding domain of some antibodies may interfere with immune cells binding to virally infected cells.
  • many antibodies that bind to the head region of HA interfere with binding to sialic acid moieties on the surface of host cells. This interaction can be measured using a hemagglutination assay and antibodies which block this interaction are referred to as HAI+.
  • HAI+ antibodies therefore inhibit the interaction between the sialic acid molecule on NKp46 with HA on the host cell.
  • HAI+ antibodies are often effective antibodies, neutralizing the virus, e.g., by inhibiting the viral attachment and therefore prevention of target cell infection.
  • the interaction between HA and NK cells is necessary for optimal Fc mediated effector function via CD 16 of anti-HA antibodies.
  • the multi-specific antibodies provided herein allow for both targeting to the virally infected cell, as well as activating NK cells via NKp46. This combination is particularly important when the antigen binding domains are HAI+ and/or bind the head region of HA.
  • 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 FcyRIIIa (CD16a).
  • FcyRIIIa 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. In some embodiments, 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 linkers 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 FcyRIIIa (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)). Behar et al.
  • sdAbs anti-FcyRIII single domain antibodies
  • sdAbs anti-FcyRIII single domain antibodies
  • the amino acid sequences of isolated anti-FcyRIII sdAbs C13, C21, C28 and C72 are displayed in Figure 1 of Behar et al, which are explicitly incorporated by reference herein. Behar et al. discloses that these sdAbs bind FcyRIIIA+ 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 influenza virus and/or influenza virus-infected cells by virtue of its binding specificity towards both an influenza 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.
  • the binding of an anti-CD 16 antigen binding fragment or the Fc domain to the CD 16 activating receptor on 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.
  • 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, CD134 (0X40), and/or CD137 (4- 1BB).
  • a second or third signalling domain e.g., 2B4, NKG2D, DAP10, DAP 12, IL21R, CD134 (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 influenza strains or subtypes.
  • the CARs described herein may also comprise several antigen recognitions regions, wherein the antigen recognitions regions recognize different and/or overlapping influenza strains or subtypes.
  • the CAR may comprise a first antigen recognitions region that recognizes HI, H2, H5, H6 and H9 influenza-based subtypes and a second antigen recognitions region that recognizes HI, H3, H5, H7, and H9 influenza-based subtypes subtype influenza strains.
  • the CAR may further comprise, e.g., a third antigen recognition region that recognizes NA and a fourth antigen recognition region that recognizes Me2.
  • 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 influenza convalescent donors.
  • the CAR or one or more antibodies recognizes at least two different strains of influenza A viruses and/or at least two different strains of influenza B viruses.
  • the CAR or one or more antibodies binds to a viral HA protein from at least two different influenza virus strains (also referred to as clades). In some embodiments, the CAR or one or more antibodies binds to a viral HA protein from at least two different influenza virus type A strains or two influenza B viruses from different lineages.
  • the CAR or one or more antibodies recognizes at least two different strains of HI influenza virus, at least two different strains of H2 influenza virus, at least two different strains of influenza H3 virus, at least two different strains of influenza H4 virus, at least two different strains of influenza H5 virus, at least two different strains of influenza H6 virus, at least two different strains of H7 influenza virus, at least two different strains of H8 influenza virus, at least two different strains of H9 influenza virus, at least two different strains of H10 influenza virus, at least two different strains of Hll influenza virus, at least two different strains of H12 influenza virus, at least two different strains of H13 influenza virus, at least two different strains of H14 influenza virus, at least two different strains of H15 influenza virus, or and at least two different strains of H16 influenza virus.
  • the CAR or one or more antibodies disclosed herein recognizes different HA subtypes of influenza.
  • said CAR or one or more antibodies binds to HI and H3 influenza subtypes.
  • 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 II 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 or one or more antibodies recognizes at least two different strains of N1 influenza virus, at least two different strains of N2 influenza virus, at least two different strains of influenza N3 virus, at least two different strains of influenza N4 virus, at least two different strains of influenza N5 virus, at least two different strains of influenza N6 virus, at least two different strains of N7 influenza virus, at least two different strains of N8 influenza virus, at least two different strains of N9 influenza virus.
  • the CAR or one or more antibodies disclosed herein recognizes different NA subtypes of influenza.
  • said CAR or one or more antibodies binds to N1 and N2 influenza subtypes.
  • the CAR or one or more antibodies recognizes at least two different strains of influenza A viruses and/or at least two different strains of influenza B viruses. In one embodiment, the CAR or one or more antibodies recognizes M2e of at least two different strains of influenza virus.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein preferably bind to an evolutionarily conserved epitope of HA, NA, or Me2.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein bind to the HA stem or stalk region.
  • the CARs and antibodies recognize the HA2 region of the stem. It is within the skill of one in the art to identify antibodies that bind to the HA stem.
  • W02013044203 describes immunogens for generating broad-spectrum HA stem antibodies.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein bind to the HA head region, for example the RBD.
  • Broad spectrum influenza antibodies have also been described in e.g., Wang et al. PLoS Pathog. 2010 Feb; 6(2): el000796.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated CR6255, CR6257, CR6260, CR6261, CR6262, CR6268, CR6272, CR6307, CR6310, CR6314, CR6323, CR6325, CR6327, CR6328, CR6329, CR6331, CR6332, CR6334, CR6336, CR6339, CR6342, CR6343, CR6344 from Table 12 of US9, 109,017.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated CR9XXX selected from Table 9 of US9,593,159 (i.e., CR9005, CR9030, CR9112, CR9114).
  • 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 antibodies.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated CR8001, CR8003, CR8015, CR8016, CR8017, CR8018, CR8019, CR8020, CR8021, CR8038, CR8039, CR8040, CR8041, CR8043, CR8049, CR8050, CR8052, CR8055, CR8057, and CR8069 depicted in Table 12 of US9,611,317.
  • 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 described above.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated F16 or F128 as described in US9,340,603 or Y 13F9 and Y6F5 described in US9,005,623.
  • 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 antibodies described above.
  • Kallewaard et al., 2016 (Cell;166(3):596-608 (2016)) describe the isolation and characterization of a broad-spectrum human monoclonal antibody (mAh), MEDI8852, which binds a HA stem region shared in viruses from all 18 influenza A virus subtypes and demonstrates coverage of both seasonal and pandemic influenza A.
  • WO2017123685 Al the contents of which are herein incorporated by reference, describe the MEDI8852 antibody.
  • Especially the heavy and light chain CDR regions identified in SEQ ID Nos: 1-3 (heavy chain CDR) and SEQ ID Nos:8-10 (light chain CDR) are herein incorporated by reference.
  • Particularly useful CARs and antibodies comprise one or more of the aforementioned heavy chain CDRs of antibody MEDI8852 as disclosed in WO2017123685 Al.
  • 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 described above.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated SD36, SD38, SD83, and SD84. In addition to the heavy chain CDRs, said CARs and antibodies may further comprise the heavy chain variable region of said single-domain antibodies.
  • particularly useful CARs and antibodies comprise at least two, at least three, or at all four of the single domain antibodies designated SD36, SD38, SD83, and SD84.
  • a particularly preferred CAR comprises MD2407 (SD38-SD36-SD83-SD84) as the antigen recognition domain.
  • a particularly preferred antibody comprises MD2407 (SD38-SD36-SD83-SD84) as the antigen binding domain. As shown in Laursen et al., MD2407 recognizes both influenza A and influenza B viruses.
  • a particularly preferred antibody of the disclosure is MD3606 (MD2407 fused to human IgGl Fc).
  • Dreyfus et al., 2012 (Science. 2012 Sep 14; 337(6100): 1343-1348) describes examples of head binding HA antibodies, including antibodies CR8033 and CR8071 which recognize distinct conserved epitopes in the head region of the influenza B HA and protect against infection with different lineages of influenza B viruses.
  • EP2822968 Al hereby incorporated by reference, describes HA head binding molecules that cross-react between different influenza B strains.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated as CR8033, CR8059, CR8071, CR10023, CR10032, CR10049, CR10051, CR11024, CR11035, CR11036, CR11038, CR11039, CR08031, CR08032, CR08034 and CR08035 in Table 5Aof EP2822968 and/or particularly useful CARs and antibodies comprise the light chain CDRs from one or more antibodies designated as CR8033, CR8059, CR8071, CR10023, CR10032, CR10049, CR10051, CR11024, CR11035, CR11036, CR11038, CR11039, CR08031, CR08032, CR08034 and CR08035 in Table 5B of EP2822968.
  • said CARs and antibodies may further comprise the light chain CDRs.
  • C139/1 (S 139/1) that binds the receptor-binding domain of HA and neutralizes many influenza subtypes such as HI, H2, H3, H5, H9 and H13.
  • Monoclonal antibody S139/1 can be obtained through Creative Biolabs (US).
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from antibody S139/1.
  • 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 described above.
  • WO 2010/010466 A2 hereby incorporated by reference, describes antibodies that bind to HA and neutralize infection of more than one subtype of influenza A virus.
  • WO 2010/010466 A2 discloses antibodies, or antibody binding fragments thereof, with a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 18; and/or a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 19.
  • Such antibodies or antibody binding fragments are incorporated herein by reference.
  • WO 2010/010466 A2 also discloses antibodies or antibody binding fragments comprising a light chain CDR1 with the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 20; a light chain CDR2 with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 21; and a light chain CDR3 with the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 22.
  • Such antibodies or antibody binding fragments are incorporated herein by reference.
  • a CAR as disclosed herein may comprise one or more of such antibody or binding fragments (such as a CDR) thereof.
  • a CAR as disclosed herein comprises one or more of the CDRs of antibody FI6 variant 1 or variant 2 as disclosed in WO 2010/010466 A2, i.e. one or more CDRs of SEQ ID Nos: 1-3 (heavy chain CDRs) and/or SEQ ID Nos:4-6 (CDRs of light chain) as disclosed in WO 2010/010466 A2.
  • C179 which neutralizes influenza A group 1 (HI, H2, H5, H6, H9) (J Virol. 1993; 67(5):2552-8).
  • US5,684,146 hereby incorporated by reference, describes the antibody C179 sequence.
  • Particularly useful CARs and antibodies comprise the heavy chain and/or light chain CDRs from C179 as depicted in SEQ ID Nos:5-10 in US5,684, 146.
  • 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.
  • Fu et al., 2016 (Nature Communications volume 7, Article number: 12780 (2016) describes a monoclonal antibody 3114 that binds to and neutralizes groups 1 and 2 influenza A viruses.
  • the heavy and light chain CDR sequences, and variable heavy and light chain sequences, of antibody 3114 are depicted in Supplementary Figure 2A and 2B of Fu et al., 2016 are hereby incorporated by reference.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs of antibody 3114.
  • 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.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies bind to the HA 90-Loop and/or the HA 220-Loop. In some embodiments, binding disrupts the integrity of the HA protein trimers.
  • Bangaru et al., 2019 (Cell, 16;177(5):1136-1152 (2019)) describe antibody (Ab) FluA-20, which reacts with most influenza A viruses and binds to the HA head domain and disrupts the integrity of HA protein trimers, thereby inhibiting spread of virus in culture, and providing protection against challenges with viruses of H INI, H3N2, H5N1, or H7N9 subtypes.
  • the epitope that FluA-20 binds to is disclosed in Table S6 of Bangaru et al., 2019, said epitope being incorporated by reference herein as a conserved HA- head epitope.
  • the epitope that FluA-20 specifically binds comprises the HA 90-Loop (HA residues 86, 96 and 98) and 220-Loop (HA residues 216, 219, 220, 221, 222, 223, 224 and 229) of which the major epitope contact residues the antibody contacts with are residues 96, 220, 221, 223 and 229.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs of antibody FluA-20.
  • 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.
  • particularly useful CARs and antibodies bind the epitope that FluA-20 binds, and which preferably disrupt the integrity of HA protein trimers.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein bind to NA, in particular evolution arily conserved regions of NA.
  • US8,383, 121 hereby incorporated by reference, describes binding molecules that bind to glycoprotein N1 subtypes.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated 6C6 or 3D4 selected from table 1 of US8,383, 121.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from the antibody designated 2B9 as disclosed in US9, 115,201.
  • a humanized heavy chain is described by SEQ ID NO:7 and 8 and a humanized light chain is described by SEQ ID NO:9 and 10.
  • 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, in particular the humanized variable domains.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more VHH domains as disclosed in Figure 2A of US10,023,629, in particular VHHs of N1-3-VHH, N1-5-VHH and N1-7-VHH.
  • said CARs and antibodies may further comprise the heavy chain variable region of said single-domain antibodies.
  • particularly useful CARs and antibodies comprise at least two, at least three, or four or more of the single domain antibodies described in Figure 2A.
  • CD6 is shown to bind to an epitope that spans two adjacent monomers of NA in a tetramer, in particular CD6 contacts amino acids 93, 94, 95, 355, 358, 375, 377, 378, 388, 389, 449, 450, 451 on a first of the adjacent monomers in an NA tetramer and amino acids 216, 217, 219, 220, 221, 250, 251, 252, 254, 262, 263, 264, 265, 266, 267, 268, 270 on the second of the adjacent monomers on an NA tetramer of an N1 subtype influenza virus.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated HF5, CD6, 4E9, 1H5 as disclosed in US 10,072,070.
  • 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, in particular the humanized variable domains.
  • particularly useful CARs and antibodies bind the epitope that CD6 binds.
  • US2020/0223905 hereby incorporated by reference, describes binding molecules that bind to glycoprotein N of different strains of Influenza B virus.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated 1F2, 1F4, 3G1, 4B2 or 4F11 as disclosed in figure 8-17 of US2020/0223905.
  • 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.
  • Wilson et al. describe a binding molecule binding glycoprotein N9 subtypes (Wilson et al. 2016 Antiviral Research 135: 48-55). Epitope mapping indicated that antibody 3c 10- 3 binds near the enzyme active site of NA.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from the antibody designated 3cl0-3 as disclosed in Wilson et al. In addition to the heavy chain CDRs, said CARs and antibodies may further comprise the light chain CDRs. In some embodiments, said CARs and antibodies may comprise the heavy and/or light chain variable domains of the antibodies.
  • Monoclonal antibody 1G01 binds to all group 1 NAs (Nl, N4, N5 and N8) and all group 2 NAs (N2, N3, N6, N7 and N9) as well as NAs from both Influenza B strains.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated 1G04, 1E01 or 1G01.
  • 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.
  • the CAR antigen recognition domains and the antigen binding regions of the antibodies described herein bind to M2.
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated 8il0, 23K12, 2 IB 15 described in US8,057,796.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from the antibody designated 14C2 described in table 2 of US8,080,244.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from the antibody designated Z3G1 described in US8, 124,091.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated TCN-031 or TCN-032 described in US8,916,160.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from one or more antibodies designated C40G1 or C40G4 described in US8,916, 160.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from one or more of the antibodies designated 8C6 and IB 12 as disclosed in Liu et al. and Zou et al.
  • 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.
  • Kolpe et al. describe a binding molecule binding M2e (Kolpe et al. 2018, Antiviral Res. 158:244-254).
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from the antibody designated Mab65 as disclosed in Kolpe et al.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from one or more of the antibodies designated L18, 019 and SI as disclosed in Fu et al..
  • 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.
  • Fernandez-Sesma et al. describe a binding molecule binding M2e (Fernandez-Sesma et al. Journal of Virology 1996, 70:7:4800-4804).
  • Particularly useful CARs and antibodies comprise the heavy chain CDRs from the antibody designated 3F12 as disclosed in Fernandez-Sesma et al.
  • 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.
  • CARs and antibodies comprise the heavy chain CDRs from the antibody designated M2-7A disclosed in Wei et al. In addition to the heavy chain CDRs, said CARs and antibodies may further comprise the heavy chain variable region.
  • 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 light 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 acid 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.
  • Human bone marrow-derived NK cells may further be characterized by the CD2+CD16+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 expresses a chimeric antigen receptor (CAR) as disclosed herein.
  • 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.
  • 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.
  • primary 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.
  • 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.
  • naive 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 CD 11a, NKG2D, and/or NKp46.
  • 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 (NKEt) NKp30, NKp44, NKp46, NKp80 and/or NKG2D.
  • SLAM signaling lymphocytic activation molecule
  • DNAM-1 DNAX accessory molecule
  • NKEt 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 initi l 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.
  • 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,
  • 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.
  • 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 ah, 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 a , 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 a , 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 a , Cytotherapy. 2009; ll(3):341-55).
  • K562 NK cell feeder cell line 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).
  • mIL-21 membrane bound IL- 21
  • Such a feeder cell line can very beneficially be used to expand and activate NK cells (Denman et ab, 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.
  • 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-expanded 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-expanded 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 are at the disposal of the skilled person.
  • antibody- coated beads Reim et a , Cancer Res 69: 8058- 8066
  • bisphosphonate-capped dendrimers Portevin et ah, J Transl Med 7: 82
  • m e !h y I - P - cy c I o d e x I ri n Li et ah, Hum Immunol, (2011 72: 538-546.
  • NK-92 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).
  • NK cells as disclosed herein.
  • primary NK cells such as (naive) primary NK cells isolated from blood of a human donor, can be transduced with nucleic acid molecules, including vectors, that encode the CAR as disclosed herein.
  • 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.
  • viral vectors 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.
  • 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.
  • NK cell electroporation with plasmid DNA encoding a CAR is for instance reported in Igegnere et ah, 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, influenza antibodies, and pharmaceutical compositions for the treatment and prevention of influenza in an individual.
  • Immune cells in particular NK cells, are provided for the treatment and prevention of influenza.
  • 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 influenza 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. Preferably, they are provided in separate pharmaceutical compositions, e.g., as a kit of parts. When used as a combined treatment, the antibodies may be administered simultaneously or separately from the immune cells. For example, the immune cells maybe 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 influenza virus.
  • an immunogenic composition preferably a vaccine composition, that elicits, raises or induces an immune response, preferably a humoral immune response, against influenza virus.
  • an immunogenic composition preferably a vaccine composition, that elicits, raises or induces an immune response, preferably a humoral immune response, against influenza virus.
  • an immunogenic composition preferably a vaccine composition, that elicits, raises or induces an immune response, preferably a humoral immune response, against influenza virus.
  • said immunogenic composition comprises at least one influenza antigen, more preferably at least one HA, NA and/or Me2 antigen, against which a humoral immune response is to be
  • 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 influenza types, subtypes and/or strains.
  • an evolutionary conserved epitope is bound by an antibody and/or CAR as disclosed herein.
  • said immunogenic composition comprises an HA antigen, more preferably an HA antigen that comprises at least part, or all, of the HA stem region.
  • the HA antigen is a headless HA antigen which may also be referred to as (i) an HA antigen that lacks the HA head domain or an HA antigen that lacks the HA globular region or (ii) an HA stem-only antigen.
  • Sagawa et al. J Gen Virol; 77 (Pt 7): 1483-7 (1996)
  • the term “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.
  • the immune cells can be administered once monthly.
  • the flu season may differ per jurisdiction. For instance, in the US, the flu season is typically October through May, the apex typically being reached in February. In Australia, the flu season is considered May to October with a peak in August.
  • influenza vaccines that have currently received market approval contain either inactivated or live attenuated influenza viruses.
  • the two most predominant types of inactivated vaccines are based on subunit influenza antigens or split viruses.
  • Split virus vaccines are prepared by disrupting viral influenza particles by treatment with detergents or other chemicals.
  • Currently licensed subunit vaccines contain viral HA, NA and/or M2e proteins that are at least partially purified after splitting.
  • Live- attenuated influenza vaccines are typically produced from viruses that do not replicate well at body temperature for instance because they are cold-adapted influenza viruses.
  • Other influenza vaccine types are vaccines that comprise recombinantly produced HA, NA and/or M2e.
  • influenza vaccine platforms include nucleic acid-based vaccines such as vaccines that comprise DNA or mRNA encoding HA, NA and/or M2e proteins.
  • nucleic acid-based vaccines such as vaccines that comprise DNA or mRNA encoding HA, NA and/or M2e proteins.
  • a vector-based, preferably viral vector-based, influenza vaccine such as a vaccine that comprises an alphavirus vector that expresses HA, NA and/or M2e proteins or an adenovirus vector that expresses HA, NA and/or M2e proteins.
  • Chimpanzee adenovirus and modified vaccinia virus Ankara are other examples of suitable vectors for expression of influenza antigens.
  • VLP virus-like particles
  • influenza vaccines that can be employed as an immunogenic composition as disclosed herein are the following:
  • Afluria (Seqirus; STN BL 125254), which is a split virus vaccine indicated for active immunization against influenza disease caused by influenza virus types A and type B that are present in said vaccine in split virus form.
  • Afluria is available in both a trivalent and quadrivalent formulation containing three influenza strains or four influenza strains respectively in split virus form.
  • Afluria is formulated as a sterile suspension for intramuscular injection.
  • FLUVIRIN® An example of a licensed influenza subunit vaccine is FLUVIRIN® (STN: BL 103837).
  • FLUVIRIN® is a trivalent, subunit (purified surface antigen) influenza virus vaccine that contains strains from influenza types A and B.
  • FLUVIRIN® is formulated as a suspension for intramuscular injection.
  • An example of a licensed live-attenuated influenza virus vaccine is FluMist (AstraZeneca/Medlmmune; STN: 125020), which is a quadrivalent influenza vaccine for active immunization for the prevention of influenza disease caused by influenza A subtype viruses and type B viruses contained in the vaccine.
  • An example of a licensed recombinant influenza protein vaccine is FluBlok (Sanofi Pasteur; STN 125285), which contains recombinant HA proteins from three influenza viruses for intramuscular injection.
  • Another example of a suitable recombinant protein influenza vaccine comprises a headless HA protein as disclosed in Sagawa et al. 1996, the contents of which are incorporated herein by reference.
  • the headless HA protein described in Sagawa et al. is a deletion mutant of influenza virus HA lacking the globular region and neutralizes all HI and H2 subtypes of influenza.
  • nucleic acid based influenza vaccine is an influenza mRNA vaccine such as the influenza mRNA vaccine from Moderna (VAL 339851; mRNA 1851) that is currently in clinical trials.
  • This mRNA influenza vaccine comprises a lipid nanoparticle-encapsulated influenza mRNA encoding an HA protein.
  • AVX502 Alphaviral vector -based influenza vaccine
  • NCT00440362 Alphavirus vector that expresses an influenza HA protein.
  • Kaneyiko et al. (Nature, 4;499(7456): 102-6 (2013)) describes another example of a VLP influenza vaccine that is a self- assembling influenza nanoparticle based on ferritin protein genetically fused to viral influenza HA. Ferritin protein naturally self- assembles into a nanoparticle. This VLP elicited neutralizing antibodies to two highly conserved haemagglutinin structures, one on the stem and one on the receptor binding site on the head.
  • NA polypeptides as subunit vaccines have been reported to provide broad protection, especially compared to HA, which is prone to antigenic drift (Eichlberger and Monto 2019, J. of Inf. Dis. 219(Sl):S75-80.)
  • NA has several conserved epitopes which can be used in subunit vaccines (Wan et al. 2013, Journal of Virology, 87(16):9290-9300, Wan et al. 2015, Nature Communications 6:6114).
  • Exemplary vaccines comprising NA polypeptides are described in US2011/0059130 and WO2019/191261.
  • M2 polypeptides can also be used in subunit vaccines.
  • the extracellular domain of influenza viral M2 (M2e) is highly conserved, thus raising broad cross-protection (Wei et al. 2020, Nature Reviews, 19:239-252).
  • Exemplary vaccines based on M2e polypeptides are described in US9,889, 189. This reference describes fusion proteins comprising tandem repeats of multiple M2e domains.
  • Neirynck describes an influenza vaccine comprising the extracellular domain of M2 to the hepatitis B core protein (HBc), which provides broad spectrum protection against influenza A (Naure Medicine 1999 Oct;5(10): 1157-63). This vaccine has already begun clinical trials (ACAM-FLU-A vaccine; NCT00819013, Sanofi).
  • the immunogenic composition is an inactivated influenza virus vaccine such as a subunit vaccine comprising influenza antigens or a split influenza virus vaccine; a live attenuated influenza virus vaccine; an influenza recombinant protein vaccine comprising a recombinant protein of at least one of the influenza antigens disclosed herein; an influenza vaccine comprising a nucleic acid, such as a DNA or mRNA, encoding at least one influenza antigen such as at least one of HA,
  • an influenza vaccine comprising a vector, preferably a viral vector, that expresses an influenza antigen as disclosed herein, such as at least one of HA, NA and or M2e; or an influenza vaccine comprising a VLP that displays on its outer surface at least one influenza antigen such as at least one of HA, NA and/or M2e.
  • the immunogenic composition as disclosed herein elicits, raises or induces antibodies that bind to an evolutionary conserved epitope of HA, NA and/or M2e, preferably an epitope that is universal (or shared) between different influenza types such as types A and B, subtypes of influenza types and/or influenza strains or genotypes.
  • the antibodies elicited by an immunogenic composition as disclosed herein bind to an evolutionary conserved epitope of HA, NA and/or M2e 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-reacts with multiple influenza strains, subtypes or types.
  • the antigen binding regions recognize epitopes that are evolutionarily conserved across multiple influenza strains, subtypes or types.
  • epitopes are generally present on the stem region of HA, but may also occur on the head region and may interfere with receptor binding or HA trimer integrity.
  • 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 intrade rmal 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, are preferably expanded and/or activated as disclosed herein.”
  • 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 subop timal. This may result in more severe symptoms.
  • NK cell CAfts 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 CAfts 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 maybe 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 maybe provided prophylactically.
  • the compositions may be provided at the beginning of the seasonal flu season.
  • 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. As discussed above, the pharmaceutical compositions may comprise engineered CAEt-expressing immune cells. Typically, virus infected individuals have low NK cell counts with an exhausted phenotype.
  • 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 upon target recognition NK cells can produce and secrete IFNy which can then stimulate humoral responses, specifically the production of IgG2a and IgG3.
  • IFNy 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 IFNy release. While not wishing to be bound by theory, the induced IFNy 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 influenza virus infection.
  • 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 a 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 lxlO 5 cells/dose to lxlO 12 cells/dose, in particular from lxlO 7 cells/dose to 2xl0 9 cells/dose, preferably administered intravenously.
  • to comprise and its conjugations is used in its nondimiting 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.
  • an element means one element or more than one element.
  • the word “approximately” or “about” when used in association with a numerical value preferably means that the value may be the given value of 10 more or less 1% of the value.

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Abstract

L'invention concerne des compositions pharmaceutiques pour traiter ou prévenir une infection par la grippe. L'invention concerne des traitements comprenant des cellules immunitaires, telles que des cellules tueuses naturelles, et des anticorps ou des vaccins ciblant la grippe. L'invention concerne également des compositions ainsi que des "kits de pièces" comprenant lesdites cellules immunitaires et des anticorps ou des vaccins. L'invention concerne en outre des cellules immunitaires modifiées exprimant un récepteur antigénique chimérique (CAR). De telles cellules exprimant un CAR sont utiles pour le traitement. Des exemples de cibles de la grippe selon la présente invention grippe comprennent la protéine hémagglutinine (HA) du virus de la grippe, le domaine extracellulaire d'une protéine M2 du virus de la grippe (M2e) et la protéine neuraminidase (NA) du virus de la grippe, en particulier des épitopes de celui-ci conservés dans l'évolution.
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