Small molecule ligand-targeted drug conjugates for anti-influenza chemotherapy and immunotherapy

Abstract

Small molecule targeted drug conjugates for anti-influenza chemotherapy and immunotherapy are disclosed herein. The disclosed drug conjugates can form linkers to recruit additional CAR T cells or other immune cells for precise elimination of influenza virus-infected cells in a subject. In a subject infected with influenza virus, the co-administered antibody or pre-existing immunity functions well together with the targeted conjugate to eliminate virus-infected cells, saving valuable time for rescuing patients in advanced stages.

Description

Technical Field

[0001] The present disclosure provides targeted delivery for anti-influenza therapy. In particular, a small molecule ligand that specifically binds to influenza virus is conjugated to a payload of drug to effect direct killing or immunomodulation of cells infected with influenza virus. Background Art

[0002] Influenza, an acute febrile respiratory disease caused by influenza virus infection (also known as "flu"), remains one of the most life-threatening disseminated diseases. According to the Influenza Fact Sheet released by the World Health Organization (WHO), influenza spreads seasonally throughout the world, resulting in approximately 3 to 5 million severe illnesses and approximately 250,000 to 500,000 deaths per year. 1 In the United States, there are 12,000 to 56,000 deaths and 140,000 to 710,000 hospitalizations directly related to influenza each year. 2 In addition to causing high morbidity and mortality, influenza imposes a huge socio-economic burden due to productivity losses and medical prevention and treatment. In the United States, the annual total cost associated with influenza has exceeded $10 billion. 3 Current Anti-Influenza Chemotherapy Against Influenza

[0003] Since influenza viruses are constantly changing through antigenic shift and drift, vaccines often become ineffective against mutant strains. Therefore, anti-influenza chemotherapy still plays an important role in the prevention and treatment of influenza. 4 Currently, the U.S. Food and Drug Administration (FDA) has approved two classes of anti-influenza drugs: M2 ion channel inhibitors and neuraminidase inhibitors. M2 ion channel inhibitors include amantadine and rimantadine. The mechanism of action of these drugs is due to blocking the acid-activated viral M2 ion channel and thus inhibiting the release of viral ribonucleoprotein from the virion into the host cytosol. 4 However, both the H1N1 and H3N2 viruses currently circulating in humans are resistant to these inhibitors. Therefore, due to the rapid emergence of drug resistance, the Centers for Disease Control and Prevention (CDC) recommends not using them. 5 Commonly used neuraminidase inhibitors include oseltamivir and zanamivir. They act as competitive inhibitors that compete with sialic acid for binding to the active site of neuraminidase. 4Although these inhibitors are effective against both influenza A virus and influenza B virus, they have two major limitations. First, only small benefits of neuraminidase inhibitors have been observed in terms of the reduction of symptom severity and the decrease in disease duration (0.6 to 0.7 days out of 7 days). 6 Second, this class of antiviral drugs also suffers from drug resistance problems. Since the 2007-2008 season, an increase in the number of oseltamivir-resistant strains has been noticed. Given the current limitations of anti-influenza chemotherapy, there is an urgent need to develop new anti-influenza drugs with novel mechanisms of action. 7 Summary of the Invention

[0004] The present disclosure provides conjugates comprising a targeting ligand (TL) against an influenza virus envelope protein, a linker (L), and a drug payload (D), wherein the TL is a molecule that binds to the envelope protein, the linker covalently binds to both D and TL, and D is an imaging agent, a therapeutic drug, an immunomodulator, or a combination thereof.

[0005] In some preferred embodiments, the above linker comprises a spacer and a cleavable or noncleavable bridge between the TL and D.

[0006] In some preferred embodiments, the above influenza virus envelope protein is neuraminidase (NA) or hemagglutinin (HA).

[0007] In some preferred embodiments, the above TL is zanamivir.

[0008] In some preferred embodiments, the above TL is selected from: oseltamivir, zanamivir, peramivir, and laninamivir.

[0009] In some preferred embodiments, the above conjugate comprises an imaging agent for quantifying the intensity of influenza infection.

[0010] In some preferred embodiments, the above imaging agent comprises a chelaton complex containing technetium-99m ( 99m Tc).

[0011] In some preferred embodiments, the binding affinity of the above conjugate to NA is from about 1 nM to about 15 nM.

[0012] In some preferred embodiments, the above D is selected from: tubulysin B hydrazide, pimodivir, ozanimod, and SN38.

[0013] In some preferred embodiments, the above conjugate is one of the following:

[0014]

[0015] zanamivir-EC20,

[0016]

[0017] zanamivir-tubulysin B hydrazide,

[0018]

[0019] zanamivir-pimodivir,

[0020]

[0021] zanamivir-ozanimod,

[0022]

[0023] zanamivir-SN38,

[0024]

[0025] zanamivir-DNP,

[0026]

[0027] zanamivir-rhamnose,

[0028]

[0029] zanamivir-FITC, or

[0030]

[0031] zanamivir-rhodamine.

[0032] In some preferred embodiments, the above cleavable bridge contains a disulfide bond or an acid-labile bond.

[0033] In some preferred embodiments, the above acid-labile bond contains an ester, hydrazone, oxime, acetal, ketal, phenol ether, or Schiff base bond.

[0034] The present disclosure also provides a method for treating influenza virus infection in a subject, the method comprising providing to the subject a conjugate, wherein the conjugate comprises a targeting ligand (TL) of neuraminidase (NA) of an influenza virus, a linker (L), and a drug payload (D), wherein TL is a molecule that binds to NA, L is covalently bound to both D and TL, and D is an imaging agent, a therapeutic drug, an immunomodulator, or a combination thereof.

[0035] In some preferred embodiments, the method uses zanamivir as TL.

[0036] In some preferred embodiments, the method uses a therapeutic drug to kill cells infected with an influenza virus in the subject or to inhibit influenza virus replication.

[0037] In some preferred embodiments, the method uses a therapeutic drug selected from: tubulysin B hydrazide, pimodivir, and SN38.

[0038] In some preferred embodiments, the method uses a therapeutic drug comprising an adaptor molecule (i.e., a fluorescein covalently bound to TL) and an anti-fluorescein CAR T cell, wherein after binding to the adaptor molecule, the CAR-T cell kills influenza virus-infected cells expressing neuraminidase that binds to TL in the subject and thereby inhibits influenza virus replication.

[0039] In some preferred embodiments, the method uses an immunomodulator to inhibit an early cytokine storm induced by an influenza virus.

[0040] In some preferred embodiments, the method uses the immunomodulator ozanimod or a hapten recognized by an autoantibody.

[0041] In some preferred embodiments, the hapten comprises a dinitrophenyl (DNP), trinitrophenyl (TNP), rhamnose, or α-galactosyl moiety.

[0042] In some preferred embodiments, the method uses a zanamivir conjugate to initiate an immune response that leads to clearance of antibody-coated viruses or virus-infected cells by antibody dependent cellular phagocytosis (ADCP), antibody dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC).

[0043] In some preferred embodiments, the above method uses an antigen or another moiety to conjugate with zanamivir, where the subject has pre-existing immunity to the antigen or moiety, or an effective dose of an antibody against the antigen or moiety is co-administered to the subject. For example, the antigen or moiety can be a toxin (such as tetanus toxoid).

[0044] The present disclosure also provides a system comprising at least two components. The first component comprises a conjugate comprising a targeting ligand (TL) for an influenza virus envelope protein, a linker (L), and a drug payload (D), where TL is a molecule that binds to the envelope protein, L is covalently bound to both D and TL, and D is fluorescein; the second component comprises an anti-fluorescein CAR T cell that binds to the fluorescein of the first component, wherein the system is facilitated to kill cells infected with influenza virus.

[0045] In vitro binding assays of representative zanamivir-DNP conjugates have shown that they have high binding affinities for both N1 and N2 classes of neuraminidases. The conjugate is much more potent than zanamivir or oseltamivir; it is effective even when added after the infection has progressed further in patients; the infection can be cured with a single injection of our drug, and it is effective against all influenza strains.

[0046] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following drawings in conjunction with the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Mechanism of action of zanamivir-therapeutic drug conjugate, (a) Schematic diagram of the drug conjugate, (b) Proposed mechanism of action.

[0048] Figure 2 Therapeutic drug payloads selected for zanamivir-targeted therapeutic drug conjugate.

[0049] Figure 3 Mechanism of action of zanamivir-hapten conjugate targeted immunotherapy, (a) Schematic diagram of zanamivir-DNP conjugate, (b) Proposed mechanism of action.

[0050] Figure 4 Design of zanamivir-based targeting ligand. The 7-OH group of zanamivir is highlighted in yellow.

[0051] Figure 5 Crystal structure of zanamivir complexed with neuraminidase.

[0052] Figure 6Binding of zanamivir-rhodamine conjugate to MDCK cells infected with influenza virus A / Puerto Rico / 8 / 34 (H1N1). (a) Confocal microscopy images of the drug group. Influenza virus-infected MDCK cells incubated with 50 nM zanamivir-rhodamine conjugate; (b) Confocal microscopy images of the competition group. Influenza virus-infected MDCK cells incubated with 50 nM zanamivir-rhodamine conjugate in the presence of 5 μM zanamivir; (c) Binding saturation curve.

[0053] Figure 7 99m Binding of Tc-chelated zanamivir-EC20 head conjugate to MDCK cells infected with influenza virus A / Puerto Rico / 8 / 34 (H1N1). (a) Binding saturation curve; (b) 99m Structure of Tc-chelated zanamivir-EC20 head conjugate.

[0054] Figure 8 99m Biodistribution of Tc-chelated zanamivir-EC20 head conjugate in influenza virus A / Puerto Rico / 8 / 1934 (H1N1)-infected mice / uninfected mice.

[0055] Figure 9 In vitro cytotoxicity of zanamivir-tubulysin B hydrazide conjugate and its component parts against neuraminidase-transfected HEK 293 cells. Cytotoxicity of zanamivir-tubulysin B hydrazide conjugate (red circles), free tubulysin B hydrazide (orange triangles), and zanamivir-tubulysin B hydrazide conjugate in the presence of 100-fold excess zanamivir (blue squares) is depicted.

[0056] Figure 10 Competitive binding of zanamivir-DNP conjugate to neuraminidase-transfected HEK 293 cells. (a) Log (dose)-response curve of zanamivir-DNP conjugate; (b) Log (dose)-response curve of zanamivir. Zanamivir-rhodamine conjugate was used as the labeled ligand.

[0057] Figure 11 Flow cytometry analysis demonstrating the ability of zanamivir-DNP conjugate to bind simultaneously to cell surface neuraminidase and anti-DNP antibody. (a) Schematic description of the antibody recruitment assay based on flow cytometry; (b) Flow cytometry analysis using neuraminidase-transfected HEK293 cells (293tn NA); (c) Flow cytometry analysis using untransfected HEK293 cells (293tn).

[0058] Figure 12In vivo protective efficacy of zanamivir-DNP conjugate against influenza virus A / Puerto Rico / 8 / 1934 (H1N1) infection in BALB / c mice when administered 2 hours after infection. (a) Body weight curve; (b) Survival curve.

[0059] Figure 13 Schematic diagram of the proposed targeted CAR-T therapy for influenza-infected cells.

[0060] Figure 14 In vitro anti-FITC CAR-T killing profile of fluorescein linker-mediated FITC-zanamivir conjugate against cells expressing influenza surface protein NA. (A) CAR T:293NA = 5:1 had 41% killing of 293NA cells; (B) CAR T:293NA = 10:1 had 61% killing of 293NA cells.

[0061] Figure 15 Zanamivir-FITC does not bind to normal 293T cells.

[0062] Figure 16 Zanamivir-FITC specifically induces cytotoxicity against NA.

[0063] Figure 17 In vitro assays using authentic virus

[0064] Figure 18 LDH assay of T cell killing of influenza-infected MDCK relative to CAR-T killing of influenza-infected MDCK.

[0065] Figure 19 Proposed mouse model for testing CAR T cell therapy in influenza-infected mice.

[0066] Figure 20 Mechanism of action of anti-influenza immunotherapy, including: A. Small molecule ligand-targeted drug conjugate; B. Structure of zanamivir-DNP conjugate: Zanamivir (target ligand) is conjugated to a hapten (2,4-dinitrophenyl) via a linker; C. After the conjugate binds to viral neuraminidase, innate antibodies against DNP inhibit influenza virus replication. Thus, the system redirects anti-dinitrophenyl (anti-DNP) antibodies to influenza virus / virus-infected cells, inducing immune-mediated destruction of influenza virus / virus-infected cells.

[0067] Figure 21 Multiple in vitro binding assays in MDCK cells infected with influenza virus A / Puerto Rico / 8 / 34 (H1N1). A. Binding of zanamivir-rhodamine conjugate; B. Binding of free zanamivir and C. Binding of zanamivir-DNP-conjugate.

[0068] Multiple in vitro binding assays in MDCK cells infected with influenza virus A / Aichi / 2 / 1968 (H3N2). A. Binding of zanamivir-rhodamine conjugate; B. Binding of free zanamivir and C. Binding of zanamivir-DNP-conjugate.

[0069] Anti-DNP antibody recruitment assay performed in MDCK cells infected with influenza virus A / Puerto Rico / 8 / 34 (H1N1). A. Flowchart of the assay; B. Cell staining results from addition of various conjugates (PE - red, TO-RPO-3lolide - blue shows nuclei) and binding curves.

[0070] Anti-DNP antibody recruitment assay performed in MDCK cells infected with influenza virus A / Aichi / 2 / 1968 (H3N2). A. Flowchart of the assay; B. Cell staining results from addition of various conjugates (PE - red, TO-RPO-3lolide - blue shows nuclei) and binding curves.

[0071] Complement-dependent cytotoxicity assay (CDC). A. Flowchart of the complement-dependent cytotoxicity assay performed in NA-transfected 293 cells. B. Only 10 nM of the drug conjugate is required to mediate maximal cell killing.

[0072] Antibody-dependent cell phagocytosis assay (ADCP). A. ADCP workflow. B. THP-1 cells (human macrophages) were treated with PMA and labeled with DiD before use. 293tnNA (GFP+) and THP-1 cells (ratio 1:1) were incubated with different concentrations of zanamivir-DNP conjugate and anti-DNP antibody (100 nM) at 4 °C for 30 minutes. ADCP effect was analyzed by flow cytometry.

[0073] Figure 27 Mouse protection study procedure: Mice were immunized by subcutaneous injection of 2,4-dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH); in the 5th week, the mice were infected with a lethal dose of influenza virus (100 LD50, A / Puerto Rico / 8 / 1934 (H1N1)); treatment with zanamivir-DNP conjugate and other drugs was started after infection and the mice were monitored for 2 weeks; mice were considered dead when they had lost 25% of their initial weight or when they were moribund. B. Surgery was performed on the mice.

[0074] Figure 28 Dose escalation study (intranasal administration). On day 0, 50 μL of H1N1 PR8 virus (100 LD 50 , 4.2×10 5Mice (5 mice per group) were infected with H1N1 PR8 virus (100 LD₅₀, 4.2×10⁶ PFU). Twenty-four hours after infection, mice were administered PBS / zanamivir / zanamivir-DNP conjugate intranasally twice a day for 5 days. A mouse was considered dead when it lost 25% of its initial body weight or when it was moribund. A. Plot of percentage of body weight. B. Percentage of surviving mice as defined.

[0075] Figure 29 Comparison of the efficacy between zanamivir-DNP conjugate and its components (intranasal administration). Procedure: On day 0, mice (5 mice per group) were infected with 50 μL of H1N1 PR8 virus (100 LD₅₀, 4.2×10⁶ PFU). 50 ,4.2×10 5 Mice (5 mice per group) were infected with H1N1 PR8 virus (100 LD₅₀, 4.2×10⁶ PFU). Twenty-four hours after infection, mice were administered zanamivir-DNP conjugate and its components intranasally twice a day for 5 days. A mouse was considered dead when it lost 25% of its initial body weight or when it was moribund. A. Plot of percentage of body weight. B. Percentage of surviving mice as defined.

[0076] Figure 30 Delayed-start-to-treat study (intranasal administration). Procedure: On day 0, mice immunized with DNP-KLH were infected with 50 μL of H1N1 PR8 virus (100 LD₅₀, 4.2×10⁶ PFU). 50 ,4.2×10 5 Forty-eight hours / seventy-two hours / ninety-six hours after infection, mice were administered 1.5 μmol / kg of zanamivir-DNP conjugate intranasally twice a day for 7 days. A mouse was considered dead when it lost 25% of its initial body weight or when it was moribund. A. Plot of percentage of body weight. B. Percentage of surviving mice as defined.

[0077] Figure 31 Single-dose treatment (intranasal administration). Procedure: On day 0, mice (5 mice per group) were infected with 50 μL of H1N1 PR8 virus (100 LD₅₀, 4.2×10⁶ PFU). 50 ,4.2×10 5 Twenty-four hours after infection, mice were administered PBS / zanamivir-DNP conjugate intranasally only once. A mouse was considered dead when it lost 25% of its initial body weight or when it was moribund. A. Plot of percentage of body weight. B. Percentage of surviving mice as defined.

[0078] Figure 32 Biodistribution and SPECT / CT imaging. A. Structure of the new zanamivir-E20 head conjugate. B. Cellular binding radioactivity according to the following procedure:

[0079] · Mice were infected with influenza virus (H1N1) 3 days before the experiment.

[0080] · Intravenous injection of 10 nmol of zanamivir-EC20 head conjugate (150 μCi) into mice.

[0081] · Radioactivity was calculated 4 hours after injection.

[0082] C. Binding curve of zanamivir-E20 head in the presence of 100× free zanamivir. D. SPECT / CT imaging after injection of the conjugate without (left) or with 100× free zanamivir according to the following procedure:

[0083] · Mice were infected with influenza virus (H1N1) 3 days before the experiment.

[0084] · Intravenous injection of 50 nmol of zanamivir-EC20 head conjugate (750 μCi) into mice.

[0085] · SPECT / CT imaging was performed 4 hours after injection

[0086] Figure 33 Dose escalation study (intraperitoneal administration) performed according to the following procedure: On day 0, mice (5 mice per group) were infected with 50 μL of H1N1 PR8 virus (100 LD 50 , 4.2×10 5 PFU). 24 hours after infection, mice were given PBS / zanamivir / zanamivir-DNP conjugate intraperitoneally twice a day for 5 days. Mice were considered dead when they lost 25% of their initial body weight or when they were moribund. A. Body weight percentage plot B. Percentage of surviving mice as defined.

[0087] Figure 34 Comparison of the potency between zanamivir-DNP conjugate and its components (intraperitoneal administration) performed according to the following procedure: On day 0, mice (5 mice per group) were infected with 50 μL of H1N1 PR8 virus (100 LD 50 , 4.2×10 5 PFU). 24 hours after infection, mice were given PBS / zanamivir / zanamivir-DNP conjugate intraperitoneally twice a day for 5 days. Mice were considered dead when they lost 25% of their initial body weight or when they were moribund. A. Body weight percentage plot B. Percentage of surviving mice as defined.

[0088] Figure 35 Antibody-dependent cellular cytotoxicity assay (ADCC). A. The ADCC Reporter Bioassay uses engineered Jurkat cells stably expressing the FcγRIIIa receptor and the NFAT (nuclear factor of activated T-cell) response element that drives firefly luciferase expression as effector cells. The biological activity of the antibody in the ADCC MOA is quantified by luciferase generated as a result of NFAT pathway activation. B. ADCC protocol and results for DNP-zanamivir.

[0089] Figure 36 In vitro antiviral assay of MDCK cells infected with H1N1 and H3N2. A. A / Puerto Rico / 8 / 34 (H1N1) and B. A / Aichi / 2 / 1968 (H3N2).

[0090] Figure 37 Single-dose treatment (intranasal administration) of mice infected with H3N2 virus. A. Treatment efficacy measured by body weight maintenance. B. Treatment efficacy measured by percentage survival.

[0091] Figure 38 Single-dose treatment (intraperitoneal administration) of mice infected with H1N1 PR8 virus. A. Treatment efficacy measured by body weight maintenance. B. Treatment efficacy measured by percentage survival.

[0092] Figure 39 Single-dose treatment (intraperitoneal administration) of mice infected with H3N2 virus. A. Treatment efficacy measured by body weight maintenance. B. Treatment efficacy measured by percentage survival.

[0093] Figure 40 Anti-DNP antibody and zana-DNP-treated naive mice infected with H1N1 PR8 virus. One day after infection with a lethal dose of H1N1 PR8 virus, naive mice were given different doses of anti-DNP antibody intravenously and immediately treated with a single-dose intraperitoneal administration of the zanamivir-DNP conjugate. A. Treatment efficacy measured by body weight maintenance. B. Treatment efficacy measured by percentage survival.

[0094] Figure 41 Synthesis protocol of zanamivir-rhamnose conjugate, a different conjugate that utilizes anti-rhamnose generated by the innate immune system to label cells infected with influenza virus and induce an immune attack against cells infected with influenza virus.

[0095] Figure 42 Competitive binding of zanamivir-DNP rhamnose conjugate with zanamivir-rhodamine conjugate as labeled ligand to neuraminidase-transfected HEK293 cells. A. The Kd of zanamivir was approximately 0.77 nM. B. The Kd of zanamivir-rhamnose was approximately 3.57 nM.

[0096] Figure 43 Immunotherapy study with zanamivir-rhamnose conjugate. Mice immunized with rhamnose-OVA (5 mice per group) were infected with 50 μL of H1N1 PR8 virus (100 LD 50 , 4.2×10 5 PFU) on day 0. At 24 hours after infection, mice were administered 1.5 / 0.5 / 0.17 μmol / kg of zanamivir-rhamnose conjugate / zanamivir / PBS intranasally twice a day for 5 days, and mice were considered dead when they lost 25% of their initial body weight or when they were moribund. A. Therapeutic effect measured by body weight maintenance. B. Therapeutic effect measured by percentage survival. Detailed Description

[0097] Although the concepts of the present disclosure are shown and described in detail in the drawings and the specification herein, the results and the description in the drawings should be considered to be exemplary rather than restrictive in nature; it is understood that only some exemplary embodiments are shown and described, and all changes and modifications falling within the spirit of the present disclosure are desired to be protected.

[0098] Unless otherwise defined, scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0099] Overview of Influenza Viruses

[0100] Influenza viruses are enveloped viruses. The overall structure of all influenza subtypes is very similar. The diameter of the virus particles is 80 to 120 nanometers and they are generally roughly spherical, but filamentous forms can occur. These filamentous forms are more common in influenza C, which can form ribbon-like structures up to 500 micrometers long on the surface of infected cells. However, despite these variable shapes, the virus particles of all influenza viruses are similar in composition. They consist of a viral envelope surrounding a central core, which contains two main types of glycoproteins. The central core contains the viral RNA genome and other viral proteins that package and protect this RNA. The RNA tends to be single-stranded, but in special cases it is double-stranded. Unusually for a virus, its genome is not a single piece of nucleic acid; instead, it consists of seven or eight segments of segmented negative-sense RNA, each RNA segment containing one or two genes that encode one gene product (a protein). For example, the influenza A genome contains 11 genes on eight RNA segments, encoding 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP: nuclear export protein), PA, PB1 (polymerase basic 1), PB1-F2, and PB2.

[0101] Hemagglutinin (HA) and neuraminidase (NA) are two large glycoproteins on the outside of the virus particle. HA is a lectin that mediates the binding of the virus to target cells and the entry of the viral genome into the target cells, while NA is involved in the release of progeny virus from infected cells by cleaving the sugars that bind to the mature virus particles. Thus, these proteins are targets for antiviral drugs. In addition, they are antigens against which antibodies can be produced. Based on the antibody response against HA and NA, influenza A viruses can be divided into subtypes. These different types of HA and NA form the basis for the differences in H and N, for example, in H5N1. Sixteen H subtypes and nine N subtypes are known, but usually only H1, 2, and 3 and N1 and 2 are found in humans.

[0102] Small Molecule Ligand-Targeted Drug Conjugates for Antiviral Therapy

[0103] Small molecule ligand-targeted drug conjugates that combine a receptor-specific ligand and a therapeutic payload have shown promise in the treatment of many diseases, especially in cancer chemotherapy. By specifically delivering the therapeutic payload to cells recognized by the targeting ligand, these drug conjugates exhibit high selectivity towards malignant cells and reduced associated collateral toxicity. To date, many cancers have been addressed by small molecule ligand-targeted drug conjugates that target receptors overexpressed on tumor cells. These overexpressed receptors include folate receptor (FR), prostate-specific membrane antigen (PSMA), cholecystokinin 2 receptor (CCK2R), carbonic anhydrase IX (CAIX), and others. 8

[0104] For enveloped viruses, the last step in replication involves the assembly of viral components on the membrane of the infected cell and budding from the surface of the infected cell. 9 Meanwhile, some viral envelope glycoproteins (such as HIV gp120 and influenza neuraminidase / hemagglutinin) are expressed on the outer surface of the infected cell. 10 Given the fact that these exogenous viral proteins are expressed only on infected cells, they have the potential to be targeted by ligand-targeted drug conjugates.

[0105] Design of Zanamivir-Therapeutic Drug Conjugates

[0106] The general scheme of the present disclosure is to provide a specific targeting ligand conjugated to a payload of a therapeutic drug or modulator to treat viral infections. The targeting ligand will specifically recognize the envelope protein of the virus that is expressed only on the surface of infected cells. In some cases, the payload of the therapeutic drug or modulator can be an engineered T cell expressing a chimeric antigen receptor (CAR T cell). For example, if the drug payload is a fluorescein linker, anti-fluorescein CAR T cells can be administered together with the targeting ligand-directed payload drug to kill virus-infected cells or inhibit virus replication in infected cells. For a detailed description of linker molecule-mediated CAR T cell therapy and its preparation, see U.S. Application 15 / 296,666, filed October 16, 2016, the entire content of which is incorporated herein by reference.

[0107] Here, we designed a series of small molecule ligand-targeted drug conjugates that target influenza virus envelope proteins, particularly neuraminidase (NA). Without being bound by any theory, other small molecule ligands that specifically target hemagglutinin (HA) can also work under this principle. For example, compounds that inhibit HA-mediated influenza virus entry can be considered potential targeted ligands that affect HA in infected cells.

[0108] Therefore, the use of the high-affinity neuraminidase inhibitor zanamivir was repurposed in this article to carry therapeutic drugs and specifically deliver them to virus-infected cells and virus replication sites (e.g., the nose, throat, and lungs). This presents a unique mechanism of action by which virus-infected cells can be killed before the release of progeny virus, hindering virus replication or inhibiting the early cytokine storm induced by virus infection ( Figure 1 ).

[0109] The therapeutic drug payloads selected for this project are shown in Figure 2. (1) Tubulysin B hydrazide is an anti-mitotic tetrapeptide that inhibits tubulin polymerization. It kills influenza virus-infected cells by inducing apoptosis or inhibits the transport of viral components by disrupting the microtubule network of influenza virus-infected cells. 11 (2) Pimodivir is an RNA-dependent RNA polymerase (RdRp) inhibitor that blocks the m 7 GTP-binding pocket in the PB2 subunit of the influenza A virus polymerase complex. It interferes with virus replication by inhibiting the PB2 cap-snatching activity. 12,13 Given the fact that the high morbidity and mortality caused by influenza are the result of both virus-induced tissue damage and overinduction of pro-inflammatory cytokine production (cytokine storm), two immunomodulatory drugs, ozanimod and SN38, were selected to improve the outcome of influenza treatment. 14,15 (3) Ozanimod is an investigational immunomodulatory drug that acts as a sphingosine-1-phosphate (S1P) receptor agonist. 16 Researchers found that S1P receptor agonists can blunt but not eliminate excessive virus-induced cytokine production, providing significant protection against influenza virus infection in mice. 17,18 (4) SN38 is a topoisomerase I inhibitor that is the active metabolite of irinotecan (a camptothecin analogue). 19 It was demonstrated that SN38 restricts the overexpression of influenza virus-induced inflammatory genes by inhibiting the recruitment of RNA polymerase II to innate immune genes. 20In addition, SN38 can also kill influenza virus-infected cells by inducing apoptosis.

[0110] When these molecules (including but not limited to tubulysin B hydrazide, pimodivir, ozanimod, or SN38) are conjugated to the targeting ligands of viral envelope proteins, they can exert multiple effects of killing virus-infected cells or inhibiting virus replication in infected cells.

[0111] Design of zanamivir-hapten conjugate targeted immunotherapy for the treatment of influenza

[0112] In addition to therapeutic drugs that can directly kill influenza virus-infected cells or inhibit virus replication in infected cells, immunotherapy can effectively trigger the immune system through antibodies present in the body to resist specific infections. A possible candidate for such immunotherapy is to wake up circulating anti-DNP antibodies by preparing a conjugate of TL and dinitrophenyl (DNP).

[0113] Since zanamivir has potential targeting ability for influenza virus or virus-infected cells, the zanamivir-dinitrophenyl (DNP) conjugate has also been developed in our laboratory ( Figure 3 ). As Figure 3 shown in 21-23

[0114] b, it is considered that the zanamivir-DNP conjugate forms a bispecific molecular "bridge" between influenza virus / virus-infected cells and endogenous circulating anti-DNP antibodies. This "labeling" step initiates an immune response that leads to the clearance of antibody-coated viruses or virus-infected cells through mechanisms such as antibody-dependent cell phagocytosis (ADCP), antibody-dependent cell cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). 7 Second, since anti-DNP antibodies already exist in human blood, there is no need for pre-vaccination for this treatment. 24

[0115] Without being bound by any theory, zanamivir conjugated with any other moiety (such as trinitrophenyl (TNP), rhamnose, or α-galactosyl) can recruit their respective antibodies to influenza-infected cells to initiate an antibody-dependent immune response. Thus, the immunotherapies disclosed herein can target influenza virus-infected cells via zanamivir conjugate markers.

[0116] Influenza virus-induced tumor types for targeted CAR T cell therapy

[0117] In the present disclosure, it is contemplated to combine with our newly developed linked CAR T cell therapy as described in U.S. Application 15 / 296,666 or its related applications, the content of which is hereby expressly incorporated by reference, to target CAR T cells to influenza virus-infected cells to perform the immune response function of CAR T cells.

[0118] Figure 13 Depicts a CAR T cell strategy for treating influenza virus-infected cells. In Figure 13 , a zanamivir-FITC conjugate is produced and attached to influenza virus-infected cells expressing viral neuraminidase on the surface. Notably, zanamivir-FITC can perform at least two different functions in this process. One is to act as an imaging agent to show the infection intensity of the influenza virus; the other is to label virus-infected cells with zanamivir, which is an NA inhibitor that blocks virus budding from the envelope. The presence of the zanamivir-FITC conjugate on the surface of infected cells can direct T cells conjugated with anti-FITC antibodies to virus-infected cells and form an immunological synapse. As known to those skilled in the art, such anti-FITC CAR T cells can be activated by binding to the zanamivir-FITC conjugate. Thus, the activated CAR T cells can secrete cytokines and subsequently kill virus-infected cells, preventing virus replication.

[0119] Without being bound by any theory, the advantages of using small molecule targeting drugs or immunomodulator conjugates to treat influenza-infected cells can be seen in several aspects. Currently, vaccines are prepared based on annual predictions of which strains are likely to circulate during the next season. However, such a strategy occasionally fails and thus renders the vaccine ineffective against the major virus strains in circulation. The exemplified zanamivir is an NA inhibitor effective against all 11 influenza NA subtypes, which blocks virus budding from infected cells. Thus, the effectiveness applies to all subtypes of influenza virus. Meanwhile, zanamivir conjugated with a payload drug (therapeutic agent or immunotherapeutic modulator) or a linker molecule (i.e., fluorescein) mediates anti-fluorescein CAR T cells, specifically labeling influenza-infected cells to initiate the necessary immune response to clear virus-infected cells.

[0120] Example

[0121] Example 1. Design of the targeting ligand

[0122] Influenza neuraminidase (NA) is a transmembrane glycoprotein anchored in the lipid raft domain of the influenza virus envelope. NA accounts for 20% (about 80) of the membrane glycoproteins, and the head of NA is a homotetramer. It helps the progeny virus to be released from the infected cells by cleaving sialic acid from membrane glycoproteins or glycolipids (during the virus budding process, the influenza virus hemagglutinin can bind to the sialic acid receptor on the host cell membrane, which hinders the release of the newly formed virus). 9 Since neuraminidase is expressed on both the surface of the influenza virus and the surface of the infected cell membrane, our group selected it as a potential target for designing targeting ligands to target the influenza virus and virus-infected cells.

[0123] To date, four neuraminidase inhibitors have been developed as anti-influenza drugs: oseltamivir (Tamiflu; Glide / Roche), zanamivir (Relenza; GlaxoSmithKline), peramivir (Rapivab; BioCryst), and laninamivir (Inavir; Daiichi Sankyo). 25 Since zanamivir is an inhibitor derived from naturally occurring sialic acid and has the lowest functionalization, zanamivir-resistant viruses are rarely found clinically. 7 Therefore, zanamivir was selected as a candidate for targeting ligand design among the neuraminidase inhibitors. Honda et al. reported that C-7 alkyl-modified analogs of zanamivir retained their inhibitory activity against neuraminidase ( Figure 4 ), which indicates that the C-7 position is tolerant to being modified as a linker attachment site. 26 Honda's conclusion was supported by the results of the X-ray crystallographic structure of zanamivir complexed with neuraminidase: the 7-OH group of zanamivir is exposed to the solvent surface region and does not cause direct interaction with the active site of neuraminidase ( Figure 5 ). 27 In addition, several research groups have also used the C7 position as a linker site to construct a set of multimeric analogs of zanamivir and zanamivir derivatives with enhanced anti-influenza activity. 28 In summary, we designed a new NA targeting ligand based on zanamivir by modifying the 7-OH group of zanamivir as a linker attachment site ( Figure 4 ).

[0124] Example 2. Compound synthesis

[0125]

[0126] Synthesis of the targeting ligand of Scheme 1. Reagents and conditions: (a) Amberlite IR-120B (H + form), MeOH; (b) acetic anhydride, 4-(dimethylamino)pyridine (DMAP), pyridine; (c) trimethylsilyl trifluoromethanesulfonate, ethyl acetate; (d) azidotrimethylsilane, tert-butanol; (e) triphenylphosphine, H 2 O, THF; (f) N,N’-bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboximidamide, triethylamine, THF; (g) sodium methoxide solution, MeOH; (h) 2,2-dimethoxypropane, p-toluenesulfonic acid, acetone; (i) 4-nitrophenyl chloroformate, DMAP, pyridine; (j) azido- -amine, DMAP, pyridine; (k) 1 M NaOH(aq), THF; (l) TFA.

[0127]

[0128] General synthetic protocol for small molecule drug conjugates of Scheme 2.

[0129]

[0130] Synthesis of the zanamivir-rhodamine conjugate of Scheme 3. Reagents and conditions: (a) DBCO-amine, DIPEA, DMF; (b) DMSO.

[0131]

[0132] Synthesis of the zanamivir-EC20 conjugate of Scheme 4. Reagents and conditions: (a) 1. wet resin in DMF, 2. Fmoc-Asp(OtBu)-OH, PyBop, DIPEA, DMF; (b) 1. 20% piperidine in DMF, 2. Fmoc-DAPA-OH, PyBop, DIPEA, DMF; (c) 20% piperidine in DMF; (d) 1. DBCO-acid, PyBop, DIPEA, DMF, 2. TFA / TIPS / EtSH / H 2 O (92.5:2.5:2.5:2.5); (e) DMSO.

[0133]

[0134] Synthesis of the zanamivir-tubulysin B-hydrazide conjugate of Scheme 5. Reagents and conditions: (a) THF / NaHCO 3 buffer.

[0135]

[0136] Scheme 6 Synthesis of zanamivir-DNP conjugate. Reagents and conditions: (a) TEA, EtOH; (b) DBCO-NHS, DIPEA, DMSO; (c) DMSO.

[0137]

[0138] Scheme 7 Synthesis of zanamivir-pimodivir conjugate. Reagents and conditions: (a) N-Boc-diethanolamine, EDC, DMAP, DCM; (b) 20% TFA in DCM; (c) DBCO-NHS, DIPEA, DMSO; (d) DMSO.

[0139]

[0140] Scheme 8 Synthesis of zanamivir-ozanimod conjugate. Reagents and conditions: (a) DBCO-acid, EDC, DMAP, DCM; (b) DMSO.

[0141]

[0142] Scheme 9 Synthesis of zanamivir-SN38 conjugate. Reagents and conditions: (a) Di-tert-butyl dicarbonate, pyridine, DCM; (b) DBCO-acid, EDC, DMAP, DCM; (c) 20% TFA in DCM; (d) DMSO.

[0143] Example 3. In Vitro Binding of Small Molecule Ligand-Targeted Drug Conjugates for Anti-Influenza Chemotherapy to Influenza Virus-Infected MDCK Cells

[0144] (1) Confocal Microscopy Study with Zanamivir-Rhodamine Conjugate

[0145] Method: MDCK cells were seeded in a confocal plate and incubated overnight. The next day, when the cells reached 80% confluence, they were infected with 100 TCID 50 influenza virus A / Puerto Rico / 8 / 34 (H1N1). On the third day, the infected MDCK cells were incubated with 50 nM zanamivir-rhodamine conjugate in the presence or absence of 5 μM zanamivir. After incubation at 37 °C for 1 h, the cells were washed with cell culture medium and sent to confocal microscopy.

[0146] As Figure 6 shown in a, strong fluorescence signals were observed when influenza virus-infected MDCK cells were incubated with 50 nM zanamivir-rhodamine conjugate. When the binding of zanamivir-rhodamine conjugate to neuraminidase was competed by 100-fold excess of zanamivir, the fluorescence emission signal disappeared (Figure 6 b), indicating that the cellular uptake of the conjugate is receptor-mediated. In short, the results indicate that the zanamivir-rhodamine conjugate can bind to and be internalized into influenza virus-infected MDCK cells.

[0147] (2) Binding affinity study with zanamivir-rhodamine conjugate

[0148] Method: MDCK cells were seeded in 24-well plates and incubated overnight. The next day, when the cells reached 80% confluence, they were infected with 100 TCID 50 influenza virus A / Puerto Rico / 8 / 34 (H1N1). On the third day, the infected MDCK cells were incubated with different concentrations of zanamivir-rhodamine conjugate in the presence or absence of 100-fold excess of zanamivir. After incubation at 37 °C for 1 h, the cells were washed with cell culture medium and the remaining fluorescence was quantified by fluorescence spectroscopy. The apparent K d .

[0149] As Figure 6 shown in c, it was found that the binding of the zanamivir-rhodamine conjugate to neuraminidase expressed on virus-infected cells saturated at a Kd of 10.98 nM, and this binding of the zanamivir-rhodamine conjugate could be competed by 100-fold excess of zanamivir.

[0150] Based on the above confocal and binding affinity studies, it was demonstrated that the zanamivir derivative is a good candidate as a targeting ligand against influenza virus neuraminidase.

[0151] Example 4. In vivo biodistribution

[0152] (1) Binding affinity study with 99m Tc-chelated zanamivir-EC20 head conjugate

[0153] Method: MDCK cells were seeded in 24-well plates and incubated overnight. The next day, when the cells reached 80% confluence, they were infected with 100 TCID 50 influenza virus A / Puerto Rico / 8 / 34 (H1N1). On the third day, the infected MDCK cells were incubated with different concentrations of 99m Tc-chelated zanamivir-EC20 head conjugate in the presence or absence of 100-fold excess of zanamivir. After incubation at 37 °C for 1 h, the cells were washed with cell culture medium and the remaining 99mRadioactivity of the Tc-chelated zanamivir-EC20 head conjugate. The apparent K was calculated by plotting the radioactivity of cell binding against the concentration of the radiotracer using GraphPad Prism 4 d .

[0154] It was found that the binding of technetium-99m ( 99m Tc)-chelated zanamivir-EC20 head conjugate to neuraminidase expressed on virus-infected cells reached saturation with a Kd of 15.09 nM, and 99m this binding of the Tc-chelated zanamivir-EC20 head conjugate could be competed by 100-fold excess of zanamivir ( Figure 7 a). This binding affinity value was consistent with the value measured by the zanamivir-rhodamine conjugate, further demonstrating that the zanamivir derivative is a good targeting ligand for influenza virus neuraminidase.

[0155] (2) Biodistribution study with 99m the Tc-chelated zanamivir-EC20 head conjugate

[0156] Method: First, BALB / c mice (6 to 7 weeks old) were infected intranasally with 50 μL of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) to develop influenza symptoms. Three days later, 100 μL of 10 nmol zanamivir-EC20 head conjugate (containing 20 pM 99m Tc-chelated conjugate) was injected intravenously into the mice in the presence or absence of 100-fold excess of zanamivir. Five hours after injection, the major tissues / organs were removed and the amount of radioactivity was determined by a gamma counter.

[0157] To test the ability of the zanamivir derivative to specifically deliver a therapeutic agent or imaging agent to influenza virus-infected lungs, the biodistribution profiles of the 99m Tc-chelated zanamivir-EC20 head conjugate in virus-infected mice / uninfected mice were measured. As Figure 8 shown, the conjugate showed the highest uptake in the lungs of virus-infected mice (the main organ where influenza virus proliferates). In addition, there was no 99m lung uptake of the Tc-chelated zanamivir-EC20 conjugate in the competition group or the uninfected group, indicating that the lung uptake of the conjugate was receptor-mediated. Except for the virus-infected lungs, the kidney was the only organ showing a significant radioactive signal. However, the signal was not eliminated in the competition group or the uninfected group, indicating 99mThe accumulation of the Tc-chelated zanamivir-EC20 head conjugate in the kidney is not neuraminidase-mediated. In summary, the results provide strong evidence that the zanamivir derivatives designed in this project can be used as targeting ligands for the specific delivery of therapeutic agents or imaging agents to virus-infected lungs.

[0158] Example 5. In vitro cytotoxicity study with zanamivir-tubulysin B hydrazide conjugate

[0159] Method: Neuraminidase-transfected HEK 293 cells were seeded in 96-well plates and incubated with zanamivir-tubulysin B hydrazide conjugate, free tubulysin B hydrazide, or zanamivir-tubulysin B hydrazide conjugate in the presence of 100-fold excess of zanamivir for 2 hours at 37 °C. The cells were then washed with fresh medium and incubated for an additional 48 hours at 37 °C. Cell viability was measured using ATP detection (CellTiter Glo, Promege Inc., Madison, WI). The EC 50 values were calculated by plotting % luminescence intensity against the log concentration of the drug using GraphPad Prism 4.

[0160] To determine the cytotoxicity and targeting specificity of the zanamivir-tubulysin B hydrazide conjugate, an in vitro cytotoxicity assay using neuraminidase-transfected HEK293 cells was performed. As Figure 9 shown, the EC 50 of the zanamivir-tubulysin B hydrazide conjugate was 5.2 nM, which was comparable to that of free tubulysin B hydrazide 50 (9.9 nM). Blocking of the neuraminidase binding site with 100-fold excess of zanamivir reduced the cytotoxicity by >30-fold, indicating that most of the cell killing was receptor-mediated.

[0161] Example 6. Small molecule ligand-targeted drug conjugates for anti-influenza chemotherapy and immunotherapy Competitive binding of zanamivir-DNP conjugate to neuraminidase-transfected HEK293 cells using zanamivir-rhodamine conjugate as the labeled ligand

[0162] Method: Neuraminidase-transfected HEK 293 cells were seeded in 24-well plates and incubated overnight. The next day, the cells were incubated with a single concentration of labeled ligand (15 nM zanamivir-rhodamine conjugate) and different concentrations of zanamivir-DNP conjugate or zanamivir. After 1 hour of incubation, the cells were washed with cell culture medium and the remaining fluorescence was quantified by fluorescence spectroscopy. The apparent K was calculated by plotting the fluorescence intensity of cell binding against the log concentration of added zanamivir-DNP conjugate or zanamivir using the competitive binding equation in GraphPad Prism 4 d .

[0163] The binding affinity of zanamivir-DNP conjugate to cell membrane-bound neuraminidase was measured in a competitive binding assay Figure 10 The measured binding affinities of zanamivir-DNP conjugate and zanamivir were 12.81 and 0.45 nM, respectively. Even though the binding affinity of the targeting ligand decreased 28-fold after conjugation with the DNP moiety, its binding affinity remained in the low nanomolar range, indicating the potential of zanamivir-DNP conjugate as a hapten conjugate for ligand targeting

[0164] Example 7. Anti-DNP Antibody Recruitment Assay with Zanamivir-DNP Conjugate

[0165] As Figure 11 shown, zanamivir-DNP conjugate was able to specifically bind to neuraminidase on the cell membrane of neuraminidase-transfected HEK293 cells (293tn NA) and recruit anti-DNP antibody at approximately 10 nM. No binding was observed in untransfected HEK293 cells (293tn).

[0166] Method: 293tn NA (neuraminidase-transfected) and control (untransfected 293tn) cells were incubated with different concentrations of zanamivir-DNP conjugate at 4 °C for 30 minutes, followed by washing 3 times with PBS. After that, the cells were stained with anti-DNP-biotin and streptavidin-PE at 4 °C for 30 minutes. The cells were washed 3 times with PBS and flow cytometry was performed

[0167] Example 8. Mouse Protection Study

[0168] Method: BALB / c mice (4 weeks old) were immunized twice with DNP-KLH at week 1 and week 3. At week 4, 50 μL LD 50Influenza virus A / Puerto Rico / 8 / 1934 (H1N1) was used to infect both immunized and non-immunized mice intranasally to develop influenza symptoms. Two hours later, mice were given PBS / zanamivir / zanamivir-DNP conjugate (1.5 μmol / kg) intranasally once a day for 5 days. A mouse was considered dead when it lost 25% of its initial body weight or when it was near death.

[0169] As Figure 12 shown, the zanamivir-DNP conjugate (red inverted triangles) had superior efficacy compared to zanamivir (green triangles) at a dose of 1.5 μmol / kg. For immunized mice treated with the zanamivir-DNP conjugate, no weight loss or influenza symptoms were observed ( Figure 12 a, red line). The zanamivir-DNP conjugate protected all immunized mice from a lethal viral challenge ( Figure 12 b, red line). In contrast, zanamivir only saved 60% of the immunized mice ( Figure 12 b, green line). It is worth noting that when the zanamivir-DNP conjugate was given to non-immunized mice, its efficacy was significantly reduced (blue line), which emphasizes the importance of the immunomodulatory function of the zanamivir-DNP conjugate.

[0170] Example 9. Influenza virus-induced tumor types for targeted CAR T therapy

[0171] In this example, in vitro studies demonstrating the killing of NA-expressing HEK 293 cells by CAR-T cells were performed.

[0172] Figure 14 shows the use of NA-expressing HEK 293 cells (293NA) to mimic cells infected with influenza virus. Figure 14A And B respectively show

[0173] CAR-T (100,000 cells): 293NA (20,000) = 5:1, resulting in 41% killing

[0174] CAR-T (200,000 cells): 293NA (20,000) = 10:1, resulting in 61% killing

[0175] Example 10. Zanamivir-FITC does not bind to normal 293T cells

[0176] Figure 15It is shown that zanamivir-FITC does not bind to normal 293T cells. Due to the specific binding of zanamivir to NA on the cell surface, if 293 cells are not transfected with NA expression, zanamivir-FITC will not be present on the cell surface and thus cannot be detected when normal 293 cells are gated in a flow cytometer. Only cells expressing NA on their surface will be detected by the zanamivir-FITC conjugate. Therefore, zanamivir-FITC can be used as a probe to identify NA-expressing cells.

[0177] Example 11. Cytotoxicity against NA is specifically induced by zanamivir-FITC

[0178] Figure 16 It is shown that anti-FITC CAR T specifically exerts its cytotoxicity against HEK 293NA cells.

[0179] Three different groups (HEK-293 + FITC-zanamivir, 293NA + EC17, 293NA + free zanamivir) were co-cultured with human CAR-T cells, and the killing percentage was measured by the LDH test.

[0180] In this table / Figure 16 HEK-293 are normal 293T cells that do not express NA, while 293NA are 293T cells that express NA. FITC-zanamivir is a linker designed for CAR-T to target cells expressing NA (essentially influenza-infected cells; 293NA in this experiment). For EC17, the FITC side can bind to CAR-T cells, but the other side does not bind to 293NA. Therefore, these three groups can be considered as: 1. non-target cells with the correct linker; 2. target cells with the wrong linker; 3. target cells with free drug. The results of these three groups are expected to show no killing, and Figure 16 the experimental results in support this hypothesis (the 3 to 5% killing from the 293NA + EC17 group may be fluctuations and is not significant compared to the 40 to 60% killing from the experimental group (293NA + FITC-zanamivir) in Figure 14.

[0181] Example 12. In vitro assay using live virus

[0182] Similar to Example 10, live virus-infected MDCK cells can be identified by the zanamivir-rhodamine conjugate to examine the expression level of NA, as Figure 17 shown in.

[0183] Generally, confluent MDCK cells are infected with 100 TCID 50 influenza virus (H1N1). In the illustrated Figure 17In this case, cells were stained with 100 nM zanamivir-rhodamine conjugate to examine the expression level of NA on the cell surface.

[0184] Approximately 15 hours after influenza virus-infected MDCK cells, these cells were co-cultured with CAR-T cells for several hours. Then, the efficacy of CAR-T killing was examined by at least two different methods: one is to examine cell lysis, such as LDH assay. Figure 18 Zanamivir-FITC conjugate-linked anti-FITC CAR-T cells killing H1N1-infected MDCK are provided. The left figure shows the literature report: after 18 hours of co-culture, conventional T cells kill MDCK cells at a maximum ratio of 10%, while the linked CAR-T cells cause approximately 8.4% killing of influenza-infected MDCK cells after only 7 hours of co-culture. In addition, the linked CAR-T cells do not kill uninfected MDCK, indicating that CAR T is highly specific for infected T cells. Another method to examine CAR T killing is to measure the virus titer in the supernatant (e.g., measuring viral genetic material using qPCR to quantify viral replication).

[0185] Example 13. In vivo mouse model for studying influenza-induced tumor types for targeted CAR-T therapy

[0186] Figure 19 A mouse model is provided for studying influenza virus-induced tumor types using the targeted linker-mediated anti-FITC CAR-T therapy described in the previous examples. First, influenza virus-infected NSG mice were studied to determine the LD of the virus titer 50 . After establishing an appropriate virus titer and infected NSG mice, zanamivir-FITC conjugate-linked linker and anti-FITC CAR-T cells were applied to the infected NSG mice for rescue. It is expected that the survival of the mice will be improved after zanamivir-FITC conjugate and anti-FITC CAR-T rescue.

[0187] Example 14. Binding affinity of zanamivir-DNP conjugate for both group 1 neuraminidase (represented by N1) and group 2 neuraminidase (represented by N2) of influenza A

[0188] Figure 21 provides an in vitro binding assay of MDCK cells infected with influenza virus A / Puerto Rico / 8 / 34 (H1N1).

[0189] Figure 22 provides an in vitro binding assay of MDCK cells infected with influenza virus A / Aichi 2 / 1968 (H3N2).

[0190] Example 15. Ability of zanamivir-DNP conjugate to induce killing of influenza virus-infected cells via CDC and ADCP

[0191] Figures 23 to 24 show that anti-DNP antibodies can be recruited to MDCK cells infected with virus for both H1N1 and H3N2 strains.

[0192] Figure 25 shows the complement-dependent cytotoxicity assay of zanamivir-DNP conjugate, where only 10 nM of the drug conjugate is required to achieve maximum killing. This indicates that the zanamivir-DNP conjugate is much more potent than zanamivir alone.

[0193] Figure 26 shows that the zanamivir-DNP conjugate enables anti-DNP antibodies to effect antibody-dependent phagocytosis (ADCP).

[0194] Example 16. A series of live mouse studies showed that:

[0195] a. Efficacy of intranasal administration of the drug in the treatment of influenza virus infection ( Figure 27 to 29)

[0196] b. Dose escalation studies showed the optimal dose after intranasal administration ( Figure 27 )

[0197] c. Dose frequency studies showed that a single dose was sufficient to produce a complete cure after intranasal administration (Figures 28, 31)

[0198] d. The treatment could be delayed until 72 hours after the detection of influenza symptoms and a complete cure could still be achieved after intranasal administration (Figures 29 to 30)

[0199] e. The same drug could be administered by intraperitoneal injection and still achieve a complete cure (Figures 33 to 34)

[0200] f. In all of the above assays, our drug significantly outperformed zanamivir ( Figure 27 to 3 4)

[0201] g. BioD data and spect / CT imaging showed specificity for infected lung tissue (Figure 32)

[0202] Example 17. Analysis of ligand-targeted immunotherapy for the treatment of influenza using an antibody-dependent cellular cytotoxicity reporter gene bioassay ( Figure 35A to B)

[0203] In this example, ADCC reporter bioassays (VVariant, catalog number: G7010, Promega) using firefly luciferase reporter gene were applied to monitor the ADCC response induced by zanamivir-DNP conjugate. Briefly, engineered Jurkat cells were used to stably express the FcγRIIIa receptor, and the NFAT (nuclear factor of activated T cells) response element driving the expression of firefly luciferase was used as effector cells, as Figure 35A shown. The biological activity of the antibody in the ADCC mode of action was quantified by luciferase resulting from the activation of the NFAT pathway, indicating that the zanamivir-DNP conjugate was involved in mediating ADCC against target cells, as Figure 35B shown. This example demonstrated that the zanamivir-DNP conjugate played an important role in mediating the ADCC effect.

[0204] Example 18. In vitro antiviral assay against MDCK cells infected with H1N1 and H3N2 for ligand-targeted immunotherapy for the treatment of influenza (Figures 36A to B)

[0205] In this example, the protective effect of zanamivir-DNP conjugate on MDCK cells infected with H1N1 or H3N2 virus was studied. As shown in Figures 36A (H1N1) and 36B (H3N2), EC50 represents the inhibitor concentration for 50% protection of virus-infected MDCK.

[0206] To ensure that the zanamivir-DNP conjugate could still inhibit the neuraminidase activity required for it to inhibit the proliferation of influenza virus, we compared the efficacy of zanamivir and zanamivir-DNP conjugate in inhibiting the replication of influenza virus in MDCK-influenza virus co-cultures.

[0207] Example 19. Single-dose treatment (intranasal administration) for mice infected with H3N2 virus (Figures 37A to B)

[0208] In this example, on day 0, mice (5 mice per group) immunized with DNP-KLH were infected with 50 μL of A / Aichi / 2 / 1968 (HA, NA), x-31b (H3N2) virus (100 LD 50 ).

[0209] Twenty-four hours after infection, the mice were given zanamivir-DNP conjugate / zanamivir / PBS intranasally only once, and the mice were considered dead when they lost 25% of their initial weight or when they were near death.

[0210] As shown in FIGS. 37A and B, intranasal administration of zanamivir-DNP provided good protection 14 days after infection (all mice were cured), while zanamivir alone did not provide the same protection.

[0211] Example 20. Single-dose treatment (intraperitoneal administration) of mice infected with H1N1 virus (FIGS. 38A to B)

[0212] In this example, on day 0, mice immunized with DNP-KLH (5 mice per group) were infected with 50 μL of A / Puerto Rico / 8 / 34 (100 LD 50 , 4.2×10 5 PFU).

[0213] The mice were given zanamivir-DNP conjugate / zanamivir / PBS intraperitoneally only once 24 hours after infection, and the mice were considered dead when they lost 25% of their initial body weight or when they were moribund.

[0214] As shown in FIGS. 38A and B, intraperitoneal administration of zanamivir-DNP provided good protection 14 days after infection (all mice were cured), while zanamivir alone did not provide the same protection.

[0215] Example 21. Single-dose treatment (intraperitoneal administration) of mice infected with H3N2 virus (FIGS. 39A to B)

[0216] In this example, on day 0, mice immunized with DNP-KLH (5 mice per group) were infected with 50 μL of A / Aichi / 2 / 1968 (HA, NA), x-31b (H3N2) virus (100 LD 50 ).

[0217] The mice were given zanamivir-DNP conjugate / zanamivir / PBS intraperitoneally only once 24 hours after infection, and the mice were considered dead when they lost 25% of their initial body weight or when they were moribund.

[0218] As shown in FIGS. 39A to B, intraperitoneal administration of zanamivir-DNP provided good protection 14 days after infection (all mice were cured), while zanamivir alone did not provide the same protection.

[0219] Example 22. Anti-DNP antibody-treated non-immunized mice infected with H1N1 virus (FIGS. 40A to B)

[0220] In this example, one day after infection with a lethal dose of H1N1 virus, unimmunized mice were given anti-DNP antibody intravenously and immediately treated with different doses of zanamivir-DNP conjugate administered intraperitoneally.

[0221] In a pre-study, on day 0, mice (3 mice per group) were infected with 50 μL of A / Puerto Rico / 8 / 34 virus (100 LD 50 , 4.2×10 5 PFU).

[0222] The mice were given anti-DNP antibody (polyclonal rabbit IgG) intravenously only once 24 hours after infection, and zanamivir-DNP conjugate was given intranasally only once 24 hours after infection.

[0223] A mouse was considered dead when it had lost 25% of its initial weight or when it was moribund.

[0224] As shown in FIGS. 40A to B, if administered simultaneously 24 hours after infection, as little as 1 mg / kg of intravenously administered anti-DNP antibody and 1.5 μmol / kg of intranasally administered zana-DNP conjugate were able to provide the necessary protection against lethal dose H1N1 virus infection.

[0225] Example 23. Synthetic scheme of zanamivir-rhamnose conjugate ( Figure 41 )

[0226] In this example, different conjugates of zanamivir-rhamnose were synthesized for inducing an immune response against influenza virus infection. Figure 41 The synthetic scheme is provided in.

[0227] Example 24. Competitive binding of zanamivir-rhamnose conjugate with neuraminidase-transfected HEK293 cells using zanamivir-rhodamine conjugate as a labeled ligand (FIGS. 42A to B)

[0228] In this example, neuraminidase-transfected HEK 293 cells were seeded in a 24-well plate and incubated overnight. The next day, the cells were incubated with a single concentration of labeled ligand (15 nM zanamivir-rhodamine conjugate) and different concentrations of zanamivir-rhamnose conjugate or zanamivir. After 1 hour of incubation, the cells were washed with cell culture medium and the remaining fluorescence was quantified by fluorescence spectroscopy. The apparent K was calculated by plotting the fluorescence intensity of cell binding against the log concentration of added zanamivir-DNP conjugate or zanamivir using the competitive binding equation in GraphPad Prism 4. d . The K of free zanamivir dThe K of zanamivir-rhodamine conjugate is approximately 0.77 nM d compared to approximately 11.71 nM, the K drawn for zanamivir-rhamnose conjugate d is approximately 3.57 nM.

[0229] Example 25. Immunotherapy study with zanamivir-rhamnose conjugate (Figs. 43A to B)

[0230] In this example, protection of zanamivir-rhamnose conjugate in mice was observed in a dose escalation study. Briefly, on day 0, mice immunized with rhamnose-OVA (5 mice per group) were infected with 50 μL of A / Puerto Rico / 8 / 34 virus (100 LD 50 , 4.2×10 5 PFU).

[0231] Twenty-four hours after infection, mice were administered 1.5 / 0.5 / 0.17 μmol / kg of zanamivir-rhamnose conjugate / zanamivir / PBS intranasally twice a day for 5 days, and mice were considered dead when they lost 25% of their initial weight or when they were moribund.

[0232] As shown in Figs. 43A to B, zanamivir-rhamnose conjugate at as low as 0.17 μmol / kg b.i.d. was able to provide at least 50% protection to mice lethally infected with A / Puerto Rico / 8 / 34 (H1N1) virus.

[0233] References

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[0235] 2. Centers for Disease Control and Prevention. Estimating seasonal influenza-associated deaths in the United States. Available at: http: / / www .cdc..gov / flu / about / disease / us_flu-related_deaths.htm 2015.

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Claims

1. A conjugate comprising a targeting ligand (TL) against an influenza virus envelope protein, a linker (L), and a drug payload (D), wherein the TL is oseltamivir, zanamivir, peramivir, or laninamivir, the linker is covalently bound to both the D and the TL, and D is dinitrophenyl or rhamnosyl.

2. The conjugate according to claim 1, wherein the linker comprises a spacer and a cleavable or non-cleavable bridge between the TL and the D.

3. The conjugate according to claim 1, wherein the influenza virus envelope protein is neuraminidase (NA) or hemagglutinin (HA).

4. The conjugate according to claim 1, wherein the TL is zanamivir.

5. The conjugate according to claim 1, which has the following formula:

6. The conjugate according to claim 3, wherein the binding affinity of the conjugate to the NA is from 1 nM to 15 nM.

7. Use of a conjugate for the preparation of a medicament for treating influenza virus infection in a subject, wherein the medicament is formulated to provide the conjugate to the subject, wherein the conjugate comprises a targeting ligand (TL) for neuraminidase (NA) of the influenza virus, a linker (L), and a drug payload (D), wherein the TL is oseltamivir, zanamivir, peramivir, or laninamivir, L is covalently bound to both the D and the TL, and D is dinitrophenyl or rhamnosyl.

8. The use according to claim 7, wherein the TL is zanamivir.

9. The use according to claim 7, wherein the D inhibits an early cytokine storm induced by the influenza virus.

10. The use according to claim 7, wherein the zanamivir conjugate elicits an immune response that results in the clearance of antibody-coated viruses or virus-infected cells through antibody-dependent cell phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC).

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