Bihapten and trihapten conjugates, compositions, methods of preparation and methods of treatment thereof

Abstract

A conjugate having the formula TL-L-Hn, wherein TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; l is a linker; h is a hapten; and n is an integer of 2 or more. Compositions comprising the conjugates and methods of use are also provided.

Description

priority

[0001] This application is related to and claims priority to: (1) U.S. Provisional Patent Application No. 63 / 332,521, filed April 19, 2022; (2) U.S. Provisional Application No. 63 / 392,744, filed July 27, 2022; and (3) U.S. Provisional Application No. 63 / 429,030, filed November 30, 2022. The contents of the above applications are incorporated herein by reference in their entirety. Technical Field

[0002] The present invention includes conjugates comprising two or more haptens, and compositions (e.g., pharmaceutical compositions) comprising such conjugates and methods for preparing such conjugates, wherein the haptens are linked to targeting ligands of target proteins on viruses or cells. In addition, the present disclosure also includes methods for treating viral infections, fibrosis, and cancer. Background Art

[0003] The aspects introduced in this section may help to better understand the present disclosure. Therefore, these statements should be read with this in mind and should not be understood as an admission of what is or is not prior art.

[0004] Well-known Fc-mediated (fragment crystallizable domain-mediated) antibody effector functions are antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In addition, antibodies have been found to mediate inflammation and immunomodulation by inducing cell differentiation and activation. These mechanisms can either protect against viral replication or enhance the clearance of infected cells through antibody-mediated effector functions against virus-infected cells (e.g., viral glycoprotein shedding, viral glycoprotein internalization, antibody synergy, and antibody glycosylation). The family of receptors that recognize the Fc domain of IgG (immunoglobulin G) molecules is known as the FcRn and FcγR families. Several studies have demonstrated a correlation between Fc:FcγR affinity and selectivity and the cytotoxic function of immune effector cells involved in immune complexes (ICs).

[0005] Human gamma receptors (FcγRs) include FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), which are expressed at different levels on the surface of various immune cells. FcγRIIIa is a key surface receptor that promotes ADCC activity and is present on the surface of natural killer (NK) cells, macrophages, monocytes, mast cells, eosinophils, and dendritic cells. However, it is the only FcγR expressed by NK cells.

[0006] Humans express two FcγRIIIa allotypes that differ at a single amino acid at position 158; the residue can be either valine (V) or phenylalanine (F), with the isotype with V at position 158 having high affinity for the Fc domain of IgG1 and the isotype with F at position 158 having low affinity. Binding of immune complexes (ICs) to the high affinity FcγRIIIa-V158 resulted in a greater in vitro cytotoxic potency than that of FcγRIIIa-F158. Fc-engineered antibodies with increased affinity for FcγRIIIa show greater therapeutic efficacy than native Fc, as they can more effectively prime and activate NK cells. It is known that ADCP activity is triggered by FcγRIIa intracellular signaling, which has been demonstrated by enhanced affinity of glycoengineered antibodies for FcγRIIa, resulting in enhanced ADCP activity. Mainly, Fc variants that exhibit high affinity for the FcγRIIa-R131 isoform, high selectivity for FcγRIIa over FcγRIIb, have been shown to mediate stronger ADCP activity. Either complement receptor can participate in the mechanism of phagocytic cells. Infected cells can also be cleared by CDC as well as ADCC and / or ADCP mediated by FcγR-bearing effector cells.

[0007] Almost all FDA-approved therapeutic antibodies are full-size IgG1 antibodies, which are approximately 150 kDa in size. Dimitrov, Engineered CH2 domains (nanoantibodies), mAbs 1(1):26-28 (2009). This is also true for the vast majority of antibodies in clinical trials. A few exceptions are antigen-binding fragments (Fab).

[0008] Full-sized antibodies present fundamental problems for therapeutics. One problem is poor tissue penetration, such as in solid tumors. Dimitrov (2009), supra. Another problem is poor or no binding to regions on the surface of certain molecules, such as the envelope glycoprotein of the human immunodeficiency virus (HIV). Ibid.

[0009] In view of the above, what is needed are some materials and methods to bring antibodies (Abs), whether naturally occurring, exogenously administered autologous Abs or exogenously administered IgG abs, into close proximity with viruses, virus-infected cells, cancer cells, immune cells and / or fibroblasts, for example to improve Ab function. This and other objects and advantages and inventive features will become apparent from the detailed description provided herein. Summary of the invention

[0010] In one aspect of the present disclosure, there is provided a TL-LHn The invention relates to a conjugate of the invention and a pharmaceutically acceptable salt thereof, wherein TL is a targeting ligand of a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; L is a linker; H is a hapten; and n is an integer of 2 or 3.

[0011] In another aspect of the present disclosure, a conjugate having the following formula IA is provided: and pharmaceutically acceptable salts thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell or fibroblast; L a , L b and L c is a linker; C is a carbon atom; R4 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl or C1-C5 alkynyl groups; H1 and H2 are haptens.

[0012] In another aspect of the present disclosure, a conjugate having the following formula IB is provided: and pharmaceutically acceptable salts thereof, wherein TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; L a , L b , L c and L d is a linker; C is a carbon atom; H1, H2 and H3 are haptens.

[0013] In another aspect of the present disclosure, a conjugate having the following structure of Formula II is provided: or a pharmaceutically acceptable salt thereof, wherein L1, L2 and L3 are each independently a linker.

[0014] In certain embodiments, the conjugate has the formula: TL-LH n Or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand of a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; L is a linker; H is a hapten; and n is an integer of 2-3; optionally, at least two of the Hs can each bind to a different antibody when in contact therewith.

[0015] At least two of the Hs can each be bound by an antibody. Each H can be bound by a different antibody. In certain embodiments, when contacted with an antibody in vivo, at least two of the Hs can each be bound to a different antibody. In certain embodiments, when contacted with an antibody in vitro, at least two of the Hs can each be bound to a different antibody.

[0016] Each H may be independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof. At least one H may be an influenza virus antigen selected from hemagglutinin and neuraminidase. At least one H may be a hepatitis virus antigen selected from L-HBsAg, S-HBsAg, M-HBsAg, and preS. At least one H may be gp120 or gp160. At least one H may be a glycoprotein.

[0017] n can be 2. n can be 3.

[0018] Each H can be independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen, wherein the virus is selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus.

[0019] In certain embodiments, n is 2, the first H is a DNP fragment, and the second H is a rhamnose fragment.

[0020] The target protein may be an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell. The target protein may be an influenza virus neuraminidase or an influenza virus hemagglutinin. The target protein may be a respiratory syncytial virus fusion protein F. The target protein may be a coronavirus spike protein. The target protein may be a hepatitis B virus surface antigen or an HBV core antigen. The target protein may be a cell surface receptor on a cancer cell. The target protein may be a folate receptor. The target protein may be a folate receptor alpha or a folate receptor beta. The target protein may be a prostate-specific membrane antigen.

[0021] The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is carbonic anhydrase 9. The target protein may be a luteinizing hormone releasing hormone receptor. The target protein may be a neurokinin 1 receptor. The target protein may be a cell surface receptor on tumor-associated macrophages. The target protein may be a cell surface receptor on myeloid-derived suppressor cells. The target protein may be a cell surface receptor on cancer-associated fibroblasts. The target protein may be a fibroblast activation protein. The targeting ligand may be a neuraminidase inhibitor. The targeting ligand may be an oseltamivir fragment, a zanamivir fragment, a peramivir fragment or a laninamivir fragment. The targeting ligand may be a zanamivir fragment. The targeting ligand may be a folic acid fragment or an analog thereof. The targeting ligand may be 5-methyltetrahydrofolate.

[0022] L may contain (-CH2CH2-O-) n , peptide, peptidoglycan or a combination of two or more thereof, wherein n is an integer between and including 1 and 32. L may be a branched linker, and at least two of the haptens are attached to different branches of the linker, wherein the different branches optionally extend from different atoms of the linker.

[0023] The targeting ligand may be a folic acid fragment or a derivative thereof, at least the first H may comprise a rhamnose fragment, and at least the second H may comprise a dinitrophenyl fragment.

[0024] The conjugates described herein may be formulated as prodrugs.

[0025] In certain embodiments, the conjugate has the formula or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; L a , L b and L c Each is a linker and may be the same or different; C is a carbon atom; R4 is selected from hydrogen, a C1-C5 alkyl, a C1-C5 alkenyl or a C1-C5 alkynyl group; H1 and H2 are each a hapten; and, optionally, wherein H1 and H2 can each bind to a different antibody.

[0026] In certain embodiments, the conjugate has the formula or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; L a , L b , Lc and L d Each is a linker and may be the same or different; C is a carbon atom; H1, H2 and H3 are each a hapten; and, optionally, each of H1, H2 and H3 may each bind a different antibody.

[0027] H1 and H2 can each be bound by an antibody. H1, H2 and H3 can each be bound by an antibody. H1 and H2 can each be independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment or a combination thereof. H1, H2 and H3 can each be independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment or a combination thereof. H1 or H2 can each independently be selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxin, biotin or a viral antigen, wherein the virus is selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus. H1, H2 or H3 can be each independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxin, biotin or a viral antigen, the virus being selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus. In certain embodiments, H1 is a DNP fragment and H2 is a rhamnose fragment.

[0028] At least one hapten may be an influenza virus antigen selected from hemagglutinin and neuraminidase. At least one hapten may be a hepatitis virus antigen selected from L-HBsAg, S-HBsAg, M-HBsAg and preS. At least one hapten may be gp120 or gp160. At least one hapten may be a glycoprotein.

[0029] The target protein may be an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell. The target protein may be an influenza virus neuraminidase or an influenza virus hemagglutinin. The target protein may be a respiratory syncytial virus fusion protein F. The target protein may be a coronavirus spike protein. The target protein may be a hepatitis B virus surface antigen or an HBV core antigen. The target protein may be a cell surface receptor on a cancer cell. The target protein may be a folate receptor. The target protein may be a folate receptor α or a folate receptor β. The target protein may be a prostate-specific membrane antigen. The target protein may be carbonic anhydrase 9. The target protein may be a luteinizing hormone-releasing hormone receptor. The target protein may be a neurokinin 1 receptor. The target protein may be a cell surface receptor on a tumor-associated macrophage. The target protein may be a cell surface receptor on a myeloid-derived suppressor cell. The target protein may be a cell surface receptor on a cancer-associated fibroblast. The target protein may be a fibroblast activation protein. The targeting ligand may be a neuraminidase inhibitor. The targeting ligand may be an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment. The targeting ligand may be a zanamivir fragment. The targeting ligand may be a folic acid fragment or an analog thereof. The targeting ligand may be 5-methyltetrahydrofolate.

[0030] In certain embodiments of the conjugate, L a , L b , L c and L d At least one of each independently comprises: (-CH2CH2-O-) n , an alkyl group, a peptide, a peptidoglycan, or a combination of two or more thereof, wherein n is an integer between and including 1 and 32. In certain embodiments of the conjugate, L a , L b and L c At least one of each independently comprises: (-CH2CH2-O-) n , an alkyl group, a peptide, a peptidoglycan, or a combination of two or more thereof, wherein n is an integer between and including 1 and 32. n can be an integer between and including 1 and 16.

[0031] L a , L b , L c and L d At least one of the may comprise a peptide fragment or a peptidoglycan fragment. a , L b and L c At least one of the may comprise a peptide fragment or a peptidoglycan fragment. a , L b and L c , and Ld Can each independently contain C2-C 18 Alkyl group. a , L b and L c Can each independently contain C2-C 18 Alkyl group.

[0032] In certain embodiments, the conjugate has the formula: or a pharmaceutically acceptable salt thereof.

[0033] In certain embodiments, the conjugate has the formula: or a pharmaceutically acceptable salt thereof.

[0034] One or more -OH groups of the conjugate may be independently substituted by thiol, phosphate or phosphonate. One or more of the -OH groups may be substituted by -OC(=O)R, wherein R is an alkyl group. One or more of the -OH groups may be substituted by -OC(=O)R, wherein R is a C1-C6 alkyl group. The amino (-NH2) group may be substituted by -OC(=O)R2, and R2 may be an alkyl group. The amino (-NH2) group may be substituted by -OC(=O)R2, and R2 may be a C1-C6 alkyl group. The carboxyl (-COOH) group may be substituted by -OC(=O)R3, wherein R3 is an alkyl group. The carboxyl (-COOH) group may be substituted by -OC(=O)R3, wherein R3 is a C1-C6 alkyl group.

[0035] In certain embodiments, the conjugate has the formula: Or a pharmaceutically acceptable salt thereof, wherein L1, L2 and L3 are linkers. One or more of L1, L2 and L3 may contain (-CH2CH2-O-) n , wherein n may be an integer between and including 1 and 16. L1, L2 and L3 may each independently comprise C2-C 18 Alkyl groups, peptide fragments or peptidoglycan fragments.

[0036] In certain embodiments, the conjugate has the formula: or a pharmaceutically acceptable salt thereof.

[0037] The conjugates may also be complexed with one or more antibodies in vivo.

[0038] A pharmaceutical composition is also provided. The pharmaceutical composition may include a conjugate (eg, any conjugate described herein) and a pharmaceutically acceptable excipient.

[0039] Also provided is a method of treating a viral infection in a subject. The method of treating a viral infection in a subject may comprise administering to the subject an effective amount of a conjugate herein or a pharmaceutical composition thereof. The method may further comprise administering to the subject an autologous antibody or an allogeneic immunoglobulin G (IgG) antibody. The viral infection may be influenza. The conjugate or the pharmaceutical composition may be administered orally. The conjugate or the pharmaceutical composition may be administered once a day. The conjugate or the pharmaceutical composition may be administered more than once a day. The conjugate or the pharmaceutical composition may be administered twice a day.

[0040] A method for treating cancer in a subject is provided. The method for treating cancer in a subject may comprise administering to the subject an effective amount of a conjugate herein or a composition thereof (e.g., a pharmaceutical composition). The method may also comprise administering to the subject an autologous antibody or an allogeneic IgG antibody. The cancer may be a hot cancer. The cancer may be renal cancer, lung cancer, or colorectal cancer.

[0041] The conjugate or the pharmaceutical composition can be administered orally or intravenously. The conjugate or the pharmaceutical composition can be administered once a day.

[0042] The method of treating cancer may further comprise administering a second therapy to the subject, wherein the second therapy comprises chemotherapy, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor.

[0043] Also provided is a method of activating an immune response in a subject, in certain embodiments, such method comprises administering to the subject an effective amount of a conjugate herein or a pharmaceutical composition thereof. The immune response may be an innate immune response. The immune response may be activated in a targeted area of ​​the subject (e.g., a tumor microenvironment or a location of a viral replication site).

[0044] The method of activating an immune response may further comprise administering an autologous antibody or an allogeneic IgG antibody to the subject.Administering an effective amount of the conjugate or the pharmaceutical composition may induce reprogramming of M2 macrophages into M1 macrophages in the targeted area. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] According to the following detailed description of various exemplary embodiments of the present disclosure, the embodiments disclosed herein and other features, advantages and aspects and matters for achieving these embodiments and features, advantages and aspects will become apparent. Such detailed description will be better understood when combined with the accompanying drawings.

[0046] Figure 1A is a schematic diagram of antibody recruitment.

[0047] Figure 1B Schematic diagram of antibody recruitment using the conjugate embodiment described herein, wherein 10 comprises a targeting ligand (TL) and two haptens (each H), wherein the targeting ligand is specific for the neuraminidase (i.e., receptor) of a virus or a virus-infected cell, and the hapten H of the conjugate 10 binds to human endogenous anti-DNP and anti-Rha antibodies (Abs 12).

[0048] Figures 2A-2H Compound 7 ( Figure 2A )、Compound 8( Figure 2B )、Compound 9( Figure 2C )、Compound 12( Figure 2D )、Compound 18( Figure 2E )、Compound 22( Figure 2F )、Compound 23( Figure 2G ) and compound 24 ( Figure 2H ) liquid chromatography-mass spectrometry (LC-MS), mass spectrometry, and UV spectroscopy data.

[0049] Figure 3 Is infected 100LD 50 Influenza A H1N1 / PR8, anti-influenza antibody removal, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 48 hpi.

[0050] Figure 4 Yes Figure 3 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0051] Figure 5 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 48 hpi.

[0052] Figure 6 Yes Figure 5 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0053] Figure 7 Is infected 100LD 50Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 96 hpi.

[0054] Figure 8 Yes Figure 7 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0055] Fig. 9 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 48 hpi.

[0056] Fig.10 Yes Fig. 9 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0057] Fig.11 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 96 hpi.

[0058] Fig.12 Yes Fig.11 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0059] Fig.13A Virus titers (fold change) at 48 hpi are shown for the conjugate and the commercial drug.

[0060] Fig. 13B Virus titers (fold change) at 96 hpi are shown for the conjugate and the commercial drug.

[0061] Fig.14A Virus titers (fold change) at 48 hpi are shown for the conjugate and the commercial drug.

[0062] Fig. 14B Virus titers (fold change) at 96 hpi are shown for the conjugate and the commercial drug.

[0063] Fig.15A It is a graph showing the relationship between the number of days after infection and the survival rate (%).

[0064] Fig. 15B It is a graph showing the relationship between the number of days after infection and body weight (%).

[0065] Fig.16 It is a bar graph of administration route (SC = subcutaneous; OG = oral gavage; IV = intravenous; IN = intranasal; PBS = phosphate buffered saline) and virus titer (PFU / ml / per ng RNA).

[0066] Fig.17 Is a bar graph of OG and PBS versus virus titer (PFU / ml per ng RNA).

[0067] Fig.18 It is a plot of time (h = hours) versus concentration (ng / mL) for a single intravenous (IV) dose of 1.5 μmol / kg (n = 2 mice / time point) related to the pharmacokinetic study of compound 24 (Zan-DNP-rhamnose).

[0068] Fig.19 is a plot of time (h = hour) versus concentration (ng / mL) for a single oral dose of 4.5 μmol / kg (n = 2 mice / time point) related to the pharmacokinetic study of compound 24 (Zan-DNP-rhamnose) administration.

[0069] Fig. 20A is a plot of days post infection versus survival (%) associated with a dose escalation study in which mice were infected with 100×LD 50 Influenza A / H1N1 / PR8 / 1934, hIVIg was used as a source of anti-hapten antibodies (injected at a dose of 8 g / kg 24 hours before drug administration), and doses of compound 24 (Zan-DNP-rhamnose) were administered at 48 hpi, with a single dose of 1.5 μmol / kg Zan-DNP-rhamnose (IV) or two doses of 4.5 μmol / kg Zan-DNP-rhamnose (oral).

[0070] Fig. 20B is with Fig. 20A The relationship between days post infection and body weight % for the dose escalation study, Fig. 20A The legend in also applies to Fig. 20B .

[0071] Fig.21 Figure 2 is a graph showing the relationship between the number of days post infection and the viral titer (PFU / ml per ng RNA) for PBS and zanamivir-DNP-rhamnose (Compound 24) IV and OG administration.

[0072] Fig. 22 It is a graph showing the relationship between the number of days after infection and the survival rate (%).

[0073] Fig.23 It is a graph showing the relationship between the number of days after infection and body weight (%).

[0074] Fig.24 The figure shows that Balb / c mice 24 hours after treatment (this is again infected with 10LD 50 The lung titers of A / H1N1 / PR8 / 1934 virus (day 0) and 48 hours after infection were compared between compound 24 and PBS, of treated and uninfected mouse cohorts (uninfected).

[0075] Fig.25 The figure shows that Balb / c mice infected with 10LD 50 A / H1N1 / PR8 / 1934 virus (day 0) and the survival rate of compound 24 (C.24) administered by various routes (i.e., IN route, IV route, and OG route) on day 4 after infection was compared with the survival rate of PBS alone.

[0076] Fig.26 The graph shows that based on Fig.25 Days post infection versus % body weight in dosing data.

[0077] Fig.27A and Fig.27B Shown are graphs of the concentration of compound 24 (in nM) versus the percentage of killing by antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) on N1-transfected HEK293 cells (NA-HEK) and wild-type (WT) HEK 293 cells in vitro, respectively.

[0078] Fig.28A and Fig.28B Shown are graphs showing the relationship between days post infection and survival rate (%) and days post infection and body weight (%) in a dose escalation study of compound 24 administered IV to Balb / c mice, respectively.

[0079] Fig.29A and Fig.29B Shown are graphs of days post infection versus survival (%) and days post infection versus body weight (%), respectively, in a study comparing Balb / c mice administered the conjugates described herein with single haptens (96 hpi treatment).

[0080] Fig. 30A and Fig. 30BShown are graphs of the relationship between days post infection and survival rate (%) and days post infection and body weight (%) in a study comparing administration of the conjugate to Balb / c mice infected with various influenza virus strains, wherein (A) is the influenza A / California / 07 / 2009 (H1N1) pdm09+Compound 24 treatment group; (B) is the influenza A / Wisconsin / 67 / 2005 (H3N2)+Compound 24 treatment group; (C) is the influenza B / Florida / 04 / 2006+Compound 24 treatment group; (D) is the influenza A / California / 07 / 2009 (H1N1) pdm09+PBS control group; (E) is the influenza A / Wisconsin / 67 / 2005 (H3N2)+PBS control group; and (F) is the influenza B / Florida / 04 / 2006+PBS control group.

[0081] Fig.31 Graphs showing cytokine and chemokine levels measured in the lungs of mice in the treated or control groups are shown.

[0082] Fig.32 Graphs of serum cytokine and chemokine levels measured in the treatment or control groups are shown to observe whether systemic inflammation was present in the test subjects.

[0083] Fig.33 Shown are images of qualitative histological analysis of lung tissue biopsies collected from mice infected with influenza A / H1N1 / PR8 / 1934 and treated with 1) PBS, 2) Compound 24 by IV, or 3) Compound 24 by OG.

[0084] Fig.34 Shown are histological images of lung or kidney tissues of virus-infected mice treated with compound 24 (oral or intravenous) or left untreated.

[0085] Fig.35 A synthetic scheme for the folate bihapten conjugate Compound 150 is shown.

[0086] Fig.36 LC-MS, mass spectrometry and UV spectroscopy data of compound 3' are shown.

[0087] Fig.37 LC-MS, mass spectrometry and UV spectroscopy data of compound 5' are shown.

[0088] Fig.38 LC-MS, mass spectrometry and UV spectroscopy data of compound 10' are shown.

[0089] Fig.39LC-MS, mass spectrometry and UV spectroscopy data of compound 11' are shown.

[0090] Fig.40 LC-MS, mass spectrometry and UV spectroscopy data of compound 13' are shown.

[0091] Fig.41 LC-MS, mass spectrometry and UV spectroscopy data of compound 14' are shown.

[0092] Fig.42 LC-MS, mass spectrometry and UV spectroscopy data of compound 18' are shown.

[0093] Fig.43 LC-MS, mass spectrometry and UV spectroscopy data of compound 150 are shown.

[0094] Fig.44 Data related to the CDC assay are shown, represented by a graph of folate double hapten (Compound 150 = FDH) concentration (nM) versus percent killing by CDC, showing the effect of Compound 150 on mouse lung cancer cells (M109) when incubated with anti-hapten antibodies and human serum (M109_comp = folate-glucosamine competitor).

[0095] Figures 45A-45D Data related to the ADCC assay are shown, in a graph of FDH concentration (nM) versus percent killing by ADCC, showing the effect of Compound 150 on a variety of mouse and human cancer cell lines in vitro (4T1-FR = Compound 150 and 4T1-FR_comp = folate-glucosamine competitor, acting on 4T1 cells; MDA-MB-231 = Compound 150 and MDA-MB-231_comp = folate-glucosamine competitor, acting on MDA-MB-231 cells; M109 = Compound 150 and M109_comp = folate-glucosamine competitor, acting on M109 cells; THP-1 = Compound 150 and THP-1_comp = folate-glucosamine competitor, acting on THP-1 cells (a human leukemic monocytic cell line used as an in vitro model of acute myeloid leukemia (AML))).

[0096] Fig.46 Data related to the in vivo mouse lung cancer efficacy study of Compound 150 are shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control; anti-PD1 + carboplatin + paclitaxel = SOC test article; and Compound 150 test article). Abs = antibody, in Fig.46In the studies shown, the antibody was a mouse anti-PD1 antibody (InVivoMAb Anti-Mouse PD-1 Catalog #BE0146; InVivoGen, San Diego, CA).

[0097] Fig.47 Shows the Fig.46 Data related to the in vivo lung cancer efficacy study of Compound 150 are shown in the graph of the percentage of subject body weight versus days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + carboplatin + paclitaxel = SOC test article; Fol dual hapten (+Abs) = Compound 150 test article). Abs = anti-PD1 antibody.

[0098] Fig.48 Data related to the in vivo human lung cancer efficacy study of Compound 150 are shown, represented by a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control; anti-PD1 + radiotherapy = SOC test article; and Compound 150 test article).

[0099] Fig.49 Shows the Fig.48 Safety data related to the in vivo human lung cancer efficacy study of Compound 150 are shown in the relationship between the percentage of subject body weight and the number of days after tumor implantation (PBS (+Abs) = control group; anti-PDL1 + radiotherapy = SOC test article; and Compound 150 test article).

[0100] Fig.50 Data related to the in vivo mouse colorectal cancer efficacy study of Compound 150 are shown, as shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control; leucovorin + 5-FU + oxaliplatin = SOC test article; Fol dual hapten (+Abs) = Compound 150 test article).

[0101] Fig.51 Shows the Fig.50 Safety data related to the in vivo colorectal cancer efficacy study of Compound 150 are shown in the relationship between the percentage of subject body weight and the number of days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + carboplatin + paclitaxel = SOC test article; and Compound 150 (+Abs) test article).

[0102] Fig.52 Data related to an in vivo mouse lung cancer combination therapy study are shown, plotted as tumor volume versus days after tumor implantation.

[0103] Fig.53 Data related to an in vivo mouse lung cancer combination therapy study are shown, plotted as tumor volume versus days after tumor implantation.

[0104] Fig.54 Data related to an in vivo mouse lung cancer combination therapy study are shown, plotted as tumor volume versus days post tumor implantation (PBS (+Abs) = control; leucovorin + 5FU + OXH = SOC test article; Fol dual hapten (+Abs) = Compound 150 test article).

[0105] Fig.55 Shows the Fig.54 Data related to the in vivo human lung cancer efficacy study of Compound 150 are shown in the graph of the relationship between the percentage of subject body weight and the number of days after tumor implantation (PBS (+Abs) = control group; folinic acid + 5FU + OXH = SOC test article; Fol dual hapten (+Abs) = Compound 150 test article).

[0106] Fig.56 Data related to an in vivo mouse colorectal cancer combination therapy study are shown, plotted as tumor volume versus days after tumor implantation.

[0107] Fig.57 Data related to an in vivo mouse colorectal cancer combination therapy study are shown, plotted as tumor volume versus days post tumor implantation (PBS (+Abs) = control; leucovorin + 5FU + OXH = SOC test article; Fol bihapten (+Abs) = Compound 150 test article; Fol bihapten + sunitinib = Compound 150 + sunitinib; Fol bihapten + FA-TLR7 = Compound 150 + FA-TLR 7).

[0108] Fig.58 Data from an in vivo comparative efficacy study of Compound 150 versus anti-PD1 + chemotherapy in mouse lung cancer are shown, as shown in a graph of tumor volume versus days after tumor implantation (PBS (+Abs) = control group; anti-PD1 + chemotherapy = SOC test article; Fol dual hapten (+Abs) = Compound 150 test article).

[0109] Fig.59 Data related to an in vivo mouse lung cancer (cold) efficacy study of Compound 150 are shown, as a graph of tumor volume versus days after tumor implantation.

[0110] Fig.60 Data related to in vivo mouse renal carcinoma (Renca cell line) efficacy studies are shown, represented by a graph of tumor volume versus days after tumor implantation.

[0111] Fig.61Shown are graphs of data comparing immune cell populations measured in Y856, LLC-1, and M109 cell lines (cold vs. hot) following treatment with PBS (control), Compound 150 (FDH), or SOC.

[0112] While the disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are herein described in detail. DETAILED DESCRIPTION

[0113] Although the concepts of the present disclosure are shown and described in detail in the description herein, the results of the description should be regarded as illustrative rather than restrictive; it should be understood that only exemplary embodiments are shown and described, and all changes and modifications that are consistent with the spirit of the present disclosure are desired to be protected.

[0114] The present disclosure provides materials and methods for recruiting antibodies (e.g., to the surface of viruses, virus-infected cells, cancer cells, immune cells, or fibroblasts). See, e.g., a schematic diagram of antibody recruitment. Figure 1A .

[0115] More specifically, a targeted therapy strategy with a dual mechanism of action is disclosed, which elicits an immune response in the host to a target (e.g., a virus, a virus-infected cell, a cancer cell). The conjugates described herein can be trivalent, such that they comprise a targeting ligand that binds (e.g., via a linker) at least two haptens that bind naturally occurring antibodies (e.g., antibodies in the human body). Once recruited, these anti-hapten antibodies bind and activate the innate immune system against the target. Pharmaceutical compositions comprising such conjugates and methods comprising administering such conjugates and compositions are also provided.

[0116] Conjugate

[0117] The conjugate described herein can be a drug conjugate targeted by a small molecule ligand, which binds a receptor-specific ligand to at least two haptens. Therefore, the conjugate can be a trivalent drug, which can be targeted to the desired cells by the receptor-specific ligand (e.g., targeting ligand), and can also be combined with two or more antibodies in the subject by a dual payload. The general scheme is to provide a specific targeting ligand conjugated with a payload of two or more haptens to treat viral infection or cancer. The targeting ligand can specifically recognize a target receptor (e.g., an envelope protein of a virus (which can be expressed only on the surface of infected cells), or a receptor overexpressed on a target cell (e.g., a folate receptor on a cancer cell, etc.)). In certain embodiments, one or more of the haptens are selected to activate the innate immune system of the subject (e.g., adjacent target cells) to recruit immune cells and / or otherwise utilize the subject's own immune system to fight against viruses or cancer.

[0118] In certain embodiments, the conjugate has the formula: TL-L-H n , or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a target cell; L is the connector; H is a hapten; and n is an integer of 2 or greater. Optionally, at least two of the Hs can be bound by different antibodies, for example, when contacted with antibodies present in vivo. In certain embodiments, n is an integer greater than or equal to 2 (e.g., 2, 3, 4, 5, ... 10, etc.). n can be 2 or 3.

[0119] In many embodiments, the conjugate has the formula: or Including pharmaceutically acceptable salts thereof, wherein TL is a targeting ligand for a target protein on the surface of a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast; L a , L b , L c and L d Each is a linker; C is a carbon atom; H1, H2 and H3 are each a hapten.

[0120] In these and other embodiments, for example, when contacted with antibodies present in vivo, at least two haptens are bound by different antibodies. In these and other embodiments, H1 and H2 can each bind to a different antibody. In these and other embodiments, H1, H2, and H3 can each bind to a different antibody.

[0121] Optionally, the conjugate may be complexed with an antibody via one or more haptens of the conjugate. When bound, each hapten typically binds independently to a different antibody. Each antibody may be a naturally occurring autoantibody, an exogenously administered autoantibody, or an exogenously administered immunoglobulin G (IgG) antibody. Binding to the antibody is typically by non-covalent interactions, such as hydrogen bonding, Coulomb bonding, Ca bonding, or the like. ++ bridges and Lifshitz-van der Waals bonds, etc. Such binding usually occurs in vivo.

[0122] Each H or H1, H2 or H3 (if applicable) can be independently selected from different haptens. In certain embodiments, when n=2, the haptens are different haptens. When n=3 or greater, two or more of the haptens can be the same. The haptens that can be used include, but are not limited to, rhamnose fragments, α-galactosyl moieties, dinitrophenyl (DNP) fragments, trinitrophenyl (TNP) fragments, fluorescein fragments, digoxigenin fragments, biotin fragments or viral antigens, the viruses selected from diphtheria, herpes zoster virus, human papillomavirus (HPV), influenza virus antigens (e.g., hemagglutinin or neuraminidase), SARS-COV-2, yellow fever virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), varicella virus, hepatitis A virus (HAV) antigens. progen (e.g., L-HbsAg, S-HbsAg, M-HbsAg and preS), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis G virus (HGV), rotavirus, mumps virus, tetanus, human immunodeficiency virus (HIV) (e.g., gp120 or gp160), cytomegalovirus (CMV), vesicular stomatitis virus (e.g., glycoprotein), rubella virus, smallpox virus, monkeypox virus, poliovirus, dengue virus and measles virus.

[0123] In some embodiments, the haptens are each independently selected from a rhamnose fragment, an α-galactosyl moiety, and a DNP fragment. In some embodiments, when n is 2, the haptens are a rhamnose fragment and a DNP fragment.

[0124] The hapten may be gp120 or gp160. The hapten may be a glycoprotein.

[0125] The targeting ligand of the conjugate can specifically recognize a targeting receptor, such as a target protein on the surface of a target cell. The target cell can be a virus, a virus-infected cell, a cancer cell, an immune cell or a fibroblast.

[0126] By specifically delivering the hapten to the cells recognized by the targeting ligand, the conjugates described herein can show high selectivity for malignant cells, virus-infected cells, immune cells, and any other cells with target proteins on their surfaces, while also reducing incidental toxicity. To date, many cancers have been treated by small molecule ligand-targeted drug conjugates targeting overexpressed receptors on tumor cells. These overexpressed receptors include, but are not limited to, folate receptor (FR), prostate-specific membrane antigen (PSMA), cholecystokinin 2 receptor (CCK2R), carbonic anhydrase IX (CA IX), etc. The targeting ligand of the conjugate can be specific for any such overexpressed receptor.

[0127] The targeting ligand can also be specific to a viral envelope protein. For example, the target protein can be a viral envelope protein or a viral envelope protein on the surface of a virally infected cell.

[0128] For example, influenza viruses are enveloped viruses. All influenza subtypes are very similar in overall structure and composition; that is, the virus particle is 80-120 nanometers in diameter, and its viral envelope contains two main types of glycoproteins wrapped around a central core. The central core contains the viral ribonucleic acid (RNA) genome and other viral proteins that package and protect such RNA. Unlike viruses, the genome of influenza viruses is not a single segment of nucleic acid, but seven or eight segments of segmented negative-sense RNA, each containing one or two genes encoding gene products (proteins). For example, the influenza A genome contains 11 genes on 8 segments of RNA, encoding 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP: nuclear export protein), PA, PB1 (polymerase basic protein 1), PB1-F2, and PB2.

[0129] HA and NA are two large glycoproteins on the outside of the virus particle. HA is a lectin that mediates the binding of the virus to the target cell and the entry of the viral genome into the target cell, while NA is usually involved in the release of progeny viruses from infected cells by cleaving sugars that bind to mature virus particles. The target protein can be influenza NA or influenza HA.

[0130] In some embodiments of the conjugates described herein, the targeting ligand is a NA or HA inhibitor, which can be used to deliver the conjugate to virus-infected cells and / or virus replication sites (e.g., nose, throat, and lungs). This can kill virus-infected cells before the progeny virus is released, thereby hindering viral replication, and / or inhibiting early cytokine storms caused by viral infection. In certain embodiments, the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment. In certain embodiments, the targeting ligand is zanamivir.

[0131] In other embodiments, the target protein is selected from respiratory syncytial virus (RSV) fusion protein F, coronavirus spike protein, hepatitis B virus (HBV) surface antigen or HBV core antigen, cell surface receptors on cancer cells, folate receptor alpha, prostate-specific membrane antigen (PSMA), carbonic anhydrase 9 (CAIX), luteinizing hormone-releasing receptor (LHRH), neurokinin 1 receptor (NK1R), surface receptors on tumor-associated macrophages (TAM) or myeloid-derived suppressor cells, folate receptor beta, surface receptors on cancer-associated fibroblasts, and fibroblast activation protein (FAP).

[0132] In certain embodiments, the targeting ligand is folic acid (folate) or an analog thereof, such as 5-methyltetrahydrofolate (5-MTHF). "Folic acid (folate)" refers to a folate receptor binding molecule, including, for example, folic acid and folic acid analogs and derivatives, such as, but not limited to, folinic acid, pteroyl polyglutamic acid, pteroyl-D-glutamate, and folate receptor-bound pteridines (e.g., tetrahydropterin), dihydrofolate, tetrahydrofolate, and deaza and dideaza analogs thereof.

[0133] The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that replace one or two nitrogen atoms with carbon atoms in the naturally occurring folate structure, or analogs or derivatives thereof. For example, the deaza analogs can include 1-deaza analogs, 3-deaza analogs, 5-deaza analogs, 8-deaza analogs, and 10-deaza analogs of folate, folinic acid, pteroylpolyglutamate, and folate receptor binding pteridines (e.g., tetrahydropterin), dihydrofolate, and tetrahydrofolate. The dideaza analogs include, for example, 1,5-dideaza analogs, 5,10-dideaza analogs, 8,10-dideaza analogs, and 5,8-dideaza analogs of folate. Other folates that can be used as complex-forming ligands in the context of the present disclosure are the folate receptor binding analogs pemetrexed, proguanil, pyrimethoprim, trimethoprim, pralatrexate, raltitrexed, aminopterin, methotrexate (also known as methotrexate), N 10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethotrexate or 3-deazamethotrexate, and 3′,5′-dichloro-4-amino-4-deoxy-N 10 -Methylpteroylglutamate (dichloromethotrexate).

[0134] Folic acid and the above analogs and / or derivatives are also referred to as "folates", "the folates" or "folates", reflecting their ability to bind to the folate receptor. As described herein, when conjugated to exogenous molecules, these molecules can effectively enhance transmembrane transport, such as through folate-mediated endocytosis. The above substances can be used as folate receptor targeting ligands described herein.

[0135] In many embodiments, the conjugate comprises a linker (L) coupling or otherwise connecting each hapten to the targeting ligand. In many embodiments, the conjugate comprises a linker L a , L b , L c In some embodiments, the conjugate comprises a linker L a , L b , L c and L d When the conjugate contains more than one linker (e.g. L a , L b , L c and optionally L d ), the linkers may be the same or different. For example, but not limited to, L a , L b , L c and L d Each of L can have the same structure. Alternatively, one or more of the linkers can have a different structure than at least one other linker. a Contains the first structure, and L b , L c and L d (when present) comprises a second structure. In certain embodiments, L b , L c and L d (when present) contain structures that are different from each other.

[0136] As used herein, the term "linker" includes a chain of atoms that is biologically functionally suitable for forming a chemical bond with a TL and / or a hapten and / or a carbon atom, and connects two or more functional parts of a molecule to form a conjugate herein. Exemplarily, the chain of atoms can be selected from carbon (C), nitrogen (N), oxygen (O), sulfur (S), silicon (Si) and phosphorus (P). The chain of atoms can covalently connect different functional capabilities of the conjugate, such as the targeting moiety and the hapten. The linker can include a variety of connections, such as a connection ranging from about 2 to about 100 atoms in a continuous backbone. The linker can include a sustained release linker (e.g., a non-hydrolyzable linker).

[0137] In certain embodiments where the conjugate comprises two haptens, the conjugate may comprise at least three linkers, a first linker connecting the targeting moiety to a carbon atom, a second linker connecting the carbon atom to a first hapten, a third linker connecting the carbon atom to a second hapten, as shown in Formula IA and Formula IB haptens, and the second linker connecting the targeting moiety to the second hapten. Alternatively, the conjugate may comprise a single branched linker connecting all haptens to the targeting moiety (see, e.g., Formula I). ​​In certain embodiments, the linker is a branched linker, and at least two haptens of the conjugate are connected to different branches of the linker. For example, the linker may comprise a main chain comprising at least two branches extending therefrom (e.g., 2 branches, 3 branches, etc.). The different branches may comprise the same structure or different structures as the main chain and / or other branches. For example, the different branches may extend from different atoms of the main chain linker, or extend from the same atom (e.g., as shown in Formula IA and Formula IB).

[0138] In many embodiments, the linker comprises a polyethylene glycol (PEG) linker, a PEG derivative linker, a peptide, an alkyl group, a peptidoglycan, or a combination of two or more of the foregoing. In some embodiments, the PEG linker may comprise (-CH2CH2-O-) n , wherein n is an integer between and including 1 and 32. In certain embodiments, n of the PEG linker is an integer between and including 1 and 16. In certain embodiments, the linker is an optionally substituted heteroalkyl.

[0139] In some embodiments, the linker is a substituted heteroalkyl comprising at least one substituent selected from the group consisting of alkyl, hydroxy, oxo, PEG, carboxylate, and halo. Unless otherwise indicated, "halo" or "halogen" by itself or as part of another substituent refers to a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0140] In some embodiments, the linker comprises a spacer. In some embodiments, the spacer comprises peptidoglycan or a sugar.

[0141] The linker may comprise C2-C 18 Alkyl groups, peptide fragments or peptidoglycan fragments. As used herein, the term "fragment" refers to a molecule that has been modified to enable it to be linked in the conjugate in a monovalent connection (such as in the case of a targeting ligand or hapten) or a divalent connection (such as in the case of a linker (L)). For example, the term "zanamivir fragment" refers to zanamivir that has been chemically modified so that it can be prepared as a targeting ligand covalently bound to a linker. In the case of zanamivir, a proton (such as a proton from an alcohol) is missing, thereby creating an open valence state on the corresponding oxygen, which in turn binds to the linker L. Typically, the alcohol oxygen closest to the dihydropyran ring of zanamivir forms a bond with the linker L. Similarly, hapten fragments are based on parts of the corresponding molecules that are already suitable for connection to the linker L. In the case of the linker L, the corresponding fragment has an open valence state to enable binding to other linker parts, targeting ligands and / or haptens. For example, in the case of PEG, the term (-CH2CH2-O-) n Refers to a fragment that is bound on both sides to another moiety (e.g., another linker component). The term "fragment" does not necessarily need to be used from a synthetic perspective; it refers to a molecule that is generated during the preparation of the conjugate. It describes the portion of the conjugate, regardless of how it is prepared.

[0142] Optionally, the conjugate can be complexed with an antibody, for example in vivo. Such complexation can be performed with different antibodies according to the hapten.

[0143] In many embodiments of the present disclosure, the conjugate has the structure of Formula I: Or a pharmaceutically acceptable salt thereof, wherein L1, L2 and L3 are each a linker. In certain embodiments, L1, L2 and L3 each independently comprise a PEG moiety, an alkyl group, a peptide group and a peptidoglycan group. In certain embodiments, the PEG moiety (-CH2CH2-O-) n The range of n is between and including 1 to 16. The alkyl group can be an alkyl chain between and including 2 to about 18 carbons (e.g., 18 carbons). For example, L1, L2, and L3 can each independently comprise C2-C 18 L1, L2 and L3 may each independently comprise a peptide fragment or a peptidoglycan fragment.

[0144] In certain embodiments, L1 comprises an amide (which may be optionally substituted with an alkyl group), a PEG moiety, an optionally substituted or unsubstituted triazole moiety wherein, if substituted, the substitution may be a carbonylalkyl group and an amide which may be optionally substituted with an alkyl group. In addition, in certain embodiments, the amide in L1 is directly bonded to the oxygen of the zanamivir fragment shown in Formula I, thereby forming a carbamate moiety with the oxygen. In certain embodiments, the tertiary carbon at the intersection of L1, L2, and L3 is bonded to the nitrogen of the amide of L1. In certain embodiments, one side of the PEG moiety is bonded to the amide and the other side is bonded to the amide or a substituted or unsubstituted triazole moiety. The PEG moiety may be represented as (-CH2CH2-O-) n , wherein n can be between and including 1 and 32, between and including 2 and 16, between and including 3 and 8, between and including 4 and 7. In some embodiments, n is 6.

[0145] In certain embodiments, L2 comprises an amide moiety optionally substituted with an alkyl group and a PEG moiety. The PEG moiety can be represented by (-CH2CH2-O-) n , where n can generally be between and including 1 and 32, between and including 2 and 16, between and including 3 and 8, and between and including 3 and 5. In some embodiments, n is 4.

[0146] In certain embodiments, L3 comprises an alkylamide group, a carbonylalkyl group, an etheralkyl group, and an amino group optionally substituted with an alkyl group. In some embodiments, L3 comprises PEG. In some embodiments, L3 comprises a PEG represented by (-CH2CH2-O-) n A PEG, wherein n may be between and including 1 and 32.

[0147] In certain embodiments, a dual hapten conjugate having the structure of Compound 24 is provided: or a pharmaceutically acceptable salt thereof.

[0148] In certain embodiments, a tri-hapten conjugate having the structure of compound 100 is provided: or a pharmaceutically acceptable salt thereof.

[0149] In certain embodiments, the conjugate is a dual hapten conjugate having the structure of Compound 101: Or a pharmaceutically acceptable salt thereof, wherein Ac is acetate.

[0150] In certain embodiments, the conjugate comprises a folate targeting moiety. In certain embodiments, the conjugate is a folate dihapten conjugate. In certain embodiments, the conjugate is a folate dihapten conjugate and has the structure of compound 150: or a pharmaceutically acceptable salt thereof.

[0151] In some embodiments, one or more hydroxyl (-OH) groups in the structures of compounds 24, 100, 101 and / or 150 can be independently replaced by sulfhydryl, phosphate or phosphonate or -OC(=O)R1, wherein R1 is an alkyl group. In these embodiments, the amino (-NH2) group can be replaced by -OC(=O)R2, wherein R2 is an alkyl group. In these and other embodiments, the carboxyl (-COOH) group can be replaced by -OC(=O)R3, wherein R3 is an alkyl group. R2 and R3 can each be, for example, a C1 to C6 alkyl group.

[0152] Optionally, a conjugate described herein (eg, Compound 24, Compound 100, Compound 101, or Compound 150) is complexed with an antibody in vivo. This complexation can be performed with different antibodies per hapten.

[0153] In certain embodiments, methods for preparing the conjugates described herein are provided. For example, the conjugate of compound 24 can be prepared as described in Example 1, and / or the conjugate of compound 150 can be prepared as described in Example 19. Similarly, other conjugates herein can be prepared according to the methods of Examples 1 and / or 19 and such other methods known in the art.

[0154] In certain embodiments, the conjugate is formulated as a prodrug. The term "prodrug" refers to a derivative of a conjugate that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound or conjugate, particularly a polyhapten conjugate disclosed herein. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the conjugate herein, including biohydrolysis moieties, such as biohydrolysis amides, biohydrolysis esters, biohydrolysis carbamates, biohydrolysis carbonates, biohydrolysis ureides, and biohydrolysis phosphate analogs. The specific prodrug of a compound with a carboxyl functional group is a low alkyl ester of a carboxylic acid. Carboxylic acid esters are conveniently formed by esterifying any carboxylic acid moieties present on the molecule. Prodrugs can generally be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).

[0155] Formulating the conjugate as a prodrug may add protecting groups that may further slow the hydrolysis of the conjugate in vivo. This may be beneficial in cases where the prodrug is administered to reprogram immune cells in a targeted area and / or slow cytokine activation in a targeted area.

[0156] The compound and optionally one or more other therapeutic agents can be administered in the form of their own (pure form) or in a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare its pharmaceutically acceptable salt. Such salts include, but are not limited to, salts prepared by the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. In addition, such salts can be prepared into alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts of carboxylic acid groups.

[0157] Suitable buffers include acetic acid and salts (1-2% w / v); citric acid and salts (1-3% w / v); boric acid and salts (0.5-2.5% w / v); and phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

[0158] Salts (eg, pharmaceutically acceptable salts) of compounds described herein can be prepared by various methods, such as including the acid in the mobile phase during chromatographic purification or stirring the chromatographically purified product with an acid solution.

[0159] The conjugates described herein may be "deuterated," meaning that one or more hydrogen atoms may be replaced by deuterium. Since deuterium and hydrogen have almost identical physical properties, deuterium substitution is the smallest structural change that can be made. Deuteration is well known to those of ordinary skill in the art.

[0160] In some embodiments, the conjugate may contain one or more asymmetric centers, thereby producing enantiomers, diastereomers and other stereoisomeric forms, which are defined as (R)- or (S)- in terms of absolute stereochemistry. In certain embodiments, the conjugate has an R- configuration. In certain embodiments, the conjugate has an S configuration. Unless otherwise indicated, all stereoisomeric forms of the conjugate should be considered. When the conjugate contains an olefin double bond, unless otherwise indicated, E and Z geometric isomers (e.g., cis or trans) and / or optical isomers should be included. In certain embodiments, for example, the D and A of the conjugate are arranged in a relative cis orientation. In certain embodiments, the D and A of the conjugate are arranged in a relative trans orientation. Similarly, it is also intended to include all possible isomers, as well as their racemic forms and optically pure forms, and all tautomeric forms. The term "geometric isomer" refers to an E or Z geometric isomer (e.g., cis or trans) of an olefin double bond. The term "positional isomers" refers to structural isomers around a central ring, for example, ortho-, meta-, and para-isomers around a benzene ring.

[0161] In addition, in each of the above and following embodiments, it is understood that the formula includes and represents not only all pharmaceutically acceptable salts of the conjugate, but also any and all hydrates and / or solvates of the conjugate formula or its salts. In fact, hydrates, solvates and N-oxides of the conjugate are also contemplated. The term "solvate" refers to a conjugate or its salt that also includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0162] It should be understood that certain functional groups (e.g., hydroxyl, amino, etc.) form complexes and / or coordination compounds with water and / or various solvents, existing in various physical forms of the conjugate. Therefore, the above formula should be understood to include and represent these various hydrates and / or solvates.

[0163] In each of the foregoing and following embodiments, it is also to be understood that the formula includes and represents any and all crystalline forms, partially crystalline forms, and non-crystalline and / or amorphous forms of the conjugate.

[0164] Pharmaceutical composition

[0165] Also provided are pharmaceutical compositions. The pharmaceutical compositions may include any conjugate described herein (e.g., a conjugate of Formula I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients or carriers. The term "composition" generally refers to any product comprising more than one component, including the conjugate. The composition may be prepared from an isolated conjugate or from salts, solutions, hydrates, solvates, and other forms of the conjugate.

[0166] In certain embodiments, the pharmaceutical composition comprises a plurality of conjugates (e.g., two or more) and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition further comprises at least one additional pharmaceutically active agent. The at least one additional pharmaceutically active agent may be an agent for treating viral infection, fibrosis, or cancer.

[0167] Pharmaceutical compositions can be prepared by combining one or more conjugates with pharmaceutically acceptable excipients and optionally one or more additional pharmaceutically active agents according to methods known in the art and described below.

[0168] The pharmaceutical composition described herein may include one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients and / or vehicles (e.g., conventional nontoxic pharmaceutically acceptable carriers, adjuvants and vehicles) and combinations thereof. Any pharmaceutically acceptable carrier, diluent and excipient known in the art may be used. Examples include, but are not limited to, excipients, colorants, preservatives and stabilizers. More specific examples include crystalline cellulose, carboxymethylcellulose calcium, sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose and magnesium stearate.

[0169] Solutions of the conjugate or pharmaceutical composition may be aqueous, optionally mixed with a non-toxic surfactant, and / or may contain carriers or excipients such as salts, carbohydrates and buffers (preferably at pH 3 to 9), but for some applications they may be more suitably formulated as sterile non-aqueous solutions or dry forms for use in combination with a suitable vehicle (e.g., sterile, pyrogen-free water or phosphate-buffered saline). For example, dispersions may be prepared in glycerol, liquid PEG, triacetin, mixtures thereof, and oils. Under normal conditions of storage and use, these preparations may also contain preservatives to prevent the growth of microorganisms.

[0170] The conjugates can be formulated into pharmaceutical compositions and administered to a mammalian host, such as a human patient, in various forms suitable for the selected route of administration. The pharmaceutical compositions can be formulated, for example, for a given route of administration, and manufactured according to methods in the art and, for example, in Remington, The Science and Practice of Pharmacy, 22 nd The composition may be an infusion or injectable composition, such as a composition that can be injected subcutaneously or intravenously.

[0171] The pharmaceutical composition can be administered to a mammalian host, such as a human patient, in various forms suitable for the selected route of administration. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In certain embodiments, the pharmaceutical composition is formulated for oral administration. In certain embodiments, the pharmaceutical composition is formulated for intramuscular administration, intravenous administration, arterial administration, intraperitoneal administration, or administration with any other art-recognized parenteral administration route.

[0172] The pharmaceutical composition can be combined with a pharmaceutically acceptable vehicle for systemic administration. The composition and formulation component percentages can vary and can be about 1 to about 99% by weight of the active ingredient (e.g., the compound or conjugate) and binders, excipients, disintegrants, lubricants, and / or sweeteners (e.g., known in the art). In such therapeutic compositions, the amount of active conjugate can achieve an effective dose level (e.g., in serum or target tissue or target cells).

[0173] Exemplary modes of parenteral administration include needle (including microneedle) syringes, needle-free syringes and infusion techniques, as well as any other parenteral administration methods recognized in the art. Parenteral formulations are typically aqueous solutions that may contain excipients such as salts, carbohydrates and buffers (preferably in the pH range of about 3 to about 9), but for some applications, they may be more suitable for formulation into sterile non-aqueous solutions or dry forms for use in combination with suitable vehicles (e.g., sterile, pyrogen-free water). Preparation of parenteral formulations under sterile conditions, such as by lyophilization, can be easily achieved using standard pharmaceutical techniques well known to those skilled in the art.

[0174] The pharmaceutical dosage form that is suitable for using can comprise sterile aqueous solution or dispersion or sterile powder, comprise the active component that is suitable for preparing sterile injectable or infusible solution or dispersion instantly, and described composition is optionally encapsulated in liposome, nanocrystal or polymer nanoparticle.In all cases, final dosage form should be sterile, mobile and stable under production and storage conditions.Liquid carrier or vehicle can be solvent or liquid dispersion medium, comprise for example but not limited to water, electrolyte, sugar, ethanol, polyol (for example glycerine, propylene glycol, liquid polyethylene glycol etc.), vegetable oil, nontoxic glyceride and / or its suitable mixture.In at least one embodiment, can by forming liposome, keep required particle diameter or use surfactant to keep required fluidity under the situation of dispersion.

[0175] Sterile injectable solutions can be prepared by combining the pharmaceutical composition with one or more of the above-mentioned other ingredients, as required, in an appropriate solvent in the required amount, followed by filtered sterilization. In the case of sterile powders for preparing sterile injectable solutions, vacuum drying and freeze drying techniques can be used, which can produce a powder of the active ingredient and any other desired ingredients present in the previously sterile filtered solution.

[0176] method

[0177] In addition, in view of the above, a method for treating viral infection in a subject is also provided. The method may include administering an effective amount of the above conjugate, prodrug or composition and a pharmaceutically acceptable excipient orally (e.g., about 0.01 μm / kg to about 10 μm / kg of the conjugate) or intravenously to the subject. In certain embodiments, "effective amount" refers to an amount that effectively treats viral infection (e.g., influenza). The method may also include administering autologous antibodies or allogeneic immunoglobulin G (IgG) antibodies.

[0178] The viral infection may be influenza. The viral infection may be influenza A. The viral infection may be influenza B. The method may induce an immune response to remove antibody (Ab)-coated viruses or antibody-coated infected cells by antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0179] In view of the above, a method for treating cancer in a subject is provided. The method comprises administering to the subject an effective amount of the conjugate, prodrug or composition and a pharmaceutically acceptable excipient. The method may also comprise administering an autologous antibody, an allogeneic IgG antibody or IVIG.

[0180] The cancer can be lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, epithelial cancer, leiomyosarcoma, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, carcinoma of the endometrium, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's Disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, gastric cancer, Cancer is selected from the group consisting of: primary CNS lymphoma, primary CNS lymphoma, glioma of the spinal axis, glioma of the brain stem, pituitary adenoma, cholangiocarcinoma, Hurthle cell thyroid cancer, and gastroesophageal junction adenocarcinoma. The cancer is lung cancer, triple-negative breast cancer, colon cancer, gastric cancer, bladder cancer, prostate cancer, or pancreatic cancer.

[0181] In certain embodiments, the cancer is a hot cancer. Cancers that are not responsive to treatment with checkpoint inhibitors and that typically have low immune infiltration are often referred to as "cold" cancers. In contrast, cancers or tumors that respond to treatment are often referred to as "hot" cancers, typically have receptors on the surface of cancer cells, and may elicit a strong immune response in the subject. The hot cancer may be kidney cancer. The hot cancer may be lung cancer. The hot cancer may be colorectal cancer.

[0182] Combination therapy is also provided. In certain embodiments of the method for treating a viral infection or cancer, the method further comprises administering a second therapy to the subject. In the case of a method for treating cancer, the second therapy may comprise administering chemotherapy (e.g., an effective amount of chemotherapy). The second therapy may comprise administering (e.g., an effective amount of) sunitinib. The second therapy may comprise administering (e.g., an effective amount of) a PD-1 inhibitor. The second therapy may comprise administering (e.g., an effective amount of) a PDL-1 inhibitor.

[0183] The second therapy may comprise administering (e.g., an effective amount) a folate toll-like receptor 7 (FA-TLR7) agonist conjugate. Since FA-TLR7 agonists can reprogram activated macrophages in the tumor microenvironment (TME) by converting pro-tumor macrophages into anti-tumor macrophages, this may act synergistically with compound 150 to further reduce tumor growth.

[0184] In certain embodiments, the second therapy comprises one or more of chemotherapy, radiotherapy, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor. In certain embodiments, the method further comprises imaging the subject (e.g., using a known imaging modality) to capture an image of the cancer (e.g., a cancerous tumor).

[0185] A method for treating fibrosis in a subject is also provided. The method comprises administering to the subject an effective amount of the above conjugate, prodrug or composition and a pharmaceutically acceptable excipient. The method may also comprise administering an autologous antibody, an allogeneic IgG antibody or IVIG.

[0186] Also provided is a method for activating an immune response of a subject (e.g., a target region of a subject, such as TME). In certain embodiments, the method for activating an immune response of a subject comprises administering an effective amount of the above-mentioned conjugate, prodrug or composition and a pharmaceutically acceptable excipient to the subject. The immune response may be an innate immune response. The immune response may be activated in the target region of the subject (e.g., the location of the TME or viral replication site (e.g., near one or more virally infected cells)). In the case of administering a prodrug, this prodrug may further slow down the hydrolysis of the basic conjugate and / or slow down the activation of cytokines in the target region of the subject.

[0187] The method of activating an immune response may further comprise administering to the subject autologous antibodies, allogeneic IgG antibodies, or human IVIG.

[0188] Administration of an effective amount of the conjugate or composition can induce M2 macrophages to be reprogrammed to M1 macrophages in the targeted area. Generally, without any expected limitations, the novel conjugates, compositions and methods herein can target the subject's innate immune system and reprogram the polarization of macrophages from M2 to M1 to favor the pro-inflammatory properties of the M1 phenotype. For example, in at least one exemplary embodiment, such conjugates and compositions include a targeting portion targeting a folate receptor (e.g., FRβ), such as a folate receptor binding ligand coupled to two or more haptens via a linker, or an analog, functional fragment, derivative or free radical thereof (e.g., pteroyl amino acid). Such an embodiment utilizes the limited expression of the folate receptor to directly localize the systemically administered conjugate and composition to folate-expressing cells (e.g., cells of cancer tissue), so that the hapten component can then bind to the desired antibody, recruit immune cells to the target site, and reprogram activated myeloid cells (e.g., M2-like macrophages) to pro-inflammatory M1 polarization. This targeted design advantageously prevents systemic activation of the immune system (i.e., reduces systemic exposure to such conjugates / compositions), thereby avoiding toxicity, while promoting activation of the subject's own immune system at or near the target site. As described above, in certain embodiments, the conjugates or compositions described herein may be administered together with a second therapy comprising administering an effective amount of a FA-TLR7 agonist, which may synergize with the conjugates / compositions described herein by further promoting the conversion of activated macrophages into anti-tumor (M1-like) macrophages.

[0189] The terms "treat," "treating," "treated," or "treatment" (with respect to a disease or condition) are an approach to obtaining beneficial or desired results, including and preferably clinical results, and include, but are not limited to, one or more of the following: improving symptoms associated with the disease, curing the disease, lessening the severity of the disease, slowing the progression of the disease, alleviating one or more symptoms associated with the disease, improving the quality of life of the patient, prolonging survival, and / or providing prophylactic or preventative treatment.

[0190] "Effective amount" and "therapeutically effective amount" refer to any amount sufficient to achieve the desired biological effect. In conjunction with the teachings provided herein, by selecting among various active conjugates and weighing factors (e.g., efficacy, relative bioavailability, patient weight, severity of adverse side effects, and mode of administration), effective preventive or therapeutic regimens can be planned that do not cause serious unnecessary toxicity but are effective in treating specific subjects. The effective amount for any particular application can vary according to factors such as the infection, cancer, or other condition being treated, the specific conjugate or composition being administered, the treatment being performed simultaneously or continuously, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound and / or other therapeutic agent without the need for excessive experimentation. A maximum dose, i.e., the highest safe dose according to some medical judgment, can be used. Multiple doses per day can be used to achieve appropriate systemic levels of the compound. Appropriate systemic levels can be determined, for example, by measuring the peak or sustained levels of the drug in the patient's plasma. "Dose" and "dosage" are used interchangeably herein.

[0191] Typically, for human subjects, the daily oral dose of the compound is about 0.01 mg / kg per day to 1,000 mg / kg per day. Oral doses range from 0.5 to 50 mg / kg, administered once or more per day, to produce a therapeutic effect. Depending on the mode of administration, the dose can be appropriately adjusted to achieve the desired local or systemic drug level. For example, the dose for intravenous administration can vary from one order of magnitude lower to several orders of magnitude lower per day. If the subject's response at this dose is insufficient, a higher dose (or an effective higher dose using a different, more localized delivery route) can be used, within the limits permitted by the patient's tolerance. In order to achieve appropriate systemic levels of the conjugate, multiple doses per day, such as twice a day, are considered.

[0192] An "effective amount" (or "therapeutically effective amount") of a conjugate (used interchangeably herein with a "compound") for use in therapy refers to that amount of the compound in a formulation which, when administered as part of a desired dosage regimen (to a mammal, e.g., a human), alleviates symptoms, ameliorates the condition, or slows the onset of a disease condition, according to clinically acceptable standards for the disorder or condition being treated, or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0193] For any conjugate, the therapeutically effective amount can be initially determined from an animal model. For compounds that have been tested on humans and compounds known to exhibit similar pharmacological activity (e.g., other related active agents), therapeutically effective doses can also be determined from human data. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. It is entirely within the capabilities of those skilled in the art to adjust the dose to achieve the highest efficacy based on the above methods and other methods known in the art.

[0194] For clinical use, the amount of any compound administered may be equal to or equivalent to 0.2-2,000 milligrams (mg) of compound per kilogram (kg) of body weight per day of the subject. The dose of the compound administered may be equal to or equivalent to 2-2,000 mg of compound per kg of body weight per day of the subject. The dose of the compound administered may be equal to or equivalent to 20-2,000 mg of compound per kg of body weight per day of the subject. The dose of the compound administered may be equal to or equivalent to 50-2,000 mg of compound per kg of body weight per day of the subject. The dose of the compound administered may be equal to or equivalent to 100-2,000 mg of compound per kg of body weight per day of the subject. The dose of the compound administered may be equal to or equivalent to 200-2,000 mg of compound per kg of body weight per day of the subject. If a precursor or prodrug of a compound is to be administered, its administration amount is equivalent to, i.e., sufficient to deliver the above amount of the compound.

[0195] The formulation of the compound can be administered to a human subject in a therapeutically effective amount. Typical dosage ranges are from about 0.01 micrograms / kg to about 2 mg / kg body weight per day. The drug dosage to be administered is likely to depend on a variety of variables, such as the type and extent of the condition, the overall health of the particular subject, the particular compound administered, the excipient used to prepare the compound, and its route of administration. Routine experimentation can be used to optimize the dosage and dosing frequency of any particular compound.

[0196] The conjugate can be administered at a concentration ranging from about 0.001 microgram / kg to greater than about 500 mg / kg. For example, the concentration can be 0.001 microgram / kg, 0.01 microgram / kg, 0.05 microgram / kg, 0.1 microgram / kg, 0.5 microgram / kg, 1.0 microgram / kg, 10.0 microgram / kg, 50.0 microgram / kg, 100.0 microgram / kg, 500 microgram / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, 30.0 mg / kg, 35.0 mg / kg In some embodiments, the present invention relates to an amount of at least about 1 mg / kg of an aqueous solution of at least about 500 mg / kg, or any incremental value thereof. The amount of at least about 1 mg / kg of an aqueous solution of at least about 500 mg / kg, or any incremental value thereof, may be used in an aqueous solution of at least about 500 mg / kg, or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0197] The dosage range for administration of the conjugate can be about 0.2 mg / kg / day to greater than about 100 mg / kg / day. For example, the dosage can be 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 2 ... day to 10mg / kg / day, 0.25mg / kg / day to 7.5mg / kg / day, 0.25mg / kg / day to 5mg / kg / day, 0.5mg / kg / day to 50mg / kg / day, 0.5mg / kg / day to 25mg / kg / day, 0.5mg / kg / day to 20mg / kg / day, 0.5mg / kg / day to 15mg / kg / day, 0.5mg / kg / day to 10mg / kg / day, 0.5mg / kg / day to 7.5mg / kg / day, 0.5mg / kg / day to 5mg / kg / day, 0.75mg / kg / day day to 50mg / kg / day, 0.75mg / kg / day to 25mg / kg / day, 0.75mg / kg / day to 20mg / kg / day, 0.75mg / kg / day to 15mg / kg / day, 0.75mg / kg / day to 10mg / kg / day, 0.75mg / kg / day to 7.5mg / kg / day, 0.75mg / kg / day to 5mg / kg / day, 1.0mg / kg / day to 50mg / kg / day, 1.0mg / kg / day to 25mg / kg / day, 1.0mg / kg / day to 20mg / kg / day, 1.0mg / kg / day to 15mg / kg / day, 1.0mg / kg / day to 10mg / kg / day, 1.0mg / kg / day to 7.5mg / kg / day, 1.0mg / kg / day to 5mg / kg / day, 2mg / kg / day to 50mg / kg / day, 2mg / kg / day to 25mg / kg / day, 2mg / kg / day to 20mg / kg / day, 2mg / kg / day to 15mg / kg / day, 2mg / kg / day to 10mg / kg / day, 2mg / kg / day to 7.5mg / kg / day, or 2mg / kg / day to 5mg / kg / day.

[0198] The conjugate may be administered at a dose ranging from about 0.25 mg / kg / day to about 25 mg / kg / day. For example, the dose may be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, / kg / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day, 33mg / kg / day, 34mg / kg / day, 35mg / kg / day, 36mg / kg / day, 37mg / kg / day, 38mg / kg / day, 39mg / kg / day, 40mg / kg / day, 41 mg / kg / day, 42mg / kg / day, 43mg / kg / day, 44mg / kg / day, 45mg / kg / day, 46mg / kg / day, 47mg / kg / day, 48mg / kg / day, 49mg / kg / day, or 50mg / kg / day.

[0199] The conjugate or its precursor can be administered at a concentration ranging from 0.01 micromolar to greater than or equal to 500 micromolar. For example, the dose can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40.0 micromolar, 45.0 micromolar, 46.0 micromolar, 47.0 micromolar, 48.0 micromolar, 49.0 micromolar, 50.0 micromolar, 51.0 micromolar, 52.0 micromolar, 53.0 micromolar, 54.0 micromolar, 56.0 micromolar, 57.0 micromolar, 58.0 micromolar, 59.0 micromolar, 60.0 micromolar, 61.0 micromolar, 62.0 micromolar, 63.0 micromolar, 64.0 micromolar, 65.0 micromolar, 66.0 micromolar, 67.0 micromolar, 68.0 micromolar, 69.0 micromolar, 70.0 micromolar, 71.0 micromolar, 72.0 micromolar, 73.0 micromolar, 74.0 micromolar, 75.0 micromolar, 76.0 micromolar, 77.0 micromolar, 78.0 micromolar, 79.0 micromo .0 micromolar, 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar, to greater than about 500.0 micromolar or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be covered.

[0200] The conjugate or precursor thereof may be administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, the concentration can be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 microgram / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, to greater than about 500.0 micrograms / mL or any incremental value thereof. It should be understood that all values ​​and ranges between these values ​​and ranges are intended to be encompassed.

[0201] The formulation can be applied in a pharmaceutically acceptable solution, which generally contains pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients. For therapy, an effective amount of the conjugate can be administered to a subject by any mode of delivering the conjugate to a desired surface. Administration of the pharmaceutical composition can be achieved by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., injection into a tumor or abscess), mucosal (e.g., topical application to the eye), inhalation, and topical application.

[0202] For oral administration, the conjugate can be easily formulated by combining the active conjugate with pharmaceutically acceptable excipients well known in the art. These excipients enable the conjugate to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated. Oral pharmaceutical preparations can be obtained as solid excipients, optionally grinding the resulting mixture, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or dragee cores. Suitable excipients include fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants such as cross-linked PVP, agar or alginic acid or a salt thereof, such as sodium alginate, can be added. Optionally, the oral formulation may also be formulated in saline or buffer (eg, EDTA) for neutralization of internal acidic conditions, or may be administered without any excipients.

[0203] Oral dosage forms of the conjugates are also contemplated. The conjugates may be chemically modified to render the derivatives effective for oral delivery. Typically, the chemical modification contemplated is the attachment of at least one moiety to the compound itself, wherein the moiety is capable of (a) inhibiting acid hydrolysis; and (b) being absorbed from the stomach or intestine into the bloodstream. It is also desirable that the overall stability of the compound be improved and that the in vivo circulation time be prolonged. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, PVP, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts," In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., Preparation and properties of adducts of streptokinase and streptokinase-plasmin complex with polyethylene glycol and pluronic polyol F38, J Appl Biochem 4: 185-189 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-trioxocane. As mentioned above, for pharmaceutical use, the polyethylene glycol portion is suitable.

[0204] The release site of the compound herein can be the stomach, small intestine (e.g., duodenum, jejunum, or ileum), or large intestine. Those skilled in the art can provide formulations that will not dissolve in the stomach but will release the substance elsewhere in the duodenum or intestinal tract. The release can avoid the harmful effects of the gastric environment by protecting the compound or releasing the compound into the extragastric environment (e.g., in the intestinal tract).

[0205] To ensure complete gastric tolerance, coatings that tolerate at least pH 5.0 are usually used. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragi S, and shellac. These coatings can be used as mixed films.

[0206] Coatings or coating mixtures can also be used for tablets that are not intended to protect from the stomach. This can include sugar coatings, or coatings that make the tablet easier to swallow. Capsules can consist of a hard shell (e.g., gelatin) for delivery of dry therapeutic drugs (e.g., powders); for liquid forms, a soft gelatin shell can be used. The shell material for flat capsules can be thick starch or other edible paper. For pills, lozenges, molded tablets, or tablet triturates, a moist massing technique can be used.

[0207] The conjugate may be included in the formulation as fine multiparticulates in the form of granules or pellets with a particle size of about 1 mm. Formulations of capsule administration materials may also be as powders, lightly compressed plugs or even tablets. The therapeutic agent may be prepared by compression.

[0208] Colorants and flavor enhancers may be included. For example, the compound may be formulated (eg, by liposome or microsphere encapsulation) and then contained in an edible product, such as a refrigerated beverage containing a colorant and a flavor enhancer.

[0209] The volume of the compound can be diluted or increased with an inert material. These diluents can include carbohydrates, particularly mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Certain inorganic salts can also be used as fillers, including tricalcium phosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell.

[0210] In the solid dosage form in which the therapeutic agent is formulated, a disintegrating agent may be included. Materials used as disintegrating agents include, but are not limited to, starch, including the commercial disintegrating agent Explotab based on starch. Sodium carboxymethyl starch, Amberlite, sodium carboxymethyl cellulose, super branched starch, sodium alginate, gelatin, orange peel, acidic carboxymethyl cellulose, natural sponge and bentonite may be used. Another form of the disintegrating agent is an insoluble cation exchange resin. Powdered gums may be used as disintegrating agents and binders, which may include powdered gums, such as agar, carrageenan or tragacanth. Alginic acid and its sodium salt may also be used as disintegrating agents.

[0211] Binders can be used to fix the compound together to form a hard tablet, including materials from natural products, such as gum arabic, gum tragacanth, starch and gelatin. Other binders include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Both PVP and HPMC can be used for granulated therapeutic agents in alcoholic solutions.

[0212] Friction reducers may be included in the formulation of the therapeutic agent to prevent sticking during formulation. Lubricants may be used as a layer between the therapeutic agent and the mold wall and may include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000.

[0213] Glidants can be added to improve the flow properties of the drug during formulation and to aid rearrangement during compression. The glidants may include starch, talc, pyrogenic silica, and hydrated aluminosilicates.

[0214] In order to help the therapeutic agent dissolve in the aqueous environment, a surfactant can be added as a wetting agent. The surfactant can include anionic detergents, such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and dioctyl sodium sulfonate. Useable cationic detergents include benzalkonium chloride and benzethonium chloride. Potential nonionic detergents that can be included in the formulation as a surfactant include lauromacrogol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. These surfactants can be present in the formulation of the compound or its derivatives alone or as a mixture of different proportions.

[0215] Orally available pharmaceutical preparations include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer (e.g., glycerol or sorbitol). The push-fit capsules may contain the active ingredient mixed with a filler (e.g., lactose), a binder (e.g., starch), and / or a lubricant (e.g., talc or magnesium stearate), and optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid (e.g., a fatty oil, liquid paraffin, or liquid polyethylene glycol). In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. The dosage of all orally administered formulations should be suitable for such administration.

[0216] For buccal administration, the composition may take the form of tablets or lozenges formulated in conventional manner.

[0217] For administration by inhalation, the compound may be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer, using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base (e.g., lactose or starch).

[0218] Pulmonary delivery of the compounds (or salts thereof) is also contemplated. The compounds are delivered to the lungs of mammals upon inhalation and cross the lung epithelial lining into the bloodstream. Other reports on inhaled molecules include Adjei & Garren, Pulmonary delivery of peptide drugs: Effect of particle size on bioavailability of leuprolide acetate in healthy male volunteers, J Pharmaceutical Research 7:565-569 (1990); Adjei et al., Bioavailability of leuprolide following intratracheal administration to beagledogs, International J Pharmaceutics 63:135-144(1990)(leuprolide acetate); Braquet et al., Effect of endothelin-1 on blood pressure and bronchopulmonary system of the guinea pig, J Cardiovascular Pharmacology13(suppl.5):143-146(1989)(endothelin-1); Hubbard et al., Annals of Internal Medicine 3:206-212(1989)(a1-antitrypsin); Smith et al.,Pulmonary deposition and clearance of aerosolized alpha-1-proteinaseinhibitor administered to dogs and to sheep,J Clinical Investigation 84:1145-1146(1989)(a-1-proteinase); Oswein et al.,Aerosolization of Proteins, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, 1990 (recombinant hepatocyte growth hormone); Debs et al., Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats, J Immunology 140:3482-3488 (1988) (interferon-gamma and tumor necrosis factor alpha) and US Patent No. 5,284,656 (granulocyte colony stimulating factor; incorporated herein by reference). A method and composition for pulmonary delivery of drugs for systemic effects is described in U.S. Patent No. 5,451,569 (specifically incorporated herein by reference to disclose its relevant contents). .

[0219] Contemplated for use are a wide variety of mechanical devices designed for pulmonary delivery of therapeutic products including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0220] Nasal delivery of pharmaceutical compositions is also contemplated. Nasal delivery allows the pharmaceutical composition to enter the bloodstream directly after the therapeutic product is applied to the nose, without allowing the product to be deposited in the lungs. Formulations for nasal delivery include formulations containing dextran or cyclodextran.

[0221] When systemic delivery of the conjugate is desired, it can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injectable formulations can be in unit dosage form, such as ampoules or multi-dose containers, with the addition of preservatives. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulating agents, such as suspending agents, stabilizers, and / or dispersants.

[0222] Pharmaceutical formulations for parenteral administration include aqueous solutions of water-soluble active compounds. In addition, the suspension of the active compound can be prepared into suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil), or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or reagents to improve the solubility of the compound, thereby preparing a high concentration solution.

[0223] Alternatively, the active conjugate may be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) before use.

[0224] The conjugates may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0225] In addition to the above formulations, the conjugates can also be formulated as a depot preparation. Such depot preparations can be formulated with suitable polymers or hydrophobic materials (such as emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, such as sparingly soluble salts.

[0226] The pharmaceutical composition may also comprise a suitable solid or gel phase or excipient. Examples of such excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers (e.g., polyethylene glycol).

[0227] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous solutions or saline solutions for suction, microencapsulation, wrapping, coating on extremely small gold particles, contained in liposomes, atomization, aerosol, pellets implanted in the skin, or dried on sharp objects to scrape into the skin. The pharmaceutical composition also includes granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, ointments, drops or preparations of long-term release active compounds, in which excipients and additives and / or adjuvants are usually used as described above, such as disintegrants, adhesives, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers. The pharmaceutical composition is suitable for various drug delivery systems. For a brief review of drug delivery methods, see Langer, New methods of drug delivery, Science 249 (4976): 1527-1533 (1990).

[0228] The conjugate and optionally one or more other therapeutic agents can be administered as such (pure form) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. In addition, such salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts of carboxylic acid groups.

[0229] Suitable buffers include acetic acid and salts (1-2% w / v); citric acid and salts (1-3% w / v); boric acid and salts (0.5-2.5% w / v); and phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).

[0230] The pharmaceutical composition contains an effective amount of a compound as described herein and optionally one or more other therapeutic agents, which are included in a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates. The term "excipient" means a natural or synthetic organic or inorganic ingredient that is combined with the active ingredient to facilitate application. The components of the pharmaceutical composition can also be mixed with the compound, as well as with each other, in a manner that does not substantially affect the desired drug efficacy.

[0231] The therapeutic agent can be provided in the form of particles, specifically including but not limited to compounds. As used herein, "particles" refer to nanoparticles or microparticles (or in some cases larger particles) that can constitute in whole or in part the compounds described herein or other therapeutic agents. The particles can contain therapeutic agents in a core surrounded by a coating (including but not limited to an enteric coating). The therapeutic agent can also be dispersed throughout the particles. The therapeutic agent can also be adsorbed into the particles. The release kinetics of the particles can be of any order, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. In addition to the therapeutic agent, the particles can also include any of those materials commonly used in the pharmaceutical and medical fields, including but not limited to erodible, non-erodible, biodegradable or non-biodegradable materials or combinations thereof. The particles can be microcapsules containing the compound solution or semi-solid compound. The particles can be of almost any shape.

[0232] Both non-biodegradable and biodegradable polymer materials can be used to make particles for delivering therapeutic agents. Such polymers can be natural polymers or synthetic polymers. The selection of the polymer is based on the time period of desired release. Bioadhesive polymers of particular interest include bioerodible hydrogels, which are described in Sawhney et al., Bioerodible hydrogels based on photopolymerized poly(ethylene glycol)-co-poly(.alpha.-hydroxy acid)diacrylate macromers, Macromolecules 26(4):581-587 (1993), the teachings of which are specifically incorporated herein by reference. These polymers include polyhyaluronic acid, casein, gelatin, gelatin protein, polydehydratase, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0233] The conjugate may be contained in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation in which the mode and profile of drug release from the formulation is controlled. This refers to immediate and non-immediate release formulations, which include, but are not limited to, sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is traditionally used to refer to a drug formulation that gradually releases the drug over a long period of time and can keep the blood level of the drug approximately constant over a long period of time. The term "delayed release" traditionally refers to a drug formulation in which there is a time delay between the administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve the gradual release of the drug over a long period of time, and therefore may or may not be "sustained release".

[0234] The use of long-term sustained release implants is particularly suitable for treating chronic conditions. As used herein, "long-term" release refers to the implant being constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, up to 30-60 days. Long-term sustained release implants are well known to those of ordinary skill in the art, including some of the above-mentioned release systems.

[0235] Some definitions

[0236] As used herein, the term "about" can allow a value or range to vary to a certain degree, for example, within 10%, 5% or 1% of a stated value or a stated limit of a range.

[0237] Numerical values ​​expressed in range format should be interpreted in a flexible manner to include not only the numerical values ​​explicitly indicated as the limits of the range, but also all individual numerical values ​​or sub-ranges encompassed within the range, as if each numerical value and sub-range were explicitly stated. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual numerical values ​​(e.g., 1%, 2%, 3% and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. Unless otherwise specified, the expression "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise specified, the expression "about X, Y or Z" has the same meaning as "about X, about Y or about Z".

[0238] In this document, the terms "a", "an" or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise stated. In addition, it should be understood that the phraseology or terminology used herein, unless otherwise defined, is for the purpose of description only and not for the purpose of limitation.

[0239] An "alkyl group" is a saturated, partially saturated or unsaturated straight or branched chain acyclic hydrocarbon having 1 to 10 carbon atoms (C1-C 10 In some embodiments, the alkyl group has a monovalent alkyl group. Examples of monovalent alkyl groups include -CH3, -CH2CH3, etc. For example, a monovalent alkyl group may be present on a substituent in the linker L chain. In some embodiments, the alkyl group has a divalent alkyl group, such as in the linker L chain. Examples of divalent alkyl groups include, but are not limited to, -CH2-, -CH2CH2-, etc. In some embodiments, the alkyl group is a saturated alkyl group. In some embodiments, the alkyl group is an unsaturated alkyl group, also referred to as an alkenyl group or an alkynyl group.

[0240] Unless otherwise stated, the term "heteroalkyl" by itself or in combination with another term means a stable straight or branched chain, or combination of both, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom O, N, P, S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3.

[0241] Any use of section headings and subheadings is intended solely for ease of reference and is not intended to limit any disclosure in a section to that section; rather, any disclosure under one section heading or subheading is intended to constitute disclosure under every other section heading or subheading.

[0242] Without departing from the scope and spirit of the technology, it will be clear to those skilled in the art that various modifications and variations of the compositions, methods and uses of the technology. Although the technology has been described in conjunction with specific exemplary embodiments, the invention claimed should not be overly limited to these specific embodiments. In fact, various modifications to the modes of implementing the invention that are obvious to those skilled in the art are intended to fall within the scope of the appended claims.

[0243] The terms and expressions used are intended to be descriptive terms only and not limiting terms. In this regard, where certain terms are defined and described or discussed elsewhere, the definitions and all descriptions and discussions are intended to be attributed to these terms. Nor is the use of such terms and expressions intended to exclude any equivalent features or portions of the features shown and described or portions thereof.

[0244] In addition, all publications and patents mentioned herein are incorporated by reference in their entirety for all purposes. In the event of an inconsistency between the usages of this document and a document incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls. Implementation

[0245] The following embodiments, presented in clauses, are not meant to be limiting but rather to further describe the invention of the present disclosure.

[0246] Item A. A conjugate having the formula: TL-LH n or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell, or fibroblast; L is the connector; H is a hapten; and n is an integer of 2-3; and optionally, wherein at least two of said H can each bind to a different antibody when contacted therewith.

[0247] Clause B. The conjugate according to clause A, wherein at least two of said H are each bound by an antibody.

[0248] Clause C. The conjugate according to clause A or B, wherein each H is bound by a different antibody.

[0249] Clause D. The conjugate according to any one of clauses AC, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

[0250] Clause E. The conjugate according to clause A, wherein n is 2.

[0251] Clause F. The conjugate according to clause A, wherein n is 3.

[0252] Clause G. A conjugate according to any one of clauses AC, E or F, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen, wherein the virus is selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus.

[0253] Clause H. The conjugate of any one of clauses AC, wherein n is 2, the first H is a DNP fragment, and the second H is a rhamnose fragment.

[0254] Clause I. The conjugate according to clause A, wherein at least one H is an influenza virus antigen selected from hemagglutinin and neuraminidase.

[0255] Clause J. The conjugate according to clause A, wherein at least one H is a hepatitis virus antigen selected from L-HBsAg, S-HBsAg, M-HBsAg and preS.

[0256] Clause K. The conjugate according to clause A, wherein at least one H is gp120 or gp160.

[0257] Clause L. The conjugate according to clause A, wherein at least one H is a glycoprotein.

[0258] Clause M. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell.

[0259] Clause N. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is influenza virus neuraminidase or influenza virus hemagglutinin.

[0260] Clause O. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is respiratory syncytial virus fusion protein F.

[0261] Clause P. A conjugate according to clause AC, E, F or IL, wherein the target protein is a coronavirus spike protein.

[0262] Clause Q. The conjugate of clause AC, E, F or IL, wherein the target protein is hepatitis B virus surface antigen or HBV core antigen.

[0263] Clause R. The conjugate of clause AC, E, F or IL, wherein the target protein is a cell surface receptor on a cancer cell.

[0264] Clause S. The conjugate according to clause A, wherein the target protein is a folate receptor.

[0265] Clause T. The conjugate according to clause S, wherein the target protein is folate receptor alpha or folate receptor beta.

[0266] Clause U. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is prostate specific membrane antigen.

[0267] Clause V. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is carbonic anhydrase 9.

[0268] Clause W. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is the luteinizing hormone releasing hormone receptor.

[0269] Clause X. The conjugate according to any one of clauses AC, E, F or IL, wherein the target protein is a neurokinin 1 receptor.

[0270] Clause Y. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

[0271] Clause Z. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is a cell surface receptor on myeloid-derived suppressor cells.

[0272] Clause AA. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

[0273] Clause BB. The conjugate of any one of clauses AC, E, F or IL, wherein the target protein is fibroblast activation protein.

[0274] Clause CC. A conjugate according to any one of clauses AC, E, F or IL, wherein the targeting ligand is a neuraminidase inhibitor.

[0275] Clause DD. The conjugate of any one of clauses AC, E, F or IL, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment or a laninamivir fragment.

[0276] Clause EE. The conjugate of clause A, wherein the targeting ligand is a zanamivir fragment.

[0277] Clause FF. A conjugate according to any one of clauses AC, E, F, IL, S or T, wherein the targeting ligand is a folate fragment or an analog thereof.

[0278] Clause GG. A conjugate according to any one of clauses AC, E, F, IL, S or T, wherein the targeting ligand is 5-methyltetrahydrofolate.

[0279] Clause HH. A conjugate according to any one of clauses AC, E, F, IL, S, T or EE, wherein L comprises (-CH2CH2-O-) n , peptide, peptidoglycan or a combination of two or more of the above, wherein n is an integer between and including 1 and 32.

[0280] Clause II. A conjugate according to any one of clauses AC, E, F, IL, S, T or EE, wherein L is a branched linker and at least two of the haptens are attached to different branches of the linker, wherein the different branches optionally extend from different atoms of the linker.

[0281] Clause JJ. A conjugate according to clause A, wherein the targeting ligand is a folate fragment or a derivative thereof, at least the first H comprises a rhamnose fragment, and at least the second H comprises a dinitrophenyl fragment.

[0282] Clause KK. The conjugate of any one of clauses A-JJ, formulated as a prodrug.

[0283] Article LL. A conjugate having the formula or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell, or fibroblast; L a , L b and L c Each is a linker, which may be the same or different; C is a carbon atom; R4 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl or C1-C5 alkynyl groups; H1 and H2 are each haptens; and Optionally, H1 and H2 can each bind to a different antibody.

[0284] Clause MM. A conjugate having the formula and pharmaceutically acceptable salts thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell, or fibroblast; L a , L b , L c and L d Each is a linker, which may be the same or different; C is a carbon atom; H1, H2, and H3 are each haptens; and Optionally, each of H1, H2 and H3 can each bind a different antibody.

[0285] Clause NN. The conjugate according to clause MM, wherein H1 and H2 are each bound by an antibody.

[0286] Clause OO. The conjugate according to clause MM, wherein H1, H2 and H3 are each bound by an antibody.

[0287] Clause PP. The conjugate of clause LL or NN, wherein H1 and H2 are each independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

[0288] Clause QQ. The conjugate of clause MM or PP, wherein H1, H2 and H3 are each independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment or a combination thereof.

[0289] Clause RR. A conjugate according to clause LL or NN, wherein H1 or H2 is each independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen, wherein the virus is selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus, and measles virus.

[0290] Clause SS. A conjugate according to clause LL or OO, wherein H1, H2 or H3 is each independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen, wherein the virus is selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus.

[0291] Clause TT. A conjugate according to clause LL or OO, wherein H1 is a DNP fragment and H2 is a rhamnose fragment.

[0292] Clause UU. The conjugate of clause LL or MM, wherein at least one hapten is an influenza virus antigen selected from hemagglutinin and neuraminidase.

[0293] Clause VV. The conjugate of clause LL or MM, wherein at least one hapten is a hepatitis virus antigen selected from L-HBsAg, S-HBsAg, M-HBsAg and preS.

[0294] Clause WW. The conjugate of clause LL or MM, wherein at least one hapten is gp120 or gp160.

[0295] Clause XX. The conjugate according to clause LL or MM, wherein at least one hapten is a glycoprotein.

[0296] Clause YY. The conjugate of any one of clauses LL-OO, wherein the target protein is an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell.

[0297] Clause ZZ. The conjugate of any one of clauses LL-OO, wherein the target protein is influenza virus neuraminidase or influenza virus hemagglutinin.

[0298] Clause AAA. A conjugate according to any one of clauses LL-OO, wherein the target protein is respiratory syncytial virus fusion protein F.

[0299] Clause BBB. A conjugate according to any of clauses LL-OO, wherein the target protein is a coronavirus spike protein.

[0300] Clause CCC. A conjugate according to any of clauses LL-OO, wherein the target protein is hepatitis B virus surface antigen or HBV core antigen.

[0301] Clause DDD. The conjugate of any one of clauses LL-OO, wherein the target protein is a cell surface receptor on a cancer cell.

[0302] Clause EEE. A conjugate according to any one of clauses LL-OO, wherein the target protein is a folate receptor.

[0303] Clause FFF. A conjugate according to any one of clauses LL-OO, wherein the target protein is folate receptor alpha or folate receptor beta.

[0304] Clause GGG. A conjugate according to any one of clauses LL-OO, wherein the target protein is prostate specific membrane antigen.

[0305] Clause HHH. A conjugate according to any one of clauses LL-OO, wherein the target protein is carbonic anhydrase 9.

[0306] Clause III. The conjugate according to any one of clauses LL-OO, wherein the target protein is the luteinizing hormone releasing hormone receptor.

[0307] Clause JJJ. A conjugate according to any one of clauses LL-OO, wherein the target protein is a neurokinin 1 receptor.

[0308] Clause KKK. A conjugate according to any one of clauses LL-OO, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

[0309] Clause LLL. The conjugate of any one of clauses LL-OO, wherein the target protein is a cell surface receptor on myeloid-derived suppressor cells.

[0310] Clause MMM. The conjugate of any of clauses LL-OO, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

[0311] Clause NNN. The conjugate of any one of clauses LL-OO, wherein the target protein is fibroblast activation protein.

[0312] Clause OOO. A conjugate according to any one of clauses LL-OO, wherein the targeting ligand is a neuraminidase inhibitor.

[0313] Clause PPP. A conjugate according to any of clauses LL-OO, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment or a laninamivir fragment.

[0314] Clause QQQ. A conjugate according to any of clauses LL-OO, wherein the targeting ligand is a zanamivir fragment.

[0315] Clause RRR. A conjugate according to any of clauses LL-OO, wherein the targeting ligand is a folate fragment or an analog thereof.

[0316] Clause SSS. A conjugate according to any of clauses LL-OO, wherein the targeting ligand is 5-methyltetrahydrofolate.

[0317] Clause TTT. A conjugate according to clause MM, wherein L a , L b , L c and L d At least one of them independently comprises: (-CH2CH2-O-) n , an alkyl group, a peptide, a peptidoglycan, or a combination of two or more of the foregoing, wherein n is an integer between and including 1 and 32.

[0318] Clause UUU. A conjugate according to clause NN, wherein L a , L b and Lc At least one of them independently comprises: (-CH2CH2-O-) n , an alkyl group, a peptide, a peptidoglycan, or a combination of two or more of the foregoing, wherein n is an integer between and including 1 and 32.

[0319] Clause VVV. A conjugate according to clause TTT or UUU, wherein n is an integer between and including 1 and 16.

[0320] Clause WWW. A conjugate according to clause MM or OO, wherein L a , L b and L c and L d At least one of the peptide fragments or peptidoglycan fragments.

[0321] Clause XXX. A conjugate according to clause MM or NN, wherein L a , L b and L c At least one of the peptide fragments or peptidoglycan fragments.

[0322] Clause ZZZ. A conjugate according to clause MM or OO, wherein L a , L b and L c , and L d Each independently contains C2-C 18 Alkyl group.

[0323] Clause AAAA. A conjugate according to clause LL or NN, wherein L a , L b and L c Each independently contains C2-C 18 Alkyl group.

[0324] Clause BBBB. A conjugate of the formula: or a pharmaceutically acceptable salt thereof.

[0325] Clause CCCC. A conjugate of the formula: or a pharmaceutically acceptable salt thereof.

[0326] Clause DDDD. The conjugate of clause AAAA or BBBB, wherein one or more of the -OH groups are independently substituted with thiol, phosphate or phosphonate.

[0327] Clause EEEE. The conjugate according to clause AAAA or BBBB, wherein one or more of the -OH groups are substituted with -OC(=O)R, wherein R is an alkyl group.

[0328] Clause FFFF. A conjugate according to clause AAAA or BBBB, wherein one or more of the -OH groups are substituted with -OC(=O)R, wherein R is a C1-C6 alkyl group.

[0329] Clause GGGG. A conjugate according to clause AAAA or BBBB, wherein the amino (-NH2) group is substituted with -OC(=O)R2, R2 is an alkyl group.

[0330] Clause HHHH. A conjugate according to clause AAAA or BBBB, wherein the amino (-NH2) group is substituted with -OC(=O)R2, R2 is a C1-C6 alkyl group.

[0331] Clause IIII. The conjugate of clause AAAA or BBBB, wherein the carboxyl (-COOH) group is substituted with -OC(=O)R3, wherein R3 is an alkyl group.

[0332] Clause JJJJ. A conjugate according to clause AAAA or BBBB, wherein the carboxyl (-COOH) group is substituted with -OC(=O)R3, wherein R3 is a C1-C6 alkyl group.

[0333] Clause KKKK. A conjugate of the formula: or a pharmaceutically acceptable salt thereof, wherein L1, L2 and L3 are linkers.

[0334] Clause LLLL. A conjugate according to clause JJJJ, wherein one or more of L1, L2 and L3 comprises (-CH2CH2-O-) n , where n is an integer between 1 and 16, inclusive.

[0335] Clause MMMM. A conjugate according to clause JJJJ or KKKK, wherein L1, L2 and L3 each independently comprise C2-C 18 Alkyl groups, peptide fragments or peptidoglycan fragments.

[0336] Clause MMMM. A conjugate of the formula: or a pharmaceutically acceptable salt thereof.

[0337] Clause NNNN. The conjugate of any one of clauses AAAA-MMMM, further complexed with one or more antibodies in vivo.

[0338] Clause OOOO. A pharmaceutical composition comprising the conjugate of any one of clauses A-MMMM and a pharmaceutically acceptable excipient.

[0339] Clause PPPP. A method of treating a viral infection in a subject, comprising administering to the subject an effective amount of the conjugate of any one of clauses A-MMMM or the pharmaceutical composition of clause OOOO.

[0340] Clause QQQQ. The method according to clause PPPP, further comprising administering autologous antibodies or allogeneic immunoglobulin G (IgG) antibodies to the subject.

[0341] Clause RRRR. The method of clause PPPP, wherein the viral infection is influenza.

[0342] Clause SSSS. The method according to any one of clauses PPPP-RRRR, wherein the conjugate or the pharmaceutical composition is administered orally.

[0343] Clause TTTT. The method according to clause PPPP, wherein the conjugate or the pharmaceutical composition is administered once a day.

[0344] Clause UUUU. The method according to clause PPPP, wherein the conjugate or the pharmaceutical composition is administered more than once a day.

[0345] Clause VVVV. The method according to clause PPPP, wherein the conjugate or the pharmaceutical composition is administered twice a day.

[0346] Clause WWWW. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the conjugate of any one of clauses A-MMMM or the pharmaceutical composition of clause OOOO.

[0347] Clause XXXX. The method according to clause WWWW, further comprising administering to the subject an autologous antibody or an allogeneic IgG antibody.

[0348] Clause YYYY. The method of clause WWWW, wherein the cancer is a thermal cancer.

[0349] Clause ZZZZ. The method of clause WWWW, wherein the cancer is renal cancer, lung cancer, or colorectal cancer.

[0350] Clause AAAAA. The method according to any one of clauses PPPP-ZZZZ, wherein the conjugate or the pharmaceutical composition is administered orally or intravenously.

[0351] Clause BBBBB. The method according to any one of clauses PPPP-ZZZZ, wherein the conjugate or the pharmaceutical composition is administered once a day.

[0352] Clause CCCCC. The method of any one of clauses PPPP-ZZZZ, further comprising administering to the subject a second therapy, wherein the second therapy comprises chemotherapy, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor.

[0353] Clause DDDDD. A method of activating an immune response in a subject, comprising administering to the subject an effective amount of the conjugate of any one of clauses A-MMMM or the pharmaceutical composition of clause OOOO.

[0354] Clause EEEEE. The method of clause DDDDD, wherein the immune response is an innate immune response.

[0355] Clause FFFFF. The method of clause DDDDD, wherein the immune response is activated in a targeted region of the subject, wherein the targeted region is the location of a tumor microenvironment or a viral replication site.

[0356] Clause GGGGG. The method of any one of clauses DDDDD-FFFFF, further comprising administering to the subject an autologous antibody or an allogeneic IgG antibody.

[0357] Clause HHHHH. The method according to clause FFFFF, wherein administration of an effective amount of the conjugate or the pharmaceutical composition induces reprogramming of M2 macrophages to M1 macrophages in the targeted area.

[0358] Item IIIIII. The conjugate according to Item A, wherein at least two of the Hs can each bind to different antibodies when contacted with the antibody in vivo.

[0359] Clause JJJJJ. The conjugate according to clause A, wherein the two Hs can each bind to different antibodies when contacted with the antibody in vitro. Abbreviations Example

[0360] The following examples are used to illustrate the present disclosure. These examples are not intended to limit the scope of the claims in any way. Example 1 Synthesis of Zan-PEG6-DNP-rhamnose (zan double hapten) drug conjugate (compound 24) Solution 1 Synthesis scheme of compound 9:

[0361] Reagents and conditions:

[0362] a) PPh3, THF and H2O; b) N,N'-di-Boc-1H-pyrazole-1-carboximidamide, tetrahydrofuran (THF) and triethylamine (TEA); c) NaOMe, MeOH; d) 2,2-dimethoxypropane, anhydrous acetone, p-TsOH; e) 4-nitrophenyl chloroformate, 4-dimethylaminopyridine (DMAP) and pyridine at room temperature; f) N3-PEG6-NH2, N,N-diisopropylethylamine (DIPEA) and THF; g) 1M NaOH (aqueous solution), THF; h) trifluoroacetic acid (TFA) at room temperature. Synthesis scheme of compound 12: Synthesis scheme of compound 16: Synthesis scheme of zan-DNP-rhamnose (Compound 24): Wherein Ac is acetate.

[0363] Synthesis of compound 2: According to Chandler et al., Synthesis of the potent influenzaneuraminidase inhibitor 4-guanidino Neu5Ac2en. 1:1173-1180(1995); Shidmoossavee et al., Chemical insight into the emergence of influenza virus strains that are resistant to Relenza, J Am Chem Soc 135:13254-13257(2013); and Ying&Gervay-Hague, One-Bead–One-Inhibitor–One-Substrate Screening of NeuraminidaseActivity,ChemBioChem 6:1857-1865 (2005), a zanamivir derivative 1 was prepared from sialic acid. Triphenylphosphine (3.67 g, 14 mmol, 1.27 equiv) was added to a solution of zanamivir intermediate (1) (5 g, 11 mmol) in THF (40 mL), and the resulting solution was stirred at room temperature for 12 hours. Subsequently, water (10 mL) was added, and the solution was stirred at room temperature for another 24 hours. The reaction solution was then concentrated, and the crude product was purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOAc / hexane) to give a yellow powder 2. The product 2 was isolated, 2.89 g, with a yield of 61%.

[0364] Synthesis of compound 3: Triethylamine (1.5 mL) was added to a solution of compound 2 (2.74 g, 6.37 mmol) and N,N'-di(tert-butoxycarbonyl)-1H-pyrazole-1-carboximidamide (2.57 g, 8.28 mmol, 1.30 equiv) dissolved in THF (20 mL). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOA / hexane) to give compound 3 (4.14 g, 97%) as a white solid.

[0365] Synthesis of compound 5: NaOMe (2.9 mL, 0.5 M, 1.414 mmol) was added to a stirred solution of compound 3 (4.225 g, 6.281 mmol) in anhydrous methanol (70 mL). The reaction mixture was then stirred for 1 hour. Dowex 50XW8 (H + ) resin to neutralize the reaction mixture, filter and concentrate to give the lead compound 4, which was used in the next step without further purification.

[0366] 2,2-dimethoxypropane (7.7 mL, 6.54 g, 62.81 mmol, 10 equiv) was added to a solution of compound 4 in anhydrous acetone (70 mL), followed by p-toluenesulfonic acid (120 mg, 0.628 mmol, 0.1 equiv), and the resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated and purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOAc / hexane) to give compound 5 as a white solid, isolating 2.4 g of the product with a yield of 65%.

[0367] Synthesis of compound 6: 4-dimethylaminopyridine (2.13 g, 17.43 mmol) and 4-nitrophenyl chloroformate (3.51 g, 17.43 mmol) were added to a solution of compound 5 (1.46 g, 2.49 mmol) in pyridine (30 mL). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on Teledyne CombiFlash Rf + Lumen (silica gel column, 0-100% EtOAc / hexane) to give compound 6 (1.62 g, 87%) as a white solid.

[0368] Synthesis of compound 7: N3-PEG6-NH2 (0.05 g, 0.14 mmol, 1.05 eq.) was added to a solution of activated zanamivir-compound 6 (0.1 g, 0.13 mmol) in THF (2.7 mL) at room temperature under argon, followed by DIPEA (0.12 mL, 0.67 mmol, 5.0 eq.) and stirred overnight. The reaction progress was monitored by thin layer chromatography (TLC) and LC / MS.

[0369] After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-10% MeOH / DCM) to give a sticky solid compound 7 (0.12 g, 93%) (see Figure 2A ).

[0370] Synthesis of compound 9: Compound 7 (75 mg, 0.078 mmol) was dissolved in THF:MeOH (6:1, 1.5 mL) and treated dropwise with 1 M NaOH (100 μl). The reaction mixture was stirred at room temperature for 1 hour, at which time LC-MS analysis showed that the deprotection of the methyl ester was complete. 50WX8(H + ) resin and the reaction mixture was neutralized, filtered and concentrated under reduced pressure. The crude intermediate product 8 was used directly in the next step without further purification. (See Figure 2B ).

[0371] TFA (0.5 mL) was added to the intermediate crude compound 8. The reaction mixture was stirred at room temperature for 1 hour, and LC-MS indicated that the reaction was complete. TFA was removed by rotary evaporation under reduced pressure, and the crude product was placed under vacuum to obtain compound 9. The total yield of the two steps was 91%. (See Figure 2C ).

[0372] Synthesis of DNP-PEG1-CO2H (Compound 12): TEA (1.38 mL, 9.87 mmol) was added to a solution of 1-chloro-2,4-dinitrobenzene, Compound 10 (0.5 g, 2.47 mmol) and 3-(2-aminoethoxy)propionic acid, Compound 11 (0.33 g, 2.47 mmol) dissolved in ethanol (25 mL). The reaction mixture was heated to 55°C for 16 hours, and the progress of the reaction was monitored by LC-MS. After the reaction was complete, which was confirmed by the disappearance of one of the starting materials (i.e., dinitrobenzene), the reaction mixture was cooled and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-20% methanol / DCM) to give a yellow solid Compound 12 in 90% yield). LC-MS [M+H]+ = 299.98. (See Figure 2D ).

[0373] Synthesis of compound 14: α-L-rhamnose monohydrate (compound 13, 1.0 g, 5.49 mmol) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath and purged with nitrogen before the addition of acetic acid dehydratase (4.15 mL, 43.92 mmol, 8.0 equivalents). The reaction mixture was slowly warmed to room temperature and monitored by TLC (hexane / EtOAc, 65:35) and LC-MS, indicating that α-L-rhamnose monohydrate was completely consumed after 20 hours of reaction under an inert atmosphere. The reaction mixture was poured into ethyl acetate and extracted twice with 1.0 M HCl. The organic layer was washed with saturated sodium carbonate solution, water and brine, and dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting crude oil product (compound 14) (yield, 98%) was used for the next step.

[0374] Synthesis of compound 16: Under an inert atmosphere, 1,2,3,4-tetra-O-acetyl-α-L-rhamnose (compound 14, 0.50 g, 1.50 mmol) was dissolved in DCM (7.5 mL), and amino-PEG4-OH (compound 15, 0.35 g, 1.81 mmol, 1.2 equivalents) was added. The reaction flask was placed in an ice bath, and boron trifluoride ether (0.56 mL, 4.51 mmol, 3.0 equivalents) was added dropwise over 30 minutes. The reaction mixture was stirred in an ice bath for 2 hours, and then the reaction was allowed to reach room temperature. TLC (hexane / EtOAc, 30:70, Rf=0.30) and LC-MS monitoring showed that after 16 hours of reaction, 1,2,3,4-tetra-O-acetyl-α-L-rhamnose was completely consumed. The reaction mixture was poured into ice water and extracted with DCM. The combined organic layers were washed twice with saturated sodium bicarbonate solution, water and brine, and dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexane / EtOAc, 20:80) to isolate the product (Compound 16) in 90% yield.

[0375] Synthesis of compound 18: Under argon atmosphere, 1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate - HATU (0.11 g, 0.28 mmol, 0.85 equiv) was added to a solution of 3-(2-((2,4-dinitrophenyl)amino)ethoxy)propionic acid (compound 12, 0.1 g, 0.33 mmol) in dimethyl sulfoxide (2 mL), followed by DIPEA (0.29 mL, 1.67 mmol, 5.0 equiv) and stirred at room temperature for 10 minutes. Fmoc-Lys-OH.HCl (compound 17, 0.11 g, 0.27 mmol, 0.8 equiv) was added to the reaction mixture and stirred at room temperature for 2-3 hours. The progress of the reaction was monitored by LC-MS. After the reaction was confirmed to be complete by LC-MS, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexane / EtOAc, 20:80), and the product (Compound 18) was isolated with a yield of 70%. (See Figure 2E ).

[0376] Synthesis of compound 19: Benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, PyBOP (0.05 g, 0.10 mmol, 1.1 equiv) was added to a solution of acid (compound 18, 0.06 g, 0.09 mmol) and Rham(OAc)3-PEG4-NH2 (compound 16, 0.04 g, 0.09 mmol, 1.0 equiv) in dimethyl sulfoxide (1.5 mL) at room temperature under argon atmosphere, followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 equiv). The progress of the reaction was monitored by LC / MS.

[0377] After the reaction was completed by LC-MS confirmation, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layer was washed with salt water, dried over anhydrous sodium sulfate, and concentrated in vacuo. The obtained crude product was purified by silica gel column chromatography (0-10% MeOH / DCM), and the isolated product (compound 19) was in a yield of 93%.

[0378] -Deprotection of Fmoc group (Compound 20): DEA (100 uL) was added to a solution of compound 19 (0.01 g, 0.01 mmol) in anhydrous DCM (0.2 ml) at room temperature under argon. The solution was stirred at room temperature for 1 hour until the reaction was complete, as confirmed by LC-MS. DEA was removed by rotary evaporation under reduced pressure, and the crude product was precipitated in ether to give a yellow solid compound 20 in quantitative yield. LC-MS [M+H] + = 876.1.

[0379] Synthesis of compound 22: To a solution of DBCO-NHS (compound 21, 0.012 g, 0.029 mmol, 1.05 equiv) and compound 20 (0.024 g, 0.027 mmol, 1.0 equiv) dissolved in DCM was added N,N-DIPEA (0.048 μl, 0.27 mmol, 10 equiv) dropwise under argon. The reaction mixture was stirred at room temperature for 2-3 hours, and the progress of the reaction was monitored by LC-MS. After completion of the reaction, the solvent was evaporated under reduced pressure using a rotary evaporator, and the crude product was purified by silica gel column chromatography on Teledyne CombiFlash Rf+Lumen (0-10% methanol / DCM) to give a pale yellow solid compound 22 in 93% yield. LC-MS [M+H]+ = 1163.2. (See Figure 2F ).

[0380] Hydrolysis of OAc group (Compound 23): Compound 22 (0.025 g, 0.022 mmol) was dissolved in anhydrous MeOH (1 mL) and treated dropwise with 0.5 M NaOH (25 μl). The reaction mixture was stirred at room temperature for 1 hour, at which time LC-MS analysis showed product formation. After the reaction was complete, the product was purified by adding 50WX8(H + ) resin and the reaction mixture was neutralized, filtered and concentrated under reduced pressure. The pale yellow crude product (Compound 23) was used directly in the next step without further purification. LCMS [M+H] + = 1037.3. (See Figure 2G ).

[0381] Synthesis of zan-PEG6-DNP-rhamnose (Compound 24): DIPEA (6 μl, 0.03 mmol, 10 equiv) was added dropwise to a solution of zanamivir-PEG6-azide (Compound 9) (2.4 mg, 0.003 mmol) and Compound 23 (3.5 mg, 0.003 mmol, 1.0 equiv) in anhydrous DCM:DMSO (10:1, 0.2 mL) under argon. The reaction mixture was stirred at room temperature (RT) for 2 hours, and the progress of the reaction was monitored by LC-MS. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (HPLC) on a C18 column (5-95% acetonitrile / 20 mM NH4OAc aqueous solution, pH 7 buffer, 60 minutes, flow rate 7 mL / minute) to obtain Zan-PEG6-DNP-rhamnose (Compound 24, 50% yield) as a yellow powder. LC-MS [M+H] + = 1745.9. (See Figure 2H ). Example 2 Comparison of compound 24 with zan-Fc-WT and zan-Fc-DLE

[0382] On day 0 of the experiment, 100 LD 50Female BALB / c mice (n=5 / group) aged 6-8 weeks were infected with influenza A virus / Puerto Rico / 8 / 1934 (H1N1) (Catalog No. NR-348, BEI Resources, NIAID, NIH). At 24 hours post-infection (hpi), mice (except the group administered only phosphate-buffered saline (PBS)) were injected intraperitoneally with 8 g / kg human intravenous immunoglobulin (IVIg) to achieve humanized titers of anti-DNP and anti-rhamnose antibodies when the test article was administered. Mice were treated with the test article at 48 hpi. Zan-DNP-rhamnose was administered in a single intranasal dose of 1.5 μmol / kg. zan-Fc-WT and zan-Fc-DLE were administered in a single intravenous dose of 10 μg / mouse (synthesis details are as follows). Mice in the two control groups received PBS as a placebo.

[0383] To evaluate drug efficacy, mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund. Figure 3 and Figure 4 shown.

[0384] Figure 3 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of IVIg at 24 hpi ( -C) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) to which the conjugate was administered at 48 hpi.

[0385] Figure 4 Yes Figure 3 Days post infection versus body weight (%) of the treated mice. Solution 2 Synthesis of zan-Fc-WT and zan-Fc-DLE

[0386] Zanamivir-azide, i.e. compound 4", was prepared according to the previously reported literature methods (Chandler et al., Synthesis of the potent influenza neuraminidase inhibitor 4-guanidinoNeu5Ac2en. X-ray molecular structure of 5-acetamido-4-amino-2,6-anhydro-3,4,5-trieoxy-D-erythro-L-gluco-nononic acid, J Chemical Society, Perkin Transactions 1: 1173-1180 (1995); Shidmoossavee et al., Chemical insight into the emergence of influenza virus strains that are resistant to Relenza, J American Chemistry Society 135(36): 13254-13257 (2013); Ying, L. & Gervay-Hague, One-bead-one-inhibitor-one-substrate screening of neuraminidase activity, ChemBioChem 6(10):1857-1865(2005)) prepared from sialic acid.

[0387] Synthesis of compound 5″: To a solution of zanamivir-azide (compound 4″) (5 g, 11 mmol) in THF (40 mL) was added triphenylphosphine (3.67 g, 14 mmol, 1.27 equiv.), and the resulting solution was stirred at room temperature for 12 hours. Subsequently, water (10 mL) was added, and the solution was stirred for another 24 hours at room temperature. The reaction solution was then concentrated, and the crude product was purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOAc / hexane) to give yellow powder compound 5″. The isolated product (compound 5″), 2.89 g; yield 61.1%.

[0388] Synthesis of compound 6″: To a solution of compound 5″ (2.74 g, 6.37 mmol) and N,N′-di(tert-butoxycarbonyl)-1H-pyrazole-1-carboximidamide (2.57 g, 8.28 mmol, 1.30 equiv) dissolved in THF (20 mL) was added triethylamine (1.5 mL). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOAc / hexane) to give compound 7″ (4.14 g, 97%) as a white solid.

[0389] Synthesis of Compound 8″: NaOMe (2.9 mL, 0.5 M, 1.414 mmol) was added to a stirred solution of compound (Compound 6) (4.225 g, 6.281 mmol) in anhydrous methanol (70 mL). The reaction mixture was then stirred for 1 hour. Dowex 50XW8 (H + ) resin to neutralize the reaction mixture, and the mixture was filtered and concentrated to give the lead compound 7", which was used in the next step without further purification.

[0390] To a solution of compound 7″ in anhydrous acetone (70 mL) was added 2,2-dimethoxypropane (7.7 mL, 6.54 g, 62.81 mmol, 10 equiv) and then p-toluenesulfonic acid (120 mg, 0.628 mmol, 0.1 equiv). The resulting mixture was stirred at room temperature overnight. The reaction mixture was then concentrated and purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOAc / hexane) to give compound 8″ (2.4 g, 65.2%) as a white solid.

[0391] Synthesis of compound 9″: To a solution of compound 8″ (1.46 g, 2.49 mmol) in pyridine (30 mL) were added 4-dimethylaminopyridine (2.13 g, 17.43 mmol) and 4-nitrophenyl chloroformate (3.51 g, 17.43 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated and purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-100% EtOAc / hexane) to give activated zanamivir (compound 9″) (1.62 g, 87%) as a white solid.

[0392] Synthesis of compound 10″: To a solution of activated zanamivir (compound 9″) (0.2 g, 0.27 mmol) in THF (12.0 mL) was added tert-Boc-N-amide-PEG6-amine (0.124 g, 0.29 mmol, 1.1 equiv) and then DIPEA (0.23 mL, 1.33 mmol, 5.0 equiv) at room temperature under argon and stirred for 6-12 hours. The progress of the reaction was monitored by TLC and LC / MS. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel column, 0-20% MeOH / DCM) to give a sticky solid compound 10″ (234 mg, 85%).

[0393] Synthesis of Compound 12″: Compound 10″ (0.22 g, 0.21 mmol) was dissolved in THF (1.5 mL) and treated dropwise with 1 M NaOH (0.6 mL). The reaction mixture was stirred at room temperature for 1 hour, at which time LC-MS analysis showed that the deprotection of the methyl ester was complete. 50WX8(H + ) resin, filtered and concentrated under reduced pressure. The crude intermediate compound 11" was used directly in the next step without further purification.

[0394] TFA (1.5 mL) was added to the intermediate crude compound 11". The solution was stirred at room temperature for 1 hour until the reaction was complete as confirmed by LC-MS. TFA was removed by rotary evaporation under reduced pressure, treated with ether (3×2 mL), and dried under vacuum to give compound 12" as a viscous product. The total yield for two steps was 79%.

[0395] Synthesis of compound 13″: To a solution of compound 12″ (4 mg, 5.86 μmol) in DMSO (200 μl) at room temperature under argon, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate and CBTF (2.60 mg, 6.15 μmol, 1.05 equiv.) were added, followed by triethylamine (8.20 μl, 58.59 μmol, 10.0 equiv.) and stirred for 10-15 minutes. The progress of the reaction was monitored by TLC and LC-MS. After completion of the reaction, compound 13″ was directly used in the next step without any purification (preferably using a silica gel column for purification for conjugation with Fc protein).

[0396] For LC-MS evaluation, the conditions were as follows: Column: XBridge BEH C18 column, 3.5 μm, 3 mm×100 mm; Mobile phase: A: 20 mM ammonium bicarbonate buffer, pH 7; B: acetonitrile (HPLC grade); Method run: 5-95% B, 7 minutes, 0.75 mL / minute.

[0397] WT-Fc expression, purification and QC analysis: Synthesize IgG1 C using known methods H 2-C H 3 wild type and subcloned into an expression vector. Confirmed plasmid DNA was prepared and used for transient transfection of CHO-S cells. 5 to 6 days after transfection, the cell suspension was centrifuged at 8,000 rpm for 30 minutes to recover the supernatant fraction. IgG1 Fc wild type (WT-Fc) was purified by protein A affinity chromatography.

[0398] In brief, the culture supernatant was passed through a 0.22 μm filter and then loaded onto a polypropylene column filled with protein A high-capacity agarose resin. The resulting flow-through was collected and passed through the column twice again, and any unbound protein was then washed away with>10CV (column volume) of 1×PBS. WT-Fc was eluted with 3ml 100mM citrate buffer (pH 3.0) and immediately neutralized with 1ml of 1M Tris (pH 8.0). The sample buffer was exchanged into 1×PBS using an AmiconUltra-4 (Millipore) spin column with a molecular weight cutoff of 10kDa, and the purity of the purified sample was assessed by 4-20% gradient SDS-PAGE gel. The final Fc protein in monomeric form was approximately 26kDa. The yield was approximately 30 to 40mg per 100ml culture.

[0399] Final cysteine ​​conjugation of Zan-PEG6-Fc-WT: Compound 13″ (10-12 equivalents) was added to a solution of purified Fc protein in PBS at pH 7.2 with slow stirring at 4°C for 10 min. After the addition was complete, the reaction mixture was stirred for 12-72 h and the progress of the reaction was monitored by SDS-PAGE and matrix-assisted laser desorption / ionization (MALDI) mass spectrometry analysis.

[0400] After the reaction is complete, the conjugated crude product is purified using a molecular weight cutoff (MWCO; 10 kDa, Vivaspin 500, catalog number GE28-9322-25) filter and centrifuged at 4°C, 15,000 / RPM for 10 minutes to remove all unreacted linkers and low molecular weight impurities. The process was repeated 3-5 times (concentration of 5 mg / mL, yield: 60-70%).

[0401] The purity and molecular weight of the zan-PEG6-Fc-WT conjugate were confirmed by SDS-PAGE and MALDI analysis, respectively. The molecular weight of the final product (zan-Fc-WT) was ~29 kDa, while the molecular weight of the Fc protein was only 26 kDa.

[0402] The synthesis of the second cysteine-based conjugate, zan-Fc-DLE, followed the above protocol and the product was confirmed by SDS-PAGE and MALDI analysis. As used herein, the term "zan-Fc-WT" is used interchangeably with the term "Zan-PEG6-Fc-WT". Example 3 Comparison of single hapten and double hapten at 48hpi and 96hpi

[0403] On day 0 of the experiment, the first group of 6-8 week old female BALB / c mice (n=5 / group) were infected with 10 LD 50 Influenza A virus / Puerto Rico / 8 / 1934 (H1N1) (Catalog No. NR-348, BEI Resources, NIAID, NIH). Mice (except for the PBS-only group) were injected intraperitoneally with 8g / kg human IVIg at 24hpi to achieve humanized titers of anti-DNP and anti-rhamnose antibodies when the test article was administered. Mice were treated with the test article at 48hpi. All three test articles were administered in a single intranasal dose of 1.5umol / kg. Mice in the two control groups received PBS as a placebo.

[0404] To evaluate drug efficacy, mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0405] The results of this experiment are as follows Figure 5 and Figure 6 shown. Figure 5 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 48 hpi. Figure 6 is in accordance with Figure 5 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0406] On day 0 of the experiment, a second group of 6-8 week old female BALB / c mice (n=5 / group) were infected with 10 LD 50Influenza A virus / Puerto Rico / 8 / 1934 (H1N1) (Catalog No. NR-348, BEI Resources, NIAID, NIH). Mice (except for the PBS-only group) were injected intraperitoneally with 8g / kg human IVIg at 72hpi to achieve humanized titers of anti-DNP and anti-rhamnose antibodies when the test article was administered. Mice were treated with the test article at 96hpi. All three test articles were administered in a single intranasal dose of 1.5umol / kg. Mice in the two control groups received PBS as a placebo.

[0407] As in previous studies, to assess drug efficacy, mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0408] The results of this experiment are as follows Figure 7 and Figure 8 shown. Figure 7 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 96 hpi. Figure 8 is in accordance with Figure 7 Figure 2. Relationship between days post infection and body weight (%) of the treated mice. Example 4 Comparison of dual haptens with commercially available drugs at 48 and 96 hours post-infection

[0409] On day 0 of the experiment, the first group of 6-8 week old female BALB / c mice (n=5 / group) were infected with 10 LD 50 Influenza A virus / Puerto Rico / 8 / 1934 (H1N1) (Catalog No. NR-348, BEI Resources, NIAID, NIH). Mice (except the group only administered PBS) were injected intraperitoneally with 8 g / kg human IVIg at 24 hpi to achieve humanized titers of anti-DNP and anti-rhamnose antibodies when the test article was administered.

[0410] Treatment of infected mice started at 48 hpi. Zan-DNP-rhamnose (Compound 24) was administered as a single intranasal dose of 1.5 μmol / kg. Tamiflu was administered at a dose of 5 mg / kg twice a day for five consecutive days, and Xofluza was administered at a dose of 1.5 mg / kg twice a day for five days. Two control groups of mice received PBS as a placebo.

[0411] To evaluate drug efficacy, mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund. Fig. 9 and Fig.10 Shown in. Fig. 9 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg( -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 48 hpi. Fig.10 is in accordance with Fig. 9 Figure 2. Relationship between days post infection and body weight (%) of the treated mice.

[0412] On day 0 of the experiment, another group of 6-8 week old female BALB / c mice (n=5 / group) were infected with 10 LD 50 Influenza A virus / Puerto Rico / 8 / 1934 (H1N1) (Catalog No. NR-348, BEI Resources, NIAID, NIH). Mice (except the group only administered with PBS) were intraperitoneally injected with 8 g / kg human IVIg at 72 hpi to achieve humanized titers of anti-DNP and anti-rhamnose antibodies when the test article was administered.

[0413] Treatment of infected mice began at 96 hpi. Zan-DNP-rhamnose was administered as a single intranasal dose of 1.5 umol / kg. Tamiflu was administered at a dose of 5 mg / kg twice a day for five consecutive days, and Xofluza was administered at a dose of 1.5 mg / kg twice a day for five days. Two control groups of mice received PBS as a placebo. To evaluate drug efficacy, mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund. The results of this experiment are presented in Fig.11 and Fig.12 Shown in. Fig.11 Is infected 100LD 50 Influenza A H1N1 / PR8, removal of anti-influenza antibodies, intraperitoneal administration of human IgG (IVIg -C)) is a graph showing the relationship between the days after infection and the survival rate (%) of mice (n=5 / group) administered the conjugate at 96 hpi. Fig.12 is in accordance with Fig.11 Figure 2. Relationship between days post infection and body weight (%) of the treated mice. Example 5 Comparison of the double-hapten virus titer at 48 hpi with the virus titer of commercially available drugs

[0414] On day 0 of the experiment, 6-8 week old female BALB / c mice (n=5 / group) were infected with 10 LD 50 Influenza A virus / Puerto Rico / 8 / 1934 (H1N1) (Catalog No. NR-348, BEI Resources, NIAID, NIH). 24 hours before the test article (TA) administration, mice (except for the PBS-only group) were intraperitoneally injected with 8 g / kg human IVIg to achieve humanized titers of anti-DNP and anti-rhamnose antibodies when the test article was administered.

[0415] Treatment of infected mice started at 48 hpi or 96 hpi. Zan-DNP-rhamnose (ZDR) (Compound 24), Zan-DNP (ZD) and Zan-rhamnose (ZR) were administered as a single intranasal dose of 1.5 μmol / kg. Tamiflu was administered twice a day for five consecutive days at a dose of 5 mg / kg, and Xofluza was administered twice a day for five days at a dose of 1.5 mg / kg. Two control groups of mice received PBS as a placebo.

[0416] To evaluate the rate of reduction of viral titer within 24 h of TA administration, 2 mice in each cohort were sacrificed by CO2 asphyxiation 24 h after TA administration, and their lungs were collected and immediately homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Viral titers in lung homogenates were measured by real-time reverse transcription polymerase chain reaction (RT-PCR). Quick-RNA TM Microrep kit (ZymoResearch Corporation, Irvine, California) was used to extract RNA from the homogenate. cDNA synthesis and reverse transcription were performed according to standard protocols. Synthetic primer / probe sets were used to identify two highly conserved regions of the influenza matrix (M) gene. In order to construct a standard curve for calculating viral titers, 10-fold dilutions of influenza A virus / Puerto Rico / 8 / 1934 (H1N1) stock solutions with known viral titers were run simultaneously with the lung homogenate.

[0417] The results of this experiment are Fig.13A , Fig. 13B , Fig.14A and Fig. 14B Shown in. Fig.13AViral titers (fold change) at 48 hpi are shown for the conjugates (compound 24 = ZDR and zan-DNP = ZD) and marketed drugs (Xofusa = XO and Tamiflu = TAMI). Fig. 13B Viral titers (fold change) at 96 hpi are shown for the conjugates (compound 24 = ZDR and zan DNP = ZD) and marketed drugs (Xofusa = XO and Tamiflu = TAMI). Fig.14A Viral titers (fold change) at 48 hpi are shown for the conjugates (compound 24 = ZDR and zan-DNP = ZD) and marketed drugs (Xofusa = XO and Tamiflu = TAMI). Fig. 14B Viral titers (fold change) at 96 hpi are shown for the conjugates (compound 24 = ZDR and zan-DNP = ZD) and marketed drugs (Xofusa = XO and Tamiflu = TAMI). Example 6 Oral administration of compound 24

[0418] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 100×LD 50 of influenza A / H1N1 / PR8 / 1934. Human IVIg was used as a source of anti-hapten antibodies and was injected at a dose of 8 g / kg 24 hours before drug administration. The test article zanamivir-DNP-rhamnose (Compound 24) was administered at 48 hours post-infection by intravenous (IV) and oral routes as a single dose of 1.5 or 4.5 μmol / kg. Mice were weighed and monitored daily for 14 days post-infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0419] When the same dose of the test article was administered orally, the survival rate dropped to 60%, whereas at a 3-fold higher dose, the survival rate rose to 80%. Fig.15A is a graph showing the relationship between days after infection and survival rate. Fig. 15B It is a graph showing the relationship between the number of days after infection and body weight (%). Example 7 Quantification of viral titers in lungs

[0420] Female Balb / c mice (2 mice / group) aged 6-8 weeks were inoculated intranasally (IN) with 100×MLD 50The mice were euthanized 3 days after infection, and the lungs were harvested and immediately homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). The viral titers of the lung homogenates were measured by real-time RT-PCR. The viral titers of the lung homogenates were measured by Quick-RNA sequencing. TM RNA was extracted from the homogenate using the Microrep kit (ZymoResearch Corporation, Irvine, California). RNA was extracted using the One Step PrimeScript TM Equal amounts of RNA were used for qrt-PCR using a RT-PCR kit (Takara Bio, Inc., Kusatsu, Japan). Primer / probe sets were synthesized to recognize two highly conserved regions of the influenza matrix (M) gene. To construct a standard curve for calculating viral titers, 10-fold dilutions of influenza A virus / Puerto Rico / 8 / 1934 (H1N1) stock solutions with known viral titers were tested simultaneously with lung homogenates.

[0421] Oral administration of compound 24 to mice did not reduce viral titers as rapidly as intranasal or IV administration of the same dose or a 3-fold higher dose. Reduced bioavailability may result in a slower reduction in lung viral titers with oral administration compared to IV and IN routes of administration.

[0422] Fig.16 Is a bar graph of route of administration (SC = subcutaneous; OG = oral gavage; IV = intravenous; IN = intranasal; PBS = phosphate buffered saline) versus viral titer (PFU / ml per ng RNA). Fig.17 Is a bar graph of OG and PBS versus virus titer (PFU / ml per ng RNA). Example 8 Pharmacokinetics of oral compound 24

[0423] Female Balb / c mice (3 mice / group) aged 6-8 weeks were administered a single intravenous (IV) dose or oral dose of 1.5 μmol / kg (2.6 mg / kg), 1.5 μmol / kg (2.6 mg / kg), or 13.5 μmol / kg of compound 24, and blood samples were collected at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 12 hours after drug administration. The plasma concentration of compound 24 was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). 24 hours before drug administration, IVIg was administered as a source of anti-hapten antibodies. Compared with IV administration, oral administration increases C max (maximum plasma concentration) was significantly lower, even at a 9-fold higher dose, but at a 9-fold higher dose the half-life (t 1 / 2 ) increased from approximately 0.4 hours to 4 hours.

[0424] Fig.18 is a graph of time (h = hours) versus concentration (ng / mL) for a single IV dose of 1.5 μmol / kg (n = 3 mice / time point). Fig.19 is a graph of time (h = hour) versus concentration (ng / mL) following oral administration of a single dose of 13.5 μmol / kg (n = 3 mice / time point). Example 9 Viral challenge and mouse therapy studies with increasing frequency of oral dosing

[0425] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 100×LD 50 of influenza A / H1N1 / PR8 / 1934. Human IVIg was used as a source of anti-hapten antibodies and was injected at a dose of 8 g / kg 24 hours before drug administration. The test compound 24 was administered at 48 hpi, with a single dose of 1.5 μmol / kg of zan-DNP-rhamnose (IV) or two doses of 4.5 μmol / kg of zan-DNP-rhamnose (oral). Mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0426] Increasing the dosing frequency to two doses, 12 hours apart, achieved 100% survival (compared to 80% survival with a single oral dose of 4.5 [mu]mol / kg zan-DNP). Fig. 20A is a graph showing the relationship between days after infection and survival rate. Fig. 20B is a graph showing the relationship between days post infection and % body weight. Example 10 Quantification of viral titers in lungs

[0427] Female Balb / c mice aged 6-8 weeks (2 mice / group) were inoculated with 100×MLD 50 Influenza A / H1N1 / PR8 / 1934 was treated 48 hours later with a single IV dose of 1.5 μmol / kg (2.6 mg / kg) or two oral doses of 4.5 μmol / kg (7.8 mg / kg) (12 hours apart) of compound 24 or PBS. Mice were euthanized 3, 5, and 8 days after infection, and their lungs were collected and immediately snap-frozen in RNA lysis buffer. Lung homogenates were prepared using a gentleMACS Octo Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Viral titers of lung homogenates were measured by qrt-PCR. Quick-RNA TM RNA was extracted from the homogenate using a Microprep kit (Zymo Research Corporation, Irvine, California). RNA was extracted using One Step PrimeScript PCR according to the manufacturer's protocol. TM Equal amounts of RNA were used for qrt-PCR using a RT-PCR kit (Takara Bio, Inc., Kusatsu, Japan). Primer / probe sets were synthesized to recognize two highly conserved regions of the influenza matrix (M) gene. To construct a standard curve for calculating viral titers, 10-fold dilutions of influenza A virus / Puerto Rico / 8 / 1934 (H1N1) stock solutions of known viral titers were tested simultaneously with lung homogenates.

[0428] Oral administration reduced viral titers more slowly than IV administration, but was able to completely eliminate viral titers by day 8 post infection. Both IV and oral administration of compound 24 completely eliminated viral infection in the lungs, with the IV route acting faster than the oral route. Fig.21 Is a graph of the number of days post infection versus viral titer (PFU / ml per ng RNA) for IV and oral (OG = oral gavage) administration of PBS and zanamivir-DNP-rhamnose. Embodiment 11 Comparison of the efficacy of compound 24 with standard of care (SOC) (96 hpi treatment)

[0429] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 10×LD 50of influenza A / H1N1 / PR8 / 1934 (day 0). A control group of healthy mice (not infected on day 0) was also tested as a control.

[0430] Human IVIg was used as a source of anti-hapten antibodies and was injected at an optimized dose of 6 g / kg 24 hours prior to drug administration in each cohort. The test article and SOC were administered at 96 hpi, with a single IV dose of 2.6 mg / kg of compound 24 ( Fig. 22 and Fig.23 C.24(A) in the experiment), a single oral dose of 23.4 mg / kg of compound 24 ( Fig. 22 and Fig.23 C.25(B)), a single oral dose of 12.3 mg / kg of baloxavir marboxil ( Fig. 22 and Fig.23 Xofluza (C) in the 5-day oral administration of oseltamivir phosphate 5 mg / kg twice a day ( Fig. 22 and Fig.23 Tamiflu (D) or PBS ( Fig. 22 and Fig.23 E) (Healthy mouse data not shown Fig. 22 and Fig.23 Mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0431] Both single IV (A) and oral administration of compound 24 (B) resulted in 100% survival of virus-infected mice, which was not achieved by the two SOC drugs tested. When treated at 96 hpi, compound 24 was superior to the current standard of care antiviral drugs. Fig. 22 It is a graph showing the relationship between the number of days after infection and the survival rate (%). Fig.23 This is a graph showing the relationship between days after infection and body weight (%). Fig.23 and Fig.24 Compound 24 = C.24).

[0432] 24 hours after drug administration (120 hpi), mice were anesthetized with isoflurane and then cervical dislocation was performed. Lung tissues were fixed with 10% neutral buffered formalin (NBF), embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and then imaged under a Nikon Eclipse light microscope at 10x magnification. Qualitative histological analysis of the lungs was performed, and images are shown in Fig.33Untreated virus-infected mice showed signs of diffuse alveolar damage, pulmonary edema, and excessive infiltration of inflammatory cells, while virus-induced histopathological changes in the lung tissues of mice treated with compound 24, either intravenously or orally, were significantly reduced compared with the untreated group.

[0433] At the same time, two healthy mouse cohorts were injected with compound 24IV and compound 24OG, respectively, and the other mouse cohort did not receive intervention. 24 hours after drug administration, the mice were anesthetized with isoflurane and then cervical dislocation was performed. The lung, kidney, liver, stomach, and small intestine tissues were fixed with 10% neutral buffered formalin (NBF), embedded in paraffin, sectioned, stained with H&E (except kidney, kidney tissue was stained with periodic acid Schiff (PAS)), and then imaged at 10x magnification under a Nikon Eclipse optical microscope. Qualitative histological analysis of the lungs was performed, and the images are shown in Figure 2. Fig.33 Compared with the untreated group, no abnormal changes were observed in the tissue morphology of mice injected with compound 24. Example 12 Comparison of the efficacy of compound 24 and single hapten (96 hpi treatment)

[0434] A targeted therapy strategy with a dual mechanism of action is disclosed that can elicit a host immune response to viruses and virus-infected cells. The neuraminidase inhibitor zanamivir (as a fragment) is used as a targeting ligand. Neuraminidase is present on the influenza virus envelope and on the surface of infected cells. Conjugates containing zanamivir fragments bind to haptens, which bind to antibodies naturally occurring in humans. Once recruited, these anti-hapten antibodies bind to and activate the innate immune system to fight viruses and virus-infected cells.

[0435] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 10×LD 50 of influenza A / H1N1 / PR8 / 1934 (day 0). Human IVIg was used as a source of anti-hapten antibodies and was injected at an optimized dose of 6 g / kg 24 hours before drug administration. The test articles were administered at 96 hpi as a single IV dose of 1.5 μmol / kg as follows: (a) Compound 24 ( Fig.29A and Fig.29B (A) in (b) zanamivir-DNP (single hapten conjugate; Fig.29A and Fig.29B (B) in (c) zanamivir-rhamnose (single hapten conjugate; Fig.29A and Fig.29B (C) in; (d) zanamivir ( Fig.29A and Fig.29B (D) in; or (e) PBS (control) ( Fig.29A and Fig.29B Mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0436] When tested in BALB / c mice supplemented with IVIg and infected with influenza A virus (H1N1, A / Puerto Rico / 8 / 1934), the zanamivir-double hapten conjugate (Compound 24) showed superior antiviral activity to the single hapten conjugate in both early and late infection. In addition, the double hapten conjugate showed better activity in late infection compared to a single dose of the single hapten conjugate. Therefore, Compound 24 can treat both early and late influenza infections. Example 13 Comparison of 24-hour lung titers

[0437] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 10×LD 50 The influenza A / H1N1 / PR8 / 1934 was treated 48 hours later with: (a) a single IV dose of 1.5 μmol / kg of compound 24; (b) a single OG dose of 13.5 μmol / kg of compound 24; (c) a single OG dose (d) Single OG dose 10 mg / kg or (e) a single IV dose of 100 μl PBS.

[0438] After 24 h of treatment, the virus titer of lung homogenates was measured by real-time RT-PCR, and the results were as follows Fig.24 As shown, the titer of compound 24 (Compound 24 = C.24) delivered orally was the lowest. Embodiment 14 Comparison of compound 24 administration routes

[0439] like Fig.25 As shown, Balb / c mice were infected on day 0. Four days after the infection challenge, compound 24 was administered by three different routes (intranasal, IV, and oral) as shown, with the highest oral dose of 13.5 μmol / kg and the other doses of 1.5 μmol / kg. The results showed that all three compound 24 cohorts had a 100% survival rate after 14 days, while mice treated with PBS did not survive past day 10. Infection / body weight % is shown in Figure 2 Fig.26 shown. Embodiment 15 Antibody-mediated effector function in vitro

[0440] To test the proof of concept of the mechanism of action of compound 24 in vitro, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were performed. The ADCC assay was performed using an ADCC kit (Promega, catalog number G7010; Promega Corporation, Madison, WI) containing influenza virus neuraminidase (N1) transfected (NA-HEK) and wild-type (WT) human embryonic kidney (HEK 293) cells. To this end, cells were plated in triplicate (100 μl, 5000 cells / well) in 96-well black-walled plates (Corning Life Sciences, Corning, NY) and then treated with serial dilutions of compound 24 in the presence or absence of 100-fold excess zanamivir. After incubation at 37°C for 2 hours, human IVIg was added to each well and the plate was incubated for another 30 minutes at 37°C. Finally, ADCC effector cells were added at 75,000 cells / well and incubated overnight at 37°C and 5% CO2.

[0441] The amount of firefly luciferase produced by the ADCC effector cells was then quantified using the Bio-Glo™ Luciferase Assay Reagent (included in the kit).Luminescence was measured using a Synergy Neo2 HTS Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT).

[0442] Analysis of the killing of virus-infected cells by the complement system was performed by complement-dependent cytotoxicity (CDC) assay. N1-transfected (NA-HEK) and wild-type (WT) HEK 293 cells were harvested, plated in triplicate (100 μl, 5,000 cells / well) in 96-well black-walled plates (Corning LifeSciences, Corning, NY), and then treated with serial dilutions of compound 24 in the presence or absence of a 100-fold excess of zanamivir. (Zanamivir is the free drug and competes with compound 24 when used in excess). After incubation at 37°C for 2 hours, human IVig and human serum were added to each well, and the plates were incubated overnight at 37°C under 5% CO2. CellTiter Cell viability was measured using an aqueous non-radioactive cell proliferation kit (Promega Corporation, Madison, WI).

[0443] Compound 24 induces antibody-mediated effector cell function in vitro with high potency and selectivity by interacting with Fc receptors expressed on the effector cells and complement proteins in the presence of antibodies. Fig.27A and Fig.27BThe relationship between the concentration of compound 24 (in nM) and the ADCC percentage and CDC percentage are shown respectively. Example 16 Dose escalation of IV administration of compound 24

[0444] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 10×LD 50 of influenza A / H1N1 / PR8 / 1934 (day 0). Human IVIg was used as a source of anti-hapten antibodies and was injected at an optimized dose of 6 g / kg 24 hours before drug administration. Mice in each test group were administered a single IV dose of the following at 96 hpi: (a) 0.17 μmol / kg of compound 24 ( Fig.28A and Fig.28B (A)); (b) 0.5 μmol / kg compound 24 ( Fig.28A and Fig.28B (B)); (c) 1.5 μmol / kg of compound 24 ( Fig.28A and Fig.28B (C)); (d) 4.5 μmol / kg of compound 24 ( Fig.28A and Fig.28B (D) in; and (e) PBS ( Fig.28A and Fig.28B (e)). Mice were weighed and monitored daily for 14 days after infection and were counted as dead when they lost 25% of their body weight or were diagnosed as moribund.

[0445] A single IV dose of 1.5 μmol / kg of Compound 24 (C) and a single IV dose of 4.5 μmol / kg of Compound 24 (D) both resulted in 100% survival of mice, which was not achieved with the other doses tested or the control (PBS). Fig.28A and Fig.28B Graphs showing the relationship between days post infection and survival rate (%) and days post infection and body weight (%) are shown, respectively. Embodiment 17 Efficacy against multiple influenza virus strains

[0446] On day 0, 6-8 week old female Balb / c mice (5 mice / group) were infected with 10×LD 50Mice infected with influenza A / California / 07 / 2009(H1N1)pdm09 were given a single IV dose of 1.5 μmol / kg of compound 24 ( Fig. 30A and Fig. 30B (A)) or PBS ( Fig. 30A and Fig. 30B Mice infected with influenza A / Wisconsin / 67 / 2005 were given a single IV dose of 1.5 μmol / kg of compound 24 at 96 hpi ( Fig. 30A and Fig. 30B (B)) or PBS ( Fig. 30A and Fig. 30B Mice infected with influenza B / Florida / 04 / 2006 were given a single IV dose of 1.5 μmol / kg of compound 24 at 96 hpi ( Fig. 30A and Fig. 30B (C)) or PBS ( Fig. 30A and Fig. 30B (F) in the figure.

[0447] Compound 24 was equally effective against all influenza virus strains tested, with 100% survival after 14 days for all three compound 24 cohorts, whereas no mice treated with PBS survived past day 11. Embodiment 18 Inflammatory cytokine and chemokine levels

[0448] Female Balb / c mice (5 mice / group) aged 6-8 weeks were infected with 10×LD 50 Influenza A / H1N1 / PR8 / 1934 (Day 0). Mice in each test group were administered a single dose of one of the following treatments at 48 hpi or 96 hpi: 100 μl PBS by IV (A), 2.6 mg / kg compound 24 by single dose IV (B); 23.4 mg / kg compound 24 by single dose OG (C), 12.3 mg / kg compound 24 by single dose OG (D). (D) and OG 5mg / kg Twice a day for 5 days (E). Two groups of mice were not infected with the virus but were given compound 24 by IV at 96 hpi (F) (relative to the other groups), and a third group of mice was neither infected nor received any treatment (G). TMBead-based immunoassays Cytokines and chemokines were measured in lung tissue samples for each group, and cytokine and chemokine levels were determined using standard protocols provided by the manufacturer (BioLegend, San Diego, CA).

[0449] Fig.31 Shown are lung-specific cytokine and chemokine expression level graphs measured in each group in the 48hpi and 96hpi treatment groups. If the cytokine or chemokine levels of the control group are not shown in the figure, there is no detectable level. Whether it is oral or intravascular administration, compound 24 does not cause a cytokine storm in the lungs in either case, but by rapidly eliminating the root cause of the cytokine storm (i.e., viral infection), it is prevented from occurring. These data confirm that the administration of compound 24 can prevent lung inflammation and damage caused by influenza virus infection.

[0450] Serum cytokine and chemokine levels were also measured to assess the presence of systemic inflammation in the subjects. Fig.32 A graph showing the expression levels of specific cytokines and chemokines in the serum of each treatment group measured at 48 hpi and 96 hpi is shown, wherein A is the PBS control group, B is the group treated with compound 24 by a single IV dose, and C is the group treated with compound 24 by a single OG dose.

[0451] The compound 24-treated group exhibited lower levels of inflammatory cytokines compared to the other treatment groups and the control. Embodiment 19 Synthesis of folate double hapten conjugate (Compound 150)

[0452] Fig.35 A synthetic scheme for the synthesis of compound 150 is shown.

[0453] Synthesis of compound 3': Compounds 1' and 2' were prepared according to the procedure specified in Ref Chemistry–An Asian Journal 7(2):272-276 (Theresa Kueckmann et al. eds.) (2012). Thereafter, TEA (1.38 mL, 9.87 mmol) was added to a solution of 1-chloro-2,4-dinitrobenzene (compound 1') (0.5 g, 2.47 mmol) and 3-(2-aminoethoxy)propionic acid (compound 2', 0.33 g, 2.47 mmol) dissolved in EtOH (25 mL). The reaction mixture was heated to 55°C for 16 hours and the progress of the reaction was monitored by LC-MS. After the reaction was confirmed to be complete by the disappearance of one of the starting materials (i.e., dinitrobenzene), the reaction mixture was cooled and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on Teledyne CombiFlash Rf+Lumen (silica gel, 12 g column, 0-20% methanol / DCM) to afford yellow solid compound 3' in 90% yield. LC-MS [M+H]+ = 300.24 (see Fig.36 ).

[0454] Synthesis of compound 5': Under argon atmosphere, HATU (0.11 g, 0.28 mmol, 0.85 equiv) was added to a solution of 3-(2-((2,4-dinitrophenyl)amino)ethoxy)propanoic acid (compound 3', 0.1 g, 0.33 mmol) in dimethyl sulfoxide (2 mL), followed by DIPEA (0.29 mL, 1.67 mmol, 5.0 equiv) and stirred at room temperature for 10 minutes. Fmoc-LysOH.HCl (purchased from Chem-Impex International, Wood Dale, IL; compound 4', 0.11 g, 0.27 mmol, 0.8 equiv) was added to the reaction mixture, stirred at room temperature for 2-3 hours, and the reaction progress was monitored by LC-MS.

[0455] After the reaction was confirmed to be complete by LC-MS, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel (4 g) column chromatography on Teledyne CombiFlashRf + Lumen (0-10% methanol / DCM), and the fractions were analyzed by LC-MS. The solvent was evaporated from the combined pure fractions using a rotary evaporator under reduced pressure to isolate compound 5' with a yield of 70%. LC-MS [M + H] + = 650.67 (see Fig.37 ).

[0456] Synthesis of 2,3,4-tetra-O-acetyl-α-L-rhamnose (Compound 7'): Compound 7' was prepared according to the procedure specified in De Coen et al., Synthetic rhamnose glycopolymer cell-surface receptor for endogenous antibody recruitment, Biomacromolecules 21(2):793-802 (2020). Briefly, α-L-rhamnose monohydrate (6, 1.0 g, 5.49 mmol) was dissolved in 9.2 mL of anhydrous pyridine. The solution was stirred in an ice bath and purged with nitrogen for 15 minutes by keeping the internal temperature below 10 °C before adding acetic anhydride (4.15 mL, 43.92 mmol, 8.0 equiv). The reaction was slowly warmed to room temperature over 2 hours, and the progress of the reaction was monitored by TLC (hexane / EtOAc, 65:35) and LC-MS, indicating that α-L-rhamnose monohydrate was completely consumed after 20 hours of reaction under an inert atmosphere. The reaction mixture was poured into ethyl acetate and extracted twice with 1.0M HCl. The combined organic layer was washed with saturated sodium carbonate solution, water and brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The resulting crude oil product compound 7' (yield 98%) was used in the next step. LC-MS [M+H] + = 333.32 and / or LC-MS [M+H2O] = 350.32.

[0457] Synthesis of compound 9': Compound 9' was prepared according to the procedure specified in De Coen et al. (2020) above. Briefly, 1,2,3,4-tetra-O-acetyl-α-L-rhamnose (7, 0.50 g, 1.50 mmol) was dissolved in DCM (7.5 mL) before adding -H2N-PEG4-OH (purchased from BroadPharm, San Diego, CA; 8, 0.35 g, 1.81 mmol, 1.2 equiv) under an inert atmosphere. The reaction flask was placed in an ice bath and boron trifluoride etherate (0.56 mL, 4.51 mmol, 3.0 equiv) was added dropwise at 4 ° C for 30 minutes. The reaction mixture was stirred at ice bath temperature for 2 hours, and then the reaction was allowed to warm to room temperature. The progress of the reaction was monitored by TLC (hexane / EtOAc, 30:70, Rf=0.30) and LC-MS, indicating that 1,2,3,4-tetra-O-acetyl-α-L-rhamnose (compound 7') was completely consumed after 16 hours of reaction. The reaction mixture was poured into ice water and extracted with DCM (3×10 mL). The combined organic layer was washed twice with saturated sodium bicarbonate solution, water and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel (12 g) column chromatography on Teledyne CombiFlash Rf+Lumen (0-10% methanol / DCM) to isolate the product (compound 9') with a yield of 90%. LC-MS [M+H] + = 466.51.

[0458] Synthesis of compound 10': PyBOP (0.05 g, 0.10 mmol, 1.1 equiv) was added to a solution of acid (compound 5', 0.06 g, 0.09 mmol) and rhamnose (OAc) 3-PEG4-NH2 (compound 9', 0.04 g, 0.09 mmol, 1.0 equiv) in dimethyl sulfoxide (1.5 mL) at room temperature under argon atmosphere, followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 equiv). The progress of the reaction was monitored by LC-MS. After the reaction was confirmed to be complete by LC-MS, the reaction mixture was quenched by adding water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel (4 g) column chromatography on Teledyne CombiFlash Rf + Lumen (0-10% MeOH / dichloromethane) to isolate the product (compound 10') with a yield of 93%. LC-MS [M+H] + = 1098.15 (see Fig.38 ).

[0459] Deprotection of -Fmoc group to synthesize compound 11': DEA (100 uL) was added to a solution of compound 10' (0.01 g, 0.01 mmol) in anhydrous DCM (0.2 ml) at room temperature under argon. The solution was stirred at room temperature for 1 hour until the reaction was complete, as confirmed by LC-MS. DEA was removed by rotary evaporation under reduced pressure, and the crude product was precipitated in ether to give a yellow solid in quantitative yield, which was used in the next step without further purification. LC-MS [M+H] + = 875.91 (see Fig.39 ).

[0460] Synthesis of compound 13': DIPEA (0.048 μl, 0.27 mmol, 10 equiv) was added dropwise to a solution of DBCO-NHS (purchased from BroadPharm, San Diego, CA; compound 12', 0.012 g, 0.029 mmol, 1.05 equiv) and compound 11' (0.024 g, 0.027 mmol, 1.0 equiv) dissolved in DCM:DMSO (10:1, 0.6 mL) under argon. The reaction mixture was stirred at room temperature for 2-3 hours, and the progress of the reaction was monitored by LC-MS. After the reaction was completed (confirmed by the complete consumption of compound 11'), the solvent was evaporated by rotary evaporator under reduced pressure, and the crude product was purified by silica gel (4 g) column chromatography on Teledyne CombiFlash Rf+Lumen (0-10% methanol / DCM) to give a light yellow solid compound 13' with a yield of 93%. LC-MS [M+H]+ = 1163.21 (see Fig.40 ).

[0461] Synthesis of intermediate 14': Compound 13' (0.025 g, 0.022 mmol) was dissolved in anhydrous MeOH (1 mL) and treated dropwise with 0.5 M NaOH (25 μl). The reaction mixture was stirred at room temperature for 1 hour, at which time LC-MS analysis showed no starting material. 50WX8(H + ) resin to neutralize the reaction, filter and concentrate under reduced pressure. The pale yellow crude product (Compound 14') was used directly in the next step without further purification. LC-MS [M+H] + = 1037.11 (see Fig.41 ).

[0462] Synthesis of folate-NHS ester (Compound 16): DCC (0.281 g, 1.359 mmol, 1.2 equiv) was added to a DMSO (12 mL) solution of compound 15' (0.50 g, 1.133 mmol) and N-hydroxysuccinimide (0.143 g, 1.246 mmol, 1.10 equiv) at room temperature under argon, and the solution was stirred for 12 hours. The progress of the reaction was monitored by LC / MS. After the reaction was confirmed to be complete by LC-MS, the reaction mixture was precipitated in acetone (15x) and the product was isolated by centrifugation. In addition, the crude product was washed with acetone (2×50 mL) and EtOAc (1×30 mL), dried in vacuo, and the crude product (Compound 16) was used in the next step without further purification.

[0463] Synthesis of folate-PEG6-azide (Compound 18'): N3-PEG6-NH2 (Compound 17', purchased from BroadPharm, San Diego, CA; 0.032 g, 0.093 mmol, 1.05 equiv) was added to a solution of folate-NHS ester (Compound 16, 0.05 g, 0.093 mmol) in anhydrous DMSO (1.0 mL) at room temperature under argon, followed by DIPEA (0.081 mL, 0.46 mmol, 5.0 equiv) and stirred for 12 hours. The reaction progress was monitored by LC / MS. After the reaction was confirmed to be complete by LC / MS, the reaction mixture was precipitated by adding ether and centrifuged. The yellow precipitate was collected and washed with ether (2×15 mL), and the crude product was purified by preparative HPLC on a C18 column (5-95% B in 60 minutes, flow rate 7 mL / min; B: acetonitrile; A: 20 mM NH4OAc, pH 7 buffer, UV@280 nm) to give compound 18′. LC-MS [M+H]+=774.83 (see Fig.42 ).

[0464] Synthesis of compound 150: DIPEA (12 μl, 0.065 mmol, 5.0 equiv) was added dropwise to a solution of compound 18' (10 mg, 0.013 mmol) and compound 14' (13.4 mg, 0.013 mmol, 1.0 equiv) in anhydrous DMSO (1.0 mL) under argon. The reaction mixture was stirred at room temperature for 2-4 hours, and the progress of the reaction was monitored by LC-MS. After completion of the reaction, the crude product was purified by preparative HPLC on a C18 column (5-95% B in 60 minutes, flow rate 7 mL / min; B: acetonitrile; a: 20 mM NH4OAc, pH 7 buffer, UV @ 360 nm and 280 nm) to give the final compound 150 with a yield of 24%. Purity: 97% (HPLC purification), and LC-MS [M+H] + = 1810.93 (see Fig.43 ). Embodiment 20 In vitro functional assay of compound 150

[0465] Mouse lung cancer cells expressing folate receptor (cell line M109) were seeded at a density of 500 cells / well in 96-cell plates and allowed to adhere overnight at 37° C. The cells were then incubated with serial dilutions of the folate double hapten conjugate Compound 150 for 2 hours at 37° C., and human serum was then added to assess the effect, if any, on complement dependent cytotoxicity (CDC).

[0466] The results are Fig.44 , which demonstrates that the addition of compound 150 induces CDC, possibly by recruiting anti-hapten antibodies on the surface of cancer cells, which are then activated by antibodies of the complement system in human serum. In the control group, 100-fold excess of folate-glucosamine ( Fig.44 In order to confirm that the observed immunogenic effect was indeed due to the binding of the folate moiety on the folate receptor on the target cells and the bihapten conjugate, a competitor was added (labeled as M109_comp in FIG).

[0467] In addition, the ADCC activity of compound 150 was measured on cancer cells expressing folate receptors (4T1 (mouse breast cancer cells), MDA-MB-231 (human breast cancer cells), M109 (mouse lung cancer cells)) and THO-1 (AML model). After incubation with serial dilutions of compound 150 for 2 hours, the cells were incubated with IVIg at 37°C for 2 hours and with effector cells expressing human Fc-γ-RIII at 37°C, 5% CO2 overnight. The results were Figure 45A-Figure 45DAs shown in , it was demonstrated that compound 150 induced ADCC by recruiting anti-hapten antibodies on the surface of cancer cells and then activating Fc-γ-RIII receptors expressed on the effector cells. Embodiment 21 In vivo efficacy study of compound 150 in a mouse lung cancer model

[0468] Female Balb / c mice aged 6-8 weeks were inoculated subcutaneously on the right side with M109 mouse lung cancer cells (mouse lung cancer cells expressing folate receptor) at a dose of 2×10 6 cells / mouse. Ten days after inoculation, when the tumor size reached 50 mm 3 At 4 pm, treatment was initiated with the following test articles: (a) PBS alone at 100 μl / mouse; (b) SOC treatment (i.e., 200 μg / mouse, 3 days / week anti-PD1 + chemotherapy (carboplatin 50 mg / kg intraperitoneally on day 18 after tumor inoculation + paclitaxel 36 mg / kg intravenously on days 18-22 after tumor inoculation); or (c) 10 nmol / mouse Compound 150, one dose per day, for 5 days / week. The PBS and Compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study.

[0469] Tumors were measured every other day, and mice were sacrificed on day 25 after tumor implantation. Fig.46 and Fig.47 The tumor volumes (mm 3 ) and the number of days after tumor implantation, and the relationship between the subject's body weight percentage and the number of days after tumor implantation. This data confirms that compound 150 inhibits tumor growth in the M109 model and shows promise as a monotherapy for lung cancer treatment. Embodiment 22 In vivo efficacy study of compound 150 in a human lung cancer (cold tumor) model

[0470] Female C57BL / 6 mice aged 6-8 weeks were inoculated subcutaneously on the right flank with LLC-1 human lung cancer cell line (LLC-1) at a volume of 5 × 10 6 cells / mouse. Ten days after inoculation, when the tumor size reached 50 mm 3At 1:100 μl / mouse, treatment was started with the following test articles: (a) PBS alone at 100 μl / mouse; (b) SOC treatment (i.e., 200 μg / mouse intraperitoneal injection of anti-PDL1, 3 days / week + 2 Gy / mouse, 3 days / week radiotherapy); or (c) 10 nmol / mouse of compound 150, one dose per day, for 5 days / week. The PBS and compound 150 groups were also intraperitoneally injected with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 after implantation to achieve humanized titers of anti-hapten antibodies within a two-week period.

[0471] Tumors were measured every other day, and mice were sacrificed on day 25 after tumor implantation. Fig.48 and Fig.49 The tumor volumes (mm 3 ) versus days after tumor implantation, and the relationship between the subject's body weight percentage and days after tumor implantation. The data demonstrate that Compound 150 may benefit from combination with therapies that convert cold tumors to hot tumors. Embodiment 23 In vivo efficacy study of compound 150 in a mouse colorectal cancer model

[0472] CT26 mouse colorectal cancer cell line was implanted subcutaneously in the right flank of female Balb / c mice aged 6-8 weeks at a dose of 2×10 6 cells / mouse. Ten days after inoculation, when the tumor size reached 50 mm 3 At 14:00, treatment was started with the following test articles: (a) PBS alone at 100 μl / mouse; (b) SOC treatment (i.e., leucovorin 100 mg / kg intraperitoneally once a week + 5-FU 50 mg / kg once a week + oxaliplatin 6 mg / kg on days 8 and 10 after tumor inoculation); or (c) 10 nmol / mouse of compound 150, one dose per day, for 5 days / week. The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study.

[0473] Tumors were measured every other day, and mice were sacrificed on day 25 after tumor implantation. Fig.50 and Fig.51 The tumor volumes (mm 3 ) and the number of days after tumor implantation, and the relationship between the subject's body weight percentage and the number of days after tumor implantation. Mice with a weight loss of more than 25% were euthanized as a humane endpoint.

[0474] In the leucovorin+5-FU+oxaliplatin group, all mice died by day 17, but in the compound 150 cohort, all mice survived and tumors grew more slowly compared to untreated controls. Embodiment 24 Evaluation of multiple combination therapies in a mouse lung cancer model

[0475] Female Balb / c mice (n=5 / cohort) aged 6-8 weeks were implanted subcutaneously with 2-5×10 6 M109 mouse lung cancer cell line cells (mouse lung cancer cells expressing folate receptors) (day 0). On day 11 after implantation, treatment was started with the following test articles: (a) PBS alone at 10 μl / mouse; (b) chemotherapy (carboplatin + paclitaxel); (c) 10 nmol / mouse of compound 150 + anti-PD1; (d) 10 nmol / mouse of compound 150 (+Abs); and (e) 10 nmol / mouse of compound 150 + chemotherapy. The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study. Tumors were measured every other day.

[0476] Fig.52 Tumor volume (mm 3 ) and the number of days after tumor implantation. Two of the five animals in the chemotherapy treatment cohort lost their tumors on day 17 after implantation (all animals in this cohort survived until the end of the study). Three of the five animals in the combination therapy compound 150 + anti-PD1 cohort lost their tumors (one of the remaining animals did not survive to day 22 after implantation). Four of the five animals in the combination therapy (compound + chemotherapy) cohort lost their tumors (3 of the 5 animals did not survive to day 22 after implantation). The data confirm that the combination of compound 150 with anti-PD-1 or chemotherapy significantly improves the therapeutic efficacy.

[0477] A second study was conducted using the same protocol as above with the following test articles: (i) PBS alone at 100 μl / mouse; (ii) SOC treatment (anti-PD1 + chemotherapy (carboplatin + paclitaxel)); (iii) 10 nmol / mouse of compound 150 + sunitinib; (iv) 10 nmol / mouse of compound 150 (+Abs); and (v) 10 nmol / mouse of compound 150 + folate-toll-like receptor 7 agonist conjugate (FA-TLR7). The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study.

[0478] Fig.53 Tumor volume (mm 3 ) versus days after tumor implantation. Four of five animals in the SOC treatment cohort lost their tumors (all animals in this cohort survived until the end of the study). One of five animals in the combination therapy compound 150 + sunitinib cohort was about to lose their tumors, but died on day 22 after implantation. One of five animals in the combination therapy compound + FA-TLR7 cohort lost their tumors (all animals in this cohort survived until the end of the study). The data confirm that the combination of compound 150 and sunitinib slightly enhanced the anti-tumor efficacy of the treatment, but at these doses, the combination of compound 150 and FA-TLR7 did not achieve any statistically relevant improvement in efficacy. Embodiment 25 Comparison of in vivo efficacy of compound 150 in mouse colorectal cancer model

[0479] Female Balb / c mice (n=5 / cohort) aged 6-8 weeks were subcutaneously implanted with CT-26 mouse colorectal cancer cells (day 0) at a dose of 2×10 6 cells / mouse. On day 11 after implantation, treatment with the following test articles was initiated: (a) PBS alone; (b) leucovorin + 5FU + oxaliplatin (OXH) (SOC cohort); or (c) 10 nmol / mouse of compound 150, one dose per day. Treatment ended on day 22 after implantation. The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study.

[0480] Tumors were measured every other day, and mice were sacrificed on day 25 after tumor implantation (all animals survived until the end of the study). Fig.54 and Fig.55 The tumor volumes (mm 3 ) versus days after tumor implantation, and the subject weight percentage versus days after tumor implantation. Three of five animals in the SOC cohort lost their tumors, and all animals were below 75% of their body weight on day 19 after implantation and were euthanized. Embodiment 26 Evaluation of multiple combination therapies in mouse colorectal cancer models

[0481] Female Balb / c mice (n=5 / cohort) aged 6-8 weeks were implanted subcutaneously with 2-5×10 6CT-26 mouse colorectal cancer cell line cells (day 0). On day 11 after implantation, treatment was initiated with the following test articles: (a) PBS alone at 100 μl / mouse; (b) chemotherapy (carboplatin + paclitaxel); (c) 10 nmol / mouse of compound 150 + anti-PD1; (d) 10 nmol / mouse of compound 150 (+Abs); (e) 10 nmol / mouse of compound 150 + chemotherapy; and (f) SOC (leucovorin + 5FU + oxaliplatin). The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study. Tumors were measured every other day.

[0482] Fig.56 Tumor volume (mm 3 ) versus days after tumor implantation. Four of five animals in the combination therapy Compound 150 + anti-PD1 cohort lost their tumors. Three of five animals in the combination therapy Compound 150 + chemotherapy cohort lost their tumors. Two of five animals in the chemotherapy (alone) cohort lost their tumors. The data confirm that the combination of Compound 150 and chemotherapy performed better than chemotherapy alone, and the combination of Compound 150 and anti-PD1 also performed better than SOC.

[0483] A second study was conducted using the same protocol as above with the following test articles: (i) PBS alone at 100 μl / mouse; (ii) leucovorin + 5FU + OXH (SOC); (iii) 10 nmol / mouse of compound 150 + sunitinib; (iv) 10 nmol / mouse of compound 150 (+Abs); and (v) 10 nmol / mouse of compound 150 + folate-TLR7 conjugate. The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study.

[0484] Fig.57 Tumor volume (mm 3 ) versus days after tumor implantation. One of five animals in the combination therapy treatment cohort Compound 150 + sunitinib cohort lost the tumor. One of five animals in the combination therapy Compound 150 + FA-TLR7 cohort lost the tumor. The data confirm that the combination therapy of Compound 150 with sunitinib or FA-TLR7 significantly improved the anti-tumor efficacy of the treatment.

[0485] Fig.58 The graphs shown compare Fig.56 and Fig.57Data were collected to highlight the difference between the combination therapy of compound 150 with anti-PD1 / sunitinib / chemotherapy and SOC treatment alone. Embodiment 27 Comparison of in vivo efficacy of compound 150 in mouse lung cancer model Y856 (cold tumor model)

[0486] Female C57BL / 6 mice aged 6-8 weeks (n=5 / cohort) were implanted subcutaneously with 2×10 6 Y856 mouse lung cancer cell line cells (day 0). On day 11 after implantation, treatment was started with the following test articles: (a) PBS (100 μl / mouse IV) + anti-DNP / anti-rhamnose antibody; (b) anti-PD1 (200 μg / mouse, IP, 3 days / week) + carboplatin (18 days after tumor inoculation, IP, 50 mg / kg) + paclitaxel (once a day from day 18-22 after tumor inoculation, IV, 36 mg / kg); or (c) FDR (Compound 150) (10 nmol / mouse, once a day) + anti-DNP / anti-rhamnose antibody.

[0487] Tumors were measured every other day, and mice were sacrificed on day 25 after tumor implantation. Fig.59 Tumor volume (mm 3 ) versus days after tumor implantation. The PBS and compound 150 groups were also injected intraperitoneally with anti-DNP and anti-rhamnose antibodies on days 10, 14, 17, and 21 to achieve humanized titers of anti-hapten antibodies throughout the study period. Embodiment 28 In vivo efficacy study of compound 150 in a mouse renal cancer model

[0488] Female Balb / c mice aged 6-8 weeks were implanted subcutaneously with 2×10 6 Renca renal cancer cell line cells. Treatment with the following test articles began 12 days after implantation: (a) PBS (100 μl / mouse, IV) + anti-DNP / anti-rhamnose antibody; (b) anti-PD1 (200 μg / mouse, IP, 3 days / week) / PD-L1 (200 μg / mouse, IP, 3 days / week) + sunitinib (20 mg / kg, OG, once a day); or (c) FDR (Compound 150) (10 nmol / mouse, once a day) + anti-DNP / anti-rhamnose antibody, one dose per day for each mouse.

[0489] Tumors were measured every other day, and mice were sacrificed on day 25 after tumor implantation. Fig.60 Tumor volume (mm 3 ) and the relationship between the number of days after tumor implantation. Embodiment 29 Immune response assessment

[0490] Balb / c mouse tumors from the above studies using Y856, LLC-1, and M109 cell lines were minced and digested using a tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Single cell suspensions were washed, fixed, and stained for the corresponding immune cell markers and analyzed by flow cytometry. Fig.61 A comparison of immune cell populations in Y856, LLC-1 and M109 tumors measured following the above evaluations is shown. (FDH = Compound 150, SOC is anti-PD1 + chemotherapy (carboplatin + paclitaxel)).

[0491] Compound 150 treatment resulted in a significant increase in anti-tumor macrophage and natural killer (NK) cell populations, while reducing the number of tumor-associated macrophages (TAMs) in a hot tumor model (M109), but not in a cold tumor model (Y8569 and LLC-1). The data confirm that compound 150 monotherapy can induce macrophage reprogramming, thereby reducing the number of TAMs and enhancing the NK cell population.

Claims

1. A conjugate having the following formula: <h2 style=";text-align:left;direction:ltr">TL-LH<h2 style=";text-align:left;direction:ltr"> n or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell, or fibroblast; L is the connector; H is a hapten; and n is an integer of 2-3; and optionally, wherein at least two of said H can each bind to a different antibody when contacted therewith.

2. The conjugate according to claim 1, wherein at least two of said H are each bound by an antibody.

3. The conjugate according to claim 1 or 2, wherein each H is bound by a different antibody.

4. The conjugate according to any one of claims 1 to 3, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof. The conjugate according to claim 1 , wherein n is 2. The conjugate according to claim 1 , wherein n is 3.

7. The conjugate of any one of claims 1-3, 5 or 6, wherein each H is independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus.

8. The conjugate according to any one of claims 1 to 3, wherein n is 2, the first H is a DNP fragment, and the second H is a rhamnose fragment.

9. The conjugate of claim 1, wherein at least one H is an influenza virus antigen selected from hemagglutinin and neuraminidase.

10. The conjugate according to claim 1, wherein at least one H is a hepatitis virus antigen selected from L-HBsAg, S-HBsAg, M-HBsAg and preS.

11. The conjugate of claim 1, wherein at least one H is gp120 or gp160.

12. The conjugate of claim 1, wherein at least one H is a glycoprotein.

13. The conjugate according to any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell.

14. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is influenza virus neuraminidase or influenza virus hemagglutinin.

15. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is respiratory syncytial virus fusion protein F.

16. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is a coronavirus spike protein.

17. The conjugate according to any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is hepatitis B virus surface antigen or HBV core antigen.

18. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is a cell surface receptor on a cancer cell.

19. The conjugate of claim 1, wherein the target protein is a folate receptor.

20. The conjugate of claim 19, wherein the target protein is folate receptor alpha or folate receptor beta.

21. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is prostate specific membrane antigen.

22. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is carbonic anhydrase 9.

23. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is luteinizing hormone releasing hormone receptor.

24. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is a neurokinin 1 receptor.

25. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

26. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is a cell surface receptor on myeloid-derived suppressor cells.

27. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

28. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the target protein is fibroblast activation protein.

29. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the targeting ligand is a neuraminidase inhibitor.

30. The conjugate of any one of claims 1-3, 5, 6 or 9-12, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment or a laninamivir fragment.

31. The conjugate of claim 1, wherein the targeting ligand is a zanamivir fragment.

32. The conjugate of any one of claims 1-3, 5, 6, 9-12, 19 or 20, wherein the targeting ligand is a folate fragment or an analog thereof.

33. The conjugate of any one of claims 1-3, 5, 6, 9-12, 19 or 20, wherein the targeting ligand is 5-methyltetrahydrofolate.

34. The conjugate of any one of claims 1-3, 5, 6, 9-12, 19, 20 or 31, wherein L comprises (-CH2CH2-O-) n , peptide, peptidoglycan or a combination of two or more of the above, wherein n is an integer between and including 1 and 32.

35. The conjugate of any one of claims 1-3, 5, 6, 9-12, 19, 20 or 31, wherein L is a branched linker and at least two of the haptens are attached to different branches of the linker, wherein the different branches optionally extend from different atoms of the linker.

36. The conjugate of claim 1, wherein the targeting ligand is a folate fragment or a derivative thereof, at least the first H comprises a rhamnose fragment, and at least the second H comprises a dinitrophenyl fragment.

37. The conjugate according to any one of claims 1 to 36, formulated as a prodrug.

38. A conjugate having the formula or a pharmaceutically acceptable salt thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell, or fibroblast; L a , L b and L c Each is a linker, which may be the same or different; C is a carbon atom; R4 is selected from hydrogen, C1-C5 alkyl, C1-C5 alkenyl or C1-C5 alkynyl groups; H1 and H2 are each haptens; and Optionally, H1 and H2 can each bind to a different antibody.

39. A conjugate having the formula and pharmaceutically acceptable salts thereof, wherein: TL is a targeting ligand for a target protein on the surface of a virus, virus-infected cell, cancer cell, immune cell, or fibroblast; L a , L b , L c and L d Each is a linker, which may be the same or different; C is a carbon atom; H1, H2, and H3 are each haptens; and Optionally, each of H1, H2 and H3 can each bind a different antibody.

40. The conjugate of claim 38, wherein H1 and H2 are each bound by an antibody.

41. The conjugate of claim 39, wherein H1, H2 and H3 are each bound by an antibody.

42. The conjugate of claim 38 or 40, wherein H1 and H2 are each independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment, or a combination thereof.

43. The conjugate of claim 39 or 41, wherein H1, H2 and H3 are each independently selected from a rhamnose fragment, an α-galactosyl moiety, a dinitrophenyl fragment, a trinitrophenyl fragment or a combination thereof.

44. The conjugate of claim 38 or 40, wherein H1 or H2 is each independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus.

45. The conjugate of claim 38 or 41, wherein H1, H2 or H3 is each independently selected from a rhamnose fragment, an α-galactosyl moiety, a DNP fragment, a TNP fragment, fluorescein, digoxigenin, biotin or a viral antigen selected from diphtheria, herpes zoster virus, human papillomavirus, influenza virus, SARS-COV-2, yellow fever virus, respiratory syncytial virus, herpes simplex virus, varicella virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis G virus, rotavirus, mumps virus, tetanus, human immunodeficiency virus, cytomegalovirus, vesicular stomatitis virus, rubella virus, smallpox virus, monkeypox virus, polio virus, dengue virus and measles virus.

46. ​​The conjugate of claim 38 or 41, wherein H1 is a DNP fragment and H2 is a rhamnose fragment.

47. The conjugate of claim 38 or 39, wherein at least one hapten is an influenza virus antigen selected from hemagglutinin and neuraminidase.

48. The conjugate of claim 38 or 39, wherein at least one hapten is a hepatitis virus antigen selected from L-HBsAg, S-HBsAg, M-HBsAg and preS.

49. The conjugate of claim 38 or 39, wherein at least one hapten is gp120 or gp160.

50. The conjugate of claim 38 or 39, wherein at least one hapten is a glycoprotein.

51. The conjugate of any one of claims 38-41, wherein the target protein is an envelope protein of a virus or a viral envelope protein on the surface of a virus-infected cell.

52. The conjugate of any one of claims 38-41, wherein the target protein is influenza virus neuraminidase or influenza virus hemagglutinin.

53. The conjugate of any one of claims 38-41, wherein the target protein is respiratory syncytial virus fusion protein F.

54. The conjugate of any one of claims 38-41, wherein the target protein is a coronavirus spike protein.

55. The conjugate according to any one of claims 38-41, wherein the target protein is hepatitis B virus surface antigen or HBV core antigen.

56. The conjugate of any one of claims 38-41, wherein the target protein is a cell surface receptor on a cancer cell.

57. The conjugate of any one of claims 38-41, wherein the target protein is a folate receptor.

58. The conjugate of any one of claims 38-41, wherein the target protein is folate receptor alpha or folate receptor beta.

59. The conjugate of any one of claims 38-41, wherein the target protein is prostate specific membrane antigen.

60. The conjugate of any one of claims 38-41, wherein the target protein is carbonic anhydrase 9.

61. The conjugate of any one of claims 38-41, wherein the target protein is luteinizing hormone releasing hormone receptor.

62. The conjugate of any one of claims 38-41, wherein the target protein is the neurokinin 1 receptor.

63. The conjugate of any one of claims 38-41, wherein the target protein is a cell surface receptor on tumor-associated macrophages.

64. The conjugate of any one of claims 38-41, wherein the target protein is a cell surface receptor on myeloid-derived suppressor cells.

65. The conjugate of any one of claims 38-41, wherein the target protein is a cell surface receptor on cancer-associated fibroblasts.

66. The conjugate of any one of claims 38-41, wherein the target protein is fibroblast activation protein.

67. The conjugate of any one of claims 38-41, wherein the targeting ligand is a neuraminidase inhibitor.

68. The conjugate of any one of claims 38-41, wherein the targeting ligand is an oseltamivir fragment, a zanamivir fragment, a peramivir fragment, or a laninamivir fragment.

69. The conjugate of any one of claims 38-41, wherein the targeting ligand is a zanamivir fragment.

70. The conjugate of any one of claims 38-41, wherein the targeting ligand is a folate fragment or an analog thereof.

71. The conjugate of any one of claims 38-41, wherein the targeting ligand is 5-methyltetrahydrofolate.

72. The conjugate of claim 39, wherein L a , L b , L c and L d At least one of each independently comprises: (-CH2CH2-O-) n , where n is an integer between 1 and 32, Alkyl groups, Peptides, Peptidoglycan, or A combination of two or more of the above.

73. The conjugate of claim 38, wherein L a , L b and L c At least one of each independently comprises: (-CH2CH2-O-) n , where n is an integer between 1 and 32, Alkyl groups, Peptides, Peptidoglycan, or A combination of two or more of the above.

74. The conjugate of claim 72 or 73, wherein n is an integer between and including 1 and 16.

75. The conjugate of claim 39 or 41, wherein L a , L b and L c and L d At least one of the peptide fragments or peptidoglycan fragments.

76. The conjugate of claim 39 or 40, wherein L a , L b and L c At least one of the peptide fragments or peptidoglycan fragments.

77. The conjugate of claim 39 or 41, wherein L a , L b and L c , and L d Each independently contains C2-C 18 Alkyl group.

78. The conjugate of claim 38 or 40, wherein L a , L b and L c Each independently contains C2-C 18 Alkyl group.

79. A conjugate of the formula: or a pharmaceutically acceptable salt thereof.

80. A conjugate of the formula: or a pharmaceutically acceptable salt thereof.

81. The conjugate of claim 79 or 80, wherein one or more of the -OH groups are independently substituted with a thiol, a phosphate or a phosphonate.

82. The conjugate of claim 79 or 80, wherein one or more of the -OH groups are substituted with -OC(=O)R, wherein R is an alkyl group.

83. The conjugate of claim 79 or 80, wherein one or more of the -OH groups are substituted with -OC(=O)R, wherein R is a C1-C6 alkyl group.

84. The conjugate of claim 79 or 80, wherein the amino (-NH2) group is substituted with -OC(=O)R2, and R2 is an alkyl group.

85. The conjugate of claim 79 or 80, wherein the amino (-NH2) group is substituted with -OC(=O)R2, and R2 is a C1-C6 alkyl group.

86. The conjugate of claim 79 or 80, wherein the carboxyl (-COOH) group is substituted with -OC(=O)R3, wherein R3 is an alkyl group.

87. The conjugate of claim 79 or 80, wherein the carboxyl (-COOH) group is substituted with -OC(=O)R3, wherein R3 is a C1-C6 alkyl group.

88. A conjugate of the formula: or a pharmaceutically acceptable salt thereof, wherein L1, L2 and L3 are linkers.

89. The conjugate of claim 88, wherein one or more of L1, L2, and L3 comprises (-CH2CH2-O-) n , where n is an integer between 1 and 16, inclusive.

90. The conjugate of claim 88 or 89, wherein L1, L2 and L3 each independently comprise C2-C 18 Alkyl groups, peptide fragments or peptidoglycan fragments.

91. A conjugate of the formula: or a pharmaceutically acceptable salt thereof.

92. The conjugate of any one of claims 79 to 91, further complexed with one or more antibodies in vivo.

93. A pharmaceutical composition comprising the conjugate of any one of claims 1-91 and a pharmaceutically acceptable excipient.

94. A method of treating a viral infection in a subject, comprising administering to the subject an effective amount of the conjugate of any one of claims 1-91 or the pharmaceutical composition of claim 93.

95. The method of claim 94, further comprising administering to the subject an autologous antibody or an allogeneic immunoglobulin G (IgG) antibody.

96. The method of claim 94, wherein the viral infection is influenza.

97. The method of any one of claims 94-96, wherein the conjugate or the pharmaceutical composition is administered orally.

98. The method of claim 94, wherein the conjugate or the pharmaceutical composition is administered once a day.

99. The method of claim 94, wherein the conjugate or the pharmaceutical composition is administered more than once a day.

100. The method of claim 94, wherein the conjugate or the pharmaceutical composition is administered twice daily.

101. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the conjugate of any one of claims 1-91 or the pharmaceutical composition of claim 93.

102. The method of claim 101, further comprising administering autologous antibodies or allogeneic IgG antibodies to the subject.

103. The method of claim 101, wherein the cancer is a thermal cancer.

104. The method of claim 101, wherein the cancer is renal cancer, lung cancer, or colorectal cancer.

105. The method of any one of claims 94-104, wherein the conjugate or the pharmaceutical composition is administered orally or intravenously.

106. The method of any one of claims 94-104, wherein the conjugate or the pharmaceutical composition is administered once a day.

107. The method of any one of claims 94-104, further comprising administering to the subject a second therapy, wherein the second therapy comprises chemotherapy, sunitinib, a PD-1 inhibitor, or a PDL-1 inhibitor.

108. A method of activating an immune response in a subject, comprising administering to the subject an effective amount of the conjugate of any one of claims 1-91 or the pharmaceutical composition of claim 93.

109. The method of claim 108, wherein the immune response is an innate immune response.

110. The method of claim 108, wherein the immune response is activated in a targeted region of the subject, wherein the targeted region is a tumor microenvironment or a location of a viral replication site.

111. The method of any one of claims 108-110, further comprising administering to the subject an autologous antibody or an allogeneic IgG antibody.

112. The method of claim 110, wherein administering an effective amount of the conjugate or the pharmaceutical composition induces reprogramming of M2 macrophages to M1 macrophages in the targeted area.

113. The conjugate of claim 1, wherein at least two of said H's can each bind to a different antibody when contacted with an antibody in vivo.

114. The conjugate of claim 1, wherein the two H's can each bind to a different antibody when contacted with an antibody in vitro.

Images