WO2017197624A1 - Ligands de tlr7 à squelette de purine et leur conjugué - Google Patents

Ligands de tlr7 à squelette de purine et leur conjugué Download PDF

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WO2017197624A1
WO2017197624A1 PCT/CN2016/082643 CN2016082643W WO2017197624A1 WO 2017197624 A1 WO2017197624 A1 WO 2017197624A1 CN 2016082643 W CN2016082643 W CN 2016082643W WO 2017197624 A1 WO2017197624 A1 WO 2017197624A1
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tlr7
cells
conjugates
tumor
compound
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PCT/CN2016/082643
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English (en)
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Dong GAO
Wang Li
Yanyuan CHEN
Chihchang WEI
Yuhuan Wang
Xiaoling Luo
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Shenzhen Hornetcorn Biotechnology Company,Co., Ltd
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Priority to PCT/CN2016/082643 priority Critical patent/WO2017197624A1/fr
Publication of WO2017197624A1 publication Critical patent/WO2017197624A1/fr

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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
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Definitions

  • the invention described herein was made with Shenzhen Hornetcorn Biotechnology Co., Ltd. support.
  • the Shenzhen Hornetcorn Biotechnology Co., Ltd. has certain rights in the invention.
  • TLRs Toll like receptors
  • B cells B cells
  • specific types of T cells DCs and macrophages.
  • Some synthetic small molecules can activate certain TLR pathways.
  • TLR ligands can control the activation of dendritic cells (DCs) , trigger the maturation program of DCs and lead to the secretion of proinflammatory cytokines.
  • DCs dendritic cells
  • TLR7 agonist imiquimod
  • BCG mixed TLR2/TLR4 agonist
  • TLR4 agonist glucopyranosyl lipid adjuvant
  • TLR7 can be activated by synthetic small molecule agonists
  • several synthesized low molecular weight TLR7 agonists were found, including imidazoquinolines and purine like molecules, to activate immune cells via the TLR7-MyD88 dependent signaling pathway.
  • Imiquimod is being successfully used for the treatment of many primary skin tumors and cutaneous metastases as the single antitumor agent with immunostimulatory capacity.
  • TLR7 ligands used as single agents or conjugated to tumor antigens can effectively eradicate tumors due to their potent stimulation of innate and adaptive immunity.
  • TLR7 ligands were able to stimulate innate immune cells to release cytokines at the very high level compared with free format at the same or lower concentration.
  • Locally intratumoral conjugates injection can elicit a systemic antitumor effect on murine breast tumor model.
  • TLR7 ligands affected the frequency of intratumoral immune cell infiltration, including the percentage of CD4 + and CD8 + increase, and the ratio of Tregs decrease.
  • Our data reveal that the antitumor effect of TLR7 ligands is associated with multiple mechanisms, inducing tumor specific immune response, activation of innate immune cells and modulation of the tumor microenvironment.
  • intratumoral administration of TLR7 ligands conjugates can active the immune system, change the tumor microenvironment and delay distant tumor growth.
  • the present invention provides for the synthetic TLR7 potent ligands and the conjugates of synthetic TLR7 ligands linked via a stable covalent bond to a macromolecule and compositions having those conjugates, as well as methods of using the compounds and conjugates.
  • the conjugates may include macromolecules directly linked to a synthetic TLR7 ligands, or linked via a linker to the TLR7 ligands, for instance, linked via an amino group, a carboxy group or a succinamide group.
  • the conjugates of the invention include a synthetic TLR7 weak ligands covalently bound to a macromolecule such as, for instance, a peptide, polypeptide, or an antigen binding fragment thereof, lipid, a polymer such as polyethylene glycol, or dendrimer.
  • the conjugates of the invention are broad-spectrum, long-lasting, and non-toxic synthetic immunostemulatory agents, which are useful for activating the immune system of a mammal, e.g., a human.
  • the conjugates of the invention optimize the immune response while limiting undesirable systemic side effects associated with un-conjugated TLR7 weak ligands.
  • the synthetic TLR7 ligands may help direct the conjugate to TLR7 within the endosomes of target cells and enhance delivery of the macromolecule.
  • the synthetic TLR7 ligands may enhance the response to the macromolecule (e.g., immune response) .
  • the macromolecule may be useful for activating the immune system and/or may direct the conjugate to particular cells.
  • the macromolecule e.g., one with a primary amino group that is linked to synthetic TLR7 ligands, may enhance the activity of the synthetic TLR7 ligands or have a separate desirable activity.
  • the macromolecule may enhance the activity of the TLR7 weak ligands by helping to direct the ligands to the TLR7 within the endosomes of target cells, by enhancing signal transduction induced by the TLR7 weak ligands, or by cross-linking the receptor, or any combination thereof.
  • Vaccines are not used in acute settings because they take too long to act and they are not effective in immunocompromised patients.
  • the invention provides a method for the rapid vaccination of patients at-risk for bacterial infection or tumor, which employs TLR7 ligands and one or more antigens.
  • the use of the vaccines of the invention induces immunity in about a few days, which provides for applications not amenable to standard vaccination protocol.
  • an immunogenic composition of the invention includes a series of synthetic TLR7 ligands, e.g., compound 6, coupled to a protein.
  • synthetic TLR7 ligands may be coupled to bacterial carbohydrates using methods to attach antigen to protein carriers.
  • an immunogenic composition of the invention includes synthetic TLR7 ligands coupled to an adjuvant and preparation comprising a recombinant antigen, such as protein or a peptide thereof.
  • a single dose of the immunogenic composition may show very potent activity, e.g., provide protective immunity.
  • conjugates of synthetic TLR7 ligands of the invention is advantageous as accessible and versatile chemistry permits conjugation to any antigen, and modifiable conjugates have defined stoichiometry.
  • the conjugates are inexpensive to prepare and are potent, and so provide rapid protection, enabling use acute settings such as trauma, burn, pre-surgery, bio-terrorism or tumor therapy.
  • X is -O-, -S-, or -NR-;
  • R is (C 1-10 ) alkyl, substituted (C 1-10 ) alkyl, C 6-10 aryl, or substituted C 6-10 aryl, C 5-9 heterocyclic, substituted C 5-9 heterocyclic.
  • n 1, 2, 3, 4 alkyl or benzyl
  • A is a macromolecule
  • A can be a macromolecule comprising an adjuvant such as a heterologous or a host cell protein or peptide.
  • an adjuvant such as a heterologous or a host cell protein or peptide.
  • the macromolecule can include organic molecules, composed of carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous, or any combination thereof, so long as the macromolecule is not harmful to body tissues.
  • the macromolecule may permit targeting or enhance the immune response, e.g., the macromolecule may be an antigen such as a melanoma-specific peptide.
  • each A when more than one A is present in a molecule of any formula described herein, each A may be the same, or the A groups may be different from each other. Accordingly, when more than one A group is present, each A is independently a group as defined for each formula.
  • the invention includes the use of conjugates of synthetic TLR7 ligands and a macromolecules, as well as TLR7 ligands conjugates and another molecule.
  • the conjugate may be useful to prevent, inhibit or treat disorders including, but not limited to, allergic asthma, infectious diseases such as respiratory viral infections, or tumor therapy, and as a vaccine, for cancer or infectious diseases.
  • a single dose of the conjugate may show very potent activity in stimulating the immune response.
  • higher doses may be administered, systemically, while under other circumstances lower doses may be administered, due to localization of the conjugate.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one compound of formula, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable diluents or carrier.
  • the invention provides a method to prevent or inhibit an infection in a mammal.
  • the method includes administering to the mammal an effective amount of a composition comprising a protein or peptide and an amount of a compound having formula (II) .
  • n 1, 2, 3, 4 alkyl or benzyl
  • A is a macromolecule
  • A can be a macromolecule comprising an adjuvant such as a heterologous or a host cell protein or peptide.
  • each A when more than one A is present in a molecule of any formula described herein, each A may be the same, or the A groups may be different from each other, Accordingly, when more than one A group is present, each A is independently a group as defined for each formula.
  • Reagents and conditions (i) NH 3 -MeOH, 80°C; (ii) Methyl 3-bromopropionate, methyl 4-bromobutyrate, methyl 5-bromovalerate, methyl 6-bromohexanoate, or Methyl 4- (bromomethyl) benzoate, K 2 CO 3 /dimethylformamide/KI, 30 °C, (iii) CH 3 O (CH 2 ) 3 ONa/CH 3 O (CH 2 ) 3 OH, reflux, (iv) Br 2 /CH 2 Cl 2 , 30 °C, (v) MeOH /10 M NaOH, 1: 1, reflux.
  • FIG. 2 Conjugation of compound to protein or peptide.
  • Reagents and conditions (vi) 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDCI) , NHS, DMSO, 25°C; (vii) DMSO or PBS, 4°C.
  • FIG. 3 TLR7/8 Activities and Cytokine Induction.
  • Addition of media with no drug The data are mean ⁇ SEM of triplicate in the representative data of three independent experiments.
  • FIG. 4 The Ratio of Cytokine Production treated with Conjugated vs Un-conjugated Compound 6.
  • Cytokine production was measured in triplicate with compound 6 at 10 ⁇ M, conjugates of ovalbumin (OVA) , bovineserum albumin (BSA) , mouse serum albumin (MSA) , monoclonal gastric cancer7 antigen (MG7) and thymosin at 1.7, 2, 2, 10 and 2.5 ⁇ M considering their various conjugate ratio, respectively.
  • OVA ovalbumin
  • BSA bovineserum albumin
  • MSA mouse serum albumin
  • MG7 monoclonal gastric cancer7 antigen
  • thymosin thymosin
  • FIG. 5 The Ratio of Cytokine Production treated with Conjugated vs. Un-conjugated Antigen.
  • Cytokine production was measured in triplicate with conjugates of OVA, BSA, MSA, MG7 and thymosin or antigen alone at 1.7, 2, 2, 10 and 2.5 ⁇ M, respectively.
  • Antigen alone, cytokine production less 30 pg/mL was defined as “1” .
  • the data are mean of triplicate in the representative data of three independent experiments.
  • FIG. 6 General synthetic route for compound 6a. Reagents and conditions: (i) NH 3 -MeOH, 80°C; (ii) Methyl 3-bromopropionate, K 2 CO 3 /dimethylformamide/KI, 30 °C, (iii) CH 3 O (CH 2 ) 3 ONa/CH 3 O (CH 2 ) 3 OH, reflux, (iv) Br 2 /CH 2 Cl 2 , 30 °C, (v) MeOH /10 M NaOH, 1: 1, reflux.
  • FIG. 7 Conjugation of compound 6a to protein or peptide.
  • Reagents and conditions (vi) 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDCI) , NHS, DMSO, 25°C; (vii) DMSO or PBS, 4°C.
  • FIG. 8 In vitro cytokine release enhancement in response to protein conjugates.
  • Mouse splenocyte were treated with 6e or 6a-OVA, -BSA, -MSA conjugates at various concentrations as indicated.
  • Culture supernatants were harvested 24 h later, and cytokine levels were measured by ELISA.
  • Compound 6e was used as a control.
  • Data are means ⁇ SEM and a representative experiment of three independent experiments in triplicate per treatment is shown.
  • FIG. 9 In vitro cytokine release enhancement in response to peptide conjugates.
  • FIG. 10 Cytokine ratio in vitro treated with conjugated vs. un-conjugated antigens, 1 ⁇ 10 6 /ml cells were incubated with serially diluted TLR7 conjugates or un-conjugates for 24 h. The levels of IL-12 and IFN- ⁇ in the culture supernatants were measured by ELISA. Data are ratio of cytokine production in vitro mouse splenocyte treated with conjugated vs. un-conjugated antigens.
  • FIG. 11 Synthesis of compound 6b. Reagents and conditions: (i) NH 3 -MeOH, 80°C; (ii) Methyl 3-bromopropionate, K 2 CO 3 /dimethylformamide/KI, 30 °C, (iii) CH 3 O (CH 2 ) 3 ONa/CH 3 O (CH 2 ) 3 OH, reflux, (iv) Br 2 /CH 2 Cl 2 , 30 °C, (v) MeOH /10 M NaOH, 1: 1, reflux.
  • FIG. 12 Conjugation of compound 6b to protein or peptide.
  • Reagents and conditions (vi) 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDCI) , NHS, DMSO, 25°C; (vii) DMSO or PBS, 4°C.
  • FIG. 13 Confirmation of conjugate-mediated stimulation via TLR7 activation.
  • BMDM (1 ⁇ 10 6 /ml) derived from wild-type or TLR7 -/- mice was incubated with 10 ⁇ M compound 6b or 2 ⁇ M conjugates for 24 h.
  • the levels of IL-12 in the culture supernatants were measured by ELISA.
  • the data are mean ⁇ SEM of triplicates and are representative of three independent experiments.
  • FIG. 14 In vitro cytokine release in response to free drugs, antigens, or conjugates. 1 ⁇ 10 6 /ml mouse splenocyte cells were incubated with serially diluted free compound 6b or 6e as indicated at x-axis, respectively. Considering that the conjugation ratios are different in each antigen, the concentrations of BSA, OVA, MSA, MG7, thymosin and their conjugates were used with one-sixth, -fifth, -fifth, equal or a quarter of the number as indicated at x-axis, respectively. The levels of IFN- ⁇ (A) and IL-12 (B) in the culture supernatants were measured by ELISA. The data are mean ⁇ SEM of triplicates and are representative of three independent experiments. The colored numbers indicate the cytokine concentration ratios for conjugated vs free compound 6b.
  • FIG. 15 Kinetics of proinflammatory cytokine induction by the different conjugates and free drugs in vivo.
  • FIG. 16 Kinetics of IgG induction by 6b-BSA or 6b-OVA in vivo.
  • Serum samples were collected at 7-day intervals from days 0 to 30.
  • BSA-or OVA-specific IgG1 (A) and IgG2a (B) were measured by ELISA. The data are mean ⁇ SEM of triplicate in the representative data of three independent experiments.
  • Fig. 17 Schematic illustration on the synthesis of Compound 6e.
  • Reagents and conditions (i) NH 3 -MeOH, 80°C; (ii) Methyl 4- (bromomethyl) benzoate, K 2 CO 3 /dimethylformamide/KI, 30 °C, (iii) CH 3 O (CH 2 ) 3 ONa/CH 3 O (CH 2 ) 3 OH, reflux, (iv) Br 2 /CH 2 Cl 2 , 30 °C, (v) MeOH /10 M NaOH, 1: 1, reflux.
  • Fig. 18 Compound 6e conjugated to OCT4. Reagents and conditions: 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide (EDCI) , NHS, DMSO, 25°C for 12 h; then the mid-compound added into the protein or peptide solution at 4 °C for 12 h.
  • EDCI 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide
  • Fig. 19 In vitro cytokine release in response to 6e-OCT4 conjugates.
  • Total murine spleen lymphocytes were isolated and treated with 6e agonist or 6e conjugates at various concentrations as indicated. Culture supernatants were harvested 24 h later, and cytokine levels were measured by immunoassay. The results are a representative of at least two separate experiments in triplicate per treatment.
  • (a and b) Levels of IL-12 and IFN- ⁇ released by spleen lymphocytes treated with 6e agonist, M2e peptide and 6e-M2e conjugate.
  • (c and d) Levels of IL-12 and IFN- ⁇ released by spleen lymphocytes treated with 6e agonist, MG7 peptide and 6e-MG7 conjugate.
  • Fig. 20 6e-OCT4 conjugates inhibit xenograft tumor growth
  • Fig. 21 6e-OCT4 vaccine induced tumor-specific immune responses.
  • Mice were i. p. immunized with different vaccines on Day 1, 14, 28 and 42.
  • Three days after last immunization a: Splenocytes were isolated and total splenocytes were re-stimulated with 10 ⁇ g/ml OCT4 antigen for 24 h, and the production of IFN- ⁇ in culture supernatants was measured using ELISA.
  • b Total splenocytes were labelled with PE-anti-mouse CD8 and FITC-anti-mouse CD3, and the percentages of CD8 + /CD3 + T cells in total splenocytes were measured using flow cytometry.
  • FIG. 22 In vitro cytokine release in response to compound 6e.
  • BMDC BMDC
  • B NK cells
  • C Balb/c mice
  • D RAW264.7
  • Cytokine levels were determined by ELISA. The data are mean ⁇ SD.
  • FIG. 23 Compound 6e induced the expression of TLR in 4T1 cells and BMDC, and activation of NF- ⁇ B via TLR7.4T1 cells (A) and BMDC (B) were stimulated with 6e from 0 to 50 ⁇ M for 12 h.
  • BMDC (C) were treated with 0 and 10 ⁇ M 6e to detect the expression of P65 and I ⁇ B ⁇ .
  • Cell lysates were assessed by western blotting using the primary antibodies: I ⁇ B ⁇ , NF- ⁇ Bp65 and mTLR7.
  • FIG. 24 In vivo antitumor efficiency of compound 6e. After 30 days, mice from each group were scarified and the weight of spleen (A) and tumors (B and C) was measured. The data are mean ⁇ SD. Data are representative of three independent experiments. *P ⁇ 0.05, **P ⁇ 0.01, respectively.
  • FIG. 25 Compound 6e induces systemic cellular immune response.
  • A Total splenocytes were labeled with APC-anti-mouse CD8 and FITC-anti-mouse CD3, and the percentages of CD8 + /CD3 + T cells in total splenocytes were measured.
  • FIG. 26 Compound 6e induces humoral immune response.
  • A Anti-4T1 antibody titers.
  • B Induction of antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • C IgG1 titers in serum.
  • FIG. 27 Compound 6e effect on the frequency of intratumoral immune cell infiltration.
  • CD25 + Foxp3 + CD4 + T cells were gated and analyzed by FCM. Total tumor infiltrated T cells were labeled with PE-anti-mouse CD4, APC-anti-mouse CD25 and FITC-anti-mouse Foxp3.
  • the protein have one or more polypeptides.
  • a composition is comprised of “substantially all” of a particular compound, or a particular form a compound when a composition comprises at least about 90%, and preferably at least about 95%, 99%, and 99.9%of the particular composition on a weight basis.
  • amino acid as used herein, comprises the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pre, Ser, Thr, Tpr, Tyr, and Val) in D or L form.
  • natural amino acids e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pre, Ser, Thr, Tpr, Tyr, and Val
  • TLR toll-like receptor
  • TLR7 synthetic TLR7 ligands are chemical compounds that are designed to bind to TLR7 and activate the receptor.
  • Exemplary synthetic TLR7 ligands provided herein include “TLR7 agonist” , “TLR8 agonist” and “TLR-9 agonist” .
  • TLR7 agonists include imiquemod, resiquimod, broprimine and loxoribine.
  • TLR7 weak ligands include compound 6a-6d.
  • the pharmaceutically acceptable salts of the compounds useful in the present invention can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
  • “Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated by the present invention.
  • Vaccines are not generally used in acute settings, because they take too long to act and they are not effective in immune compromised patients. Moreover, many strains of hospital acquired infection are resistant to conventional antibiotics.
  • compositions including synthetic TLR7 agonists and conjugates with synthetic TLR7 agonists, in combination with antigens is provided herein.
  • TLR7 agonists generally have poor pharmacokinetics, and rapid systemic absorption and excretion. Due to systemic dispersal, they result in sytokine syndrome. Effective adjuvants must create an “immune gradient” of cytokines and chemokines.
  • conjugation of potent synthetic TLR7 agonists to macromolecules enhances delivery properties, improves pharmacokinetics, and avoids systemic toxicity by localized exposure.
  • a synthetic TLR7 agonist is administered with —COOH conjugated to antigens.
  • the vaccines of the invention may unexpectedly provide a rapid and effective immune response.
  • the invention provides the following compounds and conjugates.
  • the invention provides a method to prevent or inhibit an infection or cancer in a mammal.
  • the method includes administering to the mammal an effective amount of a composition comprising a protein or peptide and an amount of a compound having formula (I) .
  • X is -O-, -S-, or -NR-;
  • Y is S or NH
  • R is (C 1-10 ) alkyl, substituted (C 1-10 ) alkyl, C 6-10 aryl, or substituted C 6-10 aryl, C 5-9 heterocyclic, substituted C 5-9 heterocyclic;
  • n 1, 2, 3, 4 alkyl or benzyl
  • A is a macromolecule
  • A can be a macromolecule comprising an adjuvant such as a heterologous or a host cell protein or peptide.
  • the macromolecule I the conjugates of the invention forms a stable bond with the TLR7 weak ligands, i.e., the conjugate does not act as a prodrug.
  • the macromolecule can include organic molecules, composed of carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorous, or any combination thereof, so long as the macromolecule is not harmful to body tissues.
  • the macromolecule may permit targeting or enhance the immune response, e.g., the macromolecule may be an antigen such as a melanoma-specific peptide.
  • each A when more than one A is present in a molecule of any formula described herein, each A may be the same, or the A groups may be different from each other, Accordingly, when more than one A group is present, each A is independently a group as defined for each formula.
  • the invention provides a method to prevent or inhibit an infection or cancer in a mammal.
  • the method includes administering to the mammal an effective amount of a composition comprising a protein or peptide and an amount of a compound having formula (II) .
  • n 1, 2, 3, 4 alkyl or benzyl
  • A is a macromolecule
  • A can be a macromolecule comprising an adjuvant such as a heterologous or a host cell protein or peptide.
  • each A when more than one A is present in a molecule of any formula described herein, each A may be the same, or the A groups may be different from each other, Accordingly, when more than one A group is present, each A is independently a group as defined for each formula.
  • TLR 7/8 Toll-like receptor 7/8 have emerged as promising targets in the development and design of small-molecule immunomodulators and immunosuppressants for serving as antiviral/anticancer agents and vaccine adjuvants. They respond universally to the presence of viral genomic ssRNA within the cytoplasm. They trigger the NF- ⁇ B mediated production of proflammatory cytokines through a MyD88-dependent pathway.
  • Structure-activity relationship (SAR) analysis indicated that short alkyl (or heteroatom substitution in alkyl) chain at the C-2 position; short hydroxyl-alkyl chains at N-1; amino group at C-4; and methoxy, hydroxyl, or methyl at C-7 were required or improved for activity of TLR7 agonist analogues.
  • Compound 6 was synthesized from the 2.6-dichlonopuine (1) (Scheme 1) .
  • Compound 1 was aminated at C-6 (2) , and alkylated at N-9 position with bromo ester of different length (3) , followed by substitution to give 2-methoxyethoxy intermediate (4) .
  • Bromination at the C-8 position (5) followed by hydrolysis gave compound 6 with exposed terminal carboxylic acid which could be coupled to antigens to construct self-adjuvanting vaccine (Scheme 2) .
  • the 9-propionyloxy of compound 6a enabled us to couple it with many different macromolecules, including proteins and peptides through EDCI. It was covalently coupled to ovalbumin (OVA) , bovine serum albumin (BSA) , mouse serum albumin (MSA) , monoclonal gastric cancer7 antigen (MG7) and Thymosin (Scheme 4) .
  • OVA ovalbumin
  • BSA bovine serum albumin
  • MSA mouse serum albumin
  • MG7 monoclonal gastric cancer7 antigen
  • Thymosin Scheme 4
  • BMDM (1 ⁇ 10 6 /ml) derived from wild-type or TLR7 -/- mice was incubated with 10 ⁇ M compound 6b or 2 ⁇ M conjugates for 24 h.
  • the levels of IL-12 in the culture supernatants were measured by ELISA.
  • the data are mean ⁇ SEM of triplicates and are representative of three independent experiments.
  • the potency of all of the conjugates was evaluated using a mouse splenocyte cytokine production assay in vitro.
  • the cells were incubated with serially diluted conjugates for 24 h, and the levels of cytokines released in the media were subsequently determined by ELISA.
  • the conjugates were more potent cytokine inducers, compared with un-conjugated6b (P ⁇ 0.0001) .
  • the cytokine IFN- ⁇ production levels were 488-, 73-, 575-, 550-and 1511-fold higher for the BSA, OVA, MSA, MG7 and thymosin conjugates, respectively, compared to 6b and as normalized to the 10 ⁇ M level of each treatment.
  • the IL-12 production levels for these molecules were 16-, 175-, 42-, 52-and 409-fold higher than that of 6b, respectively.
  • the conjugates were equally potent as or within one-digit-fold potency of free 6e. Equivalent concentrations of free antigens induced minimal or undetectable cytokine levels (FIG. 14) .
  • mice were immunized with 6b, BSA, OVA, 6b-BSA or 6b-OVA, and the levels of antigen-specific IgG in the serum were assayed at various time intervals (FIG. 16) .
  • the induction of IgG was observed after 14 days in mice immunized with 6b-BSA or 6b-OVA.
  • the levels of antigen-specific IgG continuously increased during the 30-day testing interval.
  • sera from control mice that received PBS, BSA, OVA or free 6b revealed extremely low or undetectable levels of IgG.
  • TLR7 ligand we synthesized an weak TLR7 ligand and revealed its self-adjuvanting immunoregulatory activity when bound to model protein and peptide antigens.
  • 6b is a weak and versatile synthetic small-molecule TLR7 ligand.
  • the compound could be coupled to a variety of macromolecules for the enhancement of activity and a longer duration of action, compared with the free monomeric drug. This delicate design would effectively reduce the cytokine syndrome caused by agonists in vivo and broaden the potential therapeutic application of these agents.
  • compound 6e could be easily coupled to proteins and peptides antigens through the amide banding commonly used in peptide chemistry.
  • 6e-OCT4 conjugated antigen
  • M2e peptide (sequence MSLLTEVETPTRNEWECRCSDSSD) is the 24 amino acid extra-cellular domain of M2 protein (M2) which is one of the most conserved antigens of influenza virus A.
  • M2 protein M2 protein
  • MG7 peptide KPHVHTK is a mimic epitope recognized by specific monoclonal antibody against gastric cancer associated antigen MG7. As shown in FIG.
  • 6e-M2e or 6e-MG7 could stimulate substantially higher levels of cytokines release, as compared to TLR7 agonist alone (p ⁇ 0.001) , indicating that the 6e-M2e or 6e-MG7 (0.1-1.0 ⁇ M) conjugates could activate the immune response more efficiently.
  • 6e alone could stimulate lymphocytes cytokine release at concentrations over 1 ⁇ M (p ⁇ 0.001) , but not below 1 ⁇ M.
  • the 6e-OCT4 induced significantly higher levels of IL-12 and IFN- ⁇ release than that of control and 6e at indicated concentrations (0.1 ⁇ M, 0.5 ⁇ M, 1.0 ⁇ M) (p ⁇ 0.0001) .
  • 6e-OCT4 induced obviously higher levels of IL-12 than OCT4 and 6e + OCT4 at concentrations of 0.1 ⁇ M (p ⁇ 0.0001) , 0.5 ⁇ M (p ⁇ 0.0001) and 1.0 ⁇ M (p ⁇ 0.01) .
  • 6e-OCT4 induced remarkably higher levels of IFN- ⁇ than OCT4 and 6e + OCT4 at concentrations of 0.1 ⁇ M (p ⁇ 0.0001) and 0.5 ⁇ M (p ⁇ 0.01) .
  • OCT4 and 6e + OCT4 did not cause significant change in IL-12 at concentrations of 0.1 ⁇ M and 0.5 ⁇ M, and did not cause significant change in IFN- ⁇ release at concentrations of 0.1 ⁇ M, however, OCT4 and 6e + OCT4 at 1.0 ⁇ M caused a significant increase of IL-12 (p ⁇ 0.01) and IFN- ⁇ (p ⁇ 0.0001) release. Taken together, these results suggested that 6e-OCT4 at the concentrations of 0.1-1.0 ⁇ M could effectively stimulate lymphocytes immune response.
  • Fig. 22A-D The values are shown in Fig. 22A-D for IL6, IL12, TNF- ⁇ and IFN- ⁇ of each cell type.
  • IL12 and TNF- ⁇ production was concentration-dependent from 5 to 50 ⁇ M, comparing with the concentration of LPS at 100 ⁇ M, both IL12 and TNF- ⁇ production of 6e-treated group was higher than LPS positive control.
  • NK cell we detected the release of TNF- ⁇ and INF- ⁇ (FIG. 22B) .
  • 6e was able to stimulate lymphocytes to release cytokines, which were related with adopted immunity, at the very high level compared with other innate immunity cells at the same concentration (FIG. 22D) .
  • the mean spleen weight of tumor-bearing BALB/c mice was higher than the healthy mice.
  • the 4T1 tumor cells increased the spleen size (FIG. 24A) .
  • the spleen size was also large.
  • 6e could reduce both sides of the tumors, it could not reduce the size of the spleen efficiently.
  • FIG. 24C we also found that 6e inhibited the local tumor.
  • FIG. 24B The results showed that intratumoral 6e injection in local tumor was inhibited on the other side of the tumor.
  • FIG. 26A 4T1 tumor cell antibody increased significantly in 6e group compared to control, but the control group also had high antibody titers (FIG. 26A) .
  • 6e was effective in eliciting IgG responses in serum, and these responses were generally dominated by IgG2a, which is thought to reflect Th1 driven response.
  • IgG2a titers were significantly different between 6e treated group and control group (FIG. 26D) .
  • Antibody dependent cell mediated cytotoxicity (ADCC) was determined by addition of serum samples and NK cells (cytotoxic effector cells) to 4T1 tumor cells (target cells) , and measurement of released LDH activity (FIG. 26B) .
  • NK cells cytotoxic effector cells
  • FIG. 26B 4T1 tumor cells
  • FIG. 27 To examine the 4T1 tumor microenvironment in the treated mouse and to study if the treatment of tumor-bearing mice with 6e would lead to memory responses in the mice, we collected and processed distant tumors for analysis of infiltrating cells (FIG. 27) .
  • the CD4 + /CD8 + ratio was reduced in 6e treated group (FIG. 27A) .
  • the frequency of CD25 + Foxp3 + cells was significantly reduced in 6e-treated mice compared with (FIG. 27B) .
  • TLR7 like compound 6e could activated the immune activities.
  • 6e-OCT4 conjugate significantly increased the in vitro release of IL-12 and IFN- ⁇ by mouse spleen lymphocytes.
  • the release of IFN- ⁇ , the percentages of CD3 + /CD8 + T cells and the tumor-specific cytotoxicity rates in immunized mice were significantly higher.
  • immunization with 6e-OCT4 conjugate decreased the growth of the tumor dramatically up to 90 %, as compared to mice immunized with OCT4 protein or 6e alone.
  • blood routine examination demonstrated that no abnormalities of the blood cells and components in the blood fluids were detected by 6e-OCT4 and TLR7 agonist injections.
  • our invention demonstrate for the first time that conjugates of a series of weakadenine analogues trigger a profound induction ofinflammatory cytokines, and TLR7/8 agonistactivity.
  • this kind of adjuvants will effectively reduce the side effects caused by agonists themselves, although the mechanisms of their actions are currently not clear.
  • TLR7 agonists could not only elicit the non-specific antitumor responses but also strengthen the specific humoral and cellular immune responses.
  • DCs play a crucial role in linking innate and adaptive immunity, and in the generation of a protective immune response against both infectious diseases and tumors. DCs are always immature in tumor microenvironment. Here, DCs were activated and matured by TLR7 agonist in vitro, and we detected the secretion of IL12, TNF- ⁇ at a high secretive level.
  • Activited NK cells have not only the capacity to detect changes in transformed cells, but also the responsiblity for tumor rejection in a direct manner.
  • Low natural killer (NK) activity has been reported as a high risk for developing malignancy.
  • NK cells produce perforin to lyse tumor cells directly as shown in vivo and in vitro.
  • TLR7 agonists can increase NK cell activity to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • NK cells produce type 1 cytokines such as IFN- ⁇ and TNF- ⁇ during tumor occurrence to further enhance their cytotoxicity and modulate the adaptive immune cells, such as dendritic cells and T cells.
  • TLR7 agonist stimulated NK cells to produce more TNF- ⁇ and INF- ⁇ than LPS control.
  • the TLR7 agonist group had higher ADCC activity than the other groups.
  • M1 macrophage can be activated by TLR7 agonists and INF- ⁇ , and secrete high levels of TNF- ⁇ , it is related to the inflammatory response, pathogen clearance, and antitumor immunity.
  • M2 macrophage influences an anti-inflammatory response, wound healing, and protumorigenic properties.
  • Tumor-associated macrophages TAMs
  • Activation of macrophages to the M1 phenotype leads to up-regulation of several pro-inflammatory cytokines and chemokines.
  • RAW264.7 as the model in vitro. The results showed that TLR7 weak ligands stimulated RAW264.7 to produce high level of TNF- ⁇ and IL-6, which were markers of the M1 macrophage.
  • DCs professional antigen presenting cells
  • APCs professional antigen presenting cells
  • TLR7agonist stimulated systemic antitumor CD8 T cell response. It implied that TLR7agonist may induce the CD8 T cells into T memory cells and CTL to aim to distant tumor.
  • TLR7agonist may induce the CD8 T cells into T memory cells and CTL to aim to distant tumor.
  • the results showed that CD8 T cells, B cells, type I IFN, IFN- ⁇ and plasmacytoid dendritic cells contributed to efficient tumor suppression.
  • the microenvironment of solid tumor typically contains various cell subsets of the innate immune system.
  • the immune cells have been shown to influence the immune system to promote either antitumor immunity, or tumor progression in the tumor microenvironment. Diederichsen, et al, showed a significantly higher 5-yearsurvival in patients with a low CD4 + /CD8 + ratio in the tumor infiltrating lymphocytes. Bloom field et al investigated the potential of locally delivered imiquimod in a murine model of malignant mesothelioma (AB1-HA) with primary and distal tumors (dual tumor) . They found imiquimod injection locally could stimulate an effective systemic antitumor response required both CD8 T cells and NK cells, but not CD4 T cells. CD8 + T cells within cancer nests were shown to be better predictors of outcome than the same cells found in other areas of the tumor in NSCLC. Similarly, tumor specific CD8 + T cell activity determines colorectal cancer patient prognosis.
  • AB1-HA malignant mesothelioma
  • TLR7 ligands have been exploited to increase the efficacy of cancer immunotherapy.
  • Our results confirmed that TLR7 ligands and the conjugated could induce both innate and adaptive immune cells to strong Th-1bias immune responses and the release of proinflammatory cytokines, such as TNF- ⁇ , IFN- ⁇ , IL-6 and IL-12 in vitro.
  • TLR7 weak ligands to treat tumors in a murine model of T cell lymphoma.
  • TLR7 ligands could activated TLR7 NF- ⁇ B signaling in a TLR7-specific system in vitro.
  • the anticancer therapies given directly into tumors may be more effective than given systemically, because local therapies could overcome natural suppressive factors in the tumor microenvironment, and induce systemic antitumor immunity.
  • local therapies could overcome natural suppressive factors in the tumor microenvironment, and induce systemic antitumor immunity.
  • systemic antitumor immunity For solid tumors, especially breast cancer, direct intratumoral injection is safe and effective. It implied that intratumoral immune activation could induce local and systemic antitumor immunity by TLR7 ligands and the conjugated. Because the TLR7 conjugated works effectively as an adjuvant against infections and tumors that used different pathways, it can be applied as a biodefense strategy that would be useful in mixed as well as single agent attacks.
  • TLR7 agonists used as single agents especially when applied locally can effectively eradicate tumors due to their potent stimulation of innate and adaptive immunity as well as their effects on the tumor microenvironment.
  • the antitumor effect of TLR7 agonists was associated with TLR7 activation of both innate immune cells and adaptive immune cells, especially reversal of tolerance in tumor microenvironment.
  • Non-specific innate immune cells play an important role in cancer therapy, particularly in the elimination of tumor metastases and small tumors.
  • the invention could provide a safer vaccine adjuvants for self adjuvanting immunopotention based on purine-scaffold novel TLR7 ligands. It includes synthetic TLR7 ligands, compound 6, coupled to recombinant antigen, such as protein or a peptide thereof. The compound 6 or the conjugats showed very potent immunoactivity.
  • the conjugates are inexpensive to prepare and are potent, and so provide healthy protection, enabling use settings such as infection or tumor therapy.
  • the invention is directed to pharmaceutically active compounds and conjugates, pharmaceutically acceptable salts, and pharmaceutically acceptable solvates of the compounds or conjugates, and other compounds or conjugates of the invention.
  • Advantageous methods of making the compounds of the invention are also included.

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Abstract

Les ligands de TLR7 et leurs conjugués sont utiles dans des vaccins pour la prévention, l'inhibition ou le traitement de divers troubles, tels que l'infection et le cancer.
PCT/CN2016/082643 2016-05-19 2016-05-19 Ligands de tlr7 à squelette de purine et leur conjugué WO2017197624A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2020162705A1 (fr) 2019-02-08 2020-08-13 성균관대학교산학협력단 Complexe agoniste du récepteur de type toll 7 ou 8-cholestérol, et son utilisation
WO2021177679A1 (fr) 2020-03-02 2021-09-10 성균관대학교산학협력단 Nanoparticules mimétiques d'agent pathogène vivant basées sur un squelette de paroi cellulaire d'agent pathogène, et leur procédé de production
WO2022031021A1 (fr) 2020-08-04 2022-02-10 성균관대학교산학협력단 Vaccin à arnm comprenant un adjuvant permettant un contrôle cinétique
WO2022031057A1 (fr) 2020-08-04 2022-02-10 성균관대학교산학협력단 Conjugué d'un médicament fonctionnel et d'un agoniste de récepteur de type toll 7 ou 8, dont le site actif est temporairement inactivé et utilisation associée
WO2022031011A1 (fr) 2020-08-04 2022-02-10 성균관대학교산학협력단 Ensemble adjuvant à action cinétique

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