Pharmaceutical composition comprising TLR4 and TLR7 agonists

Abstract

The present invention provides a pharmaceutical composition comprising solid particles comprising at least one toll-like receptor 4 (TLR4) agonist and micelles comprising at least one toll-Iike receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent. Also provided is a method of preparing the pharmaceutical composition, a kit or kit of parts and the medical use of the pharmaceutical composition and the kit or kit of parts.

Description

[0001] GRAF VON STOSCH

[0002] PATENTANWALTSGESELLSCHAFT MBH

[0003] Our sign / Our ref. Date KU02P004WO 27 December 2024

[0004] Applicant:

[0005] Kupando GmbH

[0006] 5 Pharmaceutical composition comprising TLR4 and TLR7 agonists

[0007] Field of the Invention

[0008] The present invention relates to pharmaceutical compositions comprising solid particles comprising at least one toll-like receptor 4 (TLR4) agonist and micelles comprising at least one toll-like receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent. Also provided is a method of preparing the pharmaceutical composition and its medical use.

[0009] Background of the Invention

[0010] Innate immunity is not only the first defense system against invading pathogens but also important in various diseases, such as cancer, antibiotic resistance, inflammatory conditions or diseases, 5 autoimmune or infectious diseases. Pattern recognition receptors (PRR), among them Toll-like receptors (TLRs) agonists, can induce innate immunity upon their stimulation. TLRs are a class of receptors expressed on various cell types and play a key role in the innate immune system. Upon activation, TLRs activate signal transduction pathways involved in immune activation. TLRs do not only recognize pathogen associated molecules but also endogenous ligands (alarmins, also called danger-associated molecular patterns or DAMPs). PRR including TLRs have revolutionized the field of immunology by filling the gap between the initial recognition of pathogens by innate immune cells and the activation of the adaptive immune response, by secretion of chemokines and cytokines and activation of antigen-presenting cells.

[0011] Anti-tumor immunity and also immune reactions in the diseases and disorders mentioned above 5 can be enhanced through stimulation of innate immunity. For example, the anti-tumor effect was shown to be associated with the memory function of the innate immune system called trained immunity. The [3-glucan-induced innate immunity was associated with transcriptomic andepigenetic rewiring of granulopoiesis and neutrophil reprogramming towards an anti-tumor phenotype (Kalafati et al., 2020, Cell 183:771-785, e712).

[0012] TLRs play another important role in the context of vaccinations. The most effective way to protect individuals from the insidious threat of many infectious diseases is through vaccination. Effective vaccination requires the use of antigens that can elicit an immune response in the host capable of providing subsequent protection against that particular infectious agent, for which the vaccine is specific. Thus, the vaccine antigen must be immunogenic enough to induce a level of immune response - humoral and / or cell-mediated - to be protective in the host.

[0013] The use of adjuvants in vaccines is a well-established method to promote a stronger immune response to weakly immunogenic antigens. In addition, adjuvants may also enhance and potentially broaden the immune response by promoting the immunogenicity of weakly immunogenic antigens.

[0014] Until recently, the development of new human adjuvants was held back by a poor understanding of their mechanisms of action. The field was revolutionized by the discovery that TLRs as innate immune receptors are directly or indirectly responsible for detecting pathogen-associated molecular patterns (PAMPs) and respond to them by activating innate and adaptive immune pathways (Kaur A et al. Curr Opin in Chem Biol 2022, 70:102172). Hundreds of ligands targeting various TLRs have been identified and characterized as vaccine adjuvants. Each TLR has its own specific tissue localization and downstream gene signaling pathways, providing the opportunity to precisely tailor adjuvants with specific immune effects.

[0015] Several mammalian TLRs and a number of their agonists have been identified. For example, guanine and uridine rich single-stranded RNA has been identified as a natural ligand for TLR7. In addition, several low molecular weight activators of TLR7 have been identified, including imidazoquinolines, and purine-like molecules.

[0016] Several TLR7 and TLR4 agonists are currently being developed as treatments for cancer. Agonists for both receptors have been approved for the treatment of cancer: BCG (OncoTICE®) likely acts via TLR2 and TLR4 stimulation and is indicated fortreatment of primary or concurrent carcinoma- in-situ of the urinary bladder and for the prevention of recurrence of high grade and / or relapsing superficial papillary transitional cell carcinoma of the urinary bladder (Stage Ta (grade 2 or 3) or T1 (grade 1, 2 or 3) after transurethral resection. BCG is also marketed as vaccine for tuberculosis. Imiquimod (i.e. Aldara®) acts via TLR7 and is approved for treatment of external genital andperianal warts (condylomata acuminata) in adults, small superficial basal cell carcinomas (sBCCs) in adults and for clinically typical, nonhyperkeratotic, non-hypertrophic actinic keratoses (AKs) on the face or scalp in immunocompetent adult patients.

[0017] However, TLRs not only play a role in cancer but also, e.g., in infectious diseases.

[0018] While TLR stimulation initiates a common signaling cascade (involving the adaptor protein MyD88, the transcription factor NF-KB, proinflammatory and effector cytokines), different TLRs are expressed by different cell types. For example, TLR7 is mainly expressed in monocytes, plasmacytoid dendritic cells, myeloid dendritic cells and B-cells and is localized to the endosome membrane. TLR7 has been shown to play a significant role in the pathogenesis of autoimmune disorders such as Systemic Lupus Erythematosus (SLE) as well as in the regulation of antiviral immunity. Due to their ability to induce robust production of anti-cancer cytokines, such as interleukin-12, TLR7 agonists have also been investigated for cancer immunotherapy.

[0019] TLR4, the most explored member of the TLR family recognizes lipopolysaccharides (LPS), a component of the outer membrane of bacteria. TLR4 is located on the plasma membrane and is predominantly expressed on myeloid lineage cells, as pDCs and naive B cells do not express TLR4 (Vuere C, Liu Y, Front Immunol 2014, 5:316). TLR4 recognizes LPS with its co-receptor myeloid differentiation factor-2 (MD-2) and CD14 (Park BS, Lee J-O, Exp Mol Med 2013, 45:e66). Substituted pyrimido [5, 4-b] indoles are known to induce TLR4-dpendent NF-KB activation and IL-6 release (Chan M et al., J. Med Chem 2013, 56:4206-4223). Recent studies have further demonstrated that TLR4, through TRIF-mediated pathways can activate cellular response characterized by polyfunctional CD8+ / CD4+ cells and enhanced CTL activity against both cancers and infectious diseases (Kim WS et al., Vaccines 2020, 8:633; Sunay MM et al., Vaccine 2019, 37:3902-3910; Reintjens NR et al., J Med Chem 2020, 63:11691-11706).

[0020] Recent work on TLR4 agonists has focused on the development and evaluation of modified products, such as monophosphoryl lipid A (MPLA) (Kim WS et al. supra) and glucopyranosyl lipid A (GLA) (Sunay MM et al., supra) that are structurally related to LPS, but devoid of high pyrogenicity and maintaining strong immunopotentiating characteristics, thereby increasing their feasibility for clinical applications.

[0021] A synergistic effect after activation of these two TLR receptors was shown in animal models of viral infection demonstrating that the combined stimulation of both receptors enhances the strength and breadth of T and B cell activity (Sato-Kaneko et al., Frontiers in Immunology 2020,11:1207). The inventors believe that a synergistic effect will also be effective in the treatment of cancer and other diseases or disorders, such as antibiotic resistance, inflammatory conditions or diseases, autoimmune diseases or infectious diseases. The inventive combination could be a standalone immunotherapy or an adjunct therapy to established treatment options, i.e., chemotherapy, radiation therapy or checkpoint inhibitor-based therapy.

[0022] Despite the recent advances in the development of TLR agonists, their formulation for pharmaceutical application is challenging. In particular, when different TLR agonists are to be administered in combination in a single pharmaceutical composition in order to achieve an optimal delivery to the target cells and synergistic effect, subsequently.

[0023] WO 2020 / 186229 describes a combination of TLR4 and TLR7 agonists as vaccine adjuvant combination in liposomal formulations with liposomes having a (large) particle size between 100 nm and 200 nm. US 2015 / 0366962 describes emulsions and suspensions for vaccination comprising a first aqueous component and a second component, which comprises the TLR4 and TLR7 agonists.

[0024] However, since repeated injections of therapeutic nanoparticle compositions apparently have been shown to trigger an accelerated blood clearance (ABC) phenomenon by antibodies, in particular for polyethylene glycol P(EG)-based liposomes, which tampers the utility of therapeutic nanoparticle delivery systems, WO 2021 / 053163 suggested a micelle formulation of TLR7 agonist alone and explains that toxicology profiles are improved when compared to liposomal compositions. However, this publication is silent with respect to a successful combination formulation of TLR4 and TLR7 and, thus, does not provide for a pharmaceutical composition, optionally, for use as a medicament comprising a TLR4 agonist and TLR7 agonist. From the prior art it seems that both, TLR4 and TLR7, need to be complexed or in association with a lipid in order to be pharmaceutically available.

[0025] In particular, the TLR4 agonist, however, turned out to be difficult to be incorporated into micelles, liposomes or other lipid-based nanoparticles and the inventors had to look for other ways to develop a combination product for the effective delivery of both, TLR4 and TLR7 agonists, to the cells of a patient in need of the combination treatment and in order to achieve a synergistic therapeutic effect as shown in the Example section of the present invention's pharmaceutical formulation. Due to the hydrophobic nature of the TLR4 agonist, a simple mixture of complexed TLR7 agonist and TLR4 in solution is not feasible.Hence, there is still a need in the art for pharmaceutical compositions for parenteral administration comprising a combination of TLR4 and TLR7 agonists, which are effective in delivering the two agonists to target cells of therapy, stable and easy to manufacture.

[0026] of the invention

[0027] The present invention aims at a pharmaceutical composition comprising both, TLR4 and TLR7 agonists, to achieve a synergistic effect and, thus, a more potent pharmacological activity. Stimulating the innate immunity, e.g., cancer patients are provided with a treatment option that has fewer side effects and improved efficacy. With respect to cancer, more particularly solid tumors, the inventive pharmaceutical compositions will be, in particular, important for the huge number of patients, who do not respond to checkpoint inhibitors. The cross reactivity of the two TLR agonists is particularly suitable for tumors with a high mutational burden and because of the germinal center activation, the inventive concept has the potential to overcome poor germinal center response in immune compromised patients, like organ transplant recipients, older patients and patients with autoimmune diseases.

[0028] The above problems are solved by the provision of a pharmaceutical composition comprising i. solid particles comprising at least one toll-like receptor 4 (TLR4) agonist and

[0029] ii. micelles comprising at least one toll-like receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent.

[0030] Since it turned out difficult to incorporate the TLR4 agonist in liposomes, micelles or other lipid-based nanoparticles on the one hand and impossible to simply add the hydrophobic TLR4 agonist to the solution on the other hand, the inventors investigated other, more sophisticated and unusual ways of formulating the TLR agonists together. Surprisingly, they found out that a combination of the TLR7 agonist associated or complexed with micelles (optionally at least partially incorporated into micelles) could be stably and pharmacologically actively formulated in combination with solid particles comprising the TLR4 agonist. In a preferred embodiment, the at least one TLR4 agonist is comprised in (nano) crystals. Without being bound by a particular theory, the inventors found out that the micelles comprising the at least one TLR7 agonist of the invention seem to stabilize the solid particles comprising the TLR4 agonist of the invention. Nanocrystals are taught to have a number of drawbacks, such as premature leakage and clearance by mononuclear phagocytosis systems, which may be overcome by surface modifications (Patel et al., Future Journal of Pharmacological Sciences 2024, 10:4). However, the inventors decided not to use any surface-modification in order to achievevery high drug-loading, while being carrier-free and, in addition, not to introduce any additional substances, which might lead to toxicology or allergic issues or else. While the use of stabilizers seems to be necessary from the prior art, the stability of the at least one TLR4 agonist solid particles, optionally (nano) crystals, seems to be sufficiently provided by the presence of the micelles comprising at least one TLR7 agonist and at least one amphiphilic micelle-forming agent. Thereby, both agonists seem to be stably dispersed in a colloidal form, optionally, with help of the surfactant / wetting properties of (functionalized) PEGs, which provides for a pharmaceutically active combination of both active agents. This is a very surprising and unexpected advantage of the inventive pharmaceutical composition.

[0031] In a preferred embodiment, the at least TLR4 agonist is has a formula according to formula (II):

[0032]

[0033] wherein z1 is an integer from 1 to 4, wherein z2 is an integer from 0 to 5, wherein R5is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R6is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R7is hydrogen, or substituted or unsubstituted alkyl, and wherein each R8is independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCI3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a tautomer thereof; or a pharmaceutically acceptable salt or solvate thereof, optionally, z1 is 1 and / or R8is substituted or unsubstituted heteroaryl.

[0034] In a more preferred embodiment, the at least one TLR4 agonist has a formula according to formula (Ila):

[0035]

[0036] prolyl, R1Sis selected from the group consisting of hydrogen, F and methyl, and R16is selected from the group consisting of cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 4-fluorophenyl, isopropyl, isobutyl, and 2,2-dimethylbutyl.

[0037] Most preferably, the at least one TLR4 agonist has a formula according to formula (lib):

[0038]

[0039] (lib).

[0040] The TLR4 agonist according to the formula (lib) is also denoted as TLR4B.

[0041] In another highly preferred embodiment, the at least one TLR4 agonist has a formula according to formula (lib):

[0042]

[0043] (Hb),

[0044] and the at least one TLR7 agonist has a formula according to formula (la):

[0045]

[0046] (la), and, optionally, the at least one amphiphilic micelle-forming agent comprises DSPE-mPEG2000. Most preferably, the TLR7 agonist has the structure according to formula (la) and is also denoted as TLR7A.

[0047] The present invention provides a pharmaceutical composition comprising

[0048] i. solid particles comprising at least one toll-like receptor 4 (TLR4) agonist and

[0049] ii. micelles comprising at least one toll-like receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent.

[0050] In the pharmaceutical composition for use according the invention at least about 80%, at least about 85% or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% or at least 97%, 98% or 99% of the at least one TLR7 agonist, based on the total amount of TLR7 agonist in the pharmaceutical composition, is incorporated into in the micelles.In one embodiment, the solid particles do not comprise the at least TLR7 agonist and / or the micelles do not comprise the at least one TLR4 agonist.

[0051] The at least one TLR4 agonist and the at least TLR7 agonist may be present in the pharmaceutical composition in a molar ratio of about 10:1 to about 1:10. In another preferred embodiment, the at least one TLR4 agonist and the at least TLR7 agonist are present in the pharmaceutical composition in a molar ratio of about 5:1 to about 1:5, preferably in a molar ratio of about 2:1 to about 1:2, more preferably in a molar ratio of about 2:1 to about 1:1 or in a molar ratio of about 1:1 to about 1:2, most preferably in a molar ratio of about 1:1.

[0052] The at least one amphiphilic micelle-forming agent may be selected from the group consisting of poloxamer, poloxamine, polyethylene glycol (PEG)-polyester, PEG-polyanhydride, PEG-poly-amino acid, phospholipid, polysorbate, polyoxyethylene alkyl ether and combinations thereof.

[0053] PEG-polyester may be selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), PEG-poly(s-caprolactone) (PCL) and combinations thereof. Optionally, the PEG-polyanhydride is a PEG-polysebacic anhydride (PSA).

[0054] The PEG-poly-amino acid may suitably be selected from the group consisting of PEG-poly(L-histidine), PEG-poly(L-aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), PEG-poly(L-lysine) and combinations thereof.

[0055] In a preferred embodiment, the phospholipid is conjugated to polyethylene glycol (PEG), optionally, the phospholipid is conjugated to PEG via a carbonyl group.

[0056] Preferably, the phospholipid comprises one or more alkyl chains being independently selected from C8-C24 alkyl(s), preferably selected from C10-C22 alkyl(s), more preferably selected from C12-C20 alkyl(s), even more preferably selected from Ci4-C18alkyl(s), and most preferably selected from Ci6-Ci8alkyl(s); wherein the alkyl chains are independently selected from saturated alkyl chains and unsaturated alkyl chains.

[0057] The phospholipid conjugated to PEG may be selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG, preferably it is DSPE-PEG.

[0058] In a preferred embodiment, the at least one amphiphilic micelle-forming agent is DSPE-PEG. The PEG, optionally in DSPE-PEG, may have a molecular weight in the range from about 350-6000 Da,preferably in the range of about 550-5000 Da, more preferably in the range of about 750-4000 Da, even more preferably in the range of about 1000-3000 Da, and most preferably PEG has a molecular weight of about 2000 Da (PEG2000).

[0059] More preferably, DSPE-PEG is DSPE-methoxy PEG (DSPE-mPEG). And even more preferred, the pharmaceutical composition comprises, preferably the micelles comprise, DSPE-(m)PEG2000. In a highly preferred embodiment, the at least one amphiphilic micelle-forming agent is DSPE-PEG2000 or DSPE-mPEG2000, more preferably, DSPE-mPEG2000. In another preferred embodiment, the micelles comprise only one amphiphilic micelle-forming agent, preferably being DSPE-PEG2000 or DSPE-mPEG2000, more preferably, DSPE-mPEG2000.

[0060] The amphiphilic micelle-forming agent may also be selected from the group consisting of DMPE-(m)PEG2000, DSPE-(m)PEG2000, and DPPE-(m)PEG5000.

[0061] In a preferred embodiment, the at least one TLR7 agonist has a formula according to formula (I):

[0062]

[0063] wherein X1may be O, S, or NRC;

[0064] R1may be hydrogen, alkoxyalkyl, alkyl, substituted alkyl, C6aryl or Cio aryl or substituted C& aryl or substituted Cio aryl;

[0065] Rcmay be hydrogen, alkoxyalkyl, alkyl, or substituted alkyl; or Rcand R1taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; each R2may be independently selected from -H, -OH, (Ci-C&)alkyl, substituted (Ci-C6)alkyl, (Ci-Ce)alkoxy, substituted (Ci-C&)alkoxy, -C(O)-(Ci-C&)alkyl (alkanoyl), substituted -C(O)-(Ci-C6)alkyl, -C(O)-(C6-Ci0)aryl (aroyl), substituted -C(0)-(C6-Cio)aryl, -C(O)OH (carboxyl), -C(O)O(Ci-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(Ci-C6)alkyl, -NRaRb, -C(O)NRaRb(carbamoyl), halo, nitro, or cyano, or R2may be absent;each Raand Rbmay independently be selected from hydrogen, (Ci-C6)alkyl, substituted (Ci-Cs)alkyl, (C3-Ca)cycloalkyl, substituted (Cj-Cslcycloalkyl, (Ci-C6)alkoxy, substituted (Ci-C6)alkoxy, (Ci-Ce)alkanoyl, substituted (Ci-Ce)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het (Ci-C6)alkyl, or (Ci-C6)alkoxycarbonyl;

[0066] wherein the substituents on any alkyl, aryl or heterocyclic group may be hydroxy, Ci.6alkyl, hydroxyl Ci-ealkylene, Ci-6 alkoxy, C3-6 cycloalkyl, Ci-6alkoxy, Ci-6alkylene, amino, cyano, halo, or aryl;

[0067] n may be 0, 1, 2, 3 or 4;

[0068] X2may be a bond, -C(O)- or -C(O)-NH-CH2-CH2-; and

[0069] R3may be a phospholipid comprising one or two carboxylic esters; or a tautomer thereof; or a pharmaceutically acceptable salt or solvate thereof.

[0070] Preferably, n is 0, X1is O, and R1is methoxyethyl.

[0071] In another preferred embodiment, R3in formula (I) comprises

[0072]

[0073] wherein R11and R12are each independently a hydrogen or an acyl group, R13is a hydrogen, and m is 1 to 8, preferably 1, and wherein a wavy line indicates a position of bonding, wherein the absolute configuration at the carbon atom bearing OR12is R, S, or any mixture thereof.

[0074] Optionally, m is 1, and / or R11and R12are each oleoyl groups.

[0075] And further optionally, the phospholipid of R3comprises two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof.

[0076] The phospholipid of R3may comprise two carboxylic esters and the carboxylic esters of are the same or different. Each carboxylic ester of the phospholipid may be a C17 carboxylic ester with a site of unsaturation at C8-C9. Alternatively, each carboxylic ester of the phospholipid is a C18 carboxylic ester with a site of unsaturation at C9-C10.

[0077] Optionally, R3comprises dioleoylphosphatidyl ethanolamine (DOPE). Optionally, R3may be 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine and X2may be C(O).In another optional embodiment, X1is oxygen, or X1is O, R1is methoxyethyl, n is 0, X2is -C(O)-, and R3is 1,2-dioleoylphosphatidyl ethanolamine (DOPE).

[0078] In a highly preferred embodiment, the at least one TLR7 agonist has a formula according to formula (la):

[0079]

[0080] The at least one TLR4 agonist has, preferably, a formula according to formula (II):

[0081]

[0082] wherein zl is an integer from 1 to 4, wherein z2 is an integer from 0 to 5, wherein R5is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R6is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R7is hydrogen, or substituted or unsubstituted alkyl, and wherein each R8is independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCI3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a tautomer thereof; or a pharmaceutically acceptable salt or solvate thereof.

[0083] Optionally, z1 is 1.

[0084] Optionally, R8is substituted or unsubstituted heteroaryl.

[0085] In a more preferred embodiment, the at least one TLR4 agonist has a structural formula according to formula (Ila):

[0086]

[0087] prolyl, R15is selected from the group consisting of hydrogen, F and methyl, and R16is selected from the group consisting of cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 4-fluorophenyl, isopropyl, isobutyl, and 2,2-dimethylbutyl.

[0088] In a highly preferred embodiment, the at least one TLR4 agonist has a structural formula according to formula (lib):

[0089]

[0090] (Hb).In a particularly preferred embodiment of the invention, in the pharmaceutical composition, the at least one TLR4 agonist has a structural formula according to formula (lib):

[0091]

[0092] (Hb),

[0093] and the at least one TLR7 agonist has a structural formula according to formula (la):

[0094]

[0095] and the at least one amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0096] More preferably, the solid particles are (nano) crystals, which may also be denoted as nanocrystals or crystals.

[0097] Optionally, the at least one amphiphilic micelle-forming agent and the at least one TLR7 agonist are present in the pharmaceutical composition in a molar ratio in the range of about 20:1 to about 4:1, preferably in the range of 10:1 to about 4:1, more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0098] Optionally, the pharmaceutical composition comprises DSPE-mPEG2000, the at least one TLR4 agonist and at least one TLR7 agonist in a molar ratio in the range of about 20:1:1 to about 4:1:1,preferably in the range of 10:1: 1 to about 4:1:1, more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0099] In a preferred embodiment, the micelles have a particle size in the range of about 2 to about 50 nm, more preferably about 5 to about 30 nm, and even more preferably about 8 to about 20 nm.

[0100] The micelles may have a polydispersity index (PDI) greater than 0 and below 0.2.

[0101] The solid particles may be (nano) crystals having at least one side having an average length in the range of 50 to 220 nm, preferably in the range of 75 to 200 nm, more preferably in the range of 100 to 190 nm.

[0102] Preferably, the pharmaceutical composition comprises an effective amount of the at least one TLR4 agonist and of the at least one TLR7 agonist.

[0103] In an optional embodiment, the pharmaceutical composition further comprises at least one additional active agent.

[0104] The at least one additional active agent may be selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, a further TLR7 agonist, and combinations thereof.

[0105] Optionally, the chemotherapeutic agent is selected from a group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine, streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyl lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

[0106] Optionally, the antimicrobial agent is selected from a group consisting of antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.

[0107] Optionally, the immune checkpoint inhibitor is selected from a group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

[0108] Optionally, the antibiotic is selected from the group consisting of amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, levofloxacin, and combinations thereof.

[0109] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, preferably water for injection or a pharmaceutically acceptable buffer.

[0110] Further provided is a method of preparing the pharmaceutical composition of the invention comprising

[0111] step a) of providing a mixture comprising at least one amphiphilic micelle-forming agent, the at least one TLR4 agonist, the at least one TLR7 agonist and an organic solvent,

[0112] step b) of lipid film hydration, including evaporating the organic solvent and, optionally, step c) of filtration,

[0113] thereby preparing the pharmaceutical composition comprising

[0114] i) solid particles comprising the at least one toll-like receptor 4 (TLR4) agonist and

[0115] ii) micelles comprising the at least one toll-like receptor 7 (TLR7) agonist and the at least one amphiphilic micelle-forming agent.

[0116] Preferably, the organic solvent comprises chloroform, methanol, tetrahydrofuran, or a mixture thereof.

[0117] Optionally, in step b) phosphate buffered saline was used for the hydration of the lipid film.

[0118] In another embodiment, step c) is performed using a filter threshold of about 0.1 to 0.3 pm, preferably of about 0.2 pm.

[0119] Preferably, step c) is performed using a polyethersulfone (PES), a polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF) membrane.

[0120] Further provided is a kit or kit of parts comprising the pharmaceutical composition and a pharmaceutically acceptable carrier, preferably water for injection or a pharmaceutically acceptable buffer, and, optionally, comprising handling instructions.

[0121] The pharmaceutical composition as described herein or the kit or kit of parts as described herein is suitable for use as a medicament.Preferably, the pharmaceutical composition or the kit or kit of parts as described herein are suitable for use in the treatment of cancer, an antibiotic resistance, an inflammatory condition or disease, an autoimmune disease or an infectious disease. Preferably, the pharmaceutical composition or the kit or kit of parts are for use in the treatment of cancer.

[0122] Preferably, the cancer is selected from a group consisting of head and neck cancer, including head and neck squamous cell carcinoma, melanoma, basal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, colon carcinoma, bladder cancer, gastric cancer, stomach cancer, colorectal cancer, liver cancer, breast cancer, lung cancer, and (squamous) skin cancer.

[0123] In a preferred embodiment, the pharmaceutical composition, optionally, as part of the kit or kit of parts, is administered to a subject in need of a TLR4 and / or TLR7 agonist treatment, preferably the subject is a human subject.

[0124] Optionally, the pharmaceutical composition is administered orally, intranasally, via aerosol, via inhalation, parenterally, intramuscularly, intraperitoneally, intravenously, rectally, intravesically or subcutaneously, preferably, the pharmaceutical composition is administered intravenously.

[0125] In another embodiment, the pharmaceutical composition is administered together with at least one additional active agent.

[0126] The at least one additional active agent may be selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, a further TLR7 agonist, and combinations thereof.

[0127] The chemotherapeutic agent may be selected from the group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyl lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

[0128] The antimicrobial agent may be selected antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.The immune checkpoint inhibitor may be selected from the group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

[0129] Detailed description of the invention

[0130] Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0131] The term "toll-like receptor agonist" (TLR agonist) refers to a molecule that binds to a Toll-Like receptor (TLR). Synthetic TLR agonists are chemical compounds that are designed to bind to a TLR and activate the receptor.

[0132] Throughout this specification and the claims which follow, unless the context requires otherwise, the term "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step but not the exclusion of any other nonstated member, integer or step. The term "consist of" is a particular embodiment of the term "comprise", wherein any other non-stated member, integer or step is excluded. In the context of the present invention, the term "comprise" encompasses the term "consist of". The term "comprising" thus encompasses "including" as well as "consisting" e.g., a composition "comprising" X may consist exclusively of X or may include something additional e.g., X + Y. The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.The word "substantially" does not exclude "completely"; e.g. a composition which is "substantially free" from Y may be completely free from Y. Similarly, the word "substantially" does not exclude "exactly"; e.g. a diagram which "substantially" corresponds to an indicated figure may exactly correspond to said figure. Where necessary, the word "substantially" may be omitted from the definition of the invention. A composition is comprised of "substantially all" of a particular compound, or a particular form a compound (e.g., an isomer) when a composition comprises at least about 90%, and at least about 95%, 99%, and 99.9%, of the particular composition on a weight basis. A composition comprises a "mixture" of compounds, or forms of the same compound, when each compound (e.g., isomer) represents at least about 10% of the composition on a weight basis. A TLR7or TLR4 agonist of the invention, or a conjugate thereof, can be prepared as an acid salt or as a base salt, as well as in free acid or free base forms. In solution, certain of the compounds of the invention may exist as zwitterions, wherein counter ions are provided by the solvent molecules themselves, or from other ions dissolved or suspended in the solvent.

[0133] The term "about" in relation to a numerical value x means x + 10%, for example, x + 5%, or x ± 7%, or x ± 10%, or x ± 12%, or x + 15%, or x + 20%.

[0134] The phrase "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. The term explicitly is used in connection with "pharmaceutically acceptable excipient", which includes any excipient, including any chemical entity, which may be used within a composition of the invention and is, according to the definition above, pharmaceutically acceptable. An "effective amount" may be a "prophylactically effective amount" or a "therapeutically effective amount" being sufficient to show benefit to the individual. In particular, the pharmaceutical composition of the invention may comprise an "effective amount" of the TLR4 and / or TLR7 agonist, preferably of both, the TLR4 and TLR7 agonist. The term "therapeutically effective amount" as used herein refers to an amount of a compound, or an amount of a combination of compounds, to treat or prevent a disease or disorder, or to treat a symptom of the disease or disorder, in a subject. In this sense, a "pharmaceutical composition" is generally a composition, which may be administered to asubject, patient, and in particular, a human patient or human subject, often in the course of a medical treatment.

[0135] As used herein, the terms "subject" and "patient" generally refers to an individual, who will receive or who has received treatment (e.g., administration of a compound) according to a medical use described herein. The terms "subject," "patient" or "subject / patient in need thereof' refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound, pharmaceutical composition, mixture or vaccine as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In a preferred embodiment, the patient is human. In some embodiments, a patient is a domesticated animal. In some embodiments, a patient is a newborn animal. In some embodiments, a patient is a newborn human. In some embodiments, a patient is a newborn mammal. In some embodiments, a patient is an elderly animal. In some embodiments, a patient is an elderly human. In some embodiments, a patient is an elderly mammal. In some embodiments, a patient is a geriatric patient. In another preferred embodiment, the patient or subject is an immunocompromised patient of subject. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, behenic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[0136] 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. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile may be employed. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.

[0137] 1418 (1985), the disclosure of which is hereby incorporated by reference.

[0138] The compounds of the (structural) formulas described herein can be solvates, and in some embodiments, hydrates. The term "solvate" refers to a solid compound that has one or more solvent molecules associated with its solid structure. Solvates can form when a compound is crystallized from a solvent. A solvate forms when one or more solvent molecules become an integral part of the solid crystalline matrix upon solidification. The compounds of the (structural) formulas described herein can be solvates, for example, ethanol solvates. Another type of a solvate is a hydrate. A "hydrate" likewise refers to a solid compound that has one or more water molecules intimately associated with its solid or crystalline structure at the molecular level. Hydrates can form when a compound is solidified or crystallized in water, where one or more water molecules become an integral part of the solid crystalline matrix.

[0139] The following definitions are used, unless otherwise described: halo or halogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Het can be heteroaryl, which encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (Ci-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

[0140] It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optical ly-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form byrecrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine agonist activity using the standard tests described herein, or using other similar tests which are well known in the art. It is also understood by those of skill in the art that the compounds described herein include their various tautomers, which can exist in various states of equilibrium with each other.

[0141] The terms "treat" and "treating" as used herein refer to (i) preventing a pathologic condition from occurring (e.g., prophylaxis); (ii) inhibiting the pathologic condition or arresting its development; (iii) relieving the pathologic condition; and / or (iv) ameliorating, alleviating, lessening, and removing symptoms of a condition. A candidate molecule or compound described herein may be in an amount in aformulation or medicament, which is an amount that can lead to a biological effect, or lead to ameliorating, alleviating, lessening, relieving, diminishing or removing symptoms of a condition, e.g., disease, for example. The terms also can refer to reducing or stopping a cell proliferation rate (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor). These terms also are applicable to reducing a titre of a microorganism (microbe) in a system (e.g., cell, tissue, or subject) infected with a microbe, reducing the rate of microbial propagation, reducing the number of symptoms or an effect of a symptom associated with the microbial infection, and / or removing detectable amounts of the microbe from the system. Examples of microbe include but are not limited to virus, bacterium and fungus.

[0142] " 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.

[0143] The term " TLR" refers to Toll-like receptors which are components of the innate immune system that regulate NFKB activation.

[0144] The terms " TLR modulator," " TLR immunomodulator" and the like as used herein refer, in the usual and customary sense, to compounds which agonize or antagonize a toll-1 ike-Receptor (see e.g., PCT / US2010 / 000369, Hennessy, E. J., et al., Nature Reviews 2010, 9:283- 307; PCT / US2008 / 001631; PCT / US2006 / 032371; PCT / US2011 / 000757. Accordingly, a " TLR agonist"is a TLR modulator which agonizes a TLR, and a " TLR antagonist" is a TLR modulator which antagonizes a TLR.

[0145] The term " TLR4" as used herein refers to the product of the TLR4 gene, and homologs, isoforms, and functional fragments thereof: Isoform 1 (NCBI Accession NP_612564.1); Isoform 2 (NCBI Accession NP_003257.1 ); Isoform 3 (NCBI Accession NP_612567.1). Agonists of TLR4that may be included in the disclosed formulations include but are not limited, a compound of formula (II). Further TLR4 agonists are, e.g., a pyrimidoindole, aminoalkyl glucosaminide phosphates, e.g., CRX-601 and CRX-547), RC-29, monophosphorul lipid A (MPL), glucopyranosyl lipid adjuvant (GLA and SLA), OM-174, PET Lipid A, ONO-4007, INI-2004 (a di-amine allose phosphate), and E6020.

[0146] The term " TLR7" as used herein refers to the product (NCBI Accession AAZ99026) of the TLR7 gene, and homologs, and functional fragments thereof. Agonists of TLR7 that may be included in the disclosed formulations include but are not limited, a compound of formula (I). Further TLR7 agonists are, e.g., imidazoquinolines, e.g., imiquimod, CL097 or gardiquimid, CL264, adenine analogs such as CL087, thiazoloquinolines such as 3M002 (CL075), guanosine analogs such asloxoribine, or thioquinoline.

[0147] Toll-like receptors (TLRs) are pattern recognition receptors that recognize conserved microbial products, known as pathogen-associated molecular patterns (PAMPs). TLR4 recognizes LPS. TLR4 signaling activates MyD88 and TRIF-dependent pathways. MyD88 pathway activates NF-KB and JNK to induce inflammatory response. TRIF pathway activates IRF3 to induce IFN-a production. TLR4 is expressed predominately on monocytes, mature macrophages and dendritic cells, mast cells and the intestinal epithelium. TLR modulators (antagonists) for TLR4 include NI-0101 (Hennessy 2010, Id.), 1A6 (Ungaro, R., et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2009, 296: G1167-G1179), AV411 (Ledeboer, A., et al., Neuron Glia Biol. 2006, 2:279-291; Ledeboer, A., et al., Expert Opin. Investig. Drugs 2007, 16:935-950), Eritoran (Mullarkey, M., et al., J. Pharmacol. Exp. Ther. 2003, 305:1093-1102), and TAK-242 (Li, M., et al., Mol. Pharmacol. 2006, 69:1288-1295). TLR modulators (agonists) for TLR4 include Pollinex® Quattro (Baldrick, P., et al., J. Appl. Toxicol. 2007, 27:399-409; DuBuske, L., et al., J. Allergy Clin. Immunol. 2009, 123: S216).

[0148] TLR7 signaling activates MyD88-dependent pathway and IRF7-dependent signaling. IRF7 pathway induces IFN-a production. TLR7 senses ss-RNA or synthetic chemicals (Imiquimod,R848). TLR7 and TLR8 are found in endosomes of monocytes and macrophages, with TLR7 also being expressed on plasmacytoid dendritic cells, and TLR8 also being expressed in mast cells. Both these receptors recognize single stranded RNA from viruses. Synthetic ligands, such as R-848 and imiquimod, can be used to activate the TLR7 and TLR8 signaling pathways. See e.g., Caron, G., et al., J. Immunol. 2005, 175:1551 -1557. TLR9 is expressed in endosomes of monocytes, macrophages and plasmacytoid dendritic cells, and acts as a receptor for unmethylated CpG islands found in bacterial and viral DNA. Synthetic oligonucleotides that contain unmethylated CpG motifs are used to activate TLR9. For example, class A oligonucleotides target plasmacytoid dendritic cells and strongly induce IFNa production and antigen presenting cell maturation, while indirectly activating natural killer cells. Class B oligonucleotides target B cells and natural killer cells and induce little interferon-a (IFNa). Class C oligonucleotides target plasmacytoid dendritic cells and are potent inducers of IFNa. This class of oligonucleotides is involved in the activation and maturation of antigen presenting cells, indirectly activates natural killer cells and directly stimulates B cells. See e.g., Vollmer, J., et al., Eur. J. Immunol. 2004, 34:251-262; Strandskog, G., et al., Dev. Comp. Immunol. 2007, 31:39- 51.

[0149] Reported TLR modulators (agonist) for TLR7 include ANA772 (Kronenberg, B. & Zeuzem, S., Ann. Hepatol. 2009, 8:103-112), Imiquimod (Somani, N. & Rivers, J. K., Skin Therapy Lett. 2005, 10:1-6), and AZD8848 (Hennessey 2010, Id.) TLR modulators (agonist) for TLR8 include VTX-1463 (Hennessey 2010, Id.) TLR modulators (agonist) for TLR7 and TLR8 include Resiquimod (Mark, K. E., et al., J. Infect. Dis. 2007, 195:1324-1331; Pockros, P. J., et al., J. Hepatol. 2007, 47:174-182). TLR modulators (antagonists) forTLR7 and TLR9 include IRS-954 (Barrat, F. J., et al., Eur. J. Immunol. 2007, 37:3582-3586), and IMO-3100 (Jiang, W., et al., J. Immunol. 2009, 182:48.25). TLR9 agonists include SD-101 (Barry, M. & Cooper, C., Expert Opin. Biol. Ther.

[0150] 2007, 7:1731-1737), IMO-2125 (Agrawal, S. & Kandimalla, E. R., Biochem. Soc. Trans. 2007, 35:1461 -1467), Bio Thrax plus CpG-7909 (Gu, M., et al., Vaccine 2007, 25:526-534), AVE0675 (Parkinson, T., Curr. Opin. Mol. Ther. 2008, 10:21-31), QAX-935 (Panter, G., et al., Curr. Opin. Mol Ther. 2009, 11:133-145), SAR-21609 (Parkinson 2008, Id.), and DIMS0150 (Pastorel I i, L., et al., Expert Opin. Emerg. Drugs 2009, 14:505-521).

[0151] The pharmaceutical composition of the invention comprises (i.) solid particles comprising at least one toll-like receptor 4 (TLR4) agonist and (ii.) micelles comprising at least one toll-like receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent.In a preferred embodiment of the pharmaceutical composition according to the invention, the at least one TLR7 agonist has a structural formula according to formula (I):

[0152]

[0153] wherein X1is O, S, or NRC;

[0154] R1is hydrogen, alkoxyalkyl, alkyl, substituted alkyl, C6aryl or Cio aryl or substituted C& aryl or substituted Cio ary I;

[0155] Rcis hydrogen, alkoxyalkyl, alkyl, or substituted alkyl; or Rcand R1taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; each R2is independently selected from -H, -OH, (Ci-C6)alkyl, substituted (Ci-C6)alkyl, (Ci-C6)alkoxy, substituted (Ci-C6)alkoxy, -C(O)-(Ci-C&)alkyl (alkanoyl), substituted -C(O)-(Ci-C&)alkyl, -C(0)-(C6-Cio)aryl (aroyl), substituted -C(0)-(C6-Cio)aryl, -C(O)OH (carboxyl), -C(O)O(Ci-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(Ci-C6)alkyl, -NRaRb, -C(O)NRaRb(carbamoyl), halo, nitro, or cyano, or R2is absent;

[0156] each Raand Rbis independently selected from hydrogen, (Ci-C6)alkyl, substituted (Ci-C6)alkyl, (C3-Cs)cycloalkyl, substituted (C3-C8)cycloalkyl, (Ci-Cefalkoxy, substituted (Ci-Ct)alkoxy, (Ci-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(Ci-C6)alkyl, Het, Het (Ci-C6)alkyl, or (Ci-Ce)alkoxycarbonyl;

[0157] wherein the substituents on any alkyl, aryl or heterocyclic group are hydroxy, Ci.6al ky I, hydroxyl Ci-6alkylene, Ci.6alkoxy, C3.6cycloalkyl, C, alkoxy, CM alkylene, amino, cyano, halo, or aryl; n is 0, 1, 2, 3 or 4;

[0158] X2is a bond, -C(O)- or -C(O)-NH-CH2-CH2-; and

[0159] R3is a phospholipid comprising one or two carboxylic esters;

[0160] or a tautomer thereof;or a pharmaceutically acceptable salt or solvate thereof. In another preferred embodiment, n is 0, X1is O, and R1is methoxyethyl.

[0161] R3in formula (I) may optionally comprise

[0162]

[0163] wherein R11and R12are each independently a hydrogen or an acyl group, R13is a hydrogen, and m is 1 to 8, preferably 1, and wherein the wavy line indicates a position of bonding, wherein the absolute configuration at the carbon atom bearing OR12is R, S, or any mixture thereof. The integer m may be 1, and / or R'1and R12may, individually or both, be oleoyl groups. In another preferred embodiment, the phospholipid of R3comprises two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof. Optionally, the phospholipid of R3comprises two carboxylic esters and the carboxylic esters of are the same or different.

[0164] In formula (I), each carboxylic ester of the phospholipid may be a C17 carboxylic ester with a site of unsaturation at C8-C9 or each carboxylic ester of the phospholipid may be a C18 carboxylic ester with a site of unsaturation at C9-C10. In another preferred embodiment, R3comprises dioleoylphosphatidyl ethanolamine (DOPE) or is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and X2is C(O).

[0165] In another preferred embodiment of the invention, X1in formula (I) is oxygen. More preferably, in formula (I) X1is O, R1is methoxyethyl, n is 0, X2is -C(O)-, and R3is 1,2-dioleoylphosphatidyl ethanolamine (DOPE).

[0166] In a particularly preferred embodiment of the invention, the at least one TLR7 agonist has a structural formula according to formula (la):

[0167]

[0168] (la). The TLR7 agonist according to formula (la) is also denoted as TLR7A.

[0169] Preferably, the at least one TLR4 agonist has a formula / structural formula according to formula (11):

[0170]

[0171] wherein z1 is an integer from 1 to 4, wherein z2 is an integer from 0 to 5, wherein R5is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R6is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R7is hydrogen, or substituted or unsubstituted alkyl, and wherein each R8is independently halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CC, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a tautomer thereof; or a pharmaceutically acceptable salt or solvate thereof. Preferably, z! is 1. Preferably, R8is a substituted or unsubstituted heteroaryl.In a more preferred embodiment of the invention, the at least one TLR4 agonist has a structural formula according to formula (Ila):

[0172]

[0173] (Ha),

[0174] wherein X is O or S, R14is selected from the group consisting of hydrogen, methyl, ethyl and prolyl, R15is selected from the group consisting of hydrogen, F and methyl, and R16is selected from the group consisting of cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 4-fluorophenyl, isopropyl, isobutyl, and 2,2-dimethylbutyl.

[0175] In a particularly preferred embodiment, the at least one TLR4 agonist has a structural formula according to formula (lib):

[0176]

[0177] (Hb).

[0178] A TLR4 agonist according to the structural formula of formula (lib) is also denoted as TLR4B.In a highly preferred embodiment, the pharmaceutical composition according to the invention comprises at least one TLR4 agonist comprising or having a structural formula according to formula

[0179]

[0180] (Hb) and the at least one TLR7 agonist comprising or having a structural formula according to formula (la):

[0181]

[0182] (la).

[0183] In a most preferred embodiment, the pharmaceutical composition according to the invention comprises at least one TLR4 agonist comprising or having a structural formula according to formula

[0184]

[0185] (lib): (lib),at least one TLR7 agonist comprising or having a structural formula according to formula (la):

[0186]

[0187] (la). and comprising at least one amphiphilic micelle-forming agent comprising DSPE-mPEG2000.

[0188] The terms "alkyl, " "alkenyl" and "alkynyl" may include straight-chain, branched-chain and cyclic monovalent hydrocarbyl radicals, and combinations of these, which contain only C and H when they are unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2 propenyl, 3 butynyl, and the like. The total number of carbon atoms in each such group is sometimes described herein, e.g., when the group can contain up to ten carbon atoms it can be represented as 1-1 OC or as C1-C10 or CMO. When heteroatoms (N, O and S typically) are allowed to replace carbon atoms as in heteroalkyl groups, for example, the numbers describing the group, though still written as e.g. G-G, represent the sum of the number of carbon atoms in the group plus the number of such heteroatoms that are included as replacements for carbon atoms in the backbone of the ring or chain being described.

[0189] Typically, the alkyl, alkenyl and alkynyl substituents of the invention contain one 10C (alkyl) or two 10C (alkenyl or alkynyl). For example, they contain one 8C (alkyl) or two 8C (alkenyl or alkynyl). Sometimes they contain one 4C (alkyl) or two 4C (alkenyl or alkynyl). A single group can include more than one type of multiple bond, or more than one multiple bond; such groups are included within the definition of the term "alkenyl" when they contain at least one carboncarbon double bond, and are included within the term "alkynyl" when they contain at least one carbon-carbon triple bond.

[0190] Alkyl, alkenyl and alkynyl groups are often optionally substituted to the extent that such substitution makes sense chemically. Typical substituents include, but are not limited to, halo, =O, =N-CN, =N-OR, =NR, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, and NO2, wherein each R is independently H, Ci-C8alkyl, C2-C8heteroalkyl, Ci-C8acyl, C2-C8heteroacyl, C2-C8alkenyl, C2-C8heteroalkenyl, C2- C8alkynyl, C2-C8heteroalkynyl, C6-Cio aryl, or C5-C heteroaryl, and each R is optionally substituted with halo, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, wherein each R' is independently H, Ci-C8alkyl, C2-C8heteroalkyl, C,-C8acyl, C2-C8heteroacyl, C6-C10aryl or C5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups can also be substituted by Ci-C8acyl, C2-C8heteroacyl, Cs-Cw aryl or C5-C10 heteroaryl, each of which can be substituted by the substituents that are appropriate for the particular group.

[0191] " Acetylene" substituents may include 2-1 OC alkynyl groups that are optionally substituted, and are of the (structural) formula -C=C-Ri, wherein Ri is H or Ci-C8alkyl, C2-C8heteroalkyl, C2-C8alkenyl, C2-C8heteroalkenyl, C2-C8alkynyl, C2-C8heteroalkynyl, Ci-C8acyl, C2-C8heteroacyl, Ce-Cio aryl, C5-C10 heteroaryl, C7-Ci2arylalkyl, or C6-Ci2heteroarylalkyl, and each Ri group is optionally substituted with one or more substituents selected from halo, =O, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, wherein each R' is independently H, Ci-C6alkyl, C2-C6heteroalkyl, Ci-C8acyl, C2-C6heteroacyl, Ce-Cio aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6. i2heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, C1-C4 heteroalkyl, Ci-C6acyl, Ci-C6heteroacyl, hydroxy, amino, and =O; and wherein two R' can be linked to form a 3-7 membered ring optionally containing up to three heteroatoms selected from N, O and S. In some embodiments, Ri of -C=C-Ri is H or Me.

[0192] " Heteroalkyl", "heteroalkenyl", and "heteroalkynyl" and the like are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the 'hetero' terms refer to groups that contain one to three O, S or N heteroatoms or combinations thereof within the backbone residue; thus at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group. The typical sizes for heteroforms of alkyl, alkenyl and alkynyl groups are generally the same as for the corresponding hydrocarbyl groups, and the substituents that may be present on the heteroforms are the same as those described above for the hydrocarbyl groups. For reasons of chemical stability, it is also understood that, unless otherwise specified, such groups do not include more than two contiguous heteroatoms except where an oxo group is present on N or S as in a nitro or sulfonyl group.While "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl groups, the term "cycloalkyl" may be used herein to describe a carbocyclic non-aromatic group that is connected via a ring carbon atom, and "cycloalkylalkyl" may be used to describe a carbocyclic non-aromatic group that is connected to the molecule through an alkyl linker. Similarly, "heterocyclyl" may be used to describe a non-aromatic cyclic group that contains at least one heteroatom as a ring member and that is connected to the molecule via a ring atom, which may be C or N; and "heterocyclylalkyl" may be used to describe such a group that is connected to another molecule through a linker. The sizes and substituents that are suitable for the cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. As used herein, these terms also include rings that contain a double bond or two, as long as the ring is not aromatic.

[0193] As used herein, "acyl" encompasses groups comprising an alkyl, alkenyl, alkynyl, aryl or arylalkyl radical attached at one of the two available valence positions of a carbonyl carbon atom, and heteroacyl refers to the corresponding groups wherein at least one carbon other than the carbonyl carbon has been replaced by a heteroatom chosen from N, O and S. Thus heteroacyl includes, for example, -C(=O)OR and -C(=O)NR2as well as -C(=O)-heteroaryl.

[0194] Acyl and heteroacyl groups are bonded to any group or molecule to which they are attached through the open valence of the carbonyl carbon atom. Typically, they are Ci-C8acyl groups, which include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8heteroacyl groups, which include methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups, aryl groups, and heteroforms of such groups that comprise an acyl or heteroacyl group can be substituted with the substituents described herein as generally suitable substituents for each of the corresponding component of the acyl or heteroacyl group.

[0195] " Aromatic" moiety or "aryl" moiety refers to a monocyclic or fused bicyclic moiety having the well-known characteristics of aromaticity; examples include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to such monocyclic or fused bicyclic ring systems which contain as ring members one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits aromaticity in 5 membered rings as well as 6 membered rings. Typical heteroaromatic systems include monocyclic C5-C6aromatic groups such as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl and the fused bicyclic moieties formed by fusing one of these monocyclic groups with a phenyl ring or with any of the heteroaromatic monocyclic groups to form a Cg-Cw bicyclic group such as indolyl,benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like. Any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups where at least the ring which is directly attached to the remainder of the molecule has the characteristics of aromaticity. Typically, the ring systems contain 5-12 ring member atoms. For example, the monocyclic heteroaryls may contain 5-6 ring members, and the bicyclic heteroaryls contain 8-10 ring members.

[0196] Aryl and heteroaryl moieties may be substituted with a variety of substituents including Ci-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C5-C12 aryl, Ci-C8acyl, and heteroforms of these, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halo, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, and NO2, wherein each R is independently H, Ci-C8alkyl, C2-C8heteroalkyl, C2-C8alkenyl, C2-C8heteroalkenyl, C2-C8alkynyl, C2-C8heteroalkynyl, C6-Cio aryl, C5-C10 heteroaryl, C?-Ci2arylalkyl, or C6-C12 heteroarylalkyl, and each R is optionally substituted as described above for alkyl groups. The substituent groups on an aryl or heteroaryl group may of course be further substituted with the groups described herein as suitable for each type of such substituents or for each component of the substituent. Thus, for example, an arylalkyl substituent may be substituted on the aryl portion with substituents described herein as typical for aryl groups, and it may be further substituted on the alkyl portion with substituents described herein as typical or suitable for alkyl groups.

[0197] Similarly, "arylalkyl" and "heteroarylalkyl" refer to aromatic and heteroaromatic ring systems which are bonded to their attachment point through a linking group such as an alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers. Typically the linker is Ci-C8alkyl or a hetero form thereof. These linkers may also include a carbonyl group, thus making them able to provide substituents as an acyl or heteroacyl moiety. An aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group may be substituted with the same substituents described above for aryl groups. For example, an arylalkyl group includes a phenyl ring optionally substituted with the groups defined above for aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane. Similarly, a heteroarylalkyl group may include aCs-Ce monocyclic heteroaryl group that is optionally substituted with the groups described above as substituents typical on aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, or it includes an optionally substituted phenyl ring or C5-C5monocyclic heteroaryl and a C1-C4 heteroalkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane.

[0198] Where an arylalkyl or heteroarylalkyl group is described as optionally substituted, the substituents may be on either the alkyl or heteroalkyl portion or on the aryl or heteroaryl portion of the group. The substituents optionally present on the alkyl or heteroalkyl portion are the same as those described above for alkyl groups generally; the substituents optionally present on the aryl or heteroaryl portion are the same as those described above for aryl groups generally.

[0199] " Arylalkyl" groups as used herein are hydrocarbyl groups if they are unsubstituted, and are described by the total number of carbon atoms in the ring and alkylene or similar linker. Thus, a benzyl group is a Cz-arylalkyl group, and phenylethyl is a C8-arylalkyl.

[0200] " Heteroarylalkyl" as described above refers to a moiety comprising an aryl group that is attached through a linking group, and differs from "arylalkyl" in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S. The heteroarylalkyl groups are described herein according to the total number of atoms in the ring and linker combined, and they include aryl groups linked through a heteroalkyl linker; heteroaryl groups linked through a hydrocarbyl linker such as an alkylene; and heteroaryl groups linked through a heteroalkyl linker. Thus, for example, C7-heteroarylalkyl would include pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.

[0201] " Alkylene" as used herein refers to a divalent hydrocarbyl group; because it is divalent, it can link two other groups together. Typically it refers to -(CH2)n- where n is 1-8 and for instance n is 1-4, though where specified, an alkylene can also be substituted by other groups, and can be of other lengths, and the open valences need not be at opposite ends of a chain. Thus -CH(Me)- and -C(Me)2- may also be referred to as alkylenes, as can a cyclic group such as cyclopropan- 1,1 -diyl. Where an alkylene group is substituted, the substituents include those typically present on alkyl groups as described herein.In general, any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group or any heteroform of one of these groups that is contained in a substituent may itself optionally be substituted by additional substituents. The nature of these substituents is similar to those recited with regard to the primary substituents themselves if the substituents are not otherwise described. Thus, where an embodiment of, for example, R2is alkyl, this alkyl may optionally be substituted by the remaining substituents listed as embodiments for R2where this makes chemical sense, and where this does not undermine the size limit provided for the alkyl per se; e.g., alkyl substituted by alkyl or by alkenyl would simply extend the upper limit of carbon atoms for these embodiments, and is not included. However, alkyl substituted by aryl, amino, alkoxy, =O, and the like would be included within the scope of the invention, and the atoms of these substituent groups are not counted in the number used to describe the alkyl, alkenyl, etc. group that is being described. Where no number of substituents is specified, each such alkyl, alkenyl, alkynyl, acyl, or aryl group may be substituted with a number of substituents according to its available valences; in particular, any of these groups may be substituted with fluorine atoms at any or all of its available valences, for example.

[0202] As used herein, the term "alkyl" by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched chain, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., Ci-C means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, ( cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1 - and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).

[0203] The term "alkylene", by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms. In one embodiment those groups have! 0 or fewer carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.The term "heteroalkyl", by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) 0, 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 remainder of the molecule. Examples include, but are not limited to:

[0204] -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, -CH=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, such as, for example, -CH2-NH-OCH3.

[0205] The term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the (structural) formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. AS described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', - NR'R", -OR1, -SR', and / or -SO2R'. Where "heteroalkyl" is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.

[0206] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to, 1 -(1,2,5,6-tetrahydropyridyl), 1 -piperidinyl, 2-piperidinyl, 3-piperidi nyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively.

[0207] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci-C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0208] The term "acyl" means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalky], substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0209] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (e.g., from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to 15 multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to aryl groups (or rings) that contain at least one heteroatom selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. Thus, the term "heteroaryl" includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, 1- pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4- oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2 -thiazolyl, 4-thiazolyl, 5-thiazolyl, 2 -furyl, 3-furyl, 2 -thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An "arylene" and a "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0210] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1 -naphthyloxy)propyl, and the like).

[0211] The term "oxo", as used herein, means an oxygen that is double bonded to a carbon atom. The term "alkylsulfonyl", as used herein, means a moiety having the formula -S(O2)-R', where R' is an alkyl group as defined above. R1may have a specified number of carbons (e.g., " C1-C4 alkylsulfonyl").

[0212] Each of the above terms (e.g., "alkyl", "heteroalkyl", "aryl" and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical.

[0213] As used herein, the terms "heteroatom" or "ring heteroatom" are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0214] A "substituent group", as used herein, means a group selected from the following moieties: -OH, -NH2, -SH, -CN, -CFJ, -CCI3, -NO2, oxo, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from: (i) oxo, -OH, -NH2, -SH, -CN, -CF3, -CCI3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, substituted with at least one substituent selected from: (a) oxo, -OH, -NH2, -SH, -CN, -CF3, -CCI3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, substituted with at least one substituent selected from: oxo,-OH, -NH2, -SH, -CN, -CF3, -CCI3, -NO2, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.

[0215] A "size-limited substituent" or" size-limited substituent group," as used herein, means a group selected from all of the substituents described above for a "substituent group," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C4-C8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.

[0216] A "lower substituent" or "lower substituent group", as used herein, means a group selected from all of the substituents described above for a "substituent group", wherein each substituted or unsubstituted alkyl is, for example, a substituted or unsubstituted Ci-C8alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloal ky 1.

[0217] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0218] The pharmaceutical composition of the invention comprises solid particles comprising at least one toll-like receptor 4 (TLR4) agonist as defined herein. In the sense of the present invention, "solid particle" describes a compound of the pharmaceutical composition, in particular, the at least one TLR4 agonist, which is not a liquid or dissolved within a liquid (including a mixture of liquids) but clearly distinguishable from a surrounding liquid phase of the pharmaceutical composition, whichmay be considered a liquid, including an aqueous or non-aqueous solution. A solid particle may, for example, be separated from the liquid phase in the pharmaceutical composition by centrifugation or filtration using suitable means adapted to the size of the solid particles and the liquid phase, which in the present case also includes the micelles comprising the at least TLR7 agonist. Preferably, the solid particles of the invention are in the form of (nano) crystals.

[0219] The solid particles of the at least one TLR4 agonist, preferably of TLR4B, are (nano) crystals (including nanocrystals and crystals) having at least one side having an average length, i.e. in at least one dimension, in the range of 50 to 220 nm, preferably in the range of 75 to 200 nm, more preferably in the range of 100 to 190 nm. The solid particles or (nano) crystals may have a diamond / brick shape or orthorhombic appearance or may appear less faceted and more irregular. The appearance of the crystals do not have any influence on the bioavailability, solubility or pharmaceutical activity of the TLR4 agonist. The solid particles, preferably (nano) crystals, are preferably polymorphic form I. Without being bound by a particular theory, the solid particles of the invention or (nano) crystals seem to be stabilized by the micelles or lipids present in the pharmaceutical composition of the invention. It could not have been foreseen by the skilled person from the information available from the prior art that the specific combination of micelles and solid particles, more particularly micelles comprising TLR7, preferably TLR7A, and solid particles comprising TLR4, preferably TLR4B, is particularly stable, when compared to other formulations in the art comprising TLR4 and / TLR7 agonists. This particular stability is even more pronounced when solid particles in polymorphic form I are used.

[0220] The presence of solid particles, preferably (nano) crystals, as well as micelles may be determined by cryo-TEM or Dynamic light scattering as described in the example section. Both methods are well known to the skilled person.

[0221] In the pharmaceutical composition of the invention, at least about 80%, at least about 85% or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% of the at least one TLR7 agonist, preferably TLR7A, based on the total amount of TLR7 agonist in the pharmaceutical composition, is incorporated into in the micelles. In another preferred embodiment, about 90-95% of the at least one TLR7 agonist, preferably TLR7A, based on the total amount of TLR7 agonist in the pharmaceutical composition, is incorporated into micelles. The term "total amount in the pharmaceutical composition" denotes all respective molecules, in particular all at least TLR7 orTLR4 agonists, within the pharmaceutical composition.Preferably, the solid particles do not comprise the at least TLR7 agonist and / or the micelles do not comprise the at least one TLR4 agonist. In other words, only the TLR4 agonist, particularly TLR4B, forms solid particles, more particularly (nano) crystals, during the manufacturing process, or is present in the form of solid particles or (nano) crystals in the pharmaceutical composition of the invention. And, preferably, only the TLR7 agonist, more particularly only TLR7A, is comprised in the micelles or associated with the micelles or complexed with the micelles or incorporated within the micelles. TLR7A is the most preferred TLR7 agonist. TLR4B is the most preferred TLR4 agonist. The terms "associated with" or "complexed with", "complexed within", "encapsulated" in or "comprised" in with respect to the at least one TLR7 agonist (TLR7A) and micelles is to be understood according to its general meaning in the art of micelle loading or encapsulating of compounds within micelles (and other lipid structures). In this context, the terms "complexed" or "associated" refer to the essentially stable combination of TLR7 agonist with the at least one amphiphilic micelle-forming agent into larger complexes or assemblies without covalent binding. In the present invention micelles are formed comprising the at least one TLR7 agonist and the at least one amphiphilic micelle-forming agent. The pharmaceutical composition of the present invention may also comprise more than one at least one amphiphilic micelle-forming agent and mixtures of amphiphilic micelle-forming agents. In general, a "micelle" is understood by the skilled person as an aggregate (or supramolecular assembly) of surfactant amphipathic lipid molecules dispersed in a liquid, forming a colloidal suspension (also known as associated colloidal system). A typical micelle in water forms an aggregate with the hydrophilic regions in contact with surrounding aqueous solvent, sequestering the hydrophobic regions in the micelle center.

[0222] In the pharmaceutical composition according to the invention, optionally, the at least one TLR4 agonist (TLR4B) and the at least TLR7 agonist (TLR7A) are present in the pharmaceutical composition in a molar ratio of about 10:1 to about 1:10. Preferably, the at least one TLR4 agonist (TLR4B) and the at least TLR7 agonist (TLR7A) are present in the pharmaceutical composition in a molar ratio of about 5:1 to about 1:5, more preferably in a molar ratio of about 2:1 to about 1:2, even more preferably in a molar ratio of about 2:1 to about 1:1 or in a molar ratio of about 1:1 to about 1:2, and most preferably in a molar ratio of about 1:1.

[0223] The pharmaceutical composition of the present invention comprises at least one amphiphilic micelleforming agent. Preferably, this at least one amphiphilic micelle-forming agent will, according to its name, form at least one and usually many micelles within the pharmaceutical composition. The skilled person knows, which manufacturing methods need to be employed in order to allow the atleast one amphiphilic micelle-forming agent to form micelles within a composition and which other conditions need to be met in order to obtain micelles in a pharmaceutical composition. The skilled person also knows typical micelle-forming agents, which are useful in the frame if the present invention and which are not explicitly mentioned herein. Further information on how to manufacture a pharmaceutical composition according to the present invention are given in the example section. The at least one amphiphilic micelle-forming agent may be selected from the group consisting of poloxamer, poloxamine, polyethylene glycol (PEG)-polyester, PEG-polyanhydride, PEG-poly-amino acid, phospholipid, polysorbate, polyoxyethylene alkyl ether and combinations thereof. Optionally, the PEG-polyester is selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), PEG-poly(s-caprolactone) (PCL) and combinations thereof. Optionally, the PEG-polyanhydride is a PEG-polysebacic anhydride (PSA). Optionally, the PEG-poly-amino acid is selected from the group consisting of PEG-poly(L-histidine), PEG-poly(L-aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), PEG-poly(L-lysine) and combinations thereof. Preferably, the phospholipid is conjugated to polyethylene glycol (PEG), and even more preferably, the phospholipid is conjugated to PEG via a carbonyl group.

[0224] The phospholipid optionally comprises one or more alkyl chains being independently selected from C8-C24alkyl(s), preferably selected from C10-C22alkyl(s), more preferably selected from C12-C20alkyl(s), even more preferably selected from C14-C18alkyl(s), and most preferably selected from C16-C18 alkyl(s), wherein the alkyl chains are independently selected from saturated alkyl chains and unsaturated alkyl chains. In a preferred embodiment, the phospholipid conjugated to PEG is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG, preferably DSPE-PEG. Most preferably, the at least one amphiphilic micelle-forming agent is DSPE-PEG. Preferably, the PEG has a molecular weight in the range from about 350-6000 Da, more preferably in the range of about 550-5000 Da, even more preferably in the range of about 750-4000 Da, particularly more preferably in the range of about 1000-3000 Da, and most preferably PEG has a molecular weight of about 2000 Da (PEG2000).

[0225] Most preferably, in the pharmaceutical composition of the invention DSPE-PEG is DSPE-methoxy PEG (DSPE-mPEG). Even more preferred is DSPE-mPEG associated or complexed with the at least one TLR7 agonist, preferably TLR7A. Most preferably, DSPE-mPEG forms micelles incorporating or having incorporated, associated with or being complexed with at least about 80%, at least about 85%or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% of the at least one TLR7 agonist (TLR7A), based on the total amount of TLR7 agonist (TLR7A) in the pharmaceutical composition the at least one TLR7 agonist, preferably TLR7A.

[0226] The pharmaceutical composition according to any one of the preceding claims, wherein the pharmaceutical composition comprises, preferably the micelles comprise, DSPE-(m)PEG2000, preferably DSPE-mPEG2000, wherein 2000 denotes the average molecular weight of the PEG moiety in the at least one amphiphilic micelle-forming agent. The average molecular weight of the PEG moiety in the at least one amphiphilic micelle-forming agent may range from 500 to 8000 Daltons (e.g., from 1000 to 4000 Daltons). Preferably, the average molecular weight of the PEG moiety is about 2000 Daltons. The term "average molecular weight" with respect to PEG moieties is well understood by the skilled person in the art.

[0227] Most preferably, in the pharmaceutical composition of the invention DSPE-PEG2000 is DSPE-methoxy PEG2000 (DSPE-mPEG). Even more preferred is DSPE-mPEG2000 associated or complexed with the at least one TLR7 agonist, preferably TLR7A. Most preferably, DSPE-mPEG2000 forms micelles incorporating or having incorporated, associated with or being complexed with at least about 80%, at least about 85% or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% of the at least one TLR7 agonist (TLR7A), based on the total amount of TLR7 agonist (TLR7A) in the pharmaceutical composition the at least one TLR7 agonist, preferably TLR7A.

[0228] Most preferably, in the pharmaceutical composition of the invention DSPE-PEG is DSPE-PEG2000 (DSPE-PEG). Even more preferred is DSPE-PEG2000 associated or complexed with the at least one TLR7 agonist, preferably TLR7A. Most preferably, DSPE-PEG2000 forms micelles incorporating or having incorporated, associated with or being complexed with at least about 80%, at least about 85% or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% of the at least one TLR7 agonist (TLR7A), based on the total amount of TLR7 agonist (TLR7A) in the pharmaceutical composition the at least one TLR7 agonist, preferably TLR7A.

[0229] In another preferred embodiment, the amphiphilic micelle-forming agent is selected from the group consisting of DMPE-(m)PEG2000, DSPE-(m)PEG2000, and DPPE-(m)PEG5000.

[0230] In another preferred embodiment, the at least one amphiphilic micelle-forming agent and the at least one TLR7 agonist, preferably TLR7A, are present in the pharmaceutical composition in a molar ratio in the range of about 20:1 to about 4:1, more preferably in the range of 10:1 to about4:1, even more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0231] In another preferred embodiment, the pharmaceutical composition comprises DSPE-mPEG2000, the at least one TLR4 agonist, preferably TLR7A, and at least one TLR7 agonist, preferably TLR4B, in a molar ratio in the range of about 20:1:1 to about 4:1:1, more preferably in the range of 10:1:1 to about 4:1:1, even more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0232] The micelles may have a particle size in the range of about 2 to about 50 nm, preferably about 5 to about 30 nm, and more preferably about 8 to about 20 nm. Optionally, the micelles have a polydispersity index (PDI) greater than 0 and below 0.2.

[0233] The pharmaceutical composition according to the invention may further comprise at least one additional active agent, which may optionally be selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, e.g. according to formula (II), a further TLR7 agonist, e.g. according to formula (I), and combinations thereof.

[0234] The chemotherapeutic agent may be selected from a group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine, streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophy I lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

[0235] The antimicrobial agent may be selected from a group consisting of antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.

[0236] The immune checkpoint inhibitor may be selected from a group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

[0237] The antibiotic may be selected from the group consisting of amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, levofloxacin, and combinations thereof.

[0238] Also provided is a kit or kit of parts comprising the pharmaceutical composition of the invention and a pharmaceutically acceptable carrier, preferably water for injection or a pharmaceutically acceptable buffer, and, optionally, comprising handling instructions.

[0239] The pharmaceutical composition of the present invention or the kit or kit of parts of the present invention is in particular suitable for use as a medicament. More preferably suitable for use in the treatment of cancer, an antibiotic resistance, an inflammatory condition or disease, an autoimmune disease or an infectious disease. Particularly, the pharmaceutical composition of the invention or the kit or kit of parts of the invention are for use in the treatment of cancer, more particularly solid tumors.

[0240] The cancer may be selected from a group consisting of head and neck cancer, including head and neck squamous cell carcinoma, melanoma, basal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, colon carcinoma, bladder cancer, gastric cancer, stomach cancer, colorectal cancer, liver cancer, breast cancer, lung cancer, and (squamous) skin cancer.

[0241] Administration of the inventive compositions can be via any of suitable route of administration, particularly parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecal ly, intraventricularly, intraurethrally, intrasternally, intracranially, intravesically, intramuscularly, or subcutaneously. More particularly, the pharmaceutical composition is administered to a subject in need of a TLR4 and / or TLR7 agonist treatment, preferably the subject is a human subject, preferably, the pharmaceutical composition is administered orally, intranasally, via aerosol, via inhalation, parenterally, intramuscularly, intraperitoneally, intravenously, rectally, intravesically, or subcutaneously. More preferably, the pharmaceutical composition is administered parenterally, even more preferably intravenously.

[0242] Administration may be as a single bolus injection, multiple injections, or as a short- or long- duration infusion. Implantable devices (e.g., implantable infusion pumps) may also be employed for the periodic parenteral delivery over time of equivalent or varying dosages of the particular formulation. For such parenteral administration, the compounds (a conjugate or other activeagent) may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances such as salts, sugars (particularly glucose or mannitol), to make the solution isotonic with blood, buffering agents such as acetic, citric, and / or phosphoric acids and their sodium salts, and preservatives. Hence, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable excipient, preferably water for injection or a pharmaceutically acceptable buffer, e.g. phosphate, citrate etc. buffered solution.

[0243] Also provided is a method of preparing the pharmaceutical composition (for medical use) of the invention comprising

[0244] step a) of providing a mixture comprising at least one amphiphilic micelle-forming agent, the at least one TLR4 agonist, preferably TLR4B, the at least one TLR7 agonist, preferably TLR7A, and an organic solvent,

[0245] step b) of lipid film hydration, including evaporating the organic solvent and, optionally, step c) of filtration,

[0246] thereby, optionally, preparing the pharmaceutical composition comprising

[0247] i) solid particles comprising the at least one toll-like receptor 4 (TLR4) agonist and ii) micelles optionally comprising the at least one toll-like receptor 7 (TLR7) agonist and the at least one amphiphilic micelle-forming agent.

[0248] Also provided is a method of preparing the pharmaceutical composition (for medical use) of the invention comprising

[0249] step a) of providing a mixture comprising at least one amphiphilic micelle-forming agent, the at least one TLR4 agonist, preferably TLR4B, the at least one TLR7 agonist, preferably TLR7A, and an organic solvent,

[0250] step b) of lipid film hydration, including evaporating the organic solvent and, optionally, step c) of filtration,

[0251] thereby preparing the pharmaceutical composition comprising

[0252] i) solid particles comprising the at least one toll-like receptor 4 (TLR4) agonist andii) micelles comprising the at least one toll-like receptor 7 (TLR7) agonist and the at least one amphiphilic micelle-forming agent.

[0253] In a preferred embodiment, the organic solvent comprises chloroform, methanol, tetrahydrofuran, or a mixture thereof. More preferably, chloroform and methanol are used in a mixture and, optionally, in a volumetric ratio in the range of about 10:1 to about 7:1, preferably about 9:1. In step b) of the method, phosphate buffered saline or another pharmaceutically acceptable buffer known to the skilled person may be used for the hydration of the lipid film, preferably phosphate buffered saline.

[0254] In an optional embodiment, step c) is performed using a filter threshold of about 0.1 to 0.3 pm, preferably of about 0.2 pm. Preferably, step c) is performed using a polyethersulfone (PES), a polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF) membrane. The filtration step should be performed according to the manufacturer's protocol and as known in the art. One example on how the pharmaceutical formulation of the invention may be produced is given in the example section.

[0255] In a very preferred embodiment, the pharmaceutical composition comprises DSPE-mPEG2000, the at least one TLR4 agonist, preferably TLR7A, and at least one TLR7 agonist, preferably TLR4B, in a molar ratio in the range of about 8:1:1 to about 6:1:1, and preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0256] In a more preferred embodiment, the pharmaceutical composition comprises DSPE-mPEG2000, TLR7A, and TLR4B, in a molar ratio in the range of about 8:1:1 to about 6:1:1, and preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0257] Most preferably, the pharmaceutical composition comprises DSPE-mPEG2000, TLR7A and TLR4B in a molar ratio of about 7:1:1.Embodiments

[0258] 1. A pharmaceutical composition comprising

[0259] i. solid particles comprising at least one toll-like receptor 4 (TLR4) agonist and

[0260] ii. micelles comprising at least one toll-like receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent.

[0261] 2. The pharmaceutical composition according to item 1, wherein at least about 80%, at least about 85% or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% of the at least one TLR7 agonist, based on the total amount of TLR7 agonist in the pharmaceutical composition, is incorporated into in the micelles.

[0262] 3. The pharmaceutical composition according to item 1 or 2, wherein the solid particles do not comprise the at least TLR7 agonist and / or wherein the micelles do not comprise the at least one TLR4 agonist.

[0263] 4. The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR4 agonist and the at least TLR7 agonist are present in the pharmaceutical composition in a molar ratio of about 10:1 to about 1:10.

[0264] 5. The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR4 agonist and the at least TLR7 agonist are present in the pharmaceutical composition in a molar ratio of about 5:1 to about 1:5, preferably in a molar ratio of about 2:1 to about 1:2, more preferably in a molar ratio of about 2:1 to about 1:1 or in a molar ratio of about 1:1 to about 1:2, most preferably in a molar ratio of about 1:1.

[0265] 6. The pharmaceutical composition according to any one of the preceding items, wherein the at least one amphiphilic micelle-forming agent is selected from the group consisting of poloxamer, poloxamine, polyethylene glycol (PEG)-polyester, PEG-polyanhydride, PEG-poly- amino acid, phospholipid, polysorbate, polyoxyethylene alkyl ether and combinations thereof.

[0266] 7. The pharmaceutical composition according to item 6, wherein the PEG-polyester is selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), PEG-poly(s-caprolactone) (PCL) and combinations thereof.The pharmaceutical composition according to item 6 or 7, wherein the PEG-polyanhydride is a PEG-polysebacic anhydride (PSA).

[0267] The pharmaceutical composition according to any one of items 6 to 8, wherein the PEG-poly-amino acid is selected from the group consisting of PEG-poly(L-histidine), PEG-poly(L-aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), PEG-poly(L-lysine) and combinations thereof.

[0268] The pharmaceutical composition according to any one of items 6 to 9, wherein the phospholipid is conjugated to polyethylene glycol (PEG).

[0269] The pharmaceutical composition according to item 10, wherein the phospholipid is conjugated to PEG via a carbonyl group.

[0270] The pharmaceutical composition according to any one of items 6 to 11, wherein the phospholipid comprises one or more alkyl chains being independently selected from C8-C24alkyl(s), preferably selected from C10-C22alkyl(s), more preferably selected from C12-C20alkyl(s), even more preferably selected from C14-C18alkyl(s), and most preferably selected from C16-C18 alkyl(s);

[0271] wherein the alkyl chains are independently selected from saturated alkyl chains and unsaturated alkyl chains.

[0272] The pharmaceutical composition according to any one of items 10 to 12, wherein the phospholipid conjugated to PEG is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG, preferably DSPE-PEG.

[0273] The pharmaceutical composition according to any one of the preceding items, wherein the at least one amphiphilic micelle-forming agent is DSPE-PEG.

[0274] The pharmaceutical composition according to any one of items 6 to 14, wherein PEG has a molecular weight in the range from about 350-6000 Da, preferably in the range of about 550-5000 Da, more preferably in the range of about 750-4000 Da, even more preferably in the range of about 1000-3000 Da, and most preferably PEG has a molecular weight of about 2000 Da (PEG2000).The pharmaceutical composition according to item 14 or 15, wherein DSPE-PEG is DSPE-methoxy PEG (DSPE-mPEG).

[0275] The pharmaceutical composition according to any one of the preceding items, wherein the pharmaceutical composition comprises, preferably the micelles comprise, DSPE-(m)PEG2000.

[0276] The pharmaceutical composition according to any one of the preceding items, wherein the amphiphilic micelle-forming agent is selected from the group consisting of DMPE- (m)PEG2000, DSPE-(m)PEG2000, and DPPE-(m)PEG5000.

[0277] The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR7 agonist has a structural formula according to formula (I):

[0278]

[0279] (R2)n

[0280] wherein X1is O, S, or NRC;

[0281] R1is hydrogen, alkoxyalkyl, alkyl, substituted alkyl, Ce aryl or Cw aryl or substituted Cbaryl or substituted Cw aryl;

[0282] Rcis hydrogen, alkoxyalkyl, alkyl, or substituted alkyl; or Rcand R1taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring;

[0283] each R2is independently selected from -H, -OH, (Ci-C6)alkyl, substituted (Ci-C6)alkyl, (Ci-C6)alkoxy, substituted (Ci-C6)alkoxy, -C(O)-(Ci-Cfi)alkyl (alkanoyl), substituted -C(O)- (Ci-Cejalkyl, -C(0)-(C6-Cio)aryl (aroyl), substituted -C(O)-(C6-Cw)aryl, -C(O)OH (carboxyl), -C(O)O(Ci-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(Ci-C6)alkyl, -NRaRb, - C(O)NRaRb(carbamoyl), halo, nitro, or cyano, or R2is absent;

[0284] each Raand Rbis independently selected from hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted(C1-C6)alkoxy, (Ci-C6)alkanoyl, substituted (Ci-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het (Ci-C6)alkyl, or (Ci-C6)alkoxycarbonyl;

[0285] wherein the substituents on any alkyl, aryl or heterocyclic group are hydroxy, Ci-6alkyl, hydroxyl Ci.6alkylene, Ci-6alkoxy, C3-6 cycloalkyl, C1-6 alkoxy, C1-6 alkylene, amino, cyano, halo, or aryl;

[0286] n is 0, 1, 2, 3 or 4;

[0287] X2is a bond, -C(O)- or -C(O)-NH-CH2-CH2-; and

[0288] R3is a phospholipid comprising one or two carboxylic esters;

[0289] or a tautomer thereof;

[0290] or a pharmaceutically acceptable salt or solvate thereof.

[0291] 20. The pharmaceutical composition according to item 19, wherein n is 0, X1is O, and R1is methoxyethyl.

[0292] 21. The pharmaceutical composition according to item 19 or 20, wherein R3in formula (I) comprises

[0293]

[0294] wherein R11and R12are each independently a hydrogen or an acyl group, R13is a hydrogen, and m is 1 to 8, preferably 1, and wherein a wavy line indicates a position of bonding, wherein the absolute configuration at the carbon atom bearing OR12is R, S, or any mixture thereof.

[0295] 22. The pharmaceutical composition according to item 21, wherein m is 1, and / or wherein R11and R12are each oleoyl groups.

[0296] 23. The pharmaceutical composition according to any one of items 19 to 22, wherein the phospholipid of R3comprises two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof.The pharmaceutical composition according to any one of items 19 to 23 wherein the phospholipid of R3comprises two carboxylic esters and the carboxylic esters of are the same or different.

[0297] The pharmaceutical composition according to item 23 or 24 wherein each carboxylic ester of the phospholipid is a C17 carboxylic ester with a site of unsaturation at C8-C9.

[0298] The pharmaceutical composition according to item 23 or 24 wherein each carboxylic ester of the phospholipid is a C18 carboxylic ester with a site of unsaturation at C9-C10.

[0299] The pharmaceutical composition according to any one of items 19 to 26, wherein R3comprises dioleoylphosphatidyl ethanolamine (DOPE).

[0300] The pharmaceutical composition according to any one of items 19 to 27, wherein R3is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and X2is C(O).

[0301] The pharmaceutical composition according to any one of items 19 to 28, wherein X1is oxygen.

[0302] The pharmaceutical composition according to any one of items 19 to 29, wherein X1is O, R1is methoxyethyl, n is 0, X2is -C(O)-, and R3is 1,2-dioleoylphosphatidyl ethanolamine (DOPE).

[0303] The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR7 agonist has a structural formula according to formula (la):

[0304]

[0305] The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR4 agonist has a structural formula according to formula (II):

[0306]

[0307] wherein z! is an integer from 1 to 4, wherein z2 is an integer from 0 to 5, wherein R5is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R6is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R7is hydrogen, or substituted or unsubstituted alkyl, and wherein each R8is independently

[0308] halogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCl3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a tautomer thereof; or a pharmaceutically acceptable salt or solvate thereof.

[0309] 33. The pharmaceutical composition according to item 32, wherein z1 is 1.

[0310] 34. The pharmaceutical composition according to item 32 or 33, wherein R8is substituted or unsubstituted heteroaryl.

[0311] 35. The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR4 agonist has a structural formula according to formula (Ila):

[0312]

[0313] and prolyl, R15is selected from the group consisting of hydrogen, F and methyl, and R16is selected from the group consisting of cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 4-fluorophenyl, isopropyl, isobutyl, and 2,2-dimethylbutyl.

[0314] The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR4 agonist has a structural formula according to formula (lib):

[0315]

[0316] (lib).7. The pharmaceutical composition according to any one of the preceding items, wherein the at least one TLR4 agonist has a structural formula according to formula (lib):

[0317]

[0318] (lib), wherein the at least one TLR7 agonist has a structural formula according to formula (la):

[0319]

[0320] (Ia), and wherein the at least one amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0321] 38. The pharmaceutical composition according to any one of the preceding items, wherein the solid particles are (nano) crystals.

[0322] 39. The pharmaceutical composition according to any one of the preceding items, wherein the at least one amphiphilic micelle-forming agent and the at least one TLR7 agonist are present in the pharmaceutical composition in a molar ratio in the range of about 20:1 to about 4:1, preferably in the range of 10:1 to about 4:1, more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.0. The pharmaceutical composition according to any one of the preceding items, wherein the pharmaceutical composition comprises DSPE-mPEG2000, the at least one TLR4 agonist and at least oneTLR7 agonist in a molar ratio in the range of about 20:1:1 to about 4:1:1, preferably in the range of 10:1:1 to about 4:1:1, more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

[0323] 41. The pharmaceutical composition according to any one of the preceding items, wherein the micelles have a particle size in the range of about 2 to about 50 nm, preferably about 5 to about 30 nm, and more preferably about 8 to about 20 nm.

[0324] 42. The pharmaceutical composition according to any one of the preceding items, wherein the micelles have a polydispersity index (PDI) greater than 0 and below 0.2.

[0325] 43. The pharmaceutical composition according to any one of the preceding items, wherein the solid particles are (nano) crystals having at least one side having an average length in the range of 50 to 220 nm, preferably in the range of 75 to 200 nm, more preferably in the range of 100 to 190 nm.

[0326] 44. The pharmaceutical composition according to any one of the preceding items, comprising an effective amount of the at least one TLR4 agonist and of the at least one TLR7 agonist.

[0327] 45. The pharmaceutical composition according to any one of the preceding items, further comprising at least one additional active agent.

[0328] 46. The pharmaceutical composition according to item 45, wherein the at least one additional active agent is selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, a further TLR7 agonist, and combinations thereof.

[0329] 47. The pharmaceutical composition according to item 46, wherein the chemotherapeutic agent is selected from a group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine, streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycinC, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyl lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

[0330] The pharmaceutical composition according to item 46, wherein the antimicrobial agent is selected from a group consisting of antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.

[0331] The pharmaceutical composition according to item 46, wherein the immune checkpoint inhibitor is selected from a group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

[0332] The pharmaceutical composition according to item 48, wherein the antibiotic is selected from the group consisting of amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, levofloxacin, and combinations thereof.

[0333] The pharmaceutical composition according to any one of the preceding items, further comprising a pharmaceutically acceptable excipient, preferably water for injection or a pharmaceutically acceptable buffer.

[0334] A method of preparing the pharmaceutical composition according to any one of the preceding items comprising

[0335] step a) of providing a mixture comprising at least one amphiphilic micelle-forming agent, the at least one TLR4 agonist, the at least one TLR7 agonist and an organic solvent, step b) of lipid film hydration, including evaporating the organic solvent and, optionally, step c) of filtration,

[0336] thereby preparing the pharmaceutical composition comprising

[0337] i) solid particles comprising the at least one toll-like receptor 4 (TLR4) agonist and ii) micelles comprising the at least one toll-like receptor 7 (TLR7) agonist and the at least one amphiphilic micelle-forming agent.

[0338] The method according to item 52, wherein the organic solvent comprises chloroform, methanol, tetrahydrofuran, or a mixture thereof.The method according to item 52 or 53, wherein in step b) phosphate buffered saline was used for the hydration of the lipid film.

[0339] The method according to any one of items 52 to 54, wherein step c) is performed using a filter threshold of about 0.1 to 0.3 pm, preferably of about 0.2 pm.

[0340] The method according to any one of items 52 to 55, wherein step c) is performed using a polyethersulfone (PES), a polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF) membrane.

[0341] A kit or kit of parts comprising the pharmaceutical composition according to any one of items 1 to 51 and a pharmaceutically acceptable carrier, preferably water for injection or a pharmaceutically acceptable buffer, and, optionally, comprising handling instructions.

[0342] The pharmaceutical composition according to any one of items 1 to 51 or the kit or kit of parts according to item 57 for use as a medicament.

[0343] The pharmaceutical composition according to any one of items 1 to 51 or the kit or kit of parts according to item 57 for use in the treatment of cancer, an antibiotic resistance, an inflammatory condition or disease, an autoimmune disease or an infectious disease.

[0344] The pharmaceutical composition or the kit or kit of parts for use according to item 58 or 59 in the treatment of cancer.

[0345] The pharmaceutical composition or the kit or kit of parts for use according to item 59 or 60, wherein the cancer is selected from a group consisting of head and neck cancer, including head and neck squamous cell carcinoma, melanoma, basal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, colon carcinoma, bladder cancer, gastric cancer, stomach cancer, colorectal cancer, liver cancer, breast cancer, lung cancer, and (squamous) skin cancer.

[0346] The pharmaceutical composition or the kit or kit of parts for use according to any one of items 58 to 61, wherein the pharmaceutical composition is administered to a subject in need of a TLR4 and / or TLR7 agonist treatment, preferably the subject is a human subject.

[0347] The pharmaceutical composition or the kit or kit of parts for use according to any one of items 58 to 62, wherein the pharmaceutical composition is administered orally, intranasally, via aerosol, via inhalation, parenterally, intramuscularly, intraperitoneally, intravenously, rectally, intravesically or subcutaneously.The pharmaceutical composition or the kit or kit of parts for use according to any one of items 58 to 63, wherein the pharmaceutical composition is administered intravenously. The pharmaceutical composition or the kit or kit of parts for use according to any one of items 58 to 64, wherein the pharmaceutical composition is administered together with at least one additional active agent.

[0348] The pharmaceutical composition or the kit or kit of parts for use according to item 65, wherein the at least one additional active agent is selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, a further TLR7 agonist, and combinations thereof.

[0349] The pharmaceutical composition or the kit or kit of parts for use according to item 66, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyl lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

[0350] The pharmaceutical composition or the kit or kit of parts for use according to item 66, wherein the antimicrobial agent is selected antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.

[0351] The pharmaceutical composition or the kit or kit of parts for use according to item 66, wherein the immune checkpoint inhibitor is selected from the group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

[0352]

[0353] ion of the

[0354] Figure 1: Synergistic effect, TLR4B and TLR7A in THP1 cells (PMA or PMA plus IFNg(y)).

[0355] A: IL-1 B(P) response, B: IL-6 response, C: TNF-a(a) response.

[0356] Figure 2: Cytokine secretion of human PBMCs stimulated with KUP-101. A: IL-6 Donor 1.

[0357] B: IL-6 Donor 2. C: IL-1 p Donor 1. D: IL-1 p Donor 2. LPS = Lipopolisaccharide Figure 3: Tumor killing for SK-MEL28 mediated by human blood cells stimulated with the TLR7A / TLR4B formulation at the end of the experiment (72 h) for 3 donors (A, B, C) and mean for 3 donors (Figure D).

[0358] Figure 4: Tumor killing for A549 mediated by human blood cells stimulated with the TLR7A / TLR4B formulation at the end of the experiment (72 h) for 3 donors (A, B, C) and mean for 3 donors (Figure D).

[0359] Figure 5: Experimental set-up Melanoma PDX Model.

[0360] Figure 6: Tumor size and statistical calculations. A: Donor 1, B: Donor 2.

[0361] Figure 7: Tumor Volume Measurements. Mean absolute tumor volume (mm3) ± SEM.

[0362] Asterisks (*) above the treatment group symbols denotes significant differences between groups within single days. The asterisks to the right of the treatment group symbols on the final day denotes overall differences between groups. Figure 8: Measurement of area of tumor metastasis in lung.

[0363] Figure 9: Number of tumor foci. A: Tumor foci in lung, B: Tumor foci density.

[0364] Figure 10: Anti metastatic effect of the TLR7A / TLR4B composition in the 4T1 model.

[0365] Statistical analysis of lung metastasis. Bar graph of tumor area percent at terminal time point at Day 23. Statistical analysis results of Group 1: Vehicle (no agonist); Group 2: Paclitaxel; Group 3: TLR7A / TTR4B A (1:1 molar ratio low dose combo); Group 4: TLR7A / TLR4B B (1:1 molar ratio med. dose combo); Group 5: TLR7A / TLR4B C (1:1 molar ratio high dose combo); Group 6: D (TLR4 alone med. Dose); Group 7: E (TLR7A alone med. Dose). In the bar graph, green line indicates mean, orange line indicates median and * indicates p<0.05 compared to Group 1 vehicle control.Figure 11: Synergy of formulated compounds (TLR7A / TLR4B in DMSO and the TLR7A / TLR4B A formulation) in the THP1 in vitro experiment. A: IL-10, B: TNF- a, C: IL-6. Statistical analysis was conducted using a one-way ANOVA, followed by Dunnett's multiple comparisons test *: p<0.05; **: p < 0.01; ***: p<0.001; ****: p<0.0001.

[0366] Figure 12: Effect of the TLR7A / TLR4B composition on primary tumor size and number of metastases of M001 Melanoma cells administered with PBMCs and monocytes from Donor 1 and Donor 2. A: primary tumor, Donor 1. B: metastases, Donor 1. C: primary tumor, Donor 2. D: primary tumor, Donor 2

[0367] Figure 13: In Figure 13, micellar structures can be seen in the background. These micelles appear to be very small, which is caused by the hydration layer of the micelles (hydrophilic part) that is not visible using cryo-TEM analysis. The average diameter of the micelles was determined by measuring the center-to-center distance for approximately 100 particles per picture via Image). The active TLR7A / TLR4B A formulations contained small micelles (A), which had an average size of approximately 14 nm. In addition to the small micelles, larger rhomboid / brick- shaped crystals were present in the active TLR7A / FLR4B A batches. Using Image), the average size of the crystals was determined by measuring the shortest and longest (diagonal) side, as depicted in Figure 13 (B, C). It was found that for all samples, the Z-avg size determined by DLS lies in between these values. Although in some batches the shape of the crystals was less faceted and more irregular, and in some images, there was more ice contamination, cryo-TEM analysis displayed the same type of particles in all active TLR7A / TLR4B A batches.

[0368] Figure 14: XRPD pattern of 2B182C Pattern I, batch CH1857-2-7A of the nano crystals of TLR4B in the composition obtained in Example 1.

[0369] Figure 15: DSC thermogram of compound 2B182C Pattern I, batch CH1857-2-7A of the nano crystals of TLR4B in the composition obtained in Example 1.

[0370] Example section

[0371] It should be noted that in some instances in the example section and the Figures and Figure legends e.g. IL-0 is denoted as IL-b, TNFa is denoted as TNFa and INFy is denoted as IN Fg.

[0372]

[0373] 1.1 Lipid film

[0374]

[0375]

[0376] of the formulation Lipid film formation and hydration of 56 100 mL sub batches was prepared. For one 100 mL sub batch, 1966 + 20 mg DSPE-mPEG2000, 107 + 1.1 mg TLR7 agonist (here TLR7A), and 51 + 0.5 mg TLR4 agonist (here TLR4B) were weighed and 167 + 17 mL of organic solvent (CHCI3 / MeOH or THF)(9:1 vol) was added to the flask, and the content was swirled until all components were dissolved. Thereafter, the flask was attached to a rotary evaporator, and the temperature of the water bath was increased to 50 + 5 °C. The flask was spun at 280 rpm at atmospheric pressure, while the temperature of the water bath was heating up. When the temperature of the water baths reached 50 + 5 °C, the pressure was decreased gradually from 1000 mbar to 200 mbar in 7 ± 2 min. The flask was left spinning at 200 mbar for a total of 30 + 3 min. During this time, the stirring speed was decreased to 10 rpm twice to distribute the lipid film around the flask, after which the stirring speed was increased to 280 rpm. After 30 + 3 min, the pressure was decreased to 0 mbar and the flask was left spinning at 280 rpm for 60 ± 6 min. After 60 min, the lipid film was visibly dry and then removed from the rotary evaporator and stored under reduced pressure at room temperature (RT) overnight. The next day, the flask was removed from the vacuum. 100 ± 1 mL PBS was heated to 50 + 5 °C using a Buchi water bath for 30 ± 3 min. At 50 °C, the PBS was added to the 2 L flask containing the lipid film, and the flask was immediately attached to the rotary evaporator. The flask was spun at 50 °C for 15 + 2 min at a stirring speed of 140 rpm. When the film was visually hydrated, the stirring speed was increased to 280 rpm for 1 min to ensure that the top part of the lipid film was also rehydrated. Thereafter, the stirring speed was decreased to 140 rpm and the flask was left spinning. After 15 min of spinning, the flask was removed from the rotary evaporator and was immediately placed in an ice bath for 30 + 3 min. Thereafter, the crude product was transferred to an Erlenmeyer flask.

[0377] Filtration: Filtration of the 56 formulation sub batches was performed in series. For each sub batch, this was done on the same day as the film hydration. The crude sub batches were filtered over PES filter of 0.2 pm into 250 mL or 500 mL Duran bottles. The filtered products were stored at 2 - 8 °C until further analysis / use.1.2 IPC an

[0378]

[0379] characterization

[0380] Before combining the filtered sub batches, the size and PDI of the crude and filtered particles were determined via Dynamic Light Scattering (DLS). In addition, the DSPE-mPEC2000 TLR7, and TLR4 contents were determined via Ultra Potent Liquid Chromatography (UPLC), and the residual chloroform and methanol contents were determined via Gas Chromatography Headspace (GC-HS).

[0381] The Z-average particle size of the 56 sub-batches varied between 119 and 169 nm (target size 100 - 200 nm), and the PDI varied between 0.09 and 0.14. Most formulation sub-batches had low residual solvent contents and only few sub-batches had chloroform contents of > 60 ppm, which is the limit given by the ICH guidelines. However, the theoretical average residual chloroform content would be 20 ppm upon combination of all 56 sub batches. The TLR7 content of the other sub-batches was within the target values of 0.93 - 1.27 mg / mL, however, slightly higher than expected. All batches had a DSPE-PEG2000 concentration of 18.4 - 19.8 mg / mL, which is within target values (16.7 - 22.7 mg / mL). Lastly, most batches had a TLR4 content of 0.43 - 0.51 mg / mL (target concentration 0.43 - 0.58 mg / mL).

[0382] 1.3 Combination and low bioburden filtration

[0383] The 56 formulation sub batches were combined in a 5-L Duran bottle, obtaining 5.05 L formulation. Thereafter, the material was filtered over a PES 0.2 pm filter into another 5-L Duran bottle under low bioburden conditions in the BioSafety Cabinet (BSC). After filtering 3.8 L, the filter was changed to a new filter as the pressure started to rapidly increase (pmax= 2.0 bar). The remaining material was filtered over the second filter, resulting in a total volume of 4.99 L, from which the samples were taken for endotoxin and LC-UV CAD analysis and for stability study. Thereafter, 4.5 L of formulation was transferred to five 1 L Duran bottles under low bioburden conditions. This material was stored at 2 -8 °C until it was shipped at the request of the customer. The remaining material was transferred to two 250-mL Duran bottles and stored at 2 - 8 °C until further use. The PES filters were tested for integrity post-use and passed the test.Table 1: Analytical results for the formulation

[0384] Test Method Result

[0385] Yield - 5.0 L

[0386] DLS Z-average diameter 139 nm

[0387] PDI 0.1

[0388] LC-UV-CAD TLR7A content 1.19 mg / mL

[0389] TLR4B content 0.46 mg / mL

[0390] DSPE-PEG2000 content 19.1 mg / mL

[0391] GC-HS Chloroform content <25 ppm

[0392] Methanol content <300 ppm

[0393]

[0394] 1.4 Conclusion

[0395] The manufacture of the tox batch was successfully finalized. 56 sub-batches of the formulation were made. Although for some formulation sub-batches the chloroform content was higher than allowed by the ICH guidelines (> 60 ppm) and the TLR4 content was lower than target values, it was decided to combine all sub batches, as the average values of all 56 sub-batches were within specifications. After combination of the batches, a final filtration was executed in the BSC. From the release analysis, it is concluded that the formulation met all release criteria. The manufacture was finalized with a yield of 5.0 L formulation with TLR4B and TLR7A.

[0396] 1.5 Particles and crystals were analyzed according to the following two parameters:

[0397] Shape of particles and crystals, diameter of particles and largest diameter of crystals were measured. Two different classes of particles are observed on the images (Figure 13). One class consists of small micellar particles with a size of -10-20 nm featuring a globular or elongated rectangular shape. The second particle class consists of nano crystals within in a size range of 120 - 200 nm.

[0398] Pharmaceutical formulation obtained as in Example 1 comprising micelles comprising TLR7A and TLR4B (nano) crystal structures is denoted as TLR7A / TLR4B formulation. It is a colloidaldispersion containing DSPE-PEG2000 as excipient. Preliminary stability studies show that the colloidal dispersion is at least stable over 3 months at 5±3°C.

[0399] 1.6 Cryo-TEM analysis

[0400] Cryo-TEM analysis was performed for several TLR7A / TLR4B A formulation batches and empty vehicle (placebo) batches of only DSPE-mPEG2000. An exemplary picture is depicted in Figure 13. In Figure 13, micellar structures can be seen in the background (Figure 13 A). These micelles appear to be very small, which is caused by the hydration layer of the micelles (hydrophilic part) that is not visible using cryo-TEM analysis. The average diameter of the micelles was determined by measuring the center-to-center distance for approximately 100 particles per picture via Image]. For the placebo batches, the average micelle size was approximately 13 nm, which is very close to the size given by DLS. Remarkably, the active TLR7A / TLR4B A formulations also contained small micelles, which had an average size of approximately 14 nm. In addition to the small micelles, larger diamond / brick-shaped crystals were present in the active TLR7A / TLR4B A batches. Using Image], the average size of the crystals was determined by measuring the shortest (C) and longest (diagonal)(B) distance, as depicted in Figure 13. It was found that for all samples, the Z-avg size determined by DLS lies in between these values. Although in some batches the shape of the crystals was less faceted and more irregular, and in some images, there was more ice contamination, cryo-TEM analysis displayed the same type of particles in all active TLR7A / TLR4B A batches.

[0401] Cryo-TEM analysis, thus, showed that the active TLR7A / TLR4B A formulation consists of small micelles and larger crystals instead of only one type of particle. To further investigate the structure and content of the small micelles and the crystals, they were separated via different techniques, and new cryo-TEM measurements were performed as described in the next paragraphs.

[0402] Cryo-TEM analysis demonstrated that the TLR7A + DSPE-PEG batch consisted of small micelles, whereas the batch containing TLR4B + DSPE-PEG consisted of small micelles and crystals. Therefore, it can be concluded that TLR7A can be successfully loaded in DSPE-PEG micelles whereas the TLR4B exists as separate crystals between 100-200 nm next to the small DSPE-PEG micelles.e 2: TLR7 to TLR4 molar ratio selection for human use -

[0403]

[0404] ine secretion from TH P-1 cells

[0405] 2.1 THP-1

[0406]

[0407] Aim of the The goal of this project was to qualify an in vitro THP-1 cell assay for assessing the activation of innate immunity by TLR4B and TLR7A and to obtain an understanding of the optimal range of both agonists to promote the differentiation of M1 macrophages (Mcp).

[0408] Methodology: THP-1, a human derived monocyte cell line (ATCC # TIB-202) was treated with phorbol myristate acetate (PMA) to differentiate the monocytes to non-polarized macrophages. Serially diluted concentrations of TLR4B and TLR7A in DMSO were co-cultured with the M<p for 1, 2, 3, 6 and 24-hour time periods (Bagaev, et al., 2018, J Immunol, 200(8): 2656-2669). At each time period, the media was collected for IL-1, IL-8, IL-6, IL-12p70, IFNa, MIP-1 p, MCP-1, and TNFa analysis. At 0, 1, 2, 3, 6 and 24-hour timepoints Mrp were assessed for polarization via flow cytometry for CD68 (pan macrophage marker), CD80 (M1 Mrp marker), CD163 (M2 M<p marker), membrane TLR4 expression and for intracellular TLR7 receptor expression. A separate cohort of M<p was assessed via flow cytometry for intracellular NFKB and p-actin following methanol fixation.

[0409] Results: The TLR4B had the stronger or dominant effect on cytokine secretion and NFkB activation. TLR7 had a less pronounced effect. Some synergy between TLR4 / 7 agonists was observed for NFkB and IL-1 b expression. NFkB expression was the highest with the TLR4B / TLR7A agonist combinations, in which the TLR4 was 10 pM. A mainly TLR4 concentration dependent increase for IFN-y, IL1-P, IL-6, IL-12, MCP-1, and TNFa was observed. No effect was observed for IL-8 at 24 hours, however, a limited effect was seen after 3 hours of stimulation. Viability slowly decreases with a concentration of TLR4 agonist of >10 mM. CD80 expression (M1 M<p marker) was observed, in response to TLR4 stimulation. No change was observed for CD163 (M2 macrophage). Strong MCP-1 (CCL2) and some MIP-1 b secretion was observed. Due to the strong TLR4 and minimal TLR7 agonist effects, synergistic effects were observed but were minimal. Conclusion: TLR4B triggered a dose dependent increase of M1 related cytokines which was the highest after 24 hours of incubation. At TLR4 concentration of > 10 mM, the cell viability decreased. A mainly TLR4 agonist-induced and dose dependent increase of NFKB activation was observed with a maximum effect after 1h. Although some synergy for TLR4 / 7 combination wasobserved for NFsB(p65) and IL-10, the effects were modest, likely due to the relatively low activity observed for the TLR7 agonist in THP1 cells treated with PMA.

[0410] 2.2 THP1 IFN-v

[0411] Aim of the study: The aim was to evaluate effects of TLR4B and TLR7A in human THP1 cells treated with PMA (see above) and to compare this with the effects when cells were further stimulated with IFN-y, to restore functional responsiveness of THP-1 cells to TLR7 (Gantier et al., 2008., The Journal of Immunology 180:2117-2124). TLR7 is involved in sequence-specific sensing of single-stranded RNAs in human macrophages.

[0412] Methodology: THP1 cells were treated with PMA or PMA plus IFN-y to create macrophages responsive to both TLR4 and TLR7 stimulation. THP-1 were differentiated with PMA for 16 hours then washed and incubated for an additional 6 hours with or without IFN-y in StemCell ImmunoCult SF media. TLR4B and TLR7A in DMSO solution were added simultaneously in molar ratios of 1:1, 1:10, 1:30, and 10:1 to these THP-1 cells. The effects of single agonists alone were also tested. Positive controls were imiquimod and LPS.

[0413] Results: THP1 cells pre-treated with both PMA and IFN-y had higher cytokine expression levels than THP1 cells pre-treated with PMA alone. Treatment for 24 hours with the combined TLR7: TLR4 agonists stimulated higher expression levels than the 0 baseline for all measured cytokines. It was also noted that the addition of IFN-y with PMA during the pre-treatment period resulted in an overall decrease in cell viability of approximately 10-30 percent.

[0414] TLR7A alone exhibited a dose responsive increase in cytokines IL-10, IL-6, and TNF-a with the IFN-y stimulated THP-1 / PMA cells. Treatment with TLR4B alone produced an increase in these same cytokines only at the highest concentration tested in IFN-y stimulated THP-1 / PMA cells.Table 2: EC50 values for single agonists

[0415] EC50THP1 / PMA EC50THP1 / PMA and IFNy Cytokine

[0416] TLR4B (mM) TLR7A (mM) TLR4B (mM) TLR7A (mM)

[0417] IL1-p 2* 2.8* 1.9* 5.7*

[0418] IL-6 2.7* 1.8* 2.4 0.4*

[0419] TNFa 2.7* 0.2 0-9 2.8*

[0420]

[0421] * As the concentrations did not span a wide range, the response often did not meet a maximal or minimal limit (top or bottom plateau) typical of a sigmoidal dose response curve; thus, the fitting of EC50 may be imprecise.

[0422] A more than additive effect was observed when TLR4B and TLR7A were provide in combination in DMSO solution for IL-1 p, TNFa (all molar ratios except 10 TLR7 to 1 TLR4). Strongest combination results were obtained for:

[0423] • IL-6: 1 mM to 1 mM (1:1)

[0424] • IL-1 (IL-1 B): 0.3 mM TLR7 to 3mM (1:10) and 0.1 mM TLR7 to 3 mM TLR4 (1:30) • TNFa: 1 mM to 1 mM (1:1 ) and 0.1 mM TLR7 to 3mM TLR4 (1:30)

[0425] Results are summarized in Figure 1: Molar ratios: 1 mM to 1 mM (1:1), 0.3 mM TLR7 to 3 mM (1:10), 1 to 30 (0.1 mM TLR7 to 3 mM TLR4), 0.3 mM TLR4 to 3 mM TLR7 (10:1).

[0426] Conclusion: Treatment of cells with PMA and IFNy resulted in macrophages with an enhanced response to TLR7 stimulation when compared to cells treated with PMA only. Statistically significant synergistic increases were observed when treatment was done with both compounds together. Depending on the molar ratio, the synergetic effects differed for the individual cytokines investigated (TNFa, IL-6 and IL-1 p). For IFN-y, the cytokine expression was reduced with the combination. Trends of synergy for cytokine secretion for PMA and PMA plus IFN-y stimulated cells were similar.e 3: TLR7 to TLR4 molar ratio selection - ine excretion studies in hPBMC derived cells

[0427] 3.1 hPBMC

[0428]

[0429] Aim of the The purpose of this experiment was to evaluate the potential of TLR4B and TLR7A alone and in combination to stimulate cytokine production in adherent cells (monocytes) derived from cryopreserved hPBMCs of 2 healthy donors (19-45 years old).

[0430] Methodology: Two vials of each donor (11 and 29) were thawed, washed and incubated overnight in X-Vivo 10 cell culture media with 5% AB serum. Non-adherent cells were removed by gentle washing with complete media (5% AB / X-Vivo 10). Adherent cells were removed, counted, and plated in a 96 well plate in complete media. On day 4 of cell incubation, the TLR agonists (in DMSO solution or controls were added in molar ratios of 1:1, 1:10, 1:30, and 10:1 and incubated for 24 hours. TLR7: TLR4 agonist combinations were added simultaneously. Media was collected and analyzed for IL-6, IL-1 p, IFN-y, and TNFa.

[0431] Results: Both the TLR4 and TLR7 agonists individually showed a dose-dependent increase of cytokine expression. Several boosted synergistic cytokine stimulation effects were seen for the combination of the TLR4 and TLR7 agonists.

[0432] Cytokine expression results: The effects on IL-6 and TNF-a were more pronounced for both donor 11 and 29 at the TLR7 / 4 agonist molar ratios of 1 to 10 (0.3 pM TLR7 agonist to 3 pM TLR4 agonist) and 1 to 30 (0.1 pM TLR7 to 3 pM TLR4). Only small effects for IL-1 p were seen. The LPS (10 mg / ml) controls exhibited good cytokine responses over the baseline control while the imiquimod (10 pM) control showed little to no response.

[0433] Conclusion: Similar to the results observed in the experiments with THP1 cells, synergistic effects on cytokine expression (IL-6, IL-ip, TNFp) were observed in adherent cells isolated from cryopreserved PBMCs from two healthy individuals stimulated with the TLR4 / 7 agonists.

[0434] 3.2 Inotiv Study HPBMC-KPD-1 Effects of Compounds and Stimulation on Secretion of IL- 1 b(3) and IL-6 on Vitro Isolated Human Peripheral Blood Monocytes

[0435] Aim of the study: The aim was to evaluate secretion of cytokines IL-1 p and IL-6, from adherent monocyte / macrophages cultured from cryopreserved PBMCs from two healthy individuals (>45 years old) after incubation with micellar encapsulated TLR7A and TLR4B crystals at molar ratios of 1:1 or 1:10 versus unformulated agonists dissolved in DMSO. A second aim was toevaluate the effects of serum on the ability of the TLR4 / TLR7 formulation of Example 1 to induce cytokine secretion. IL-1 p is of particular interest since its secretion by innate immune cells is associated with the phenomenon of trained immunity.

[0436] Methodology: Two separate cryopreserved human PBMC samples were obtained and resuspended in culture media. Cells were diluted to 2x106cells / ml in complete X-Vivo media supplemented with 5% Human AB serum. Cells were incubated overnight to allow monocytes to adhere to the plate. After overnight incubation non-adherent cells will be washed away and adherent cells will be collected by washing with 10 mM EDTA / PBS. Isolated monocytes will be resuspended seeded into 96 well plates. Cells will be treated with the agonist formulation of Example 1 and free TLR7 / 4 agonists at 2 concentrations and 2 molar ratios for 24 hours in the presence of 5% Human AB Serum. The 1:1 molar ratio will be tested at 1 pM:1 pM and 3 pM:3 pM and the 1:10 molar ratio will be tested at 0.3 pM:3 pM and 3 pM:9 pM agonist concentrations. To evaluate the effects of serum on the ability of the TLR4 / 7 agonist formulation to induce cytokine expression, one set of cells (plate 2) will be pre-treated with the TLR4 / 7 agonist formulation of Example 1 for 2 hours prior to the addition of 5% Human AB Serum. Following the addition of agonists, supernatants will be collected for measurement of IL-1 b(0) and IL-6 and images of representative wells will be acquired to assess cell morphology.

[0437] Results: Cytokine secretion for the TLR7A / TLR4B formulation as well as for the unformulated (in DMSO) occurred with the 1 to 1 and the 1 to 10 molar ratio.

[0438] Conclusions: Stimulation of monocytes with the 1 to 1 or 1 to 10 molar ratio of the TLR7A / TLR4B formulation results in a strong increase of cytokine secretion.

[0439] 3.3 Summary

[0440] Statistically significant synergies regarding cytokine secretion and activation of NFKB induced by TLR4B and TLR7A were observed for several molar ratios of this combination in human derived cells. Cytokine results are shown in Figure 2.

[0441] In vitro testing with the TLR7A / TLR4B formulation as produced in Example 1 provided a similar pattern of synergy, albeit with lower absolute levels of secreted cytokines compared to the unformulated TLR agonists. This may be due to methodological constraints of the in vitro experiments. Data obtained in the preliminary tox study in mice suggests that synergistic effects (boosted cytokine responses) with both compounds are observed in vivo.The best molar ratios of TLR7 to TLR4 agonists as derived from cytokine secretion data using human immune cells and PBMC are 1 to 1 and 1 to 10 as determined in a human monocyte cell line. An excess of TLR7 agonist over TLR4 seems not favorable in terms of boosting synergistic cytokine production.

[0442] Example 4: Ex vivo studies - 2D and 3D Tumor platform

[0443] 4.1 Introduction

[0444] Ex vivo tumor 2D cell line derived and 3D patient-derived xenograft (PDX) models were used to test the molar ratios which were shown to be active in the in vitro cellular assays.

[0445] Tumor spheres from PDX derived cell lines isolated from solid tumors of different origins were embedded into layers of Cypre's VersaGel® (a biocompatible and chemically defined extracellular matrix hydrogel) using the Symphony® 3D gel patterning technology for standard 96-well plates. Human PBMCs and matching monocytes were layered above the 3D tumor sphere culture as a model of the patient's immune system to allow for the evaluation of immunotherapies on tumor growth.

[0446] 4.2 2D tumor killing experiment

[0447] Tumor killing mediated by the TLR7A / TLR4B formulation (molar ratio agonist 1 to1) was investigated with 2 tumor cell lines, the melanoma cell line SK-MEL-28 and the epithelial derived lung cancer cell line A549 over a time period of 72 h using the Incucyte system.

[0448] Aim of the study: The goal of the study was to investigate whether human PBMC plus autologous monocytes stimulated by the TLR7A / TLR4B formulation could kill tumor cells.

[0449] Methodology: Fresh PBMCs from 3 donors plus autologous monocytes were isolated. 50000 / well of PBMC and 5000 / well of monocyte cells were co-added to wells containing Cell Tracker labelled tumor cells treated with the TLR7A / TLR4B formulation of Example 1 (1:1 molar ratio at three concentrations), Pembrolizumab (Pembro) or the TLR7A / TLR4B formulation plus Pembrolizumab. A caspase 3 / 7 activated dye was added to the co-culture system to detect apoptosis. Anti-CD3 / CD28 treated cells were used as a positive control. Adherent tumor cells and total apoptotic cells were tracked and quantified continually for 72 hours using an Incucyte device.Results Target cells were co-cultured with PBMCs and monocytes from 3 different donors Cell Tracker Red dye-stained cells represent only tumor cells. The killing of tumor cells over time is reflected by a decrease in the Red Object Count (Per Image).

[0450] When using the data of total tumor cells at the end point (72 h), all treatment groups were compared with vehicle, the TLR7A / TLR4B formulation showed significant killing effect on all 3 random donors, the TLR7A / TLR4B formulation+Pembro only showed an effect on donor Y1814 and had no effect on donor Z0604 or Z0605. The results are shown in Figure 3.

[0451] The killing effect of the TLR7A / TLR4B formulation was also observed for the tumor cell line A549 (Figure 4). The tumor cell killing effect of TLR7A / TLR4B against the A549 tumor cell line was not as strong as the effect seen in the SK-MEL tumor cell line, but it was still significant. Similarly, also the positive control (CD3+IL2) shows a less significant tumor killing effect against the A549 tumor cell line compared to the SK-MEL tumor cell line. Response rates were similar across the 3 donors meaning that the tumor killing effect induced by TLR7A / TLR4B was significant for all three donors.

[0452] Conclusion: The TLR7A / TLR4B formulation (1 to 1 molar ratio) and the positive control CD3+IL2, showed killing for both cell lines. Effects were larger for SK-MEL_28 than for A549. Pembrolizumab showed no effect on its own. The killing pattern observed was relatively consistent for all 3 donors in all experiments. The TLR7A / TLR4B formulation mediated killing was concentration dependent. The killing effect started at around 30 h. The effect of the TLR7A / TLR4B formulation alone was stronger than the effect of TLR7A / TLR4B formulation+Pembrolizumab.

[0453] For the SK-MEL-28 cell line, the TLR7A / TLR4B formulation at the concentration of 3 pM showed a stronger effect than observed for the positive control, CD3+IL2. At 1 or 0.3 pM, similar killing effect as with CD3+IL2 were observed for Donors Y1814 and Z0605. The effect of TLR7A / TLR4B formulation+Pembro showed less killing of tumor cells for these two donors in comparison with the positive control.

[0454] On A549 cell line, the killing effect of TLR7A / TLR4B formulation and TLR7A / TLR4B formulation+Pembro was observed starting from around 48 h. The killing effect of TLR7A / TLR4B formulation was dose dependent and consistent for all 3 donors. There was no significant difference between TLR7A / TLR4B formulation and TLR7A / TLR4B formulation+Pembro treatmentat the same concentration for all 3 donors. The observed killing mediated by TLR7A / TLR4B formulation treatment was more pronounced in comparison to the positive control CD3 plus IL2.

[0455] 4.3 3D IO Melanoma PDX Model

[0456] Aim of the study: The goal of this study was to screen the TLR4 agonist TLR4B, the TLR7 agonist TLR7A and combinations of the two formulated in DMSO (non-micelle / crystal compounds) using the Cypre 3D immuno-oncology coculture assay with one melanoma PDX model (MEXF 622) using two PBMC donors and supplementing with matching monocytes. The PBMCs and monocytes act as a proxy for the immune system in this 3D tumor model system. The principle behind this assay is that immunotherapies can be evaluated for their anti-tumor effects via their ability to activate antigen presenting cells, which in turn actives T and B cells and results in tumor cell death. It is also possible that immunotherapy directly activates anti-tumor T cells.

[0457] Methodology: Tumor sphere size (as measured by high content Hoechst staining) and tumor killing (as measured by high content Caspase 3 / 7 staining) were assessed.

[0458] Tumor cells from the melanoma PDX model were embedded into layers of Cypre's extracellular matrix (ECM) hydrogel to generate 3D tumor spheres. After the spheres were established, TLR4B and TLR7A agonists dissolved in DMSO, PBMCs and matching monocytes were layered onto the surface of the hydrogel in 96 well plate. IL-2 was added to the media to maintain the viability of PBMCs. Imiquimod and Pembrolizumab served as positive controls. On day 6 following drug treatment, the model was assayed for tumor sphere size and tumor cell death using Cypre's high content image analysis algorithms (cf. Figure 5)

[0459] Results: Efficacy was determined by analyzing tumor sphere size, total tumor area and tumor cell death with high content imaging of Hoechst stained cells. The tumor size analysis suggests both the combination of TLR4B and TLR7A agonists (1 pM+1 pM) reduced tumor size and total tumor area for MEXF622 more than single agent TLR4B or TLR7A (1pM) agonist only with PBMCs / monocytes from donor 1 and 2 (cf. Figure 6).

[0460] Conclusion: The results suggest that TLR4B and TLR7A agonists synergistically promote tumor killing by human PBMCs cultured with matching monocytes in the MEXF622 melanoma model.1A

[0461] Example 5: In vivo studies

[0462] 5.1 Introduction

[0463] Effects of the TLR7A / TLR4B composition on primary tumors and metastasis were investigated in syngeneic mouse experiments.

[0464] 5.2 In Vivo Efficacy Study of the Effects of the TLR7A / TLR4B composition in the 4T1 Mouse Syngeneic Tumor Model

[0465] Aims of the study: This study was designed to test the effects of two molar ratios (1:1 and 1:10) of the TLR7A / TLR4B composition at two concentrations on tumor growth in syngeneic model of breast cancer (4T1 ) over 21 days.

[0466] Methodology: The tumor cell line was expanded prior to subcutaneous inoculation into the rear flank of each C57BL6 or BALB / C mouse. Per mouse 300000 tumor cells were injected. 8 mice per group were used. Following inoculation, tumors were allowed to grow until they can be palpitated prior to randomization. At that point, mice were treated IP twice a week for 6 weeks with the TLR7A / TLR4B composition or a checkpoint inhibitor or negative control (vehicle). Tumor size was measured twice a week for the duration of the study. Lungs were collected and analyzed histologically for the presence of metastatic nodules. The study was terminated at Day 28 and lungs were collected for H& E staining to look at the extent of lung metastases of the 4T1 tumor. Some lungs were collected prior to study termination from moribund mice (See Table 3 for the test groups). Metastases were quantified by counting the number of tumor foci in the lung tissue as well as the total area of tumor tissue in the lungs. All analyses were performed using the HALO imaging software.

[0467] Table 3: Summary on test groups

[0468] Group N Treatment Dose route Dose / kg body weight (two doses used) 1 8 Vehicle IP N / A

[0469] 2 8 Anti-PD-L1 IP 10 mg / kg

[0470] 3 8 TLR7A / TLR4B IP 1 and 0.5 mg / kg

[0471] (1:1; TLR7: TLR4

[0472]

[0473] 4 8 TLR7A / TLR4B IP 4 and 2 mg / kg

[0474] (1:1 TLR7: TLR4)

[0475] 5 8 TLR7A / TLR4B IP 1 and 5 mg / kg

[0476] (1:10; TLR7: TLR4)

[0477] 6 8 TLR7A / TLR4B IP 2 and 10 mg / kg

[0478] (1:10; TLR7: TLR4)

[0479]

[0480] Results:

[0481] Statistically significant decreases in primary tumor volume over time were observed for the TLR7A / TLR4B composition treatment groups in this 4T1 model. The results are depicted in Figure 6 and Table 4.

[0482] Table 4: Mean % Inhibition, Tumor Volume. Mean % inhibition of tumor volume for each group in comparison to the group 1 (vehicle) tumor volume by study days. Statistically significant values are underlined.

[0483] Treatment Group Day1 Day4 Day7 Day11 Day14 Day18 Day21

[0484] P value P value P value P value P value P value P value 1: Vehicle vs. 2: aPD-L1 >0.9999 0.9998 0.997 0.6926 0.0416 0.2948 <0.0001 1: Vehicle vs. 3: >0.9999 >0.9999 >0.9999 >0.9999 >0.9999 0.4378 0.047 TLR7A / TLR4B (1:1)

[0485] 1: Vehicle vs. 4: >0.9999 >0.9999 0.9954 0.2744 0.0355 0.047 0.0254 TLR7A / TLR4B (1:1)

[0486] 1: Vehicle vs. 5: >0.9999 0.9973 0.8866 0.0339 0.0027 0.0002 <0.0001 TLR7A / TLR4B (1:10)

[0487] 1: Vehicle vs. 6: >0.9999 0.9698 0.9757 0.0205 0.0014 0.0003 <0.0001 TLR7A / TLR4B (1:10)

[0488]

[0489] Survival: A tendency to prolonged survival was observed for groups receiving the composition prepared according to Example 1. Checkpoint treatment appeared to be toxic in this experiment.Lun metastasis assessment: All groups had 100% occurrence of tumor metastasis except for Group 4, which had only 87.5% of the animals showing tumor metastasis. Group 2 had the highest tumor area percentage (tumor area / whole lung tissue area) of all the groups at 17.44 + / -9.52%. This is very similar to that of Group 1 at 16.44 + / - 5.63%. It should be noted that only 3 animals were assessed in Group 2 due to animals dying on study. All other groups had at least 7 animals assessed for tumor metastasis. Group 5 and Group 4 had the lowest tumor area percentages at 1.77 + / -0.49% and 2.69 + / -1.02% respectively. For tumor foci density (number of tumor foci / whole lung area), Group 1 had the highest tumor foci density at 0.922 + / - 0.22 mm'2. Of the treated groups, Groups 2 and 6 had the highest tumor foci density 0.66 + / - 0.25 and 0.64 + / - 0.25 mm'2respectively. Group 5 had the lowest tumor density at 0.27 + / - 0.05 mm'2(cf. Figures 8 and 9).

[0490] Conclusion: T reatment with the TLR7A / TLR4B composition showed statistically significant effects on the primary tumor as well as on metastasis formation and size of metastasis in the 4T1 breast cancer syngeneic mouse model.

[0491] 5.3 In Vivo Efficacy Study of the 4T1 Model in BALB / c Mice

[0492] Aim: This second 4T1 tumor model was performed to show the synergy of the 2 agonists of the TLR7A / TLR4B composition.

[0493] Table 5: Summary on test items.

[0494] Group Treatment Dose / kg body weight

[0495] 1 Vehicle na

[0496] 2 Paclitaxel 10 mg / kg

[0497] 3 TLR7A / TLR4B 1:1 molar ratio 1 and 0.5 mg / kg

[0498] 4 TLR7A / TLR4B 1:1 molar ratio 4 and 2 mg / kg

[0499] 5 TLR7A / TLR4B 1:1 molar ratio 8 and 4 mg / kg

[0500] 6 TLR4B 2 mg / kg

[0501]

[0502] 7 TLR7A 4 mg / kgTable 6: Methodology.

[0503] 4T1 Breast cancer Mouse i.p. 2 x per week for 1 to 1 molar ratio

[0504] model (BALB / c) 3 weeks TLR7A & TLR4B 200 pL per Female animals, Day -1 inoculation injection

[0505] Investigating of tumors cells on Group 1: vehicle

[0506] primary tumours day -4, Dosing start Group 2: paclitaxel

[0507] and metastasis, at day 1. Group 3: 20 and 10 pg / mouse, single agonists 3 animals per group: Group 4 80 and 40 g / mouse and combination. sacrifice and Group 5: 160 and 80 pg / mouse 11 animals per immunostaining. Group 6: 80 pg TLR7A / mouse group. Day 24 terminal Group 7: 40 pg TLR4B / mouse timepoint

[0508]

[0509] Primary tumor size was assessed every 3 days approximately.

[0510] Metastasis analysis: Lung tissues were collected and fixed in 10% NBF for 24-48 hours before processing for FFPE embedding. One lung section / mice at 4 pM thickness was stained with H& E. Images of stained slides were acquired using Hamamatsu Nanozoomer image scanner. Images were analyzed with HALO3.1.1076.379. HALO is an image analysis software assisting pathologists to quantify objects identified regarding numbers of cells, area, staining intensity and so on. In this study, tumor and normal lung area presented in the section were annotated manually based on pathologist examination. Total lung tissue area, total tumor area and number of tumor foci were subsequently quantified using HALO.

[0511] Results: Primary tumor: No consistent statistically significant tumor volume reduction of Groups 2-7 in comparison to Group 1 (only for day 6 Group 4 versus group 1 and on day 20 Group 7 versus Group 1 ). At the terminal time point (day 24), Groups 2-7 showed significant reduction in tumor area percentages of lung metastasis compared to Group 1. Group 4 showed the lowest percentage. Synergy of the 2 agonists was observed regarding the anti metastatic activity (Groups 4 better than Groups 6 and Groups 7).Table 7: Tumor Area Percentage - Terminal

[0512] Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 1 N / A 0.98106 0.99814 1.00000 1.00000 0.99650 0.99814 Group 2 0.02652 N / A 0.73209 0.97296 0.87034 0.73252 0.52246 Group 3 0.00295 0.30629 N / A 0.93481 0.73209 0.56674 0.11725 Group 4 0.00016 0.03605 0.08026 N / A 0.26791 0.16783 0.00521 Group 5 0.00029 0.15880 0.30629 0.76830 N / A 0.26748 0.04693 Group 6 0.00554 0.31002 0.47754 0.85952 0.77214 N / A 0.19845 Group 7 0.00295 0.52246 0.90256 0.99650 0.96395 0.83217 N / A Non-normal data, one-tailed Mann-Whitney U test used. p<0.05 shows significance that the row group has lower tumor area percentage than the column group. Significant values are highlighted in bold.

[0513]

[0514] Metastasis: Metastasis formation was strongly reduced by TLR7A / TLR4B treatment. The 1 to 1 molar ratio showed the strongest reduction in the middle group (Group 4). The single separately formulated drug substances had also effects on their own (Group 6 and Group 7), however the combination of both agonists had a synergetic effect and further reduced metastasis formation (cf. Figure 10).

[0515] 5.4 Summary

[0516] Anti-tumor effects of the TLR7A / TLR4B composition of Example 1 were observed in vivo in the 4T1 model. Effects on the primary tumor were statistically significant in the first 4T1 experiment. Strong statistically significant effects on metastasis formation could be observed and confirmed. A synergy between both agonists could be shown for the metastasis formation. Spleenocytes from animals treated with the TLR7A / TLR4B composition of Example 1 showed specific tumor cell killing activity after re-stimulation with 4T1 cells.Example 6: In vitro testing of micelle / crystal formulations

[0517] 6.1 Synergy in THP1 cells

[0518] Aim of the study: The aim was to evaluate effects of the formulations TLR4B and TLR7A in human THP1 cells treated with PMA and IFN-y (Gantier et al., 2008, The Journal of Immunology 180:2117-2124).

[0519] Methodology: THP1 cells were treated with PMA plus IFN-y to create macrophages responsive to both TLR4 and TLR7 stimulation. TLR4B and TLR7A in DMSO solution or formulated in different formulations were added, all in the in molar ratio of 1:1 to these THP-1 cells. The effects of single agonists alone were also tested. Positive control was LPS. It needs to be emphasized, that the concentration of LPS was by mistake 1000-fold lower than in the experiments regarding the molar ratio in oncology. This most likely explains the lack of cytokine production seen for this condition. The incubation period of TLR7A / TLR4B formulations with the cells was 24 h. Formulations tested were: DMSO control (diagonal black stripes), The TLR7A and TLR4B compounds formulated in DMSO (white) and the TLR7A / TLR4B A formulation, either as single agonists or combined (black). TLR7A / TLR4B A comprises micelles and crystals and the composition comprises 7.5 mg / ml (2.7 mM) DSPE-PEG2000, 0.4 mg / ml (0.4 mM TLR7A, and 0.2 mg / ml (0.4 mM) TLR4B.

[0520] Results: Cytokine secretion (IL-1 p, TNFa and IL-6) was induced after stimulation of all formulations tested (Figure 11 A-C). TLR4 related signaling was low in all cases. Both the DMSO formulated combination of TLR4B and TLR7A and the TLR7A / TLR4B A formulation showed synergy or additive effects in comparison to single compounds in the respective formulation. Vehicle control did not increase any background cytokine production when compared to DMSO only.

[0521] Conclusion: TLR7A / TLR4B A and the combination of TLR7A / TLR4B in DMSO (Table 8) show the expected synergistic / additive effects in vitro as observed previously at the molar ratio of 1:1 for both agonists (TLR4B and TLR7A).Table 8: Tested formulations

[0522] Unformulated agonists in DMSO

[0523] 1 DMSO control

[0524] 2 LPS positive control

[0525] 3 TLR40.3 pM

[0526] 4 TLR4 1 pM

[0527] 5 TLR43 pM

[0528] 6 TLR70.3 pM

[0529] 7 TLR7 1 pM

[0530] 8 TLR73 pM

[0531] 9 TLR4 / TLR70.3 pM

[0532] 10 TLR4 / TLR7 1 pM

[0533] 11 TLR4 / TLR7 3 pM

[0534] TLR4B / TLR7A A, agonists formulated with micelles / crystals A Vehicle TLR7A / TLR4B A

[0535] B TLR7A / TLR4B A- TLR40.3 pM

[0536] C TLR7A / TLR4B A- TLR4 1 pM

[0537] D TLR7A / TLR4B A- TLR43 pM

[0538] E TLR7A / TLR4B A- TLR70.3 pM

[0539] F TLR7A / TLR4B A- TLR7 1 pM

[0540] G TLR7A / TLR4B A- TLR73 pM

[0541] H TLR7A / TLR4B A- TLR4 / TLR70.3 pM

[0542] I TLR7A / TLR4B A- TLR4 / TLR7 1 pM

[0543] J TLR7A / TLR4B A- TLR4 / TLR73 pM

[0544]

[0545] 6.2 Trained immuni

[0546]

[0547] No HPBMC-KPD-3)

[0548] Aims of the study: Trained immunity is a functional state of the nnate immune response and is characterized by long-term epigenetic reprogramming of innate immune cells. This concept originated in the field of infectious diseases -training of innate immune cells, such as monocytes, macrophages and / or natural killer cells, by infection or vaccination enhances immune responses against microbial pathogens after re-stimulation. Immune progenitor cells in the bone marrow can also be trained (that is, central trained immunity), which explains the long-term innate immunity-mediated protective effects of vaccination against heterologous infections. Neutrophils, natural killer (NK) cells and non- leukocytes, such as epidermal cells, also display innate immune memory features. Trained immunity can last for months to a few years and is systemically regulated through myelopoiesis. Monocytes and macrophages have pivotal roles in innate immune memory, and the molecular mechanisms underlying trained immunity in these cells have been thoroughly investigated nanomaterials inherently interact with phagocytic myeloid cells and are thus ideal tools to regulate trained immunity by delivering drugs to myeloid cells and their progenitors in the bone marrow, including small molecules and other.

[0549] Tumors escape destruction by the immune system through a process called immunoediting, which interferes with several aspects of normal immune activation. First, tumor cells lose their ability to effectively present antigen owing to downregulation of major histocompatibility complex molecules or presentation of mutated and thus unrecognizable antigens. Functional antigen presentation would be required to trigger anti-tumor T cell responses. Second, tumors disrupt the delicate balance of co-stimulatory and co-inhibitory signals, further impairing T cell activation. This loss of immunogenicity is exemplified by the upregulation of PDL1 by tumor cells. Immunotherapies, such as chimeric antigen receptor T cell therapy and checkpoint inhibition, focus on the adaptive arm of the immune system. However, components of the innate immune system are also involved, generating an immunosuppressive tumor microenvironment consisting of tumor-associated myeloid cells and myeloid-derived suppressor cells, reducing the effectiveness of the abovementioned immunotherapies. The imbalance of anti-tumor and protumor myeloid cells in the tumor microenvironment stems, in part, from their aberrant production in the bone marrow. In response to cytokines and growth factors produced by tumor cells, the bone marrow produces immature cells, which are 'polarized' following their arrival in the tumor microenvironment to assist in sustaining the immunosuppressive milieu, in essence creating an inescapable positive feedback loop. This cancer-mediated immune cell modulation by HSPCs inthe bone marrow may be counteracted and / or reversed by trained immunity induction. Rebalancing innate immune cell production bias from aberrant hematopoiesis to 'trained' myelopoiesis may overcome the immunosuppressive tumor microenvironment (van Leent et al., 2022, Nature Reviews Materials 7:465-481).

[0550] The aim of the study was to see whether TLR7A / TLR4B formulations could also induce trained immunity. This can experimentally be shown in vitro, using monocytes derived from human blood which are first trained with an immune stimulus and after 6 days exposed again to a heterogeneous 2ndstimulus. In case training took place, the second signal produces a stronger cytokine production by monocytes (Dominguez-Andres et al., 2021, STAR protocols 2:100365).

[0551] Methodology: Monocytes were isolated from fresh human blood from 2 donors basically by using Ficoll gradient centrifugation followed by enrichment of monocytes by placing them in plastic wells and after their adherence removal of non-adherent cells. The protocol was adapted from a published standard protocol (Dominguez-Andres et al., 2021, STAR protocols 2:100365).

[0552] On day 0, cells are stimulated with a challenge for the first time for 24 h. After 24 hours (day 1 ), training stimulation media was washed off with PBS (without Ca / Mg) and 200 pl of fresh complete X-Vivo media was added to each well. Cells were incubated for six days at 37 °C / 5% CO2. Media was exchanged for fresh media on Day 4. On Day 7 all wells received Test Articles or vehicle control and were incubated at 37 °C / 5% CO2overnight.

[0553] After a total of 24 hours of treatment plates will be spun down at 400 x G for 5 min, supernatants from replicates were isolated for TNFa, IL-6, IL-10, IL-1 Ra, IP-10. Test were done in triplicate. The trained immunity effect is shown for monocytes mainly for TNFa and IL-6 stimulation. In this experiment, -Glucan is used as a positive control for the induction of trained immunity. LPS is used as a secondary heterogenous stimulus on day 6. TLR7A / TLR4B formulations were again used with a molar ratio of 1:1.

[0554] Results: The results show that in particular for IL-6 but also for TNFa a trained immunity effect can be observed for the positive control (p-Glucan) and for the TLR7A / TLR4B formulation TLR7A / TLR4B A and for TLR7A / TLR4B drug substances combined in DMSO. The negative control is represented by medium, medium restimulation or by media with the respective vehicle.Table 9: Trained immunity in vitro in human derived monocytes from 2 donors.

[0555] Cytokines (pg / mL) (Means of 3 technical replicates)

[0556] Donor 1 Monocytes Donor 2 Monocytes Stimulus IL- IP- IL-1 p IL-6 TNF-a IL-10 IP-10 IL-6 TNF-a IL-10 Challenge 1p 10 Media 230.

[0557] 1.6 3688.7 2314.4 260.3 1.8 2922.0 1635.8 70.3 41.0 LPS 0

[0558] Media

[0559] — 799.6 731.2 59.7 57.9 — 442.1 336.4 12.5 5.8 Pam3CSK

[0560] TLR7A / TLR4B in DMSO

[0561] O.3: O.3pM

[0562] 4.6 4113.3 706.3 37.6 66.2 7.5 3148.7 496.7 40.4 28.1 LPS

[0563] 3:3pM 123. 137. 104.

[0564] 10.4 8124.7 2595.1 751.5 7.0 6913.3 2629.3 LPS 3 5 2 0.3:0.3pM

[0565] __ 332.6 59.7 8.8 43.9 __ 396.2 63.6 7.8 14.8 Pam3CSK

[0566] 3:3pM

[0567] — 573.5 199.3 21.6 52.7 — 572.7 220.9 19.2 10.2 Pam3CSK

[0568] TLR7A / TLR4B A

[0569] Vehicle 150.

[0570] 2.5 3286.7 2302.5 249.7 2.3 2532.3 1427.0 75.9 25.0 LPS 5

[0571] 0.3:0.3pM 244. 222.

[0572] 6.6 4773.3 1599.9 153.7 2.5 5433.3 1916.6 61.8 LPS 3

[0573] 3:3pM 296. 152.

[0574] 9.8 6066.0 2492.0 278.7 1.8 4368.7 2419.3 46.2 LPS 1 6 Vehicle

[0575] — 1073.0 838.6 94.8 57.8 — 302.5 331.2 16.7 4.7 Pam3CSK

[0576] 0.3:0.3pM 181.

[0577] — 1362.7 502.2 50.2 — 879.9 308.6 47.2 5.6 Pam3CSK 5

[0578] 3:3pM 208.

[0579] — 1486.8 883.9 60.9 — 825.3 364.3 30.6 5.7 Pam3CSK 5

[0580]

[0581] For TLR7A / TLR4B A training effects could be shown for both donors.

[0582] Conclusion: This experiment shows that TLR7A / TLR4B A and the TLR7A / TLR4B in DMSO can induce trained immunity in vitro.6.3 In vivo

[0583]

[0584] formulation

[0585] ine stimulation in mouse

[0586] Aims of the

[0587]

[0588] In this in vivo study, the pharmacodynamic profile in terms of cytokine secretion after administration of different formulations of the two test compounds (TLR4B and TLR7A) among them TLR7A / TLR4B A, encapsulated in micelle nanoparticles / crystal form, were evaluated in non-tumor-bearing female BALB / c mice.

[0589] Methodology: Mice were injected i.v. with test compounds, TLR7A / TLR4B formulations with 0.2mg / mL (TLR4B) and 0.2mg / mL of 0.4mg / mL of (TLR7A), final dose were 2 and respectively 4 mg / kg. Both TLR7A / TLR4B A and TLR7A / TLR4B D and one further formulation were included and their respective vehicle controls as well. For pharmacodynamic analyses, blood was collected 2, 6 and 24 hours after treatment on Day 0 and final blood was collected 48 hours after treatment on Day 2 from all animals. Also, the liver, left kidney and spleen were collected at final necropsy on Day 2. Blood samples were analyzed via a Meso Scale Discovery (MSD) multiplex assay kit to determine the cytokine levels of CCL5 (RANTES), CXCL10 (IP-10), IL-6, IL-10, IFN-y, IFN-a, IFN-p, and TNF-a. Body weight, hematology and blood chemistry were determined. Results: All formulations showed strong increases of cytokine production following administration. The vehicle of TLR7A / TLR4B A did not show any activity in terms of cytokine production.

[0590] TLR7A / TLR4B A showed a strong IP10, RANTES, IL6 and TNFa increase and some increase for INFa, INF.Table 10: Statistical significance of cytokine induction relative to the respective vehicle Cytokine Group comparison Formulation / group 2 hours 6 hours 24 hours IFN-Y Group 2 vs. Group 5 TLR7A / TLR4B A ** ** *

[0591] Group 3 vs. Group 6 TLR7A / TLR4B D ** ** ***

[0592] IL-6 Group 2 vs. Group 5 TLR7A / TLR4B A ** ns ns Group 3 vs. Group 6 TLR7A / TLR4B D ** ns ns

[0593] IP10 Group 2 vs. Group 5 TLR7A / TLR4B A ** * ns Group 3 vs. Group 6 TLR7A / TLR4B D **** **

[0594] IFNa Group 2 vs. Group 5 TLR7A / TLR4B A ns ns Group 3 vs. Group 6 TLR7A / TLR4B D * ns ns

[0595] 1L10 Group 2 vs. Group 5 TLR7A / TLR4B A * ** ****

[0596] Group 3 vs. Group 6 TLR7A / TLR4B D ** **** **** TNF-a Group 2 vs. Group 5 TLR7A / TLR4B A *** ns *

[0597] Group 3 vs. Group 6 TLR7A / TLR4B D ** **** ** RANTES Group 2 vs. Group 5 TLR7A / TLR4B A *** ns **

[0598] Group 3 vs. Group 6 TLR7A / TLR4B D * *** **

[0599] IFN-p Group 2 vs. Group 5 TLR7A / TLR4B A ns ns ns Group 3 vs. Group 6 TLR7A / TLR4B D ** ns ns

[0600]

[0601] Toxicology: No animal died or needed to be sacrificed. There was no weight loss observed after administration of TLR7A / TLR4B A and TLR7A / TLR4B D administration. Histology and blood chemistry results were variable in each group. Mostly there was no difference between TLR7A / TLR4B A and TLR7A / TLR4B D and their controls.

[0602] Conclusion: From this experiment it appears that TLR7A / TLR4B A and similarly also TLR7A / TLR4B D are suitable formulations for i.v. administration when used as previously determined a molar ratio of 1 to 1 of the 2 agonists.

[0603]

[0604] 7: Anti-Tumor in nic Zebrafish Tumor

[0605]

[0606] Model

[0607] Aims of the study: The aim of this study was to evaluate the anti-tumor efficacy of the composition prepared according to Example 1 using ZTX-ONCOLEADS, a registered platform based on the zebrafish tumor xenograft in vivo model for drug discovery. Melanoma cancer cell line M-001, in the presence of human PBMCs and monocytes, was used to evaluate the effect of the TLR7A / TLR4B A composition on tumor size and metastasis.

[0608] Methodology: Transgenic Tg(fH!: EGFP ’ zebrafish embryos, which express enhanced green fluorescent protein on endothelial cells, were used in the study. This transgenic line is widely used for tumor angiogenesis studies and testing of novel anti-angiogenic compounds. Tumor cells of the M-001 melanoma cancer cell line, as well as human PBMCs and monocytes derived from 2 donors were used. Cells were subcutaneously implanted into the perivitelline space (PVS) of 2 days old zebrafish larvae (in groups of 10 larvae per donor). In total, 700 cells were administered in a molar ratio of 10:1:4 (PBMC:monocytes:tumor cells). Subsequently, approximately 2 nL of test item formulation or vehicle control or PBS solutions were injected intravenously into the 2 days old larvae which were followed up for 3 days (to Day 5 age). TLR7A / TLR4B composition concentration in the injection solution was 79 pM or 400 pM of TLR7A and TLR4B, respectively. Accounting for the wet weight of a zebrafish embryo at 4-5 days of age (5 day of age is at 3 days after treatment, when the experiment was terminated) of 1 mg (approximately 1000 nL), TLR7A / TLR4B A composition concentration in the larvae was 1.58 or 8 pM, respectively.

[0609] Anti-cancer efficacy of test item formulation was determined by evaluating the change in primary tumor size (i.e., tumor growth or reduction) and the number of tumor cells disseminated to the distal caudal venous plexus (CVP) three days after implantation. For evaluation of tumor size, images were obtained right after implantation of tumor cells (Day 0 = 2 days old larvae) and after 72 hours of incubation of larvae at termination of the experiment (Day 3). Tumor size was analyzed as the pixels of labeled tumor cells at Day 0 and Day 3, and the number of disseminated cells to the CVP was manually counted. Relative tumor size (Day 3 to Day 0) was normalized to the vehicle control (empty formulation) for statistical analysis and graphical presentation.

[0610] Results: In a pre-study, it was determined that the TLR7A / TLR4B A composition did not show any toxicity in the dose range tested (up to 400 pM in injection solution). Regarding the effect ontumor size, in the groups administered with PBMCs and monocytes from donor 1, treatment with TLR7A / TLR4B A composition at 79 or 400 pM (1.58 or 8 pM in larvae) showed a reduced primary tumor size, but the effect did not reach statistical significance versus vehicle control. In the groups administered with PBMCs and monocytes from donor 2, TLR7A / TLR4B A composition at 79 pM (1.58 pM in larvae) significantly reduced the primary tumor size by 58% after 3 days of treatment, compared to vehicle control. However, 400 pM of TLR7A / TLR4B A composition concentration (8 pM in larvae) did not show a statistically significant reduced primary tumor size, but it also showed a trend towards a decrease in primary tumor size. It was considered that this might be due to a bi-modal response of the pathway that the product is targeting, where higher doses could possibly lead to the activation of the pathway with a different phenotype. Furthermore, treatment with TLR7A / TLR4B A composition at both concentrations did not affect metastasis of melanoma cells to the CVP compared to respective vehicle control.

[0611] Effect on primary tumor size and number of metastases is shown Figure 12 for the experiment using PBMCs and monocytes from donor 1 and donor 2, respectively.

[0612] Example 8: Evaluation of the Haemolytic Potential of the TLR7A / TLR4B A composition in Minipig Whole Blood In Vitro

[0613] The objective of this study was to evaluate in vitro whether the TLR7A / TLR4B A (1:1 molar ratio of TLR7A and TLR4B) composition may induce red blood cell (RBC) haemolysis and / or RBC and platelet clumping as well as its compatibility with minipig plasma from minipig blood.

[0614] For assessment of haemolysis, blood samples collected in lithium heparin anti-coagulant tubes from 5 individual minipigs were incubated with either the TLR7A / TLR4B A composition (at final concentrations in blood of 23.1 and 2.31 pg / mL) or control (vehicle control, positive control - saponin or negative control - phosphate buffered saline (PBS)) solutions. After centrifugation, the amount of haemoglobin in the supernatant was determined spectrophotometrically according to the method described by Cripps C. M. (1968). This was expressed as a percentage of the total blood haemoglobin. The TLR7A / TLR4B A composition was graded as non-haemolytic in minipig blood as defined in ASTM F756-17, Standard Practice for Assessment of Haemolytic Properties of Materials.

[0615] For assessment of red blood cell (RBC) and platelet clumping, the TLR7A / TLR4B A composition solutions (at final concentrations in blood of 23.1 and 2.31 pg / mL), vehicle control and negativecontrol (PBS) were added to whole blood samples from 5 individual animals. No clumping of red blood cells was observed microscopically. No abnormal platelet clumping was exhibited when samples were compared with whole blood only controls after microscopic evaluation.

[0616] For assessment of compatibility with minipig plasma, the TLR7A / TLR4B A composition solutions (at final concentrations in blood of 23.1 and 2.31 pg / mL), vehicle control, positive control (acetonitrile) and negative control (PBS) were added to plasma samples from 8 individual animals. No precipitation was observed macroscopically for the the TLR7A / TLR4B A composition, vehicle or negative control when compared with the positive control.

[0617] In conclusion, the TLR7A / TLR4B A composition is compatible with minipig whole blood and plasma up to the highest tested assay concentration, 23.1 pg / mL.

Claims

Claims1. A pharmaceutical composition comprisingi. solid particles comprising at least one toll-like receptor 4 (TLR4) agonist andii. micelles comprising at least one toll-like receptor 7 (TLR7) agonist and at least one amphiphilic micelle-forming agent.

2. The pharmaceutical composition according to claim 1, wherein at least about 80%, at least about 85% or 90%, preferably at least about 93%, 94%, 95%, and more preferably 97% of the at least one TLR7 agonist, based on the total amount of TLR7 agonist in the pharmaceutical composition, is incorporated into in the micelles.

3. The pharmaceutical composition according to claim 1 or 2, wherein the solid particles do not comprise the at least TLR7 agonist and / or wherein the micelles do not comprise the at least one TLR4 agonist.

4. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR4 agonist and the at least TLR7 agonist are present in the pharmaceutical composition in a molar ratio of about 10:1 to about 1:10.

5. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR4 agonist and the at least TLR7 agonist are present in the pharmaceutical composition in a molar ratio of about 5:1 to about 1:5, preferably in a molar ratio of about 2:1 to about 1:2, more preferably in a molar ratio of about 2:1 to about 1:1 or in a molar ratio of about 1:1 to about 1:2, most preferably in a molar ratio of about 1:1.

6. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one amphiphilic micelle-forming agent is selected from the group consisting of poloxamer, poloxamine, polyethylene glycol (PEG)-polyester, PEG-polyanhydride, PEG-poly- amino acid, phospholipid, polysorbate, polyoxyethylene alkyl ether and combinations thereof.

7. The pharmaceutical composition according to claim 6, wherein the PEG-polyester is selected from the group consisting of PEG-poly(lactic acid) (PEG-PLA), PEG-poly(lactic-co-glycolic acid) (PLGA), PEG-poly(s-caprolactone) (PCL) and combinations thereof.

8. The pharmaceutical composition according to claim 6 or 7, wherein the PEG-polyanhydride is a PEG-polysebacic anhydride (PSA).

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the PEG- poly-amino acid is selected from the group consisting of PEG-poly(L-histidine), PEG-poly(L- aspartic acid), PEG-poly(L-asparagine), PEG-poly(L-glutamic acid), PEG-poly(L-glutamine), PEG-poly(L-lysine) and combinations thereof.

10. The pharmaceutical composition according to any one of claims 6 to 9, wherein the phospholipid is conjugated to polyethylene glycol (PEG).

11. The pharmaceutical composition according to claim 10, wherein the phospholipid is conjugated to PEG via a carbonyl group.

12. The pharmaceutical composition according to any one of claims 6 to 11, wherein the phospholipid comprises one or more alkyl chains being independently selected from C8-C24alkyl(s), preferably selected from C10-C22alkyl(s), more preferably selected from C12-C20alkyl(s), even more preferably selected from C14-C18alkyl(s), and most preferably selected from C16-C18 alkyl(s);wherein the alkyl chains are independently selected from saturated alkyl chains and unsaturated alkyl chains.

13. The pharmaceutical composition according to any one of claims 10 to 12, wherein the phospholipid conjugated to PEG is selected from the group consisting of 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE)-PEG, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE)-PEG, 1,2-dimyristoyi-sn-glycero-3-phosphoethanolamine (DMPE)-PEG, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-PEG, preferably DSPE-PEG.

14. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one amphiphilic micelle-forming agent is DSPE-PEG.

15. The pharmaceutical composition according to any one of claims 6 to 14, wherein PEG has a molecular weight in the range from about 350-6000 Da, preferably in the range of about 550-5000 Da, more preferably in the range of about 750-4000 Da, even more preferably in the range of about 1000-3000 Da, and most preferably PEG has a molecular weight of about 2000 Da (PEG2000).

16. The pharmaceutical composition according to claim 14 or 15, wherein DSPE-PEG is DSPE- methoxy PEG (DSPE-mPEG).

17. The pharmaceutical composition according to any one of the preceding claims, wherein the pharmaceutical composition comprises, preferably the micelles comprise, DSPE- (m)PEG2000.

18. The pharmaceutical composition according to any one of the preceding claims, wherein the amphiphilic micelle-forming agent is selected from the group consisting of DMPE- (m)PEG2000, DSPE-(m)PEG2000, and DPPE-(m)PEG5000.

19. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR7 agonist has a structural formula according to formula (I):wherein X1is O, S, or NRC;R1is hydrogen, alkoxyalkyl, alkyl, substituted alkyl, C6aryl or C aryl or substituted C6aryl or substituted Cw aryl;Rcis hydrogen, alkoxyalkyl, alkyl, or substituted alkyl; or Rcand R1taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring;each R2is independently selected from -H, -OH, (Ci-C5)alkyl, substituted (Ci-C6)alkyl, (Ci-C6)alkoxy, substituted (Ci-C6)alkoxy, -C(O)-(Ci-C6)alkyl (alkanoyl), substituted -C(O)- (Ci-C6)alkyl, -C(O)-(Ce-Cw)aryl (aroyl), substituted -C(O)-(C6-Ci0)aryl, -C(O)OH (carboxyl), -C(O)O(Ci-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(Ci-C6)alkyl, -NRaRb, - C(O)NRaRb(carbamoyl), halo, nitro, or cyano, or R2is absent;each Raand Rbis independently selected from hydrogen, (Ci-C6)alkyl, substituted (Ci-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (Ci-C6)alkoxy, substituted (Ci-C5)alkoxy, (Ci-C6)alkanoyl, substituted (Ci-C6)alkanoyl, aryl, aryl(Ci-C6)alkyl, Het, Het (Ci-C6)alkyl, or (C1-C6)alkoxycarbonyl;wherein the substituents on any alkyl, aryl or heterocyclic group are hydroxy, Ci -6alkyl, hydroxyl Ci.6alkylene, C1-6 alkoxy, Oi ftcycloalkyl, Ci.6alkoxy, C1-6 alkylene, amino, cyano, halo, or aryl;n is 0, 1, 2, 3 or 4;X2is a bond, -C(O)- or -C(O)-NH-CH2-CH2-; andR3is a phospholipid comprising one or two carboxylic esters;or a tautomer thereof;or a pharmaceutically acceptable salt or solvate thereof.

20. The pharmaceutical composition according to claim 19, wherein n is 0, X1is O, and R1is methoxyethyl.

21. The pharmaceutical composition according to claim 19 or 20, wherein R3in formula (I) comprises12wherein R11and R12are each independently a hydrogen or an acyl group, R13is a hydrogen, and m is 1 to 8, preferably 1, and wherein a wavy line indicates a position of bonding, wherein the absolute configuration at the carbon atom bearing OR12is R, S, or any mixture thereof.

22. The pharmaceutical composition according to claim 21, wherein m is 1, and / or wherein R11and R12are each oleoyl groups.

23. The pharmaceutical composition according to any one of claims 19 to 22, wherein the phospholipid of R3comprises two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, epoxidation, hydroxylation, or a combination thereof.

24. The pharmaceutical composition according to any one of claims 19 to 23 wherein the phospholipid of R3comprises two carboxylic esters and the carboxylic esters of are the same or different.

25. The pharmaceutical composition according to claim 23 or 24 wherein each carboxylic ester of the phospholipid is a C17 carboxylic ester with a site of unsaturation at C8-C9.

26. The pharmaceutical composition according to claim 23 or 24 wherein each carboxylic ester of the phospholipid is a C18 carboxylic ester with a site of unsaturation at C9-C10.

27. The pharmaceutical composition according to any one of claims 19 to 26, wherein R3comprises dioleoylphosphatidyl ethanolamine (DOPE).

28. The pharmaceutical composition according to any one of claims 19 to 27, wherein R3is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and X2is C(O).

29. The pharmaceutical composition according to any one of claims 19 to 28, wherein X1is oxygen.

30. The pharmaceutical composition according to any one of claims 19 to 29, wherein X1is O, R1is methoxyethyl, n is 0, X2is -C(O)-, and R3is 1,2-dioleoylphosphatidyl ethanolamine (DOPE).

31. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR7 agonist has a structural formula according to formula (la):(la).

32. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR4 agonist has a structural formula according to formula (II):wherein z1 is an integer from 1 to 4, wherein z2 is an integer from 0 to 5, wherein R5is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R6is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein R7is hydrogen, or substituted or unsubstituted alkyl, and wherein each R8is independentlyhalogen, -CN, -SH, -OH, -COOH, -NH2, -CONH2, nitro, -CF3, -CCI3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; or a tautomer thereof; or a pharmaceutically acceptable salt or solvate thereof.

33. The pharmaceutical composition according to claim 32, wherein z1 is 1.

34. The pharmaceutical composition according to claim 32 or 33, wherein R8is substituted or unsubstituted heteroaryl.

35. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR4 agonist has a structural formula according to formula (Ila):wherein X is O or S, R14is selected from the group consisting of hydrogen, methyl, ethyl and prolyl, R15is selected from the group consisting of hydrogen, F and methyl, and R16is selected from the group consisting of cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 4- fluorophenyl, isopropyl, isobutyl, and 2,2-dimethylbutyl.

36. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR4 agonist has a structural formula according to formula (lib):(Hb).

37. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one TLR4 agonist has a structural formula according to formula (lib):(Hb), wherein the at least one TLR7 agonist has a structural formula according to formula (la):(la), and wherein the at least one amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

38. The pharmaceutical composition according to any one of the preceding claims, wherein the solid particles are (nano) crystals.

39. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one amphiphilic micelle-forming agent and the at least one TLR7 agonist are present in the pharmaceutical composition in a molar ratio in the range of about 20:1 to about 4:1, preferably in the range of 10:1 to about 4:1, more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

40. The pharmaceutical composition according to any one of the preceding claims, wherein the pharmaceutical composition comprises DSPE-mPEG2000, the at least one TLR4 agonist and at least one TLR7 agonist in a molar ratio in the range of about 20:1:1 to about 4:1:1, preferably in the range of 10:1:1 to about 4:1:1, more preferably in the range of about 8:1:1 to about 6:1:1, and most preferably in a molar ratio of about 7:1:1; preferably, the amphiphilic micelle-forming agent comprises DSPE-mPEG2000.

41. The pharmaceutical composition according to any one of the preceding claims, wherein the micelles have a particle size in the range of about 2 to about 50 nm, preferably about 5 to about 30 nm, and more preferably about 8 to about 20 nm.

42. The pharmaceutical composition according to any one of the preceding claims, wherein the micelles have a polydispersity index (PDI) greater than 0 and below 0.2.

43. The pharmaceutical composition according to any one of the preceding claims, wherein the solid particles are (nano) crystals having at least one side having an average length in the range of 50 to 220 nm, preferably in the range of 75 to 200 nm, more preferably in the range of 100 to 190 nm.

44. The pharmaceutical composition according to any one of the preceding claims, comprising an effective amount of the at least one TLR4 agonist and of the at least one TLR7 agonist.

45. The pharmaceutical composition according to any one of the preceding claims, further comprising at least one additional active agent.

46. The pharmaceutical composition according to claim 45, wherein the at least one additional active agent is selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, a further TLR7 agonist, and combinations thereof.

47. The pharmaceutical composition according to claim 46, wherein the chemotherapeutic agent is selected from a group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine,chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine, streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyl lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

48. The pharmaceutical composition according to claim 46, wherein the antimicrobial agent is selected from a group consisting of antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.

49. The pharmaceutical composition according to claim 46, wherein the immune checkpoint inhibitor is selected from a group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

50. The pharmaceutical composition according to claim 48, wherein the antibiotic is selected from the group consisting of amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, levofloxacin, and combinations thereof.

51. The pharmaceutical composition according to any one of the preceding claims, further comprising a pharmaceutically acceptable excipient, preferably water for injection or a pharmaceutically acceptable buffer.

52. A method of preparing the pharmaceutical composition according to any one of the preceding claims comprisingstep a) of providing a mixture comprising at least one amphiphilic micelle-forming agent, the at least one TLR4 agonist, the at least one TLR7 agonist and an organic solvent, step b) of lipid film hydration, including evaporating the organic solvent and, optionally, step c) of filtration,thereby preparing the pharmaceutical composition comprisingi) solid particles comprising the at least one toll-like receptor 4 (TLR4) agonist and ii) micelles comprising the at least one toll-like receptor 7 (TLR7) agonist and the at least one amphiphilic micelle-forming agent.

53. The method according to claim 52, wherein the organic solvent comprises chloroform, tetrahydrofuran, methanol or a mixture thereof.

54. The method according to claim 52 or 53, wherein in step b) phosphate buffered saline is used for the hydration of the lipid film.

55. The method according to any one of claims 52 to 54, wherein step c) is performed using a filter threshold of about 0.1 to 0.3 pm, preferably of about 0.2 pm.

56. The method according to any one of claims 52 to 55, wherein step c) is performed using a polyethersulfone (PES), a polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF) membrane.

57. A kit or kit of parts comprising the pharmaceutical composition according to any one of claims 1 to 51 and a pharmaceutically acceptable carrier, preferably water for injection or a pharmaceutically acceptable buffer, and, optionally, comprising handling instructions.

58. The pharmaceutical composition according to any one of claims 1 to 51 or the kit or kit of parts according to claim 57 for use as a medicament.

59. The pharmaceutical composition according to any one of claims 1 to 51 or the kit or kit of parts according to claim 57 for use in the treatment of cancer, an antibiotic resistance, an inflammatory condition or disease, an autoimmune disease or an infectious disease.

60. The pharmaceutical composition or the kit or kit of parts for use according to claim 58 or 59 in the treatment of cancer.

61. The pharmaceutical composition or the kit or kit of parts for use according to claim 59 or 60, wherein the cancer is selected from a group consisting of head and neck cancer, including head and neck squamous cell carcinoma, melanoma, basal cell carcinoma, pancreatic cancer, ovarian cancer, prostate cancer, colon carcinoma, bladder cancer, gastric cancer, stomach cancer, colorectal cancer, liver cancer, breast cancer, lung cancer, and (squamous) skin cancer.

62. The pharmaceutical composition or the kit or kit of parts for use according to any one of claims 58 to 61, wherein the pharmaceutical composition is administered to a subject in need of a TLR4 and / or TLR7 agonist treatment, preferably the subject is a human subject.

63. The pharmaceutical composition or the kit or kit of parts for use according to any one of claims 58 to 62, wherein the pharmaceutical composition is administered orally, intranasally,via aerosol, via inhalation, parenterally, intramuscularly, intraperitoneally, intravenously, rectally, intravesically or subcutaneously.

64. The pharmaceutical composition or the kit or kit of parts for use according to any one of claims 58 to 63, wherein the pharmaceutical composition is administered intravenously.

65. The pharmaceutical composition or the kit or kit of parts for use according to any one of claims 58 to 64, wherein the pharmaceutical composition is administered together with at least one additional active agent.

66. The pharmaceutical composition or the kit or kit of parts for use according to claim 65, wherein the at least one additional active agent is selected from a group consisting of chemotherapeutic agent, antimicrobial agent, immune checkpoint inhibitor, a further TLR4 agonist, a further TLR7 agonist, and combinations thereof.

67. The pharmaceutical composition or the kit or kit of parts for use according to claim 66, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, doxil, epirubicin, cyclophosphamide, bortezomib, oxalipltin, altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, melphalan flufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine streptozotocin, temozolomide, thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, uramustine, cisplatin, mitomycin C, bleomycin, topotecan, irinotecan, docetaxel, paclitaxel, podophyl lotoxin, vincristin, plicamycin, daunorubicin, dactinomycin, adriamycin, 5-fluorouracil, hormones, hormone antagonists, cytokines, and combinations thereof.

68. The pharmaceutical composition or the kit or kit of parts for use according to claim 66, wherein the antimicrobial agent is selected antiseptic, antibiotic, antiviral, antifungal, antiparasitic and combinations thereof.

69. The pharmaceutical composition or the kit or kit of parts for use according to claim 66, wherein the immune checkpoint inhibitor is selected from the group consisting of programmed cell death protein-1 (PD-1), programmed cell death protein-ligand 1 (PD-L1) inhibitors, cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) inhibitors, lymphocyte activation gene-3 (LAG-3) inhibitors, and combinations thereof.

Images