A pH‐sensitive Macromolecular Prodrug as TLR7/8 Targeting Immune Response Modifier

Abstract The chemical synthesis and biological activity of novel functionalized imidazoquinoline derivatives (ImQ) to generate Toll‐like receptor (TLR) 7/8 specific prodrugs are presented. In vivo activity of ImQs to induce inflammation was confirmed in zebrafish larvae. After covalent ligation to fully biodegradable polyphosphazenes (ImQ‐polymer), the macromolecular prodrugs were designed to undergo intracellular pH‐sensitive release of ImQs to induce inflammation through binding to endosomal TLR7/8 (danger signal). We showed ImQ dissociation from prodrugs at a pH 5 pointing towards endosomal prodrug degradability. ImQ‐polymers strongly activated ovalbumin‐specific T cells in murine splenocytes as shown by increased proliferation and expression of the IL‐2 receptor (CD25) on CD8+ T cells accompanied by strong IFN‐γ release. ImQ prodrugs presented here are suggested to form the basis of novel nanovaccines, for example, for intravenous or intratumoral cancer immunotherapeutic applications to trigger physiological antitumor immune responses.

Abstract: The chemical synthesis and biological activity of novel functionalized imidazoquinolined erivatives (ImQ) to generate Toll-like receptor (TLR) 7/8 specific prodrugs are presented. In vivo activityo fI mQs to inducei nflammation was confirmed in zebrafish larvae. After covalentl igation to fully biodegradable polyphosphazenes (ImQ-polymer), the macromolecular prodrugs were designedt ou ndergo intracellularp H-sensitive release of ImQs to inducei nflammation throughb inding to endosomal TLR7/8 (danger signal). We showedI mQ dissociation from prodrugs at ap H5 pointing towardse ndosomal prodrug degradability.I mQ-polymers strongly activated ovalbumin-specific Tcells in murine splenocytes as shown by increased proliferation and expression of the IL-2 receptor (CD25) on CD8 + Tcells accompanied by strongI FN-g release. ImQ prodrugs presentedh erea re suggestedt of orm the basis of novel nanovaccines,for example, for intravenous or intratumoral canceri mmunotherapeutic applications to trigger physiologicala ntitumori mmune responses.
Agonistso ft oll-like receptors( TLRs) are being actively pursued for their ability to activate the immune system for ah ost of therapeutica pplications,i ncluding canceri mmunotherapy. [1] Murine TLR7 [2] and human TLRs 7a nd 8 [3] are known to sense guanosine-based drugst hat induce an antiviral responsei n vivo, stimulating the development of tolerogenic antigen-presenting dendritic cells (DCs), producing selective cytokines that drive Tcell responses. To date, most DC-basedt umor immunetherapeutics trategies involvee xv ivol oading of DCsw ith tumor-associated antigens and immune-stimulatory agents (adjuvants) and subsequent re-injection into the patient for in vivo Tcell activation. [4] This represents ah ighly promising but labor and cost intensivem ethodology.T hus, am ore direct, pharmaceutical approach could be of significant value to the field. [5] However,l ow molecular weightm olecular adjuvants share ap oor pharmaceuticalp rofile and rapid diffusion from the local tissue, even via intratumoral delivery,a nd hence the risk of systemic immune response represents as evere limitation of the applicable dose to levels below that required for activation. One of the most widely investigated groups of TLR agonists are imidazoquinolines, af amily of synthetic small molecules, includingi miquimod (R837), resiquimod (R848), gardiquimod, and other variants.H erein we present an ovel imidazo[4,5-c]quinolin-4-amine agonist, chemically modified as such to enable reversible covalentl igationt os ynthetic and/or biological macromolecules, that is, macromolecular prodrugs. The rational for am acromolecularp rodrug approach, alongside potential gains in therapeutic efficacy [6,7] is the location of the TLR7/8 receptors in the endosome, well understood to be the majori ntracellularg ateway for macromolecules. [8] During the last decade, polymer-based nanomedicines or polymer therapeutics [9,10] have become an important tool for drug delivery [6] and theranostics. [11] Although there can be clinicali ssues with heterogeneity and reproducibility the use of macromolecular prodrugs has been shown in aw ide number of cases to increaset he efficacy and positivelyi nfluencing the biodistribution of therapeutics. [6,9] Poly(organo)phosphazenes are av ersatile class of polymers with immense potentialf or application in nanomedicine. [12,13] Recent advances in polyphosphazenes ynthesisa llow simple access to controlled molecularw eights, controlled hydrodynamic volumes and high water solubility. [14] One of relativelyf ew fully degradable, [13,15] water soluble polymers available, essential design features in avoiding the deleterious effects associated with post-drug-release accumulationo f high molecularw eightm acromoleculesi nt he organism. [7] Moreover,c ertain poly(organo)phosphazenes themselves appear as potent immunoadjuvantsi nv accine delivery in terms of their complexes with protein antigens leading to activation and antigen presentation of DCs. [16] Recently,ap Hr eleasing nanogel approach was successfully appliedt oi nduce superiora ntibody and Tc ell responses against at uberculosis antigen. [17] Herein, we demonstrate the design of macromolecular prodrugs, capable of undergoing ac ontrolled intracellular releaseo ft he low-molecularw eightT LR agonist as as o-called "danger signal" to induce inflammation through binding to endosomal TLR7/8. However,s ince known imidazoquinoline based agonists lack appropriate sites for conjugation to suitable pH release systems, the implementation of novel synthetic imidazoquinoline analogues is necessary.Wereport the synthesis of novel ai midazo[4,5-c]quinoline derivative with an aliphatic ketone moiety attached to the benzylic group, which was suitable for couplingt om acromolecules equipped with hydrazide linkerst og ive pH cleavable hydrazone linkage, [18] well-proven to provideaclean rapid releaseint he acidic endosomal environment, thus to potentially produce ac ontrolled, intracellular presentation of the TLR agonist. The group of David et al. have investigated the structure-activity relationships of imidazoquinoline analogues. [19] It was demonstrated that maintaining high agonistic activity of imidazoquinolines narrows the possibilities to introduce functional groups down to af ew certain positions of the 1Himidazo[4,5-c]quinolinea ctive core. Most notably,p ositionC 4 is required to hold ap rimary amine and C2 as hort aliphatic chain. In short, position N1 would appear to be the preferred site for functionalization for subsequent conjugation, since a wide varietyo fs ubstitution patterns are tolerated at this position. Our initial concept was to synthesize halogenated 1benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amines, which then act as substrates for cross-couplingr eactions to install various keto-functionalities. To ensure rapid pH-drivend issociation,a n aliphatic ketone was introduced. Considering the circumstances mentioned above,asynthesis route was designed starting with 4-chloro-3-nitroquinoline, ac ommon precursor for fused quinoline-type heterocyclic frameworks (Scheme 1). [20] Af unctionalized benzylic group can be installed at this stage by substitutiono ft he C4-chlorine with ab enzylamine. Treatmento f 4-chloro-3-nitroquinoline with 2-bromob enzylamine using triethylamine in THF solutiong ave quinolin-4-amine 1 in excellent yield. By combininga nd adjusting two published one-pot procedures fort he formation of benzimidazoles starting from ortho-nitroaniline, [21] 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinoline 2 could be synthesized in 83 %y ield. This transformation consisted of the reduction of the nitro group with iron powder aided by ammonium chloride in iso-butanol combined with the annulationo fa1 H-2-butyl-imidazole motif employing valeraldehyde. The established route to carry out amino-functionalizationsw ith quinoline-derivatives is to firstly subjectc ompoundss uch as 3 to N-oxidization( compareS cheme 1, 4), before conversionwith nitrogen-bearing reagents. Aliphatic ketones could be successfully installed in as ingles tep via a Heck-type reaction using an allyl alcohol along with triethylamine, [22] to give a3 -oxo-butyl-chain attached to the benzylgroup. By subjecting varioush alobenzyl-imidazoquinoline derivatives to thisp alladium acetate-catalyzed coupling, suitable conversionswere observed. By far the highest-yielding reaction was shown to be preparation of 3 from 2 with but-3-en-2-ol (81 %, Scheme 1). The N5-oxide 4 could be accessed in very good yield by employing meta-chloroperoxybenzoic acid as an oxidizinga gent, without the requirement for hitherto unsuccessfulk etal protection of the ketones.T hese formations proceeded slowly,t aking 60 h. The same was true for the final preparation of C4-amines, where the reactionc onditions also involved refluxing in dichloromethane for two to three days. For this purpose, benzoyl isocyanate was added as the nitrogen source.T he concluding step consisted of refluxing the intermediate with sodium methoxidei nm ethanolic solution to releaset he primary amine. The synthesis of 5 resulted in good yield (53 %). In order to assess the effect of chemical modification on the activity of the novel TLR agonist, as eries of tests were carried out in livingz ebrafish larvae as shown in Figure 1. Stimulation with 5 resulted in NF-kB signaling activation in zebrafish macrophages visualized in Tg(mpeg1:mCherry) gl23 [23] / Tg(6 Hsa. NFKB:E GFP) nc1 [24] double transgenic animals, which have macrophages demarcated by mCherrye xpression and report NF-kBa ctivity by EGFPe xpression ( Figure 1A).
5 showed stronger induction of NF-kBt han the known TLR7/8 agonist R848 as quantified by flow cytometry (Figure 1B). QPCR furtherc onfirmed induction of inflammatory cytokines IL1b, IL6 and Tnf-a upon 5 stimulationf or 6h (Figure 1C). In addition, IL1b induction upon stimulation with 5 was reducedi nM yD88 mutant zebrafish,i ndicatingaMyD88 dependent signal pathway as expected for specific TLR7/8 activation ( Figure 1D). [25] In the next step, water soluble polyphosphazenes decorated with hydrazide linkers( 43 wt %) were prepared asp reviously reported (see ref. [26] and Supporting Information FigureSI-2). The novel TLR agonist 5 could be successfully bound to the polymer due to hydrazone formation with the carbonyl groups (Scheme 2). After purification by dialysis, UV/Vis, GPC and DLS measurements were used to prove covalent drug loading. The percentage of loading was calculated using the UV/Vis absorbance at 324 nm ( Figure SI-3). Conjugation between3and 6weight percentoft he macromolecular conjugate was achieved representing al oading yield of between 10 and 25 percent of the linkers( Figure SI-3).
The GPC curves of the polymer conjugatesa fter drug loading, measured in DMF,a re shiftedt ol ower retention volumes (lower hydrodynamic volume), presumably due to the hydrophobicity of the conjugated drugs, while still possessing ar elatively narrow of 1.3 ( Figure SI-4). The hydrodynamic volume is ac rucial parameter in terms of bio-distribution and cell uptake. Dynamic light scattering studies showed that whilst the hydrophilic polymers show hydrodynamic diameters in the 10 nm range, they form supramolecular associates in the 100 nm range upon conjugation of the hydrophobic drug as a result of hydrophobic interactions (Figure 2). This phenomenon which has been previously reportedw ith similar phosphazene based systems [27] is thought to be assisted by the high backbone flexibility relative to carbon based polymers. Furthermore, in aw ork with different polymer-TLR7/8 agonistc onjugates, Lynn et al. recently showed that increased densities of To ll-like receptor agonists arrayed on polymer carriers is associated with particle formation and enhanced cytokinep roduction. [28] Double internalization of the agonist, via its chemical binding location close to the backbone of the brush type  [29] reducing the probability of agonist presentation prior to intracellular release.T he release of 5 from the conjugates was analyzed by HPLC. Within ap eriod of 24 ha t3 78C, 100 %r eleasef rom the polymer-drug conjugates could be observedf or the samples exposed to pH 5a nd only 50 %f or the conjugates stored at pH 7.4 (Figure 2b).
The release rate is comparable to publishedd ata using similar hydrazide based polymer systems. [26,30] At pH 5, approximately 50 %r elease of compound 5 was observed after 2.5 hours and full releasew as observed after 30 hours, whereas at pH 7.4 only approx. 50 %r eleaseo f5 waso bservedd uring the whole period measured. As complete clearance of the macromolecular carrieri sastringent requirement for subsequenti n vivo applications, the degradation profileso ft he polymers were also investigated. The degradation studies of the conjugates at 37 8C, pH 5a nd 7m easured by size exclusion chromatography,s howed that the polymers are stable over as hort period of time in an aqueous environmentb ut degrade significantly to small moleculesu nder theses imulated physiological conditions within 10 weeks (SI-5). These resultsare comparable to previous degradation studies of amino substituted poly(organo)phosphazenes but could be easily accelerated or decelerated as andw hen required. [11] Compound 5,u pon releasef rom polymer-ImQ conjugate is expectedt os timulate Tc ells through internalization and activation of APCs such as DCs. Thus, in ordert oa nalyse the immunological activity of polymeric prodrugs we started investigating cellular uptake and localization of ImQ-conjugated within murineD Cs. Compound 5conjugated ImQ polymera ccumulated in generated DCs and co-localized with intracellular endosomal andl ysosomal vesicles ( Figure 3A-D). Overall, at otal of 87 %o fa ll DCs internalized ImQ-based macromolecular prodrug.
The capacity of ImQ-conjugate activated DCs to stimulate T cells via DCswas furtheranalysedi nTcell/DC co-cultures. Figure 2. a) Schematic representation of the intra-and intermolecular agglomeration and self-assemblyo fthe polymers uponconjugation of the hydrophobicdrug in aqueouss olvents. b) Molecular size distribution by intensity as detected by dynamic light scattering for polymers 2-5 in phosphate buffer at pH 7.4 (polymer concentration1mg mL À1 ,d h -hydrodynamic diameter). c) Release of 5 from the conjugatea t378Ci nacidic environment (acetate buffer,pH5), and an eutral solution (pH 7.4,tris buffer). The amount of the released drug was estimatedu sing ac alibration curve for the free drug. Figure 3. A-D:Confocal microscopy images of dendritic cells incubated with polymer-I mQ conjugates. MurineB MDCs (A) were stained with LysoTracker Green (504 nm/511 nm) for 1hour (B) and with polymer-ImQ (390 nm/ 415 nm). LysoTracker Green accumulates in cellular compartments with low internalpHa nd stains lysosomes (green, B). Polymer-ImQ accumulates in lysosomes and in endosomes (C and D, purple regionsindicated by arrows). E-F) Conjugates activate Tcells and IFN-g release. Splenocytes were isolated from transgenic OT-I mice, labeled with CFSE and stimulated for 4days with R837,R 848orc onjugate, together with the ovalbumin derived SIINFEKLpeptide, which stimulates ovalbumin specific CD8 + Tcells from OT-I mice. On day 4, CD8 + Tcells were analyzed for CD25 expression and CFSE diluted cells,c onsideredasp roliferatingTcells by flow cytometry. E) CD25 expression depicted as mean fluorescence intensity (MFI) is shown for splenocytes culturescontaining 1 mm conjugate. F) Absolute cell numbers of proliferating CD8 + Tcells per splenocyte culture were calculated in CFSE diluted dividing Tcells using ad osageranging from 0.1 to 5 mm.G)Supernatants from splenocytecultures were analyzed for IFN-g secretion.Mean AE standard deviation from triplicate analysis is shownf or CD25 expression, *) p < 0.05, **) p < 0.01. Polymer alone and SIINFEKL peptidealone dissolved in DMSO served as negative controls.
We used splenocytes from OT-I mice, which among other immune cells comprise APCs and predominantly CD8 + Tcells. Such Tcells entirely express at ransgenic Tcell receptor,w hich is specific for as hort ovalbumin related peptides equence (SIINFEKL) primarily presented by APCs. Thus strongA PC-mediated Tcell responses can be raised in an OT-I splenocyte culture upon TLR7/8 ligationt ogether with SIINFEKL-peptide.I n order to investigate the effect on Tcells, we measuredt he degree of Tcell activation, as well as the secretion of IFN-g,a cytokinet hat is associated with activation of CD8 + effector T cells ( Figure 3E-F). The activation marker CD25 was significantly increased on CD8 + Tcells in stimulation cultures using conjugates, R837 or R848 compared to the pristine polymer alone. The dose response curve using the conjugate was comparable to the course of proliferation induced by R837, between 0.1 and 5 mm.T he amount of IFN-g released into the culture was between 200 and 600 ng mL À1 after the stimulation with 0.1 to 5 mm using conjugate, R837 or R848c ompared to 200 ng mL À1 for the polymer backboneo rS IINFEKL-peptide alone. The conjugates inducedt he highestI FN-g releasea t5 mm,w hich strongly declined with decreasing conjugatec oncentrations in the splenocytes cultures down to levels of the negative controls as shown by polymer or SIINFEKL peptide alone. Based on our observation the conjugate strongly stimulates murine CD8 + Tcellsp resumablyb yD C-mediated Tcell priming involving TLR7/8signaling in DCs.
In conclusion, the chemical synthesis and biological activity of an ovel functionalized imidazoquinoline (ImQ) derivative bearing a3 -(3-oxobutyl)-benzyl moiety at the N1-position of the ImQ was presented. The keto-group was introduced to provide as ite for reversible binding to hydrazide groups attached to modified, water soluble and biodegradable polyphosphazenes.T he resulting hydrazone functionality served as an acid labile linkageb etween the polymeric carrier system and the immune response modifier.C ompound 5-conjugated ImQ polymer accumulated in generated DCsa nd co-localized with endosomal and intracellular lysosomal vesicles. The capacity of ImQ-conjugate activated DCs to stimulate Tcells via DCs was further analysed in Tcell/DC co-cultures.
In splenocytesf rom OT-I mice stimulated with polymer-ImQ conjugates in the presence of SIINFEKL-antigen, CD8 + Tc ells proliferated and strongly released IFN-g into the supernatant when compared to known immune response modifiers resiquimod (R848) and imiquimod (R837), used as positivec ontrols. In addition, CD8 + Tc ells showed high expression of the IL-2 receptor (CD25), considered as an activation marker forTcells after polymer-ImQ conjugate stimulation. Ongoing in vivo studies on murine tumor models are in progress. Oure fforts will focus to target the tumor microenvironment of tumors to induce local inflammation, which might lead to tumor rejecting immune reactions.

Experimental Section
Full experimental details, including details on the materials used, synthetic procedures and structural characterisation can be found in the Supporting Information.