Novel drug conjugates for testing cellular drug uptake

Abstract

The present invention relates to a drug conjugate of formula (I) drug – cleavable linker – oligonucleotide (formula (I)), wherein the oligonucleotide comprises from the 5'-end to the 3'-end (a) a PCR handle, (b) a barcode allowing to detect the drug within a cell by sequencing, preferably next generation sequencing, and (c) a poly A-tail.

Description

[0001] New PCT-Patent Application

[0002] Deutsches Krebsforschungszentrum Stiftung des offentlichen Rechts

[0003] Vossius Ref.: AJ2141 PCT

[0004] Novel drug conjugates for testing cellular drug uptake

[0005] The present invention relates to a drug conjugate of formula (I) drug - cleavable linker - oligonucleotide (formula (I)), wherein the oligonucleotide comprises from the 5'-end to the 3'-end (a) a PCR handle, (b) a barcode allowing to detect the drug within a cell by sequencing, preferably next generation sequencing, and (c) a poly A-tail.

[0006] Currently, the most widely used method for the assessment of the cellular uptake of a drug efficacy in vivo is based on the effect of the drug, on the size of the tumour and the survival of treated animals. Immunohistochemistry and immunofluorescence staining of tumour tissue sections are commonly used to determine whether the drug has had the desired effect on signalling in the tumour. These methods are limiting because (i) they rely on previous knowledge about the proteins which are likely to be affected by the drug, (ii) they do not consider the heterogeneity of signalling pathway activation within the different tumour cell populations and (iii) even with the use of marker staining to identify different cell types in a section, it is difficult to discriminate between different cell types, and impossible to get detailed information about the regulatory state of a particular cell on a tumour section.

[0007] The efficacy of the cellular uptake of a drug can be determined in vitro at the transcriptional level looking for effects on gene / pathway expression, and at the post-translational level by immunohistochemical analyses of proteins. Single-cell sequencing can be used to increase the resolution of such analyses, the data generated is circumstantial; capturing information as to drug concentrations per cell would lead to transformational improvements in our understanding of the origins of drug resistance development.

[0008] The present invention aims at addressing the fundamental limitations of the discussed in vivo and in vitro studies of tumour therapy resistance and in particular the difficulty of determining whether a drug was actually taken up by a target cell, if so at what concentration, and the implications thereof for the development of drug resistance. Accordingly, the present invention relates in a first aspect to a drug conjugate of formula (I) drug - cleavable linker - oligonucleotide (formula (I)), wherein the oligonucleotide comprises from the 5'-end to the 3'-end (a) a PCR handle, (b) a barcode allowing to detect the drug within a cell by sequencing, preferably next generation sequencing, and (c) a poly A-tail.

[0009] As used herein, a drug is a chemical substance (typically of known structure) which, when administered to a living organism, produces a biological effect. The drug is preferably a chemical substance that can be used to treat, cure, or prevent a disease but can also be a chemical substance that can be used to diagnose a disease. The drug is preferably a chemotherapeutic agent (also known as cytotoxic agent or cytostatic drugs). The drug can also be, for example, a drug against a neurodegenerative disease, such as Alzheimer, Parkinson, Huntington's disease (HD), or amyotrophic lateral sclerosis (ALS), or can be an immunosuppressive drug (i.e., a drug that inhibit or prevent the activity of the immune system). Non-limiting but preferred examples of drugs will be provided herein below and shown in Figure 9.

[0010] As used herein, a cleavable linker is a linker that can be or is cleaved, in particular once the drug conjugate has entered a cell. By the cleavage of the linker within the cell the drug is released from the oligonucleotide. Cleavable linkers may rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases within the cell. Non-limiting but preferred examples of cleavable linkers will be provided herein below.

[0011] As used herein, oligonucleotides are short nucleic acid (e.g. DNA or RNA) molecules, (oligomers). Oligonucleotides can be chemically synthesized using building blocks, protected phosphoramidites of natural or chemically modified nucleosides (or, to a lesser extent, of non- nucleosidic compounds). The oligonucleotide chain assembly proceeds in the 3' to 5' direction by following a routine procedure referred to as a "synthetic cycle". Completion of a single synthetic cycle results in the addition of one nucleotide residue to the growing chain. A less than 100% yield of each synthetic step and the occurrence of side reactions set practical limits of the efficiency of the process. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (U), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA.

[0012] The oligonucleotide according to the invention comprises from the 5'-end to the 3'-end (a) a PCR handle, (b) a barcode allowing to detect the drug within a cell by sequencing, preferably next generation sequencing, and (c) a poly A-tail. The optimum length of the PCR handle depends on the sequencing technology to be used and it is a matter of routine to fit the length of the PCR handle to the sequencing technology. Preferably the PCR handle is 15 to 25 bp and most preferably 20 to 22 bp, such as, for example, 21 bp. (b) Also the design of a barcode sequence is matter of routine. It is generally ideal to use barcodes that are as unique and as short as possible. The barcode has preferably a length of 10 to 25 bp, more preferably 12-20 bp and most preferably 15-18 bp, such as, for example, 16 bp. (c) Also the optimum length of the poly-A tail can be selected as a matter of routine. The preferred length is 20-45 bp, more preferably 25-40 bp and most preferably 30-35 bp, such as, for example, 32 bp.

[0013] As used herein, a PCR handle is a constant sequence that allows PCR amplification during sequencing. The same PCR handle can be used for a plurality of oligonucleotides with different barcodes and thereby all oligonucleotides can be PCR amplified by the same primers. On the other hand, the barcode is a variable barcode sequence that is suitable to distinguish a particular drug from (all) other drugs, so that the sequencing of the barcode within the oligonucleotide can uniquely identify (unique identifier) the drug, such as uniquely identify the drug inside a cell. The sequencing reads as obtained from the sequencing process can be grouped thereby assigning barcodes and consequently drugs to particular cells. This can be done on a single-cell basis or for a plurality of cells, as needed. The poly(A) tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA that has only adenine bases. In eukaryotes, polyadenylation is part of the process that produces mature mRNA for translation.

[0014] Non-limiting but preferred features of the oligonucleotide according to the invention and its subparts will be provided herein below.

[0015] As used herein sequencing refers to the process of determining the nucleotide order of the oligonucleotide according to the invention, in particular the barcode sequence thereof. Sequencing involves PCR amplification via the PCR handle. Most prior art sequencing is performed by using the chain termination method developed by Frederick Sanger. This technique uses sequence-specific termination of a DNA synthesis reaction using modified nucleotide substrates. However, other sequencing technologies, such as pyrosequencing can also be used.

[0016] In contrast to conventional sequencing techniques, next generation sequencing (NGS) enables the simultaneous sequencing of hundreds of millions of DNA fragments. NGS is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed. The technology can be used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA. Illumina NGS technology utilizes a fundamentally different approach from the classic Sanger chain-termination method. It leverages sequencing by synthesis (SBS) technology - tracking the addition of labeled nucleotides as the DNA chain is copied - in a massively parallel fashion. Next-generation sequencing generates masses of DNA sequencing data and is less expensive and less time-consuming than traditional Sanger sequencing.

[0017] As can be taken from the appended examples, in particular Example 1 the drug conjugate of the invention is illustrated by an engineering of a barcoded drug (temozolomide). The drug conjugate of the invention enables to determine drug cellular penetration and effect on targeted molecular pathways by sequencing (i.e., single-cell RNAseq in the examples). In the examples, a drug (such as temozolomide or palbociclib) is conjugated to a 69-bp single stranded oligo containing a barcode (unique identifier). To ensure that the functionality of temozolomide is not affected by the conjugation a cleavable linker is used. In Example 1 valine- citruline(VC)-PABC is used as linker that is cleaved by the protease cathepsin once it has penetrated the cell (Figure 1). The conjugation of the DNA barcode to temozolomide is performed by an antibody drug conjugate (ADC) kit by Cel I Mosaic® Inc. Further examples of particular drug conjugates are provided in Figure 9. The broad applicability and versatility of the technology of the present invention is further demonstrated by Examples 2 to 4. Example 2 shows application of the technology to complex cell structures and tissues based on patient- derived glioblastoma stem-like cells (GSCs; NCH705), grown as neurospheres. In Example 3 a disulfide linker is shown as the linker that is cleaved by glutathione once it has penetrated the cell, instead of the valine-citruline(VC)-PABC cleaved by protease cathepsin as in Example 1. Finally, in Example 4 palbociclib is illustrated as a drug instead of temozolomide. In addition, Example 4 shows multiplexing of the technology, as in Example 4 10,000 cells per sample were loaded onto a GEM-X OCM ("On-Chip Multiplexing") chip.

[0018] In accordance with a preferred embodiment of the first aspect the oligonucleotide comprises at least two, preferably at least three and most preferably at least four phosphorothioate internucleotide linkages at the 5'-terminus and / or at least two, preferably at least three and most preferably at least four phosphorothioate internucleotide linkages at the 3'-terminus.

[0019] Several phosphate backbone variants have been developed in an attempt to alter the chemical properties of native-state DNA and therefore overcome the two major challenges involved with using oligonucleotides in vivo, including: 1) delivery to the interior of the cell through the plasma membrane, a lipid bilayer that without transport proteins, is mostly impermeable to polar molecules, and 2) extension of the effective molecular lifetime by minimizing extra and intracellular nuclease degradation.

[0020] One of the most widely-used backbone variants is phosphorothioate. Phosphorothioate has been found to help alleviate the second major challenge associated with using oligonucleotides in vivo by reducing the activity of a variety of extra and intracellular nucleases (Putney et al. (1981), PNAS,78(12):7350-7354, de Noronha et al. (1992), PCR Methods Appl., 2(2):131-136 and Eckstein (2014), Nucleic Acid Therapeutics, 24(6):374-387). Hence, phosphorothioate internucleotide linkages are advantageous because they are resistant to nucleases. It is in particular preferred that the oligonucleotide comprises at least three phosphorothioate internucleotide linkages at the 5'-terminus and at least three phosphorothioate internucleotide linkages at the 3'-terminus, and even more preferred that the oligonucleotide comprises at least four phosphorothioate internucleotide linkages at the 5'-terminus and at least four phosphorothioate internucleotide linkages at the 3'-terminus.

[0021] In accordance with another preferred embodiment of the first aspect the oligonucleotide comprises one or more modified nucleotides, preferably being selected from a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, a 2'- deoxy-2'-fluoro modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'- alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

[0022] Modified nucleotides (such as nucleotides with a modification at the 2' position of the ribose ring) are commonly used in the art to help increase oligonucleotide stability and improve resistance to nuclease activity in vivo. The incorporation of the modified nucleotides according to the above preferred embodiment into the oligonucleotide according to the invention might therefore help to stabilize the oligonucleotide according to the invention in cells.

[0023] In accordance with a preferred embodiment of the first aspect the cleavable linker is cleaved if the drug conjugate is present in a cell, preferably by a lysosomal enzyme or an intracellular reducing molecule, such as glutathione.

[0024] As discussed herein above, cleavable linkers may rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases within the cell. The above preferred embodiment of the first aspect is directed to such linkers.

[0025] Lysosomal-cleavable peptide linkers are used in the prior art for antibody-drug conjugates (ADCs) (Balamkundu and Liu (2023), Biomedicines, 11(11) :3080) and the same linkers can also be used as linkers in accordance with the present invention. Also Glutathione induced cleavage of linkers is known from the prior art (Su et al. (2021), Acta Pharm Sin B. 2021 Dec; 11(12): 3889-3907).

[0026] In accordance with a further preferred embodiment of the first aspect the cleavable linker is a peptide (preferably comprising or consisting of GGFG, FRRG, FRRL or FRRLL or being ValAla, AlaAla, cBuCit or ValCit-based linker, such as a valine-citrulline-p-aminobenzylcarbamate (VC- PABC)-based linker) that can be selectively cleaved by an intracellular hydrolytic enzyme; an acid-sensitive hydrazone, carbonate or silyl ether that can be selectively cleaved in endosomes (pH = 5-6) or lysosomes (pH = 4.8); or a disulfide that can be cleaved by intracellular reducing molecule, such as glutathione.

[0027] In accordance with a yet further preferred embodiment of the first aspect the cleavable linker is a cathepsin degradable linker, preferably a VC-PABC-based linker, or is a glutathionesensitive linker, preferably disulfide (i.e., comprising - S-S - moiety), or monomethyl disulfide (i.e. comprising -CH(CH3)-S-S-CH(CH3)- moiety) linker.

[0028] For example, the MC-VC-PABC linker, i.e., one of the preferred VC-PABC-based linkers, may be based on the following chemical structure

[0029] The "curved lines" at the left and right end of the above VC-PABC-based linker structure show the sites where the drug and the oligonucleotide are attached. Preferably the oligonucleotide is attached at the left side and the drug at the right side. An example is shown in Figure 1.

[0030] The above linker is comprised in the exemplified drug conjugates as shown In Figure 1 and Figure 9, compounds la, 2a, 3a, 4a, and 5a. In the exemplified drug conjugates as shown in Figure 9, compounds lb, 2b, 3b, 4b, and 5b the linker is the above-mentioned -CH(CH3)-S-S- CHfCHs)- moiety.

[0031] Linker reactants (i.e., the unreacted, isolated linkers to which the drug and the oligonucleotide can be attached by standard chemistry that is as used for the production of antibody drug conjugates) that can be used to prepare drug conjugates according to the invention are commercially available. For example, the unreacted, isolated MC-VC-PAB-PNP peptide linker has the chemical structure (CAS No. 159857-81-5) the unreacted, isolated Val-cit-PAB-OH peptide linker has the chemical structure (CAS. No.

[0032] 159857-79-1)

[0033] The unreacted, isolated cBu-Cit-OH (Mal-cyclobutane-1) linker has the chemical structure (Cas

[0034] No. 1799663-03-8)

[0035] The above Linker reactants are commercially available, for example, from MedChem Express and in total 450 cleavable linkers are commercially available via MedChem Express. These 450 cleavable linkers were used in the art to prepare ADCs and can likewise be used to prepare the conjugates according to the present invention by standard chemistry.

[0036] For instance, any drug containing a free NH group can be converted to a carbamate and any drug containing a free hydroxyl OH can be converted to a carbonate and conjugated to one or the cleavable linkers. Likewise, a free NH group or a free OH group can be used to attach the oligonucleotide to the linkers. This is illustrated by the conjugates in Figures 1 and 9. As an illustration, various conjugates according to the invention may be synthesized according to the schemes provided on figure 10.

[0037] Custom designed ADCs can be commercially ordered from companies, such as CellMosaic, Inc. and these companies can also prepare and deliver the conjugates of the invention. The herein exemplified conjugates were prepared in collaboration with CellMosaic, Inc. by standard chemistry tha was know from the ADC field. As mentioned above, the conjugation of the DNA barcode to temozolomide was performed by an antibody drug conjugate (ADC) kit by CellMosaic® Inc.

[0038] The linkers according to the above preferred embodiments are designed to be suitable in the bloodstream and then release the payload once in the cell. Such cleavable linkers include as type enzymatically-cleavable peptide linkers, acid sensitive hydrazone linkers, and glutathione-sensitive disulfide linkers. Among these suitable linkers a VC-PABC-based linker is most preferred. VC-PABC and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma and related linkers are readily cleaved by cathepsins in the lysosome while remaining reasonably stable in human plasma. The tetra peptidyl-aminomethoxy linkers FRRG, FRRL and FRRLL are also cathepsin-cleavable.

[0039] The glutathione-sensitive linker is most preferably a disulfide (e.g. a monomethyl disulfide bond; Figure 9). Disulfides are thermodynamically stable at physiological pH and are designed to release the conjugated payload drug upon internalization into cells, where the cytosol provides a much stronger reducing environment compared to the extracellular environment (blood circulation). The scission of disulfide bridge requires the presence of a cytoplasmic thiol co-factor, such as (reduced) glutathione (GSH) or the intracellular enzyme protein disulfide isomerase.

[0040] In accordance with a yet further preferred embodiment of the first aspect the drug is a small molecule, preferably selected from temozolomide (Examples 1-3, Figures 6-8, 11-12), palbociclib (Example 4, Figures 13 and 14), alectinib, idasanutlin, temsirolimus, amanitin, auristatin, calicheamicin, camptothecin, cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, esperamicin, geldanamycin, maytansinoid, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoid, erlotinib, bortezomib, fulvestrant, sunitinib (sutent), letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5- fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, , topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, neocarzinostatin chromophore, aclacinomysins, actinomycin, antramycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6- diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2, 2', 2"- trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, , paclitaxel, albumin-engineered nanoparticle formulation of paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates or acids of any of the foregoing.

[0041] The small molecule is preferably a low molecular weight organic compound. The molecular weight of the small molecule is preferably mass range of 50 - 1500 Daltons (Da), more preferably 50 - 1000 Daltons (Da).

[0042] Organic molecules relate or belong to the class of chemical compounds having a carbon basis, wherein the carbon atoms are linked together by carbon-carbon bonds. The original definition of the term organic related to the source of chemical compounds, with organic compounds being those carbon-containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources. Organic compounds can be natural or synthetic. The organic molecule is preferably an aromatic molecule and more preferably a heteroaromatic molecule. In organic chemistry, the term aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms. Aromatic molecules are very stable, and do not break apart easily to react with other substances. In a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g., N, S, or O.

[0043] The list of preferred small molecules in the above preferred embodiments comprises small molecules that are in medical use for various diseases, in particular cancer and immune diseases.

[0044] In a preferred embodiment of the first aspect, the drug may be an anti-cancer cytotoxic drug or an immunosuppressive drug.

[0045] The present invention relates in a second aspect to a lipid particle comprising the drug conjugate of the first aspect.

[0046] The definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second aspect of the invention as far as being amenable therewith.

[0047] The lipid particles serve as drug delivery system mediating the entry of the drug conjugate of the first aspect into cells. The lipid particle is preferably a lipid nanoparticle (LN P).

[0048] A lipid nanoparticle is typically spherical with an average diameter between 10 and 1000 nanometers. Solid lipid nanoparticles possess a solid lipid core matrix that can solubilize lipophilic molecules. The lipid core is stabilized by surfactants (emulsifiers). The emulsifier used depends on administration routes and is more limited for parenteral administrations. The term lipid is used in this context in a broader sense and includes triglycerides (e.g. tristearin), diglycerides (e.g. glycerol bahenate), monoglycerides (e.g. glycerol monostearate), fatty acids (e.g. stearic acid), steroids (e.g. cholesterol), and waxes (e.g. cetyl palmitate). All classes of emulsifiers (with respect to charge and molecular weight) have been used in the art to stabilize the lipid dispersion. It has been found that the combination of emulsifiers might prevent particle agglomeration more efficiently.

[0049] The present invention relates in a third aspect to an in vitro or ex vivo method for testing whether a drug is taken up by cells comprising (a) contacting at least one drug conjugate of the first aspect and / or lipid particle of the second aspect with the cells, (b) isolating one or more cells after step (a), and (c) detecting the at least one barcode of the at least one drug conjugate of the first aspect and / or lipid particle of the second aspect within the one or more cells of (b) by sequencing, preferably next generation sequencing, wherein the detection of a barcode within the one or more cells of (b) by sequencing indicates that the respective drug entered the one or more cells.

[0050] The definitions and preferred embodiments of the first and second aspect of the invention apply mutatis mutandis to the third aspect of the invention as far as being amenable therewith.

[0051] Phagocytosis and diffusion through the cell membrane are principal drug uptake mechanisms. One of the most obvious mechanisms for drug resistance is a low drug uptake in cells. For this reason, in vitro or ex vivo methods fortesting whether a drug is taken up by cells are important tests to be carried out in the process of developing new medicaments (Zhang et al. (209), Drug Deliv. 2019; 26(1): 328-342).

[0052] The method of the second aspect of the present invention is a particular advantageous method for testing cellular drug uptake, because it is no longer necessary to check whether the drug per se has entered the cell. Instead, the barcode of the drug conjugate can be sequenced, thereby identifying whether the drug entered the cells. As discussed above, the barcode serves as a unique identifier of the drug. In sequencing barcoding is a method used to identify a particular species by analysing a specific region of a nucleic acid molecule. This region is called the barcode. The nucleotide sequence of this barcode is then compared to a reference library or reference list which contains information of many species linked to their respective barcodes.

[0053] In accordance with a preferred embodiment of the third aspect in step (a) at least two, preferably at least five and most preferably at least 50 different drug conjugates and / or lipid particles are contacted with the cells.

[0054] Hence, the method is preferably carried out as a multiplex method (Example 4), wherein a plurality of drug conjugates and / or lipid particles are tested in parallel (for example, in a multiwell plate format). The plurality is with increasing preference at least 5, at least 10 at least 20, at least 50, at least 1000 an at least 1000 different conjugates and / or lipid particles.

[0055] In accordance with a further preferred embodiment of the third aspect in step (a) known amounts of the at least one drug conjugate of the first aspect and / or lipid particle of the second aspect are contacted with a known number of cells, and in step (c) the at least one barcode of the at least one drug conjugate and / or lipid particle is detected in a quantitative manner, thereby allowing to quantify the amount of the respective drug that entered the one or more cells.

[0056] Thus, the method is preferably carried out in a quantitative manner, such that the amount of the respective drug that entered the one or more cells can be determined.

[0057] In accordance with another preferred embodiment of the third aspect the method further comprises (d) analyzing the transcriptome, proteome and / or metabolome of the one or more cells of (b), and optionally (c) comparing the transcriptome, proteome and / or metabolome with the transcriptome, proteome and / or metabolome of one or cells of the same cell type(s) that were not contacted with the at least one drug conjugate and / or lipid particle, thereby allowing the detection of changes of the transcriptome, proteome and / or metabolome as induced by the uptake of the respective drug by the one or more cells. Transcriptome, proteome and metabolome analysis provide insights on how the uptake of the drug into cells influences gene expression, protein expression and the formation and / or processing of metabolites of a cell, respectively.

[0058] In accordance with a preferred embodiment of the third aspect the cells in step (a) are all of the same cell-type or comprise cells of different cell-types.

[0059] In the case of different cell-types the different cell-types may be derived from different tissues, different diseases, and / or different subjects / patients (preferably human subjects / patients).

[0060] The present invention relates in a fourth aspect to at least one drug conjugate of the first aspect and / or lipid particle of the second aspect for use in a method of diagnosis in vivo whether a drug is taken up by cells within a subject, wherein the subject is preferably a nonhuman subject.

[0061] The definitions and preferred embodiments of the first to third aspect of the invention apply mutatis mutandis to the fourth aspect of the invention as far as being amenable therewith.

[0062] The subject herein is preferably a vertebrate, more preferably a mammal and most preferably human. The mammal can be a human, a domestic animal or a pet animal such as a horse, cattle, pig, sheep, goat, dog or cat.

[0063] While the method according to the third aspect of the invention is an in vitro or ex vivo method, the fourth aspect is directed to an in vivo application for testing whether at least one drug conjugate of the first aspect and / or lipid particle of the second aspect is / are taken up by cells within a subject.

[0064] Also described herein is an method for testing whether a drug is taken up by cells of a subject comprising (a) administering the at least one drug conjugate of the first aspect and / or lipid particle of the second aspect to the subject, (b) isolating one or more cells after step (a) from the subject, and (c) detecting the at least one barcode of the at least one drug conjugate of the first aspect and / or lipid particle of the second aspect within the one or more cells of (b) by sequencing, preferably next generation sequencing, wherein the detection of a barcode within the one or more cells of (b) by sequencing indicates that the respective drug entered the one or more cells of the subject.

[0065] The present invention relates in a fifth aspect to at least one drug conjugate of the first aspect and / or lipid particle of the second aspect for use in treating a disease, wherein the disease is preferably a cell proliferative disease, an inflammatory disease or a neurodegenerative disease.

[0066] The definitions and preferred embodiments of the first to fourth aspect of the invention apply mutatis mutandis to the fifth aspect of the invention as far as being amenable therewith.

[0067] The treatment of a disease as referred to herein comprises a curative treatment as well as a disease preventive treatment and is preferably a curative treatment. The curative treatment ameliorates at least one symptom of the disease and / or stops / slows down disease progression.

[0068] In connection with the fifth aspect of the invention it is to be understood that the drug is selected such that it is suitable to treat the target disease. For example, in the case of the treatment of a cell proliferative disease the drug can be a compound inhibiting cell proliferation, such as a cytostatic drug.

[0069] The cell proliferative disease is preferably a cancer. Cancer is an abnormal malignant new growth of tissue that possesses no physiological function and arises from uncontrolled usually rapid cellular proliferation. The cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulva cancer, bladder cancer, salivary gland cancer, pancreatic cancer, thyroid cancer, kidney cancer, lung cancer, cancer concerning the upper gastrointestinal tract, colon cancer, colorectal cancer, prostate cancer, squamous-cell carcinoma of the head and neck, cervical cancer, glioblastomas, malignant ascites, lymphomas and leukemias.

[0070] Inflammatory diseases are diseases in which excess inflammation plays a key role. The inflammatory disease may be an autoimmune disease or a disease that is most likely of autoimmune origin (e.g., rheumatoid arthritis, multiple sclerosis, myasthenia gravis, psoriasis, vitiligo, systemic lupus erythematosus, sarcoidosis, focal segmental glomerulosclerosis, Crohn's disease, Bechet's Disease, pemphigus, ankylosing spondylitis, and ulcerative colitis) or another non-autoimmune inflammatory disease (e.g., long term allergic asthma control, or ankylosing spondylitis).

[0071] Neurodegenerative diseases are caused by the progressive loss of neurons, in the process known as neurodegeneration. Neurodegenerative diseases include amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, tauopathies, and prion diseases.

[0072] Also described herein is a pharmaceutical composition comprising the at least one drug conjugate of the first aspect and / or lipid particle of the second aspect that can be used, for example, in connection with the fifth aspect.

[0073] Herein, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the invention comprises the compounds recited above. It may, optionally, comprise further molecules capable of altering the characteristics of the compounds of the invention thereby, for example, stabilizing, modulating and / or activating their function. The pharmaceutical composition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable drug dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 pg to 5 g units per day. However, a more preferred dosage might be in the range of 0.01 mg to 100 mg, even more preferably 0.01 mg to 50 mg and most preferably 0.01 mg to 10 mg per day. The length of treatment needed to observe changes and the interval following treatment for responses to occur vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art.

[0074] Further described herein is a method treating a disease in subject, wherein the disease is preferably a cell proliferative disease, an inflammatory disease or a neurodegenerative disease by administering to the subject a therapeutically effective amount of at least one drug conjugate of the first aspect and / or lipid particle of the second aspect.

[0075] The present invention relates in a sixth aspect to a kit comprising a

[0076] (I)

[0077] (a) a library of drugs,

[0078] (b) a library of oligonucleotides, wherein each kind of oligonucleotide in the library of (b) comprises from the 5'-end to the 3'-end a PCR handle, a barcode allowing to specifically detect one kind of drug in the library of (a) within a cell by sequencing, preferably next generation sequencing, when being linked with the one kind of drug in the library of (a), and a poly A-tail, and

[0079] (c) one or more kinds of cleavable linkers for linking each kind of drug in the library to a specific kind of oligonucleotide in the library; or

[0080] (ID

[0081] (a) a library of drugs, wherein the drugs are linked to one or more kinds of cleavable linkers for linking each kind of drug in the library to a specific kind of oligonucleotide in the library of

[0082] (b), and

[0083] (b) a library of oligonucleotides, wherein each kind of oligonucleotide in the library of (b) comprises from the 5'-end to the 3'-end a PCR handle, a barcode allowing to specifically detect the drugs in the library of (a) within a cell by sequencing, preferably next generation sequencing, when being linked with the drugs in the library of (a), and a poly A-tail; or

[0084] (HI) (a) a library of drugs,

[0085] (b) a library of oligonucleotides, wherein each kind of oligonucleotide in the library of (b) comprises from the 5'-end to the 3'-end a PCR handle, a barcode allowing to specifically detect the drugs in the library of (a) within a cell by sequencing, preferably next generation sequencing, when being linked with the drugs in the library of (a), and a poly A-tail, and wherein the oligonucleotides are linked to one or more kinds of cleavable linkers for linking each kind of oligonucleotides in the library to a specific kind of drug in the library of (a).

[0086] The definitions and preferred embodiments of the first to fifth aspect of the invention apply mutatis mutandis to the sixth aspect of the invention as far as being amenable therewith.

[0087] The kit of the sixth aspect of the invention implements a / the means required for preparing the drug conjugate of the first aspect and / or lipid particle of the second aspect.

[0088] The various components of the kit may be packaged into one or more containers such as one or more vials. The vials may, in addition to the components, comprise preservatives or buffers for storage. The kit may comprise instructions how to use the kit, which preferably inform how to use the components for preparing the drug conjugate of the first aspect and / or lipid particle of the second aspect and optionally how to use them in connection with the third to fifth aspect.

[0089] In accordance with a preferred embodiment of the sixth aspect the drugs and the oligonucleotides in the libraries can be linked via the linker by a click-chemistry reaction.

[0090] In chemical synthesis, click chemistry is a class of simple, atom-economy reactions commonly used for joining two molecular entities of choice. Click-chemistry reactions are known in the art and a review can be found, for example, in Devaraj and Finn (2023), Chemical reviews,

[0091] 21:6697-6698. The figures show:

[0092] Figure 1 - Representation of the exemplary oligo conjugated Temozolomide that was developed with the collaboration of CellMosaic Inc. We have conjugated Temozolomide with a 69bp DNA barcode that can be read by single-cell RNA high-throughput sequencing. For the conjugation ofTMZ to the oligonucleotide, we used a protease cleavable VC-PABC linker which is similar to those currently used for antibody-drug conjugates (ADC), allowing the release of the small molecule into the cell.

[0093] Figure 2 - Design of the oligos used to perform single cell RNAseq. (*) represents a phosphorothioate bond between the nucleotides to ensure the stability of the oligo within the cells. The unique identifierthat acts as a barcode to detect the drug within the cell is shown in bold. A PCR handle from TotalSeq A (https: / / www.biolegend.com / en-us / totalseq) that is compatible with Illumina sequencing technology is underlined.

[0094] Figure 3 - LN229 cells transfected with biotinylated oligos and fixed at 4, 8, 12, 18 and 24 hours after transfection. Circles: DAPI; Arrows: Streptavidfin-FITC. Scale bar: 50p.m.

[0095] Figure 4 - LN229 cells transfected with phosphorothioate internucleotide linkages and fixed 24 hours after transfection. Scale bar: 50pm.

[0096] Figure 5 - (A) cDNA amplification by PCR as determined by gel electrophoresis, depicting 1-kb ladder (1), 100 bp ladder (2), amplification of cDNA without primers (3), amplification without cDNA (4), amplification of cDNA with forward and reverse primers (5), amplification of cDNA with 1 / 3 of forward and reverse primers (6), primers only (7). (B) cDNA melting curves as determined by qRT-PCR of LN229 transfected with the oligos.

[0097] Figure 6 - Representation of the exemplary oligo conjugated Temozolomide with a disulfide linker. Figure 7 - Determination of temozolomide efficacy with and without lipofectamine. (A) Cell viability of LN229 treated for 24 hours with and without lipofectamine with different concentration of temozolomide. (B) Temozolomide IC50 determination of LN229 and LN308.

[0098] Figure 8 - (A) GEX library preps prior to single cell RNAseq. A non-treated sample (Blank) as well as GFP-labelled cells transfected with Temozolomide only (GFP-TMZ) were used as control to ensure the efficacy of the conjugated Temozolomide (Oligo). (B) Single cell populations identified by RNAseq, highlighting the efficacy of our experimental set-up to discriminate between cells that receive Temozolomide and cells that weren't treated. Cells within the right cluster that were treated with Temozolomide were also shown to have received a lower concentration of the drug (C).

[0099] Figure 9 - Examples of drug conjugates.

[0100] Figure 10 - Exemplary synthesis schemes for conjugating various drugs according to the invention.

[0101] Figure 11 - Upper plots: UMAP showing single cell populations identified by RNAseq on the basis of transcriptomic changes induced by conjugated or unconjugated TMZ in glioblastoma patient-derived NCH705 cells, highlightingthe efficacy of the invention in an alternative (more heterogenous) model. Cells are shaded according to cell cycle phase, with the percentage of the number of cells in each phase included in the legend. Note the depletion of the cells in cluster 1, and corresponding enrichment of cells in clusters 2 and 3 upon treatment with unconjugated or oligo-conjugated TMZ compared with the mock treated sample.

[0102] Lower plots: Cells shaded according to the amount of oligo captured in each cell population, showing differential compound uptake across different cell states.

[0103] Figure 12 - UMAP showing single cell populations identified by RNAseq on the basis of transcriptomic changes induced by TMZ-oligo conjugated via a disulfide linker in patient- derived NCH705 cells, showing use of the invention with a reducible disulfide linker. Cells are shaded according to the amount of oligo captured in each cell population, showing differential compound uptake across different cell states. Figure 13 - Cell viability of MCF7 (breast) cancer cell line incubated with increasing concentrations of palbociclib. Data are normalized to the DMSO control.

[0104] Figure 14 - Upper plots: UMAPs showing single cell populations identified by RNAseq on the basis of transcriptomic changes induced by oligo-conjugated and unconjugated palbociclib (CDK4 / 6 inhibitor) and corresponding conjugated-palbociclib oligo levels in breast cancer cell line MCF7. This figure shows use of the invention with a different class of drugs and in different cancer entities. Cells are shaded according to cell cycle phase, with the percentage of the number of cells in each phase included in the legend. Note the expected cell cycle arrest induced by unconjugated palbociclib and to palbociclib-oligo, as indicated by an enrichment of cells in the G1 cell cycle phase (cluster 2).

[0105] Lower plots: Cells are shaded according to the amount of oligo captured in each cell population, showing differential compound uptake across cells.

[0106] The examples illustrate the invention.

[0107] Example 1

[0108] The oligos were designed to be compatible with current high-throughput RNAseq technology. In addition to a PCR handle, oligos harbored a unique identifier of 16-bp (Figure 2).

[0109] In order to test whether the large stretch of oligos could penetrate the cells, we used biotinylated oligos that enable the tracing overtime via immunofluorescence.

[0110] In that respect, HEK293 and LN229 cell lines were seeded in a 6-well plate onto coverslips (200000 cells per well) and filled up to a volume of 2000 pl. After 48 hours, the cells were transfected with the biotinylated oligos. Per well, 250 pl of the reduced serum medium Opti- MEM™ (Thermofischer), along with 7.5 pl TranslT®-LTl Transfection Reagent (Mirus), and 1.125 pl of 100 pM biotinylated oligos along a pre-prepared serial dilution (50nM, 25nM, 10 nM, 5 nM, 2.5 nM), including 1.125 pl of water for a mock reaction. This was done by firstly mixing Opti-MEM™ and TranslT®-LTl 5 mins at 37°C under low frequency shaking. Then biotinylated oligos were added to the mix. After 20 minutes, the mix was then added to the cells. After transduction cells were washed twice with PBS, fixed with 4 % PFA for 15 mins at RT then wash three times with PBS. Cells were fixed at 4, 8, 12, 18 and 24 hours after transfection (Figure 3).

[0111] Although oligos could penetrated the cells, fluorescent signals strongly decreased 4 hours after transfection. In order to increase stability, we added four phosphorothioate internucleotide linkages at the 5'-terminus and six phosphorothioate internucleotide linkages at the 3'-terminus. This ensured the stability of the oligo within the cells for at least 24 hours (Figure 4).

[0112] Next, we measured the amount of oligo within the cells by qRT-PCR. RNA was extracted from LN 229 cells (after Mock transfection, transfection with 50 nM DNA oligo for 1 day, 4 days and 7 days) using the RNAeasy Mini Kit (Qiagen) according to the manufacturer's protocol. To measure the effect of DNAase on the oligos, each cell lysates were split into two samples: One sample was treated with DNAase, the second was not treated with. Samples were then examined for integrity using an Agilent 2100 Bioanalyzer. Next, 1 pg RNA of each sample was reverse transcribed by Superscript II (Life Technologies) according to the manufacturer's protocol using the following forward and reverse primers, respectively: 5'- CCTTGGCACCCGAGAATTCC-3' (SEQ ID NO: 1) and 5'-TTGCAGAAACAGTCACGTTG-3' (SEQ ID NO: 2). Complementary cDNA was measured using Absolute SYBR Green ROX Mix (ABgene) in technical triplicate using the Lightcycler® 480 (Roche). cDNA levels were quantified based on the standard curves of a serial dilution of cDNA transcribed from LN229 RNA and normalization to the housekeepergenes ARFl and HPRT. cDNA of the oligo template was used as a positive control, H2O and genomic DNA of NCH644 were used as negative controls (Figure 5A and 5B).

[0113] In order to allow the readout of any compound of our choice by single cell RNA sequencing, we have designed a system using a linker that is cleavable once penetrating the cells. In that respect, we cooperate with CellMosaic Inc. who designed and performed the chemistry.

[0114] At first, we used a cathepsin cleavable linker that consists of a maliemidocaproyl that binds to the 5' of the oligo, followed by valine and citrulline peptides bound to the self-immolating para-amino benzyl carbamate that releases the compound (Temozolomide [TMZ] in this example) enzymatically based on similar mechanism of anti-Cd30 dipeptide-linked auristatin immunoconjugate (Sanderson, R. J.; et. Al. Clinical Cancer Research, 2005, 11, 843-852) (Figure 1). Mc-VC-PAB-AE-TMZ compound was obtained from CellMosaic Inc. (Calculated exact mass: 835.37. Obtained: [M+H]+: 836.4.). Disulfide oligo with 5' thiol modifier C6 S-S was obtained from Integrated DNA Technologies (Coralville, IA, USA). TCEP was obtained from Chem-lmpex (Wood Dale, IL, USA). HPLC analyses were done in an acetonitrile / O.lM TEAA gradient system with a flow rate of 0.8 mL / min at 45 °C. Buffer A: 0.1M TEAA, pH 7.0, Buffer B: 80% Acetonitrile in 0.1M TEAA, pH 7.0. Gradient: 5 to 50% B within 8 minutes, then to 95% within 2 minutes and finally hold at 95%B for another 2 minutes. Briefly, disulfide oligo was first reduced by TCEP to release the free SH group by standard reducing protocol (Bioconjugation Techniques by Greg T. Hermanson, 2ndedition, 2008, pages 95-96). The reducing reagents were removed by passing through a lOKda filter and then alkylated with Mc-VC-PAB-AE-TMZ by standard maleimide thiol conjugation protocol (Bioconjugation Techniques by Greg T. Hermanson, 2ndedition, 2008, pages 283-286). The progress of the reaction was monitored by C18 HPLC. Upon completion, excess Mc-VC-PAB-AE-TMZ was removed by passing through a short Sephadex G- 25 column (around 8 mL settle down resin) pre-equilibrium with PBS buffer. Fractions were analyzed by reversed phase C18 HPLC using the standard TEAA buffer system. Fractions containing the products were combined and quantified by UV absorbance at 260 nm assuming the extinction coefficient of the product is the same as the disulfide oligo (Calculated Ext. Coefficient at 260 nm: 721,600 M-1cm-1.

[0115] Next, oligo-TMZ conjugate with a disulfide bond linker as alternative to the enzymatically cleavable linker was designed and custom synthesized at CellMosaic Inc. (Woburn, MA, USA). The reducible disulfide linker is the same as SSNPP linker for trastuzumab DM1 described in Philips et al. 2008 (Phillips G.D.L.; et. Al. Cancer Res., 2008, 68, 9280-9290) (Figure 6).

[0116] HPLC analyses were done in an acetonitrile / O.lM TEAA gradient system with a flow rate of 0.8 mL / min at 45 °C. Buffer A: 0.1M TEAA, pH 7.0, Buffer B: 80% Acetonitrile in 0.1M TEAA, pH 7.0. Gradient: 5 to 50% B within 8 minutes, then to 95% within 2 minutes and finally hold at 95%B for another 2 minutes. The preparative HPLC purification was done using the same TEAA buffer system with a slightly shallower gradient than the analytical.

[0117] TMZ-MM-S-S-MM-Osu was obtained from CellMosaic Inc. Oligo with 5'-C6 amino modifier was obtained from TriLink biotechnologies (San Diego, CA, USA). Around 70% of the oligos contained the C6 amino modifier. C6 amino oligo (100 nmol) was mixed directly with 15 equivalents of TMZ-MM-S-S-MM-Osu in a 0.1 M solidum phosphate buffer, pH 7.2 containing some organic solvent at 37 °C. The progress of the reaction was monitored by C18 HPLC. After half an hour, over 65% of oligo was converted to the product. Further addition of TMZ-MM-S- S-MM-Osu into the reaction mixture did not increase the conversion. This indicates the leftover unreacted oligo does not contain any C6 amino group. Excess TMZ-MM-S-S-MM-Osu and unreacted oligo (non-amino, truncated components) were removed by C18 HPLC purification. Fractions containing products were combined, concentrated, and buffer exchanged with 0.1 M citrate buffer, pH 6 using tangential flow filtration with 10 Kda filter. The final product was quantified by UV absorbance at 260 nm assuming the extinction coefficient of the product is the same as the oligo (Calculated Ext. Coefficient at 260 nm: 721,600 M4cm4).

[0118] Because the drug is linked to a 69-bp oligo, lipofectamine was used to efficiently transfect the oligo-conjugated drug within the cells. To ensure that lipofectamine had no effect on the efficacy of the drug, we measured cell viability upon temozolomide treatment with and without the transfection reagent. LN229 and LN308 cells seeded at a density of 3000 cells per well in a 96-well plate and filled up with 90 pl of cell culture medium. No significant effect of lipofectamine on temozolomide efficacy could be observed (Figure 7A). Next, to determine the IC20 and IC50 of LN229 and LN308, a Temozolomide serial dilution (500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 3.9 pM, and a DMSO vehicle control) was applied in a 96-well plate in technical and biological triplicates up to a total well volume of 100 pl. In both cases, the inhibition was only applied once, with a medium change after 3 days in the 6-day-group. Viability was measured using the CellTiter-Glo assay (Promega), according to the manufacturer's protocol and using a Mithras LB 940 plate reader (Berthold Technologies). After normalizing to the DMSO control, IC20 and IC50 values were determined by applying a four-parameter logistic curve using Graphpad Prism 10 at 3 days and 6 days after inhibition. (Figure 7B).

[0119] Having ensured the stability of the oligo over time and the efficiency of the transfection, we then treated the cell line LN229 with the oligo-conjugated temozolomide. To be able to assess the reliability of our system within a cellular entity, we used LN229 that stably express GFP and that were treated with native temozolomide as positive control. LN229 treated with lipofectamine only were used as negative control.

[0120] Single-cell RNA-seq libraries were generated using the 10X Genomics 3' v3.1 chemistry with the following adpatations: 1 pl ADT primer (0.2 pM) was added to the cDNA amplification mix. cDNA was purified with Ampure beads in two steps by adding additional beads to the supernatant after the first 0.6x purification to run a 1.8x purification for shorter cDNA fragments. Short RNA library synthesis was performed using HotStart Kapa mix, indexed truseq primers and the SI primer in a 100 pl reaction (Figure 8A). The different sequences for library prep are listed in Table 1.

[0121] Table 1 (SEQ. ID NOs 3 to 7)

[0122] = PTO backbone

[0123] As shown in Figure 8B, two clusters could be identified upon treatment. Interestingly, the untreated LN229 cluster on the right side while the cells treated with temozolomide either native or conjugated are to be found in both clusters. There was no difference in the number of treated cells (both conjugated and native temozolomide) to be found in both clusters, indicating that the conjugated temozolomide harboured the same effect as the native temozolomide. Thanks to the developed technology, the number of molecules of temozolomide could now be assessed and showed that treated cells that clustered with untreated cells received less temozolomide as compared to the treated cells in the left clusters (Figure 8C).

[0124] Thanks to the conjugation of a molecule of drug with a stretch of oligo that is compatible with high-throughput sequencing methods it is now possible to determine how many molecules of the drug of interest penetrates the cell. In addition, it is possible to study the effect of the drug at the single cell level and identify populations of resistant cells, which will help to understand the mechanism of resistance.

[0125] Example 2

[0126] In order to illustrate use of the technology of the present invention in an alternative (more heterogenous) model, patient-derived glioblastoma stem-like cells (GSCs; NCH705), grown as neurospheres, were seeded into a 48 well plate at a density of 70,000 cells per well. The next day, cells (two wells per sample) were transfected with 3.5 pM (IC20) of the TMZ-VC-PAB-oligo (the oligo used forthe conjugation is depicted in Table 4, SEQ ID NO: 18), 3.5 pM unconjugated TMZ or PBS (mock) using 0.6 pl TransIT and 20 pl OPTI-MEM per well. 20 hours later, cells were harvested, singularized using accutase and washed twice in PBS. 40,000 cells were loaded onto a GEM-X 3' chip and GEMs generated using a Chromium X. Library preparation and enrichment of the oligo sequence was carried out as described in example 1, with the implementation of v4 of the lOx Genomics 3' chemistry. The gene expression library and oligo library were mixed at a ratio of 1:19 and sequenced on one lane of a NovaseqX Plus 25B. In addition, the method was tested for compatibility with RNAseq multiplexing by transfecting cells with PBS (mock), unconjugated TMZ or TMZ-VC-PAB-oligo as described in example 1. Read 2 primers that were used are depicted in Table 2. After harvesting, 10,000 cells per sample were loaded onto a GEM-X OCM Chip. Subsequent library generation and sequencing followed as detailed in example 1 (Figure 11).

[0127] Table 2 (SEQ ID NOs 9 to 16)

[0128] * = PTO backbone

[0129] Example 3

[0130] In this example, use of a conjugate comprising a disulfide linker is shown. The oligo used for the conjugation is depicted in Table 3 (SEQ ID NO: 17). GSCs were seeded into a 48 well plate at a density of 70,000 cells per well. The next day, the provided TMZ-S-S-oligo conjugated was first dialyzed into PBS for 1 hour using a ReadyLyzer dialysis column. Then cells were transfected with 3.5 pM (IC20) of the conjugate using 0.6 pl TransIT and 20 pl OPTI-MEM per well. Cell harvesting, library generation and sequencing followed as detailed in Examples 1 and 2 (Figure 12).

[0131] Table 3 (SEQ ID NO: 17)

[0132] * = PTO backbone

[0133] Example 4

[0134] The sensitivity of breast cancer (MCF7) to palbociclib was determined by seeding 7,500 cells into each well of a 96-well plate and applying increasing concentrations of palbociclib. Cell viability was assessed after 72 hours using the Cell Titer Gio assay (Promega), whereby the substrate and the buffer were mixed and 90 pl of the solution was applied to each well. After shaking for 2 minutes at 200 rpm, and a further incubation for 8 minutes, luminescence detected with an integration time of 1 second using a Mithras 940B plate reader. The luminescence corresponds to cell viability. Data were normalized to the DMSO control, and dose response curves were generated using a 4 parameter non-linear regression model in GraphPad 10 (Figure 13). Further, palbociclib was conjugated to a modified oligo sequence (SEQ ID NO 18, Table 4) via a VC-PAB linker as described in Example 1. MCF7 cells were seeded at a densitiy of 70,000 cells per well in 48-well plates. Then cells were transfected with the IC20 of conjugated palbociclib (4 pM), unconjugated palbociclib or PBS (mock) using 0.6 pl TransIT and 20 pl OPT I -MEM per well. 20 hours later, cells were harvested, singularized using accutase and washed twice in PBS. 10,000 cells per sample were loaded onto a GEM-X OCM chip, and subsequent library generation and sequencing followed as detailed in example 1 (Figure 14).

[0135] Table 4 (SEQ ID NO: 18)

[0136] * = PTO backbone

Claims

1. Claims1. A drug conjugate of formula (I) drug - cleavable linker- oligonucleotide (formula (I)), wherein the oligonucleotide comprises from the 5'-end to the 3'-end(a) a PCR handle,(b) a barcode allowing to detect the drug within a cell by sequencing, preferably next generation sequencing, and(c) a poly A-tail.

2. The drug conjugate of claim 1, wherein the oligonucleotide comprises at least two, preferably at least three and most preferably at least four phosphorothioate internucleotide linkages at the 5'-terminus and / or at least two, preferably at least three and most preferably at least four phosphorothioate internucleotide linkages at the 3'- terminus.

3. The drug conjugate of claim 1 or 2, wherein the cleavable linker is cleaved if the drug conjugate is present in a cell, preferably by a lysosomal enzyme or an intracellular reducing molecule, such as glutathione.

4. The drug conjugate of any one of claims 1 to 3, wherein the cleavable linker is a peptide (preferably comprising or consisting of GGFG, FRRG, FRRL or FRRLL or being ValAla, AlaAla, cBuCit or ValCit-based linker, such as a valine-citrulline-p- aminobenzylcarbamate (VC-PABC)-based linker) that can be selectively cleaved by an intracellular hydrolytic enzyme; an acid-sensitive hydrazone, carbonate or silyl ether that can be selectively cleaved in endosomes (pH = 5-6) or lysosomes (pH = 4.8); or a disulfide that can be cleaved by intracellular reducing molecule, such as glutathione.

5. The drug conjugate of any one of claims 1 to 4, wherein the cleavable linker is a cathepsin degradable linker, preferably a VC-PABC-based linker, or is a glutathionesensitive linker, preferably disulfide or monomethyl disulfide.

6. The drug conjugate of any one of claims 1 to 5, wherein the drug is a small molecule, preferably selected from temozolomide, palbociclib, alectinib, idasanutlin, temsirolimus, amanitin, auristatin, calicheamicin, camptothecin, cryptophycin, daunomycin, dolastatin, doxorubicin, duocarmycin, epothilone, esperamicin, geldanamycin, maytansinoid, methotrexate, monomethyl auristatin E ("MMAE"), monomethyl auristatin F ("MMAF"), pyrrolobenzodiazepine, rhizoxin, SG2285, tubulysin, vindesine, toxoid, erlotinib, bortezomib, fulvestrant, sunitinib (sutent), letrozole, imatinib mesylate, PTK787 / ZK 222584, oxaliplatin, 5-fluorouracil, leucovorin, rapamycin, lapatinib, lonafarnib, sorafenib, gefitinib, AG1478, AG1571, thiotepa, cyclophosphamide, busulfan, improsulfan, piposulfan, benzodopa, carboquone, ethylenimine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine, bullatacin, bullatacinone, topotecan, bryostatin, callystatin, CC-1065, adozelesin, carzelesin, bizelesin, cryptophycin 1, cryptophycin 8, KW-2189, CB1-TM1, eleutherobin, pancratistatin, sarcodictyin, spongistatin, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, calicheamicin gamma 1, calicheamicin omega 1, dynemicin, dynemicin A, clodronate, neocarzinostatin chromophore, aclacinomysins, actinomycin, antramycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubucin, 6-diazo-5-oxo- L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubucin, liposomal doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, 5-fluorouracil, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, aminoglutethimide, mitotane, trilostane, folinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone,elfornithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansine, ansamitocins, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, polysaccharide-k, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, T-2 toxin, verracurin A, roridin A, and anguidine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside, cyclophosphamide, paclitaxel, albumin- engineered nanoparticle formulation of paclitaxel, doxetaxel, chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, cisplatin, carboplatin, vinblastine, platinum, etoposide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine, retinoic acid, capecitabine, or pharmaceutically acceptable salts, solvates or acids of any of the foregoing.

7. A lipid particle comprising the drug conjugate of any one of claims 1 to 6.

8. An in vitro or ex vivo method for testing whether a drug is taken up by cells comprising(a) contacting at least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7 with the cells,(b) isolating one or more cells after step (a), and(c) detecting the at least one barcode of the at least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7 within the one or more cells of (b) by sequencing, preferably next generation sequencing, wherein the detection of a barcode within the one or more cells of (b) by sequencing indicates that the respective drug entered the one or more cells.

9. The method of claim 8 which is a multiplex method, wherein in step (a) at least two, preferably at least five and most preferably at least 50 different drug conjugates of any one of claims 1 to 6 and / or lipid particle of claim 7 are contacted with the cells.

10. The method of claim 8 or 9, wherein in step (a) known amounts of the at least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7 are contacted with a known number of cells, and in step (c) the at least one barcode of the at least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7 is detected in a quantitative manner, thereby allowing to quantify the amount of the respective drug that entered the one or more cells.

11. The method of any one of claims 8 to 10, further comprising(d) analyzing the transcriptome, proteome and / or metabolome of the one or more cells of (b), and optionally(c) comparing the transcriptome, proteome and / or metabolome with the transcriptome, proteome and / or metabolome of one or cells of the same cell type(s) that were not contacted with the at least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7, thereby allowing the detection of changes of the transcriptome, proteome and / or metabolome as induced by the uptake of the respective drug by the one or more cells.

12. At least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7 for use in a method of diagnosis in vivo whether a drug is taken up by cells within a subject, wherein the subject is preferably a non-human subject.

13. At least one drug conjugate of any one of claims 1 to 6 and / or lipid particle of claim 7 for use in treating a disease, wherein the disease is preferably a cell proliferative disease, an inflammatory disease or a neurodegenerative disease.

14. A kit comprising a(I)(a) a library of drugs,(b) a library of oligonucleotides, wherein each kind of oligonucleotide in the library of (a) comprises from the 5'-end to the 3'-end a PCR handle, a barcode allowing to specifically detect one kind of drug in the library of (a) within a cell by sequencingpreferably next generation sequencing when being linked with the one kind of drug in the library of (a), and a poly A-tail, and(c) one or more kinds of cleavable linkers for linking each kind of drug in the library to a specific kind of oligonucleotide in the library; or(ID(a) a library of drugs, wherein the drugs are linked to one or more kinds of cleavable linkers for linking each kind of drug in the library to a specific kind of oligonucleotide in the library of (b), and(b) a library of oligonucleotides, wherein each kind of oligonucleotide in the library of (a) comprises from the 5'-end to the 3'-end a PCR handle, a barcode allowing to specifically detect the drugs in the library of (a) within a cell by sequencing, preferably next generation sequencing when being linked with the drugs in the library of (a), and a poly A-tail; or(III)(a) a library of drugs,(b) a library of oligonucleotides, wherein each kind of oligonucleotide in the library of (a) comprises from the 5'-end to the 3'-end a PCR handle, a barcode allowing to specifically detect the drugs in the library of (a) within a cell by sequencing, preferably next generation sequencing when being linked with the drugs in the library of (a), and a poly A-tail, and wherein the oligonucleotides are linked to one or more kinds of cleavable linkers for linking each kind of oligonucleotides in the library to a specific kind of drug in the library of (a).

15. The kit of claim 14, wherein the drugs and the oligonucleotide in the libraries can be linked via the linker by a click-chemistry reaction.

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