Targeted protein degradation and recruitment

Abstract

The present invention pertains to the field of targeted protein degradation (TPD) and target protein recruitment (TPR) providing a versatile platform for TPD, TPR and other applications dependent on cell surface ternary complex formation. In particular, the present invention provides a compound comprising a macromolecular hydrophilic polymer scaffold which is conjugated with several copies of at least two different protein binding ligands. The polymeric scaffold of the invention enables the use of small molecule ligands for target proteins of interest. Furthermore, the invention also relates to a composition comprising said polymeric scaffolds and uses thereof, e.g. for inhibiting or removing malignant or unwanted proteins; or for use in the targeted recruitment of effector cells such as CAR-T cells.

Description

[0001] Targeted protein degradation and recruitment

[0002] FIELD OF THE INVENTION

[0003] The present invention pertains to the field of targeted protein degradation (TPD) and target protein recruitment (TPR) providing a versatile platform for TPD, TPR and other applications dependent on cell surface ternary complex formation. In particular, the present invention provides a compound comprising a macromolecular hydrophilic polymer scaffold which is conjugated with several copies of at least two different protein binding ligands. The polymeric scaffold of the invention enables the use of small molecule ligands for target proteins of interest. Furthermore, the invention also relates to a composition comprising said polymeric scaffolds and uses thereof, e.g. for inhibiting or removing malignant or unwanted proteins; or for use in the targeted recruitment of effector cells such as CAR-T cells.

[0004] BACKGROUND TO THE INVENTION

[0005] Targeted protein degradation (TPD) targeted protein recruitment (TPR) is an emerging modality in the field of drug development. Traditional drugs, such as small molecule ligands and antibodies, typically inhibit protein activity by binding catalytic pockets or preventing protein-protein interactions. In contrast, TPD disrupts the activity of disease-related target proteins of interest (POIs) by binding to them and directing them to cellular degradation machinery. Unlike conventional drugs, TPD drug design permits the use of non-blocking protein-binding ligands, expanding the spectrum of druggable target proteins. The current generation of TPD technologies leverages the degradation capacity of either the ubiquitin- proteasome system (UPS) or lysosomes. PROteolysis Targeting Chimeras (PROTACs) and molecular glues facilitate UPS-driven protein degradation, while AUtophagosome TArgeting Chimeras (AUTACs) and AuTophagosome TEthering Compounds (ATTECs) rely on lysosomal degradation. Clinical validation of TPD drugs is promising, with several PROTACs and molecular glues entering the clinical development stage. Additionally, retrospective studies unraveled that certain approved drugs act as molecular glues.

[0006] These TPD technologies primarily focus on targeting intracellular target proteins. Recently, a novel TPD concept has been introduced aimed at degrading extracellular target proteins, which constitute 40% of the human proteome. Compounds such as LYsosome TArgeting Chimeras (LYTACs) and molecular degraders of extracellular proteins through the asialoglycoprotein (ASGPR) receptor (MoDE-As) simultaneously bind to an extracellular target protein and a lysosomal trafficking membrane receptor, i.e., the cation-independent mannose-6-phosphate receptor (CI-M6PR: a lysosomal trafficking receptor ubiquitously expressed on mammalian cell surfaces) or the ASGPR (a lysosomal trafficking receptor expressed on the surface of hepatocytes) for LYTACs and ASGPR for MoDE-As. The resulting ternary complex undergoes endocytosis, leading to the trafficking and degradation of the target protein in the lysosomes. The initial design of LYTACs, relying on CI-M6PR for lysosomal trafficking, involved a monoclonal antibody as a ligand against a target POI on the cell surface. This antibody was conjugated to a polymer containing multiple repeating units of mannose 6-phosphate (or mannose 6-phosphonate) (M6P) as ligands for the CI-M6PR. On the other hand, LYTACs and MoDE-As that rely on ASGPR for lysosomal trafficking were based on the conjugation of either a monoclonal antibody (for LYTACs) or a small molecule (for MoDE-As) to trivalent N-Acetylgalactosamine (GalNAc) as a high-affinity ligand for the ASGPR.

[0007] Thus, the molecular design of these technologies is either based on the same types of small molecule ligands or (mono)clonal antibodies and has several limitations in terms of mechanism-of-action and / or structural design. For example, the current LYTAC method is based on recombinant production in for example E. coli resulting in less flexibility in the degree of ligand functionalization and molecular weight of the scaffold. Further, in the case of MoDE-As, due to their low molecular weight, these molecules have a faster body clearance and therefore may not be as efficient as larger molecules that reside longer in the body.

[0008] There is a need for an improvement in the field of TPD and TPR technology to increase binding affinity, efficiency and recruitment of proteins of interest (POIs) to lysosomes. It was therefore an object of the present invention to provide a solution to the problems listed herein above. The present invention provides a non-recombinant TPD approach, involving a multivalent display of small molecule ligands, mediating ternary complex formation between a target extracellular POI selected for degradation and a cell surface lysosomal trafficking receptor. Alternatively, the present invention also provides a nonrecombinant TPR approach, involving a multivalent display of small molecule ligands, mediating ternary complex formation between a target extracellular effector protein or effector cell and a cell surface protein expressed on a cancer cell or immune cell. The chimeras as provided herein feature an array of small molecule affinity ligands for the target POI designated for degradation, paired with an array of ligands for a lysosomal trafficking receptor, all conjugated along a macromolecular hydrophilic backbone. Alternatively, the chimeras as provided herein feature an array of small molecule affinity ligands for the target POI designated recruitment, paired with an array of ligands to bind cancer or immune cells, all conjugated along a macromolecular hydrophilic backbone. The molecular design of known technologies that promote ternary complex formation between target protein and receptors to drive protein degradation or recruitment is based on antibodies or small molecules. The structural design of the compound of the invention highly differentiates as it is based on a hydrophilic polymer scaffold conjugated with multiple copies of protein binding ligands for the target protein or target cell and multiple copies of binding ligands for the lysosomal trafficking receptor or cancer / immune cell, randomly spaced along the hydrophilic polymer. This design offers vastly more flexibility in tuning pharmacokinetics and pharmacodynamics by varying ligand density and molecular weight of the hydrophilic polymer scaffold. Besides the multivalent polymer design which increases the avidity of the ternary complex, other main advantages of the present invention are that no recombinant production is required resulting in a much higher flexibility in degree of ligand functionalization and higher molecular weight of the scaffold. The compound of the invention can be used in various molecular weights, in particular in molecular weights suitable to prolong plasma half-life. Further, in case the case of degradation, the degraded target protein molecules do not regain activity again. Since target proteins are trafficked to the lysosomal degradation pathway, a suitable target protein binding ligand does not necessarily have to bind the active site of the target protein. This opens up a much wider set of ligands, including ligands that bind undruggable targets. Further, in case of recruitment, these multivalent scaffolds provide a strong ternary complex between an effector protein such as an antibody and a targeted cell protein presented on a cancer or immune cell and thus are particularly suitable in respectively cancer or immune therapy. Hence, the present invention herewith provides a promising design for TPD or TPR applications and beyond that involve the formation of ternary complexes.

[0009] SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention provides a compound comprising a macromolecular hydrophilic polymer scaffold conjugated with

[0011] - at least one extracellular target protein binding ligand; and

[0012] - at least one further protein binding ligand for binding i) a lysosomal trafficking receptor; or ii) a target protein presented on a cancer cell or immune cell; wherein each of said binding ligands is present in at least 5 copies, and wherein said extracellular target protein binding ligand is different from said further protein binding ligand.

[0013] In another aspect, the present invention provides a compound comprising a macromolecular hydrophilic polymer scaffold conjugated with at least one lysosomal trafficking receptor binding ligand and at least one extracellular target protein binding ligand, wherein each of said binding ligands is present in at least 5 copies.

[0014] In another aspect, the present invention provides a compound comprising a macromolecular hydrophilic polymer scaffold conjugated with at least one extracellular target protein binding ligand and at least one further protein binding ligand, wherein each of said binding ligands is present in at least 5 copies.

[0015] In a specific embodiment, said extracellular target protein binding ligand is different from said lysosomal trafficking receptor binding ligand or said further protein binding ligand.

[0016] In a particular embodiment, the binding ligands and copies thereof are arranged on the polymer scaffold such that a ternary complex can be formed between the extracellular target protein binding ligand and the target protein of interest and between the lysosomal trafficking receptor binding ligand and the lysosomal receptor.

[0017] In another embodiment, the ratio of the further protein binding ligands to the extracellular target protein binding ligand conjugated to the polymer scaffold ranges from about 1 :20 to about 20:1 , in particular about 1 :10 to about 10:1 , more in particular about 1 :5 to about 5:1 , even more in particular about 1 :3 to about 3:1 , most in particular 1 :1 .

[0018] In another particular embodiment, the density of the ligands conjugated per 100 monomer repeating units of hydrophilic polymer scaffold ranges about and between 1 % to 50%.

[0019] In a specific embodiment, the hydrophilic polymer has a molecular weight of at least about 40 kDa, in particular at least about 70 kDa, more in particular at least about 100 kDa, even more particular at least about 150 kDa.

[0020] In yet a specific embodiment, said hydrophilic polymer scaffold is a linear and / or branched polymer, in particular a linear polymer.

[0021] In yet another embodiment, the hydrophilic polymer scaffold is a polysaccharide, in particular dextran.

[0022] In another embodiment, each of the binding ligands is present in at least 10 copies, in particular at least 20 copies, more in particular at least 50 copies.

[0023] In a particular embodiment, the compound according to the invention further comprises at least one further tissue-specific targeting ligand.

[0024] In a further embodiment, wherein in case said further protein binding ligand is a lysosomal trafficking receptor binding ligand, the extracellular target protein binding ligand has affinity to a target protein to be degraded.

[0025] In yet a particular embodiment, the lysosomal trafficking receptor binding ligand is Mannose-6- Phosphate (M6P) and / or N-Acetyl Galactosamine (GalNAc).

[0026] In a specific embodiment said target protein presented on a cancer cell can be, but is not limited to, CAIX (Carbonic Anhydrase IX), PSMA, uPAR, folate receptor, and avp3 integrins, HER2 / neu (ERBB2), EGFR (ERBB1), VEGFR, FGFR, CD20, CD19, CD22, CD33, CD38, CD52, CD70, CD123, PD-L1 (CD274), PD-L2 (CD273), CTLA-4, EpCAM (CD326), N-cadherin, ICAM-1 , VCAM-1 , MUC1 , CEA, PSA, CA-125, GD2, MDR1 (P-glycoprotein, ABCB1), BCRP (ABCG2), MMPs (Matrix Metalloproteinases), ALK (Anaplastic Lymphoma Kinase), BRAF, Mesothelin.

[0027] In another embodiment, wherein in case said further protein binding ligand is a ligand binding a target protein presented on a cancer or immune cell, the extracellular target protein binding ligand has affinity to an effector protein, in particular an antibody or an effector cell, in particular a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and a eosinophil or a chimera thereof such as a CAR T cell, a CAR B cell, a CAR natural killer (NK) cell, a CAR macrophage, a CAR monocyte, a CAR neutrophil, a CAR dendritic cell, a CAR mast cell, a CAR basophil, and a CAR eosinophil.

[0028] In a further embodiment, the ligands are attached to the polysaccharide via linkers.

[0029] In even a further embodiment, the present invention provides a compound wherein said lysosomal trafficking receptor binding ligand binds a lysosomal trafficking receptor on a cell membrane of a cell. In a particular embodiment the cell membrane can be the membrane of an immune cell or a cancer cell.

[0030] In a further embodiment, the present invention provides a pharmaceutical composition comprising the compound according to the invention and a pharmaceutically acceptable excipient.

[0031] In a particular embodiment, the present invention provides a compound or pharmaceutical composition according to the invention for use in human and / or veterinary medicine.

[0032] In a specific embodiment, the present invention provides a compound or pharmaceutical composition according to the invention for use in the treatment of oncology, inflammatory and / or autoimmune diseases.

[0033] In yet a specific embodiment, the compound or pharmaceutical composition according to the invention wherein the further protein binding ligand is a lysosomal trafficking said lysosomal trafficking receptor binding ligand, can be used in targeted protein degradation.

[0034] In even a further aspect, the present invention provides a method, in particular an in vivo method, of degrading a target protein of interest comprising: contacting the compound or the pharmaceutical composition according to the invention, with the target protein of interest and the lysosomal trafficking receptor of a cell membrane under conditions in which the lysosomal trafficking receptor of said cell shuttles said target protein to the lysosome of said cell for degradation.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. When reference is made to the term ‘DEXTRAC’ it is to be understood as a trafficking chimera wherein Dextran is used as a backbone.

[0037] Fig. 1 : Mechanism of action. The trafficking chimeras according to the invention mediate ternary complex formation between a target protein of interest (POI) for degradation and the cation-independent mannose-6-phosphate receptor (CI-M6PR) as a lysosomal shuttling receptor on the cell surface. This connection directs the POI into the lysosomes for degradation. The trafficking chimeras according to the invention consist of multiple copies of synthetic small molecule ligands for both the POI and the Cl- M6PR, arrayed on the backbone of a hydrophilic polymer scaffold. The formation of a ternary complex involving the trafficking chimeras, the POI, and the CI-M6PR initiates endocytosis, transporting the POI into lysosomes where it is subsequently degraded through lysosomal proteolysis.

[0038] Fig. 2:1H-NMR spectroscopy confirms successful synthesis of trafficking chimeras arrayed on the backbone of dextran. Integration of the1H-NMR spectra confirm the transformation of native 150 kDa dextran (A) through consecutive functionalization steps: alkyne functionalization aiming for a DS of 20 (B), incorporation of a small fraction of Cy5 for fluorescence tracking (C), introduction of DNP-PEGa- azide aiming for a DS of 10 (D), and addition of M6P-PEG3-azide aiming for a DS of 10 (E). To determine the DS for each functional group, the integration value of the a-C1 proton of the glucose repeating units was normalized to 100. The alkyne DS was calculated by integration the alkyne proton, while the DNP DS was determined by integrating DNP protons. Full functionalization was confirmed by the complete disappearance of alkyne proton peaks in the final DEXTRAC spectrum (E). As all M6P proton peaks overlapped with proton peaks of dextran, M6P DS was calculated by subtracting the calculated DNP DS from the calculated alkyne DS. Fig. 3: DEXtran TRAfficking Chimeras (DEXTRACs) mediate potent and selective ternary complex formation. (A) Experimental set-up for evaluating DEXTRAC-mediated ternary complex formation. Jurkat cells were co-incubated with DEXTRAC and AF488-labeled anti-DNP antibodies for 24 hours, followed by analysis using flow cytometry. (B) Cellular association of AF488-labeled anti-DNP antibodies with DEXTRAC. Bell-shaped curve fitting was applied. (C) Graphical illustration of the Hook effect; i.e., at high DEXTRAC concentration, binary complex formation between the binding partners is favored of overternary complex formation. (D) Different molecularweights and concentrations of dextran fitted with AF488 fold-increasemax values. (E) Overview of experimental and fitted AF488 fold-increasemax values for each DEXTRAC. One-way ANOVA was used for statistical analysis, ns: not significant, ***: p = 0.0004,****: p < 0.0001 . (F) Assessment of the association of AF488-labeled anti-DNP antibodies with Jurkat cells after 24 hours of co-incubation with either DEXTRAC 150 kDa DS 20 or the control (150 kDa Cy5 / DNP-dextran), at a dextran concentration of 10 nM. This was conducted in the absence or presence of 10 mM M6P, and measurements were performed using flow cytometry.

[0039] Fig. 4: DEXTRACs exhibit high avidity and specificity ligand-receptor binding. (A) Experimental set-up for free M6P ligand competition experiments. Jurkat cells were incubated for 24 hours with varying concentrations of Cy5-labeled DEXTRAC 40 kDa DS 10 or 150 kDa DS 20, in the presence of competing free M6P ligand. After 24-hour of incubation, cellular DEXTRAC association was quantified by flow cytometry. (B1) Inhibition curves obtained for DEXTRAC 40 kDa DS 10 (left) and 150 kDa DS 20 (right). (B2) Inhibition curve obtained at 500 nM DEXTRAC 40 kDa DS 10 (left) and 50 nM of 150 kDa DS 20 (right). Note that 500 nM of DEXTRAC 40 kDa DS 10 and 50 nM of DEXTRAC 150 kDa DS 20 correspond to a similar concentration of dextran-bound M6P. (C) Experimental set-up for free DNP ligand competition experiments. Biotinylated anti-DNP antibody was immobilized on streptavidin beads. These beads were incubated for 30 minutes with Cy5-labeled DEXTRAC 40 kDa DS 10 or 150 kDa DS 20, respectively, in presence of competing free DNP ligand. After the 30-minute of incubation, DEXTRAC association to the beads was analysed by flow cytometry. (D) Inhibition curve obtained at 500 nM of DEXTRAC 40 kDa DS 10 (left) and 50 nM of 150 kDa DS 20 (right). Note that 500 nM of DEXTRAC 40 kDa DS 10 and 50 nM of DEXTRAC 150 kDa DS 20 correspond to a similar concentration of dextran-bound DNP.

[0040] Fig. 5: DEXTRACs mediate lysosomal trafficking and degradation of target protein. (A) Experimental outline of the Western blot analysis experiment. (B) Western blot on lysates of Jurkat cells incubated for 24 hours with 20 nM AF488-labeled anti-DNP antibody alone or combined with 20 nM DEXTRAC 150 kDA DS 20. As controls, 100 nM bafilomycin A1 (Baf) or 10 mM M6P were co-incubated to inhibit lysosomal activity or ternary complex formation, respectively. (C) Western blot band intensity analysis using ImageJ software. For each sample, the band intensity was plotted against the distance along the y-axis. The reported values represent the percentage of the total area under the curve (AUC) for each plotted curve.

[0041] Fig. 6: DNP / FAPI-dextran outperforms single ligand FAPI-PEG-DNP in mediating ternary complex formation between FAP+ cells and anti-DNP antibodies. HT-1080.hFAP cells were seeded in a 24- well plate at a concentration of 50 000 cells in 500 pL DMEM per well and were left to adhere overnight before treatment. Cells were washed once with PBS containing 1% BSA and resuspended in 200 pL of PBS, after which Cy5 / DNP / FAPI-dextran or FAPI-PEG12-DNP stock solution was added to achieve a DNP well concentration of 10 pM. The AF488 signal was measured by flow cytometry. The results demonstrate selective ternary complex formation between FAP+ cells, Cy5 / DNP / FAPI-dextran and aDNP antibody.

[0042] Fig. 7: Antibody recruitment capacity of single ligand antibody recruiting molecules (SL-ARMs). Flow cytometry analysis of SK-RC-52 cells treated with SL-ARMs (10 uM) followed by the addition of AF488-anti-DNP antibodies. Excess AAZ was added as competitor to SL-ARMs in binding with cells. DNP-PEGX-N3 (10 uM) is a control construct lacking the AAZ ligand. (n=3, mean SD).

[0043] Fig. 8: Binding of multivalent dextran antibody recruitment molecules (dex-ARMs) through AAZ- mediated anchoring to SK-RC-52 cells. Flow cytometry analysis of SK-RC-52 cells treated with Cy5- labeled dex-ARMs. (n=3, mean + SD).

[0044] Fig. 9: Dex-ARMs show superior antibody recruiting capacity versus SL-ARMs. (A, left panel) Flow cytometry analysis of SK-RC-52 cells treated with SL-ARM-2 and dex-ARM, followed by the addition of AF488-anti-DNP antibodies (n=3, mean + SD). (A, right panel) AF488 signal was measured using flow cytometry. The results demonstrate selective ternary complex formation between CAIX+ cells, Cy5 / DNP / AAZ-dextran (i.e. dex-arm) and aDNP antibody. (B) Confocal microscopy imaging of SK-RC-52 cells treated with dex-ARM, followed by staining with AF488-anti-DNP antibody (green fluorescence). (C) Flow cytometry analysis of SK-RC-52 cells treated dex-ARM with different ratio between AAZ to DNP ratio, followed by staining with AF488-anti-DNP antibody (n=3, mean + SD). (D) Flow cytometry analysis of SK-RC-52 cells treated with SL-ARM-2 and dex-ARM, in presence of AAZ as a competing ligand, followed by the addition of AF488-anti-DNP antibodies (n=3, mean + SD).

[0045] Fig. 10: Serum from immunized mice. (A) Dilution curves of IgG, lgG1 and lgG2a antibody titers measured via ELISA (n=3; mean + SD). (B) Flow cytometry analysis of SK-RC-52 cells treated with SL- ARM-2 and dex-ARM, in presence of AAZ as a competing ligand, followed by the addition of 1% of immune serum and staining with AF488-anti-lgG1 antibody. (n=3, mean + SD).

[0046] Fig. 11 : FITC / AAZ-dextran mediates ternary complex formation between CAIX+ cells and anti- FITC antibodies. SK-RC-52 cells were seeded in a 24-well plate at a concentration of 250 000 cells in 500 pL RPMI per well and were left to adhere overnight before treatment. 25 pL of PBS or FITC / AAZ- dextran stock solution was added to each well to achieve a FITC / AAZ-dextran concentration of 15, 75, or 150 nM. The cellular binding of dextran and aFITC antibody was assessed by flow cytometry, respectively. The results demonstrate selective association of FITC / AAZ-dextran to CAIX+ cells (A) and ternary complex formation between CAIX+ cells, FITC / AAZ-dextran and aFITC antibody (B).

[0047] Fig. 12: Transduction of mouse CD8+ T cells with anti-FITC scFv CAR. (a) Experimental protocol for transduction of mouse CD8+ T cells with retroviral particles, (b) Flow cytometry histograms of anti- Myc stained mouse CD8+ T cells, (c) Quantitative analysis of anti-Myc surface staining of mouse CD8+ T cells (n=3, mean ± sd). Statistical analysis by Student' t-test.

[0048] Fig. 13: FITC / AZA-dextran binds to mouse anti-FITC scFv CAR T cells, (a) Experimental protocol for flow cytometry analysis of FITC / AZA-dextran binding to mouse anti-FITC scFv CAR T cells, (b) Flow cytometry histograms of FITC / AZA-dextran binding to mouse anti-FITC scFv CAR T cells, (c) Quantitative analysis of FITC / AZA-dextran binding to mouse anti-FITC scFv CAR T cells. (n=3, mean ± sd). Statistical analysis by Students’ t-test.

[0049] Fig. 14: FITC / AZA-dextran mediates killing of CAIX+ SK-RC-52 cells by anti-FITC scFv CAR T cells (a) Experimental protocol for flow cytometry analysis killing of CAIX+ SK-RC-52 cells by scFv anti- FITC scFv CAR T cells in presence of FITC / AZA-dextran. (b) Quantitative analysis of SK-RC-52 cell killing by anti-FITC scFv CAR T cells in presence of FITC / AZA-dextran, and control constructs. (n=6, mean ± sd). Statistical analysis by One-way ANOVA.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0052] The term "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 10 % or less, preferably + / - 5 % or less, more preferably + / - 1 % or less, and still more preferably + / - 0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0053] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0054] When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless a context dictates otherwise.

[0055] The present invention provides trafficking chimeras for the degradation of extracellular proteins or for the recruitment of effector proteins or cells to a target immune cell or cancer cell. The trafficking chimeras feature multiple small-molecule ligands customized for a specific protein of interest (POI) and a lysosomal trafficking receptor, such as CI-M6PR or ASGPR or ligand for a target protein presented on a cancer cell or immune cell. These ligands are randomly positioned on a highly biocompatible polymeric scaffold. The trafficking chimeras form potent ternary complexes with the target POI and a lysosomal trafficking receptor, demonstrate high selectivity, and guide the POI to the lysosomes for degradation. The recruitment chimeras form potent ternary complexes with the target effector protein or cell and a target protein expressed on a cancer or immune cell, demonstrate high selectivity, and guide the effector protein or cell to the cancer or immune cell or interest. The multivalent display of the ligands enhances avidity towards their binding partners and serves as a pivotal driving force behind the biological activity. The design of the trafficking chimeras offers notable flexibility through alterations in the scaffold chain length and / or ligand density, both of which distinctly impact biological activity. In essence, the described invention emphasizes trafficking chimeras as a promising platform for Targeted Protein Degradation (TPD), opening avenues for exploration with other POI and lysosomal trafficking binding ligands. Moreover, the applicability of the trafficking chimeras extends beyond TPD, particularly in scenarios involving ternary complex formation such as recruitment of effector proteins such as antibodies (Targeted Protein Recruitment; TPR). Accordingly, such specific chimera are also encompassed by the invention.

[0056] In a first aspect, the present invention provides a ‘trafficking chimera’ in the form of a compound comprising a polymer scaffold conjugated with at least one lysosomal trafficking receptor binding ligand and at least one extracellular target protein binding ligand, wherein each of said binding ligands is present in at least 5 copies. In one embodiment, the polymer scaffold is a hydrophilic scaffold. In a further embodiment, the polymer scaffold is a macromolecule.

[0057] As used herein, the term “macromolecular hydrophilic polymer scaffold” refers to a three-dimensional structure composed of polymer molecules that have an affinity for water. The scaffold is designed to provide a supportive framework for binding ligands that specifically bind cells surface molecules or extracellular molecules. The scaffold serves the purpose as a ‘carrier’ for the ligand and binding moieties as provided herein, in particular the lysosomal trafficking ligands and extracellular target binding moieties, wherein after binding with the target molecules, the scaffold carrier is trafficked towards the lysosomes for degradation and removal of the target molecules. Within the context of the invention, it will thus be appreciated that the extracellular target protein binding ligands are different types of ligands compared to the lysosomal trafficking receptor binding ligands meaning that both types of ligands present on the scaffold do not have the same binding affinity to a specific target molecule.

[0058] As used herein, the polymer scaffold according to the invention is a macromolecular scaffold, preferably a linear and / or branched scaffold, more preferably a linear macromolecular scaffold.

[0059] The molecular weight of the polymer scaffold can be varied from 1 Da to 3,000,000 Da fortuning ternary complex avidity and pharmacodynamic and pharmacokinetic properties. In a specific embodiment, the hydrophilic polymer has a molecular weight of at least about 40 kDa, such as at least 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa. In a particular embodiment, the hydrophilic polymer has a molecular weight of about and between 5 kDa to about 500 000 kDa, about and between 10 kDa to about 100 000 kDa, about and between 20 kDa to about 50 000 kDa, about and between 50 kDa to about 10 000 kDa, about and between 100 kDa to about 5 000 kDa, in particular about and between 100 kDa to about 200 kDa, more in particular about 150 kDa.

[0060] In one embodiment, the scaffold is a hydrophilic polymer, in particular a biocompatible and / or biodegradable polymer. The hydrophilic polymer scaffold according to the invention can be any of the following macromolecular polymer scaffolds selected from the group consisting of: dextran, hyaluronic acid (HA), hydroxyethyl starch (HEs), gelatin, poly (vinyl alcohol) (PVA), poly (acrylic acid) (PAA), poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-oxazolines) (Pox), poly (sulfobetaine methacrylate (PSBMA), poly (2-methacryloyloxyethyl phosphorycholine (PMPC), poly (sulfo betaine) pSB), polyethylene glycol (PEG) , polyglutamate, poly aspartate, alginate, and other zwitterion ic / ampholyte polymers. The choice of polymer depends on factors such as desired properties, intended application, biocompatibility, and biodegradability requirements. In this context, particularly suitable polymer scaffolds forthe invention are dextran, hyaluronic acid (HA), hydroxyethyl starch (HEs) and gelatin which are biodegradable polymers.

[0061] In one embodiment, the scaffold is a linear and / or branched hydrophilic polymer, in particular a linear hydrophilic polymer. In the context of the present invention, the term “linear polymer” is meant to be a type of polymer characterized by a chain-like structure where repeating monomer units are linked together in a single, continuous chain, without any branching or cross-linking. As used herein, “branched polymers” are a type of polymer that contains a main chain with side chains or branches extending from it and have a more irregular and diverse range of branch structures (e.g. in the form of a graft copolymer having a main chain of one polymer with branches of another; or in the form of a star polymer having several polymer chains connected to a central core). These branches are made of the same repeating units as the main chain and often synthesized through polymerization reactions.

[0062] In another embodiment, the scaffold of the present invention is not a dendrimer. Dendrimers differ significantly in their structure and synthesis compared to branched polymers. Dendrimers are highly symmetrical, monodisperse, and possess a well-defined, tree-like structure with multiple generations of branches emanating from a central core.

[0063] In one other embodiment, the hydrophilic polymer scaffold is a neutral or a non-cationic polymer scaffold. The disadvantage of using cationic polymers (e.g. PAMAM dendrimers) is the reduction of binding selectivity to cell membranes via protein-ligand interactions, as these polymers can interact with negatively charged phospholipid bilayers. Cationic macromolecules often cause toxicity and immunogenicity, hindering the clinical translation. Further, PAMAM dendrimers consist of non- biodegradable amide bonds, which may pose further safety risks apart from their cationic charge.

[0064] Contrary, neutral or non-cationic polymers, such as for example but not limited to Dextrans, provide beneficial stealth effects similar to those of PEG. These type of polymers do not bind to cell membranes, leading to more selective ligand interactions. Neutral or non-cationic macromolecules such as Dextrans have been used clinically as plasma expanders and are therefore much more favourable forthe intended application according to the invention. The glycosidic bonds of Dextrans forming the linear backbone are biodegradable, which may generate hydrophilic metabolites that are more easily removed by the body.

[0065] Thus, in a particular embodiment, the hydrophilic polymer scaffold is a polysaccharide, in particular dextran or a modified version therefore such as for example alkyne-dextran, Cy5-dextran, DNP / 5- dextran, of MeP / DNP / Cy5-dextran. Dextran is a biocompatible polysaccharide made up of glucose units linked together by a-1 ,6 glycosidic bonds, with occasional a-1 ,3 or a-1 ,4 linkages. Dextrans are hydrophilic, non-ionic polymers that exhibit minimal intrinsic interaction with proteins. The hydroxyl groups on the glucopyranose units are accessible for chemical conjugation, facilitating the creation of a multivalent ligand display. Dextran is particularly suitable to conjugate multiple copies of different binding ligands and this multivalency can improve target binding affinity and selectivity, which attributes to the avidity effect. Furthermore, owing to their high water solubility, more hydrophobic affinity ligands can be used while still maintaining good overall solubility, and the adjustable size of dextrans could be harnessed to modulate pharmacokinetics. These properties have prompted the widespread adoption of dextran, including its utilization as a plasma expander in medical settings. The molecular weight of dextran can vary widely depending on the polymerization degree, which refers to the number of repeating units of glucose in the polymer chain. Dextran can have molecular weights ranging from a few thousand Daltons (Da) to several million Daltons. As such, the design of the compound according to the invention offers vastly more flexibility in tuning pharmacokinetics and pharmacodynamics by varying ligand density and molecular weight of the polymer scaffold. Compared to other polymers, dextran has good water-solubility, low immunogenicity and flexible design in a way that ligand density, ligand ratio and dextran molecular weight can easily be varied. As evident from the example section, a higher molecular weight (MW) of trafficking chimera is more effective in mediating ternary complex formation than their lower MW counterparts. In addition, an increase in the density degree of the binding ligands also attributes to this effect. Co-incubation with a 40 kDa hydrophilic polymer scaffold with a ligand density (DS) of 10 achieved a maximum fold-increasemax value of 6, while an analogous 150 kDa DEXTRAC (10 DS) mediated a markedly higher fold-increasemax of 65 at a similar concentration (Cmax) of polymer-bound ligand.

[0066] Thus, the ternary complex formation is even further increased when the trafficking chimera has a particular ligand density (i.e. Degree of Substitution; the number of ligands conjugated per 100 monomer repeating units of a hydrophilic polymer backbone). Doubling the ligand density (i.e. 20 DS versus 10 DS) on a polymer scaffold of equal MW leads to an increase in fold-increasemax value without compromising the Cmax value. Non-limiting examples to achieve a particular DS is for example through alkyne functionalization, subsequent introduction of DNP-PEGs-azide, and through addition of M6P- PEGs-azide.

[0067] In a particular embodiment, the density degree of the lysosomal trafficking receptor binding ligands conjugated per 100 monomer repeating units of hydrophilic polymer scaffold range about and between 1 % to 50%, in particular about and between 2% to 45%, more in particular about and between 3% to 40%, even more in particular about and between 4% to 30%, most in particular about and between 5% to 20% while the density degree of the extracellular target protein binding ligands conjugated per 100 monomer repeating units of hydrophilic polymer scaffold range about and between 1 % to 50%, in particular about and between 2% to 45%, more in particular about and between 103 to 40%, even more in particular about and between 4% to 30%, most in particular about and between 5% to 20%. In a specific embodiment, the density degree of the lysosomal trafficking receptor binding ligands and the extracellular target protein binding ligands is 10%-20% binder density, in particular 10% binder density.

[0068] In a specific embodiment, the hydrophilic polymer scaffold, such as dextran, has a molecular weight of at least 10 kDa such as at least at least 40 kDa, at least 60 kDa, at least 80 kDa, at least 100 kDa, at least 120 kDa, at least 140 kDa, or at least 150 kDa and a degree of substitution (of ligand binding density) of at least 5% such as at least 10%, preferably at least 20% in case of overall binding ligand density and preferably at least 10% in case of lysosomal trafficking receptor binding ligand or extracellular target protein binding ligands. In a preferred embodiment, the molecular weight of the hydrophilic scaffold is at least 40 kDa such as at least 70 kDa, at least 150 kDa, at least 550 kDa, at least 2150 kDa. The advantage of such a molecular weight combined with an optimal degree of substitution is that it results in a multivalent display of both types of ligands that increases the avidity of the ternary complex between the hydrophilic polymer backbone, target protein and lysosomal trafficking receptor, and thereby increases the efficiency of target protein degradation. The examples as used herein reveal that enhancing the lysosomal trafficking receptor binding ligand density and extending the hydrophilic polymer scaffold length leads to an improvement in binding avidity between cell surface lysosomal receptor and the trafficking chimera according to the invention.

[0069] In the context of the present invention, the hydrophilic polymer scaffold, can additionally be modified with various functional groups, signalling molecules, or incorporated with bioactive molecules to enhance their biocompatibility, mechanical properties, and specific biological functions.

[0070] The trafficking chimeras according to the invention mediate ternary complex formation between a target protein of interest (POI) for degradation and a lysosomal shuttling receptor on a cell surface. This connection directs the POI into the lysosomes for degradation. The trafficking chimeras according to the invention consist of multiple copies of synthetic small molecule ligands for both the POI and the lysosomal shuttling receptor, arrayed on the backbone of a hydrophilic polymer scaffold. The formation of a ternary complex involving the trafficking chimeras, the POI, and the lysosomal shuttling receptor initiates endocytosis, transporting the POI into lysosomes where it is subsequently degraded through lysosomal proteolysis.

[0071] Accordingly, the hydrophilic polymer scaffold of the invention can be conjugated with at least one, such as at least two, three, four, ... different lysosomal trafficking receptor binding ligands. As used herein, the term “lysosomal trafficking receptor binding ligand” is to be understood as a molecule that binds a lysosomal trafficking receptor and mediates subsequent lysosomal trafficking and degradation of a target protein. Lysosomal trafficking receptors play a crucial role in directing cargo molecules to lysosomes for degradation or recycling, thereby regulating various cellular processes such as protein turnover, nutrient sensing, and cellular homeostasis. Lysosomal trafficking receptor binding ligands can be natural or synthetic compounds that selectively interact with these lysosomal receptors, either enhancing or inhibiting their function. Thus, the lysosomal trafficking receptor binding ligands described herein may bind to any suitable lysosomal trafficking receptor. For example, ligands that can be applied herein include Mannose-6-Phosphate (M6P) and (triantennary) N-Acetyl Galactosamine (GalNAc), which bind the Cation-Independent Mannose-6-Phosphate Receptor (CI-M6PR) and ASiaGlycoProtein Receptor (ASGPR), respectively. Other non limiting examples of to which the lysosomal trafficking receptors binding ligand can bind include the lysosome-associated membrane glycoprotein (LAMP), monodansylcadaverine (MDC), sortilin, folate receptor, IFN-induced transmembrane protein 3 (IFITM3), molecules in the endosome / lysosome pathway (e.g., LIMP-1 , LIMP-2), lysosomal acid lipase receptor (LALR), FcRn, integrins, LRP1 , LDL receptors, transmembrane E3 ligases (RNF43, ZNRF3), cytokine receptors (CXCR7, IL2R) etc. It is appreciated that one skilled in the art is aware of which ligand can be suitably used to have affinity or bind to a specific lysosomal trafficking receptor.

[0072] Further, the hydrophilic polymer scaffold of the invention can be conjugated with at least one, such as at least two, three, four, ... different extracellular target protein binding ligands. As used herein, the term “extracellular target protein binding ligand” is to be understood as a molecule that binds but not necessarily blocks the target protein of interest (POI). Since the hydrophilic polymer scaffold with its conjugated lysosomal trafficking receptor binding ligand traffics target proteins to the lysosomal degradation pathway, a suitable target protein binding ligand does not necessarily have to bind the active site of the target protein. This opens up a much wider set of ligands, including ligands that bind undruggable targets. Extracellular target proteins of interest are located outside of cells, typically bound on the cell surface or within the extracellular matrix (i.e. soluble). Extracellular target protein binding ligands according to the invention can be naturally occurring molecules, such as hormones, growth factors, cytokines, or antibodies, or they can be synthetic compounds designed for specific interactions with target proteins. In a particular embodiment, the trafficking chimeras according to the invention are synthetic small molecule ligands that can bind the POI providing the advantage that multiple copies of such small molecules can be conjugated to the backbone. In a particular embodiment, the small molecule ligands according to the invention are molecules having a molecular weight of less than 60 kDa, in particular nanobodies, scFv, Fab fragments and the alike. The ligands according to the invention in general possess high specificity and affinity for their target proteins, enabling them to be trafficked to the lysosome to be degraded.

[0073] The extracellular target protein may be any target protein molecule which is desired for targeted degradation via the lysosomal pathway. In some embodiments, the extracellular target protein is a cell surface molecule such as a cell surface receptor or a soluble target protein present in the extracellular matrix. Cell surface receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, a receptor in the epidermal growth factor receptor (EGFR) family (e.g., HER2 (human epidermal growth factor receptor 2), etc.), a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the platelet derived growth factor receptor (PDGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor in the discoidin domain receptor (DDR) family, and a mucin protein (e.g., MUC1). Within the context of oncology disease indications, the extracellular target proteins may be immunosuppressive immune checkpoint receptors and / or their respective ligands such as but not limited to PD1 , PDL1 , CTLA4, TIGIT, TIM3, LAG3, SIRPalpha. Within the context of inflammatory disease indications, the extracellular target proteins may be immune stimulatory receptors and / or their respective ligands TNFR1 , TNFR2, IL12, IL23, IL12R, IL6, IL6R, IL4, IL4R, IL13.

[0074] In some embodiment, the extracellular target protein is a soluble target protein. As used herein, soluble target proteins are proteins that reside, are secreted or released into the extracellular space and are accessible in bodily fluids such as blood, plasma, serum, urine, or cerebrospinal fluid. Soluble target proteins of interest include, but are not limited to (i) cytokines such as interleukins (e.g., IL-6, IL-10), interferons (e.g., IFN-a, IFN-y), tumor necrosis factors (e.g., TNF-a), chemokines (e.g., CXCL8, CCL2); (ii) growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-p), (iii) hormones such as insulin, cortisol, thyroid hormones, reproductive hormones (e.g., estrogen, progesterone, testosterone); (iv) enzymes such as troponin, creatine kinase, alanine aminotransferase, aspartate aminotransferase, amylase, lipase, (v) antibodies, (vi) proteases and protease inhibitors.

[0075] In a particular embodiment, herein provided is a hydrophilic polymer scaffold such as dextran, preferably a dextran with a molecular weight of at least 40 kDa, which is conjugated with multiple copies, preferably at least 5 copies, of synthetic small-molecule ligands for the cation-independent mannose-6-phosphate receptor (CI-M6PR) as the lysosomal trafficking receptor binding ligand, and multiple copies, preferably at least 5 copies, of an extracellular target protein binding ligand thereby being able to traffic a target protein of interest to the lysosome for degradation. In another embodiment, the scaffold is conjugated with multiple copies, preferably at least 5 copies, of synthetic small-molecule ligands for the asialoglycoprotein (ASGPR) receptor as the lysosomal trafficking receptor binding ligand.

[0076] In a particular embodiment, the lysosomal trafficking receptor and / or extracellular target protein can be a cell surface molecule expressed on the cell membrane of an immune cell. In a particular embodiment, the lysosomal trafficking receptor and extracellular target protein are expressed by the same immune cell. In some embodiments, the lysosomal trafficking receptor and / or extracellular target is present on an immune cell selected from a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and an eosinophil.

[0077] In a particular embodiment, the extracellular target protein present on the immune cell is an inhibitory immune receptor. As used herein, an “inhibitory immune receptor” is a receptor present on an immune cell that negatively regulates an immune response. In another particular embodiment, the cell surface molecule present on the immune cell is an immune checkpoint molecule. Non-limiting examples of immune checkpoint molecules to which the extracellular target protein binding ligand may specifically bind include PD-1 , PD-L1 , CTLA4, TIM3, LAG3, TIGIT, etc.

[0078] In another embodiment, the lysosomal trafficking receptor and / or the extracellular target protein is a cell surface molecule expressed on the cell membrane of a cancer cell. In a particular embodiment, the lysosomal trafficking receptor and extracellular target protein are expressed by the same cancer cell. In the context of the present invention, the term “cancer cell” may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semisolid tumor, a hematological malignancy (e.g., a leukemia cell, a lymphoma cell, a myeloma cell, etc.), a primary tumor, a metastatic tumor, and the like. In some embodiments, the extracellular target protein present on the cancer cell is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the extracellular target protein is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.

[0079] In a particular embodiment, the compound according to the invention further comprises at least one further tissue-specific targeting ligand that is conjugated to the scaffold in addition to the lysosomal trafficking receptor binding ligand and the extracellular target protein binding ligand as described herein. The compound according to the invention may comprise a further small molecule ligand that binds to a protein that is selectively or highly expressed by certain immune or cancer cells. Non-limiting examples of small molecule ligands are ligands that bind to carbonic anhydrase IX (CAIX) and Fibroblast Activation Protein (FAP) or Prostate-specific membrane antigen (PSMA), folate receptor alpha.

[0080] In a particular embodiment, the trafficking chimeras according to the invention consist of multiple copies of synthetic small molecule ligands for both the POI and the lysosomal shuttling receptor, arrayed on the backbone of a hydrophilic polymer scaffold. The formation of a ternary complex involving the trafficking chimeras, the POI, and the lysosomal shuttling receptor initiates endocytosis, transporting the POI into lysosomes where it is subsequently degraded through lysosomal proteolysis.

[0081] In a specific embodiment, the trafficking chimera is a hydrophilic polymer scaffold, in particular a dextran scaffold, conjugated with a suitable lysosomal trafficking receptor binding ligand such as M6P and / or GalNAc. In another specific embodiment, the conjugated M6P ligand is present in at least 2 copies such as at least 3, 4, 5, 10, 20, 50, 100, 250, 500, 750, 1000 or more copies of M6P ligand, preferably at least 5 copies to provide sufficient binding avidity towards POI and / or lysosomal trafficking receptor. In some embodiments, the copies of conjugated M6P ligand include from 1 to 1000 copies, such as from 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 75, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 copies. In an embodiment wherein the conjugated ligand is GalNAc, it is preferably present in at least 2 copies such as at least 3, 4, 5, 10, 20, 50, 100, 250, 500, 750, 1000 or more copies of GalNAc ligand. In some embodiments, the copies of conjugated GalNAc ligand include from 1 to 1000 copies, such as from 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 75, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 copies.

[0082] In another particular embodiment, the compound is a hydrophilic polymer scaffold, in particular a dextran scaffold, conjugated with a suitable extracellular target protein binding ligand wherein said ligand is present in at least 2 copies such as at least 3, 4, 5, 10, 20, 50, 100, 250, 500, 750, 1000 or more copies of extracellular target protein binding ligand, preferably at least 5 copies. In some embodiments, the copies of conjugated extracellular target protein binding ligand include from 1 to 1000 copies, such as from 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 75, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10 copies.

[0083] In a further embodiment, the present invention provides a compound comprising a macromolecular hydrophilic polymer scaffold conjugated with at least one lysosomal trafficking receptor binding ligand and at least one extracellular target protein binding ligand, wherein each of said binding ligands is present in at least 5 copies, and wherein said extracellular target protein binding ligand is different from said lysosomal trafficking receptor binding ligand.

[0084] Macromolecular hydrophilic polymer scaffolds disclosed in the prior art that have the purpose as drug delivery systems may be conjugated with multiple copies of e.g. GalNAc residues and act as a lysosomal trafficking receptor binding ligand (affinity for ASGPR) as well as an extracellular target protein binding ligand since certain hepatocytes express ASGPR. A disadvantage is that there is aspecific binding of the ligand residues leading to suboptimal trafficking as well as suboptimal binding of target proteins. Further, such scaffolds are only able to traffic extracellular target proteins that also have affinity for GalNAc (such as for example cells that express ASGPR). In contrast, the lysosomal trafficking receptor binding ligands of the scaffold of the present invention have the sole purpose to traffic the extracellular target protein to the lysosome, while the extracellular target protein binding ligands have the sole purpose of capturing POIs, without any binding affinity interference.

[0085] Within the context of the invention, it will also be understood that the chosen extracellular target protein binding ligands are different types of ligands compared to the chosen lysosomal trafficking receptor binding ligands. With the term “different types of ligands”, it is meant to be that both types of ligands present on the scaffold do not have the same binding affinity to a specific target molecule. In other words, the chosen extracellular target protein binding ligand has essentially no or minor affinity for the chosen lysosomal trafficking receptor and vice versa.

[0086] For example, and in one embodiment, this means that when M6P is used as a lysosomal trafficking receptor binding ligand in the scaffold, the extracellular target protein binding ligand has no or minor affinity for CI-M6PR. Alternatively, and in another embodiment, when GalNAc is used as a lysosomal trafficking receptor binding ligand in the scaffold, the extracellular target protein binding ligand has no or minor affinity for ASGPR. In one embodiment, it may however be the case that when M6P is used as a lysosomal trafficking receptor binding ligand in the scaffold, the extracellular target protein binding ligand has affinity for ASGPR and vice versa (GalNAc as lysosomal ligand and extracellular ligand has affinity for M6PR).

[0087] Accordingly, the binding ligands according to the invention in general possess high specificity and affinity for their target proteins, enabling them to form a ternary complex and to be trafficked in a focused manner to the lysosome to be degraded. The extracellular target protein may be any target protein molecule which is desired for targeted degradation via the lysosomal pathway but does not have affinity for the chosen lysosomal trafficking receptor binding ligand of the scaffold.

[0088] It will be appreciated that it is this multivalent character of the combination of multiple lysosomal trafficking receptor binding ligands combined with multiple extracellular target protein binding ligands that is particular advantageous for increasing binding efficiency and forming of the ternary complex between the hydrophilic polymer backbone, target protein and lysosomal trafficking receptor, thereby increasing the efficiency of target protein degradation. In particular, the binding probability of the POI with the extracellular target protein receptor increases vastly due to the presence of an entire series of ligand types for the same molecule that are able to replace or aid in reforming a bond in case one or more bonds are lost with the POI preventing the POI to completely diffusing away.

[0089] In a particular embodiment, the lysosomal trafficking receptor binding ligands and the extracellular target protein binding ligands are positioned randomly on the polymer scaffold. In the context of the present invention, it is understood that randomly positioned means that each of the ligand or copies thereof are distributed in a manner where their placement lacks any discernible pattern or regularity and are arranged on the polymer scaffold such that a ternary complex can be formed between the binding ligands, the target protein and the lysosomal receptor. In various contexts, "randomly positioned" implies that the location of each ligand or copies thereof within a given space is determined by chance rather than any predetermined arrangement or systematic organization. The advantage of such randomly distribution results in a stronger ternary complex formation while maintaining the hydrophilic property of the polymer scaffold.

[0090] In a further embodiment, the ligands are attached to the hydrophilic polymer scaffold via linkers, in particular short, hydrophilic, non-degradable linkers. Non-limiting examples of linkers include ether linkers, ester linkers, peptide linkers, amide linkers, maleimide or maleimide-based linkers; Succinimidyl- 4-(A / -maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) linkers; vinylsulfone-based linkers; linkers that include polyethylene glycol (PEG), such as, but not limited to tetraethylene glycol; linkers that include propanoic acid; linkers that include caproleic acid, and linkers including any combination thereof.

[0091] In a further embodiment, the present invention provides a pharmaceutical composition comprising the compound according to the invention and a pharmaceutically acceptable excipient. A "pharmaceutical composition" refers to a composition formulated in pharmaceutically-acceptable or physiologically- acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically-active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0092] In a particular embodiment, the present invention provides a compound or pharmaceutical composition according to the invention for use in human and / or veterinary medicine.

[0093] In a specific embodiment, the present invention provides a compound or pharmaceutical composition according to the invention for use in the treatment and / or prevention of oncology, inflammatory and / or autoimmune diseases.

[0094] As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0095] As used herein, "prevent," and similar words such as "prevented," "preventing" or "prophylactic" etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition. In a particular embodiment, the trafficking chimera according to the invention can be administered in a concentration ranging from about and between 1 nM to about 2000 nM, about and between 5 nM to about 1500 nM, about and between 10 nM to about 1000 nM, about and between 50 nM to about 750 nM, about and between 100 nM to about 500 nM, and more in particular about and between 15 nm to about 150 nM. It will be appreciated that one skilled in the art can adjust the concentration of the trafficking chimera or the pharmaceutical composition of the invention to an effective amount, or in an effective amount with respect to pg dose, to achieve a particular effect.

[0096] As used herein, the term "amount" refers to "an amount effective" or "an effective amount" of a trafficking chimera of the pharmaceutical composition according to the invention to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0097] In even a further embodiment, the present invention provides a method, in particular an in vivo method, of degrading a target protein of interest comprising: contacting the compound or the pharmaceutical composition according to the invention, with the target protein of interest and the lysosomal trafficking receptor of a cell membrane under conditions in which the lysosomal trafficking receptor of said cell shuttles said target protein to the lysosome of said cell for degradation.

[0098] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation ortransplantation. In a preferred embodiment, compositions are administered parenterally. The phrases "parenteral administration" and "administered parenterally" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0099] In yet a specific embodiment, the compound or pharmaceutical composition according to the invention can be used in targeted protein degradation. More specifically, the compound or pharmaceutical composition according to the invention can be suitably used to degrade antibodies to which the extracellular target protein binding ligand is directed to. As shown in the example section described herein, the trafficking chimera mediates potent and selective ternary complex formation between aDNP antibody and CI-M6PR showing its binding multivalency.

[0100] In another aspect of the present invention, there is provided a compound comprising a linear and / or branched macromolecular hydrophilic polymer scaffold conjugated with at least one extracellular target protein binding ligand and at least one further protein binding ligand. In essence, all embodiments as previously described are applicable to this aspect of the invention unless otherwise specified. For example, in a particular embodiment, each of said binding ligands is present in at least 2 copies, preferably at least 5 copies and said hydrophilic polymer has a molecular weight of at least about 40 kDa. Accordingly, the only difference compared to the compound wherein a lysosomal trafficking receptor ligand is conjugated to the polymer scaffold is that, here, at least one further protein binding ligand is conjugated to the scaffold which is not limited to a lysosomal trafficking receptor. This aspect of the invention has the advantage that such a compound is particularly suitable for target protein recruitment (TPR) or cell recruitment instead of trafficking for lysosomal degradation only. As exemplified in Example 3, AAZ / FITC / dextrans were manufactured demonstrating a proof-of-principle that the current platform is able to specifically recruit anti-FITC antibodies (Example 3.1) or effector cells such as CAR- T cells expressing those antibodies (Example 3.2).

[0101] Within the context of this aspect, it will be understood that the term “one further protein binding ligand” may refer to, and encompasses a ligand suitable to bind either a lysosomal trafficking receptor, or a target protein presented on a target cell such as a cancer cell of immune cell. It will also be appreciated that in case the “further protein binding ligand” is a ligand having affinity for a lysosomal trafficking receptor, the extracellular target protein binding ligand has affinity to a protein to be degraded. Alternatively, it will also be appreciated that in case the “further protein binding ligand” is a ligand having affinity for a target cell protein such as for example a target protein presented on a cancer or immune cell, the extracellular target protein binding ligand has affinity to an effector protein such as for example an antibody or an effector cell such as for example a CAR-T cell.

[0102] Accordingly in the latter case, the macromolecular hydrophilic polymer scaffold can be conjugated with (i) an array of extracellular target protein binding ligands that are able to recruit an effector protein such as for example an antibody or an effector cell; and (ii) an array of further protein binding ligands that are suitable to bind a target cell protein such as for example a target protein of a cancer or immune cell. In this way, the effector protein or effector cell is specifically directed to the target cell and can induce a biological response such as for example inducing innate immune killing of a cancer cell or activating or stimulating an immune cell.

[0103] Extracellular target protein binding ligands according to this aspect of the invention can be naturally occurring molecules, such as hormones, growth factors, cytokines, or antibodies, or they can be synthetic compounds designed for specific interactions with target proteins, in particular synthetic small molecule ligands, nanobodies, scFv Fab fragments or the alike, and may further include galactose-a- 1 ,3-galactose (a-Gal), rhamnose, nitroarenes, phosphorylcholine or (cyclic) peptides that bind IgG antibodies. Non-limiting examples of further protein binding ligands can be fibroblast-activation protein inhibitors (FAPI) or carbonic anhydrase IX inhibitors (CAIXI) such as for example acetazolamide (AAZ; also abbreviated herein as AZA), folic acid to bind folate receptor alpha, ACUPA to bind PSMA, cyclic RGD that binds avp3 integrin. In Example 3.1 , a selective association of FITC / AAZ-dextran to CAIX+ target cells and ternary complex formation between CAIX+ cells, FITC / AAZ-dextran and aFITC antibody has been demonstrated. Accordingly, these multivalent scaffolds provide a strong ternary complex between an antibody effector protein and a targeted cell protein presented on a cancer cell or an immune cell and thus are particularly suitable in respectively cancer or immune therapy.

[0104] In a specific embodiment, due to its multivalent structure, a scaffold comprising an array of FAPI as the further protein binding ligand binds to FAP which is overexpressed on the cell surface of a cancer cell, while the array of DNP binds to aDNP antibodies thereby mediating potent and selective ternary complex formation between aDNP antibody and FAP. Alternatively, a scaffold comprising an array of AAZ (or abbreviated as AZA) as the further protein binding ligand binds to CAIX which is overexpressed on the cell surface of a cancer cell, while the array of DNP binds to aDNP antibodies thereby mediating potent and selective ternary complex formation between aDNP antibody and CAIX. As such, the Fc domain of aDNP antibody induces innate immune killing of cancer cells (e.g. complement activation NK cell and macrophage recruitment).

[0105] In yet a further embodiment, this aspect of the invention can also suitably be used in the targeted recruitment of effector cells, and more in particular in CAR-T cells CAR NK cell, or CAR macrophage cells. In a specific embodiment, the macromolecular hydrophilic polymer scaffold can be conjugated with (i) an array of extracellular target protein binding ligands such as for example fluorescein isothiocyanate (FITC) thereby being able to recruit an effector protein anti-FITC which is present on CAR T-cells; and (ii) an array of further protein binding ligands such as for example AAZ (or abbreviated as AZA). In Example 3.2, successful killing of SK-RC-52 cells by anti-FITC scFv CAR T cells, in presence of FITC / AZA-dextran, but not in presence of control dextran constructs lacking FITC or AZA, respectively was demonstrated.

[0106] Accordingly, these multivalent scaffolds provide a strong ternary complex between an effector cell such as a CAR-T cell and a targeted cell protein presented on target cell. In a particular embodiment, the target cell can be a cancer cell or an immune cell, ... The advantage is that the effector cell such as a CAR T-cell activates and releases cytotoxic factors in the vicinity of a cancer cell and is thus particularly suitable in targeted cancer therapy for example in cancers such as glioblastoma, colorectal, breast cancer, renal cell carcinoma, etc. Alternatively, the effector cell can activate and releases immunomodulating or stimulating factors in the vicinity of an immune cell and is thus particularly suitable in targeted immune therapy, immuno deficiency therapy, or auto-immune disease therapy.

[0107] In this embodiment for targeted recruitment of effector cells, non-limiting examples of extracellular target protein binding ligands are FITC, DNP, biotin, FLAG peptide; while non-limiting examples of further protein binding ligands are FAPI or carbonic anhydrase IX inhibitors (CAIXI) such as for example acetazolamide (AAZ, or abbreviated as AZA), folic acid to bind folate receptor alpha, cyclic RGD to bind avp3 integrin; or ligands able to bind CD19, CD20, BCMA, BAFF, IL-23R, CD38, CD8, CD4; or ligands able to bind HIV Env glycoprotein which are present on infected cells.

[0108] In another embodiment, the target protein is a cell surface molecule expressed on the cell membrane of a cancer cell or an immune cell. In some embodiments, the target protein present on the cell membrane of the cancer cell can be, but is not limited to, CAIX (Carbonic Anhydrase IX), PSMA, uPAR, folate receptor, and avp3 integrins, HER2 / neu (ERBB2), EGFR (ERBB1), VEGFR, FGFR, CD20, CD19, CD22, CD33, CD38, CD52, CD70, CD123, PD-L1 (CD274), PD-L2 (CD273), CTLA-4, EpCAM (CD326), N-cadherin, ICAM-1 , VCAM-1 , MUC1 , CEA, PSA, CA-125, GD2, MDR1 (P-glycoprotein, ABCB1), BCRP (ABCG2), MMPs (Matrix Metalloproteinases), ALK (Anaplastic Lymphoma Kinase), BRAF, Mesothelin.

[0109] In some other embodiments, the target protein present on the cell membrane of the immune cell can be, but is not limited to, CD3, CD4, CD8, CD14, CD19, CD20, CD28, CD40, CD45, CD56, TLR2, TLR4, TLR7 / 8, TLR9, MHC Class I, MHC Class II, ICAM-1 (CD54), VCAM-1 (CD106), IL-2R (CD25), IL-4R, IFN-yR, CTLA-4, PD-1 , CD40L (CD154), Fc Receptors (FcRs), Integrins (e.g., LFA-1 , VLA-4), CCR5, CXCR4.

[0110] In another embodiment, the effector cell can be an endogenous cell such as a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, and a eosinophil or a chimera thereof such as a CAR T cell, a CAR B cell, a CAR natural killer (NK) cell, a CAR macrophage, a CAR monocyte, a CAR neutrophil, a CAR dendritic cell, a CAR mast cell, a CAR basophil, and a CAR eosinophil.

[0111] In these specific embodiments, the binding ligands and copies thereof are also arranged in a random manner on the polymer scaffold such that a ternary complex can be formed between the binding ligands and the target protein of interest. In a particular embodiment, the ratio of the further protein binding ligand to the extracellular target protein binding ligand conjugated to the polymer scaffold ranges from about 1 :20 to about 20:1 , in particular about 1 :10 to about 10:1 , more in particular about 1 :5 to about 5:1 , even more in particular about 1 :3 to about 3:1 , most in particular 1 :1.

[0112] Thus, the present invention also provides a compound comprising a macromolecular hydrophilic polymer scaffold, in particular a linear and / or branched scaffold, conjugated with at least one extracellular target protein binding ligand and at least one further protein binding ligand suitable for binding i) a lysosomal trafficking receptor; or ii) a target protein presented on a cancer cell or immune cell; wherein said extracellular target protein binding ligand is different from said further protein binding ligand. In a specific embodiment, each of said extracellular target protein binding ligand or further protein binding ligand is present in at least 2 copies, preferably at least 5 copies. In another embodiment, said hydrophilic polymer has a molecular weight of at least about 40 kDa.

[0113] Within the context of this aspect of the invention, it will also be understood that the chosen extracellular target protein binding ligands are different types of ligands compared to the chosen further protein binding ligand. With the term “different types of ligands”, it is meant to be that both types of ligands present on the scaffold do not have the same binding affinity to a specific target molecule. In other words, the chosen extracellular target protein binding ligand has essentially no or minor affinity for the chosen further protein and vice versa.

[0114] Thus, the compound or pharmaceutical composition according to this aspect of the invention can be used in targeted recruitment of bioactive proteins and (CAR-)T cells.

[0115] The compounds of each aspect of the invention can be prepared according to the reaction schemes provided in the examples hereinafter, but those skilled in the art will appreciate that these are only illustrative for the invention and that the compounds of this invention can be prepared by any of several standard synthetic processes commonly used by those skilled in the art of organic chemistry.

[0116] The invention will now be illustrated by means of the following synthetic and biological examples, which do not limit the scope of the invention in any way. EXAMPLES

[0117] EXAMPLE 1 - LYSOSOMAL DEGRADATION

[0118] MATERIALS AND METHODS

[0119] Materials

[0120] Unless otherwise specified, all materials were procured from commercial providers.

[0121] Instrumentation

[0122] All NMR spectra were acquired using a Bruker 300 / 400 / 500 MHz FT-NMR spectrometer and processed through ACDLabs Spectrus Processor (ACDLabs) and MestReNova (Bruker) software. Chemical shifts (6) were reported in ppm relative to TMS. Samples were prepared in D2O or DMSO-d6, and their signals were referenced to the residual non-deuterated signals of the solvent. Flow cytometry analysis was conducted on a BD Accuri C6 (BD Biosciences), and the data were analyzed using FlowJo software. Confocal microscopy images of 2D cell cultures were captured using a Leica DMI6000 B inverted microscope equipped with an oil immersion objective (Leica, 63x, NA 1 .40) and connected to an Andor DSD2 confocal scanner. Image processing was performed using Imaged software. Western blot imaging was performed on a iBright CL750 imaging system (Thermo Fisher Scientific) and analyzed by iBright Analysis and Imaged software.

[0123] Cell culture durkat cells (human T lymphocyte cell line) were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics (50 units / mL penicillin and 50 pg / mL streptomycin). The cells were incubated at 37 °C in a controlled, sterile environment with 95% relative humidity and 5% CO2.

[0124] SK-RC-52 cells were cultured in RPMI, and HT-1080.hFAP cells were cultured in DMEM, supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics (50 units / mL penicillin and 50 pg / mL streptomycin). The cells were incubated at 37 °C in a controlled, sterile environment with 95% relative humidity and 5% CO2.

[0125] Chemical Synthesis

[0126] DNP-PEGa-azide

[0127] In a round-bottom flask, Alpha-amino-omega-azido octa(ethylene glycol) (1.23 mmol) and 1-Chloro-2,4- dinitrobenzene (1.47 mmol) were dissolved in 25 mL of dichloromethane (DCM). Subsequently, triethylamine (3.07 mmol), and the reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. Following solvent evaporation, the crude product was purified by silica gel chromatography using DCM as the eluent. Post-purification, a yellow oil was obtained with a yield of 90%, and analyzed by NMR and LC-MS (ESI).

[0128] 1H-NMR (400 MHz, CDCI3) 6 9.14 (d, J = 2.6 Hz, 1 H; -C(NO2)-CH=C(NO2)-C(NH-CH2-R)=CH-CH=), 8.80 (s, 1 H; -C(NO2)-CH=C(NO2)-C(NH-CH2-R)=CH-CH=), 8.27 (dd, J = 9.5, 2.6 Hz, 1 H; -C(NO2)- CH=C(NO2)-C(NH-CH2-R)=CH-CH=), 6.96 (d, J = 9.5 Hz, 1 H; -C(NO2)-CH=C(NO2)-C(NH-CH2-R)=CH- CH=), 3.83 (t, J = 5.3 Hz, 2H; PEG), 3.71 - 3.58 (m, 32H; PEG), 3.38 (t, J = 5.1 Hz, 2H; PEG). LC-MS (ESI): calcd for C24H40N6OI2604.27, found 622.30 [M+NH4]+

[0129] Alkyne-functionalization of dextran

[0130] Dextran (0.030 mmol) was dissolved 0.5M sodium hydroxide (NaOH) solution at a dextran concentration of 100 mg / mL. The solution underwent nitrogen bubbling for 15 minutes before adding 0.44 or 0.88 equivalents of glycidyl propargyl ether (GPE) relative to the amount of glucose units, to achieve a DS of 10 and 20, respectively. The 4.4-fold excess of GPE was employed to compensate for GPE hydrolysis in basic aqueous conditions. Subsequently, the solution was purged with nitrogen and stirred for 24 hours at 50°C. The resulting product underwent dialysis against water (MWCO 3.5 kDa) for four days. Following freeze-drying, a white solid was obtained and subjected to NMR analysis.

[0131] Cy5-dextran

[0132] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Dextran-alkyne (150 kDa dextran;alkyne DS20) (0.006 mmol), tris(1-benzyl-1 H-1 ,2,3-triazol-4-yl-methyl)amine (TBTA; 2 equivalents relative to the amount of CuSO4), and sodium ascorbate (3 equivalents relative to the amount of CuSO4) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, Cy5-azide (0.006 mmol) and CuSO4(0.65 equivalents relative to the amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a blue solid was obtained and subjected to NMR analysis.

[0133] Cy5 / DNP-dextran

[0134] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Dextran-Cy5-alkyne (0.002 mmol), TBTA (2 equivalents relative to the amount of CuSO4), and sodium ascorbate (3 equivalents relative to the amount of CuSO4) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, DNP-PEGs-azide (0.5 equivalents relative to the amount of dextran-bound alkyne) and CuSO4(0.65 equivalents relative to the amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a green-to-yellow solid was obtained and subjected to NMR analysis.

[0135] Cy5 / DNP / M6P-dextran (DEXTRACs)

[0136] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Dextran-Cy5-alkyne (0.002 mmol), TBTA (2 equivalents relative to the amount of CuSO4), and sodium ascorbate (3 equivalents relative to the amount of CuSO4) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, M6P-PEG3-azide (1 equivalent relative to the remaining amount of dextran-bound alkyne) and CuSC (0.65 equivalents relative to the remaining amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a green-to-yellow solid was obtained and subjected to NMR analysis.

[0137] Synthesis of N1-(26-azido-3,6,9, 12, 15, 18,21,24-octaoxahexacosyl)-N4-(5-sulfamoyl-1,3,4-thiadiazol-2- yl)succinamide (AAZ-PEG8-N3)

[0138] 4-oxo-4-((5-sulfamoyl-1 ,3,4-thiadiazol-2-yl)amino)butanoic acid (0.0714 mmol, 1 equiv) and Hatu (0.0928 mmol, 1 .3 equiv) were weighed into a vial and dissolved then in anhydrous DMF. Subsequently, DIPEA (0.2854 mmol, 4 equiv) was added to the mixture and stirring 10 min at room temperature. 26- azido-3,6,9,12,15,18,21 ,24-octaoxahexacosan-1 -amine (0.1142 mmol, 1.6 equiv) were weighed and dissolved in anhydrous DMF and then added to the mixture and reacted for 3 hours. The crude was firstly evaporated on vacuum and then purified on TLC perspective (DCM:MEOH = 10:1). The product was collected and colored in white and yield 70%. The product was measured on NMR and LC-MS.

[0139] 1 H NMR (400 MHz, CDCI3) 6 12.82 (s, 1 H), 7.31 (s, 1 H), 7.16 (s, 2H), 3.79 - 3.34 (m, 36H), 2.98 (s, 2H), 2.76 (t, J = 7.6 Hz, 2H).

[0140] LCMS: [M+H]+= 701 ,26 (theoretical); found = 701 ,40

[0141] Flow cytometry analysis of cellular D EXTRAC and anti-DNP antibody association

[0142] Jurkat cells were seeded in U-bottom 96-well plates at a concentration of 150 000 cells in 89 pL RPMI per well. To each well, 1 pL of human Fc blocking reagent was added to prevent nonspecific binding of anti-DNP antibodies. After a 30-minute incubation at 37°C with 5% CO2, AF488-labeled anti-DNP antibodies were added to achieve concentrations of 0, 5, 10, 50, or 100 nM per well. Concurrently with the addition of anti-DNP antibodies, DEXTRACs were added to reach concentrations of 0, 1 , 5, 10, 50, 100, 500, 1000, or 5000 nM and were then incubated for 24 hours at 37°C with 5% CO2. All conditions were tested in triplicate. After the 24-hour incubation, cells were washed twice and resuspended in PBS. The AF488 and Cy5 signals were measured by flow cytometry.

[0143] In a subsequent experiment, Jurkat cells were seeded in U-bottom 96-well plates at a concentration of 150 000 cells in 89 pL RPMI per well. To each well, 1 pL of human Fc blocking reagent was added to prevent nonspecific binding of anti-DNP antibodies, along with PBS or M6P to achieve a concentration of 10 mM. After a 30-minute incubation at 37°C with 5% CO2, AF488-labeled anti-DNP antibodies were added to achieve a concentration 10 nM per well. Concurrently with the addition of anti-DNP antibodies, DEXTRAC 150 kDa DS 20, or its respective control (containing DNP but lacking M6P) was added to reach a concentrations of 10 nM and then incubated for 24 hours at 37°C with 5% CO2. All conditions were tested in triplicate. After the 24-hour incubation, cells were washed twice and resuspended in PBS. The AF488 and Cy5 signals were measured by flow cytometry.

[0144] Flow cytometry analysis of free M6P ligand competition experiments

[0145] Jurkat cells were seeded in U-bottom 96-well plates at a concentration of 150 000 cells in 90 pL RPMI per well. Subsequently, 0, 0.1 , 1 , 10, 100, 1000 or 10 000 pM free M6P was added, and the cells were incubated for 30 minutes at 37°C with 5% CO2. Following this, Cy5-labeled 40 kDa DS 10 or 150 kDa DS 20 DEXTRAC was added to achieve a well concentration of 1 , 5, 10, 50, 100, 500 or 1000 nM. The cells were then incubated for 24 hours at 37°C with 5% CO2. All conditions were tested in triplicate. After the 24-hour incubation, cells were washed twice and resuspended in PBS. Subsequently, the Cy5 signal was measured using flow cytometry.

[0146] Flow cytometry analysis of free DNP ligand competition experiments

[0147] Biotinylated anti-DNP antibody was immobilized on SVP-40-5 streptavidin beads following the manual instructions and subsequently aliquoted into microcentrifuge tubes, resulting in 500 000 beads per sample. After centrifugation (10 minutes at 6 000 g), the beads were resuspended in 200 pL of PBS containing 0.05, 0.5, 5, 50, 500, or 5000 pM DNP-PEGs-azide, along with 500 nM of DEXTRAC 40 kDa DS 10 or 50 nM DEXTRAC 150 kDa DS 20, and incubated for 30 minutes at room temperature with stirring. Following the incubation, the beads were centrifuged and washed three times with 1 mL of PBS, after which they were resuspended in 200 pL of fresh PBS. All conditions were tested in triplicate. The binding of Cy5-labeled DEXTRAC to the beads was assessed by flow cytometry.

[0148] Confocal microscopy

[0149] Jurkat cells were seeded in U-bottom 96-well plates at a concentration of 150 000 cells in 90 pL RPMI per well. Subsequently, Cy5-labeled DEXTRAC and AF488-labeled anti-DNP antibody were added to achieve a concentration of 100 nM and co-incubated for 24 hours at 37°C with 5% CO2. Following the 24-hour incubation period, the cells were washed twice and resuspended in fresh RPMI. The freshly resuspended cells were then introduced into 35 mm Willco wells at a concentration of 20 000 cells in 200 pL RPMI per well, after which confocal microscopy was performed. For counterstaining of lysosomes, LysoTrackerRed was added to the cell culture medium according to the manufacturer’s instructions. Note that in the confocal microscopy experiment, which included lysosomal staining, no washing steps were performed to remove free AF488-labeled anti-DNP antibody.

[0150] Western blot analysis

[0151] For immunoprecipitation, Protein A / G magnetic beads were loaded into microcentrifuge tubes, to which 10 pg of anti-AF488 antibody was coupled according to the provided manual instructions. Subsequently, the beads were crosslinked to prevent co-elution of the coupled anti-AF488 antibody during the elution of AF488-labeled proteins captured from cell lysates.

[0152] Jurkat cells were seeded in microcentrifuge tubes at a concentration of 1 000 000 cells in 900 pL RPMI.

[0153] To each sample, 10 pL of human Fc blocking reagent was added to prevent nonspecific binding of anti- DNP antibodies, along with PBS or M6P to achieve a concentration of 10 mM. Following a 30-minute incubation at 37°C with 5% CO2, PBS or bafilomycin A1 (a lysosomal inhibitor) was added to achieve a concentration of 100 nM. Next, AF488-labeled anti-DNP antibodies were added to reach a concentration of 20 nM. Concurrently, with the addition of anti-DNP antibodies, DEXTRAC 150 kDa DS 20 or PBS was added to reach a concentration of 20 nM, and the mixture was incubated for 24 hours at 37°C with 5% CO2.

[0154] After the 24-hour incubation, cells were washed twice, and the cell suspension was collected, centrifuged, and cell lysates were prepared using RIPA lysis buffer containing Halt Protease Inhibitor Cocktail and EDTA, following the manual instructions. The resulting lysates were then added to a microcentrifuge tube containing crosslinked Protein A / G magnetic beads conjugated to anti-AF488 antibody for immunoprecipitation and subsequent elution of AF488-labeled protein fragments, in accordance with the manual instructions.

[0155] Each eluate containing AF488-labeled protein fragments was combined with 4x Laemmli Sample Buffer, and DTT was introduced to achieve a concentration of 10 mM. Subsequently, these samples underwent heating to 95°C for 10 minutes, were then loaded onto an SDS-polyacrylamide gel (with 4% and 12.5% acrylamide for the stacking and separating gel, respectively), and subjected to electrophoresis at 120V for 15 minutes, followed by 150V for 65 minutes. The proteins were then transferred to a 0.45 pm nitrocellulose membrane at 250 mA for 90 minutes. After blocking with 5% BSA in TBST buffer (20 mM Tris, 137 mM NaCI, pH 7.6, 0.1 % Tween 20) for 1 hour at room temperature, the membrane was incubated overnight with anti-AF488 antibody (diluted at 1 :500) in TBST buffer + 5% BSA at 4°C with gentle shaking. Subsequently, the membrane underwent three washes with TBST buffer and was then incubated for 1 hour with HRP-conjugated goat anti-rabbit IgG antibody (diluted at 1 :7500) in TBST buffer + 5% BSA at room temperature with gentle shaking. Finally, the membranes were washed three times with TBST buffer and incubated in Clarity Max Western ECL substrate for 2 minutes, followed by visualization with the iBright CL750 imaging system.

[0156] RESULTS

[0157] Synthesis of DEXTRACs by clicking azide-functionalized ligands to alkyne substituted dextrans

[0158] We designed DEXTRACs to present an array of synthetic small-molecule ligands for the cationindependent mannose-6-phosphate receptor (CI-M6PR), as cell surface lysosomal shuttling receptor, and anti-dinitrophenol antibodies (anti-DNP) as a model target protein of interest (POI) for degradation (Scheme 1). Notably, whereas the anti-DNP antibody has sufficient affinity to bind a single DNP motif, with an estimated affinity of approximately 10 nM, the multivalent display of DNP motifs has been reported to dramatically increase the avidity of binary complex formation. Due to the low affinity of M6P for the CI-M6PR, a multivalent presentation of multiple M6P copies clustered on a covalent carrier is necessary to achieve sufficient avidity for long-term binding to the CI-M6PR and subsequent triggering of lysosomal trafficking. To this end, we first modified dextrans with molecular weights (MW) of 40, 70, and 150 kDa, respectively, by reacting them with glycidyl propargyl ether in aqueous medium in presence of sodium hydroxide (NaOH) as a catalyst. We aimed for a degree of substitution (DS) - the percentage of glucose repeating units in the dextran backbone substituted with alkynes - of 10 or 20. This approach generates a small library of dextrans for further investigations into the impact of dextran chain length and ligand density on the efficiency of ternary complex formation. For each dextran,1H- NMR analysis indicated an alkyne DS consistent with the intended values of 10 or 20, as illustrated in Figure 2 and summarized in Table 1. Additionally, DOSY analysis revealed identical diffusion patterns (data not shown) for both dextran- and alkyne-associated protons, thereby confirming the attachment of the alkyne groups to the dextran backbone.

[0159] Scheme 1. DEXTRACs are synthesized via a CuAAC reaction between alkyne-substituted dextrans and azide-functionalized ligands. In the first step, dextrans are functionalized with alkynes by reacting with glycidyl propargyl ether in an aqueous medium, aiming for a degree of substitution (DS) of 10 and 20, respectively. In three consecutive reactions, the alkyne-functionalized dextrans are reacted in a waterDMSO mixture Cy5-azide, DNP-PEGs-azide, and M6P-PEG3-azide, targeting a 1 :1 DNP:M6P ratio. Note that although DNP is an acronym for dinitrophenol, it is an aniline derivative that is commonly used in the synthesis of DNP-conjugates.

[0160] Table 1. Chemical characterization and in vitro ternary complex characterization of DEXTRACs.

[0161] DEXTRAC DS alkynea DS DNPa DS M6Pa MW (Da)b AF488 fold-increasemaxc.

[0162] DEXTRAC DS alkyneaDS DNPaDS M6PaMW (Da)bAF488 fold-increasemaxc

[0163] 40 kDa DS 10 9 5 4 50 692 6

[0164] 40 kDa DS 20 18 10 8 70 495 39

[0165] 70 kDa DS 10 9 5 4 94 949 10

[0166] 70 kDa DS 20 19 11 8 123 117 58

[0167] 150 kDa DS 10 11 5 6 205 848 65

[0168] 150 kDa DS 20 20 9 11 260 747 72aDetermined by1H-NMR (exemplified for the 150 kDa DS 20 DEXTRAC in Figure 2).bCalculated based on DS determined by1H-NMR.cCalculated based on the fitted bell-shaped curves when co incubated with 100 nM anti-DNP as target POI.

[0169] Subsequently, the alkyne-functionalized dextran backbone was reacted with azide-modified ligands and a small fraction of Cy5-azide ( / .e., 1 equivalent relative to dextran) for fluorescence tracking. In three separate, consecutive reactions in a waterDMSO mixture, Cy5-azide, DNP-PEGs-azide, and M6P- PEGs-azide were conjugated to dextran, at a 1 :1 ratio of DNP to M6P. The copper-catalyzed azidealkyne cycloaddition (CuAAC) reaction was catalyzed by copper sulfate (CuSO4) in the presence of tris(1-benzyl-1 H-1 ,2,3-triazol-4-ylmethyl)amine (TBTA). As demonstrated in Figure 2 for the 150 kDa DEXTRACs,1H-NMR analysis confirmed successful CuAAC-mediated ligand conjugation, as indicated by the appearance of triazole-associated proton signals and DNP-associated proton signals. Additionally, the reported downfield shift observed in alkyne-associated protons further verified the conjugation. DOSY analysis supported the attachment of DNP groups to the dextran backbone, demonstrated by the identical diffusion patterns of both dextran- and DNP-associated protons. While spectral overlap presented challenges for precise DOSY analysis of dextran- and M6P-associated peaks, the conjugation of M6P-PEG3-azide was corroborated through the integration of triazole- associated protons and the aforementioned downfield shift of alkyne-associated protons.

[0170] DEXTRACs mediate potent and selective ternary complex formation between cells and a target POI

[0171] We investigated whether DEXTRACs could induce ternary complex formation between the CI-M6PR on the cell surface and anti-DNP antibodies added to the extracellular medium. Jurkat cells ( / .e., a leukemic human CD4+ T cell line) express CI-M6PR and were co-incubated with varying concentrations of DEXTRACs and AF488-labeled anti-DNP antibody. After 24 hours, the cellular association of anti-DNP antibodies was assessed by flow cytometry (Figure 3A). Jurkat cells incubated solely with the anti-DNP antibody were used as a control to determine the level of nonspecific cellular anti-DNP antibody association. The AF488 fold-increase value, obtained by dividing the AF488 fluorescence by the AF488 signal of the control, reflects the potency by which DEXTRAC-mediated ternary complex formation to the cellular association of the anti-DNP antibody target POI.

[0172] Figure 3B displays the fold-increase in AF488-fluorescence of Jurkat cells as a function of the molar concentration of dextran-bound ( / .e., DNP or M6P) ligand. All graphs feature a bell-shaped curve, with a decline in cellular fluorescence at high DEXTRAC concentrations. This phenomenon is known as the Hook effect (Figure 3C). It refers to the preferential formation of binary complexes between the ligands and their binding partners rather than the intended ternary complexes. This occurs in the presence of an excess of ligand, which saturates the binding sites of the CI-M6PR and anti-DNP, thereby preventing them from interacting with each other to form a ternary complex.

[0173] Furthermore, the maximum AF488 fold-increase values (Table 1) forthe respective DEXTRACs indicate that ligand multivalency is a crucial determinant for DEXTRAC-mediated ternary complex formation. This occurs at two levels: first, higher molecular weight (MW) DEXTRACs are more effective in mediating ternary complex formation than their lower MW counterparts (Figure 3D). Indeed, when co- incubated with 100 nM anti-DNP antibody, a 40 kDa DEXTRAC with a DNP+M6P DS of 5+5 achieved a maximum AF488 fold-increase value (fold-increasemax) of 6, while an analogous ( / .e., DNP+M6P DS of 5+5) 150 kDa DEXTRAC mediated a markedly higher fold-increasemax of 65 at a similar concentration (Cmax) of dextran-bound ligand (Figure 3E). Secondly, doubling the ligand density ( / .e., DNP+M6P DS of 10+10 versus 5+5) on a dextran of equal MW leads to an increase in fold-increasemax value without compromising the Cmax value (Table 2). This phenomenon was particularly evident for 40 and 70 kDa DEXTRACs, but was less pronounced for 150 kDa DEXTRACs. This suggests that the effect of dextran chain length on DEXTRAC-mediated ternary complex formation outweighs that of ligand density for the highest molecular weight DEXTRACs. Notably, the observed maximum fold increase values are on par with those reported for conceptually analogous systems relying on CI-M6PR when considering similar total compound concentrations (j.e., a 1 pM ligand concentration corresponds to a 10 nM concentration of a 150 kDa DS 20 DEXTRAC). Interestingly, whereas the antibody-glycopolymer conjugate LYTACs did not mediate a strong increase in cellular association with a soluble extracellular target protein upon increasing the glycopolymer chain length, DEXTRACs exhibit a dramatic increase in recruitment efficiency for a soluble extracellular target, thereby advocating for the design flexibility of the DEXTRAC concept. Finally, to confirm that both binary and ternary complexes were dependent on the CI-M6PR, Jurkat cells were treated with 150 kDa DS 20 DEXTRAC or a control dextran-bound M6P in the presence of an excess of freely soluble M6P as a competing ligand. As shown in Figure 3F, the presence of competing M6P ligand or lack of dextran-bound M6P fully abolished both the cellular Cy5 and the AF488 fluorescence.

[0174] Table 2: DEXTRAC in vitro biological activity parameters. Data are derived from flow cytometry analysis of Jurkat cells incubated for 24 hours with DEXTRACs and varying concentrations (5, 10, 50, or 100 nM) of anti-DNP antibody. Activity values for each parameter are reported and visualized in a heatmap. It is important to note that comparisons should be drawn specifically between DEXTRACs that were co-incubated with the same concentration of anti-DNP antibody (outer right column) for accurate assessments.aCalculated based on the fitted bell-shaped curves of Figure 2A.bEC50 is defined as the minimum DEXTRAC concentration needed to achieve 50% of fold-increasemax.cIC50 is defined as the DEXTRAC concentration to revert back to 50% of fold-increasemax.

[0175] DEXTRACs exhibit high avidity and specif icity ligand-receptor binding

[0176] Given the design of DEXTRACs, an improved avidity towards anti-DNP antibody and CI-M6PR, respectively, by multivalent DNP- and M6P-display may contribute to the potency of ternary complex formation. To investigate this hypothesis, we assessed binary complex formation for the least and most effective DEXTRACs ( / .e., 40 kDa DS 10 and 150 kDa DS 20, respectively). To evaluate binary complex formation between DEXTRACs and CI-M6PR on cell surfaces, Jurkat cells were incubated with DEXTRACs in the absence or presence, respectively, of varying concentrations of free M6P as a competing ligand for M6P:CI-M6PR binary complex formation. The binding of Cy5-labeled DEXTRACs to Jurkat cells was evaluated by flow cytometry (Figure 4A). All DEXTRACs displayed a dose- responsive association to Jurkat cells, showing no qualitative differences after normalization . Note that normalization was needed to account for intrinsic differences in Cy5 fluorescence between the respective DEXTRACs.

[0177] Association of 40 kDa DS 10 and 150 kDa DS 20 DEXTRACs to cells could be inhibited in presence of increasing concentrations of free M6P ligand (Figure 4 B1). Notably, at high DEXTRAC concentrations, the Cy5 signal did not return completely to baseline, likely due to non-specific interactions between the DEXTRACs and Jurkat cells that result in cellular internalization, probably through fluid-phase endocytosis, a mechanism extensively reported for dextrans. To compare the avidity of binary complex formation between cell surfaces and 150 kDa DS 20 DEXTRACs and 40 kDa DS 10 DEXTRACs, respectively, we incubated Jurkat cells with Cy5-labeled DEXTRACs at a similar concentration of dextran-bound M6P (Figure 4 B2) and calculated IC50 values from the fitted inhibition curves as the concentration of free ligand required to reduce DEXTRAC binding by 50%. A slightly higher IC50 was found for 150 kDa DS 20 DEXTRACs (i.e. , 87 pM of free M6P) compared to the 40 kDa DS 20 DEXTRAC ( / .e., 50 pM of free M6P), indicating a modestly higher avidity for the high MW DEXTRAC.

[0178] For assessing the avidity of binary complex formation between DEXTRACs and the anti-DNP antibody, beads displaying anti-DNP antibody were prepared by adsorbing biotinylated anti-DNP antibodies onto streptavidin-coated polystyrene beads. These beads were then incubated with Cy5-labeled DEXTRACs at a similar concentration of dextran-bound DNP in the absence or presence of varying concentrations of free DNP as a competing ligand for DNP-anti-DNP binary complex formation. The binding of Cy5- labeled DEXTRACs to the beads was evaluated by flow cytometry (Figure 4C). IC50 values calculated from the fitted Cy5 fluorescence values as a function of competing DNP concentrations, indicated a substantially higher IC50 for 150 kDa DS 20 DEXTRAC (i.e., 896 pM of free DNP) compared to the 40 kDa DS 20 DEXTRAC (i.e., 99 pM of free DNP; Figure 4D).

[0179] Taken together, our data reveal that enhancing M6P ligand density and extending dextran chain length leads to a modest improvement in binding avidity between cell surface CI-M6PR and DEXTRACs. In contrast, augmenting DNP ligand density and dextran chain length results in a substantial increase in binding avidity between DEXTRACs and anti-DNP antibodies. Consequently, these findings suggest a more pronounced impact of enhancing DNP ligand display, as opposed to M6P display, on the avidity of the resulting ternary complex formation. The underlying reasons for this observed difference remain elusive at present and may be attributed to variations in spatial restrictions affecting the availability of ligands to bind to their respective targets.

[0180] DEXTRACs traffic a target protein to lysosomes for degradation

[0181] We next investigated whether DEXTRACs mediated the shuttling of a target protein to lysosomes for degradation. To this end, Jurkat cells were incubated with AF488-labeled anti-DNP antibody in the presence of DEXTRACs and imaged by confocal fluorescence microscopy. Co-localization of Cy5- labeled DEXTRACs and AF488-labeled anti-DNP antibody indicates the cellular internalization of the anti-DNP antibody via ternary complex formation with DEXTRACs (confocal images not shown). Lysosomal localization of the internalized complexes was confirmed by counterstaining with LysoTrackerRed (Confocal images not shown). In contrast, control experiments with Cy5-dextran showed only extracellular AF488 fluorescence (confocal images not shown).

[0182] To assess wheter antiDNP antibody as a model target POI for degradation indeed becomes degraded after transportation into lysosomes, lysates from Jurkat cells incubated with 20 nM of AF488-labeled anti-DNP antibody alone or in combination with 20 nM of DEXTRAC 150 kDa DS 20 were investigated by Western blot analysis. As controls, 100 nM bafilomycin A1 or 10 mM M6P were co-incubated to inhibit lysosomal activity or ternary complex formation, respectively. The lysates were subjected to immunoprecipitation onto magnetic beads conjugated to anti-AF488 antibody. This enabled the capture of proteins containing the AF488 label, resulting in purified lysates for subsequent analysis by reducing SDS-PAGE after elution. Following blotting, an AF488 counterstaining was performed to visualize AF488-containing proteins. Figure 5A illustrates that all lysates exhibit a specific band at 50 kDa, corresponding to the heavy chain of the intact anti-DNP antibody. Interestingly, this band is also present in samples incubated solely with the anti-DNP antibody or in the presence of DEXTRAC and excess M6P, suggesting that detection of the anti-DNP antibody in solution persists despite multiple washing steps before lysate preparation. A diffuse protein signal below 50 kDa was observed in the lysate of Jurkat cells incubated with DEXTRAC and anti-DNP antibody, indicating the presence of degraded protein fragments containing AF488 label. Band intensity analysis revealed that this signal was more faint when cells were co-incubated with a lysosomal inhibitor or in the presence of an excess of freely soluble M6P as a competing ligand, or when incubated solely with anti-DNP antibody (Figure 5B). Collectively, these data demonstrate that DEXTRACs indeed facilitate lysosomal trafficking and the degradation of the target protein.

[0183] EXAMPLE 2 - IN VITRO TERNARY COMPLEX FORMATION BETWEEN CANCER TARGET CELLS, MODIFIED SCAFFOLD ACCORDING TO THE INVENTION AND ANTIBODIES

[0184] INTRODUCTION

[0185] Antibody-recruiting molecules (ARMs) are bivalent entities that incorporate a ligand for target cell surface binding and a ligand for endogenous antibody binding. ARMs facilitate the formation of a ternary complex between cells and antibodies, thus initiating innate immune effector responses. ARMs have been crafted using small molecules, lipids, and protein-based ligands for cancer cell targeting.

[0186] The number of small molecule ligands discovered that bind selectively and with high affinity to cancer cell surface receptors has been growing. Notable targets include PSMA, uPAR, folate receptor, and avp3 integrins. Non-internalizing receptors, which facilitate prolonged cell surface antibody display, are particularly desirable for efficient antibody recruitment. Our research has identified carbonic anhydrase IX (CAIX) as a promising receptor for ARMs development. This enzyme's expression is upregulated by the hypoxia-inducible factor (HIF) signaling pathway under hypoxic conditions. CAIX collaborates with bicarbonate and other ion transporters to remove acid or CO2 from cancer cells, aiding their survival. Its regulatory role results in an acidic extracellular pH and a more basic or neutral intracellular pH in cancer cells. Overexpression of CAIX is noted in various cancers such as glioblastoma, colorectal, and breast cancer, marking it as a hypoxia indicator and an attractive anti-tumor target. In renal cell carcinoma, CAIX is often constitutively expressed and is one of the most well-characterized markers of the disease. Analogs of acetazolamide (AAZ), a commercial carbonic anhydrase inhibitor, are susceptible to chemical modification while preserving their binding affinity to CAIX. The therapeutic application of acetazolamide is grounded in its inhibition of CAIX, managing conditions like edema, glaucoma, altitude sickness, some epileptic seizures, metabolic alkalosis, and periodic paralysis. It achieves this by altering bicarbonate reabsorption in the kidneys, decreasing intraocular pressure, modifying cerebral and systemic pH levels, and impacting ion transport. Small molecule AAZ conjugates have thus far been utilized for targeting diagnostic and therapeutic agents to CAIX-expressing cells. Here we report on the first design of CAIX-targeting ARMs, using an acetazolamide analog as the targetbinding ligand and dinitroaniline as a model ligand for endogenous anti-dinitrophenol antibodies. We describe the synthesis of small molecule single-ligand ARMs and multivalent ARMs comprising multiple ligand copies displayed on the backbone of dextran. In vitro studies on cell cultures expressing CAIX showed superior antibody-recruiting activity of the multivalent ARMs.

[0187] See Example 1 for instrumentation, cell culture, alkyne-functionalization of dextran, synthesis of Cy5- dextran, Cy5 / DNP-dextran, DNP-PEGs-azide.

[0188] EXAMPLE 2.1 - ADNP AB RECRUITMENT - FAP+ CELLS

[0189] Chemical synthesis

[0190] FAPI-PEGi2-azide

[0191] In a round-bottom flask, azido-PEGi2-acid (0.03 mmol), DIPEA (0.04 mmol) and HATU (0.04 mmol) were dissolved in 0.288 mL of dry dimethylformamide (DMF) and stirred for 20 minutes at room temperature. Subsequently, 0.100 mL of a FAPI stock solution (0.06 mmol) was added dropwise, and the reaction mixture was stirred overnight at room temperature under a nitrogen atmosphere. Following solvent evaporation, the crude product was purified by preparative thin layer chromatography (TLC) using 95:5 dichloromethane:methanol as eluent. Post-purification, a light yellow oil was obtained with a yield of 52%, and characterized by LC-MS (ESI).

[0192] Synthesis of FAPI-PEGi2-azide

[0193] Cy5 / FAPI-dextran

[0194] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Cy5-dextran (0.00015 mmol), TBTA (2 equivalents relative to the amount of CUSO4), and sodium ascorbate (3 equivalents relative to the amount of CUSO4) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, FAPI-PEGi2-azide (0.5 equivalents relative to the amount of dextran-bound alkyne) and CuSO4 (0.65 equivalents relative to the remaining amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a green to yellow solid was obtained and characterized by NMR.

[0195] Cy5 / DNP / FAPI-dextran (DEXTRACs)

[0196] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Cy5 / FAPI-dextran (0.00006 mmol), TBTA (2 equivalents relative to the amount of CuSC ), and sodium ascorbate (3 equivalents relative to the amount of CuSC ) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, DNP-PEGs-azide (1 equivalent relative to the remaining amount of dextran-bound alkyne) and CuSC (0.65 equivalents relative to the remaining amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a yellow solid was obtained and characterized by NMR.

[0197] Synthesis of Cy5 / DNP / FAPI-dextran (DEXTRACs)

[0198] FAPI-PEGi2-DNP

[0199] DNP-NH-PEG12-COOH (0.0383 mmol, 1.0 eq), HATU (0.0766mmol, 2 eq) and 33 pL DIPEA (0.191 mmol, 5 eq) were dissolved in 2 mL anhydrous DMF. The mixture was allowed to react for 10 minutes. Subsequently, FAPI (0.0574 mmol, 1 .5 eq) was added to the reaction mixture. The reaction was stirred at room temperature overnight and monitored by TLC (DCM:MeOH 15:1 , Rf = 0.3). After the reaction, the mixture was washed twice with brine and dried with anhydrous sodium sulfate. The mixture was finally concentrated and purified by preparative TLC (DCM:MeOH 15:1). The product was characterized NMR and ESI-MS.

[0200] Synthesis of FAPI-PEG12-DNP

[0201] Flow cytometry analysis of in vitro ternary complex formation between FAP+ cells, Cy5 / DNP / FAPI-dextran and aDNP antibody.

[0202] HT-1080.hFAP cells were seeded in a 24-well plate at a concentration of 50 000 cells in 500 pL DMEM per well and were left to adhere overnight. Next, 200 pL of PBS or free FAPI stock solution was added to achieve a well concentration of 1000 pM. After 30 minutes of incubation at 37°C with 5% CO2, cells were washed once with PBS containing 1 % BSA and resuspended in 200 pL of PBS, after which Cy5 / DNP / FAPI-dextran or FAPI-PEG12-DNP stock solution was added to achieve a DNP well concentration of 10 pM. After 30 minutes of incubation at 4 °C, cells were washed twice with PBS containing 1 % BSA and were detached using cell dissociation buffer. Following centrifugation (350 g, 5 minutes), the cell pellets were resuspended in 200 pL of PBS containing 1 % BSA and 10 pg / mL AF488- labeled aDNP antibody, and incubated on ice for 40 minutes. Post-antibody staining, the cells underwent two additional washes and were resuspended in cold PBS containing 1 % BSA. The AF488 signal was measured by flow cytometry.

[0203] DNP / FAPI-dextran outperforms single ligand FAPI-PEG-DNP in mediating ternary complex formation between FAP+ cells and anti-DNP antibodies.

[0204] HT-1080.hFAP cells were exposed for 30 minutes to Cy5 / DNP / FAPI-dextran or FAPI-PEG12-DNP, followed by incubation with AF488-labeled anti-DNP antibodies and analysis via flow cytometry. The data in Figure 6 revealed that Cy5 / DNP / FAP I -dextran vastly outperformed FAPI-PEG12-DNP in recruiting anti-DNP antibodies to the FAP+ cell surface. Strikingly, monovalent FAPI-PEG12-DNP was not capable of generating any meaningful recruitment of anti-DNP antibody, further highlighting the superiority of a multivalent approach. On the contrary, Cy5 / DNP / FAPI-dextran mediated strong anti- DNP recruitment, boosting cellular association of AF488-labeled anti-DNP antibody approximately 100- fold compared to cells incubated with anti-DNP antibody alone, expressed by the AF488 anti-DNP Ab fold-increase value. Moreover, pre-incubation with excess of free FAPI ligand prior to addition of Cy5 / DNP / FAPI-dextran and anti-DNP antibody completely inhibited the antibody recruitment capacity of the former, demonstrating that the effect is highly selective and driven by binding of FAPI ligand, conjugated onto the dextran, to the FAP protein, expressed on the cell surface. All data taken together suggests that multivalent interactions between displayed FAPI and FAP, as well as DNP and anti-DNP antibody, are crucial for an efficient ternary complex formation.

[0205] EXAMPLE 2.2 - ADNP AB RECRUITMENT - CAIX+ CELLS

[0206] The targeted immune-killing of tumors via antibody recruitment represents a promising approach to cancer therapy. This study explores the efficacy of multivalent dextran-templated antibody-recruiting molecules (dex-ARMs) for targeting carbonic anhydrase IX (CAIX)-expressing tumors. CAIX, a marker overexpressed in hypoxic tumors, was targeted using acetazolamide (AAZ) analogs, while dinitrophenol (DNP) analogs were used to recruit endogenous antibodies. We synthesized both single-ligand ARMs (SL-ARMs) and multivalent dex-ARMs, and evaluated their antibody-recruiting efficiencies using flow cytometry. Our results showed that dex-ARMs significantly outperformed SL-ARMs in recruiting anti- DNP antibodies to the cell surface of CAIX-expressing cells. The multivalent dex-ARMs demonstrated robust antibody recruitment even in the presence of competing ligands, suggesting enhanced multivalent interactions. Furthermore, dex-ARMs effectively recruited endogenous antibodies from serum, indicating their potential for clinical applications. This study establishes the foundational efficacy of dex-ARMs and suggests their utility in enhancing immune responses against hypoxia-associated tumor markers, providing a novel strategy for cancer immunotherapy.

[0207] MATERIALS AND METHODS

[0208] Chemical synthesis

[0209] Synthesis of N1-(2-(2-(2-((2,4-dinitrophenyl)amino)ethoxy)ethoxy)ethyl)-N4-(5-sulfamoyl-1,3,4- thiadiazol-2-yl)succinamide (SL-ARM-1)

[0210] 4-oxo-4-((5-sulfamoyl-1 ,3,4-thiadiazol-2-yl)amino)butanoic acid (0.0714 mmol, 1.1 equiv) and HATU (0.065, l equiv) were weighed into a round-bottom flask and dissolved in anhydrous DMF. Following this, anhydrous DIPEA (0.2142 mmol, 3equiv) was added to the mixture and stirring for 10 minutes at room temperature. Then, NH2-PEG2-DNP (0.065 mmol, l equiv) was added and stirred for 3 hours. After reaction, DMF was evaporated under vacuum. The crude product was purified using preparative TLC(10:1 DCM MeOH). The product was collected and characterized by NMR and LC-MS.

[0211] 1 H NMR (400 MHz, CDCI3) 6 8.86 (d, J = 2.7 Hz, 1 H), 8.84 (d, J = 5.2 Hz, 1 H), 8.30 (s, 2H), 8.26 (dd, J = 9.6, 2.8 Hz, 1 H), 7.96 (t, J = 5.6 Hz, 1 H), 7.26 (s, 1 H), 3.68 (m, J = 6.4, 3.4 Hz, 4H), 3.58 (td, J = 4.0, 1.1 Hz, 2H), 3.52 (td, J = 4.0, 1 .2 Hz, 2H), 3.39 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 2.77 - 2.68 (t, 2H), 2.46 (t, J = 6.9 Hz, 2H).

[0212] LC-MS: [M-H]+ = 575,11 (theoretical); found = 575,0

[0213] Synthesis of N1-(26-((2,4-dinitrophenyl)amino)-3, 6,9, 12, 15, 18,21 ,24-octaoxahexacosyl)-N4-(5- sulfamoyl-1,3,4-thiadiazol-2-yl)succinamide (SL-ARM-2) 4-oxo-4-((5-sulfamoyl-1 ,3,4-thiadiazol-2-yl)amino)butanoic acid (0.0714 mmol, 1.2equiv) and HATU (0.066, 1.1 equiv) were weighed in a round-bottom flask and dissolved in anhydrous DMF. Following this, anhydrous DIPEA (31 pL, 3equiv) was added to the mixture and stirring for 10 minutes at room temperature. Then, NH2-PEG8-DNP (0.06 mmol, l equiv) was added and stirred for 3 hours. After reaction, DMF was evaporated under vacuum. The crude product was purified using preparative TLC(13:1 DCM MeOH). The product was collected and characterized by NMR and LC-MS.

[0214] 1 H NMR (400 MHz, CDCI3) 6 10.04 (s, 1 H), 9.10 (d, J = 2.6 Hz, 1 H), 8.79 (s, 1 H), 8.25 (dd, J = 9.5, 2.7 Hz, 1 H), 7.58 (s, 1 H), 7.07 (s, 2H), 6.98 (d, J = 9.6 Hz, 1 H), 3.88 - 3.41 (m, 36H), 3.03 (t, J = 7.6 Hz, 2H), 2.81 (t, J = 7.5 Hz, 2H).

[0215] LCMS: [M+H]+ = 840,26 (theoretical); found = 841 ,05

[0216] Alkyne-functionalization of dextran, Cy5-dextran, Cy5 / DNP-dextran synthesis

[0217] See Example 1 .

[0218] Synthesis of N1-(26-azido-3.6.9.12.15.18.21.24-octaoxahexacosyl)-N4-(5-sulfamoyl-1.3.4-thiadiazol-2- yl)succinamide (AAZ-PEG8-N3)

[0219] 4-oxo-4-((5-sulfamoyl-1 ,3,4-thiadiazol-2-yl)amino)butanoic acid (0.0714 mmol, 1 equiv) and Hatu (0.0928 mmol, 1 .3 equiv) were weighed into a vial and dissolved then in anhydrous DMF. Subsequently, DIPEA (0.2854 mmol, 4 equiv) was added to the mixture and stirring 10 min at room temperature. 26- azido-3,6,9,12,15,18,21 ,24-octaoxahexacosan-1 -amine (0.1142 mmol, 1.6 equiv) were weighed and dissolved in anhydrous DMF and then added to the mixture and reacted for 3 hours. The crude was firstly evaporated on vacuum and then purified on TLC perspective (DCM:MEOH = 10:1). The product was collected and colored in white and yield 70%. The product was measured on NMR and LC-MS.

[0220] 1 H NMR (400 MHz, CDCI3) 6 12.82 (s, 1 H), 7.31 (s, 1 H), 7.16 (s, 2H), 3.79 - 3.34 (m, 36H), 2.98 (s, 2H), 2.76 (t, J = 7.6 Hz, 2H).

[0221] LCMS: [M+H]+= 701 ,26 (theoretical); found = 701 ,40

[0222] Synthesis of Cy5 / DNP / AAZ-dextran (dex-ARMs)

[0223] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Cy5 / DNP-dextran (0.0001 mmol), TBTA (2 equivalents relative to the amount of CuSC ), and sodium ascorbate (3 equivalents relative to the amount of CuSC ) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, AAZ-PEGs-azide (1 equivalent relative to the remaining amount of dextran-bound alkyne) and CuSO4 (0.65 equivalents relative to the remaining amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a yellow solid was obtained and characterized by NMR.

[0224] Synthesis of Cy5 / DNP / AAZ-dextran (DEXTRACs)

[0225] AAZ-PEGs-DNP

[0226] 4-Oxo-4-((5-sulfamoyl-1 ,3,4-thiadiazol-2-yl)amino)butanoic acid (0.07 mmol) and HATU (0.07 mmol) were weighed in a round-bottom flask and dissolved in 0.714 mL of anhydrous DMF. Next, DIPEA (0.18 mmol) was added to the mixture and stirring for 10 minutes at room temperature. Then, NH2-PEG8-DNP (0.06 mmol) was added and stirred overnight at room temperature. After solvent evaporation, the crude was purified by preparative TLC (13:1 dichloromoethane:methanol). The product was collected and characterized by NMR and LC-MS (ESI).

[0227] Synthesis of AAZ-PEGs-DNP

[0228] Dex-ARMs with 1:1 AAZ / DNP ratio

[0229] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Cy5 / DNP-d extra n (0.00011 mmol), TBTA (2 equivalents relative to the amount of CuSC ), and sodium ascorbate (3 equivalents relative to the amount of CuSC ) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSOFW mixture at a dextran concentration of 50 mg / mL. Subsequently, AAZ-PEGs-azide (1.1 equivalent relative to the remaining amount of dextran-bound alkyne) and CuSC (0.65 equivalents relative to the remaining amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a yellow solid was obtained and characterized by NMR.

[0230] Dex-ARMs with 1:3 and 3:1 AAZ / DNP ratio

[0231] For the synthesis of dex-ARMs with AAZ / DNP ratios of 1 :3 and 3:1 , we followed protocols similar to those described in sections 5.3 and 5.4. In these syntheses, we aimed for degrees of substitution (DS) of DNP-PEGs-azide / AAZ-PEGs-azide of 5 / 15 and 15 / 5, respectively.

[0232] Cell culture

[0233] SK-RC-52 renal cell carcinoma (RCC) cells were cultured in RPMI 1640 medium supplemented with standard additions of fetal bovine serum (FBS), penicillin / streptomycin, and pyruvate.

[0234] Flow cytometry

[0235] Flow cytometry analysis of in vitro anti-DNP antibody recruiting of SL-ARMs

[0236] SK-RC-52 cells were seeded in a 24-well plate at a concentration of 150000 cells in 500 pL RPMI per well and were left to adhere overnight. Following this, each well received either 2.5 pL of DMSO or 112.5 pM free AAZ stock solution, and was then incubated for 30 minutes at 37°C with 5% CO2. Subsequently, 60 pL of PBS, SL-ARM-1 , SL-ARM-2, DNP-PEG2 / 8-N3 stock solution was added to each well to achieve a DNP concentration of 10 pM. This mixture was then incubated for 2 hours at 37°C. Afterwards, the cells underwent two washes with PBS containing 1 % BSA and were detached using cell dissociation buffer. Following centrifugation (350 g, 5 minutes), the cell pellets were resuspended in 500 pL of PBS containing 1 % BSA and 20 pg / mL AF488-labeled DNP antibody, and incubated on ice for 30 minutes. Post-antibody staining, the cells underwent two additional washes and were resuspended in cold PBS. The AF488 signal was then measured using flow cytometry.

[0237] Flow cytometry analysis of in vitro anti-DNP antibody recruiting of dex-ARMs:AAZ:DNP-1 :1

[0238] SK-RC-52 cells were seeded in a 24-well plate at a concentration of 150000 cells in 500 pL RPMI per well and were left to adhere overnight. Following this, 60 pL of PBS, SL-ARM-2, or dex-ARM stock solution was added to each well to achieve a DNP concentration of 0.0001 , 0.001 , 0.01 , 0.1 , 1 , 10 or 100 pM. This mixture was then incubated for 2 hours at 37°C. Afterwards, the cells experienced method for washing and staining with anti-DNP antibody and the measurement method on flow cytometer.

[0239] Flow cytometry analysis of in vitro anti-DNP antibody recruiting of dex-ARMs with various of AAZ / DNP ratio

[0240] SK-RC-52 cells were seeded in a 24-well plate at a concentration of 150000 cells in 500 pL RPMI per well and were left to adhere overnight. Following this, 60 pL of PBS, dex-ARMs: AAZ: DNP-1 :1 , 3:1 and 1 :3 stock solution was added to each well to achieve a Dextran concentration of 0.001 , 0.01 , 0.1 , 1 , 10 or 100 nM. This mixture was then incubated for 2 hours at 37°C. Afterwards, the cells experienced method for washing and staining with anti-DNP antibody and the measurement method on flow cytometer.

[0241] Competition experiment of SL-ARM-2 and dex-ARM in presence of AAZ

[0242] SK-RC-52 cells were seeded in a 24-well plate at a concentration of 150000 cells in 500 pL RPMI per well and were left to adhere overnight. Following this, 60 pL of SL-ARM-2 or dex-ARM was added to each well to achieve a 10 pM DNP concentration. Subsequently, 2 pL AAZ stock solutions was added to each well to achieve AAZ concentration series of 0.01 , 0.1 , 1 , 10, 100, 1000 or 5000 pM. This mixture was then incubated for 2 hours at 37°C. Afterwards, the cells were washed and detached from well. The cell will be resuspended in 200 pL for measurement.

[0243] Flow cytometry analysis of in vitro serum binding of ARMs to cells

[0244] SK-RC-52 cells were seeded in a 24-well plate at a concentration of 150000 cells in 500 pL RPMI per well and were left to adhere overnight. Following this, 60 pL of PBS, SL-ARM-2, dex-ARM stock solution was added to each well to achieve a DNP concentration of 0.0001 , 0.001 , 0.01 , 0.1 , 1 , 10 or 100 pM. This mixture was then incubated for 2 hours at 37°C. Afterwards, the cells underwent two washes with PBS containing 1 % BSA and were detached using cell dissociation buffer. Following centrifugation (350 g, 5 minutes), the cell pellets were resuspended in 100 pL of PBS containing 1 % serum and 1 % BSA, and incubated on ice for 30 minutes. Subsequently, the cells underwent two washes with PBS containing 1 % BSA and were resuspended in 100 pL of PBS containing 1 % BSA and 10 pg / mL AF488-labeled lgG1 antibody. Post-antibody staining, the cells underwent two additional washes and were resuspended in cold PBS. The AF488 signal was then measured using flow cytometry.

[0245] Microscopy

[0246] 20000 SK-RC-52 cells with 200pL RPMI medium was seeded into chambered coverslip and incubated for 24 hours at 37°C. 20 pL stock solution were added to well to reach a DNP concentration of 5 pM in SL-ARMs and dex-ARM. After two hours incubation of the mixture, cells were washed with PBS for two times. Subsequently anti-DNP antibodies was added to cells and incubate on ice for 30 min. Cells were washed with PBS and then measure on confocal microscopy.

[0247] DNP immunization

[0248] LNP(poly(l:C),) were produced as reported by Lamoot et al. and was combined with 50 pg of KLH-DNP in water and administered intramuscularly to mice. RESULTS AND DISCUSSION

[0249] AAZ-DNP conjugates recruit anti-DNP antibodies to CAIX-expressing cells

[0250] Synthesis of small molecule single-ligand AAZ-DNP conjugates

[0251] An AAZ-DNP conjugate was synthesized based on literature reports of small molecule fluorescent AAZ conjugates that bind to CAIX-expressing cells. The synthesis route is depicted in Scheme 2. Initially, 1- Chloro-2,4-dinitrobenzene (1) was reacted with PEG2 diamine or amino-PEGs-azide repeating units to yield the amino-PEG-dinitroaniline derivatives 2 and 3. Then, Staudinger reduction of azide (3) to form amine-PEGs-dinitroaniline (4). It is noteworthy that DNP, although an acronym for dinitrophenol, refers here to an aniline derivative frequently utilized in the synthesis of DNP conjugates. The single-ligand ARMs, SL-ARM-1 and SL-ARM-2, were subsequently synthesized by coupling the acetazole amide analogue 5, 4-Oxo-4-((5-sulfamoyl-1 ,3,4-thiadiazol-2-yl)amino)butanoic acid (acetazolamide), to 6 via amide bond formation in the presence of HATU. SL-ARM-1 and SL-ARM-2 contain 2 and 8 ethylene glycol repeating units, respectively. NMR spectroscopy and mass spectroscopy confirmed the structures and purity of the resultant compounds.

[0252] Scheme 2. Chemical synthesis route for SL-ARMs. In a first step, 1-Chloro-2,4-dinitrobenzene was reacted with PEG2 diamine or amino-PEGs-azide followed by Staudinger reduction, yielding amino- PEG2 / 8-dinitroaniline. The final step involved the conjugation of amino-PEG2 / 8-dinitroaniline to an acetazole amide analogue CAIX-ligand via HATU-mediated amide coupling and yielded the SL-ARMs.

[0253] In vitro antibody recruitment by small molecule single-ligand AAZ-DNP conjugates

[0254] The efficacy of SL-ARM-1 and SL-ARM-2 in recruiting anti-DNP antibodies to cell surfaces was evaluated in vitro using SK-RC-52 cells, a human renal cancer cell line with notably high CAIX expression. The cells were treated with 10 -uM of the compounds for 2 h, followed by washing and incubation with AF488-labeled anti-DNP antibodies for 30 min. Flow cytometry analysis provided conclusive evidence that both ARMs were capable of mediating antibody recruitment, as indicated by a minimal AF488 background signal in untreated cells and the complete inhibition of antibody recruitment in the presence of an excess of compound (5) as a competitor (Figure 7). Moreover, PEG2 / 8- dinitroaniline modified with an azide group instead of the AAZ motif failed to recruit anti-DNP antibodies. Confocal microscopy further confirmed the localization of AF488-anti-DNP antibodies on the cell surface. Multivalent ligand ARMs outperform single ligand ARMs

[0255] Synthesis of dextrans substituted with AAZ and DNP motifs

[0256] We developed multivalent dextran-templated ARMs, hereinafter referred to as dex-ARMs, by attaching multiple copies of AAZ-analog and DNP ligands to a 150 kDa dextran backbone utilizing copper- catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry). This synthetic approach, depicted in Scheme 3, and the chosen molecular weight (i.e. 150 kDa) and degree of substitution (DS) were fostered by our prior research that delineated the influence of dextran molecular weight on the binding strength of the ternary complexes formed by dextran displaying multiple copies of two types of ligands.

[0257] Scheme 3. Dex-ARMs are synthesized via a CuAAC reaction between alkyne-substituted dextrans and azide-functionalized ligands. In a first step, both ligands were functionalized with PEGa- azide to allow for CuAAC-mediated click chemistry. For the CAIX binding ligand, an AAZ-derivative was conjugated to amino-PEG8-azide via HATU coupling. For the DNP ligand, amino-PEG8-azide was reacted with 1-Chloro-2,4-dinitrobenzene. In a second step, dextrans are functionalized with alkynes by reacting with glycidyl propargyl ether in basic aqueous medium, aiming for a degree of substitution (DS) of 20. In consecutive reactions, the alkyne-functionalized dextrans are reacted in a waterDMSO mixture with Cy5-azide, AAZ-PEGs-azide and DNP-PEGs-azide, targeting a AAZ:DNP ratio of 1 :1 , 3:1 and 1 :3. Note that although DNP is an acronym for dinitrophenol, it is an aniline derivative that is commonly used in the synthesis of DNP-conjugates.

[0258] In a first step, dextran was functionalized with glycidyl propargyl ether in an aqueous medium using sodium hydroxide (NaOH) as a catalyst. This was done to achieve a DS of 20 %, which represents the proportion of glucose repeating units in the dextran chain modified with alkynes. This specific DS was chosen based on previous findings that while a higher DS enhances the avidity of ternary complex formation, excessive modification can lead to solubility challenges. Hence, a DS of 20 was determined to be an optimal balance. Proton NMR (1H-NMR) analysis confirmed an alkyne DS in agreement with the target value of 20, as summarized in Table 2. Furthermore, DOSY analysis yielded identical diffusion patterns for protons related to both dextran and the alkyne groups, thus verifying the successful conjugation of the alkyne functionalities to the dextran backbone.

[0259] Subsequently, the dextran backbone, functionalized with alkyne groups, was subjected to consecutive reactions with azide-modified ligands. The synthesis of these ligands is outlined in Scheme 3. First, a low amount of Cy5-azide (1 equivalent per dextran molecule) was reacted with alkyne-functionalized dextran to enable synthesis and in vitro evaluation of fluorescently labelled dex-ARMs. Next, the ligands AAZ-PEGs-azide and DNP-PEGs-azide were sequentially attached to the dextran in a waterDMSO solvent mixture. The ligands were conjugated at AAZ to DNP molar ratios of 1 :1 , 3:1 , and 1 :3, respectively. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was facilitated by copper sulfate (CuSO4) with the assistance oftris(1-benzyl-1 H-1 ,2,3-triazol-4-ylmethyl)amine (TBTA) as a ligand to catalyze the process. To visualize dextran binding to CAIX by confocal microscopy, we synthesized monofunctional DEX-AAZ conjugates with enhanced Cy5 labeling, incorporating five equivalents of Cy5-azide per dextran molecule.

[0260] 1H-NMR analysis confirmed successful CuAAC-mediated ligand conjugation, as indicated by the appearance of triazole-associated proton signals and DNP- and AAZ-associated proton signals. Additionally, the decrease in AUC of the alkyne-associated protons further verified the conjugation after click chemistry. DOSY analysis supported the attachment of DNP / AAZ motifs to the dextran backbone, demonstrated by the identical diffusion patterns of both dextran- and AAZ / DNP-associated protons. While spectral overlap presented challenges for precise DOSY analysis of dextran- and AAZ / DNP- associated peaks, successful conjugation of both ligands was supported through the integration of triazole-associated protons and the aforementioned disappearance of alkyne-associated protons

[0261] Table 2. Chemical characterization of dextran conjugates. dextran DS alkyneaDS DNPaDS AAZaMW (Da)bdextran-alkyne 20 0 0 170 615 dextran-Cy5-alkyne 20 0 0 171 216Cdex-Cy5-AAZ-DNP (1 :1)d0 10 10 290 663 dex-Cy5-AAZ-DNP (3:1)d0 15 5 286 220 dex-Cy5-AAZ-DNP (1 :3)d0 5 15 295 106aDetermined by1H-NMR (cfr. Figure 3).bCalculated based on DS determined by1H-NMR.cThis MW was not quantifiable by NMR due to the low functionalization with Cy5. We aimed for the conjugation of

[0262] 1 Cy5 equivalent per dextran molecule. Hence, we added the MW of Cy5 to obtain a theoretical MW of dextran-Cy5-alkyne.dRepresenting dex-ARMs with a target AAZ:DNP molar ratio of 1 :1 , 3:1 and 1 :3, respectively.

[0263] In vitro binding of multivalent dex-ARMs to CAIX-expressing cells

[0264] Previous research has indicated that small molecule fluorescent AAZ analog conjugates can adhere to the surface of CAIX-expressing cells. However, to the best of our knowledge, such binding has not been validated for macromolecular AAZ conjugates, which may introduce steric hindrances that affect optimal CAIX binding. To investigate this, SK-RC-52 cells were incubated with 5 pM of Cy5-labeled dex-ARMs for 2 h at a temperature of 37 °C. Flow cytometry (Figure 8) analysis demonstrated that dex-ARMs successfully associate to of CAIX-expressing cells. For confocal microscopy imaging, we synthesized a Cy5-labeled dextran containing AAZ, but no DNP, to allow for increasing the density of the Cy5 label needed for proper imaging, while omitting the hydrophobicity of the DNP motifs to avoid aggregation of the final product. Subsequent confocal imaging (data not shown) of SK-RC-52 cells treated with this construct unambiguously demonstrated cell surface localization of the construct.

[0265] In vitro anti-DNP antibody recruitment by multivalent dex-ARMs conjugates

[0266] SK-RC-52 cells were exposed to a range of concentrations of SL-ARMs and dex-ARMs for 2 h at 37 °C, followed by incubation with AF488-labeled anti-DNP antibodies and analysis via flow cytometry. The data revealed that dex-ARMs significantly outperformed SL-ARMs in recruiting anti-DNP antibodies to the SK-RC-52 cell surface (Figure 9A, left panel). Both the antibody density per cell, indicated by the median AF488 fluorescence intensity (MFI), and the recruitment efficiency, indicated by the concentration of ARMs needed, were markedly improved with a more than 100-fold reduction in the calculated EC50 values from the dose-response curves. This suggests that multivalent interactions between displayed AAZ and CAIX, as well as DNP and anti-DNP, are crucial. Notably, while the anti- DNP antibody can bind a single DNP motif with an affinity of around 10 nM, the multivalent presentation of DNP motifs is known to significantly enhance the overall strength of the binary complex formation. Moreover, altering the ratio of AAZ to DNP had only a minimal effect on the antibody-recruiting capacity of the dex-ARMs. Confocal microscopy validated that AF488-labeled anti-DNP antibodies, recruited by dex-ARMs were localized on the cell surface (Figure 9B).

[0267] To further explore the role of the dex-ARM design on the antibody-recruiting efficiency, we synthesized two other dex-ARMs where we varied the AAZ-to-DNP ratio from 1 :1 to 3:1 and 1 :3, respectively (Figure 9C). Flow cytometry analysis of the antibody recruiting efficiency on dex- ARM-treated SK-RC-52 cells, indicated a minor influence of the ligand ratio, thereby pointing at a good robustness of the dex-ARMs system. We then tested antibody-recruiting efficacy of SL-ARMs and dex- ARMs at concentration series of 0.0001 , 0.001 , 0.01 , 0.1 , 1 , 10, 100 pM, corresponding to DNP, in presence of an increasing concentration of acetazolamide (5) as a competing ligand for CAIX binding (Figure 9D). Whereas again dex-ARMs greatly outperformed SL-ARMs in terms of the total amount of anti-DNP antibodies that could be recruited to the cell surface, the calculated IC50 values only showed a minor difference. These findings might suggest that multivalent interaction between AAZ motifs on the dextran backbone is possible, but likely limited, as otherwise as much higher concentration of competing ligand should be required to displace dex-ARMs-recruited anti-DNP antibodies from the cell surface, when compared to SL-ARMs-recruited anti-DNP antibodies. Hence, we postulate that the superior antibody-recruiting efficiency of dex-ARMs, likely due to an increased display of DNP motifs on the surface of dex-ARM-bound cells.

[0268] Multivalent dex-ARMs are superior in recruiting endogenous antibodies from serum

[0269] Next, we explored whether dex-ARMs could also mediate the recruitment of endogenous antibodies from serum. As mice generally do not have substantial titers of anti-DNP antibodies in their serum, we immunized mice with a commercially available Keyhole limpet hemocyanin (KLH)-DNP conjugate. In this conjugate a DNP analog is covalently bound to KLH as an immunogenic carrier protein. To amplify the antibody response, the KLH-DNP conjugate was adjuvanted with Polyinosinic:polycytidylic acid (poly(l:C)) encapsulated in lipid nanoparticles (LNP), which our laboratories recently identified as a potent vaccine adjuvant. While poly(l:C) alone activates Toll-like receptor 3 (TLR3) on endosomal membranes in antigen-presenting cells, LNP encapsulation facilitates cytoplasmic delivery, also activating cytoplasmic RIG-l-like receptors — a mechanism we found crucial for the superior adjuvanticity of LNP(poly(l:C)) over its soluble form. Following a bi-weekly immunization regimen, we detected DNP- specific antibody titers in the serum via ELISA, which used commercially available BSA-DNP conjugate- coated plates (Figure 10). Predominantly an lgG1 isotype was found in this serum.

[0270] To investigate whether SL-ARMs and dex-ARMs could recruit antibodies from serum to cells, we supplemented PBS (containing 1 %BSA) with 1 % of serum from DNP-immunized mice. Antibody recruitment efficacy was then determined by flow cytometry, with counterstaining against lgG1. Dex- ARMs again exceeded the performance of SL-ARMs, eliciting a significant increase in both the density of antibodies on the cell surface and the efficiency of recruitment, as demonstrated by the low concentration of dex-ARMs required for antibody recruitment from serum.

[0271] CONCLUSION

[0272] In this Example 2, the design and efficacy of multivalent dextran-templated antibody-recruiting molecules (dex-ARMs) for the targeted immune-killing of carbonic anhydrase IX (CAIX)-expressing or FAP+ expressing tumors was demonstrated. The findings reveal that dex-ARMs, incorporating both acetazolamide (AAZ) analogs for CAIX targeting (resp. FAPI analogs for FAP) and dinitrophenol (DNP) analogs for antibody recruitment, significantly outperform single-ligand ARMs (SL-ARMs) in both antibody recruitment and cell surface retention. Flow cytometry analysis indicated that the ligand ratio within dex-ARMs has a minor impact on their antibody-recruiting efficiency, highlighting the robustness of the dex-ARMs system. Furthermore, even in the presence of a competing ligand, dex-ARMs showed superior performance compared to SL-ARMs, suggesting that multivalent interactions enhance antibody binding and retention on the cell surface. Additionally, the in vitro studies demonstrated that dex-ARMs effectively recruit endogenous anti-DNP antibodies from serum, an important feature for potential clinical applications. This superior performance is due to the increased display of DNP motifs on the surface of dex-ARM-bound cells, facilitating more efficient antibody recruitment. Overall, the multivalent approach using dextran as a scaffold provides a versatile and effective strategy for enhancing the targeting and efficacy of antibody-recruiting molecules. The present concept of dex-ARMs is particularly suitable for therapeutic applications in cancer treatment, particularly for targeting hypoxia-associated markers such as CAIX or FAP.

[0273] EXAMPLE 3 - ANTI-FITC ANTIBODY & EFFECTOR CELL RECRUITMENT

[0274] EXAMPLE 3.1 - ANTI-FITC ANTIBODY IN VITRO EXPERIMENT

[0275] In this first experiment, the anti-FITC antibody was taken as a model for an anti-FITC CAR with regard to further development of dextran-based adaptors for anti-FITC universal CAR T-cell therapy (see Example 3.2).

[0276] MATERIAL AND METHODS

[0277] See Example 1 for instrumentation, cell culture, alkyne-functionalization of dextran and synthesis of AAZ-PEG8-N3.

[0278] Chemical synthesis

[0279] FITC-dextran

[0280] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. Dextran-alkyne (150 kDa dextran, alkyne DS 20) (0.0002 mmol), TBTA (2 equivalents relative to the amount of CuSC ), and sodium ascorbate (3 equivalents relative to the amount of CuSC ) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSOFLO mixture at a dextran concentration of 50 mg / mL. Subsequently, FITC-PEGs-azide (0.5 equivalents relative to the amount of dextran-bound alkyne) and CuSO4 (0.65 equivalents relative to the amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a yellow solid was obtained and characterized by NMR.

[0281] FITC / AAZ-dextran (DEXTRACs)

[0282] H2O and DMSO were purged with nitrogen for 10 minutes before utilization. FITC-dextran (0.0001 mmol), TBTA (2 equivalents relative to the amount of CUSO4), and sodium ascorbate (3 equivalents relative to the amount of CUSO4) were weighed in a round-bottom flask and dissolved in a 3.5:1 DMSO:H2O mixture at a dextran concentration of 50 mg / mL. Subsequently, AAZ-PEGs-azide (1 equivalent relative to the remaining amount of dextran-bound alkyne) and CuSO4 (0.65 equivalents relative to the remaining amount of dextran-bound alkyne) were sequentially added. Following these additions, the solutions were nitrogen-flushed for 10 minutes and stirred for 42 hours at 50°C, shielded from light. Upon completion of the reaction, the solution was precipitated twice in cold methanol and dialyzed against water (MWCO 3.5 kDa) for four days. After freeze-drying, a yellow solid was obtained and characterized by NMR. -M-

[0283] Synthesis of FITC / AAZ-dextran (DEXTRACs)

[0284] Flow cytometry analysis of in vitro ternary complex formation between CAIX+ cells, FITC / AAZ- d ext ran and aFITC antibody

[0285] SK-RC-52 cells were seeded in a 24-well plate at a concentration of 250 000 cells in 500 pL RPMI per well and were left to adhere overnight. Following this, each well received either 25 pL of PBS or 22 000 pM acetazolamide (AAZ.or abbreviated as AZA) stock solution, and was then incubated for 30 minutes at 37°C with 5% CO2. Subsequently, 25 pL of PBS or FITC / AAZ-dextran stock solution was added to each well to achieve a FITC / AAZ-dextran concentration of 15, 75, or 150 nM. This mixture was then incubated for 2 hours at 37°C with 5% CO2. Afterwards, the cells underwent two washes with PBS containing 1 % BSA and were detached using cell dissociation buffer. Following centrifugation (350 g, 5 minutes), the cell pellets were resuspended in 100 pL of PBS containing 1 % BSA and 2.5 pg / mL APC- labeled aFITC antibody, and incubated on ice for 40 minutes. Post-antibody staining, the cells underwent two additional washes and were resuspended in cold PBS containing 1 % BSA. The FITC and APC signals were then measured using flow cytometry.

[0286] RESULTS

[0287] FITC / AAZ-dextran mediates ternary complex formation between CAIX+ cells and anti-FITC antibodies.

[0288] CAIX+ SK-RC-52 cells were exposed for 2 hours to different concentrations of FITC / AAZ-dextran in absence or presence of excess free AAZ, followed by incubation with APC-labeled anti-FITC antibodies and analysis via flow cytometry. The data in Figure 11 revealed that FITC / AAZ-dextran strongly associates to SK-RC-25 cells, even at low nanomolar concentrations, as reflected by the intense FITC fluorescent signal. Moreover, FITC / AAZ-dextran bound to SK-RC-52 cells efficiently recruits APC- labeled anti-FITC antibodies to the cell surface, boosting cellular association of APC-labeled anti-FITC antibody more than 40-fold compared to cells incubated with anti-FITC antibody alone, as expressed by the APC anti-FITC Ab fold-increase value. Note that the anti-FITC antibody in this experiment was taken as a model for an anti-FITC CAR with regard to further development of dextran-based adaptors for anti- FITC universal CAR T-cell therapy. Moreover, pre-incubation with high concentration of free AAZ ligand prior to addition of Cy5 / DNP / FAP I -dextran and anti-FITC antibody completely inhibited the antibody recruitment capacity of the former, demonstrating that the effect is highly selective and driven by binding of AAZ ligand, conjugated onto the dextran, to the CAIX protein, expressed on the cell surface. All data taken together suggest that multivalent interactions between displayed AAZ and CAIX, as well as FITC and anti-FITC antibody, are crucial for an efficient ternary complex formation. In summary, FACS results demonstrate selective association of FITC / AAZ-dextran to CAIX+ cells (Figure 11 A) and ternary complex formation between CAIX+ cells, FITC / AAZ-dextran and aFITC antibody (Figure 11 B).

[0289] EXAMPLE 3.2 - ANTI-FITC EFFECTOR CELL IN VITRO EXPERIMENT

[0290] This second experiment builds further on the experiment performed under Example 3.1 wherein the anti-FITC antibody as such was taken as a model for an anti-FITC CAR. Here, anti-FITC scFv CAR T cells are developed which - in presence of FITC / AZA-dextran - are able to successful kill SK-RC-52 cells through a ternary complex formation. As used herein, AZA may also be abbreviated as AAZ.

[0291] MATERIAL AND METHODS

[0292] Transduction of mouse CD8+ T cells with anti-FITC scFv CAR

[0293] Retroviral particles encoding for the anti-fluorescein (anti-FITC) anti-FITC single-chain variable fragment (scFv) chimeric antigen receptor (CAR) containing an additional Myc-tag were produced by transfecting Phoenix-ECO cells with pCL-Eco plasmid and transfer plasmid, encoding for the anti-FITC scFv CAR, using the CalPhosMammalianTransfection kit at a 1 :3 ratio. Primary mouse CD8+ T cells were isolated from spleens of wild type (WT) C57BL / 6 mice and activated for 48 h on plates coated with anti-CD3 (0,5 ug / ml) and anti-CD28 (5 ug / ml) antibodies.

[0294] The experimental protocol for transduction and analysis of activated CD8+ T cells is shown in Figure 12A. Activated mouse CD8+ T cells were treated for 20 h at 37 °C with retroviral particles encoding for the anti-fluorescein anti-FITC scFv CAR containing an additional Myc-tag. Retroviral particles encoding for the anti-fluorescein anti-FITC ScFv CAR containing an additional Myc-tag were added to retronectin- coated plates and centrifuged at 2000xg for 90 min at 35°C. Supernatant was removed and activated mouse CD8+ T cells were added. After 20 h of incubation at 37 °C, transduced CD8+ T cells were stained with a fluorescent CL647-conjugated anti-Myc-tag antibody, followed by flow cytometry analysis. Non-transduced mouse CD8+ T cells served as control. Flow cytometry analysis (Figure 12B-C) confirmed expression of the anti-FITC scFv CAR on the T cell surface.

[0295] RESULTS FITC / AZA-dextran binds to mouse anti-FITC scFv CAR T cells

[0296] Experimental protocol for flow cytometry analysis of FITC / AZA-dextran binding to mouse anti-FITC scFv CAR T cells is shown in Figure 13A. Anti-FITC scFv CAR T cells were treated with FITC / AZA-dextran (2 |j.M FITC total concentration in well) for 30 min at 4°C, followed by flow cytometry analysis of the cellular FITC signal. Non-transduced mouse CD8+ T cells served as control. Flow cytometry analysis (Figure 13B-C) confirmed binding of FITC / AZA-dextran to anti-FITC scFv CAR T cells.

[0297] FITC / AZA-dextran mediates killing of CAIX+ SK-RC-52 cells by anti-FITC scFv CAR T cells

[0298] Experimental protocol for flow cytometry analysis of killing of SK-RC-52 (target cells) by anti-FITC scFv CAR T cells (effector cells) in presence of FITC / AZA-dextran is show in Figure 14Aa. SK-RC-52 cells, expressing high levels of carbonic anhydrase IX (CAIX) were labeled with Cell Proliferation Dye eFluor 450 and seeded in a 96-well U-bottom plate, followed by the addition of FITC / AZA-dextran (2 |j.M FITC total concentration in well). AZA-dextran and FITC-dextran, lacking FITC and AZA, respectively, served as controls. After 2 h incubation at 37 °C, cells were washed, transferred to a flat bottom 96-well plate and anti-FITC scFv CAR T cells, or non-transduced, T cells, respectively, were added at an effector to target (E:T) ratio of 8 and co-cultured for 24 h. Afterwards, cells were treated with Fixable Viability Dye eFluor 780 incubated for 30 minutes on ice, and analyzed by flow cytometry. The target killing was determined using the equation below

[0299] Flow cytometry analysis (Figure 14B) indicated successful killing of SK-RC-52 cells by anti-FITC scFv CAR T cells, in presence of FITC / AZA-dextran, but not in presence of control dextran constructs lacking FITC or AAZ, respectively.

Claims

CLAIMS1 . A compound comprising a macromolecular hydrophilic polymer scaffold conjugated with- at least one extracellular target protein binding ligand; and- at least one further protein binding ligand for binding i) a lysosomal trafficking receptor; or ii) a target protein presented on a cancer cell or immune cell; wherein each of said binding ligands is present in at least 5 copies, and wherein said extracellular target protein binding ligand is different from said further protein binding ligand.

2. The compound according to claim 1 , wherein the ratio of said further protein binding ligand to said extracellular target protein binding ligand conjugated to the polymer scaffold ranges from about 1 :20 to about 20:1 , in particular about 1 :10 to about 10:1 , more in particular about 1 :5 to about 5:1 , even more in particular about 1 :1.

3. The compound according to any one of claims 1 to 2, wherein the density of said ligands conjugated per 100 monomer repeating units of the polymer scaffold ranges about and between 1 % to 50%.

4. The compound according to any one of claims 1 to 3, wherein said hydrophilic polymer has a molecular weight of at least about 40 kDa, in particular at least about 70 kDa, more in particular at least about 100 kDa, even more particular at least about 150 kDa.

5. The compound according to any one of claims 1 to 4, wherein said hydrophilic polymer scaffold is a linear and / or branched polymer, in particular a linear polymer.

6. The compound according to any one of claims 1 to 5, wherein said hydrophilic polymer scaffold is a polysaccharide, in particular dextran.

7. The compound according to any one of claims 1 to 6, wherein each of said binding ligands is present in at least 10 copies, in particular at least 20 copies, more in particular at least 50 copies.

8. The compound according to any one of claims 1 to 7, wherein in case said further protein binding ligand is a lysosomal trafficking receptor binding ligand, the extracellular target protein binding ligand has affinity to a target protein to be degraded.

9. The compound according to any one of claims 1 to 8, wherein said lysosomal trafficking receptor binding ligand is Mannose-6-Phosphate (M6P) and / or N-Acetyl Galactosamine (GalNAc).

10. The compound according to any one of claims 1 to 7, wherein said target protein presented on a cancer cell is CAIX (Carbonic Anhydrase IX).11 . The compound according to any one of claims 1 to 7 or claim 10, wherein in case said further protein binding ligand is a ligand binding a target protein presented on a cancer or immune cell, the extracellular target protein binding ligand has affinity to an effector protein, in particular an antibody or an effector cell, in particular a CAR-T cell, CAR NK cell, or CAR macrophage cell.

12. The compound according to any one of claims 1 to 11 , wherein said ligands are attached to the polymer via a linker.-SI-13. A pharmaceutical composition comprising the compound according to any one of claims 1 to 12, and a pharmaceutically acceptable excipient.

14. The compound according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, for use in human and / or veterinary medicine, in particular for use in the treatment and / or prevention of cancer, inflammatory and / or autoimmune diseases.

15. The compound according to any one of claims 1 to 12, or the pharmaceutical composition according to any one of claims 13 to 14, wherein the further protein binding ligand is a lysosomal trafficking receptor binding ligand, for use in targeted protein degradation.

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