WO2023203127A1 - Alphabody-based degrader molecules - Google Patents

Abstract

The invention relates to binding agents for targeted protein degradation comprising an Alphabody protein molecule specifically targeting an intracellular protein, and a degrader molecule acting as scavenger of proteasomal activity. Said Alphabody protein-based degrader binding agent forms a ternary complex with the target protein and the E3 ligase in the cell, resulting in activation of targeted protein degradation. Particularly, the binding agent thus comprises cell-penetrating Alphabody proteins linked to a degrader moiety. More particularly, the invention relates to binding agents which are hybrid molecules wherein the degrader moiety is conjugated to the Alphabody protein. More particularly, the invention relates to binding agents comprising degrader moieties specifically binding an E3 ligase complex to enhance ubiquitination and proteasomal degradation of the target specifically binding the Alphabody protein. Most particularly, the invention relates to binding agents comprising MCL1- and MDM4/MDM2-targeting Alphabody-based degrader molecules for use in treatment of diseases associated with said target molecules, preferably for use in treatment of cancer.

Description

ALPHABODY-BASED DEGRADER MOLECULES

FIELD

The invention relates to binding agents for targeted protein degradation comprising an Alphabody protein molecule specifically targeting an intracellular protein, and a degrader molecule acting as scavenger of proteasomal activity. Said Alphabody protein-based degrader binding agent forms a ternary complex with the target protein and the E3 ligase in the cell, resulting in activation of targeted protein degradation. Particularly, the binding agent thus comprises cell-penetrating Alphabody proteins linked to a degrader moiety. More particularly, the invention relates to binding agents which are hybrid molecules wherein the degrader moiety is conjugated to the Alphabody protein. More particularly, the invention relates to binding agents comprising degrader moieties specifically binding an E3 ligase complex to enhance ubiquitination and proteasomal degradation of the target specifically binding the Alphabody protein. Most particularly, the invention relates to binding agents comprising MCL1- and MDM4/MDM2-targeting Alphabody-based degrader molecules for use in treatment of diseases associated with said target molecules, preferably for use in treatment of cancer.

INTRODUCTION

Targeted protein degradation (TDP) is an emerging therapeutic field with the potential for eliminating disease-causing proteins historically considered undruggable or difficult to target, thus providing for an extended panel of potential drugs beyond conventional small molecules or antibodies. Proteolysistargeting chimera (PROTAC™) are known as heterobifunctional small molecules consisting of two ligands joined by a linker, wherein one ligand recruits and binds a protein of interest while the other recruits and binds an E3 ubiquitin ligase. Simultaneous binding of the protein of interest and E3 ligase or E3 ligase complex by the PROTAC molecule induces ubiquitination of the protein of interest and its subsequent degradation by the ubiquitin-proteasome system, after which the PROTAC molecule is recycled to further target the proteins of interest (1). While traditional small molecule inhibitors usually inhibit the enzymatic activity of the target, PROTACs do not require binding to orthosteric sites affecting the enzymatic activity of the protein, but solely interaction with the target is sufficient to provide proximity to the E3 ligase, which is gathered to the protein of interest via the second ligand, to trigger the protein of interest its proteasomal degradation (8). This technology thereby increases the potential of discovering alternative binding pockets usable for drug development as well as expand the druggable proteome (5). Current PROTAC modalities typically require caution in view of their target-specific effects, though for some target classes, such as kinases, PROTACs can comparatively achieve greater specificity with respect to off-target effects over small molecule inhibitor modalities, and a PROTAC approach as a therapeutic may as well result in a cleaner inhibitory phenotype and better tolerability (1, 8), adding a layer of specificity to the non-selective inhibitors of a protein of interest for target classes that are inherently dealing with off-target effects (4). In general, PROTAC molecules' physicochemical property space falls beyond the 'rule of 5' owing to the bifunctional nature of the molecules, which is less advantageous for therapeutic use, even though several orally bioavailable compounds have been developed that fall outside the rule of 5 parameters (1). PROTACs thus tend to feature properties which can pose a challenge to permeability, such as a higher molecular mass and high number of hydrogen bond donors and acceptors. However, despite the perceived adversities, many PROTACs are capable of efficiently entering cells and achieve sufficient intracellular concentrations (8). In 2019, the first PROTAC molecules entered clinical testing, and currently proof-of-concept with a PROTAC against two well-established cancer targets, the estrogen receptor (ER, ARV-471) and the androgen receptor (AR, ARV-110), has been demonstrated (1). The search for effective PROTACs is hampered by their complexity and modular nature, since a PROTAC can be broken down into its three constituent parts, resulting in three variables requiring optimization. The more variants of E3 ligase ligands and linkers there are, the more laborious this becomes for PROTAC synthesis (8, 10). Even though the human genome is estimated to encode more than 600 E3 ligases, each with specificity for a different subset of proteins and/or a specific cellular expression profile (1), currently the von Hippel-Lindau tumor suppressor (VHL) and Cereblon (CRBN) ligands are the most popular and most frequently used E3 ligase ligands to design PROTAC degraders, due to their favorable features as small molecule drugs (6), including strong, specific and biophysically validated binding affinities to the targeted E3 ligase, an acceptable physicochemical profile, and well-characterized structural information of their binding modes (10). In fact, CRBN appears to be the preferred E3 ligase for the first wave of TPD therapeutics in clinical trials. Other exploitable 'generic' E3 ligases are RNF4 (ring finger protein 4), RNF114, KEAP1, FEM1B, DCAF15, DCAF16 and AhR, and several other ubiquitin ligases that await development as potential PROTACTable ligases (1,6), including MDIVI2, which is an E3 ligase that can be recruited exploiting the substrate receptor nutlin-3a, through binding MDIVI2 via the well-known nutlin compound. MDIVI2 could be particularly advantageous when used in PROTACs for its oncogenic role as a suppressor of p53 (2), inducing its ubiquitination and subsequent proteasomal degradation, though the number of MDIVI2 PROTACs has remained limited due to their challenging physicochemical profile and limited degradation activity (6). As an alternative to the successful PROTAC small molecule modalities applied in TPD, antibody and peptide drugs possess the advantages of high affinity and selectivity, but large molecular weight and poor membrane permeability make it difficult to succeed in an intracellular approach (7), thereby restricting their benefits as PROTAC-like entities for therapeutic development. Protein- or antibodybased target-binding moieties explored as PROTAC alternatives include for instance AbTACs (14) and LYTACs (15), which extend the protein degradation technology toolbox, including for example their use for target validation, but eventually also for the development of novel therapeutics, though these modalities are restricted to extracellular targets. Moreover, besides proteasomal degradation, other pathways for targeted protein degradation such as lysosomal degradation and autophagosomal targeting are explored in parallel. Takahashi et al. (16) indeed presented AUTACs as intracellularly targeted warheads to act via the autophagy-dependent degradation route, and also ATTECs (17) which link a protein-of-interest-targeting small molecule to an autophagy protein for inducing protein removal via this pathway.

The variations in protein degradation route as well as in the type of modality that is applied to bind a protein of interest seem endless, though, unexpected challenges and technical difficulties remain in finding and combining the most optimal molecule entities for translation to different therapeutic purposes. Among the explored protein-based modalities for intracellular TPD, Nanobody-based fusions referred to as the ARMeD system, for instance, provide for a target-specific Nb, coupled to the RING domain of the E3 ubiquitin ligase RNF4, which were shown to trigger degradation without off-target effects upon delivery into the cell (7, 19); further also GlueTACs as covalent antigen-binding Nanobodybased chimera targeting a membrane protein and conjugated to a cell-penetrating peptide and lysosomal sorting sequence for triggering lysosomal degradation were preclinically tested (18). Furthermore, bioPROTACs have been described which provide for fusions between a target-recognizing unit and an E3 ligase, rather than an E3 ligand (3), which can be expressed in a cell as to result in proteasomal degradation of the target via ternary complex formation. However, considerable challenges remain in the delivery of such proteins inside cells for use in the destruction of diseasecausing proteins.

Although PROTAC-based approaches are mainly established in the chemical small molecule space, exploration of new drug modalities for use in TPD is booming, although with outstanding challenges and reservations. The high engineerability of Alphabody® scaffolds has previously been demonstrated to facilitate a versatile usage of Alphabody molecules as potent target inhibitors and enabled the modification of Alphabody building blocks with additional functionalities such as cell-penetration and half-life extension features (13). These small proteins have the advantages of providing a high target specificity due to their antigen-binding domain, and allow intracellular targeting because of their small size, hydrophobic nature, and high engineerability. As for every upcoming modality, development of Alphabody therapeutics into the clinic has remained a challenge, and the suitability of Alphabody scaffolds for developing a TPD therapeutic modality is thus far from predictable.

Since there is a clear need to further explore and improve therapeutic TPD modalities capable of reaching intracellular targets for promoting their degradation, preferably through an acceptable delivery route, integrating the desired target-binding modalities, such as high selectivity and specificity, protein-based modalities are in scope. Ideally, these should be suitably combined with degrader units capable to reach their targets in a manner favorable for therapeutic use, all-in-all providing for the nextgeneration therapeutic TPD modalities.

SUMMARY OF THE INVENTION

The invention relates to a novel design of target-specific protein-based compounds which are capable of specifically binding a target protein intracellularly and promoting its degradation. The binding agents presented herein tackle the problems known for PROTACs, as mentioned herein, and overcome several hurdles for therapeutic use and efficient delivery of target-specific and target-selective degrader molecules into cells. A novel and unique modality of targeted protein degradation (TPD) is described herein by applying Alphabody proteins as target-binding building blocks as our preferred antigenbinding molecules with several attributes that favor Alphabody-based degraders over other existing TPD modalities. Indeed, Alphabody molecules are highly engineerable allowing the exploration of a large target space, highly formattable allowing the introduction of many functionalities including cell penetration moieties, half-life extension, cell targeting, bi-specificity, and highly robust allowing good survival in circulation and in intracellular environments (12, 13). The application of an Alphabody as target-binding moiety, in combination with a linked degrader entity, reveals a novel system of Alphabody-based degrader molecules. So, by delivering proof of concept for the combination of robust and highly-engineerable Alphabody technology integrated in novel formats of Alphabody-based degraders, and using thorough biological insight in molecular disease mechanisms, development of the Alphabody-based degrader molecules as intracellular TPD therapeutics is highly promising.

In a first aspect, the invention relates to a binding agent composed of one or more Alphabody molecules with a structure sequence of the formula HRS1-L1-HRS2-L2-HRS3, specifically binding a target protein or antigen, and a degrader unit, said Alphabody single chain protein and degrader moiety being connected directly or via a linker, wherein: said Alphabody protein has a structure sequence wherein each of HRS1, HRS2 and HRS3 is independently a heptad repeat sequence (HRS), forming an alpha-helix, comprising 2 to 7 consecutive heptad repeat units, said heptad repeat units being 7-residue fragments represented as 'abcdefg' or 'defgabc', the symbols 'a¹ to 'g' denoting conventional heptad positions, at least 50 % of all heptad a- and d-positions are occupied by isoleucine residues, each HRS starts and ends with an aliphatic or aromatic amino acid residue located at either a heptad a- or d-position, and/or wherein a threonine or arginine is located in the first a-position of any one of the HRS1, HRS2, and/or HRS3 and/or wherein a glutamine is positioned at the last d-position of any one of the HRS1, HRS2, and/or HRS3, wherein the Alphabody protein binds the target molecule via interaction with amino acid residues exposed at one or more of the alpha-helical HRS units, or with amino acids part of a region of a concave Alphabody groove, wherein each of LI and L2 are independently a linker fragment, which covalently connect HRS1 to HRS2 and HRS2 to HRS3, respectively, wherein said Alphabody comprises a cell-penetrating region, and wherein said binding agent is capable of entering a cell and upon binding of the target to the Alphabody binding site, triggers or enhances protein degradation of said target.

In one embodiment, the cell-penetrating region or entity of the Alphabody protein comprises or consists of: at least one positively charged internalization region mediating cellular uptake of the binding agent, wherein said internalization region is characterized by the presence of at least six positively charged amino acid residues, preferably arginine's, of which at least 50 % are comprised within said Alphabody structure sequence HRS1-L1-HRS2-L2-HRS3, and/or at least one peptide tag or fused entity for facilitating cellular entry of the binding agent.

A specific embodiment relates to said Alphabody-based degrader, wherein the cell-penetrating entity of the Alphabody protein comprises or consists of one or more peptide tags for facilitating cellular entry comprising the sequence (Arg-Pro)n, wherein n is an integer from 6 to 12. A further specific embodiment relates to said binding agent comprising the cell-penetrating entity as being a peptide tag comprising an (Arg-Pro)n sequence linked to the N- or C-terminal end of the Alphabody protein sequence, appearing as a single chain protein.

In a further embodiment, the invention relates to the Alphabody-based degrader wherein the degrader entity is a small entity, or small compound, as known in the art and defined herein, such as a small molecule or a peptide coupled to said Alphabody, wherein the coupling is made directly or via a linker, and specifically may be obtained via conjugation, for instance using maleimide or NHS-ester coupling, or enzymatic ligation. In a further embodiment, the Alphabody-based degrader described herein comprises a degrader moiety which is an E3 ligase ligand or E3 ligase complex ligand or E3 ligase complex binder (as used interchangeably herein) for scavenging the proteasomal complex through ubiquitination of the target protein bound to the Alphabody moiety and thereby resulting in proteasomal degradation of the target. In a specific embodiment, said degrader moiety specifically binds an E3 ligase binding site of any one of the E3 ligases selected from VHL or CRBN.

In a further embodiment, the binding agent, or Alphabody-based degrader molecule, as used interchangeably herein, may comprise a further entity, such as a functional moiety for detection of the molecule through a label or a tag, or a functional moiety for extending its half-life when present in a subject, or any alternative functional moiety, such as a further target binding moiety, or a further degrader entity or tag.

In a second aspect, the invention relates to nucleic acid molecules encoding any one of the Alphabodybased degrader molecules as described herein, or at least the single chain protein-moiety of the chemically conjugatable protein, wherein said nucleic acid molecule encodes the Alphabody-containing polypeptide for conjugation with a degrader moiety to result in the Alphabody-based degrader molecule as described herein.

A further aspect of the invention relates to the pharmaceutical composition comprising any of the binding agents described herein, or the Alphabody-based degrader molecules as described herein, or the nucleic acid molecule described herein, said composition optionally containing one or more pharmaceutically acceptable carriers, such as an excipient, or a diluent.

A further aspect relates to the use of the described products herein, such as Alphabody-based degrader agents, nucleic acids encoding the Alphabody-based degrader polypeptide portions, or the pharmaceutical composition described herein, for in vitro protein degradation of a target.

In a further aspect, further uses of the described products herein is disclosed, such as Alphabody-based degrader agents, nucleic acids encoding the Alphabody-based degrader polypeptide portions, or the pharmaceutical composition described herein, for use as a medicine, or for use in a method to treat a diseased subject are also aspects of the invention disclosed herein. Specifically, the use of any one of the Alphabody-based degrader agents, nucleic acids encoding the Alphabody-based degrader polypeptide portions, or the pharmaceutical composition described herein, in treatment of a disease or disorder, which is associated with said target molecule. In a specific embodiment, said Alphabodybased degrader is for use in treatment of a disease in the area of cancer, immune-oncology or inflammation. In a final aspect, a method is described to produce said Alphabody-based degrader molecule, comprising the steps of: a) providing a target-specific Alphabody protein scaffold structure, preferably obtained using an Alphabody library, or by design based on known antigen-binding proteins and Alphabody scaffolds, and/or using structural information available for the target and/or Alphabody binding agent of interest, b) formatting of the single chain Alphabody-based protein sequence for optimization of cellpenetration and/or target binding, c) introducing the nucleic acid molecule encoding said Alphabody-based protein in a cell for recombinant expression and purification of the Alphabody-based protein, and d) linking the degrader moiety, provided by chemical synthesis or recombinant production, to the Alphabody-based protein binding agent, preferably by conjugation or ligation, e) optionally, test the conjugated Alphabody-based degrader molecule for degradation activity on its target, preferably in a cell-based assay, for identifying the conjugated Alphabody-based degrader molecule design providing for degradation.

In a specific embodiment, the optional step e) provides for a parallel testing of a plurality of different Alphabody-based degrader molecules wherein the conjugation is made at a different position on the protein moiety, which may influence the ternary complex formation. The optional step e) is envisaged to allow for determining the optimal position of the conjugate on the protein-based binding agent, as to provide for functional and/or optimal Alphabody-based degrader molecule binding agents for forming the ternary complex with their target of choice and E3 ligase(complex).

DESCRIPTION OF THE FIGURES

The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Figure 1. MCL1 antibody analysis using WES system. A, monoclonal rabbit MCLl-specific Ab (clone D35A5; mAb #5453, Cell Signaling; 'MCLl CST') antibody reaches 90 % saturation at 1:30 dilution at 0.125 pg/pl, 0.5 pg/pl, and 2.0 pg/pl of HEK293T protein lysate. B, polyclonal rabbit anti-MCLl (A302- 715A-T; Bethyl Laboratories 'MCLl Bethyl') antibody shows 90 % saturation only at 1:10 at various lysate concentrations.

Figure 2. Linear increase of MCLl protein at different lysate concentrations. Measurement of Chemiluminescence value of antibody detection (Ab2 = MCLl CST antibody) at different antibody dilutions over a concentration range of 0.125 - 2.0 pg/pl concentration of HEK293T protein lysate. Figure 3. MCLl protein level in HEK cells after 24h treatment with MCL1 degraders. Normalized (using P-actin or vinculin protein levels) MCLl protein levels in MCLl-expressing HEK293T cells after a 24h treatment with a range of 100 nM - 3.5 pM concentrations of CMPX-558Ap-GR7, CMPX-558Bp-GR7, CMPX-326Ap-GR7 degrader molecules, or the negative control CPAB CMPX-321A.

Figure 4. MCLl protein levels in HEK cells after short-term treatment with MCLl degraders. HEK293T cells were treated within a range of 0- 3000 nM of the CMPX-558Ap-GR7 or CMPX-558Bp-GR7 for 4 hours. The MCLl protein levels were detected with the WES system using anti-MCLl antibody. The MCLl expression levels were normalized to COXIV protein levels.

Figure 5. MCLl protein degradation upon CMPX-558Ap-GR7 treatment. HEK293T cells were treated with the 0-3000 nM of CMPX-558Ap-GR7 for 24 or 48 hours. The MCLl protein levels were detected with the WES system using anti-MCLl antibody. The MCLl expression levels were normalized to total protein levels and shown as MEAN ± SEM, technical replicates N=2.

Figure 6. CMPX-558Ap-GR7 treatment of HEK293T cells decreases the MCLl protein level through proteasomal degradation. HEK293T cells were treated with 0, 50 or 150 nM of CMPX-558Ap-GR7 for 24 hours in the absence or presence of the proteasomal inhibitor MG132 (1 pM). The MCLl protein levels were detected with the WES system using anti-MCLl antibody. For quantification, the MCLl expression levels were normalized to total protein levels, as shown in A, and the virtual blot representations of total protein detection and immune detection of MCLl are shown in B.

Figure 7. CMPX-558Ap-GR7 treatment of myeloma H929 cells decreases the MCLl protein level. Myeloma H929 cells were treated with 0-1000 nM concentrations of CMPX-558Ap-GR7 for 24h or 48h. The MCLl protein levels were detected with the WES system using anti-MCLl antibody. The MCLl expression levels were normalized to total protein levels and shown as MEAN ± SEM, technical replicates N=3.

Figure 8. CMPX-558Ap-GR7 treatment of non-small-cell lung cancer H23 cells decreases the MCLl protein level. Non-small-cell lung cancer H23 cells treated with 0-1000 nM concentrations of CMPX- 558Ap-GR7 for 24h or 48h. The MCLl protein levels were detected with the WES system using anti- MCLl antibody. The MCLl expression levels were normalized to total protein levels and shown as MEAN ± SEM, technical replicates N=3.

Figure 9. Purified CMPX-326Ap-GR7, CMPX-558Ap-GR7 and CMPX-558Bp-GR7 proteins on SDS-PAGE.

Protein load on SDS-PAGE is 2.5 or 5 pg, and gel was stained using Coomassie brilliant blue. M, Protein molecular mass marker in kilodalton (kDa). Figure 10. Pull-down of MCL1 AlphaTAC molecules from whole cell lysate of the A549 cell line. (A) Immunoprecipitation of V5-tagged MCLl CPAB (321A; SEQ ID NO:5), MCLl CPAB-based degrader 558Ap-GR7 (conjugated protein SEQ. ID NO:2), and a mutated MCLl CPAB-based degrader 584Cp-GR7 (conjugated protein SEQ ID NO: 4; used herein as a negative control for (strong) MCLl binding) followed by immunoblotting for MCLl, VHL and V5 (input control). (B) immunoblotting for MCLl and VHL in the whole cell lysate (WCL). V5 PD: V5 pull-down, data representative of two repeats.

Figure 11. Design of hybrid CPAB-based degraders. Graphical scheme of various designs of hybrid cellpenetrating Alphabody (CPAB)-based degraders (for an MCLl binder; alternatively, a CPAB specifically targeting another protein is used instead, such as an MDM4/2 Alphabody protein as shown in Table 3); the star marks the conjugation position. Nt, Amino-terminus, Ct, Carboxy-terminus, nRP7, N-terminal cationization region; cRP7, C-terminal cationization region.

Figure 12. SDS-PAGE of purified unconjugated hybrid CPAB-based degrader constructs. 5 pg purified protein sample (prior to conjugation) in reduced or non-reducing conditions was loaded on the gel, and profiles were compared with a molecular weight marker, for which corresponding MW are indicated in kDa. The sample ID is indicated by the construct number as provided in Table 3.

Figure 13. MDM4-HiBiT protein expression upon treatment with distinct MDM2/4 AlphaTAC molecules. (Left) Relative expression level of MDM4-HiBiT after 48h of treatment with distinct MDM2/4 CPAB-based degrader molecules at a concentration range of 0 - 300 nM. Data are shown as MEAN ± SEM, biological replicates were done (N=3); data representative of two repeats. * p<0,05 and ** p<0,01 by one-way ANOVA. (Right) Graphical scheme of the designs of each respective hybrid CPAB- based degrader (for a MDM2/4 binder) evaluated in this experimental assay. 632G corresponds to SEQ ID NO: 13; 632J to SEQ ID NO:16; 632L to SEQ ID NO:18. RP7: cationization region, V5: V5 tag.

Figure 14. Hybrid AlphaTAC molecules against MDM4/2 are capable of reducing the MDIVI2 protein levels in the A549 lung cancer cell line by approximately 50 % at nM concentration. A549 lung cancer cell line was treated for 48h with different concentrations ranging from 0-1000 nM of MDM4/2 AlphaTAC molecule 632L-AHPC (conjugated SEQ ID NO:18). The MDM2 protein levels were determined via western blot. Vinculin was used as a loading control. Data representative of two repeats.

Figure 15. Pull-down of MDM2/4 AlphaTAC molecules from whole cell lysate of A549 cell line. (A) Immunoprecipitation of a V5-tagged MDM2/4 CPAB (632*, which is an unconjugated 632-based control MDM4/2 CPAB) and MDM2/4 CPAB-based degrader 632H-Thal (conjugated SEQ ID NO: 14), followed by immunoblotting for MDM4 and Cereblon (CRBN). (B) Immunoprecipitation of a V5-tagged MDM2/4 CPAB (632*) and MDM2/4 CPAB-based degrader 632H-Thal, followed by immunoblotting for MDM2 and Cereblon (CRBN). (C) immunoblotting for MDM4 and Cereblon (CRBN) in the whole cell lysate (WCL). V5 PD: V5 pull-down, Data representative of two repeat.

Figure 16. Cellular uptake of MDM2/4-specific hybrid AlphaTAC molecules. HeLa cells were treated with increasing concentrations of several different MDM2/4 CPAB-based degrader molecules (conjugated 632G -SEQ ID NO: 13 and conjugated 632L- SEQ ID NO: 18). Cellular uptake was determined by fluorescent imaging of the V5 tag. Uptake was compared to that of an MCLl specific Alphabody molecule (453E -SEQ ID NO:19) and MCLl- and MDM2/4-targeting CPAB molecules 321A (SEQ ID NO:5) and 6321 (SEQ ID No: 15), respectively. Data are shown as MEAN ± SEM. Technical replicates N=2. ** p<0,01 and *** p<0,001 by one-way ANOVA.

Figure 17. Annotated alignment of Alphabody constructs. MCLl specific Alphabody protein construct CMPX-558A (SEQ ID NO:2), and different conjugation variants of MDM4/2-specific Alphabody protein 632 constructs (SEQ ID NO: 13, 14, 16, and 18) were aligned using ClustalW to demonstrate the general consensus formula used in the application for Alphabody polypeptides and optionally N- and/or C- terminal additions or tags. Residue in bold is used for conjugation (Cys for CMPX-558A at position 22 for maleimide coupling; Lys (or N-terminal residue) for 632 Alphabody constructs for NHS coupling). Annotation of the consensus formula used for the binding agent Alphabody constructs: His8 tag (italic); TEV cleavage site (SEQ ID NO:22; italic underlined); RP7 (SEQ ID NO: 33; underlined); V5 tag (SEQ ID NO:21;°); the Alphabody structure: HRS1-L1-HRS2-L2-HRS3 as indicated in blocks per HRS, each consisting here of 4 heptad repeats (a to g; with 'a' and 'd' positions indicated per heptad). The short linkers used in between each segment or a build from a (plurality of) GlySer or GlyAla residues, as flexible linker, optionally including a Proline or Methionine, or a K (lys) or C (cys) as conjugation residue. LI and L2 linkers used in 632 are examples of 'designed' or optimized linkers used herein.

DETAILED DESCRIPTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim.

Definitions

Where an indefinite or definite article is used when referring to a singular noun e.g., "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology).

"Nucleotide sequence", "nucleic acid molecule(s)", or "DNA/RNA sequence" as used herein refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and singlestranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog. By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. "Coding sequence" is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. Such cassettes can be constructed into a "vector".

The terms "protein" or "polypeptide", are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same, preferably said polypeptide sequence having a length of at least 91, at least 100, or more than 110 amino acid residues. A "peptide" may be referred to herein as any amino acid residue sequence of small size, hence not the size of a polypeptide or protein, or may also be referred to as a partial amino acid sequence derived from its original protein for instance after tryptic digestion. The peptide length is defined herein as an amino acid sequence which can be produced in itself or exists 'as such', so is not part of a larger protein, and is limited to a peptide sequence length with a maximum of 10 amino acids, 20, 30, 40, 50, 60, 70, 80 or to maximum of 90 amino acids. These terms defined herein also apply to amino acid peptides or polypeptides or polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the (poly)peptide, such as glycosylation, phosphorylation and acetylation, and also myristoylation. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton (kDa). A "protein domain" is a distinct functional and/or structural unit in a protein. Usually, a protein domain is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions.

By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide which has been purified from the molecules which flank it in a naturally-occurring state, e.g., a binding agent or Alphabody-based degrader molecule as identified and disclosed herein which has been removed from the molecules present in a sample or mixture, such as a production host, that are adjacent to said binding agent. An isolated binding agent can be generated by amino acid chemical synthesis and optionally further chemical linkage, and in case of a single chain protein can be generated by recombinant production or by purification from a complex sample.

The term "linked to", or "fused to", as used herein, and interchangeably used herein as "connected to", "conjugated to", "ligated to" refers, in particular, to "genetic fusion", e.g., by recombinant DNA technology, as well as to "chemical and/or enzymatic conjugation" resulting in a stable covalent link. Preferably, "fused to" refers to a genetic fusion, while "conjugated to" rather refers to a chemical and/or enzymatic conjugation.

"Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison.

Amino acids are presented herein by their 3- or 1-lettercode nomenclature as defined and provided also in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138: 9-37 (1984)); as follows: Alanine (A or Ala), Cysteine (C or Cys), Aspartic acid (D or Asp), Glutamic acid (E or Glu), Phenylalanine (F or Phe), Glycine (G or Gly), Histidine (H or His), Isoleucine (I or He), Lysine (K or Lys), Leucine (L or Leu), Methionine (M or Met), Asparagine (N or Asn), Proline (P or Pro), Glutamine (Q. or Gin), Arginine (R or Arg), Serine (S or Ser), Threonine (T or Thr), Valine (V or Vai), Tryptophan (W or Trp), and Tyrosine (Y or Tyr).

Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the percentage of identity is calculated over a window of the full-length sequence referred to. A "substitution", or "mutation", or "variant" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity, which is hereby defined as a 'functional variant'.

The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified", "mutant", "engineered" or "variant" refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wildtype gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, favorably associates with another chemical entity or binding domain, such as a protein, Alphabody, or a degrader moiety, among others.

"Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders. The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding. A "binding agent", or "agent" as used interchangeably herein, relates to a molecule that is capable of binding to another molecule, via a binding region or binding domain located on the binding agent, wherein said binding is preferably a specific binding, recognizing a defined binding site, pocket or epitope. The binding agent may be of any nature or type and is not dependent on its origin. The binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as designed and synthetically produced. Said binding agent may hence be a small molecule, a chemical, a peptide, a polypeptide, an antibody, an Alphabody, or any hybrid structure derived of any one thereof, or any derivatives thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others.

A "compound" as defined herein includes but is not limited to binding agents that may be "small molecules", which refers to a low molecular weight (e.g., < 900 Da or < 500 Da) organic compound. The compounds or binding agents also include chemicals, polynucleotides, lipids or hormone analogs that are characterized by low molecular weights. Other biopolymeric organic compounds include small peptides or peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and/or larger (poly)peptides comprising from 40 to maximally 90 amino acids, such as for instance antibody mimetics, fragments, or conjugates, preferably maximally 30 amino acids.

Antibody mimetics are organic compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Examples of antibody mimetics include but are not limited to: Affibodies, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Gastrobodies, Kunitz domain peptides, Monobodies, Optimers, and Obodies among others.

Specifically, the binding agents of the present invention comprise one or more Alphabody proteins, which provide for antibody mimetics specifically binding to a target of interest via the binding moiety described herein, preferably present in the alpha-helical regions of said Alphabody scaffold, and structurally unrelated to antibodies. Alphabody polypeptides typically have a triple-stranded alphahelical coiled coil structure which is suitable as a scaffold structure. An Alphabody polypeptide surface consists of pre-shaped regions such as concave grooves, convex alpha-helical surfaces, and (flexible) linker regions. Any such region, or a combination thereof, can be modified for different purposes, including the design or selection of target binding sites, modification of global charge and/or polarity and/or posttranslational modifications, and/or inclusion of further functionalities.

As used herein, a "therapeutically active agent" or "therapeutically active composition" means any molecule or composition of molecules that has or may have a therapeutic effect (i.e., curative or prophylactic effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non-cytotoxic agent. Even more preferably, a therapeutically active agent has a curative effect on the disease. The binding agent or the composition, or pharmaceutical composition of the invention may act as a therapeutically active agent, when beneficial in treating patients with a disease related to the target of the Alphabody-based degrader as described herein, or patients suffering from another disease. The therapeutically active agent/binding agent or composition may include an agent comprising an Alphabody specifically binding the human target, such as for instance MCL1 or MDM4/2 as described herein, and/or may contain or be coupled to additional functional groups, or functional moieties advantageous when administrated to a subject. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the art as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments, for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups may for example be linked directly (for example covalently) to the Alphabody, the degrader moiety, or any further part of said binding agent, such as a tag, cationization or functional moiety present in the binding agents described herein, or optionally via a suitable linker or spacer, as will again be clear to the skilled person. One of the most widely used techniques for increasing the half-life and/or reducing immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the antibody or active antibody fragment. Another technique for increasing the half-life of a binding domain may comprise the engineering into bi-or multi-functional or bi- or multispecific domains (for example, one Alphabody against the target of interest for degradation, one degrader moiety for binding to an E3 ligase, and one moiety against a serum protein such as albumin aiding in prolonging half-life) or into fusions, with peptides (for example, a peptide against a serum protein such as albumin).

As used herein, the terms "determining," "measuring," "assessing,", "identifying", "screening", and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

The term "subject", "individual" or "patient", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, a subject is a subject with or suspected of having a disease or disorder, in particular a disease or disorder as disclosed herein, also designated "patient" herein. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.

The term "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness. Treatment may also refer to a prophylactic treatment which relates to a medication, or a treatment designed and used to prevent a disease from occurring.

A "composition" relates to a combination of one or more active molecules, and may further include buffered solutions and/or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance. Suitable conditions as used herein could also refer to suitable binding conditions, for instance when Alphabody-based degrader molecules are aimed to bind their target of interest for stimulating its degradation.

A pharmaceutical composition comprising the one or more binding agents or therapeutic agents, or nucleic acid molecule(s) as provided herein, optionally comprise a carrier, diluent or excipient. A "carrier", or "adjuvant", in particular a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non- exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavoring agents or colorants. A "diluent", in particular a "pharmaceutically acceptable vehicle", includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles. A pharmaceutically effective amount of polypeptides, or conjugates of the invention and a pharmaceutically acceptable carrier is preferably that amount which produces a result or exerts an influence on the particular condition being treated. For therapy, the pharmaceutical composition of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including orally, parenterally, topically, nasally, ophthalmically, intrathecally, intracerebroventricularly, sublingually, rectally, vaginally, and the like. Still other techniques of formulation such as nanotechnology and aerosol and inhalant are also within the scope of this invention. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counter-indications and other parameters to be considered by the clinician. The pharmaceutical composition of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. When prepared as lyophilization or liquid, physiologically acceptable carrier, excipient, stabilizer need to be added into the pharmaceutical composition of the invention (Remington's Pharmaceutical Sciences 22nd edition, Ed. Allen, Loyd V, Jr. (2012). The dosage and concentration of the carrier, excipient and stabilizer should be safe to the subject (human, mice and other mammals), including buffers such as phosphate, citrate, and other organic acid; antioxidant such as vitamin C, small polypeptide, protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as PVP, amino acid such as amino acetate, glutamate, asparagine, arginine, lysine; glycose, disaccharide, and other carbohydrate such as glucose, mannose or dextrin, chelate agent such as EDTA, sugar alcohols such as mannitol, sorbitol; counterions such as Na+, and /or surfactant such as TWEEN™, PLURONICS™ or PEG and the like.

Detailed description

The present application provides for a novel modality introduced in the field of targeted protein degradation (TPD) usable for intracellular protein targeting, acting preferably through proteasomal degradation, making use of cell-penetrating Alphabody-based molecules. The high engineerability of Alphabody scaffolds has previously been demonstrated to facilitate a versatile usage of Alphabody molecules as potent target inhibitors and enabled the modification of Alphabody building blocks with additional functionalities such as cell-penetration and half-life extension features (13). The field of PROTAC design and targeted protein degradation is growing and drug discovery on previously undruggable targets is under exploration, thereby providing opportunities beyond target inhibition. Alphabody protein products have been developed as selective, target-specific potent inhibitors, but so far, Alphabody proteins have never been tested or altered for the purpose of promoting their targets' degradation. The present invention for the first time describes Alphabody molecules which were engineered with the aim to functionally induce protein degradation of their target, while retaining also the specific and selective target-binding capacity, thereby demonstrating that functional Alphabody- mediated target inhibition can be adapted into removal or degradation of the target, and in particular, of an intracellular target, when cell-penetrating alphabodies (CPAB) are used. The combination of different features that facilitate cellular uptake in combination with fusion with E3 ligase binders to trigger or enhance degradation of the target upon binding with the Alphabody targeting moiety was for the first time shown in this application. Such a novel TPD modality may thus be further explored for therapeutic use and was also established herein as a binding agent called an 'Alphabody-based degrader molecule' modality, defined herein, and also referred to herein as AlphaTAC®, revealing protein degraders with high specificity for their target or protein of interest, and efficiently acting as potent degraders of the target.

Alphabody scaffold structure

Alphabody molecules not only have a unique and defined structure but also have several advantages over the traditional protein-based scaffolds and therapeutic modalities known in the art. These advantages include, but are not limited to, the fact that they are compact and small in size (between 10 and 14 kDa), they are extremely thermostable (i.e., they generally have a melting temperature of more than 100° C), they can be made resistant to different proteases, they are highly engineerable, specifically in the sense that multiple substitutions will generally not obliterate their correct and stable folding, and have a structure which is based on natural motifs which have been redesigned via protein engineering techniques. Furthermore, Alphabody polypeptides or Alphabody proteins, as used interchangeably herein, typically have a triple-stranded alpha-helical coiled coil structure which is suitable as a scaffold structure. An Alphabody polypeptide surface consists of pre-shaped regions such as concave grooves, convex alpha-helical surfaces, and (flexible) linker regions. Any such region, or a combination thereof, can be modified for different purposes, including the design or selection of target binding sites, modification of global charge and/or polarity and/or posttranslational modifications. In addition, the termini of the Alphabody polypeptide can be appended with different tags, such as recognition tags or cell-penetrating peptide sequence motifs for intracellular delivery, and expanded by further fusions to functional moieties such as a half-life extension, or a degrader moiety. In brief, the Alphabody protein scaffold, as used herein, consists of a contiguous polypeptide that folds into a three-helix coiled-coil with antiparallel helix topology, and its entire scaffold surface can be deployed to generate a binding surface for a given target. Second generation target-inhibitory Alphabody polypeptides have been produced as cell-penetrating Alphabody (CPAB) proteins and are efficiently taken up by cells, thereby addressing a fundamental challenge in the targeting of intracellular drug targets by protein-based therapeutics. The CPABs comprise a cell-penetrating moiety which may be composed of a cationization region or internalization region that is at least partially present within the HRS1-L1-HRS2-L2-HRS3 sequence structure of the Alphabody polypeptide, and ensures internalization through the presence of several positively charged amino acid residues, to arrive at an Alphabody sequence structure that is neutral in charge, or overall positively charged (i.e., not negatively charged). Alternatively, the CPABs presented herein may comprise a cell-penetrating moiety which may be composed of a cell-penetrating peptide, as further defined herein. Alternatively, the CPABs presented herein may comprise one or more cationization or internalization regions and one or more cell-penetrating peptides, together acting as cell-penetrating moiety of the CPAB.

The binding agent as described herein comprises an Alphabody polypeptide which functions as targetbinding moiety, wherein said Alphabody protein scaffold thus consists of the structural formula HRS1- L1-HRS2-L2-HRS3, and wherein it is preferably an anti-parallel Alphabody polypeptide wherein the HRS1, HRS2 and HRS3 together form a single-chain triple-stranded, predominantly alpha-helical, coiled coil structure, wherein each of the heptad repeat sequences (HRS) HRS1, HRS2 and HRS3 is independently a heptad repeat sequence that is characterized by at least 2, not necessarily identical, heptad repeat units of 7-residue (poly)peptide fragments represented as 'abcdefg' or 'defgabc', wherein the symbols 'a¹ to 'g' denote conventional heptad positions at which amino acid residues are located, and at least 50 %, 70 %, 90 %, or 100 % of the conventional heptad positions 'a' and 'd ' are occupied by amino acids selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, threonine, serine, alanine or derivatives thereof, wherein at least 50 %, 70 %, 90 %, or wherein 100 % of the conventional heptad positions 'b', 'c', 'e', 'f and 'g' are occupied by amino acids selected from the group consisting of glycine, alanine, cysteine, serine, threonine, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, arginine or derivatives thereof, the resulting distribution of amino acid residues enabling the identification of said heptad repeat sequences, and optionally a C-terminal partial heptad repeat ending with a heptad core residue located at an a- or d-position, and each of LI and L2 is independently a linker fragment, which covalently connects HRS1 to HRS2 and HRS2 to HRS3, respectively.

More particularly, an Alphabody structure as used in the context of the target-specific binding agent presented herein, can be defined as an amino acid sequences having the general formula HRS1-L1- HRS2-L2-HRS3, wherein each of HRS1, HRS2 and HRS3 is independently a heptad repeat sequence (HRS) comprising or consisting of 2 to 7 consecutive but not necessarily identical heptad repeat units, at least 50 % of all heptad a- and d-positions are occupied by isoleucine residues, each HRS starts and ends with an aliphatic or aromatic amino acid residue located at a heptad a-position or d-position; and/or wherein threonine or arginine is located at the first a-position of any of said HRS1, HRS2 and/or HRS3, and/or glutamine is located at the last d-position of any of said HRS1, HRS2 and/or HRS3, each of LI and L2 are independently a linker fragment, as further defined hereinafter, which covalently connect HRS1 to HRS2 and HRS2 to HRS3, respectively.

The Alphabody structure as envisaged herein is restricted to 3-stranded coiled coils. The coiled coil region in an Alphabody polypeptide can be organized with all alpha-helices in parallel orientation (corresponding to a 'parallel Alphabody' as described in EP2161278 by Complix NV) or with one of the three alpha-helices being antiparallel to the two others (corresponding to an 'antiparallel Alphabody' as described in EP2367840 by Complix NV). The alpha-helical part of an Alphabody structure (as defined herein) will usually grossly coincide with the heptad repeat sequences although differences can exist near the boundaries. For example, a sequence fragment with a clear heptad motif can be non-helical due to the presence of one or more helix-distorting residues (e.g., glycine or proline). Reversely, part of a linker fragment can be alpha-helical even though it is located outside a heptad repeat region. Further, any part of one or more alpha-helical heptad repeat sequences is also considered an alphahelical part of a single-chain Alphabody.

As used herein, an 'antiparallel Alphabody' refers to an Alphabody as defined above, further characterized in that the alpha-helices of the triple-stranded, alpha-helical, coiled coil structure together form an antiparallel coiled coil structure, i.e., a coiled coil wherein two alpha-helices are parallel, and the third alpha-helix is antiparallel with respect to these two helices. As will become clear from the further description herein, the Alphabodies envisaged herein comprise an amino acid sequence with the general formula HRS1-L1-HRS2-L2-HRS3, wherein HRS1 provides for alpha-helix A, HRS2 for alpha-helix B and HRS3 for alpha-helix C, respectively, but which in certain particular embodiments may comprise additional residues, moieties and/or groups which are covalently linked, more particularly N- and/or C-terminally covalently linked to a basic Alphabody sequence structure having the formula HRS1-L1-HRS2-L2-HRS3, and/or conjugated at any amino acid position within said Alphabody structure sequence. Thus, reference is made herein generally to 'Alphabody' or 'Alphabody polypeptides', or 'Alphabody proteins' which comprise or consist of an Alphabody as defined above, which may be covalently linked to additional sequences. The binding features described for an Alphabody herein can generally also be applied to polypeptides comprising said Alphabody. In one embodiment, the binding agent as described herein comprises at least one Alphabody protein specifically binding a target molecule linked to a degrader moiety, as described herein, wherein the Alphabody protein is an 'anti-parallel Alphabody' thus comprising a single-chain protein consisting of the formula HRS1-L1-HRS2-L2-HRS3, wherein HRS1, LI, HRS2, L2 and HRS3 represent amino acid sequence fragments that are covalently interconnected, said protein spontaneously folding in aqueous solution by way of the HRS1, HRS2 and HRS3 fragments forming a triple-stranded, antiparallel, alphahelical coiled coil structure of helix A, B, and C, resp., and wherein each of HRS1, HRS2 and HRS3 is independently a heptad repeat sequence that is characterized by a n-times repeated 7-residue pattern of amino acid types, represented as (a-b-c-d-e-f-g-)n or (d-e-f-g-a-b-c-)n and optionally a C-terminal partial heptad repeat ending with a heptad core residue located at an a- or d-position, wherein the pattern elements 'a' to 'g' denote conventional heptad positions at which said amino acid types are located and n is a number equal to or greater than 2, and at least 50 %, 70 %, 90 %, or 100 % of the conventional heptad positions 'a' and 'd' are occupied by isoleucine, wherein at least 50 %, 70 %, 90 %, or wherein 100 % of the conventional heptad positions 'b', 'c', 'e', 'f and 'g' are occupied by amino acids selected from the group consisting of glycine, alanine, cysteine, serine, threonine, histidine, asparagine, aspartic acid, glutamine, glutamic acid, lysine, arginine or derivatives thereof, the resulting distribution of amino acid types enabling the identification of said heptad repeat sequences, and and each HRS starts and ends with an aliphatic or aromatic amino acid residue located at either a heptad a- or d- position, and/or wherein the first 'a' of each HRS may be threonine or arginine and/or the last 'd' of each HRS may be glutamine, and each of LI and L2 is independently a linker, which may be a flexible linker consisting of 1 to 30 amino acid residues, this linker including any amino acid residue that cannot be unambiguously assigned to a heptad repeat sequence, and LI and L2 have an amino acid composition comprising at least 50 % amino acids selected from the group consisting of glycine, alanine, serine, threonine, proline or derivatives thereof, or alternatively LI and/or L2 may be 'designed' linkers as defined herein.

The terms 'heptad¹, 'heptad unit', or 'heptad repeat unit' are used interchangeably herein and shall herein have the meaning of a 7-residue (poly)peptide motif that is repeated two or more times within each heptad repeat sequence of an Alphabody structure, and is represented as 'abcdefg' or 'defgabc', wherein the symbols 'a' to 'g' denote conventional heptad positions. As understood by the skilled person this implies that the consecutive heptad units within a repeat need not contain the same amino acids but will contain the same type of amino acid (hydrophobic vs. polar, as detailed below) at the same position. Conventional heptad positions are assigned to specific amino acid residues within a heptad, a heptad unit, or a heptad repeat unit, present in an Alphabody structure, for example, by using specialized software such as the COILS method of Lupas et al. (Science 1991, 252:1162-1164; htt ps ://em bn et . vita I it. ch/software/COILS_form.html). However, it is noted that the heptads as present in the Alphabody structure are not strictly limited to the above-cited representations (i.e., 'abcdefg' or 'defgabc') as will become clear from the further description herein and in their broadest sense constitute a 7-residue (poly)peptide fragment per se, comprising at least assignable heptad positions a and d.

The terms 'heptad a-positions', 'heptad b-positions', 'heptad c-positions', 'heptad d-positions', 'heptad e-positions', 'heptad f-positions' and 'heptad g-positions' refer respectively to the conventional 'a', 'b', 'c', 'd ', 'e', 'f' and 'g' amino acid positions in a heptad, heptad repeat or heptad repeat unit. A heptad motif (as defined herein) of the type 'abcdefg' is typically represented as 'HPPHPPP' (SEQ ID NO: 27), whereas a 'heptad motif' of the type 'defgabc' is typically represented as 'HPPPHPP' (SEQ. ID NO: 28), wherein the symbol 'H' denotes an apolar or hydrophobic amino acid residue (including amino acids Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, and/or Tryptophan) and the symbol 'P' denotes a polar or hydrophilic amino acid residue (including Serine, Threonine, Cysteine, Asparagine, Glutamine and/or Tyrosine). Typical hydrophobic residues located at a- or d- positions include aliphatic (e.g., leucine, isoleucine, valine, alanine, methionine) or aromatic (e.g., phenylalanine, tryptophan, tyrosine, histidine) amino acid residues. Heptads within coiled coil sequences do not always comply with the ideal pattern of hydrophobic and polar residues, as polar residues are occasionally located at ' H ' positions and hydrophobic residues at 'P' positions. Thus, the patterns 'HPPHPPP' and 'HPPPHPP' are to be considered as ideal patterns or characteristic reference motifs. A 'heptad repeat sequence' ('HRS') as used herein shall have the meaning of an amino acid sequence or sequence fragment comprising or consisting of n consecutive heptads, where n is a number equal to or greater than 2.

A heptad repeat sequence (HRS) can thus generally be represented by (abcdefg)n or (defgabc)n in notations referring to conventional heptad positions, or by (HPPHPPP)n or (HPPPHPP)n in notations referring to the heptad motifs, with the proviso that a) the amino acids at positions a-g or H and P need not be identical amino acids in the different heptads, b) not all amino acid residues in a HRS should strictly follow the ideal pattern of hydrophobic and polar residues, and c) the HRS may end with an incomplete or partial heptad motif. With regard to the latter, in particular embodiments, the HRS may contain an additional sequence "a", "ab", "abc", "abed", "abede", or "abedef" following C-terminally of the (abcdefg)n sequence. However, in particular embodiments of the Alphabody structure as envisaged herein, a 'heptad repeat sequence' ('HRS') is an amino acid sequence or sequence fragment comprising n consecutive (but not necessarily identical) heptads generally represented by abcdefg or defgabc, where n is a number equal to or greater than 2, wherein at least 50 % of all heptad a- and d-positions are occupied by isoleucine residues, each HRS starting with a full heptad sequence abcdefg or defgabc, and ending with a partial heptad sequence abed or defga, such that each HRS starts and ends with an aliphatic or aromatic amino acid residue located at either a heptad a- or d-position. In order to identify heptad repeat sequences, and/or their boundaries, these heptad repeat sequences comprising amino acids or amino acid sequences that deviate from the consensus motif, and if only amino acid sequence information is at hand, then the COILS method of Lupas et al. (Science 1991, 35 252:1162-1164) is a suitable method for the determination or prediction of heptad repeat sequences and their boundaries, as well as for the assignment of heptad positions. Furthermore, the heptad repeat sequences can be resolved based on knowledge at a higher level than the primary structure (i.e., the amino acid sequence). Indeed, heptad repeat sequences can be identified and delineated on the basis of secondary structural information (i.e. alpha-helicity) or on the basis of tertiary structural (i.e., protein folding) information. A typical characteristic of a putative HRS is an alpha-helical structure. Another (strong) criterion is the implication of a sequence or fragment in a coiled coil structure. Any sequence or fragment that is known to form a regular coiled coil structure, i.e., without stutters or stammers as described in Brown et al. Proteins 1996, 26:134-145, is herein considered a HRS. Also, and more particularly, the identification of HRS fragments can be based on high-resolution 3-D structural information (X-ray or NMR structures). Finally, in particular embodiments, the boundaries to an HRS fragment may be defined as the first a- or d-position at which a standard hydrophobic amino acid residue (selected from the group valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan) is located. In particular embodiments, the boundaries to an HRS fragment can be defined by the presence of an isoleucine amino acid residue.

The heptad repeat sequences (HRS) of the Alphabody scaffold as used herein, in one embodiment specifically refers to a structural formula wherein said 'abedefg' or 'defgabc' is represented as any one of the following amino acid sequences selected from IAAISRR, IAAIERQ, TEQ.IQ.Q.R or RQAIQQA, or as exemplified in the Alphabody sequences used herein (SEQ. ID NOs: 1-20), wherein heptad sequences or heptad fragments , as used interchangeably herein, are present as annotated (for instance, but not limited to, as shown in the alignment of Figure 17 for a number of the exemplified Alphabody polypeptides used herein).

In the context of the single-chain structure of the Alphabodies (as defined herein) the terms 'linker', 'linker fragment' or 'linker sequence' are used interchangeably herein and refer to an amino acid sequence fragment that is part of the contiguous amino acid sequence of a single-chain Alphabody structure HRS1-L1-HRS2-L2-HRS3, as the LI and/or L2 linker, and covalently interconnects the HRS sequences of that Alphabody structure. The linkers within a single-chain structure of the Alphabodies (as defined herein) thus interconnect the HRS sequences, and more particularly the first to the second HRS, by LI, and the second to the third HRS, by L2, in an Alphabody structure. Each linker sequence in an Alphabody structure commences with the residue following the last heptad residue of the preceding HRS and ends with the residue preceding the first heptad residue of the next HRS. Connections between HRS fragments via disulfide bridges or chemical cross-linking or, in general, through any means of interchain linkage (as opposed to intra-chain linkage), are explicitly excluded from the definition of a LI or L2 linker fragment (at least, in the context of an Alphabody) because such would be in contradiction with the definition of a single-chain Alphabody. Further in the context of an Alphabody, 'Ll' shall denote the linker fragment one, i.e., the linker between HRS1 and HRS2, whereas ' L2' shall denote the linker fragment two, i.e., the linker between HRS2 and HRS3. The two linkers LI and L2 in a particular Alphabody structure may be the same or may be different. Suitable linkers for use in the polypeptides envisaged herein will be clear to the skilled person, and may generally be any linker used in the art to link amino acid sequences, as long as the linkers are structurally flexible (called 'flexible linkers' herein), in the sense that they do not affect the characteristic three dimensional coiled coil structure of the Alphabody. Based on the further disclosure herein, the skilled person will be able to determine the optimal linkers, optionally after performing a limited number of routine experiments. In particular embodiments, the linkers LI and L2 are amino acid sequences consisting of at least 4, in particular at least 8, more particularly at least 12 amino acid residues, with a non-critical upper limit chosen for reasons of convenience being about 30 amino acid residues. In a preferred embodiment, a linker fragment is between 1 and 35 amino acids, between 2 and 33, between 3 and 32, between 4 and 30, between 6 and 25, between 7 and 22, between 8 and 20, between 9 and 18, or between 10 and 15 amino acids long.

It is envisaged that the nature of the linker is not critical with regard to the binding properties of the polypeptides envisaged herein. A linker fragment in an Alphabody structure defined herein as LI or L2 as part of the single chain formula HRS1-L1-HRS2-L2-HRS3 may be a 'flexible linker' in conformation to ensure relaxed (unhindered) association of the three heptad repeat sequences as an alpha-helical coiled coil structure. In particular embodiments, the 'flexible' linker sequences are glycine and serine- rich sequences with a minimum length of about 2, 4, 6 or preferably 8 amino acids. In particular embodiments the linkers comprise 1 or 2 repeats of a "glycine/serine-rich" sequence such as GGSGGSGG (SEQ ID NO: 29) or GSGGGGSG (SEQ ID NO: 30). The linker connecting HRS1 and HRS2, referred to as "linker 1", may be the same or different from the linker connecting HRS2 and HRS3, referred to as "linker 2". In particular embodiments, linker 1 is (GGSGGSGG)n, wherein n = 1 or 2 and I in ker 2 is (GSGGGGSG)n, wherein n = 1 or 2. Such linkers need not be composed only of Gly/Ser residues and in addition typically contain one or more amino acids N- and/or C-terminally thereof. Such linkers include, but are not limited to TGGSGGSGGMS (SEQ ID NO: 31) and TGGSGGSGGGGSGGSGGMS (SEQ ID NO: 32), or any further alternative as also exemplified in Complix patent applications as listed herein, or as exemplified herein, for instance using Gly-Ala linkers. A linker fragment in an Alphabody structure defined herein as LI or L2 as part of the single chain formula HRS1-L1-HRS2-L2-HRS3 may further also be a 'designed' linker in a conformation to ensure suitable (unhindered) association of the three heptad repeat sequences as an alpha-helical coiled coil structure. In particular embodiments, the 'designed linker' sequence is an optimized and a short sequence of maximally 6 residues, preferably a designed 'Ll' sequence, with LI as defined in the Alphabody structure sequence used herein, HRS1-L1-HRS2-L2- HRS3, which is a designed LI sequence of 6 residues between the last HRS of the first helix (HRS1) and the first heptad repeat of the second helix (HRS2), and likewise, designed 'L2' sequence, with L2 as defined in the Alphabody structure sequence used herein, HRS1-L1-HRS2-L2-HRS3, which is a designed L2 sequence of 3 residues between the last HRS of the second helix (HRS2) and the first heptad repeat of the third helix (HRS3), as exemplified for instance for LI and L2 in the sequence of the 632-based Alphabody constructs, as shown in Figure 17.

Alphabody as target-binding moiety

The 'Alphabody-based degrader molecule' or 'AlphaTAC', or 'binding agent', as interchangeably used and presented herein, comprises at least one Alphabody polypeptide which specifically binds a target protein, thus the Alphabody structure sequence as used herein provides for a binding site with the target or protein of interest for which degradation is intended on using the binding agent. Siad target binding site of the Alphabody structure may involve an interaction of Alphabody amino acid residues exposed at one or more of the HRS units, or amino acids part of a region of a concave Alphabody groove.

A 'solvent-oriented¹ or 'solvent-exposed' region of an alpha-helix of an Alphabody structure shall herein have the meaning of that part on an Alphabody structure which is directly exposed or which comes directly into contact with the solvent, environment, surroundings or milieu in which it is present. The solvent-oriented region is largely formed by b-, c- and f-residues. There are three such regions per single-chain Alphabody, i.e., one in each alpha-helix. Any part of such solvent-oriented region is also considered a solvent-oriented region. For example, a sub-region composed of the b-, c- and f-residues from three consecutive heptads in an Alphabody alpha-helix will also form a solvent-oriented surface region. So, in one embodiment, said target binding region of the Alphabody is located or defined by amino acids in the helical part, preferably predominantly by a helical part of a single HRS, most preferably the B helix (or HRS2).

Alternatively, the target binding region of the Alphabody is located or defined by amino acids located in the groove of an Alphabody. The term 'groove of an Alphabody' shall herein have the meaning of that part on an Alphabody polypeptide as envisaged herein which corresponds to the concave, groovelike local shape, which is formed by any pair of spatially adjacent alpha-helices within said Alphabody protein. Residues implicated in the formation of (the surface of) a groove between two adjacent alphahelices in an Alphabody are generally located at heptad e- and g-positions, but some of the more exposed b- and c-positions as well as some of the largely buried core a- and d-positions may also contribute to a groove surface; such will essentially depend on the size of the amino acid side-chains placed at these positions. If the said spatially adjacent alpha-helices run parallel, then one half of the groove is formed by b- and e-residues from a first helix and the second half by c- and g-residues of the second helix. If the said spatially adjacent alpha-helices are antiparallel, then there exist two possibilities. In a first possibility, both halves of the groove are formed by b- and e-residues. In the second possibility, both halves of the groove are formed by c- and g-residues. The three types of possible grooves are herein denoted by their primary groove forming (e- and g-) residues: if the helices are parallel, then the groove is referred to as an e/g-groove; if the helices are antiparallel, then the groove is referred to as either an e/e-groove or a g/g-groove.

As detailed herein, it is envisaged that, the Alphabody polypeptides comprises, within the Alphabody structure, a binding site to an intracellular protein. Examples of intracellular target molecules to which the Alphabod polypeptides as envisaged in certain embodiments can specifically bind include for example, but are not limited to, proteins involved in cellular processes chosen from the group consisting of cell signaling, cell signal transduction, cellular and molecular transport (e.g. active transport or passive transport), osmosis, phagocytosis, autophagy, cell senescence, cell adhesion, cell motility, cell migration, cytoplasmic streaming, DNA replication, protein synthesis, reproduction (e.g. cell cycle, meiosis, mitosis, interphase, cytokinesis), cellular metabolism (e.g. glycolysis and respiration, energy supply), cell communication, DNA repair, apoptosis and programmed cell death. The binding agents or Alphabody-based degrader molecules or AlphaTACs as envisaged herein are further capable of maintaining their functionality in the intracellular environment, i.e. to specifically bind the target molecule and allow ternary complex formation via the degrader moiety, for inducing or promoting degradation of the target molecule. Indeed, it has been demonstrated herein that the polypeptides provided herein are not only capable to enter the cell, and are stable in the intracellular milieu, but are also capable of effectively binding their intracellular target and promoting the ubiquitination and subsequent proteasomal degradation thereof. Particular Alphabody-based degrader molecules as described herein are capable of specifically binding to target molecules which are classified as anti- apoptotic members of the BCL-2 family of proteins for instance. Examples of anti-apoptotic members of the BCL-2 family of proteins are MCLl, BCL-2, BCL-XL, BCL-w and BFL-1/A1. It should be understood that one Alphabody may bind to several (i.e., one or more) intracellular proteins of interest, such as exemplified herein for an Alphabody protein moiety specifically binding for target molecules MDM4 and MDM2, so providing for a specific but cross-reacting AlphaTAC. In particular embodiments, the binding of the Alphabody is driven by one of its alpha-helices, which is stabilized in the Alphabody coiled coil structure. In other embodiments, the binding of the Alphabody to its target(s) is driven by a combination of more helices, formed by the HRS1-L1-HRS2-L2-HRS3 structural formula upon spontaneous assembly in solution.

It is envisaged herein that the skilled person aiming to produce an Alphabody-based degrader molecule is aware of the design of the Alphabody, and /or the screening for an Alphabody (see below) to obtain an Alphabody sequence that encodes for a protein which can specifically bind the target protein of interest. The skilled person is also aware of different assays to determine whether a produced Alphabody is capable of specifically binding the target protein, including but not limited to protein binding assays as exemplified herein, or as known in the art.

Cell-penetrating Alphabodies (CPABs)

The binding agent comprising at least one Alphabody protein moiety linked to a degrader moiety, comprises an Alphabody polypeptide which contains a cell-penetrating moiety, which provides for uptake of the Alphabody-based degrader molecule into a cell to arrive there to promote degradation of the target bound to the Alphabody through ubiquitination and proteasomal degradation. Said cellpenetrating moiety of the Alphabody polypeptide is envisaged herein as involving one or more positively charged internalization regions ensuring internalization of the Alphabody into a cell, wherein said internalization region is characterized by the presence of at least six positively charged amino acid residues of which at least 50 % are comprised within said Alphabody structure sequence, and/or the presence of at least one peptide tag for facilitating cellular entry.

As described in W02014064092 by Complix NV, it has been found that by introducing such an internalization region at least in part into an Alphabody sequence, a polypeptide can be created which is able to penetrate the cell autonomously, i.e. without the need for any other structure enabling penetration into the cell. Moreover, this can be combined with the provision of a binding site to an intracellular target within the Alphabody structure, such that highly efficient intracellular binding agents are obtained. The CPAB polypeptides provided herein have been designed to contain certain types of amino acid residues within one or more limited regions comprising an Alphabody structure, more particularly at least in part within the Alphabody structure. More particularly, it has been found that specific positively charged (also referred to as cationic) regions work particularly well to ensure internalization of the polypeptides. Thus, in particular embodiments, the polypeptides envisaged herein comprise at least one positively charged internalization region, that is characterized by a number of positively charged amino acid residues at specific positions of the Alphabody scaffold, through which the polypeptides are provided with the capacity to enter cells. In certain embodiments, the at least one positively charged internalization region can be considered to contain a "cell penetrating motif or a "cell penetrating pattern" (also referred to herein as a "CPAB motif or "CPAB pattern"). Such a motif or pattern can be considered characteristic for providing the polypeptides envisaged herein with cell penetrating activity.

So, in particular embodiments, the binding agents comprise an Alphabody and a degrader moiety wherein the Alphabody structure sequence comprises at least one positively charged internalization region ensuring internalization of said polypeptide into a cell, wherein said internalization region typically extends between two positively charged amino acid residues, and contains a fragment of maximally about 16 amino acid residues and is characterized by the presence of at least four to six positively charged amino acid residues of which at least 50 % are comprised within said Alphabody structure sequence and wherein said internalization region comprises at least 4 arginine residues,. The term "positively charged amino acid(s)" as used herein, refers to (an) amino acid(s) selected from the group consisting of arginine and lysine. Thus, the polypeptides provided herein comprise a positively charged sequence that starts with a positively charged amino acid residue and ends with a positively charged amino acid residue and which ensures that the polypeptides are capable of entering the cell. It will be clear to the skilled person that the binding agents as envisaged herein may contain (but not necessarily contain) additional positively charged amino acid residues that are located outside an internalization region as envisaged herein. Thus, a certain number of positively charged amino acid residues may be present in the binding agents as envisaged herein, which do not form part of an internalization region as described herein and which are thus not considered to contribute to the cell penetrating capacity of the binding agent. Furthermore, the binding agents as envisaged herein, may or may not contain two or more internalization regions as described herein, which are located separate from each other or which are overlapping each other. In a specific embodiment, the at least one positively charged internalization region of the Alphabody moiety of the binding agents envisaged herein, is further characterized by the presence of at least six positively charged amino acid residues. The at least six amino acid residues can be chosen from the group consisting of arginine (R) and lysine (K). Further embodiments provide for internalization regions with at least four residues of the at least six positively charged residues in the internalization region being arginines or when at least five residues of the at least six positively charged residues in the internalization region are lysines highly efficient cell penetration is observed. In a specific embodiment, the positively charged amino acid residues used herein are not lysines, and limited to arginine residues, in order to avoid ubiquitination of the Alphabody-based degrader agent. In a preferred embodiment, the internalization region does not constitute lysine residues, and only a single lysine is present in the binding agent, providing for specific conjugation means, for instance when NHS ester coupling is aimed for, which will occur at the primary amine of Lysine in physiological conditions.

It has been demonstrated (e.g. in W02014064092 by Complix NV) that the CPAB motifs can be integrated into the Alphabody scaffold without disrupting the target binding site. In particular embodiments, the polypeptides provided herein comprise at least one Alphabody structure sequence, which (i) is capable of being internalized into a cell through the presence of at least one positively charged internalization region as described herein, which is comprised at least in part within said Alphabody structure sequence, and in addition (ii) specifically binds to an intracellular target molecule primarily through a binding site present on the Alphabody structure sequence. In these particular embodiments, the polypeptides provided herein specifically bind to an intracellular target molecule primarily through a binding site present on the B-helix of the Alphabody structure sequence.

In an alternative embodiment, the cell-penetrating moiety comprises at least one peptide tag for facilitating cellular entry of the binding agent. The mechanism of how particular sequences endow peptides with cell-penetrating capacities is still under debate. Nevertheless, it has been recognized that naturally occurring cell-penetrating sequences, and the synthetic peptides cF(PR4, mediate cell penetration by the presentation of guanidium groups to the negatively charged phospholipid bilayer. Furthermore, cell-penetrating peptides, typically 5-40 residues in length, are amino-acidic sequences classified as cationic, amphipathic and hydrophobic, with an overall net positive charge to interact with the negative charges on the cell wall, and (self-)assembly of stable secondary structures, which may contribute to internalization potential. Particular examples of known cell-penetrating peptides are known in the art, such as for instance, but not limited to TAT, CPP5, Penetratin, Pen-Arg, pVEC, M918, TP10 (see Madani et al, Journal of Biophysics, Volume 2011, Article ID 414729), and TAT-HA fusogenic peptides (Wadia et 20 al., Nat Med, 2004, 10, 310-315). Their potential as (part of) a therapeutic molecule, and their pro's and con's in pharmacological applications is reviewed for instance in Jauset and Beaulieu (2019; Curr opin. Pharmacol. 47:133-140).

A formatted Alphabody translocating across the cell membrane without compromising target binding properties, also called cell-penetrating Alphabody (CPAB), may alternatively comprise a cellpenetrating moiety in the format of a peptide tag which for instance consists of 7 consecutive arginineproline repeats ( [RP]?) that were engineered to be added co-translationally at the N- and C-terminus of the Alphabody structure as also disclosed in (13). The rationale for the choice of a proline residue flanked by arginine's, as used also in a number of the Alphabody-based degrader molecules exemplified herein is based on the knowledge of known cell-penetrating peptides inferring that a proline residue would increase uptake by reducing the electrostatic repulsion between the protein backbone amides and the lipid bilayer and their hydrophobic nature expected to enhance the efficiency of cell penetration. Alphabody CMPX-321A carrying CPAB tags at its N- and C-terminus was found to efficiently cross the mammalian cell membrane (13) and based on those results, exemplified binding agents used herein provide for CPABs with such RP7-tags as cell-penetrating moiety, or part of the cell-penetrating moiety of the binding agent used herein. So in a specific embodiment, the binding agent described herein comprises an Alphabody with a cell-penetrating moiety which comprises at least one peptide tag for facilitating cellular entry comprising the sequence (Arg-Pro)n, wherein n is an integer from 4 to 15, or more preferably from 4 to 14, or from 4 to 13, or from 4 to 12, or from 4 to 11, or from 4 to 10, or from 4 to 9 , or from 4 to 8, or from 4 to 7, or from 4 to 6, or more preferably from 5 to 15, or more preferably from 5 to 14, or from 5 to 13, or from 5 to 12, or from 5 to 11, or from 5 to 10, or from 5 to 9 , or from 5 to 8, or from 5 to 7, or from 5 to 6, or more preferably from 6 to 15, or more preferably from 6 to 14, or from 6 to 13, or from 6 to 12, or from 6 to 11, or from 6 to 10, or from 6 to 9 , or from 6 to 8, or from 6 to 7, or more preferably from 7 to 14, or from 7 to 13, or from 7 to 12, or from 7 to 11, or from 7 to 10, or from 7 to 9 , or from 7 to 8. In a further specific embodiment said peptide tag may be fused to the Alphabody sequence at the N- and/or C-terminal end, directly or via a linker. In a further embodiment, said peptide tag as described herein may be fused at the N- and/or C-terminal end of the binding agent as described herein, comprising a degrader moiety, a further tag, a linker, or another functional moiety between the Alphabody sequence and the cell-penetrating moiety. In a further embodiment, the CPAB or the binding agent of the present invention may comprise one or more peptide tags as cell-penetrating moieties, as described herein, specifically may comprise two, three, or more cell-penetrating peptide tags, which may be positioned N- and/or C-terminally of the Alphabody sequence.

In a further specific embodiment, the binding agents described herein comprise at least one CPAB with at least one cell-penetrating moiety which is composed of a cationization region or internalization region, as described herein, and which is at least partially present within the HRS1-L1-HRS2-L2-HRS3 sequence structure of the Alphabody, and ensures internalization through the presence of a number of positively charged amino acid residues, to arrive at an Alphabody sequence structure that is neutral in net charge, or has a net positive charge (i.e. not negatively charged). Alternatively, the binding agents described herein comprise at least one CPAB comprising a cell-penetrating moiety which is composed of a cell-penetrating peptide, as defined herein. Alternatively, the binding agents described herein comprise at least one CPAB comprising a cell-penetrating moiety comprising one or more cationization or internalization regions and one or more cell-penetrating peptides, together acting as cell-penetrating moiety of the CPAB or binding agents disclosed herein, and functioning as degrader molecule. So, one embodiment relates to said binding agent comprising an Alphabody targeting moiety which comprises at least one of the cell-penetrating moieties as described herein, and a degrader moiety, and said biding agent being capable to penetrate into a cell and promote degradation of an intracellular target protein, when bound to the Alphabody. With intracellular protein is meant herein a cytosolic, nuclear or membrane-bound protein that is reachable from inside the cell.

In particular embodiments, the cell-penetrating moiety comprises or consists of a cell-penetrating peptide tag, such as at least one, such as one or two, (RP)7 cationization motif(s) (i.e. a peptide consisting of an amino acid sequence RPRPRPRPRPRPRP (SEQ ID NO:33)). In particular embodiments, biding agents are provided comprising at least one Alphabody specifically binding to a target protein which comprises a cell-penetrating entity characterized by the presence of a sequence conjugated to the Alphabody structure sequence ensuring internalization of the polypeptide into the cell. In particular embodiments, the polypeptides envisaged herein are characterized by the presence of a sequence such as, but not limited to, RPRPRPRPRPRPRP (SEQ. ID NO:33). In particular embodiments, the sequence is a (RP)7 cationization motif.

Screening and design of Alphabody moieties

A next aspect relates to methods of producing the binding agents described herein, comprising as a first step the generation of the Alphabodies described herein. Said producing of Alphabodies includes generation and screening of a random library of Alphabody polypeptides and known in the art, for example as described in published international patent application WO 2014/064092 in the name of Complix NV. In a further embodiment, it is envisaged that for the production of target-specific Alphabody polypeptides, i.e. having detectable binding affinity for, or inhibitory activity on intracellular target molecules, binding sites are introduced on an Alphabody structure sequence based on mimicry. The process of producing Alphabody polypeptides based on the process of mimicry is disclosed in detail in published international patent application WQ2012/093013 in the name of Complix NV. Where the Alphabody is required to bind to a specific target, particular screening steps can further be envisaged. In particular embodiments envisaged herein, the target-specific Alphabody of the binding agents described herein can be obtained by methods which involve generating a random library of Alphabodies and screening this library for an Alphabody polypeptide capable of specifically binding to a target of interest, and in particular to an intracellular target molecule of interest. These methods are described in detail in published patent application WO 2012/092970 in the name of Complix NV. It will be understood that the selection step of the methods described in WQ2012/092970 can be performed by way of a method commonly known as a selection method or a by way of a method commonly known as a screening method. Both methods envisage the identification and subsequent isolation (i.e., the selection step) of desirable components (i.e. Alphabody library members) from an original ensemble comprising both desirable and non-desirable components (i.e. an Alphabody library). In the case of a selection method, library members will typically be isolated by a step wherein the desired property is applied to obtain the desired goal; in such case, the desired property is usually restricted to the property of a high affinity for a given intracellular target molecule of interest and the desired goal is usually restricted to the isolation of such high-affinity library members from the others. Such method is generally known as an affinity selection method and, in the context of the present disclosure, such affinity selection method will be applied to a single-chain Alphabody library for the purpose of selecting Alphabodies having a high affinity for an intracellular target molecule of interest or a subdomain or subregion thereof. Equally possible is to select for kinetic properties such as e.g. high on-rate for binding to a given an intracellular target molecule of interest, or low off-rate for library members bound to said target by adjusting the appropriate selection conditions (e.g. short incubation times or long wash cycles, or other conditions as is known by someone skilled in the art of library selection techniques). Alternatively, in the case of a screening method, library members will typically be isolated by a step wherein all library members, or at least a substantial collection of library members, are individually examined with respect to a given desired property, and wherein members having such desired property are retained whereas members not having such desired property are discarded; in such case, and in the context of the present disclosure, desired properties may relate to either a binding site on the intracellular target molecule of interest or a subdomain or subregion thereof, which is different from binding sites known for inhibitory agents, such a binding sites not located in the active pocket of the target, non-orthostheric, or even allosteric binding sites, or high to middle low affinity binders. The selection step of the methods for producing Alphabody-based proteins as envisaged herein thus may be accomplished by either an (affinity) selection technique or by an affinity-based or activity-based functional screening technique, both techniques resulting in the selection of one or more polypeptides comprising at least one single-chain Alphabody having beneficial (favorable, desirable, superior) affinity or activity properties or particular binding modes compared to the non-selected Alphabodies of the library. Specific binding of an Alphabody or binding agent to a target molecule or protein of interest can be determined in any suitable manner known per se, including, for example biopanning, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art. Thus, in particular embodiments, the Alphabody libraries envisaged herein are provided as a phage library and binding Alphabodies are identified by contacting the phage with the labeled target molecule, after which binding phages are retrieved by detection or selective collection of the labeled, bound target. Typically, a biotinylated target can be used, whereby phage which generate an Alphabody binding to the target are captured with a streptavidin-coated support (e.g. magnetic beads). The method for producing a binding agent as described herein, comprising an Alphabody protein, specifically binding a target, linked to a degrader moiety, thus comprises the steps of: a) providing an Alphabody specifically binding a target by structural design and/or by screening and selection of an Alphabody library, as described herein, and b) format said Alphabody to obtain a CPAB, by engineering the Alphabody structure sequence as to include at least one internalization region and/or by addition of a peptide tag to said Alphabody, as described herein, with retained functionality to bind the specific target of interest, and c) link said CPAB with a degrader moiety to obtain the Alphabody-based degrader molecule, wherein the linking in step c may be obtained via conjugation to a small molecule or peptide, as further described herein.

Finally, to provide for a therapeutically suitable product, the Alphabody-based degrader molecule produced by the method described herein can be recombinantly produced through conventional protein production and purification methods, and optionally chemical linkage, labelling and conjugation as known to the skilled person and as described further herein.

So, a specific embodiment relates to a method to produce the Alphabody-based degrader molecule, for degradation of a target molecule in a cell, comprising the steps of: a. identifying or selecting an Alphabody sequence structure which specifically binds a target protein of interest, wherein said identification is optionally performed through structural design and/or using a screening method of an Alphabody library as known in the art and described above; b. engineering and/or formatting of the Alphabody sequence, for instance by optimization of the target-binding region, and/or addition of a cell-penetrating moiety, preferentially at N- or C-terminus of the Alphabody sequence; c. expression of the engineered Alphabody protein in a host cell and isolation of the Alphabody protein from said host cell; and d. conjugation of a degrader moiety, and/or optionally screen for target protein degradation using conjugated Alphabody molecules with differently positioned degrader moieties.

In further specific embodiment, said method comprises to provide an engineered or formatted Alphabody sequence to obtain favorable target binding and therapeutic properties, as well as to allow and optimize for coupling of a degrader moiety (e.g. by addition or removal/substitutions of Lysine or Cysteine residues required as a linking point for a conjugate, or to avoid undesired ubiquitination at several Lysine positions). The action of engineering or formatting the original Alphabody sequence identified in step a. may specifically involve one or more of the following alterations or strategies: the introduction of one or two 'designed' rigid linkers at positions LI and/or L2, as described herein, to replace the originally obtained (flexible) linkers (which typically contain glycine, alanine and serine residues); the introduction of a cell penetrating moiety, typically a cationic tag consisting of a number of RP repeats, as described herein, either at the N- or C-terminus or both termini or ends of the Alphabody, directly linked or with a linker or tag in between; the introduction or removal of positively or negatively charged residues within the Alphabody scaffold for optimizing and/or introducing the cell penetrating properties of the Alphabody; the removal (e.g. by mutagenesis and functional or binding analysis) of excess hydrophobic residues that are not contributing substantially to binding of the target; the introduction of a tag (e.g. His-tag, V5-tag) at either the N- or C-termini, serving to aid in purification and/or detection of the Alphabody construct; the introduction of an albumin binding moiety at the C-terminus of one of the Alphabody helices, to extend the in vivo serum half-life, as described herein and for instance in ref. (13); the introduction of a lysine residue at a specific position, for conjugation purposes via NHS chemistry, and the removal of excess lysine residues that were present in the Alphabody library used to obtain the binder, as to avoid undesired ubiquitination of the Alphabody-based degrader molecule itself.

Degrader moiety

A proteolysis targeting chimera (PROTAC) is a heterobifunctional small molecule composed of two active domains and a linker, capable of removing specific unwanted proteins. Rather than acting as a conventional enzyme inhibitor, a PROTAC works by inducing selective intracellular proteolysis. Recruitment of an E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. Generally, the structural features of PROTACs are limited to small molecule entities (and optionally a linker).

The present invention relates to a novel type or modality used for TPD, so as degrader molecule, wherein at least the entity for targeting the protein of interest comprises an Alphabody™ protein, as previously described. So, degrader molecules with at least one of the entities of the type of an Alphabody protein, is herein referred to as 'Alphabody-based degrader', are also 'AlphaTAC®'. This degrader molecule thus comprises a polypeptide moiety in the form of an Alphabody, as described and defined herein, preferably capable of specifically binding a target protein, which comprises further entities, such as for instance a cell-penetrating moiety or region and a degrader moiety, to form a functional binding agent for targeted protein degradation. When the Alphabody moiety is specifically binding a target protein of interest, at least one additional moiety is required as a degrader, with said moiety linked by fusion, conjugation or another covalent linkage/linker such as a peptide linker. In the specific embodiment wherein the degrader moiety is of small molecule or peptidic in nature, and linked to the Alphabody polypeptide via conjugation, the combination of at least one target-specific Alphabody and a conjugated degrader moiety is herein called a 'hybrid degrader construct' or 'hybrid AlphaTAC' or 'hybrid Alphabody-based degrader molecule', wherein the term 'hybrid' indicates that the Alphabody protein (for target binding) and the degrader moiety (for scavenging E3 activity) are present as a combination of two different structural types. More specifically the Alphabody being a single chain polypeptide, and the degrader being a small molecule or a small peptide (often chemically or enzymatically altered, such as by hydroxylation), wherein both structures can be considered as independent moieties (for instance for their production), and can as well be considered as a linked mixed-type structure (for instance provided as a conjugate). Typically both structures of the hybridtype Alphabody-based degraders are produced separately and afterwards combined into the hybrid Alphabody-based degrader through covalent linking (e.g by chemical or enzymatic conjugation or ligation, or recombination, among others).

It's known from the established insights in the PROTAC therapeutic field that it is not enough for PROTACs orTPD modalities just to bring E3s close to target proteins for these drugs to work. The various surfaces must first come together in a ternary complex and that interaction must then trigger ubiquitination (9). Induced electrostatic surface interactions between the target and the E3 ligase in the presence of the PROTAC play a central role in the stability of the ternary complex. Repulsive interactions may therefore inhibit ternary complex formation. However, recent works pointed out that positive cooperativity is not a strict requirement for efficient protein degradation, and hypothesized that ternary complex formation does not only rely on binding affinity rates and positive cooperativity but as well depends on the absolute concentration of the target and the E3 ligase in cells (4). Another observation typical for TPD mode of action involving ternary complex formation has been called the Hook effect, and is most often visible at concentrations in the range of 1-10 pM, and refers to a bellshaped dependency on the applied PROTAC or degrader molecule concentration. At high concentrations, ineffective binary complexes (POI-PROTAC or PROTAC-E3 ligase) are observed and compete with effective ternary complexes (POI-PROTAC-E3 ligase), resulting in a negative impact on the degrader's potency in a concentration-dependent manner (4).

In a further aspect , the binding agent thus comprises an Alphabody protein moiety for targetengagement, and this target-binding moiety is linked to a 'degrader moiety' or 'degrader', or 'degrader entity' or 'degrader unit' or 'degrader building block', as used interchangeably herein, which upon binding of the target to the Alphabody moiety triggers the degradation of the target in a direct or indirect manner. The function of the degrader moiety within the binding agent or Alphabody-based degrader molecule is thus to promote or induce or stimulate the targeted protein degradation of said bound target protein in a cell. With 'promoting' or 'stimulating' or 'inducing' or 'enhancing' degradation of a target is meant that due to the Alphabody-based degrader molecule being present in the cell, the target protein level in said cell is reduced as compared to the target protein level in a control cell, which is a cell wherein no Alphabody-based degrader is present, but which may contain a negative control or vehicle construct or protein such as a CPAB that is lacking a degrader moiety, or a CPAB with a nonfunctional degrader moiety, or an Alphabody-based degrader which is not specifically binding the target protein of interest, or another negative control as known by the skilled person, which at least has no impact on the target protein of interest. With a reduced protein level is meant that the measured protein amounts in a cell or cells or tissue or solution or at least 5 % lower as compared to the control, preferably at least 10 % lower, more preferably at least 15 % lower, more preferably at least 20 % lower, more preferably at least 25 % lower, more preferably at least 30 % lower, more preferably at least 35 % lower, more preferably at least 40 % lower, more preferably at least 45 % lower, more preferably at least 50 % lower, more preferably at least 60 % lower, more preferably at least 70 % lower, more preferably at least 80 % lower, or more. Alternatively, the degrader moiety is also referred to as the "degrader", the "degradation-promoting substrate", or "degradation-promoting ligand". In one embodiment the binding agent or Alphabody-based degrader molecule, as used interchangeably herein, relates to a target-specific Alphabody linked to a degrader moiety, wherein the degrader moiety enhances, promotes, triggers or stimulates targeted protein degradation of said protein target, as used interchangeably herein.

In a preferred embodiment, the Alphabody-based degrader molecule is thus composed of at least one Alphabody with the structure as provided herein, typically comprising HRS1-L1-HRS2-L2-HRS3, and comprising a cell-penetrating region or moiety, resulting in an Alphabody moiety which as a whole is capable of penetrating through a cell membrane when administered to a cell culture or organism or subject, and intracellularly binding a specific target; and wherein said at least one Alphabody is coupled, fused or linked to a 'degrader moiety', wherein said degrader moiety thus comprises a structure that is functional in triggering or promoting, stimulating, or enhancing, as used interchangeably herein, the protein degradation of the target protein that is bound to the Alphabody entity of the binding agent, in a cell. The action through which the protein target its degradation is triggered, enhanced, promoted or stimulated may be through recruitment of a proteasomal degradation pathway component, such as an E3 ligase, or an E3 ligase complex component, to mediate poly-ubiquitination of the Alphabodybound target, or via induction, triggering or stimulation of recruitment or involvement of alternative protein degradation pathways, such as the lysosomal protein degradation or autophagosomal degradation.

In a specific embodiment, the degrader moiety is an E3 ligase complex ligand or binder for promoting ubiquitination and proteasomal degradation of the target bound to the Alphabody.

In a further specific embodiment, the degrader moiety as used herein promotes proteasomal degradation, preferably by inducing poly-ubiquitination of the Alphabody-bound target protein through an E3 ligase which specifically binds to the degrader moiety. In said embodiment, the degrader moiety may thus comprise a natural or synthetic ligand of an E3 ligase, or of an E3 ligase complex, sufficient to trigger the activity of an E3 ligase to form a ternary complex with the Alphabody-based degrader molecule or binding agent and the target, thereby promoting its ubiquitin-mediated degradation in a cell. Alternatively, the degrader moiety may comprise an E3 ligase activity itself, or may specifically bind to a portion of a E3 ligase complex, thereby leading to the same effect of ubiquitin- mediated degradation of the Alphabody-bound target in a cell.

The degrader moiety of the Alphabody-based degrader molecule is selected based on a number of preferences, one of which confers its specificity for a certain proteasomal degradation signaling pathway, or more specifically its specificity for a certain E3 ligase, or E3 ligase complex. Moreover, when present in a PROTAC or, in as specifically described herein in an Alphabody-based degrader molecule, the differences in degradation profiles conferred by different ligases are caused and driven by several factors including shape complementarity, the ability to form degradation-competent ternary complexes between the ligase and the target or protein of interest, the subcellular localization of ligase and target, and cell-type-specific expression profiles of ligase and target in a subject. So the degrader moiety may be selected based on the desired (sub)cellular location of the target, the affinity for a specific E3 ligase, and the conformational properties in relation to the Alphabody of the binding agent.

In a specific embodiment, the binding agent as described herein comprises one or more degrader moieties, wherein a combination of ligands targeting different E3 ligases or E3 ligase complex components are bound. Indeed, in tumor cells for instance, the mutation rate may provide for resistance to specific degraders which rely on non-essential ligases (e.g. CRBN and VHL) whose genomic loss or deletion results in no discernible effect on cellular viability or phenotype. In fact, preclinical studies of degraders that use CRBN or VHL to target multiple protein classes have detected emerging resistance that occurs via mutation and/or downregulation of components of the ubiquitin ligase machinery (1). Notably, no cross-resistance has been observed for PROTACs recruiting different E3 ligases, suggesting that the use of PROTACs recruiting different E3 ligases may restore the sensitivity to protein degraders (2).

Within the PROTAC field, a large number of E3 ligase ligands are known to the skilled person, and a number of E3 ligase ligands are commonly applied. The exploration of novel ligands for alternative E3 ligases is however still in its infancy, providing for ample future opportunities to diversify and increase the precision of TPD for specific purposes. Specific embodiment are included herein wherein the degrader moiety comprises a ligand or substrate for an E3 ligase specifically present in one or more cell-types or tissues. It is envisaged herein that the binding agent as described herein comprises one or more degrader moieties, wherein the specific structure and E3 ligase or E3 ligase complex binding specificity will be dictated by the presence of the E3 ligase or E3 ligase complex in the tissue or cell type of interest, which is, in the tissue or cell type where the target protein or protein of interest, specifically targeted by the Alphabody protein of the binding agent will also be present and those coupled binding actions will provide for a therapeutic effect. The skilled person is already provided with a few examples of tissue-specific ligases, as for instance described in Bekes et al (1), including for instance, but not limited to KLHL40 and KLHL41 in skeletal muscle, RNF182 and TRIM9 in the CNS. Moreover, some ligases exhibit 'reverse specificity', which indicates low expression in some tissues or cell types, such as for instance known for VHL, which has a low expression in platelets (1). Another example of cell-specific E3 ligase targeting may involve tumor-enriched or tumor-specific E3 ligases, which may coincide with the dependence of the tumor on the expression of a certain ligase, which is favorable to avoid ligase- based resistance to PROTACs, for example Cancer testis antigens (CTAs) comprise ubiquitin ligases that have restricted expression in the normal testis but are highly overexpressed across multiple cancer types (e.g. MAGE-RING ligases) (1).

In an alternative embodiment, the binding agent as described herein comprises one or more degrader moieties, wherein the degrader moiety is a ligand in a non-active state or off-status, or is auto-inhibited, or in a passive state, when present as such in the binding agent, and is activated to trigger ubiquitination of the Alphabody-bound target protein through an external stimulus, which for instance results in a post-translational modification or presence or proximity of a further binding partner.

In specific embodiments disclosed herein, the binding agent or Alphabody protein-based degrader molecule, comprises a degrader moiety that specifically binds the von Hippel-Lindau (VHL) or cereblon (CRBN) E3 ligase. The VHL and CRBN proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes, and the two most popular E3 ligases being recruited by bifunctional PROTACs to induce ubiquitination and subsequent proteasomal degradation of a target protein.

A further embodiment relates to the binding agent as described herein, wherein the degrader moiety comprises or consists of a small molecule or a peptide linked to said Alphabody moiety, wherein the linkage is made directly or via a linker, and wherein said small molecule or peptide has a structure as defined herein. More specifically, small molecule moieties as defined herein refers to compounds as defined herein including but not limited to a low molecular weight (e.g., < 900 Da or < 500 Da) organic compounds, chemicals, polynucleotides, lipids or hormone analogs characterized by low molecular weights. More specifically, peptides are described herein as any amino acid residue sequence of small size, hence not the size of a polypeptide or protein, or a partial amino acid sequence derived from its original protein for instance after tryptic digestion, all in all, the length of the peptide being limited to a maximum of 10 amino acids, 20, 30, 40, 50, 60, 70, 80 or to maximum of 90 amino acids. Peptides may comprise one or more amino acid residues that are synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as naturally- occurring amino acid polymers, post-translationally modified peptides, such as glycosylated, phosphorylated, hydroxylated, acetylated, and myristoylated residues. The small molecules or peptides as referred to herein to confer degrader functionality may as well include peptide-like molecules (peptidomimetics) comprising from about 2 to about 40 amino acids and/or larger peptides comprising from 40 to maximally 90 amino acids, such as for instance antibody mimetics, fragments, or conjugates.

The skilled person is provided with ample examples of PROTACs wherein such small molecule or peptidic ligands are applied as degrader moiety, as for instance discussed in (1, 6, 8). In specific embodiments disclosed herein, the peptidic degrader moiety may relates or include a hydroxylated HIFlalpha (Hifla) peptide (e.g. peptide sequence of SEQ. ID NO:6 with a hydroxylated proline) recognized by VHL, which through E3 ligase recruitment leads to VHL-mediated ubiquitination (see examples), or may include small molecules such as Thalidomide and AHPC for targeting CRBN and VHL, respectively, for which the generally known formula is shown hereafter:

Those commonly used degrader moiety types are used herein to provide proof of concept, and to further explore and validate the unique properties of the binding agents or Alphabody-based degrader molecules described herein.

Specifically in view of using Thalidomide, which are glutarimides most frequently used ligands to recruit E3 ubiquitin ligase cereblon (CRBN), these immunomodulatory imide drugs are often called "IMiDs" and hydrolysed spontaneously in solution. Thalidomide and its N-alkyl analogs are hydrolyzed at very similar rates, with half-lives ranging from 25 to 35 h at 32°C at pH 6.4 (H. Schumacher et al. Brit. J. Pharmacol., 1965, 25, 324-337). Thalidomide may be unstable in aqueous solution at physiological pH values and in any solution of pH greater than 7.0. The first PROTACs that progressed into clinical trials used glutarimide CRBN ligands. However, glutarimides are used as a racemic mixture because the two enantiomers can undergo rapid and spontaneous racemization in vitro and in vivo. The binding affinity of the (S)- enantiomeric thalidomide to CRBN is at least 10-fold stronger than the corresponding (R)- enantiomer. Only the (S)- enantiomer fits the binding pocket well (P. Chamberlain et al. Nat.Struct. Mol. Biol. 2014, 21, 803-809). The use of racemic glutarimide as the E3 ligase ligands in PROTACs may thus complicate drug development process and is one of the significant barriers to the therapeutic applications of CRBN. Recent development efforts have led to the synthesis of a phenyl dihydrouracil (PD)-based PROTACs, which retains cereblon affinity while eliminating the racemization-prone chiral center in phenyl glutarimide (J. Jarusiewicz et al., ACS Med. Chem. Lett. 2023, 14, 141-145). Alternatively, exploring the possibility to conjugate alternative, more stable CRBN binders, available as NHS esters is thus in scope for Alphabody-based degrader molecules.

The combination of said small molecule or peptidic degrader moiety with an Alphabody-based polypeptide by conjugation results in a hybrid conjugate which for the first time provides for cellpenetrating polypeptide or protein-based agents (including an Alphabody) that specifically trigger targeted protein degradation in a cell, more specifically proteasomal degradation, of an intracellular protein target. Indeed, typically the cell-penetrating capability of PROTAC heterobifunctional compounds was mostly limited to containing peptides, or polypeptides that are smaller than the CPAB units used herein. In a specific embodiment, the binding agent or Alphabody-based degrader molecule envisaged herein comprises a functional and folded CPAB, which is at least 90 amino acids in length or more, preferably at least 100, or at least 110, or at least 120 amino acids in length, wherein said CPAB comprises the Alphabody structure sequence as defined herein, functional in specifically binding an intracellular target protein of interest, and said CPAB protein further comprises a cell-penetrating moiety as defined herein, and optionally further polypeptidic features such as a half-life extension, a tag, or further linkers. The binding agent comprising a degrader moiety which is a small molecule or peptide, as described herein, is linked to or coupled to said Alphabody-based polypeptide of at least 90 amino acids or more, as defined herein, wherein the linking occurs through conjugation or coupling, to obtain a 'hybrid' type binding agent. Indeed, the 'hybrid type' binding agent as used herein refers to a binding agent wherein at least one degrader moiety is coupled to one or more of the amino acids of the polypeptide portion of the binding agent such that this is not obtainable by a genetic fusion encoding said polypeptide. More specifically, the linking or coupling may be obtained through conjugation, for instance using maleimide or NHS-ester coupling, or enzymatic ligation, as known in the art. The present application discloses thereby such hybrid conjugates using degrader moieties commonly known and commonly applied in the PROTAC field, however, typically conjugated to a small molecule for target binding. By conjugation of said degrader moieties to a small hydrophobic protein for target binding, as shown here for the Alphabody-based protein degraders, a unique TPD modality has been successfully designed and proven functionally, although many hurdles were to be expected. Indeed, protein-based target binders are known to have high specificity and selectivity, and are therefore more desired over small chemical molecules, but they are also more prone to certain environments, and generally not cell-permeable. CPABs have overcome all these hurdles, and were shown herein to be further engineerable to develop into hybrid protein-based degraders. Besides the observation that the production of these hybrid molecules was successful for several constructs and with different Alphabody proteins specific for several targets, and containing flexible or designed linkers, and using different degrader moieties, conjugated at different locations of the protein, their internalization in the cells was shown to be at least as efficient as for the CPAB itself, although the molecule was enlarged and showed altered properties after conjugation. Moreover, by screening or testing constructs with the degrader conjugated at different positions of the protein chain (see examples), the most optimal configurations for ternary complex formation and resulting in target protein degradation in a cell-based assay have been demonstrated herein for the first time.

In said embodiment relating to binding agents comprising one or more degrader moieties which are small molecules or peptides specifically triggering or promoting TPD of the Alphabody-bound target protein, the degrader moietie(s) are linked or conjugated directly to the Alphabody-moiety containing polypeptide chain, or are linked via a linker to the Alphabody-moiety containing polypeptide chain.

The position of the amino acid within the polypeptide chain that is conjugated with said small molecule or peptidic degrader moiety may be selected or screened for, or may be dictated by structural information, as to allow for a resulting Alphabody-based degrader molecule capable of forming a ternary complex with the protein of interest, via the Alphabody binding site, and the protein degradation component, such as the E3 ligase, and induce the target its degradation. As exemplified herein (see also Figure 11, Table 3 and Figure 17), the position of the conjugation or the conjugation site, as used interchangeably herein, may in one embodiment be at the N-terminus of the Alphabodycontaining polypeptide chain. In another embodiment, the position of the conjugation of the degrader moiety may be located at the initiation of the first HRS1 or A- helix of the Alphabody protein. In another embodiment, the position of the conjugation of the degrader moiety may be located within the Alphabody, preferably at an amino acid located in a linker (LI or L2) of the HRS1-L1-HRS2-L2-HRS3 sequence structure of the Alphabody. In another embodiment, the position of the conjugation of the degrader moiety may be located at the distal end of the third HRS3 or C-helix of the Alphabody protein. In another embodiment, the position of the conjugation of the degrader moiety may be located at the at the C-terminal end of the CPAB, i.e. when a cell-penetrating peptide, or specifically an RP7 tag, is present at the C-terminal end of the Alphabody polypeptide, the conjugation may be at the C-terminal side of said cationization tag. It will be clear to the skilled person that the introduction of a specific conjugation site in the polypeptide portion of the binding agent is possible through the engineerability of the Alphabody-containing polypeptide by retaining binding capacity for the target, but specifically include, remove, and/or replace amino acids involved in the conjugation. The simplest, most common techniques used for crosslinking or conjugation of polypeptides involve the use of chemical groups that react with primary amines (-NH2). Primary amines exist at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys, K) amino acid residues. These primary amines are positively charged at physiologic pH, therefore, they occur predominantly on the outside surfaces of native protein tertiary structures where they are readily accessible to conjugation reagents introduced into the aqueous medium. Furthermore, primary amines are typically nucleophilic, facilitating targeting those groups for conjugation with several reactive groups, such as synthetic chemical groups including isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these conjugate to amines by either acylation or alkylation. However, N-hydroxysuccinimide esters (NHS) esters and imidoesters are the most popular amine-specific functional groups that are incorporated into reagents for protein crosslinking. So a common method to chemically couple a small molecule to a protein is by using N-hydroxy succinimide (NHS) esters. These NHS esters are often described as reactive towards primary amines, although side reactions with tyrosine, serines have been reported. The primary amines known to react on proteins are the alpha-NH(2)-group of the N-terminus or the epsilon-NH(2)-group of lysine. By formatting the binding agent as disclosed herein through lysine (K) residue inclusion, replacement and/or removal, specific position in the polypeptide portion of the binding agent can be targeted for conjugations, for instance by including one lysine at the conjugation site of choice, and if necessary, remove or replace lysine residues elsewhere present in the polypeptide chain of the binding agent, and/or blocking the N-terminus, thereby preventing aspecific conjugation. So, in a specific embodiment, maximally one lysine residue is present in the Alphabody-based degrader molecule, at the site of conjugation, if required for the specific mode of conjugation, and no further lysine's are present in said Alphabody-based degrader molecule as to prevent self-ubiquitination of the Alphabody-based degrader molecule when present in a cell or subject. In a further specific embodiment, conjugation is obtained via another amino acid residue (e.g. cys), and no lysine's are present in the polypeptide chain of the Alphabody-based degrader molecule.

Besides amine-reactive compounds, chemical groups forming bonds with sulfhydryls (-SH) are the most common crosslinkers and conjugation reagents for proteins. Sulfhydryls, also called thiols, are present in the side-chain of cysteine (Cys, C) amino acids, which may occur as part of a disulfide bond within or between polypeptide chains as the basis of native tertiary or quaternary protein structures of the protein, though when free or reduced sulfhydryl groups (-SH) are available, chemical conjugation via reaction with thiol-reactive compounds is possible. Sulfhydryl-reactive chemical groups in biomolecular probes for labeling and crosslinking cysteines and other sulfhydryls include maleimides, haloacetyls and pyridyl disulfides. The number of available (i.e., free) sulfhydryl groups in a protein can be easily controlled or modified, by engineering the polypeptide portion of the binding agent, taking into account the properties required to be retained for target binding and structural stability. Generation of conjugates is performed in reducing environment to retain free suflhydril groups, and using sulfhydryl-addition reagents, such as 2-iminothiolane (Traut's Reagent), SATA, SATP, or SAT(PEG)4. Sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents. Most of these groups conjugate to sulfhydryls by either alkylation (usually the formation of a thioether bond) or disulfide exchange (formation of a disulfide bond). Maleimide conjugation may be used for instance for conjugating the small molecule or peptide degrader moiety as described herein, through engineering the polypeptide portion of the binding agent by providing a cysteine at the desired location, and remove and/or replace cys residues at further positions, taking into account the required target-binding properties, for specific conjugation. Finally, combining sulfhydryl-reactive groups with amine-reactive groups to make heterobifunctional crosslinkers provides greater flexibility and control and may also be applied to provide conjugated or hybrid binding agents or Alphabody-based degrader molecules with one or more degrader moieties as described herein.

Overall, the binding agent or Alphabody-based degrader molecule as described herein, comprising at least one target-engaging Alphabody moiety, which is a cell-penetrating Alphabody or CPAB, and fused or linked to a degrader moiety, as described herein, provides for a polypeptide-based binding agent that upon addition or administration to a cell (or cell culture or tissue or organism or subject), allows for cell-penetration of the polypeptide-based binding agent into the cell(s), and is thereby reaching its protein of interest to specifically bind this intracellular target by a binding site provided by the Alphabody moiety, as well as engaging a TPD component present in the cell, such as an E3 ligase or E3 ligase complex or component thereof, through binding with the degrader moiety of the binding agent, and as a result a ternary complex is formed and the targeted protein degradation of the protein of interest is stimulated within the cell (s), ultimately via ubiquitin-mediated proteasomal degradation of the protein of interest. Upon ubiquitination and degradation of a target molecule, the binding agent itself may be retained and recycled into an iterative process to further bind additional target molecules and repeatedly trigger TPD of the further bound target molecules within said cell. To the best of our knowledge, this application for the first time demonstrates a combination of a polypeptide-based molecule, i.e. a molecule wherein the polypeptidic portion is larger than 90 amino acids, is capable to cell-penetrate and induce degradation , rather than inhibition, of a specific protein target, specifically of an intracellular or intracellularly reachable protein target. So far, the heterobifunctional molecules that were demonstrated to induce TPD (PROTAC- molecules), only contain very small peptidic regions, or no peptides at all, when cell-penetrating properties are desired. Alternatively, protein-based TPD agents have been developed for intracellular targeting, though when larger polypeptide agents were envisaged, the polypeptides are expressed in or by the cell or only completed or made as a whole when already present in the cell. Herein, the combined modalities of a cell-penetrating protein that recognizes simultaneously a Protein of interest and a TPD component, such as an E3 ligase, is superior to any of the previously demonstrated cell-penetrating inhibitors or cell-penetrating PROTACs in that the larger polypeptidic nature allows for a number of beneficial characteristics such as more robust, rigid, water soluble, specific and stable compounds, with more straightforward manufacturability as compared to more conventional PROTAC chemical synthesis. Moreover, these polypeptide-based compounds or binding agents are suitable for oral absorption, their high specificity reduces off-target toxicity issues known for small molecule PROTACs. Moreover, the Alphabody-based polypeptide binding agents described herein mediating TPD of the protein of interest and thereby provide novel opportunities for complex drug targets, that are less likely to be druggable using inhibitors. In conclusion, the Alphabody-based degrader molecules as described herein on the one hand outperform small molecule TPD drugs on target space, discovery process and safety profile, and on the other hand, outperform alternative TPD biotherapeutics on cellular uptake, robustness, potency, and bioavailability.

Further functionalities of the binding agents

In a further aspect, the binding agent or Alphabody-based degrader molecule as described herein, comprises a further functional moiety, which may be a half-life extending moiety, or a detectable label. The term 'functional moiety' as used herein refers to a molecule or component which performs an additional function for the binding agents when used for a specific purpose. Said purpose may for instance but non-limiting include the purpose of therapeutic use, diagnostic use, the use as vehicle in targeted-delivery, the use in drug discovery or screening assays, the use in structural analysis, the use in gene therapy, among others. So, the functional moiety conjugated to or as part of the Alphabodybased degrader molecule may for instance comprise a therapeutic moiety, such as a further targetbinding moiety, a half-life extension, a small-molecule compound, a nanoparticle, a peptide, a payload, among others. The functional moiety included in the Alphabody-based degrader molecule envisaged herein may be directly coupled or coupled by a linker to the Alphabody structure moiety, the CPAB or cell-penetrating moiety of the Alphabody or to the degrader moiety, or may be chemically conjugated to the polypeptide or single chain Alphabody-based binding agent. For instance, the functional moiety may be provided via a genetic fusion encoding the binding agent wherein the functional moiety is fused to any part of the Alphabody-based degrader molecule N- or C-terminal of the Alphabody, Cell penetrating moiety or degrader moiety, directly or via a linker. Alternatively, the functional moiety may be attached to additional amino acids or moieties covalently bound to the sequence corresponding to HRS1-L1-HRS2-L2-HRS3, such as N-terminally attached to the first heptad repeat sequence, HRS1, or C- terminally attached to the last heptad repeat sequence, HRS3, or C-terminally attached to a further moiety as described herein.

Further embodiments relate to said Alphabody-based degrader molecule comprising an Alphabody that is a bivalent or multivalent Alphabody (i.e. an Alphabody and a further functional moiety that is also an Alphabody), which may be bivalent or multivalent binding agents comprising concatenated Alphabodies. The functional moiety as envisaged herein may also provide for a specific function, such as for example a protein extending the in vivo half-life of the binding agent, or a targeting peptide, which targets or directs the polypeptide to certain specific cell types. Also, the other entity can be a linker, such as a suitable peptidic linker to couple proteins, as known by the person skilled in the art.

In a specific embodiment a further functional moiety providing for a half-life extension through binding to Albumin is provided, wherein said Albumin binding moiety may be a peptide, an antibody or antibody fragment or single domain antibody such as a Nanobody, or may be an Alphabody-based binding region itself. More specifically, the albumin binding region may be conjugated to the C-terminus of HRS3, wherein said albumin binding region comprises a sequence SDFYFXXINKA (SEQ ID NO: 34) and a sequence TXEXVXALKXXILXAH (SEQ. ID NO: 35), wherein X can be any amino acid; HRS1, HRS2, HRS3 and the albumin binding region together forming a five-stranded alpha-helical bundle including the Alphabody, which also binds the target. Alternatively, such an albumin binding region comprises a sequence SDFYFXXINKAKTXEXVXALKXXILXAH (SEQ ID NO: 36), preferably a sequence SDFYFXXINKAKTCEAVXALKXXILXAH (SEQ. ID NO: 37), wherein X can be any amino acid.

In further embodiments, binding agents are provided comprising or essentially consisting of at least one Alphabody directed against a specific target, which Alphabody comprises a cell penetrating moiety, as described herein, and linked to a degrader moiety, as described herein, wherein the further moiety is a detectable label or a tag. In other words, the binding agent is labeled or tagged, or has a detectable moiety fused to it, bound to it, coupled to it, linked to it, complexed to it, or chelated to it. A "label" or "detectable moiety" in general refers to a molecule or moiety that emits a signal or is capable of emitting a signal upon adequate stimulation, or to a moiety that is capable of being detected through binding or interaction with a further molecule (e.g. a tag, such as an affinity tag, that is specifically recognized by a labelled antibody), or is detectable by any means (preferably by a non-invasive means, if detection is in vivo/ inside the human body). Furthermore, the detectable moiety may allow for computerized composition of an image, as such the detectable moiety may be called an imaging agent. Detectable moieties include fluorescence emitters, phosphorescence emitters, positron emitters, radioemitters, etc., but are not limited to emitters as such moieties also include enzymes (capable of measurably converting a substrate) and molecular tags. Examples of fluorescence emitters include cyanine dyes (e.g. Cy5, Cy5.5, Cy7, Cy7.5), FITC, TRITC, coumarin, indolenine-based dyes, benzoindolenine-based dyes, phenoxazines, BODIPY dyes, rhodamines, Si-rhodamines, Alexa dyes, and derivatives of any thereof. Further examples of labels, tags or detectable moieties, as used interchangeably herein include but are not limited to affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6x His or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II® and Twin-Strep-tag®; solubilizing tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as a FLAG-tag; epitope tags, such as V5-tag, myc-tag and HA-tag; fluorescent labels or tags (i.e., fluorochromes/-phores), such as fluorescent proteins (e.g., GFP, YFP, RFP etc.); luminescent labels or tags, such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs); phosphorescent labels; a metal chelator; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, betagalactosidase, urease or glucose oxidase).

Binding agents as described herein comprising a detectable moiety may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo imaging purposes, depending on the choice of the specific label. Further aspects of the invention relates to nucleic acid molecules encoding the binding agent of Alphabody-based degrader molecule as described herein. Specifically for a 'hybrid' Alphabody-based degrader molecule the nucleic acid molecule encoding the polypeptide for conjugation with a degrader moiety to result in the (hybrid) binding agent described herein. An alternative embodiment provides nucleic acid molecules such as isolated nucleic acids, (isolated) chimeric gene constructs, expression cassettes, recombinant vectors (such as expression or cloning vectors) comprising a nucleotide sequence, such a coding sequence, that is encoding the polypeptide binding agent or Alphabody-based degrader molecule as identified herein.

Another embodiment provides for a host cell comprising the binding agent(s), or a host cell for recombinant production of the binding agent as described herein. The host cell may therefore comprise the nucleic acid molecule encoding said polypeptide binding agent or Alphabody-based degrader molecule. The host cell may also be transfected with the binding agent or Alphabody-based degrader or nucleic acid molecule encoding the binding agent as disclosed herein. Host cells can be either prokaryotic or eukaryotic. The host cell may also be a recombinant host cell, which involves a cell which has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule encoding the polypeptide binding agent of the invention. Representative host cells that may be used to produce or be transfected with said Alphabody-based degrader molecules, are but not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for production of the binding agents of the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Yeast host cells that may be used with the binding agents of the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus- insect cell systems (e.g. review Jarvis, Virology Volume 310, Issue 1, 25 May 2003, Pages 1-7). Alternatively, the host cells may also be transgenic animals or plants.

A further aspect relates to a method to produce the binding agent or Alphabody-based degrader molecule as describe herein, comprising the steps of: a. manufacturing a protein binding agent comprising an Alphabody structure sequence as described herein, wherein the structure sequence is optionally identified by screening and/or selection from an Alphabody library as described previously, and further engineered to specifically binding a target, comprises a cell-penetrating moiety, as previously described herein, and optionally suitable for conjugation of a degrader moiety, b. further produced to obtain a 'hybrid' type binding agent by labelling or conjugating the binding agent using a suitable degrader moiety and respective labelling or conjugation method, and c. purifying the binding agent (prior to or after labelling or conjugation) as to obtain a binding agent that is capable of binding said target, as well as binding an E3 ligase, thereby forming a ternary complex in the cell and triggering targeted protein degradation of said bound target.

In a specific embodiment, the degrader moiety is a small molecule or peptide that is conjugated to the Alphabody moiety to obtain a hybrid type binding agent, as described herein, and the suitability of the Alphabody protein for conjugation in step a is resulting for instance from a Cys or Lys integration in the Alphabody sequence structure. Further suitable Alphabody polypeptides for conjugation are those made ready for maleimide coupling, NHS-ester linkage or chemical conjugation, or further conjugations methods known in the art, which allow modifications in the Alphabody sequence without altering target binding properties.

Recombinant production of the binding agents as envisaged herein, as known by someone skilled in the art, requires protein expression and purification, wherein the polypeptide binding agent is produced from an expression vector using a suitable expression system and wherein the binding agent may comprise a tag (typically at the N-terminal or C-terminal end) with e.g. a Histidine or other sequence tag for easy purification. Transformation or transfection of nucleic acids or vectors into host cells may be accomplished by a variety of means known to the person skilled in the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

Another aspect relates to the pharmaceutical composition comprising the binding agent or Alphabodybased degrader molecule as described herein, or the nucleic acid molecule encoding any of said Alphabody based degrader molecules, said composition preferably comprising one or more pharmaceutically acceptable carriers, such as an excipient, diluent or carrier known in the art, and described herein. The further therapeutic use of said Alphabody-based degrader molecule, nucleic acid molecule, or compositions described herein, is envisaged in prevention or treatment of disorders related to the target specifically bound by the Alphabody moiety of said binding agent. In a particular embodiment, the therapeutic use of said binding agents in medicine will provide a novel PROTAC modality for treating a subject to obtain targeted protein degradation of certain disease-related targets.

As exemplified herein, one target protein that is in scope of targeted protein degradation concerns Myeloid cell leukaemia 1 (MCL1), which is a member of the anti-apoptotic BCL-2 family, a group of intracellular anti-apoptotic proteins that contain up to four BCL-2 homology (BH) regions. All members of this family are characterized by their ability to bind and antagonize apoptosis-initiating proteins, such as the pore-forming pro-apoptotic BCL-2 family members BAK and BAX. Under normal conditions, programmed cell death is counteracted by anti-apoptotic proteins, such as MCL1 and BCL-2. However, upon cellular stress, the pro-apoptotic BH3-only proteins are upregulated and bind to pro-apoptotic BCL-2 family members by docking of the BH3-only domain into a hydrophobic groove at the surface of the BCL-2 family member. This interaction results in activation of BAK and BAX that will initiate the programmed cell death cascade by permeabilization of the mitochondrial outer membrane. Many tumors prevent apoptosis by the upregulation of the anti-apoptotic protein MCL1, which makes it an attractive candidate for anti-cancer therapy. Flavopiridol, a pan-cyclin dependent kinase inhibitor, and AZD-4573, a selective CDK9 inhibitor, have been developed to indirectly downregulate MCL1. In addition, chemotherapeutics such as anthracyclines are known to repress MCL1 transcription, but all are associated with considerable side-effects limiting their therapeutic potential. So far, none of the more than 20 compounds evaluated in preclinical studies or clinical trials have resulted in the approval of an MCL1 targeted therapy, despite its clear correlation with therapy resistance in various cancers. Monoclonal antibodies are currently at the forefront of clinical treatments for a wide variety of cancers. Although therapeutic monoclonal antibodies are endowed with many favorable characteristics that are important for their use as therapeutic agents, such as long serum half-life, high specificity, and immunological effector functions, they are also critically limited by low tissue penetration and inability to directly address intracellular drug targets, such as MCL1. Additionally, they impose high production or formulation costs due to low stability and the need for essential post-translational modifications, and harbor complexities in drug administration and patient care. To overcome at least in part these impediments, targeted protein degradation by novel PROTAC modalities has been tried (e.g. Wang et al J. Med. Chem. 2019, 62, 8152-8163, and WO2019/118893 disclosing specific degron (stapled) peptide sequences including MCL1 targeting). However, no protein-based degraders of MCL1 have been described, and therefore, the current Alphabody-based degrader molecules with the capacity to act intracellularly upon cell penetration provides for a unique alternative approach to tackle this target and provide proof of concept for the therapeutic use of this novel PROTAC modality.

So in a further specific embodiment the Alphabody-based degrader molecule which specifically binds a target involved in cancer is disclosed herein for use in treatment of cancer.

Furthermore, TPD has extended to inflammatory diseases and immuno-oncology, with targets as interleukin-1 receptor-associated kinase 4 (IRAK-4) currently in clinical trials to treat autoimmune diseases, bruton tyrosine kinase (BTK) as established target in both inflammation and cancer, with inhibitors available for treatment of different hematological cancers, such as leukemia and lymphoma, challenges for this target may be overcome by TPD approaches as these molecules may degrade both wide-type and mutant BTK proteins.

Finally, the use of the binding agent or Alphabody-based degrader molecule, the nucleic acid molecule, or the pharmaceutical composition described herein, for in vitro assays with the purpose of target specific protein degradation is envisaged herein, which may be a research assay, a screening assay, or a diagnostic assay.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for methods, samples and products according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

EXAMPLES

Introduction on Alphabody-based targeted protein degradation molecules.

PROTACs (Proteolysis-targeting chimeras) induce protein degradation of specific disease-causing proteins by the ubiquitin-proteasome pathway. A PROTAC molecule consists of two major parts - the target binding part and an E3 ligase interacting part. Herein we utilized Cell-Penetrating Alphabody proteins (CPABs), which bind an intracellular protein of interest to target this protein for destruction based on the PROTAC concept. The CPAB is coupled/fused to a moiety (small molecule or peptide) representing an E3 ligase ligand, recruiting an E3 ubiquitin ligase complex leading to ubiquitination of the target and priming the target for proteasomal degradation.

We put forward to turn a CPAB into a degrader of its target, via conjugation of an E3 ligase targeting moiety or E3 ligase ligand to a particular position on the CPAB. This approach covers a number of frequently applied ligands known to bind E3 ligase complex proteins which may be conjugated to the targeting moiety, in this case the CPAB. The most well-known are binders to Von-Hippel-Lindau (VHL) and Cereblon (CRBN). In the non-limiting examples as described herein, in first instance, the Hydroxylated HIFla peptide specifically binding the VHL was conjugated to CPABs via maleimide coupling. In alternative non-limiting instances described herein, NHS-ester conjugation is used as a coupling method, which is known to provide higher stability, and small molecules including thalidomide and AHPC are conjugated to the CPABs for recruiting CRBN and VHL E3 ligases, respectively, yielding hybrid molecules wherein the Alphabody-based degrader makes use of a small molecule entity to trigger the proteasomal machinery.

The examples described herein are non-limiting to the claim and help the skilled person to understand how Alphabody moieties engineered to penetrate the cells, called cell-penetrating Alphabodies (CPABs), may be further formatted through conjugation into heterobifunctional PROTAC-type molecules which trigger intracellular degradation of their target in a highly specific manner for therapeutic utilities. The Alphabody-based PROTACs or degraders described herein were shown to efficiently penetrate the cells and once inside the cell, also act in triggering proteasomal degradation of their target.

Example 1. Design and production of maleimide conjugated Alphabody-based degraders targeting MCLl.

As an initial proof-of-concept, targeting of MCLl (MCLl apoptosis regulator, BCL2 family member) was aimed for, using an MCLl-specific CPAB CMPX-321A (SEQ ID NO:5), which was previously demonstrated to be an MCLl inhibitory CPAB (13). MCLl-specific CPAB CMPX-321A was used to target MCLl, and coupled to the HIFla peptide hydroxylated at the first proline, functioning as E3 ligase ligand. The hydroxy 'GR7' peptide (SEQ. ID NO:6) is recognized by VHL (von Hippel-Lindau complex), which through E3 ligase recruitment leads to VHL-mediated ubiquitination of MCLl bound by the CPAB leading to its proteasomal degradation. According to the Phosphosite Plus® v6.6.0.4 (Hornbeck et al. 2015 Nucleic Acids Res. 43:D512-20), 14 lysines of MCLl could undergo ubiquitination. As the HIFla peptide contains a hydroxyl-proline, in a first instance, chemical coupling of the peptide was used to link it to the CPAB, which was performed via maleimide (non-reducible) conjugation onto free cysteines engineered into the CPAB at different positions: either N- terminally (CMPX-558A), C-terminally (CMPX-558B), or at an exposed f-heptad position in the first (non-target-binding) helix of the CPAB (CMPX-326A), resulting in the MCLl targeting labelled CPABs: CMPX-558Ap-GR7, CMPX-558Bp-GR7, and CMPX-326Ap-GR7, resp. In this way, we can evaluate the preferred spatial configuration of MCL1:CPAB complex relative to VHL, bound to the HIFla, as the efficiency of ternary complex formation will influence ubiquitination efficacy. In addition, a mutant variant of CMPX-558Ap-GR7 was generated by introducing 2 mutations that lower the affinity for MCLl binding, resulting in CMPX-584Cp-GR7.

The MCLl-specific alphabodies, CMPX-558A, CMPX-558B, and CMPX-326A, as well as CMPX-584C were produced and purified followed by conjugation of the GR7 peptide, as described in the Materials and methods below. Final conjugated hydroxy-peptide-CPAB proteins had a purity over 95 %, as shown on SDS-PAGE (Figure 9; Table 1).

For production and purification of the MCLl inhibitory CPAB CMPX-321A, used as negative control, we refer to the method used in Pannecoucke et al. 2021 (Science Advances, 7 (13): eabel682).

Table 1. Purified protein concentration and molecular mass.

Example 2. Initial testing of the MCLl degradation in HEK293T cells using Alphabody-based degraders.

HEK293T cells were treated with concentrations of 100 nM - 3.5 pM with the MCLl degraders CMPX- 326Ap-GR7, CMPX-558Ap-GR7, and CMPX-558Bp-GR7 for 24 hours (Figure 3). Non-conjugated CPAB CMPX-321A was used as a negative control. The MCLl protein levels were detected with the WES system using anti-MCLl monoclonal antibody (see Methods). The MCLl expression levels were normalized either to vinculin, or -actin expression.

We found that treatment of HEK293T cells with CMPX-558Ap-GR7 (HIFla peptide coupling at N- terminal end of CPAB, without G/S spacer) led to decreased MCLl protein levels. For CMX-558Bp-GR7 and CMPX-326Ap-GR7, which contain alternative configurations in the ternary complex, no conclusive data were obtained, meaning that these configurations appeared to be less favored (Figure 3). The negative control CMPX-321A - non-conjugated CPAB - had no effect on MCLl protein levels. From this experiment, it can be concluded that CMPX-558Ap-GR7 (HIFla peptide coupling at N-terminal end of CPAB, without G/S spacer) has the preferred spatial configuration of MCL1:CPAB complex relative to VHL, once bound to the HIFla.

Since the MCLl protein turnover is only about 30-40 minutes (reviewed in Adams and Cory, 2007. Oncogene 26(9):1324-37), we decided to test short-term treatment with the MCLl degraders. HEK293T cells were treated within a range of 0- 3000 nM of the MCLl degraders for 4 hours (Figure 4). The MCLl protein levels were detected with the WES system using anti-MCLl antibody. The MCLl expression levels were normalized to COXIV protein levels. As shown in Figure 4, we did not observe any decrease in MCLl protein expression.

The results described above are also consistent with the results published for the MCLl PROTAC (Wand et al. 2019; Med. Chem. 62, 17, 8152-8163) as they also were able to see the effect on MCLl expression levels with long-term treatments.

In a further test, HEK293T cells were treated with a range of 0-3000 nM of CMPX-558Ap-GR7 for 24 or 48 hours (Figure 5). The MCLl protein levels were detected with the WES system using anti-MCLl antibody. The MCLl expression levels were normalized to total protein levels and shown as MEAN ± SEM, N=2. As shown in Figure 5, a steady decline of MCLl is observed, up to a concentration of 100 nM CMPX-558Ap-GR7. At higher concentrations, the MCLl level rises again, an observation that is indicative of a typical PROTAC functionality and which is known as the hook effect (e.g. see Scheepstra et al., 2019. Comp. Struct. Biotechnol J.17:160-176).

Example 3. MCLl protein degradation occurs through the proteasomal machinery.

We next evaluated whether CMPX-558Ap-GR7 treatment decreases the MCLl expression levels through proteasomal degradation. HEK293T cells were treated with 0, 50 or 150 nM of CMPX-558Ap- GR7 for 24 hours in the absence or presence of the proteasomal inhibitor MG132 (1 pM) (Figure 6). The MCLl protein levels were detected with the WES system using anti-MCLl antibody. The MCLl expression levels were normalized to total protein levels, as shown in A, and the virtual blot representations of total protein detection and immune detection of MCLl is shown in B. We found that MG132 treatment abolished the effect of CMPX-558Ap-GR7, indicating that the observed decrease of the MCLl protein levels upon treatment with CMPX-558Ap-GR7 is due to proteasomal degradation of MCLl.

In addition to HEK293T cells expressing MCLl, we also used myeloma H929 cells and non-small-cell lung cancer H23 cell lines to analyze an initial effect of the CPAB-mediated degradation of MCLl.

Both, myeloma H929 cells (Figure 7) and non-small-cell lung cancer H23 cell line (Figure 8) treated with 0-1000 nM concentrations of CMPX-558Ap-GR7 for 48h resulted in decreased MCLl protein levels, as detected with the WES system using anti-MCLl antibody and normalized to total protein levels and shown as MEAN ± SEM, N=3. Example 4. Design and production of N-Hydroxysuccinimide conjugated Alphabody-based degraders targeting MCL1 and MDM4.

Further constructs were made to provide coverage of a broader range of modalities for E3 ligase binding as well as by applying different target binding sites, and even different targets, to explore the impact of the nature of the E3 ligand, the linker, and the conjugation site on ternary complex formation.

Since maleimide linkage may compromise stability of the Alphabody-based degraders in vivo, an alternative linking chemistry using N-Hydroxysuccinimide (NHS) is applied in novel designs to generate a (serum-stable) peptide linkage between the NHS-activated molecule (typically a small molecule) and a primary amine (such as the protein N-terminus or a lysine side chain). So, as further proof of concept for Alphabody-based degraders, which was already demonstrated to provide for functional effects in Examples 1-3 using Alphabody based MCLl degrader constructs through CPAB CMPX-321A and maleimide-mediated conjugated HIFla peptide for E3 ligase targeting, novel constructs using different Alphabodies against MCLl, and against a second target, MDM4, were designed. In view of a specific conjugation at a desired position in the Alphabody-based constructs, amino acid sequences of the applied Alphabodies were further engineered to ensure that maximally 1 lysine was present, specifically at the envisaged conjugation site (Figure 11, Table 3). Additionally, beyond the conjugation of the hydroxyl-HIFla peptide as E3 ligase ligand, alternative ligands were applied in the conjugation providing for a so-called 'hybrid approach' by conjugation of small molecule E3 ligase ligands to the CPABs. As an example of such a hybrid Alphabody-based degrader, the frequently used small molecule ligands thalidomide and (2S,4R)-l-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (AHPC), specifically binding CRBN and VHL E3 ligase components, resp., were applied herein, in parallel to the application of HIFla as E3 ligase ligand.

The hydroxyproline containing peptide derived from Hifla is obtained with a C-terminal NHS moiety for conjugation (https://www.thermofisher.com). Enzymatic ligation of the Hifla peptide to the N- terminus of a protein is another alternative to generate a (stable) peptide bond (https://enzytag.com/). Moreover, pegylated NHS-esters of the CRBN ligand Thalidomide and of the VHL ligand AHPC were purchased from Broadpharm (BP-24453-250 mg, Thalidomide-O-PEG4-NHS ester, and BP-25120-250 mg, (S, R, S)-AHPC-PEG4-NHS ester), for conjugation to CPABs. The efficacy of the resulting hybrid Alphabody-based degraders is crucially dependent on the formation of a ternary complex between the E3 ligase, the degrader and the target, for which one pivotal parameter in the design is the length of the linker between the target- and the E3 ligase-binding moieties. For example, the pegylated AHPC- NHS esters from BroadPharm are available with 2, 4, 6, or 8 PEG units, allowing for some degree of variability in the 3D space that can be 'mapped' to obtain an optimal ternary complex. Alternatively, when making use of CPAB moieties, far more variability in 3D space can be explored by choosing a particular position on the CPAB for conjugation. Since an Alphabody can be formatted into a CPAB via the introduction of cationized tags at the N- and C-termini (nRP7 and cRP7 tags, respectively), these tags can actually function as a linker in the current design. Figure 11 enumerates graphically the various design proposals (for an MCLl binder); the red star marks the conjugation position. The designed constructs made for 2 targets, MCLl and MDM4-binding CPABs, using 3 different ligands (HIFla, Thalidomide, and AHPC), thus provided for 36 differently designed hybrid-type Alphabodybased degraders, based on the conjugation of the CPAB proteins of SEQ ID NOs: 7-12 for MCLl-binding CPABs and SEQ. ID NO: 13-18 for MDM4-binding CPABs (Figure 11, Table 3). The plasmid construction, production, and purification were performed as described in Materials and Methods below, resulting in produced proteins as indicated in Table 2 and Figure 12; followed by the NHS coupling (see Methods) of their respective small molecule of peptide ligands.

Table 2. Purified protein concentration and molecular mass of unconjugated hybrid CPAB-based degrader constructs.

Example 5. Cellular uptake of AlphaTAC products is comparable to Cell-penetrating Alphabody internalization.

Previous studies have amply demonstrated the capacity of CPAB molecules to be taken up by a range of cancerous/non-cancerous cell types (e.g. as shown in WQ2022/162069). As AlphaTAC molecules were designed to bind an intracellular protein of interest and an intracellular E3 ligase, it is crucial that they retain the CPAB's capacity to efficiently enter the cell. We therefore set out to evaluate whether adding an E3 ligase ligand to the CPAB structure would interfere with its uptake. Initial uptake measurements were performed in the HeLa cell line as this cell line was also used in the degradation assays. In this cell line, the MCLl and MDM2/4 CPABs (6321 and 321A) equipped with cationization uptake motifs showed a concentration-dependent uptake that proved significantly higher than that of a non-cationized MCLl Alphabody (435E; SEQ. ID NO: 19) (Figure 16). Similarly, MDM2/4-targeting hybrid AlphaTAC molecules also showed efficient cellular uptake comparable to that of the MCLl and MDM2/4 CPABs (Figure 16). These observations indicate that coupling an E3 ligase ligand does not interfere with the AlphaTAC's capacity to enter the cell, although the molecules are enlarged by these additional ligands (small molecules or peptide conjugates). Moreover, the physicochemical properties of the conjugated Alphabody proteins were different over the non-conjugated Alphabody proteins, which further made it unpredictable whether this novel modality could still efficiently penetrate the cell membrane.

Example 6. In vitro characterization of Alphabody-based degrader modalities in reporter degradation cell lines.

The previously designed and produced CPAB-based degrader molecules are characterized first for their capability to efficiently penetrate cells as shown in Example 5, by using confocal microscopy to measure their uptake by visualization of the V5 tag by immunofluorescence.

Further, the efficacy of MCLl and MDM4 degraders was analyzed in degradation reporter cell lines to monitor the targets, MCLl and MDM4. A HiBiT cell line for a luciferase-based reporter assay was setup (see methods), and as an alternative, also a fluorescent tag Clover cell line for a fluorescence assay would be suitable as reported cell line.

The hybrid MDM2/4 AlphaTAC molecules engaging both VHL and CRBN were analyzed (n=3-6 biological replicates) using the HiBit reporter cell line and showed to significantly reduce levels of MDM4-HiBiT by approximately 50 % at nM concentrations (Figure 13). Interestingly, those MDM4-targeting AlphaTAC products where the E3 ligand was conjugated C-terminally (632L) proved far more efficacious than those where conjugation happened at the N-terminus (632G), especially in the case of thalidomide-coupled constructs (Figure 13). The opposite was true with the MCLl AlphaTACs where an N-terminally conjugated construct (CMPX558Ap-GR7) showed superior efficacy compared to a C- terminally conjugated construct (CMPX558Bp-GR7) (Figure 3). These findings highlight the importance of the versatility of our AlphaTAC design platform as each target warrants distinct binding characteristics.

These cell lines further allow to monitor the target levels in the presence or absence of the proteasomal inhibitor MG132 or bortezomib, as to measure the effect of the CPAB-based degraders. Moreover, the capacity to reduce target protein level of MDM4 degraders using the reporter cell line was determined in 5 different concentrations in a range from 10 nM till 1 pM 24h and 48h after treatment, as to confirm their functionality. Complementary to the aforementioned findings using the MDM4-HiBiT degradation reporter HeLa cell lines, 632L-AHPC (C-terminally conjugated MDM2/4 AlphaTAC) was also able to reduce levels of MDM2 in the A549 lung cancer cell line by approximately 50 % at nM concentrations (Figure 14).

Example 7. Confirmation of protein interaction between AlphaTAC moieties and targets using pulldown.

We evaluated the MDM4/2 AlphaTAC molecule's capacity to bind both target and respective E3 ligase using a pull-down assay (see methods). In this assay, the hybrid AlphaTAC product was pulled down from A549 cell lysate using its V5 TAG, followed by a western blot to detect the presence of both target (M DM 2/4) and E3 ligase (CRBN) (Figure 15). The MDM2/4-targeting hybrid AlphaTAC '632H- Thalidomide' was shown to be capable to pull-down both targets MDM4 (Figure 15A) and MDM2 (Figure 15B), as a result of the cross-reactivity of this Alphabody for its target-binding, as well as the E3 ligase (CRBN), via its conjugated ligand, within the A549 whole cell lysate, suggestive of potential ternary complex formation. An unconjugated MDM2/4 CPAB control (632*) only showed binding to MDM4 (Figure 15A) and MDM2 (Figure 15B), but not CRBN, confirming that CRBN binding is mediated by the conjugation of Thalidomide. Similar results were obtained with cell lysate from the U2OS cell line (data not shown).

Similarly, the MCLl-targeting hybrid AlphaTAC 558Ap-GR7 (HIFla conjugated SEQ. ID NO:2) showed interaction with both target (MCL1) and E3 ligase (VHL) within the A549 whole cell lysate, suggestive of potential ternary complex formation. An unconjugated MCL1 CPAB control (321A) only showed binding to MCL1 (Figure 10A), but not VHL, confirming that VHL binding is mediated by the conjugation of GR7 HIFla peptide. Moreover, a mutated version of the 558Ap CPAB molecule, 584Cp, which has been modified to lower MCL1 binding affinity only showed binding to VHL and not MCL1 (Figure 10A).

Example 8. Further in vitro characterization of Alpha-body based degrader molecules.

Furthermore, in vitro serum stability is assessed using a quantitative ELISA protocol with the V5 tag as readout, and SDS-PAGE followed by Western Blot revealing any molecular weight shift of the constructs, by visualizing the V5 tag, which would indicate breakdown of the CPAB moiety.

For potent degraders identified herein, further characterization is required for MCL1 or MDM4 specific targeting and efficacy, which is performed through a number of steps. The IC₅o of the degraders will be determined in target-dependent and target-independent hematological cell lines. For MCL1, A427 and H838 cell lines are both dependent on MCL1 expression according to the DepMap (https://depmap.org/), while MCL1 indels showed limited growth inhibition in A549 and H2009 cell lines. For MDM4, A549 is dependent on MDM4 expression, while MDM4 indels showed limited growth inhibition in H2172 and H1993. Five different concentrations in a range from 1 nM till 1 pM 24h and 48h after treatment are tested.

In addition, the EC₅o of the MCLl degraders in MCLl-dependent (A427 and H838) and MCL1- independent (A549 and H1944) cell lines is assayed via Cell titer Glow to determine whether the observed growth inhibition is MCLl dependent, as well as EC₅o values of the MDM4 degraders in MDM4-dependent (A549) and MDM4-independent (H2172 and H1993) cell lines is assayed in the same way. These in vitro characterization experiments provide for the most potent degraders of different modalities for each target (MDM4 and MCLl). Further analysis described below is envisaged to demonstrate proof of concept through in vivo analysis and PK/BD behavior and anti-tumor efficacy.

Example 9. Design and production of half-life extended (HLE) Alphabody-based protein degradation molecules.

For the MCLl targeting Alphabody constructs, a half-life extension (HLE) variant can be designed by incorporating an albumin binding moiety at the C-terminus of the protein scaffold, as described in Pannecoucke et al., which involves integration of any one of SEQ. ID NOs:34-37.

Such an approach is not an option for the MDM4 targeting degraders used herein, because the C- terminal helix is part of the MDM4-binding site. An alternative approach here is to fuse an albumin binding domain via a flexible linker (Gly-Ser) to the MDM4-binding Alphabody. For instance, such an albumin binding domain may be a modified Affibody for specific binding to albumin or a small Alphabody with C-term Albumin-binding motif. These albumin binding domain fusions can also be used in combination with other target-specific Alphabodies.

The in vitro characterization of half-life extended Alphabody-based protein degradation molecules will be performed as described in Example 6.

Example 10. In vivo characterization of potent Alphabody-based protein degradation molecules.

In an initial setup, one may assess the long-term AlphaT AC-based response in 3D organotypic tumor spheroids derived from responsive target-dependent human cancer cell lines. To obtain this, 3D tumor spheroids are cultured in the presence of vehicle control, CPAB control or the most potent AlphaTAC molecule(s) followed by quantifying the number of alive/dead cells after staining with Nexcelom ViaStain acridine orange/propidium iodide staining solution and spheroid analysis using the Zeiss Cell Discoverer 7 system.

Moreover, as to determine the in vivo biodistribution, C57BL6 mice (n=3 per group, total 7 groups) are injected intravenously with vehicle or MCL1/MDM4 degrader (dose level: 20 mg/kg). Samples (serum, lung, spleen, liver, kidney, brain, and lymph nodes) are harvested at time points: pre-dose, lOmin, lh, 3h, 6h, 12h. Half of the tissue is homogenized to quantify CPAB-degrader levels using the ELISA assay. The other half of the tissue is formalin-fixed for immunohistochemistry to assess biodistribution, using the V5 tag.

In vivo anti-tumor efficacy assessment is performed using a subcutaneous cancer model as follows: nude mice (n=10 per group, total 3 groups) are inoculated with tumor cells (human cell line, chosen based on target-dependency and whether it can establish xenograft tumors). Once tumors reach 100 mm³, mice are randomized into 3 groups and injected IV or IT with vehicle, CPAB control, or the CPAB- degraders (dose level 40 mg/kg), at a frequency of once/day. Tumor volumes are measured two times per week. Mice are euthanized once the first mouse of the control group is nearing clinical endpoints, and samples are harvested. The timepoint may be adjusted to earlier, in case the treatment groups show tumor regression, as we want to ensure there is tumor left to analyze. Samples taken are from serum, tumor, lung, spleen, liver, kidney, brain, and lymph nodes. Half of the tissue is homogenized to quantify CPAB-degrader levels using the ELISA assay. The other half of the tissue will be formalin-fixed for immunohistochemistry to assess biodistribution, using the V5 tag.

In vivo hematological cancer models are further tested as follows: nude mice (n=10 per group, total 3 groups) are inoculated with tumor cells via tail vein injections of 2 x 10⁵ cells of a human hematological cancer cell line (chosen based on whether it can establish xenograft tumors). Subsequently, mice are randomized into 3 groups and injected i.v. with vehicle, AlphaTAC (most potent AlphaTAC identified in dose level 20 mg/kg), or its respective CPAB control (dose level 20 mg/kg) at a frequency of once per day for 5 weeks, to reach an end point with overall survival. Mice are euthanized once they reach clinical endpoints, and samples harvested. Samples taken are bone marrow, serum, lung, spleen, liver, kidney, brain, and lymph nodes. The bone marrow is evaluated for the presence of human hematological cancer cells. For the remaining tissue samples, half of the tissue is homogenized to quantify AlphaTAC levels using a V5 ELISA assay. The other half of the tissue is formalin-fixed for immunohistochemistry to assess biodistribution, using the V5 tag.

Materials and methods

Production and purification of CPAB MCL1 degraders CMPX-326Ap-GR7, CMPX-558Ap-GR7, CMPX- 558Bp-GR7 and CMPX-584Cp-GR7.

The Alphabodies (SEQ. ID NO: 1-4) for the listed degraders were expressed in BL21 (DE3) pLysS cells grown in LB medium (including ampicillin) starting from an overnight grown pre-culture (ODsoo O.l). Cell cultures were grown at 37°C till ODsoo reached about 0.5-0.6, following induction of expression by the addition of ImM IPTG at 37°C for 4h, following harvesting by centrifugation (2O'at 5000 rpm at 4°C) and pellet resuspension in 50mM HEPES 500mM NaCI pH 7.2. Purification was performed starting from a resuspended pellet by adding 10 pg/mL DNase, 5mM MgCL, ImM AEBSF and incubation for 15 minutes on a rotating wheel at room temperature. Next, the suspension was sonicated for 5 min (amplitude 70, cycle 0.5) on ice, followed by clarification (incubation of the sample for 15 minutes on the rotating wheel at RT, and centrifugation for 15 min at 20000 rpm at 4°C). The inclusion bodies in the insoluble pellet fraction were washed by resolving in IBW1 buffer (50 mM HEPES pH 7.2, 0.25M GuHCI 1% Triton X100), followed by centrifugation for 15 minutes at 16000 rpm (4°C). The pellet fraction was washed by resolving in IBW2 buffer (50 mM HEPES pH 7.2, 1% Triton X100) followed by centrifugation for 15 minutes at 16000 rpm (4°C). The pellet was resuspended in solubilization buffer (50 mM HEPES pH 7.2, 500 mM NaCI, 4 M GuHCI) until the pellet was completely resolved, followed by centrifugation for 15 minutes at 16000 rpm (4°C). The CPAB was retrieved in the solubilized pellet fraction. The purification was proceeded using the solubilized pellet after addition of 2.5 mM TCEP to reduce disulfide bonds, prior to loading the sample on an IMAC column. Elution fractions were dialyzed against 50 mM Sodium Acetate 150mM NaCI pH 5, and concentrated using Amicon Ultra -15 centrifugal filter (3K / Millipore) to a volume preparative Size exclusion chromatography (SEC). Prior to SEC, 5mM TCEP was added for at least 20 minutes to generate monomers. 0.22 pm filtered sample was loaded on a Hiload 26/60 Superdex 75 pg (GE Healthcare) in 20mM HEPES, 150mM NaCI, 2.5mM TCEP, pH 7.2. Since purification of the protein was performed under reducing conditions, labeling of the CPAB on cysteine sulfhydryl group was possible in a maleimide based chemistry.

GR7 Hifla peptide was synthesized at Proteogenix (France), and samples were resolved in a 1:3 ACN:mQ solution to obtain a 20mg/mL stock solution, to further dilute with an equal volume 20mM HEPES, 150 mM NaCI pH7 to obtain a working solution of 10 mg/mL.

Conjugation of the GR7 (SEQ ID NO:6) Hifla peptide was performed by maleimide coupling to a Cysteine residue present in the MCLl-specific CPAB CMPX-321A at different positions:

-CMPX-326A: maleimide coupling to Cys at position 54 of SEQ. ID NO:1, resulting in CMPX-326Ap-GR7

-CMPX-558A: maleimide coupling to (N-terminal) Cys at position 22 of SEQ ID NO:2, resulting in CMPX- 558Ap-GR7

-CMPX-558B: maleimide coupling to (C-terminal) Cys at position 164 of SEQ ID NO:3, resulting in CMPX- 558Bp-GR7

-CMPX-584C: maleimide coupling to (N-terminal) Cys at position 22 of SEQ ID NO:4, resulting in CMPX- 584Cp-GR7.

The samples desalted on a HiPrep 26/10 desalting columns to 20mM HEPES, 150mM NaCI, ImM TCEP, pH 7.0. Labeling of CPAB was performed using lOx excess GR7 for 2h30 at room temperature, followed by concentration using an Amicon Ultra-4 centrifugal filter (3K / Millipore), to perform analytical SEC. Eluted fractions were pooled, concentrated and filtered, and purified proteins were stored in 20 mM HEPES 150 M NaCI pH 7.2. Final conjugated hydroxy-peptide-CPABs were analyzed by SDS-PAGE as shown in Figure 9, and molecular mass and protein concentration were determined as indicated in Table 1.

MCL1 protein detection.

Two different MCL1 antibodies were tested for their compatibility to apply in the WES system (Bio- techne Protein Simple): a monoclonal rabbit MCLl-specific Ab (clone D35A5; mAb #5453, Cell Signaling; 'MCL1 CST') and polyclonal rabbit anti-MCLl (A302-715A-T; Bethyl Laboratories 'MCL1 Bethyl'). For each MCL1 antibody, 3 different Ab dilutions were used (1:10, 1:50, 1:250) to test Ab saturation; 3 different lysate concentrations were used (0.125 pg/pl, 0.5 pg/pl, 2 pg/pl) to test the linearity and optimal protein lysate concentration; and two negative controls were included, the first one where no primary antibody was present and the second one where no lysate was used.

As shown in Figure 1A, MCL1 (CST) Ab reaches 90 % saturation at 1:30 dilution at 0.125 ug/ul, 0.5 ug/ul, and 2.0 ug/ul of HEK293 protein lysate, whereas for the MCL1 (Bethyl) Ab, 90 % saturation was only reached at 1:10 at various lysate concentrations (Figure IB), so the latter was not included in further experiments. Using the MCL1 mAb from Cell Signaling at saturating antibody concentration, the protein lysate concentration should be such that our protein of interest concentration is within the dynamic range of the assay. A linear increase of MCL1 levels was observed in the range of 0.125-2 pg/pl of HEK293T protein lysate concentrations, leading to the preferred conditions of HEK293 cells at 0.5 pg/pl protein lysate concentration and 1:30 MCL1 mAb from Cell Signaling when using the WES system (Figure 2).

Normalization of protein levels for MCL1 quantification using WES.

For initial experiments, we normalized to expression levels of housekeeping proteins: anti-vinculin mouse monoclonal antibody (clone hVIN-1; Sigma V9131), anti- -Actin rabbit monoclonal antibody (clone 13E5, Cell signaling technology #4970), and anti-COXIV, rabbit monoclonal antibody (clone 3E11, Cell signaling technology #4850). However, as used in later experiments, the best normalization was obtained by using total protein signal, which was obtained using the total protection detection module for chemiluminescence based total protein assays (Bio-techne Cat N°DM-TP01).

Design, production and purification of Hybrid Alphabody-based degraders

Alphabodies were recombinantly produced using the same expression protocol as described here above in the methods for the CPAB MCL1 degraders using maleimide coupling. Purification was performed using a similar method with a few differences: TCEP was only applied in the above method for allowing cysteine coupling, and not added in current productions; the first resuspension buffer contained PMSF instead of AEBSF; sonication was performed 1-2 min, at amplitude of 30-40%, cycle 0.5; the IBW1 buffer contained no GuHCI ). Further down the purification was proceeded by addition of elution buffer of Gravitrap (50mM Hepes pH 7.2, 4M GuHCI, IM Imidazole) to each supernatant to have a starting concentration of 20 mM Imidazole in the sample prior to IMAC (Ni Gravitrap) purification. 1ml Ni Sepharose Gravitrap columns were equilibrated with 10 ml equilibration buffer (50mM Hepes pH 7.2, 4M GuHCI, 20mM Imidazole) and subsequently loaded with approximately 25ml of supernatant. Subsequently, the Gravitrap columns were washed with 10 CV equilibration buffer and eluted with 3ml elution buffer. The samples were left for dialysis (Slide-A-Lyzer dialysis kit 7 MWCO 3- 12ml capacity) overnight against II of dialysis buffer (50mM Sodium acetate pH 5, 150mM NaCI). The Alphabodies were harvested by centrifugation at 4500 rpm for 10 minutes, followed by filtration of the supernatant on Millex 0.22 pm low protein binding filters.

Table 3. List of CPAB-based degrader proteins for hybrid NHS-coupling.

His6, V5, AB, Alphabody, RP7 cationization region, *insertion of Lysine residue for NHS-coupling

Conjugation through hybrid NHS-coupling

Conjugation of the small molecule ligands (AHPC and Thalidomide) was performed by NHS coupling to the N-terminus or to a specific lysine residue present in the MCLl-or-MDM4-specific CPAB at different positions (see Table 3). Pegylated NHS-esters of the CRBN ligand Thalidomide and of the VHL ligand AHPC were purchased from Broadpharm (BP-24453-250 mg, Thalidomide-O-PEG4-NHS ester, and BP- 25120-250 mg, (S, R, S)-AHPC-PEG4-NHS ester), for conjugation to the Alphabody proteins. Constructs 631D, 641A, and 632G, which require conjugation at the N-terminus were subjected to TEV cleavage of the N-terminus prior to conjugation. The other constructs, which require conjugation on a lysine residue were subjected to TEV cleavage of the N-terminus after conjugation.

TEV cleavage procedure: TEV cleavage was performed by adding TEV protease (10 % of total protein) to the Alphabody stock overnight at 4°C. Subsequently, the cleaved construct was purified using IMAC (HisTrap HP 1 ml purification column). In short, the HisTrap HP columns were loaded with the cleaved protein and subsequently washed with 10 CV PBS, followed by a wash with 5 CV of 50 mM Imidazole and eluted with 10 CV elution buffer (PBS, 400mM Imidazole). The samples were left for dialysis (Slide- A-Lyzer dialysis kit 7 MWCO 3-12 ml capacity) overnight against 1 I of PBS. The Alphabodies were harvested by centrifugation at 4500 rpm for 10 minutes, followed by filtration of the supernatant on millex 0.22pm low protein binding filters.

Conjugation protocol small molecules: a stock of 20 mg/ml of both Thalidomide-O-PEG4-NHS and (S,R,S)-AHPC-PEG4-NHS in DMSO was prepared. Both Thalidomide and AHPC conjugates were added to the Alphabody samples in a 40x excess and allowed to couple for 4 hours at RT (not shaken and not in the dark). Subsequently, the obtained samples were dialyzed to PBS overnight using the Slide-A-Lyzer dialysis kit (3kDa). PBS was refreshed after 3 hours.

Product Concentration determination: as both AHPC and Thalidomide are small molecules whose composition can interfere with spectrophotometric analysis, protein concentration determination was determined via SDS-PAGE in comparison to a reference protein sample (BSA) with a known concentration. Table 4 provides the obtained concentrations for the exemplified batches used herein.

Conjugation was confirmed via Mass spectrometric analysis (intact mass) for each construct used in further experiments.

Conjugation protocol Hifla: the hydroxyproline containing peptide derived from Hifla is obtained with a C-terminal NHS moiety for conjugation to the N-terminus of the CPAB protein product (https://www.thermofisher.com). A stock of 20 mg/ml of Hifla peptide was prepared and added to the Alphabody samples in a 40x molar excess and allowed to couple for 4 hours at RT (not shaken and not in the dark). Subsequently, the obtained samples were dialyzed to PBS overnight using the Slide-A-Lyzer dialysis kit (3kDa). PBS was refreshed after 3 hours.

Final protein concentration was determined using the Trinean DropSensel6 and a final SDS-PAGE was run to confirm sample purity. Table 4. AlphaTAC product concentration

ALPHATAC ESTIMATED CONCENTRATION

HiBiT assay

To screen the efficacy of the different MCLl and MDM2/4 AlphaTAC products, we generated distinct stable "degradation reporter" cell lines in the HeLa background (Schwinn, M.K., Steffen, L.S., Zimmerman, K. et al. A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics. Sci Rep 10, 8953 (2020)) that allow us to monitor MCLl and MDM4 protein levels. In short, MCLl and MDM4 were endogenously tagged at their C-terminus with HiBiT using the CRISPaint approach (Schmid-Burgk, J., Honing, K., Ebert, T. et al. CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nat Commun 7, 12338 (2016)). HiBiT is a very small protein tag (±1.3 kDa) that can be detected using a luciferase-based assay. These HiBiT-tagged cell lines were plated at a density of 3-5 x 10³ cells per well in 200 pl culture medium (high-glucose DMEM, 10 % FBS, 1 % Pen/Strep) in a 96-well white/clear bottom plate (Thermo Fisher (Cat N° 165306)). The cells were allowed to assimilate for 24h prior to adding the treatment. The culture medium was aspirated and replaced with culture medium containing a serial dilution of the AlphaTAC treatment. For the untreated control wells, PBS was added to the medium. After 48 hours of treatment, the level of HiBiT protein was evaluated using the Nano-Gio HiBit lytic detection system (Promega). In short, a volume of Nano- Glo HiBiT lytic reagent equal to the volume of culture medium present in each well (50 pl) was added mixed in an orbital shaker (300-600 rpm, 3-10 minutes). Subsequently the luminescence in the lysate was measured using the Victor Nivo Multimode Microplate reader (PerkinElmer).

MDM2 Western blot procedure

Protein extraction: A549 cells were plated at a density of 150,000 cells per well in a 6 well plate. 24 h after plating/when cells reached 60-70 % confluency, cells were treated with AlphaTAC product or PBS control. Prior to cell lysis, the culture medium was discarded and the cells were washed by adding 5 ml of cold PBS. The PBS was removed and the cells were lysed by adding 500 pl of lysis buffer (25mM Tris- HCI pH 7.4, 150 mM NaCI, 1 % NP40, 5 mM MgCI2, 5 % glycerol, phosphatase inhibitor cocktail (PhosSTOP, Roche), EDTA-free protease inhibitor cocktail (Merck)). The cell lysate was scraped off the plate and transferred into a fresh Eppendorf tube. The cell lysates were cleared by centrifugation (15 min, 16000 g, 4°C) after which the supernatant was collected and the pellet discarded. The obtained total protein concentration was determined using the Pierce BCA Protein Assay Kit. The lysates were stored at -80°C.

Sample preparation: the protein lysate sample was diluted with lysis buffer to obtain 30 pg of protein for WB. Subsequently, samples were reduced by adding NuPAGE 4X sample buffer (ThermoFisher, Cat N° NP0007) and NuPAGE 10 X DTT (ThermoFisher, Cat N° NP0009). The protein samples were incubated at 95°C for 5 minutes and spun down prior to loading on SDS gel (NuPAGE Bis-Tris Plus Gels 4-12 %). Samples were run alongside a pre-stained protein ladder (Cat N°. 26617).

Membrane Transfer and Blotting the gel on the membrane: the blotting sandwich was prepared according to the iBIot ThermoFisher protocol). The blot was run at 23 V for 11 minutes after which the membrane with transferred proteins transferred into 5 % milk solution (50 ml of 0.1 %TBS-T, 2.5 g milk powder) where it was left to incubate for 1 h at RT. After incubation, the milk solution was discarded and the membrane was washed with 0.1 % TBS-T for 20 min. The membrane was cut and subsequently incubated (ON, shaker, 4°C) with 3 ml of the corresponding primary antibody at the indicated dilutions; Vinculin (Sigma-Aldrich, clone hVIN-1); MDMX (Millipore, clone 04-1555, dilution 1:500); MDM2 (Cell Signaling, clone 86934, dilution 1:500); VHL (Cell signalling, clone 68547, dilution 1:500); CRBN (Cell signalling, clone 71810, dilution 1:500); MCL1 (Cell signalling, clone 5435, dilution 1:500). Primary antibodies were diluted in 0.1 % TBS-T with 3 % BSA.

Secondary incubation and Visualization: the primary antibody dilution was discarded and the membrane was washed 3 times with 0.1 % TBS-T for 1-1.5 hours at RT on a shaker (change TBS-T every 15 minutes). The secondary antibodies (goat anti-rabbit IgG-HRP (P044801-2), goat anti-mouse IgG- HRP (P044701-2)) were diluted in 0.1 %TBS-T enriched with 5 % milk powder. The secondary antibodies were added onto the membrane and incubated at RT on a shaker for 1 hour. Finally, the membrane was washed 3 times with 0.1 % TBS-T for 1- 1.5 hours at RT on a shaker (change TBS-T every 15 minutes). The membrane was covered in ECL solution (Thermo Fisher Cat N° 34577) and imaged using the Syngene software.

Pull-down assay protocol

Parental HeLa cells were cultured in 10 cm diameter dishes in high-glucose DMEM with 10 % FBS and 1 % Pen/Strep antibiotics. Prior to cell lysis, the culture medium was discarded and the cells were washed by adding 5 ml of cold PBS. The PBS was removed and the cells were lysed by adding 500 pl of lysis buffer (25 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 % NP40, 5 mM MgCL, 5 % glycerol, phosphatase inhibitor cocktail (PhosSTOP, Roche), EDTA-free protease inhibitor cocktail (Merck)). The cell lysate was scraped off the plate and transferred into fresh Eppendorf tubes. The tubes containing cell lysate were centrifuged for 5 minutes at maximum speed (4°C), the supernatant was collected whilst the pellet was discarded. The obtained total protein concentration was determined using the Pierce BCA Protein Assay Kit. 1 mg of the parental HeLa lysate was pre-incubated with the 5 pg of the V5-tagged AlphaTAC products for 2 hours at 4°C.

The V5-tagged AlphaTACs were pulled down using ant-V5-tag mAb-Magnetic beads (MBL). First, 50 pl of the beads were washed with NP40 wash buffer (25 mM Tris-HCI pH 7.4, 150 mM NaCI, 0.1 % NP40, 2.5 mM MgCL, 5 % Glycerol). Subsequently, the 1 mg of parental HeLa lysate + AlphaTAC product was added onto the V5 beads and incubated overnight at 4°C with gentle rotation. After incubation, the beads were separated from the protein lysate using a magnetic separator, the supernatant was discarded.

The beads were washed three consecutive times with 1 ml of NP40 wash buffer, followed by separation of the beads on the magnetic separator for a few seconds. Subsequently, the beads were resuspended in SDS sample buffer and heated at 70°C in a thermoshaker at 900-1000 rpm. The beads were separated via the magnetic separator and the supernatant was collected. Finally, DTT was added to the samples which were then boiled for 5 minutes at 95°C. The samples were loaded on SDS-PAGE, followed by western blotting.

Cellular uptake assay

The HeLa cell line was kept in culture in DMEM medium supplemented with 5 % FBS in filter cap flasks in a temperature and atmosphere controlled incubator (37°C, 5 % CO2). For cell detachment a Trypsin- EDTA solution was used.

Day 0: HeLa cells were resuspended in culture medium without FBS (assay medium), counted and seeded into assay plates (ThermoFisher Scientific, cat N°160376) compatible with downstream imaging (cover glass bottom, no coating), at a density of 6000 cells per well in a volume of 100 pl. For cell dispensing, the Multidrop dispenser was used (ThermoFisher Scientific) to ensure an even distribution.

Day 1: construct solutions were prepared by dilution in assay medium at desired concentrations (dose response of: 0.125 pM, 0.25 pM, 0.5 pM and 1 pM) and kept in a 96 well plate made out of polypropylene to minimize construct retention/sequestering/sticking.

Prior to the assay, all medium was removed from the assay plate using the aspiration needles of the washer/dispenser. Immediately, 100 pl of the construct stocks was pipetted over the cells using the Freedom Tecan pipetting platform. Subsequently, the cells were incubated for 30 minutes at 37°C, 5% CO2 to allow CPAB uptake. Following that, all fixation and staining steps were performed using the MultiFlo washer/dispenser at room temperature:

The cells were fixated for 20 minutes in 2 % PFA. Subsequently, the medium was removed and replaced with 4 % PFA medium and again allowed to incubate for another 10 minutes. Fixation was followed by two washes with 200 pl of PBS solution and permeabilization with 0.05 % Triton X-100 in PBS for 5 minutes. After permeabilization, the cells were blocked (1 hour) with 2 % BSA (Sigma-Aldrich, cat N°A7030-100G) and 0.05 % Triton X-100 in PBS solution, followed by incubation with the anti-V5 primary antibody (1 hour, ThermoFisher Scientific, catN° R960-25; diluted 1/500). Excess primary antibody was washed off with PBS, followed by secondary antibody staining (ThermoFisher Scientific, catN° A21202; diluted 1/1000) combined with Hoechst staining (DNA staining) (ThermoFisher Scientific, catN° 62249; at 2 pM) for 45 minutes. Excess stain was washed off using PBS, followed by staining with HCS Cell Mask (ThermoFisher Scientific, catN° H32721; diluted 1/50) for 30 minutes. Excess stain was again washed off using PBS.

After staining, the plates were either processed immediately, or kept at 4°C until imaging. For image acquisition, the OPERA Phenix (PerkinElmer) instrument was used. Images were acquired with a 20X water objective, taking a stack of 6 images (0.8 pm distance between), covering a depth of 4 pm total. For every well of the assay plate, a total of 30 fields were imaged (a 6 image Z-stack for each field). Following combinations of emission and excitation wavelengths were used:

• 375 nm excitation and 435-480 nm emission for Hoechst,

• 488 nm excitation and 500-550 nm emission for the construct staining,

• 640 nm excitation and 650-760 nm emission for HCS Cell Mask.

To estimate and compare uptake of constructs, an analysis was preformed using the Harmony software of the OPERA microscope (PerkinElmer).

Analysis was performed as follows: for each stack a maximum project image was created, followed by cells being segmented using the HCS Cell Mask and Hoechst, and measurement of the signal intensity of the construct within the cells. Intensity measures were compared between tested constructs and positive controls for uptake by comparing the measured in-cell intensity signals.

Sequence listing

>SEQ ID NO:1: amino acid sequence of CMPX-326A (including an N-terminal His & C-terminal V5 tag; the Cys in bold and underlined was applied for maleimide coupling of the GR7 peptide)

>SEQ ID NO:2: amino acid sequence of CMPX-558A (including an N-terminal His & C-terminal V5 tag; the Cys in bold and underlined was applied for maleimide coupling of the GR7 peptide) >SEQ ID N0:3: amino acid sequence of CMPX-558B (including an N-terminal His & C-terminal V5 tag; the Cys in bold and underlined was applied for maleimide coupling of the GR7 peptide)

>SEQ ID NO:4: amino acid sequence of CMPX-584C (including an N-terminal His & C-terminal V5 tag; the Cys in bold and underlined was applied for maleimide coupling of the GR7 peptide)

>SEQ ID NO:5: CMPX-321A (including an N-terminal His tag and a C-terminal V5 tag)

>SEQ ID NO: 6: GR7 amino acid sequence (Hifla peptide)

>SEQ ID NO:7: amino acid sequence of CPAB clone 631D (AB584C)

>SEQ ID NO:8: amino acid sequence of CPAB clone 631E (AB584C)

>SEQ ID NO:9: amino acid sequence of CPAB clone 631F(AB584C)

>SEQ ID NO:10: amino acid sequence of CPAB clone 631G (AB584C)

>SEQ ID NO:11: amino acid sequence of CPAB clone 631H

>SEQ ID NO:12: amino acid sequence of CPAB clone 6311

>SEQ ID NO:13: amino acid sequence of CPAB clone 632G

>SEQ ID NO:14: amino acid sequence of CPAB clone 632H

>SEQ ID NO:15: amino acid sequence of CPAB clone 6321

>SEQ ID NO:16: amino acid sequence of CPAB clone 632J

>SEQ ID NO:17: amino acid sequence of CPAB clone 632K

>SEQ ID NO:18: amino acid sequence of CPAB clone 632L (MDM)

>SEQ ID NO:19: amino acid sequence of CPAB clone 435E Alphabody

>SEQ ID NO:20: amino acid sequence of CPAB clone 641A (including an N-terminal His & C-terminal RP12 and V5-tag). For this construct, NHS coupling occurred at the N-terminus.

>SEQ ID NO: 21: V5 tag (wherein X is R or K)

>SEQ ID NO: 22: TEV protease cleavage site

>SEQ ID NO: 23-26: heptad fragments

>SEQ ID NO:27: heptad motif, wherein H refers to hydrophobic amino acid residue (Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, Tryptophan ) and P refers to Polar amino acid residue (Serine, Threonine, Cysteine, Asparagine, Glutamine, Tyrosine)

>SEQ ID NO:28: heptad motif, wherein H refers to hydrophobic amino acid residue (Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, Tryptophan ) and P refers to Polar amino acid residue (Serine, Threonine, Cysteine, Asparagine, Glutamine, Tyrosine)

>SEQ ID NO:29: Glycine-serine linker

>SEQ ID NO:30: Glycine-serine linker

>SEQ ID NO:32: Glycine-serine linker

>SEQ ID NO:33: RP7 peptide tag

>SEQ ID NO:34: albumin binding region wherein X can be any amino acid

>SEQ ID NO:35: albumin binding region wherein X can be any amino acid

>SEQ ID NO:36: albumin binding region wherein X can be any amino acid >SEQ ID NO:37: albumin binding region wherein X can be any amino acid

REFERENCES

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Claims

1. A binding agent comprising at least one Alphabody protein with a structure sequence of the formula HRS1-L1-HRS2-L2-HRS3, specifically binding a target molecule, linked to a degrader moiety, wherein: said Alphabody protein specifically binds a target molecule and has a structure sequence wherein each of HRS1, HRS2 and HRS3 is independently a heptad repeat sequence (HRS) comprising 2 to 7 consecutive heptad repeat units, wherein said heptad repeat units are 7- residue fragments represented as 'abcdefg' or 'defgabc', the symbols 'a¹ to 'g' denoting conventional heptad positions, at least 50 % of all heptad a- and d-positions are occupied by isoleucine residues, and each HRS starts and ends with an aliphatic or aromatic amino acid residue located at either a heptad a- or d-position, and/or wherein the first 'a' of each of said HRS can be threonine or arginine and/or the last 'd' of each of said HRS can be glutamine, and wherein each of LI and L2 are independently a linker fragment, which covalently connect HRS1 to HRS2 and HRS2 to HRS3, respectively, said Alphabody protein comprises a cell-penetrating moiety, and wherein said binding agent is capable to penetrate into a cell and promote degradation of the target molecule when bound to the Alphabody protein.

2. The binding agent of claim 1, wherein the cell-penetrating moiety of the Alphabody protein comprises: at least one positively charged internalization region for facilitating internalization into a cell, wherein said internalization region is characterized by the presence of at least six positively charged amino acid residues of which at least 50 % are comprised within said Alphabody structure sequence, and/or at least one peptide tag for facilitating cellular entry.

3. The binding agent of claims 1 or 2, wherein the cell-penetrating moiety comprises at least one peptide tag for facilitating cellular entry, said peptide tag comprising the sequence (Arg-Pro)n, wherein n is an integer from 6 to 12.

4. The binding agent of any one of claims 1 to 3, wherein the degrader moiety is a small molecule or a peptide coupled to said Alphabody, wherein the coupling is made directly or via a linker.

5. The binding agent of claim 4, wherein coupling is obtained via conjugation, for instance using maleimide or NHS-ester coupling, or enzymatic ligation.

6. The binding agent of any one of claims 1 to 5, wherein the degrader moiety is an E3 ligase complex ligand or binder for promoting protein degradation of the target bound to the Alphabody.

7. The binding agent of any one of claims 1 to 6, wherein the degrader moiety specifically binds the E3 ligase VHL, or CRBN.

8. The binding agent of any one of claims 1 to 7, comprising a further functional moiety, which may be a half-life extending moiety, or a detectable label.

9. A nucleic acid molecule encoding the binding agent of any one of claims 1 to 8 or encoding the Alphabody polypeptide for conjugation with a degrader moiety to result in the binding agent of any one of claims 1 to 8.

10. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 8, or the nucleic acid molecule of claim 9, optionally comprising one or more pharmaceutically acceptable carriers.

11. The binding agent of any one of claims 1 to 8, the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 10, for use as a medicament.

12. The binding agent of any one of claims 1 to 8, the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 10, for use in treatment of a disease or disorder, which is associated with said target molecule.

13. The binding agent of any one of claims 1 to 8, the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 10, for use in treatment of cancer.

14. Use of the binding agent of any one of claims 1 to 8, the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 10, for in vitro protein degradation of a target.

15. A method to produce the binding agent of any one of claims 1 to 8, for use in degradation of a target molecule in a cell, comprising the steps of: a. selection of an Alphabody protein specifically binding a target molecule, preferably through structural design and/or by screening of an Alphabody library; b. engineering of the Alphabody sequence by optimization of the target-binding region, and addition of a cell-penetrating moiety, preferably at N- or C-terminus of the Alphabody sequence; c. expression of the engineered Alphabody protein in a host cell and isolation of the Alphabody protein from said host cell; and d. conjugation of a degrader moiety, and/or optionally screen for target protein degradation using conjugated Alphabody molecules with differently positioned degrader moieties.