WO2023203127A1 - Molécules d'agent de dégradation à base d'alphabody - Google Patents

Molécules d'agent de dégradation à base d'alphabody Download PDF

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WO2023203127A1
WO2023203127A1 PCT/EP2023/060263 EP2023060263W WO2023203127A1 WO 2023203127 A1 WO2023203127 A1 WO 2023203127A1 EP 2023060263 W EP2023060263 W EP 2023060263W WO 2023203127 A1 WO2023203127 A1 WO 2023203127A1
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alphabody
protein
degrader
cell
binding
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PCT/EP2023/060263
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English (en)
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Anna SABLINA
Stefan Loverix
Karen VANDENBROUCKE
Yvonne MCGRATH
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Vib Vzw
Katholieke Universiteit Leuven
Complix Nv
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Publication of WO2023203127A1 publication Critical patent/WO2023203127A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • 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.
  • the binding agent thus comprises cell-penetrating Alphabody proteins linked to a degrader moiety.
  • the invention relates to binding agents which are hybrid molecules wherein the degrader moiety is conjugated to the Alphabody protein.
  • 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.
  • 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.
  • TDP Targeted protein degradation
  • PROTACTM Proteolysistargeting chimera
  • PROTACs 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).
  • 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.
  • many PROTACs are capable of efficiently entering cells and achieve sufficient intracellular concentrations (8).
  • VHL von Hippel-Lindau tumor suppressor
  • CRBN Cereblon
  • RNF4 ring finger protein 4
  • RNF114 ring finger protein 4
  • KEAP1B KEAP1B
  • DCAF15 DCAF16
  • AhR AhR
  • MDIVI2 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).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • E3 ligase rather than an E3 ligand (3)
  • 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.
  • 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 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 'a 1 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
  • 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.
  • 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.
  • 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.
  • 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.
  • said degrader moiety specifically binds an E3 ligase binding site of any one of the E3 ligases selected from VHL or CRBN.
  • the binding agent, or Alphabody-based degrader molecule 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.
  • 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.
  • 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
  • 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.
  • Alphabody-based degrader agents 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.
  • 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.
  • said Alphabodybased degrader is for use in treatment of a disease in the area of cancer, immune-oncology or inflammation.
  • 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
  • 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).
  • FIG. 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.
  • 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.
  • FIG. 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.
  • FIG. 1 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 1 Purified CMPX-326Ap-GR7, CMPX-558Ap-GR7 and CMPX-558Bp-GR7 proteins on SDS-PAGE.
  • MCLl CPAB-based degrader 584Cp-GR7 conjuggated protein SEQ ID NO: 4; used herein as a negative control for (strong) MCLl binding
  • B immunoblotting for MCLl and VHL in the whole cell lysate (WCL).
  • V5 PD V5 pull-down, data representative of two repeats.
  • FIG. 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.
  • CPAB Cellpenetrating Alphabody
  • FIG. 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.
  • FIG. 13 MDM4-HiBiT protein expression upon treatment with distinct MDM2/4 AlphaTAC molecules.
  • 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.
  • 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 conjuggated 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.
  • FIG. 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.
  • FIG. 1 Annotated alignment of Alphabody constructs.
  • MCLl specific Alphabody protein construct CMPX-558A SEQ ID NO:2
  • different conjugation variants of MDM4/2-specific Alphabody protein 632 constructs SEQ ID NO: 13, 14, 16, and 18
  • ClustalW ClustalW
  • 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).
  • 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).
  • LI and L2 linkers used in 632 are examples of 'designed' or optimized linkers used herein.
  • Nucleotide sequence 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.
  • 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.
  • 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.
  • 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".
  • 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.
  • polypeptide 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.
  • isolated or “purified” is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • 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.
  • 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.
  • “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.
  • amino acid identity refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison.
  • 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.
  • the percentage of identity is calculated over a window of the full-length sequence referred to.
  • substitution 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'.
  • 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.
  • modified 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.
  • binding pocket 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.
  • 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.
  • affinity 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • Such functional groups 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).
  • 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).
  • 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
  • peptides for example, a peptide against a serum protein such as albumin
  • determining As used herein, the terms “determining,” “measuring,” “assessing,”, “identifying”, “screening”, and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • subject 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.
  • the subject is a human, a rat or a non-human primate.
  • the subject is a human.
  • 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.
  • patient a disease or disorder as disclosed herein.
  • the aforementioned terms do not imply that symptoms are present.
  • treatment refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder.
  • disease or disorder refer to any pathological state, in particular to the diseases or disorders as defined herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • excipient 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.
  • 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.
  • 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 TWEENTM, PLURONICSTM or PEG and the like.
  • 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.
  • TPD targeted protein degradation
  • 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.
  • CPAB cell-penetrating alphabodies
  • TPD TPD-based degrader molecule' modality
  • AlphaTAC® a binding agent that reveals protein degraders with high specificity for their target or protein of interest, and efficiently acting as potent degraders of the target.
  • 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.
  • Alphabody polypeptides or Alphabody proteins 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.
  • 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.
  • 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).
  • the CPABs presented herein may comprise a cell-penetrating moiety which may be composed of a cell-penetrating peptide, as further defined herein.
  • 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 'a 1 to 'g' denote conventional heptad positions at which amino acid residues are located, and at least 50 %, 70 %, 90 %, or 100 % of the
  • 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,
  • 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.
  • 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).
  • helix-distorting residues e.g., glycine or proline.
  • part of a linker fragment can be alpha-helical even though it is located outside a heptad repeat region.
  • any part of one or more alpha-helical heptad repeat sequences is also considered an alphahelical part of a single-chain Alphabody.
  • 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.
  • 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.
  • '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.
  • 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 (
  • 'heptad 1 , '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.
  • 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).
  • 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.
  • '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.
  • '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 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.
  • the HRS may contain an additional sequence "a”, “ab”, “abc”, “abed”, “abede”, or “abedef” following C-terminally of the (abcdefg)n sequence.
  • a 'heptad repeat sequence' 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.
  • 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.
  • the heptad repeat sequences can be resolved based on knowledge at a higher level than the primary structure (i.e., the amino acid sequence).
  • heptad repeat sequences can be identified and delineated on the basis of secondary structural information (i.e.
  • HRS alpha-helicity
  • tertiary structural i.e., protein folding
  • 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.
  • the identification of HRS fragments can be based on high-resolution 3-D structural information (X-ray or NMR structures).
  • 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.
  • 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 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).
  • 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 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.
  • '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.
  • 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.
  • 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.
  • 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.
  • the 'flexible' linker sequences are glycine and serine- rich sequences with a minimum length of about 2, 4, 6 or preferably 8 amino acids.
  • 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".
  • 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.
  • 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.
  • 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.
  • HRS1-L1-HRS2-L2- HRS3 which is a designed LI
  • the 'Alphabody-based degrader molecule' or 'AlphaTAC', or 'binding agent' 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-oriented 1 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.
  • 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.
  • 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).
  • 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.
  • both halves of the groove are formed by b- and e-residues.
  • 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.
  • the Alphabody polypeptides comprises, within the Alphabody structure, a binding site to an intracellular protein.
  • 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.
  • 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.
  • 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.
  • anti-apoptotic members of the BCL-2 family of proteins are MCLl, BCL-2, BCL-XL, BCL-w and BFL-1/A1.
  • 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.
  • the binding of the Alphabody is driven by one of its alpha-helices, which is stabilized in the Alphabody coiled coil structure.
  • 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.
  • 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.
  • CPABs Cell-penetrating Alphabodies
  • 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.
  • a polypeptide 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.
  • 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.
  • 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”).
  • a motif or pattern can be considered characteristic for providing the polypeptides envisaged herein with cell penetrating activity.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • TAT Trigger opin. Pharmacol. 47:133-140
  • 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;
  • a formatted Alphabody translocating across the cell membrane without compromising target binding properties also called cell-penetrating Alphabody (CPAB)
  • 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).
  • 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.
  • 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 or or from 7 to 10,
  • said peptide tag may be fused to the Alphabody sequence at the N- and/or C-terminal end, directly or via a linker.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • intracellular protein is meant herein a cytosolic, nuclear or membrane-bound protein that is reachable from inside the cell.
  • 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 RPRPRPRPRPRP (SEQ ID NO:33)).
  • 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.
  • polypeptides envisaged herein are characterized by the presence of a sequence such as, but not limited to, RPRPRPRPRPRP (SEQ. ID NO:33).
  • sequence is a (RP)7 cationization motif.
  • 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.
  • 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.
  • the Alphabody is required to bind to a specific target, particular screening steps can further be envisaged.
  • 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.
  • 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).
  • desirable components i.e. Alphabody library members
  • an Alphabody library i.e. an Alphabody library
  • 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.
  • affinity selection 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • a proteolysis targeting chimera 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 AlphabodyTM protein, as previously described.
  • 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.
  • the Alphabody moiety 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.
  • 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.
  • Alphabody being a single chain polypeptide
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • the degrader moiety is also referred to as the "degrader", the “degradation-promoting substrate”, or “degradation-promoting ligand”.
  • 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.
  • 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.
  • a proteasomal degradation pathway component such as an E3 ligase, or an E3 ligase complex component
  • the degrader moiety is an E3 ligase complex ligand or binder for promoting ubiquitination and proteasomal degradation of the target bound to the Alphabody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • non-essential ligases e.g. CRBN and VHL
  • 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).
  • 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).
  • the degrader moiety comprises a ligand or substrate for an E3 ligase specifically present in one or more cell-types or tissues.
  • 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.
  • 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.
  • 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).
  • CTAs cancer testis antigens
  • MAGE-RING ligases 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 peptidic degrader moiety may relates or include a hydroxylated HIFlalpha (Hifla) peptide (e.g. peptide sequence of SEQ.
  • 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.
  • 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.
  • 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.
  • 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.
  • cellpenetrating polypeptide or protein-based agents including an Alphabody
  • 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.
  • 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.
  • CPAB comprises the Alphabody structure sequence as defined herein, functional in specifically binding an intracellular target protein of interest
  • 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.
  • the 'hybrid type' binding agent 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.
  • 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.
  • degrader moieties commonly known and commonly applied in the PROTAC field, however, typically conjugated to a small molecule for target binding.
  • 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.
  • 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.
  • 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.
  • 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.
  • the position of the conjugation or the conjugation site may in one embodiment be at the N-terminus of the Alphabodycontaining polypeptide chain.
  • 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.
  • the conjugation may be at the C-terminal side of said cationization tag.
  • 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.
  • 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.
  • 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.
  • binding agent 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.
  • K lysine
  • 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.
  • 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.
  • Sulfhydryls also called thiols
  • Cys, C cysteine amino acids
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TPD TPD
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a further functional moiety providing for a half-life extension through binding to Albumin 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.
  • 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.
  • binding agents 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.
  • 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).
  • 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.
  • 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.
  • labels, tags or detectable moieties 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 (CBP), maltose binding protein
  • 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.
  • 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.
  • 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 CHO
  • 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).
  • 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.
  • binding agent 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.
  • 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.
  • 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.
  • 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.
  • a tag typically at the N-terminal or C-terminal end
  • 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.
  • compositions 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.
  • pharmaceutically acceptable carriers such as an excipient, diluent or carrier known in the art, and described herein.
  • MCL1 Myeloid cell leukaemia 1
  • BCL-2 BCL-2 homology
  • 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.
  • 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.
  • 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.
  • Alphabody-based degrader molecule which specifically binds a target involved in cancer is disclosed herein for use in treatment of cancer.
  • 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.
  • IRAK-4 interleukin-1 receptor-associated kinase 4
  • BTK bruton tyrosine kinase
  • binding agent or Alphabody-based degrader molecule 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.
  • PROTACs Protein-Penetrating Alphabody proteins
  • 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.
  • 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.
  • CPABs cell-penetrating Alphabodies
  • Alphabody moieties engineered to penetrate the cells 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.
  • MCLl MCLl apoptosis regulator, BCL2 family member
  • MCLl-specific CPAB CMPX-321A SEQ ID NO:5
  • 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.
  • VHL von Hippel-Lindau complex
  • E3 ligase recruitment leads to VHL-mediated ubiquitination of MCLl bound by the CPAB leading to its proteasomal degradation.
  • Phosphosite Plus® v6.6.0.4 Hydrophilphosite Plus® v6.6.0.4 (Hornbeck et al. 2015 Nucleic Acids Res. 43:D512-20)
  • 14 lysines of MCLl could undergo ubiquitination.
  • HIFla peptide contains a hydroxyl-proline
  • 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.
  • 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).
  • 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.
  • CMPX-558Ap-GR7 HIFla peptide coupling at N-terminal end of CPAB, without G/S spacer
  • 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.
  • a steady decline of MCLl is observed, up to a concentration of 100 nM CMPX-558Ap-GR7.
  • 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).
  • 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.
  • 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.
  • myeloma H929 cells and non-small-cell lung cancer H23 cell lines were used to analyze an initial effect of the CPAB-mediated degradation of MCLl.
  • N-Hydroxysuccinimide N-Hydroxysuccinimide
  • 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).
  • AHPC (2S,4R)-l-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4- methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
  • 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/).
  • 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.
  • 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.
  • 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.
  • Example 5 Cellular uptake of AlphaTAC products is comparable to Cell-penetrating Alphabody internalization.
  • Example 6 In vitro characterization of Alphabody-based degrader modalities in reporter degradation cell lines.
  • 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.
  • MCLl and MDM4 degraders were 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 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).
  • 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.
  • 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.
  • 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.
  • MCL1 or MDM4 specific targeting and efficacy are performed through a number of steps.
  • the IC 5 o of the degraders will be determined in target-dependent and target-independent hematological cell lines.
  • 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.
  • 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.
  • the EC 5 o 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 EC 5 o 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.
  • 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.
  • An alternative approach here is to fuse an albumin binding domain via a flexible linker (Gly-Ser) to the MDM4-binding Alphabody.
  • 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.
  • Example 10 In vivo characterization of potent Alphabody-based protein degradation molecules.
  • 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.
  • Samples serum, lung, spleen, liver, kidney, brain, and lymph nodes
  • Samples 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.
  • tumor cells human cell line, chosen based on target-dependency and whether it can establish xenograft tumors. Once tumors reach 100 mm 3 , 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
  • 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.
  • AlphaTAC most potent AlphaTAC identified in dose level 20 mg/kg
  • CPAB control dose level 20 mg/kg
  • 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.
  • 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.
  • 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).
  • IBW1 buffer 50 mM HEPES pH 7.2, 0.25M GuHCI 1% Triton X100
  • 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.
  • Hifla peptide was performed by maleimide coupling to a Cysteine residue present in the MCLl-specific CPAB CMPX-321A at different positions:
  • CMPX-584C maleimide coupling to (N-terminal) Cys at position 22 of SEQ ID NO:4, resulting in CMPX- 584Cp-GR7.
  • MCL1 antibodies 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').
  • 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
  • 90 % saturation was only reached at 1:10 at various lysate concentrations ( Figure IB), so the latter was not included in further experiments.
  • the protein lysate concentration should be such that our protein of interest concentration is within the dynamic range of the assay.
  • 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 ).
  • 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.
  • Conjugation of the small molecule ligands 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.
  • IMAC HisTrap HP 1 ml purification column
  • 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.
  • 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.
  • BSA reference protein sample
  • 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.
  • HiBiT is a very small protein tag ( ⁇ 1.3 kDa) that can be detected using a luciferase-based assay.
  • HiBiT-tagged cell lines were plated at a density of 3-5 x 10 3 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.
  • PBS was added to the medium.
  • the level of HiBiT protein was evaluated using the Nano-Gio HiBit lytic detection system (Promega).
  • 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).
  • 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)).
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • MBL ant-V5-tag mAb-Magnetic beads
  • 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).
  • NP40 wash buffer 25 mM Tris-HCI pH 7.4, 150 mM NaCI, 0.1 % NP40, 2.5 mM MgCL, 5 % Glycerol.
  • 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.
  • 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).
  • a Trypsin- EDTA solution was used for cell detachment.
  • 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.
  • the Multidrop dispenser was used (ThermoFisher Scientific) to ensure an even distribution.
  • 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.
  • 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).
  • BSA Sigma-Aldrich, cat N°A7030-100G
  • the plates were either processed immediately, or kept at 4°C until imaging.
  • the OPERA Phenix (PerkinElmer) instrument was used for image acquisition. 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:
  • 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)
  • 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)

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Abstract

L'invention concerne des agents de liaison pour la dégradation ciblée de protéines comprenant une molécule de protéine alphabody ciblant de manière spécifique une protéine intracellulaire, et une molécule d'agent de dégradation agissant en tant qu'agent de piégeage d'activité protéasomale. Ledit agent liant l'agent de dégradation à base de protéine alphabody forme un complexe ternaire avec la protéine cible et la E3 ligase dans la cellule, conduisant à l'activation de la dégradation ciblée de protéines. En particulier, l'agent de liaison comprend ainsi des protéines alphabody pénétrant dans les cellules liées à une fraction d'agent de dégradation. Plus particulièrement, l'invention concerne des agents de liaison qui sont des molécules hybrides, la fraction de dégradation étant conjuguée à la protéine alphabody. Plus particulièrement, l'invention concerne des agents de liaison comprenant des fractions d'agent de dégradation se liant de manière spécifique à un complexe de E3 ligase pour améliorer l'ubiquitination et la dégradation protéasomale de la cible se liant de manière spécifique à la protéine alphabody. Plus particulièrement, l'invention concerne des agents de liaison comprenant des molécules d'agent de dégradation à base d'alphabody ciblant MCL1 et MDM4/MDM2 destinés à être utilisés dans le traitement de maladies associées auxdites molécules cibles, de préférence destinées à être utilisés dans le traitement du cancer.
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