Cation-independent mannose 6-phosphate receptor conjugates for targeted protein degradation

Abstract

The present invention relates to a protein binding agent that specifically binds to human cation-independent mannose 6-phosphate receptor (CI-M6PR), more specifically to a polypeptide agent that comprises an immunoglobulin single variable domain (ISVD) that specifically binds to CI-M6PR with nanomolar to picomolar affinity, fused to a further protein binding agent that specifically binds to an extracellularly accessible protein target, such as a membrane protein, an extracellular protein or a secreted protein. More specifically, the CI-M6PR specific ISVD recognizes the N-terminal domain 1, 2 and / or 3 of CI-M6PR, thereby providing a means and method for the internalization, lysosomal targeting and degradation of the agent that comprises the ISVD and the target that is bound to the protein binding agent. Thus, the CI-M6PR conjugate disclosed herein is linked or fused to a further protein binding agent, in particular linked or fused to another antigen binding protein (such as an ISVD or an antibody) that is relevant for use in therapy, more specifically for the treatment of a disease that is affected by the target antigen that is bound by the antigen binding protein. More specifically, disclosed herein is a CI-M6PR ISVD fusion to an antigen binding protein that specifically binds EGFR for the treatment of cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a protein binding agent that specifically binds to human cation-independent mannose 6-phosphate receptor (CI-M6PR), more specifically to a polypeptide agent that comprises an immunoglobulin single variable domain (ISVD) that specifically binds to CI-M6PR with nanomolar to picomolar affinity, fused to a further protein binding agent that specifically binds to an extracellularly accessible protein target, such as a membrane protein, an extracellular protein or a secreted protein. More specifically, the CI-M6PR specific ISVD recognizes the N-terminal domain 1, 2 and / or 3 of CI-M6PR, thereby providing a means and method for the internalization, lysosomal targeting and degradation of the agent that comprises the ISVD and the target that is bound to the protein binding agent. Thus, the CI-M6PR conjugate disclosed herein is linked or fused to a further protein binding agent, in particular linked or fused to another antigen binding protein (such as an ISVD or an antibody) that is relevant for use in therapy, more specifically for the treatment of a disease that is affected by the target antigen that is bound by the antigen binding protein. More specifically, disclosed herein is a CI-M6PR ISVD fusion to an antigen binding protein that specifically binds EGFR for the treatment of cancer. [Background technology]

[0002] background Small molecule drugs act by binding to well-defined pockets in disease-causing proteins and modulating their function. However, as many proteins lack such niches, as much as 85% of the human proteome is currently considered undruggable (Neklesa, et al., 2017, Pharmacol. Ther. 174, 138-144). This challenges the emergence of PROteolysis TArgeting Chimera (PROTAC) technology, a therapeutic modality that exploits the ubiquitin-proteasome system for the selective degradation of intracellular target proteins (Sakamoto, et al. (2001) Proc. Natl. Acad. Sci. 98, 8554-8559). Such degraders consist of a conjugate of E3 ligase coupled to a ligand that can bind to any site on the target protein. In addition to enabling the targeting of undruggable proteins, targeted protein degradation strategies have another advantage over inhibition-based treatment strategies: removal of the protein abolishes all of its functions, which is important, for example, when the protein acts as a signaling scaffold. However, because PROTACs use the cytosolic proteolytic machinery, they are essentially limited to target engagement in the intracellular environment.

[0003] Nanobodies are variable domains of heavy chain-only antibodies (VHHs) of camelid origin that are characterized by their small size (±15 kDa) [1]. This allows for good tissue penetration while maintaining similar potency and binding specificity as conventional antibodies [3]. As modular building blocks, VHHs can be easily tethered together in the multivalent and / or multispecific formats exploited in this approach. VHHs are also highly stable and soluble, and therefore can be easily and cost-effectively produced in lower organisms such as bacteria and yeast [4]. Among the intracellular TPD approaches investigated, a nanobody-based fusion, termed the ARMeD system, has been shown to provide Nbs coupled to the RING domain of the E3 ubiquitin ligase RNF4 that specifically target proteins of interest, thereby triggering their degradation without off-target effects upon delivery to cells (Zhong et al. Eur J Med Chem. 2022;231:114142; Ibrahim,et al. Molecular Cell,2020.79,(1),155-166.e9).

[0004] Due to their great conformational stability, they have high intrinsic pH and protease resistance [1], which are attractive properties for circulating the endosomal-lysosomal system. Moreover, VHH-based formats are suitable for various administration routes, including by intravenous injection and inhalation, positioning them as ideal components for therapeutic purposes. GlueTAC, for example, is a chimera based on a covalent antigen-binding nanobody conjugated to a cell-penetrating peptide and a lysosomal sorting sequence to target membrane proteins and trigger lysosomal degradation (Zhang, et al. J. American Chem. Society. 2021. 143(40), 16377-16382).

[0005] In fact, lysosomes are cellular acidifying organelles that contain more than 70 hydrolytic enzymes. These enzymes are responsible for the breakdown of cleavable cellular macromolecules into their original building blocks [2]. Macromolecules generally reach lysosomes via endocytosis, phagocytosis or endocytosis, after which each basic unit can be recycled and used in the synthesis of other macromolecules or further metabolized as a source of energy.

[0006] It is known that membrane-bound protein targets are ubiquitinated by the expression of membrane-bound E3 ligases, thereby inducing their endocytosis and lysosomal degradation. Novel techniques have demonstrated the use of this mechanism to apply membrane-bound E3 ligases to co-target membrane or extracellular proteins for degradation. For example, AbTAC reported by Cotton et al. (J. Am. Chem. Soc. 2021, 143, 593-598); and heterobifunctional molecules targeting membrane-bound E3 ligases and transmembrane target proteins reported by Maurice (WO2021 / 176034A1).

[0007] Degradation of extracellularly accessible proteins can also be made possible by externally exploiting lysosomes by receptor-mediated endocytosis via the cation-independent mannose 6-phosphate receptor (CI-M6PR), a P-type lectin on the plasma membrane of cells, which constantly recycles through the endolysosomal pathway, thereby efficiently internalizing proteins or targets bound to the receptor and delivering them to endosomes and lysosomes. A further application is therefore based on the acidic pH in endosomes, which leads to dissociation of cargo or complexes from the CI-M6PR receptor at a pH of around 5.8 at the late endosomal stage

[20] , allowing rapid recycling of the CI-M6PR receptor itself, which in virtually all cell types can constantly shuttle between the cell surface and the late endosomal compartment to target extracellular ligands to lysosomes (Dahms, et al. (1989), SJ Biol. Chem. 264, 12115-12118).

[0008] Thus, the CI-M6PR cargo is efficiently delivered to lysosomes via the endocytic cycle, a concept used in the design of lysosome-targeted chimeras (LYTACs)

[10] , similar to PROTACs, providing an alternative format to coupling complex chemically synthesized glycopeptide ligands of CI-M6PR to anti-targeting antibodies. LYTACS allow the depletion of secreted and membrane-associated proteins and have been shown to be agonists of CI-M6PR

[10] . LYTACs have been shown to internalize and degrade a selection of both extracellular and transmembrane proteins in vitro when administered to cells. However, a downside from the perspective of in vivo applications and large-scale production is the large size of the construct (±150 kDa of a monoclonal antibody), which can hinder its biodistribution in solid tissues

[21] , and recombinant expression in mammalian cells is required. Furthermore, when mannose 6 phosphonate (M6Pn) glycopolypeptide is used for binding CI-M6PR, the long synthetic process for producing the ligand and its subsequent conjugation to the antibody is very complicated and very expensive. In fact, the production of mannose 6 phosphonate (M6Pn) glycopolypeptide ligand and its subsequent conjugation to the antibody involves a 13-step synthetic process.

[0009] An interesting example of an extracellularly accessible protein target is, for example, the human epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase (RTK) that plays a central role in the growth and maintenance of epithelial tissues. It is frequently overexpressed and causes disease progression in many types of cancer, including an estimated 60–80% of colorectal cancers (CRC)

[23] . Chemotherapy is usually the first-line treatment for unresectable metastatic CRC (mCRC), and in patients with RAS wild-type (WT) cancer, this is combined with one of two approved anti-EGFR monoclonal antibodies (mAbs), cetuximab or panitumumab. These exert their function primarily by antagonizing EGF-stimulated activation of EGFR, thereby inhibiting its kinase function. The addition of such mAbs to chemotherapy for the treatment of RAS-WT mCRC showed an extension of overall survival by several months compared to chemotherapy alone

[24] . However, activating mutations in KRAS (the predominantly mutated RAS isoform in CRC), a downstream component of the EGFR signaling pathway, occur in approximately 35–45% of CRCs and are the major intrinsic resistance mechanism to anti-EGFR mAbs

[25] . However, among RAS-WT mCRCs, those harboring V4600E mutations in the BRAF gene also fail to respond to treatment

[26] . Furthermore, acquired resistance to anti-EGFR mAbs occurs in virtually all patients, with half of the cases caused by secondary mutations in the KRAS gene

[27] and sometimes in the EGFR extracellular domain, evading antibody binding

[28] . In this context, targeted degradation of EGFR may provide an exciting new strategy to overcome intrinsic and acquired resistance. In contrast to EGFR inhibition, downregulation of EGFR has been shown to induce cell death in a range of cancer cells, including the KRAS-mutated HCT116 cell line, which has relatively low EGFR expression and does not respond to cetuximab

[29] . Indeed, kinase-inhibited EGFR can function in several ways as a scaffolding node for interaction with survival proteins and maintenance of downstream pro-survival signaling [29–30].

[0010] Therefore, for many therapeutic applications, a lysosome targeting approach as an adjunct to targeted protein degradation would be beneficial as an alternative mechanism of action to provide novel medical modalities.Therefore, it is necessary to generate next-generation lysosome-targeting binding agents that overcome the above-mentioned obstacles of existing lysosome targeting strategies. Summary of the Invention

[0011] Summary of the Invention With the aim of providing a new type of binder capable of mediating lysosomal targeting via reversible binding to the CI-M6PR receptor, the present invention is based on the application of human-to-mouse cross-reactive immunoglobulin single variable domains (ISVDs), in particular VHHs, that bind to CI-M6PR at physiological pH and dissociate therefrom in a pH-dependent manner, resulting in lysosomal uptake (Callewaert et al., PCT / EP2022 / 054278). The covalent coupling of such anti-CI-M6PR VHHs to further conjugates specific for extracellular, secreted or transmembrane target proteins ultimately results in a novel modality for CI-M6PR-mediated lysosomal uptake and degradation. The present invention therefore relates to a new VHH-based LYTAC format, also called nanoLYTAC, where the efficacy and potency of endosomal / lysosomal targeting depends on the properties of the fusion protein provided, on the one hand, by an immunoglobulin single variable domain (ISVD) that recognizes CI-M6PR for recycling, and on the other hand on a coupled binder specific for the extracellularly accessible target protein. It was found that this new format offers several substantial advantages over existing extracellular targeted proteolytic modalities.

[0012] By using anti-CI-M6PR VHH specifically designed and characterized for lysosomal targeting, alternative nanobody-based LYTACs (or nanoLYTACs) form functional bispecific therapeutic tools to deliver antigen-binding protein domains, such as antibodies, or more specifically, other coupled binding agents, including ISVDs or VHHs, for lysosomal degradation, which can then be selected for their properties in targeting specific extracellularly accessible proteins of interest. As a proof of concept, a characterized VHH specific for CI-M6PR, as reported in Callewaert et al. (PCT / EP2022 / 054278), was coupled to an antigen-binding protein known to target the transmembrane receptor EGFR, as exemplified herein. Further POCs have been demonstrated showing that endocytic internalization and / or lysosomal degradation was obtained upon coupling with at least two types of CI-M6PR-specific VHHs disclosed herein, each type being characterized to bind to a CI-M6PR epitope located in the N-terminal domains 1-3 as characterized in Callewaert et al. (PCT / EP2022 / 054278). Indeed, this panel of VHHs has previously been characterized as a panel of CI-M6PR binders with different pH-dependencies for association with the receptor, thus providing a useful toolbox for designing customized Nb-based LYTACs taking into account the desired outcome or treatment objective.

[0013] The present invention relates to multispecific lysosomal targetable anti-CI-M6PR binders, called nanolysosomal targeting chimeras or nanoLYTACs, based on the identification of a panel of VHHs that specifically bind to the N-terminal region of human and mouse CI-M6PR present on the extracellular side of the plasma membrane, thereby enabling transport through the endolysosomal pathway. Moreover, the anti-CI-M6PR VHHs adopt specific pH-dependent dissociation properties that facilitate delivery to the lysosomal compartment. Fusion of these anti-CI-M6PR VHH moieties to additional protein binders, preferably with antigen binding for targeting other extracellular or membrane targets, allows the application of these binders for targeted lysosomal internalization and protein target degradation.

[0014] Thus, a first aspect of the present invention relates to a protein conjugate that specifically binds to the human cation-independent mannose 6-phosphate receptor (CI-M6PR; also known as IGF2R) and contains an immunoglobulin single variable domain (ISVD) that specifically recognizes a binding site located on the extracellular N-terminal domains 1, 2 and / or 3 of the human CI-M6PR, said ISVD being fused to a protein binding domain or agent that specifically binds to an extracellularly accessible target. More specifically, said CI-M6PR specific ISVD of said protein binder provides high affinity binding to the receptor in vitro or in cells, and exhibits high affinity binding to the K D The value is in the range of 100nM or less.More specifically, when the protein binding agent is bound to CI-M6P receptor, it is internalized in the cell.Preferably, when the protein binding agent is bound to CI-M6PR, it is internalized in the cell as a complex with the extracellularly accessible target that is bound to the binding agent that specifically binds to the extracellularly accessible target.

[0015] In certain embodiments, the protein binding agent (also referred to herein as nanoLYTAC) comprises an ISVD that specifically recognizes a binding site located on N-terminal domains 2 and 3 and specifically binds to CI-M6PR, defined by an epitope comprising amino acid residues 191, 194-197, 208, 219, 224, 225, 297, 357, 408-409, 431, 433, and 457 as set forth in SEQ ID NO: 23. Further particular embodiments provide such binding agents comprising an ISVD that specifically binds via interactions between residues 32, 52-57, 100-103, and 108 thereof as set forth in SEQ ID NO: 8 and residues herein represented as epitopes in N-terminal domains 2 and 3 of CI-M6PR.

[0016] Another particular embodiment relates to said protein binding agents (also referred to herein as nanoLYTACs) comprising an ISVD that specifically recognizes a binding site located on N-terminal domain 1 and specifically binds to CI-M6PR defined by an epitope comprising amino acid residues 59, 60, 85, 87, 89, 146, 147, and 148 and 118 or 119 as set forth in SEQ ID NO: 23. A further particular embodiment provides said binding agents comprising an ISVD that specifically binds via an interaction between residues 31, 33, 35, 53, 54, 56, 57, 96 and 104 thereof as set forth in SEQ ID NO: 7, or residues 31-35, 50, 52-57, 96-98 as set forth in SEQ ID NO: 24, and residues as set forth herein as epitopes in N-terminal domain 1 of CI-M6PR.

[0017] In a particular embodiment, the binding agent comprises or consists of a fusion protein comprising a CI-M6PR-specific ISVD as described herein and a binding agent that specifically binds to an extracellularly accessible protein target fused directly or via a linker, preferably the ISVD is structured according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1) and comprises CDR1, CDR2 and CDR3 regions selected from the CDR1, CDR2 and CDR3 regions of an ISVD sequence selected from the group of SEQ ID NOs: 1, 5, 7, 8, 24 or 25, wherein the CDR regions are annotated according to Kabat, MacCallum, IMGT, AbM or Chothia. In certain embodiments, the M6PR-specific ISVD comprises CDR1, CDR2 and CDR3 from SEQ ID NO:1, or CDR1, CDR2 and CDR3 from SEQ ID NO:5, or CDR1, CDR2 and CDR3 from SEQ ID NO:7, or CDR1, CDR2 and CDR3 from SEQ ID NO:8, or CDR1, CDR2 and CDR3 from SEQ ID NO:24, or CDR1, CDR2 and CDR3 from SEQ ID NO:25, wherein the CDRs may be defined according to the annotations of Kabat, MacCallum, IMGT, AbM, or Chothia, as further defined herein.

[0018] Further embodiments relate to the protein binding agents described herein, wherein the CI-M6PR specific ISVD comprises a CDR1 sequence selected from SEQ ID NOs: 36-41, a CDR2 sequence selected from SEQ ID NOs: 42-47, and a CDR3 sequence selected from SEQ ID NOs: 48-53, or comprises an ISVD having: - CDR1 consisting of SEQ ID NO: 36, CDR2 consisting of SEQ ID NO: 42 and CDR3 consisting of SEQ ID NO: 48, - CDR1 consisting of SEQ ID NO: 37, CDR2 consisting of SEQ ID NO: 43 and CDR3 consisting of SEQ ID NO: 49, - CDR1 consisting of SEQ ID NO: 38, CDR2 consisting of SEQ ID NO: 44 and CDR3 consisting of SEQ ID NO: 50, - CDR1 consisting of SEQ ID NO: 39, CDR2 consisting of SEQ ID NO: 45 and CDR3 consisting of SEQ ID NO: 51, - a CDR1 consisting of SEQ ID NO: 40, a CDR2 consisting of SEQ ID NO: 46, and a CDR3 consisting of SEQ ID NO: 52, or - a CDR1 consisting of SEQ ID NO: 41, a CDR2 consisting of SEQ ID NO: 47, and a CDR3 consisting of SEQ ID NO: 53.

[0019] A further embodiment is said protein binding agent comprising a CI-M6PR specific ISVD comprising said CDRs of SEQ ID NO: 1, 5, 7, 8, 24 or 25 annotated according to AbM, comprising a FR1 sequence corresponding to SEQ ID NO: 78, a FR2 sequence corresponding to SEQ ID NO: 79, a FR3 sequence corresponding to SEQ ID NO: 80, and a FR4 sequence corresponding to SEQ ID NO: 81, or a FR1 sequence selected from SEQ ID NOs: 54-59, a FR2 sequence selected from SEQ ID NOs: 60-65, a FR3 sequence selected from SEQ ID NOs: 66-71, and a FR4 sequence selected from SEQ ID NOs: 72-77, or a protein binding agent comprising: FR1 consisting of SEQ ID NO: 54, FR2 consisting of SEQ ID NO: 60, FR3 consisting of SEQ ID NO: 66 and FR4 consisting of SEQ ID NO: 72, FR1 consisting of SEQ ID NO: 55, FR2 consisting of SEQ ID NO: 61, FR3 consisting of SEQ ID NO: 67 and FR4 consisting of SEQ ID NO: 73, FR1 consisting of SEQ ID NO: 56, FR2 consisting of SEQ ID NO: 62, FR3 consisting of SEQ ID NO: 68 and FR4 consisting of SEQ ID NO: 74, FR1 consisting of SEQ ID NO: 57, FR2 consisting of SEQ ID NO: 63, FR3 consisting of SEQ ID NO: 69 and FR4 consisting of SEQ ID NO: 75, FR1 consisting of SEQ ID NO: 58, FR2 consisting of SEQ ID NO: 64, FR3 consisting of SEQ ID NO: 70 and FR4 consisting of SEQ ID NO: 76, or FR1 consisting of SEQ ID NO: 59, FR2 consisting of SEQ ID NO: 65, FR3 consisting of SEQ ID NO: 71 and FR4 consisting of SEQ ID NO: 77, or any humanized variant thereof as further described herein.

[0020] Another embodiment relates to said binding agent, wherein said CI-M6PR specific ISVD comprises a sequence selected from the group of SEQ ID NO: 1, 5, 7, 8, 24 or 25, or a sequence having at least 85% amino acid identity thereto and containing the same CDRs as SEQ ID NO: 5, 7, 8, 24 or 25, or a humanized variant thereof as further defined herein or as set forth in SEQ ID NOs: 26 to 35.

[0021] Further specific aspects relate to the binding agent described herein that is a multispecific or multivalent binding agent. More particularly, bivalent or bispecific agents are envisaged herein. Even more specifically, a multispecific protein binding agent is envisaged that specifically binds to human CI-M6PR, specifically recognizes the binding site located on the extracellular N-terminal domain 1, 2 and / or 3 of human CI-M6PR as defined herein, and is fused or linked to a binding agent that specifically binds to an extracellularly accessible target, said fusion or linkage being carried out by direct coupling or via a linker that may be a short peptide linker, or a polypeptide moiety such as an Fc tail or another moiety that may include an additional antigen binding domain or more specifically an ISVD. Specifically, said binding agent that includes an ISVD that specifically binds to CI-M6PR may include a binding moiety that specifically binds to a cell surface or extracellular molecule, and this binding moiety also includes an ISVD and / or an additional moiety for specifically binding to an extracellularly accessible target.

[0022] In a particular embodiment, the fusion protein or binding agent of the invention is a multispecific fusion protein comprising a CI-M6PR specific ISVD of the invention and optionally further moieties fused to a protein conjugate that specifically binds to an extracellularly accessible target, any of the components may be labeled for detection or may provide a tag or label.

[0023] Another embodiment relates to a protein binding agent of the invention comprising a multispecific fusion protein comprising a CI-M6PR specific ISVD of the invention fused to a protein conjugate that specifically binds to an extracellularly accessible target and optionally a further moiety, said target specific protein conjugate comprising or consisting of an antigen binding protein domain, more particularly comprising an ISVD, or an antibody or an active fragment thereof, or particularly an IgG, or any type of VHH-Fc fusion format. In a further particular embodiment, said further moiety is a functional moiety, preferably comprising an antigen binding domain, such as a therapeutic moiety that preferably binds to a further target, and / or a half-life extension.

[0024] In certain embodiments disclosed herein, the protein binding agent of the present invention comprises a binding agent that specifically binds to the transmembrane protein epidermal growth factor receptor (EGFR) at an extracellular site. More specifically, the fusion protein comprises an EGFR-specific binding agent comprising an antibody comprising an ISVD consisting of SEQ ID NO: 12, 17, or a homologue having at least 90% identity thereto and identical CDRs, or a heavy chain as shown in SEQ ID NO: 87 and a light chain as shown in SEQ ID NO: 86, providing EGFR-specific conventional antibody binding as an EGFR-specific binding agent, more specifically, the protein binding agent may comprise SEQ ID NO: 88 or 89 and SEQ ID NO: 86. Alternatively, the protein binding agent of the present invention which specifically binds to the extracellularly accessible protein target EGFR comprises a sequence selected from the group of sequences SEQ ID NO: 13, 14, 18, 19, 82 to 85, or functional homologues thereof having at least 90% identity and identical CDRs, or heavy chain-VHH fusions of SEQ ID NO: 88 or 89 provided as EGFR-specific antibodies together with light chain SEQ ID NO: 86.

[0025] Another aspect relates to a nucleic acid encoding a protein binding agent or fusion protein comprising a CI-M6PR specific ISVD fused to an extracellularly accessible target-specific protein binding agent as described herein, or further a combined multispecific binding agent.Furthermore, a vector comprising said nucleic acid molecule for expression of said binding agent or fusion protein is disclosed herein.

[0026] Another aspect relates to the application or use of the binding agents, multispecific binding agents, fusion proteins or nucleic acids disclosed herein in drug discovery, structural analysis, or screening assays, such as structure-based drug discovery or fragment-based screening assays.

[0027] Another aspect relates to a production method for obtaining a binding agent as described herein, comprising the steps of providing a fusion protein of the invention by recombinant expression of a nucleic acid molecule and optionally a further nucleic acid molecule (in case of antibody expression) in a host, and purifying the fusion protein from said host, optionally in the format of an antibody formed by the fusion protein and a further antibody chain.

[0028] Further embodiments relate to the application or use of the multispecific binding agent described herein, for example a bispecific agent comprising an ISVD that specifically binds to CI-M6PR and a second antigen binding domain for binding to an extracellularly accessible target protein, in a method for degrading the target, which is a cell surface molecule or an extracellular molecule or a transmembrane protein, by lysosomal uptake of the multispecific agent in lysosomes when bound to the target. Certain embodiments further disclose the use of the binding agent, multispecific binding agent or fusion protein described herein for in vitro lysosomal tracking, optionally when operably linked or chemically coupled to a label.

[0029] A further aspect relates to a pharmaceutical composition comprising any of the binding agents, multispecific binding agents, or fusion proteins described herein.

[0030] Another aspect of the present invention relates to the medical use of the binding agent, multispecific binding agent, fusion protein or pharmaceutical composition described herein. More specifically, the agent or protein is used for treating lysosomal storage disease or for enzyme replacement therapy. Another aspect of the present invention relates to the multispecific binding agent described herein or the pharmaceutical composition comprising the multispecific binding agent for use in disorders related to the target of disease caused by or related to the extracellularly accessible protein target to which the binding agent is specifically bound, more specifically the target being a cell surface or extracellular molecule. Specifically, in one embodiment, the target is EGFR, and the binding agent is provided for use in the treatment of cancer.

[0031] A final aspect of the invention relates to the binding agents, multispecific binding agents, fusion proteins, or labeled forms thereof for use as diagnostics or in vivo imaging.

[0032] The drawings described are only schematic and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn to scale for illustrative purposes. [Brief description of the drawings]

[0033] [Figure 1] Figure 1 is an SDS-PAGE analysis of LYTAC expression studies in Pichia pastoris. Constructs 14-19 (compositions shown in Table 1) were produced in wild-type Pichia pastoris (i.e., NCYC2543) and 20 μl of supernatant was analyzed by SDS-PAGE. "MM" = molecular weight marker (Precision Plus Protein Standard, Bio-Rad). [Diagram 2]Figure 2 is an SDS-PAGE analysis of endoglycosidase H (EndoH) digests of LYTAC. Constructs 14-19 (compositions shown in Table 1) were produced in wild-type Pichia pastoris (i.e., NCYC2543) and 19 μl of supernatant was incubated with EndoH overnight at 37°C and then analyzed by SDS-PAGE. "MM" = molecular weight marker (Precision Plus Protein Standard, Bio-Rad). [Diagram 3] Figure 3 is an SDS-PAGE analysis of LYTAC expression studies in Pichia pastoris. Constructs 26-29 (compositions shown in Table 1) were produced in WT Pichia pastoris and 20 μl of supernatant was analyzed by SDS-PAGE. Clones shown in red were selected for larger scale expression and purification. "MM" = molecular weight marker (Precision Plus Protein Standard, Bio-Rad). [Figure 4] Figure 4 is an SDS-PAGE of gravity-flow immobilized metal ion chromatography (IMAC) purification for LYTAC constructs. Constructs 26-29 (compositions shown in Table 1) were expressed in 50 ml cultures of wild-type Pichia pastoris (i.e., NCYC2543) and purified from the supernatant through gravity-flow IMAC and subsequent desalting. 20 μl of flow-through (FT) and wash (W) fractions and 1 μg of purified protein (P) were analyzed by SDS-PAGE. "MM" = molecular weight marker (Precision Plus Protein Standard, Bio-Rad). [Figure 5A]Figure 5. In vitro EGFR internalization efficacy of VHH-based nanoLYTAC constructs as determined by flow cytometry. HeLa cells were treated with 5 or 50 nM of nanoLYTAC constructs (26-27) or control for 24 h. Live cells were stained for cell surface EGFR (PE-AF647) and measured on a BD LSR II flow cytometer. (A) Representative flow cytometry histograms of cell surface EGFR levels measured for untreated HeLa cells or HeLa cells treated with 5 nM of nanoLYTAC constructs 26 (9G8 S54A-VHH8) or 27 (2x9G8 S54A-VHH8) or the corresponding control constructs 28 (9G8 S54A-GBP) or 29 (2x9G8 S54A-GBP), respectively. [Figure 5B] Figure 5. In vitro EGFR internalization efficacy of VHH-based nanoLYTAC constructs as determined by flow cytometry. HeLa cells were treated with 5 or 50 nM of nanoLYTAC constructs (26-27) or control for 24 h. Live cells were stained for cell surface EGFR (PE-AF647) and measured on a BD LSR II flow cytometer. (B) Representative flow cytometry histograms of cell surface EGFR levels measured for untreated HeLa cells or HeLa cells treated with 50 nM of nanoLYTAC constructs 26 (9G8 S54A-VHH8) or 27 (2x9G8 S54A-VHH8) or the corresponding control constructs 28 (9G8 S54A-GBP) or 29 (2x9G8 S54A-GBP), respectively. [Figure 5C]Figure 5. In vitro EGFR internalization efficacy of VHH-based nanoLYTAC constructs as determined by flow cytometry. HeLa cells were treated with 5 or 50 nM of nanoLYTAC constructs (26-27) or control for 24 h. Live cells were stained for cell surface EGFR (PE-AF647) and measured on a BD LSR II flow cytometer. (C) Bar graph showing the median fluorescence intensity measured for each condition. Data are the mean ± SEM of two replicates. [Figure 6] Figure 6 is a Western blot analysis for detection of total EGFR in HeLa cell lysates. HeLa cells were treated in duplicate with 50 nM of constructs 26 (9G8 S54A-VHH8), 27 (2x9G8 S54A-VHH8), 28 (9G8 S54A-GBP) or 29 (2x9G8 S54A-GBP) or left untreated (UT) for 24 hours. As a positive control for EGFR degradation, cells were treated with 50 ng / ml of recombinant human EGF. Cell lysates were obtained and immunoblotted for EGFR and β-actin. "kDa" = kilodaltons. [Figure 7] Figure 7 shows the primary images of the live cell imaging experiments. (A-F) Shown are specific VHHs fluorescently labeled with Alexa Fluor 488 (i.e., VHH7, -1, -5, -8, negative control (GBP) or recombinant human acid alpha-glucosidase (rhGAA) used as a positive control). For each image, the most appropriate Z-stack was selected over a 120 min incubation, and intracellular proteins (green) were shown together with LysoTracker (magenta) and brightfield signals. Imaging was performed on a Zeiss Spinning Disk microscope with a Plan-Apochromat 40X (1.40 oil DIC UV-Vis-IR M27) ​​objective. [Figure 8]Figure 8. Microscopic analysis of anti-CI-M6PR VHH7 and VHH8 internalized and in lysosomes. Alexa Fluor 488 (AF488) labeled VHHs were incubated (37°C) for 4 hours on HeLa cells and stained with anti-LAMP1 antibody detected using DyLight594 coupled antibody. (A) Percentage of endocytosed anti-CI-M6PR VHH-AF488 detected in LAMP1 positive lysosomes. (B) Percentage of LAMP1 stained lysosomes containing VHH7 and VHH8. (C) Images corresponding to colocalization of LAMP1 (magenta) with AF488-VHH7, AF488-VHH8 treated and untreated (medium) cells (green). Nuclei were stained with DAPI (cyan). Imaging of three fields of view was performed for each VHH-AF488 on an LSM880 Airyscan confocal microscope (Zeiss) in SR mode using a 63X objective. [Figure 9] Figure 9 shows the association-dissociation graphs of humanized VHH7 variants analyzed using Biolayer Interferometry (BLI). BLI was performed in kinetics buffer (0.2M Na2HPO4, 0.1M Na+ citrate, 0.01% bovine serum albumin, 0.002% Tween-20) on an Octet Red96 (ForteBio) instrument. Biotinylated human domain 1-3His6 was immobilized on a streptavidin SA biosensor (Sartorius) to a signal of 0.6 nm. An association phase of 120 s in serially diluted (0–200 nM) VHH7 (A), VHH7h1 (B), VHH7h2 (C), VHH7h3 (D) or VHH7hWN (E) in phosphate citrate buffer at pH 7.4 was followed by a dissociation phase of 420 s in phosphate buffer at either pH 7.4, 6.5, 6.0, 5.5 or 5.0. Between runs, the biosensor was regenerated by three exposures of 10 s to regeneration buffer (10 mM glycine pH 3). The extent of association and dissociation was measured in δ nm over time (s). The black curves represent double-baseline subtracted data fitted according to a 1:1 binding model (grey dashed line). [Figure 10]Figure 10 shows the association-dissociation graphs of humanized VHH8 variants analyzed using Biolayer Interferometry (BLI). BLI was performed on an Octet Red96 (ForteBio) instrument in kinetics buffer (0.2M Na2HPO4, 0.1M Na+ citrate, 0.01% bovine serum albumin, 0.002% Tween-20). Biotinylated human domain 1-3His6 was immobilized on a streptavidin SA biosensor (Sartorius) to a signal of 0.6 nm. An association phase of 120 s in serially diluted (0-200 nM) VHH8 (A), VHH8h1 (B), VHH8h2 (C), VHH8h3 (D) or VHH8hWN (E) in phosphate citrate buffer at pH 7.4 was followed by a dissociation phase of 420 s in phosphate buffer at either pH 7.4, 6.5, 6.0, 5.5 or 5.0. Between assays, the biosensor was regenerated by three exposures of 10 s to regeneration buffer (10 mM glycine pH 3). The extent of association and dissociation was measured in δ nm over time (s). The black curve represents the double-baseline subtracted data fitted according to a 1:1 binding model (grey dashed line). [Figure 11] Figure 11 is an amino acid sequence alignment of CI-M6PR domains 1-3 for human, mouse and bovine proteins and a representation of the VHH7 / 1H11 and VHH8 epitope residues. Multiple alignment of bovine (B / , Bos taurus), human (H / , Homo sapiens) and mouse (M / , Mus musculus) CI-M6PR domains 1-3 sequences showing three distinct domains of the antigen, domain 1 (D1; bovine residues 49-171), domain 2 (D2; bovine res. 172-325) and domain 3 (D3; bovine res. 326-476). Full circles represent core epitope residues selected based on integration of the 4 angstrom distance output of the VHH, PISA and FastContact analyses. Semicircles define additional residues within 4 angstrom distance of the VHH. [Figure 12]FIG. 12 is a diagrammatic representation of the co-crystal structure of domains 1-3 of VHH7 and hCI-M6PR. (A) VHH7 is colored black with its paratope residues (shown as sticks) facing domain 1 (D1) of CI-M6PR (gray). A detailed view of the CI-M6PR epitope of VHH7 is shown in B and C. (B) Detailed interface of CI-M6PR D1 displayed as a surface diagram, and the paratope residues of CDR1, -2 and -3 of VHH7 are shown as sticks. (C) Detailed interface of VHH7 displayed as a surface diagram, and the epitope residues of CI-M6PR D1 shown as sticks. (D) Shown are the paratope residues of VHH7 (black) within less than 4A of the epitope region on D1 (gray). [Figure 13] FIG. 13 is a diagrammatic representation of the co-crystal structure of domains 1-3 of VHH8 and hCI-M6PR. (A) VHH8 is colored black with its paratope facing domains 2 (D2) and D3 (gray) of CI-M6PR. A detailed view of the CI-M6PR epitope of VHH8 is shown in B and C. (B) Detailed interface of CI-M6PR D2 and D3 displayed as a surface diagram (light grey) and sticked paratope residues of CDR1, -2 and -3 of VHH7 (dark grey). (C) Detailed interface of VHH8 displayed as a surface diagram and epitope residues of CI-M6PR D2 and D3 shown as sticks. (D) Shown are paratope residues of VHH8 (black) within less than 4A of the epitope region on D1 (grey). [Figure 14]FIG. 14 is a diagrammatic representation of the co-crystal structure of domains 1-3 of VHH 1H11 and hCI-M6PR. (A) VHH 1H11 is colored black with its paratope residues (shown as sticks) facing domain 1 (D1) of CI-M6PR (grey). A detailed view of the CI-M6PR epitope of VHH 1H11 is shown in B and C. (B) Detailed interface of CI-M6PR D1 displayed as a surface diagram, and the paratope residues of CDR1, -2 and -3 of VHH 1H11 are shown as sticks. (C) Detailed interface of VHH 1H11 displayed as a surface diagram, and the epitope residues of CI-M6PR D1 shown as sticks. (D) Shown are the paratope residues of VHH 1H11 (black) within less than 4A of the epitope region on D1 (grey). [Figure 15] Figure 15 is a schematic of anti-CI-M6PR VHH binding to domains 1-3 of hCI-M6PR. (A) The trefoil-shaped structure of CI-M6PRD1-D3 (similar to PDB:1q25) is shown in schematic form (white), with VHH7 and VHH8 bound to either D1 and D2-D3, respectively (grey). (B) Same as A, but CI-M6PRD1-D3 is similar to PDB:6p8i, with VHH 1H11 binding to D1 (grey). [Figure 16] Figure 16. Crystal structure information of the N-terminal 3 domain of the cation-independent mannose-6-phosphate receptor in complex with anti-CI-M6PR VHH7. Observed crystal contacts in VHH7:hCI-M6PRD1-D3 structure; crystal packing enabled by the Asn112-linked glycan of one protein and the M6P binding pocket in hCI-M6PRD3 of another protein. Figure created in PyMol 2.3.3. [Figure 17]Figure 17-18: Tandem competitive BLI of purified anti-CI-M6PR VHH. Tandem competitive BLI was performed on an Octet Red96 (ForteBio) instrument in kinetics buffer (1x PBS, 1mg / ml bovine serum albumin, 0.02% Tween-20 and 0.05% sodium azide). Human CI-M6PR domain 1-3His6 (0.5mg / mL in 50mM MES, 150mM NaCl, pH6.5) was incubated with EZ-Link™ NHS-PEG4-biotin (1mg, Thermo Fischer A39259) and NaHCO3 - (100mM) for 30 minutes at room temperature. Biotinylated human domain 1-3His6 was purified using Zeba Spin Desalting Columns™ (7K MWCO, 2 mL, Thermo Fischer 89890) and immobilized on a streptavidin SA biosensor (Sartorius) to a signal of 0.5 nm. In the competition assay (left), a 60 s association phase on 400 nM purified VHH7 (top) or VHH8 (bottom) was followed by a second association phase on 400 mM one of a series of anti-CI-M6PR VHHs recombinantly produced and purified in E. coli (Figure 17) or periplasmic extracts of E. coli expressing one of a series of anti-CI-M6PR VHHs (Figure 18). In the second reversed assay (right), a 60 s association phase with either 400 nM of anti-CI-M6PR-VHH recombinantly produced and purified in E. coli (Figure 17) or with periplasmic extracts of E. coli expressing one of a series of anti-CI-M6PR VHHs (Figure 18) was followed by a second 60 s association phase with 400 nM of VHH7 or VHH8. Between assays, the biosensor was regenerated by three 10 s exposures to regeneration buffer (10 mM glycine pH 3). Data were double-base subtracted and aligned in Octet Data Analysis software v9.0 (ForteBio). Greyscale curves represent the double-base subtracted data. The competition table shows which combinations of saturating and competing VHHs (all 400 nM in Figure 17) resulted in competitive or non-blocking interactions. [Figure 18]Figure 17-18: Tandem competitive BLI of purified anti-CI-M6PR VHH. Tandem competitive BLI was performed on an Octet Red96 (ForteBio) instrument in kinetics buffer (1x PBS, 1mg / ml bovine serum albumin, 0.02% Tween-20 and 0.05% sodium azide). Human CI-M6PR domain 1-3His6 (0.5mg / mL in 50mM MES, 150mM NaCl, pH6.5) was incubated with EZ-Link™ NHS-PEG4-biotin (1mg, Thermo Fischer A39259) and NaHCO3 - (100mM) for 30 minutes at room temperature. Biotinylated human domain 1-3His6 was purified using Zeba Spin Desalting Columns™ (7K MWCO, 2 mL, Thermo Fischer 89890) and immobilized on a streptavidin SA biosensor (Sartorius) to a signal of 0.5 nm. In the competition assay (left), a 60 s association phase on 400 nM purified VHH7 (top) or VHH8 (bottom) was followed by a second association phase on 400 mM one of a series of anti-CI-M6PR VHHs recombinantly produced and purified in E. coli (Figure 17) or periplasmic extracts of E. coli expressing one of a series of anti-CI-M6PR VHHs (Figure 18). In the second reversed assay (right), a 60 s association phase with either 400 nM of anti-CI-M6PR-VHH recombinantly produced and purified in E. coli (Figure 17) or with periplasmic extracts of E. coli expressing one of a series of anti-CI-M6PR VHHs (Figure 18) was followed by a second 60 s association phase with 400 nM of VHH7 or VHH8. Between assays, the biosensor was regenerated by three 10 s exposures to regeneration buffer (10 mM glycine pH 3). Data were double-base subtracted and aligned in Octet Data Analysis software v9.0 (ForteBio). Greyscale curves represent the double-base subtracted data. The competition table shows which combinations of saturating and competing VHHs (all 400 nM in Figure 17) resulted in competitive or non-blocking interactions. [Figure 19]Figures 19-20 are association-dissociation graphs of anti-CI-M6PR VHH1H11 and VHH1H52, respectively, analyzed using BLI. BLI was performed on an Octet Red96 (ForteBio) instrument in kinetics buffer (0.2M Na2HPO4, 0.1M Na+ citrate, 0.01% bovine serum albumin, 0.002% Tween-20). Biotinylated human domain 1-3His6 was immobilized on a streptavidin SA biosensor (Sartorius) to a signal of 0.6 nm. An association phase of 120 s in VHH 1H11 (Figure 19) or VHH 1H52 (Figure 20) serially diluted (0-200 nM) in phosphate citrate buffer at pH 7.4 was followed by a dissociation phase of 420 s in phosphate buffer at either pH 7.4, 6.5, 6.0, 5.5 or 5.0. Between assays, the biosensor was regenerated by three exposures of 10 s to regeneration buffer (10 mM glycine pH 3). The extent of association and dissociation was measured in δ nm over time (s). The black curve represents the double-baseline subtracted data fitted according to a 1:1 binding model (grey dashed line). [Figure 20]Figures 19-20 are association-dissociation graphs of anti-CI-M6PR VHH1H11 and VHH1H52, respectively, analyzed using BLI. BLI was performed on an Octet Red96 (ForteBio) instrument in kinetics buffer (0.2M Na2HPO4, 0.1M Na+ citrate, 0.01% bovine serum albumin, 0.002% Tween-20). Biotinylated human domain 1-3His6 was immobilized on a streptavidin SA biosensor (Sartorius) to a signal of 0.6 nm. An association phase of 120 s in VHH 1H11 (Figure 19) or VHH 1H52 (Figure 20) serially diluted (0-200 nM) in phosphate citrate buffer at pH 7.4 was followed by a dissociation phase of 420 s in phosphate buffer at either pH 7.4, 6.5, 6.0, 5.5 or 5.0. Between assays, the biosensor was regenerated by three exposures of 10 s to regeneration buffer (10 mM glycine pH 3). The extent of association and dissociation was measured in δ nm over time (s). The black curve represents the double-baseline subtracted data fitted according to a 1:1 binding model (grey dashed line). [Figure 21] Figure 21 shows the amino acid sequences of VHH7 and VHH8 with annotated CDRs. Kabat numbering is used for numbering of amino acid residues. Complementarity determining regions 1, 2 and 3 (CDR1,2,3) are shown in grey box according to AbM, MacCallum, Chothia, IMGT or Kabat annotation. [Figure 22]Figure 22 is a Coomassie brilliant blue stained SDS-PAGE of Pichia pastoris produced VHH based anti-EGFR nanoLYTAC constructs and controls performed in Laemmli sample buffer both with (A) and without (B) dithiothreitol. "MM" = molecular weight marker. Construct 30 = VHH7-FLAG3His6. Construct 31 = VHH8-FLAG3His6. Construct 33 = 9G8 S54A-FLAG3His6. Construct 34 = 9G8 S54A-VHH7-FLAG3His6. Construct 35 = 9G8 S54A-VHH8-FLAG3His6. Construct 36 = 2x9G8 S54A-VHH7-FLAG3His6. Construct 37 = 2x9G8 S54A-VHH8-FLAG3His6. Construct 38 = 9G8 S54A-GBP-FLAG3His6. Construct 39 = 2x9G8 S54A-GBP-FLAG3His6. [Figure 23]Figure 23. In vitro EGFR internalization efficacy of VHH-based nanoLYTAC constructs as determined by flow cytometry. HeLa cells were treated with 50 nM of nanoLYTAC constructs (34-37) or control for 24 h. Live cells were stained for cell surface EGFR (PE-AF647) and measured on a BD LSR II flow cytometer. (A) Representative flow cytometry histograms of cell surface EGFR levels measured for untreated HeLa cells or HeLa cells treated with 50 nM of nanoLYTAC constructs 34 (9G8 S54A-VHH7) or 35 (9G8 S54A-VHH8) or the corresponding control construct 38 (9G8 S54A-GBP) or 50 ng / ml of recombinant human EGF (rhEGF). (B) Representative flow cytometry histograms of cell surface EGFR levels measured for untreated HeLa cells or HeLa cells treated with 50 nM of nanoLYTAC constructs 36 (2x9G8 S54A-VHH7) or 37 (2x9G8 S54A-VHH8) or the corresponding control construct 39 (2x9G8 S54A-GBP) or 50 ng / ml rhEGF. (C) Bar graph showing the median fluorescence intensity measured for each condition, normalized to the median fluorescence intensity of untreated HeLa cells and expressed as a percentage. Erbitux, an FDA / EMA approved monoclonal anti-EGFR antibody. Data are the mean ± SEM of two replicates. The indicated asterisks represent p values ​​obtained from unpaired t-tests comparing LYTAC-treated conditions to untreated (black) and control construct (38 or 39)-treated conditions (grey). *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001. ****P ≤ 0.0001. [Figure 24]Figure 24. Western blot assay to evaluate the in vitro EGFR degradation efficacy of VHH-based nanoLYTAC constructs. HeLa cells were treated with 50 nM nanoLYTAC constructs (34-37), control constructs (38-39) or 50 ng / ml recombinant human EGF (rhEGF) for 24 h. Cell lysates were obtained and immunoblotted for EGFR and β-tubulin. EGFR intensity values ​​were determined by densitometry, normalized to loading controls, and expressed relative to untreated or construct 38-treated conditions. (A) Western blot analysis of the first biological replicate. (B) Western blot analysis of the second biological replicate. (C) Western blot analysis of the third biological replicate. "kDa" = kilodaltons. "r" = biological replicate. [Diagram 25] Figure 25. In vitro inhibition of ligand-induced EGFR activation in response to treatment with VHH-based nanoLYTAC constructs. HeLa cells were treated with 50 nM of nanoLYTAC constructs (34-37), control constructs (38-39) or Erbitux (50 nM or 40 μg / ml) for 24 h, after which cells were stimulated with 50 ng / ml of recombinant human EGF (rhEGF) for 5 min. Cell lysates were obtained and immunoblotted for phospho-EGFR (Tyr1068). Ponceau S staining of membranes is shown to demonstrate total protein levels. "UT" = untreated. "Ebx" = Erbitux (FDA / EMA approved monoclonal anti-EGFR antibody). "kDa" = kilodaltons. [Figure 26] Figure 26 is a Coomassie brilliant blue stained SDS-PAGE of cetuximab-based anti-EGFR nanoLYTAC constructs produced in Chinese Hamster Ovary (CHO) cells and cetuximab performed in Laemmli sample buffer both with and without dithiothreitol. "MM" = molecular weight marker. "Ctx-VHH7" = cetuximab-VHH7 fusion construct. "Ctx-VHH8" = cetuximab-VHH8 fusion construct. "Ctx" = cetuximab. [Figure 27] Figure 27: In vitro EGFR internalization efficacy of cetuximab-VHH fusions as LYTAC constructs as determined by flow cytometry. HeLa cells were treated with 5 or 50 nM cetuximab-based nanoLYTAC constructs (Ctx-VHH7 or Ctx-VHH8) or control for 24 hours. Live cells were stained for cell surface EGFR (PE-AF647) and measured on a BD LSR II flow cytometer. (A) Representative flow cytometry histograms of cell surface EGFR levels measured for untreated HeLa cells or HeLa cells treated with 5 nM cetuximab-based nanoLYTAC constructs or cetuximab or 50 ng / ml or recombinant human EGF (rhEGF). (B) Bar graph showing the median fluorescence intensity measured for each condition, normalized to the median fluorescence intensity of untreated HeLa cells and expressed as a percentage. Data are means ± SEM of two replicates. Erbitux = FDA / EMA approved monoclonal anti-EGFR antibody. The indicated asterisks represent p-values ​​obtained from unpaired t-tests comparing LYTAC-treated conditions with untreated (black) and cetuximab-treated conditions (grey). *P ≤ 0.05. *$P ≤ 0.01. ***P ≤ 0.001. ****P ≤ 0.0001. [Figure 28]Figure 28 is a Western blot assay to evaluate the in vitro EGFR degradation efficacy of cetuximab-VHH fusions as nanoLYTAC constructs. In two independent experiments, HeLa cells were treated with 5 nM of LYTAC constructs (Ctx-VHH7 or Ctx-VHH8), cetuximab or 50 ng / ml of recombinant human EGF (rhEGF) for 24 hours. Cell lysates were obtained and immunoblotted for EGFR and β-tubulin. Intensity values ​​for EGFR were determined by densitometry, normalized to loading controls, and expressed relative to untreated or cetuximab-treated conditions. "kDa" = kilodaltons. "Ctx-VHH7" = cetuximab-VHH7 fusion construct. "Ctx-VHH8" = cetuximab-VHH8 fusion construct. "Ctx" = cetuximab. "r" = biological replicate. [Figure 29] Figure 29 is a Coomassie brilliant blue stained SDS-PAGE of anti-GFP nanoLYTAC constructs and controls based on VHH produced in Pichia pastoris. "MM" = molecular weight marker. Construct 42 = GBP-FLAG3His6. Construct 43 = GBP-VHH7-FLAG3His6. Construct 44 = GBP-VHH8-FLAG3His6. Construct 45 = GBP-VHH1-FLAG3His6. Construct 46 = GBP-VHH5-FLAG3His6. Construct 47 = GBP-VHH 1H11-FLAG3His6. Construct 48 = GBP-VHH 1H52-FLAG3His6. [Diagram 30]Figure 30 is a Western blot assay to assess in vitro GFP internalization and degradation in HeLa cells treated with anti-GFP nanoLYTAC constructs. HeLa cells were treated with 50 nM recombinant GFP (rGFP) and 50 nM nanoLYTAC constructs (43=GBP-VHH7 and 44=GBP-VHH8) or control constructs with or without chloroquine (42=GBP) for 24 hours. Cell lysates were obtained and immunoblotted for GFP and β-tubulin. As a positive control, 2.5 ng of rGFP was analyzed. "UT" = untreated. "kDa" = kilodaltons. "CQ" = chloroquine. [Diagram 31] Figure 31 is a Western blot assay to assess in vitro GFP internalization and degradation in MCF7 cells treated with anti-GFP nanoLYTAC constructs. MCF7 cells were treated with 200 nM recombinant GFP and 200 nM nanoLYTAC constructs (43 = GBP-VHH7 and 44 = GBP-VHH8) or control constructs with or without chloroquine (42 = GBP) for 24 hours. Cell lysates were obtained and immunoblotted for GFP and β-tubulin. "UT" = untreated. "kDa" = kilodaltons. "CQ" = chloroquine. [Diagram 32] Figure 32 is a Western blot assay to assess in vitro GFP internalization and degradation in HeLa cells treated with anti-GFP nanoLYTAC constructs. HeLa cells were treated with 200 nM recombinant GFP (rGFP) and 200 nM nanoLYTAC constructs (43=GBP-VHH7, 44=GBP-VHH8, 45=GBP-VHH1, 46=GBP-VHH5, 47=GBP-VHH 1H11, 48=GBP-VHH 1H52) or control constructs (42=GBP) for 24 hours. Cell lysates were obtained and immunoblotted for GFP and β-tubulin. "UT"=untreated. "kDa"=kilodaltons. "CQ"=chloroquine. [Diagram 33]Figure 33 is a Western blot assay to assess in vitro GFP internalization and degradation in HeLa cells after washout of anti-GFP nanoLYTAC treatment. HeLa cells were treated for 24 hours with 50 nM recombinant GFP (rGFP) and 50 nM of nanoLYTAC constructs (43=GBP-VHH7 and 44=GBP-VHH8) or control constructs with or without chloroquine (42=GBP). Cell lysates were obtained after treatment (+0 hours) and after an additional 3 hours (+3 hours) and 7 hours (+7 hours) of incubation in fresh growth medium. Lysates were immunoblotted for GFP and β-tubulin. As a positive control, 2.5 ng of rGFP was analyzed. "UT" = untreated. "kDa" = kilodaltons. "CQ" = chloroquine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description The present invention will be described with respect to certain embodiments and with reference to certain drawings, but the present invention is not limited thereto, but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. Of course, it should be understood that not necessarily all aspects or advantages can be achieved in accordance with any particular embodiment of the present invention. Thus, for example, one skilled in the art will recognize that the present invention can be embodied or implemented to achieve or optimize one advantage or advantages taught herein without necessarily achieving other aspects or advantages that may be taught or suggested herein. The present invention, together with its features and advantages, both as to its construction and method of operation, can be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will become apparent and elucidated with reference to the embodiment(s) described herein below. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, structure, or characteristic described with respect to the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, although they may.

[0035] definition When an indefinite or definite article (e.g., "a" or "a", "the") is used when referring to a singular noun, this includes a plural of that noun unless something else is specifically stated. When the term "comprising" is used in the specification and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third, and other similar terms in the specification and claims are used to distinguish between similar elements and are not necessarily intended to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the aspects of the invention described herein are capable of operating in sequences other than those described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning to those skilled in the art of the invention. Practitioners are particularly advised to consult Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in molecular biology, biochemistry, structural biology, and / or computational biology).

[0036] As used herein, "nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term encompasses double- and single-stranded DNA, (reverse) complementary DNA, and RNA. It also encompasses known types of modifications, such as methylation, substitution of one or more "caps" of naturally occurring nucleotides with analogs. By "nucleic acid construct" is meant a nucleic acid sequence constructed to contain one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes containing non-naturally occurring nucleic acid sequences, and the like. A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequence or genomic DNA, and introns can also be present under certain circumstances. As used herein, the term "vector construct", "expression vector" or "recombinant vector" is intended to refer to a nucleic acid molecule that can transport another nucleic acid molecule to which it is linked. More specifically, the vector can include any vector known to those skilled in the art, including any suitable type, including but not limited to, for example, plasmid vector, cosmid vector, phage vector (such as lambda phage), virus vector, and even more specifically, lentivirus, adenovirus, AAV or baculovirus vector, or artificial chromosome vector, such as bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or P1 artificial chromosome (PAC).Expression vectors include plasmids and viral vectors, and generally contain the desired coding sequence and appropriate DNA sequences required for the expression of operably linked coding sequences in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment, and may lack functional sequences required for the expression of the desired DNA fragment. The construction of expression vectors for use in cell transfection is also well known in the art, and can therefore be accomplished through standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

[0037] The terms "protein", "polypeptide" and "peptide" are further used interchangeably herein to refer to a polymer of amino acid residues and its variants and synthetic analogs. A "peptide" may also be referred to as a partial amino acid sequence derived from its original protein, for example after trypsin digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The terms also encompass post-translational modifications of polypeptides, such as glycosylation, phosphorylation and acetylation. Based on the amino acid sequence and modifications, the mass or weight of the atoms or molecules of a polypeptide is expressed in (kilo)daltons (kDa). "Isolated" or "purified" refers to a material that is substantially or essentially free of components that normally accompany it in its natural state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that has been purified from adjacent molecules in its naturally occurring state (e.g., a fusion protein or a protein-binding agent, such as an antibody or nanobody, identified and disclosed herein, removed from molecules present in a sample or mixture, such as a production host, adjacent to the polypeptide). An isolated protein or peptide can be produced by amino acid chemical synthesis, or can be produced by recombinant production or by purification from a complex sample.

[0038] A "homolog", "homologs" or "functional homolog" of a protein encompasses peptides, oligopeptides, polypeptides, proteins and enzymes that have amino acid substitutions, deletions and / or insertions compared to the unmodified protein in question and have biological and functional activity similar to the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical amino acid-by-amino acid over a comparison window. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. As used herein, a "substitution" or "mutation" or "variant" results from the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid or nucleotide sequence of a parent protein or a fragment thereof. It is understood that a protein or fragment thereof can have conservative amino acid substitutions that do not substantially affect the activity or functionality of the protein.

[0039] As used herein, amino acids are defined as defined by the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138:9-37 (1984)), are presented by their three or one letter code nomenclature as defined and provided herein; as follows: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0040] "Binding" refers to any interaction, whether direct or indirect. Direct interaction refers to contact between binding partners. Indirect interaction refers to any interaction where the interacting partners interact in a complex of more than two molecules. The interaction can be completely indirect with the aid of one or more bridging molecules, or partially indirect where there is still direct contact between the partners stabilized by the additional interaction of one or more molecules. By the term "specifically binds" as used herein, a binding domain that recognizes a specific target but does not substantially recognize or bind other molecules in the sample is meant. Specific binding does not mean exclusive binding. However, specific binding means that the protein has a particular increased affinity or preference for one or some of their binders. The term "affinity" as used herein generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide, shifting the equilibrium of a single protein monomer towards the presence of a complex formed by their binding. Affinity is the strength of binding of a single molecule to its ligand. This is typically measured using the equilibrium dissociation constant (K) used to assess and rank the order strength of bimolecular interactions. D ) is the rate constant of the reaction. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate of [antibody][antigen] complex formation is equal to the rate of dissociation into its components [antibody] + [antigen]. Measurement of the reaction rate constant is the equilibrium or affinity constant (1 / K D ) can be used to define K D The smaller the value, the greater the affinity of the antibody for its target. The rate constants for both directions of the reaction are the association rate constant (K on ), which is called the "on speed" (K on ), which is a constant used to characterize how fast an antibody binds to its target. Conversely, the dissociation rate constant (K off) is the "off rate" (K off ) is the part of the reaction used to calculate the K . In the measurements presented herein, the flatter the slope, the slower the off-rate or the stronger the antibody binding. Conversely, a steeper downside indicates a faster off-rate and weaker antibody binding. The experimentally measured ratio of the off-rate to the on-rate (K off / K on ) is K D The on-rate and off-rate are measured and used to calculate the K D Several determination methods are known to those skilled in the art for calculating K D takes into account the standard error, which is considered to be a value independent of the assay used. As used herein, the term "protein complex" or "complex" or "assembled protein(s)" refers to a group of two or more associated macromolecules, at least one of which is a protein. As used herein, a protein complex typically refers to an association of macromolecules that can be formed under physiological conditions. The individual members of a protein complex are linked by non-covalent interactions.

[0041] "Binding agent" refers to a molecule capable of binding to another molecule, said binding being preferably a specific binding recognizing a defined binding site, pocket or epitope. A binding agent can also be provided as a (covalent) complex of several molecules, such as an antibody or the like. A binding agent can be of any nature or type, independent of its origin. A binding agent can be chemically synthesized, naturally occurring, recombinantly produced (and purified), as well as designed and synthetically produced. Thus, said binding agent can be a small molecule, a chemical, a peptide, a polypeptide, an antibody, or any derivative thereof, such as a peptidomimetic, an antibody mimetic, an active fragment, a chemical derivative, among others. The protein binding agent disclosed herein is itself a polypeptide that is also composed of a fusion protein comprising a first binding agent, specifically a CI-M6PR specific ISVD as described herein, and a second binding agent that specifically binds to an extracellularly accessible target protein. In certain embodiments, said second binding agent of the fusion protein may require an additional component, such as an antibody light chain, to form a binding site of an extracellularly accessible target protein as a whole together with the fusion protein forming the protein binding agent of the present invention. The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex that, as a result of its shape and charge, favorably associates with another chemical entity, compound, protein, peptide, antibody or Nb. The term "pocket" includes, but is not limited to, a cleft, channel or site. The term "part of the binding pocket / site" refers to less than all of the amino acid residues that define the binding pocket or binding site. For example, the part of the residues may be key residues that play a role in ligand binding or may be residues that are spatially related and define the three-dimensional compartment of the binding pocket. The residues may be contiguous or discontinuous in the primary sequence. In the case of antibody-related molecules, the term "epitope" is also used to describe the binding site, which are used interchangeably herein. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography, Cryo-EM and multidimensional nuclear magnetic resonance.

[0042] The terms "antibody", "antibody fragment" and "active antibody fragment" as used herein refer to a protein that contains an immunoglobulin (Ig) domain or antigen-binding domain capable of specifically binding to an antigen, in this case the N-terminal domains 1-3 of the (human) CI-M6PR protein. An "antibody" can further be an intact immunoglobulin from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The term "active antibody fragment" refers to any antibody or part of an antibody-like structure that has high affinity for an antigenic determinant or epitope itself and contains one or more complementarity determining regions (CDRs) that account for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, nanobodies, domain antibodies, and single chain structures such as complete light chains or complete heavy chains. An additional requirement for the "activity" of the fragment in the context of the present invention is that the fragment is capable of binding to CI-M6PR or is an antibody fragment, taking into account that the binding agent specifically recognizes an extracellularly accessible target, and activity includes the ability to specifically bind to an extracellularly accessible target itself, or the ability to bind in the presence / after co-expression of an additional protein domain, such as a light chain or a light chain variable domain.Preferably, the CI-M6PR binding activity includes specifically binding and having a favorable dissociation profile at lower pH (i.e., acidic conditions such as endosomes and lysosomes below pH 7), and more preferably, being capable of dissociating at a pH around 5.8 in a subject and / or being capable of retaining binding at said pH (depending on the application / treatment).The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD"), refers to an immunoglobulin domain that essentially consists of four "framework regions" (referred to in the art and hereinafter as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively), which are interrupted by three "complementarity determining regions" or "CDRs" (referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1"; "complementarity determining region 2" or "CDR2"; and "complementarity determining region 3" or "CDR3", respectively). Thus, the general structure or sequence of an immunoglobulin variable domain can be depicted as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domain (IVD)(s) that confers specificity to the antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site. In view of the above definitions, the antigen-binding domain of a conventional four-chain antibody (e.g. an IgG, IgM, IgA, IgD or IgE molecule; known in the art), or an Fv fragment such as a Fab fragment, F(ab')2 fragment, a disulfide-linked Fv or scFv fragment, or a diabody (all known in the art) derived from such a conventional four-chain antibody, binds to a respective epitope of an antigen by means of a pair of (related) immunoglobulin domains such as a light chain variable domain and a heavy chain variable domain, i.e. a VH-VL pair of immunoglobulin domains which together bind to an epitope of the respective antigen.Immunoglobulin single variable domain (ISVD) as used herein refers to a protein having an amino acid sequence comprising four framework regions (FR) and three complementarity determining regions (CDR) according to the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The "immunoglobulin domain" of the present invention also refers to "immunoglobulin single variable" (abbreviated as "ISVD"), which is equivalent to the term "single variable domain", and defines a molecule in which an antigen binding site is present on and formed by a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Thus, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. Thus, a single variable domain may be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, provided that it is capable of forming a single antigen-binding unit (i.e. a functional antigen-binding unit that consists essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0043] In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx NV (Sanofi). For a general description of nanobodies, reference is made to the prior art cited herein, such as the further description below (as well as e.g., those described in WO 2008 / 020079). "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments and VHH antibodies, were originally described as antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e., "antibodies lacking light chains"; Hamers-Casterman et al (1993) Nature 363:446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (herein referred to as "VH domains") and the light chain variable domains present in conventional four-chain antibodies (herein referred to as "VL domains").For a further description of VHHs and nanobodies, reference is made to the review article by Muyldermans (review in Molecular Biotechnology 74:277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 to Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231 and WO 02 / 48193 to Unilever; International Publication Nos. WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 to the VIB; International Publication No. WO 03 / 050531 to Algonomics NV and Ablynx NV; International Publication No. WO 01 / 90190 to the National Research Council of Canada; International Publication No. WO 03 / 025020 (=European Patent No. 1433793) to the Antibody Association; and Ablynx Nos. WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 by Ablynx NV, and further published patent applications by Ablynx NV. As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) can be characterized in particular by the presence of one or more "hallmark residues" in one or more framework sequences.Further description of nanobodies, including humanization and / or camelization of nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions", multivalent or multispecific constructs (including some non-limiting examples of linker sequences), and different modifications to extend the half-life of nanobodies and their preparations can be found, for example, in WO 08 / 101985 and WO 08 / 142164. Nanobodies form the smallest antigen-binding fragments that fully retain the binding affinity and specificity of full-length antibodies. Nbs possess very long complementarity determining region 3 (CDR3) loops and convex paratopes, which allow them to penetrate into hidden cavities of target antigens.

[0044] As used herein, the terms "determining," "measuring," "evaluating," "identifying," "screening," and "assaying" are used interchangeably and encompass both quantitative and qualitative determinations.

[0045] A "pharmacologically or therapeutically effective amount" of a protein binding agent or binding agent composition is preferably an amount that results in or has an effect on the particular condition being treated. A "therapeutically active agent" is used to refer to any molecule that has or can have a therapeutic effect (i.e., a curative or stabilizing effect) in relation to the treatment of a disease (as described further herein). Preferably, the therapeutically active agent is a disease-modifying agent and / or an agent that has a curative effect on a disease. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual together with a 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 harmless to a patient at a concentration consistent with the effective activity of the active ingredient, and any side effects attributable to the carrier do not negate the beneficial effects of the active ingredient. Suitable carriers or adjuvants typically include 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.Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, MF et al. ("Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311), Strickley, RG ("Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1" PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349), and Nema, S. et al. ("Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51(4), 166-171). The term "excipient" as used herein is intended to include all substances that may be present in a pharmaceutical composition and that are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffer substances, stabilizers, flavorings or colorings. "Diluents", particularly "pharmaceutical acceptable vehicles", include vehicles such as water, saline, physiological saline solution, glycerol, ethanol, and the like. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.

[0046] The terms "subject", "individual" or "patient", as used interchangeably herein, refer to any organism, such as a vertebrate, particularly any mammal, including both humans and other mammals, for which diagnosis, treatment or prevention is desired (e.g., animals such as rodents, rabbits, cows, sheep, horses, dogs, cats, llamas, pigs, or non-human primates (e.g., monkeys)). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, rat, or non-human primate. Preferably, the subject is a human. In one embodiment, the subject is a subject who has or is suspected of having a disease or disorder, particularly a disease or disorder disclosed herein, also referred to herein as a "patient". However, it will be understood that the above terms do not imply that symptoms are present. The terms "treatment" or "treating" or "treat" may be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, prevents, controls, halts, reduces or reverses the progression or severity of a sign, symptom, disorder, condition or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, symptoms or disorders.

[0047] The term "medicament" as used herein refers to a substance / composition used in therapy, i.e. in the prevention or treatment of a disease or disorder. According to the present invention, the term "disease" or "disorder" refers to any pathological condition, in particular a disease or disorder as defined herein.

[0048] Detailed Description The present invention is based on the identification of CI-M6PR specific VHHs fused to additional antigen binding proteins to enable target binding at the cell surface or extracellular space and trigger internalization of the protein binder-target complex via the CI-M6P receptor endocytosis / lysosomal pathway. VHHs were selected as binders that specifically engage with CI-M6PR because they are known to be highly stable and soluble and can be easily and cost-effectively produced in low-lysosomal organisms such as bacteria and yeast. In addition, VHHs are unique in their great conformational stability, as well as high intrinsic pH and protease resistance, all of which form attractive properties for circulating the endosomal-lysosomal system. Furthermore, VHH-based formats are suitable for various routes of administration, including via intravenous injection and inhalation, thus providing a novel approach to apply lysosomal targeting of drug products, optionally in complex with their targets. More specifically, the target binding agent fused to the CI-M6PR specific ISVD or VHH described herein can be a target protein, preferably an antigen binding domain specific for a target present on the cell surface or extracellularly, and also provides antibody-based target binding, preferably ISVD-based target binding by itself. Such bispecific conjugates or ISVD fusion polypeptides, also named nanoLYTACs herein, provide CI-M6PR mediated lysosomal uptake as cargo for delivery of specific extracellular or cell surface target(s) that are ultimately degraded in lysosomes.

[0049] Since CI-M6PR constantly moves between late endosomes and cell membranes, the protein binders disclosed herein may dissociate at lower pH in these subcellular organelles or may retain binding to CI-M6PR and recycle with it. The latter may contribute to the increased half-life of such binders in subjects. Furthermore, the ability to adjust the pH dissociation of antigen-binding domains is known in the art, and may allow, for example, to generate multispecific conjugates in which CI-M6PR-specific ISVDs can maintain their binding throughout the recycling process, while additional antigen domain conjugates may dissociate from their targets at pH values ​​corresponding to the pH in endosomes and lysosomes to release their targets for degradation. This increases their target degradation effectiveness and thus potency. However, high protease resistance is also required for the recycling of such ISVD-based anti-CI-M6PR conjugates.

[0050] The present invention discloses at least two types of CI-M6PR specific ISVDs, based on their binding to specific epitopes on the N-terminal domain of CI-M6PR.As exemplified herein, which ISVD is selected as part of the protein binder as described herein depends on the combination and selection of extracellularly accessible target and its binder, because epitope location can be related to the efficacy and pH-dependence profile of both CI-M6PR binding and extracellularly accessible target binding.By providing two types of CI-M6PR ISVDs, each of which is covered by several VHH examples, a toolbox is provided for those skilled in the art who aim to obtain targeted protein degradation via CI-M6PR mechanism to select from.

[0051] Thus, a first aspect of the present invention provides a protein binding agent, preferably comprising a fusion protein, comprising an ISVD-based binding agent that specifically binds to the N-terminal extracellular part of the CI-M6PR protein linked (either directly or via a spacer or linker) to a binding agent that specifically binds to a target protein that is accessible from the outside of the cell, more particularly a protein that is secreted by the cell or is a membrane protein or is present outside the cell, more particularly a protein binding specifically to a conformational epitope present on domains 1, 2 and / or 3 as defined herein.

[0052] The binding agent or fusion protein of the present invention is called "fusion" because different binding agents are connected by direct fusion, which is made through a peptide bond between the amino acid residues of the chain and the ISVD itself, or by indirect fusion, which is made by a linker. The fusion site is preferably designed to result in a flexible fusion protein, where the different paratopes do not interfere with each other in binding to their respective targets or antigens. A preferred "linker molecule", "linker" or "short polypeptide linker" is a peptide having a length of about 10 amino acids. Non-limiting examples of suitable linker sequences are known to those skilled in the art. The linker can be selected to maintain a fixed distance between the structural domains as well as to maintain the independent functions (e.g., antigen binding) of the fusion partners.

[0053] In certain embodiments, the "linker" between the CI-M6PR-specific ISVD and the target-specific binding agent of the protein binding agent of the present invention (where "target" as used herein is an "extracellularly accessible target protein") may be a longer polypeptide linker so that at least two different binding sites can be reached or bound by the protein binding agent simultaneously. For example, a CI-M6PR-specific ISVD as described herein may be fused at its N- or C-terminus to an Fc domain (e.g., the Fc tail of an Ig) and a target-specific binding agent may be fused via its N- or C-terminus to an identical or compatible Fc tail, resulting in a bispecific format protein binding agent in which two of the Fc fusions form a dimer in the same way as in antibody-type molecules via disulfide bridges in the hinge region of the Fc portion. Alternatively, the Fc tail can be fused at its N- or C-terminus to the CI-M6PR-specific ISVD and at the other terminus to a target-specific binder, resulting in a CI-M6PR-ISVD-Fc-target binder protein binder, which can be formed as a dimeric molecule to provide a bivalent bispecific agent. Additional linker formats also include Fc with knob-into-hole linkage possibilities, where again the CI-M6PR-ISVD and target-specific binder or N- or C-terminus are fused to the Fc to provide a dimeric bispecific binder.

[0054] In a further embodiment, the linker between the CI-M6PR specific ISVD and the target-specific binding agent of the protein binding agent of the present invention can be provided by an additional functional group or moiety that is advantageous when administered to a subject. Examples of such functional groups and the techniques for introducing them are clear to those skilled in the art and can generally include all functional groups and techniques mentioned in the art, as well as functional groups and techniques known per se for the modification of pharmaceutical proteins, in particular the modification of antibodies or antibody fragments, for which reference is made, for example, to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, PA (1980). Such functional groups can be, for example, directly (e.g., covalently) linked to the ISVD and / or the target-specific binding agent, or can be optionally linked via an additional suitable linker or spacer, as will again be clear to those skilled in the art. The functional groups can also be applied as additional moieties linked to the CI-M6PR specific ISVD or the target-specific binding agent. One of the most widely used techniques for increasing the half-life and / or reducing the immunogenicity of pharmaceutical proteins involves the attachment of a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof, such as methoxypoly(ethylene glycol) or mPEG. For example, for this purpose, PEG can be attached to a cysteine ​​residue naturally present in the immunoglobulin single variable domain of the invention, the immunoglobulin single variable domain of the invention can be modified to appropriately introduce one or more cysteine ​​residues for the attachment of PEG, or an amino acid sequence comprising one or more cysteine ​​residues for the attachment of PEG can be fused to the N-terminus and / or C-terminus of the ISVD or active antibody fragment of the invention, all using techniques of protein engineering known per se to the skilled artisan. Another, usually less preferred, modification involves N-linked or O-linked glycosylation, usually as part of a co-translational and / or post-translational modification, depending on the host cell used to express the protein-binding agent.Another technique for increasing the half-life of a binding domain may involve engineering into a bifunctional or bispecific domain (e.g., at least one target-specific binder, one ISVD or active antibody fragment against CI-M6PR, and one against a serum protein such as albumin that helps extend the half-life), or into a fusion of an antibody fragment, particularly an immunoglobulin single variable domain, with a peptide (e.g., a peptide against a serum protein such as albumin). Thus, the half-life extension can be applied as a linker between the CI-M6PR-specific ISVD and the target-specific binder, or can be coupled to any one of them.

[0055] Binding to CI-M6PR protein on the extracellular surface of cells requires a certain affinity to maintain its binding during the internalization of the receptor in endosomes.When the threshold binding affinity, which may be in the micromolar, nanomolar or picomolar range, is reached and the target-specific conjugate binds to its target, the internalization and uptake of the bispecific agent in complex with the target in cells results in the protein binder / target complex being present in cellular compartments from early endosomes to late endosomes, and finally moving to the lysosomes of cells.For the CI-M6PR conjugate of the present invention, a binding affinity in the nanomolar to picomolar range is envisaged when determined at neutral pH, more specifically pH 7.4, to allow efficient uptake and / or recycling by CI-M6PR protein in cells.

[0056] Binding to CI-M6PR in the CI-M6PR-specific ISVD of the protein binding agent of the invention, specifically N-terminal domains 1-3, is defined herein as binding to an epitope present in at least one or more of the three N-terminal domains comprising amino acid residues 1-161 present in SEQ ID NO:23 for N-terminal domain 1, amino acid residues 162-313 present in SEQ ID NO:23 for N-terminal domain 2, and amino acid residues 314-467 present in SEQ ID NO:23 for N-terminal domain 3 (see, e.g., FIG. 11). In one embodiment, the CI-M6PR-specific ISVD provides the necessary biophysical and binding properties at different pH values ​​to retain binding to the N-terminal portion of the CI-M6P receptor upon internalization to endosomes and / or lysosomal trafficking on or within a cell. In more specific embodiments, the internalization efficiency is defined as the minimum internalization rate of the CI-M6PR specific binding agent in voxels / minute in a live cell imaging experimental method (see Examples), and is considered herein to be "internalized" at an internalization rate of at least 15 voxels / minute, or at least 35, or at least 50, or at least 65, or at least 80, or at least 100, or at least 120 voxels / minute.

[0057] In certain embodiments, the binder provides retained binding to the CI-M6P receptor upon internalization, as shown by its pH-dependent binding profile (demonstrated for the ISVD by BLI), and dissociates from the receptor only at a pH below the pH of the endosomal compartment, and thus below pH 6. Thus, the ISVD-based binder provides strong binder at neutral pH and endosomes (pH 6-5.5), but allows clear dissociation from the receptor at lower pH, which likely allows the ISVD binder to be at least partially recycled back to the outer membrane. This may result in functional ISVD-based removal of surface or extracellular molecules from outside the cell to the endosomal compartment. Such pH-dependent dissociation profiles have been observed, for example, for the previously disclosed VHH8 (SEQ ID NO: 8), VHH5 (SEQ ID NO: 5) and VHH1H52 (SEQ ID NO: 25) ISVDs (Callewaert et al., PCT / EP2022 / 054278). Although these VHHs belong to different VHH families, they compete for the same binding site on CI-M6PR, and based on co-crystal analysis of VHH8 with CI-M6PR domains 1–3, the epitope was determined to be located in the N-terminal domains 2 and 3.

[0058] Thus, in certain embodiments, the ISVD specifically binds to CI-M6PR and specifically recognizes a binding site located on N-terminal domains 2 and 3, which can be more specifically described as an epitope (also called a VHH8-petiole) or an ISVD contacting the following amino acid residues of CI-M6PR Lys191, Gly194, Ala195, Tyr196, Leu197, Phe208, Arg219, Gln224, Leu225, Ile297, Lys357, Gly408, Asp409, Asn431, Glu433, and Phe457 as shown in SEQ ID NO:23.

[0059] As used herein, "epitope" or "binding site" refers to an antigenic determinant of a polypeptide that constitutes a binding site or binding pocket on a target molecule, such as the extracellular portion of the CI-M6P receptor protein, more specifically, a binding pocket on the N-terminal domain (1-3) accessible to the ISVD or VHH. An epitope can include 3 amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually at least 8, 9, 10 or more such amino acids. These residues are "in contact" with the binding agent. An epitope is defined herein as amino acids that are in contact with each other based on integrated analysis, PISA and FastContact analysis at a distance of 4 angstroms or less from the VHH residues, as described in Callewaert et al. (PCT / EP2022 / 054278).

[0060] In a further embodiment, said CI-M6PR specific binding agent may be defined as an agent that competes for binding to said VHH8 epitope as described herein.

[0061] The binder residues that constitute the essential residues for specifically binding to a target or binding to an epitope of a target are defined herein as paratopes, as known in the art. Thus, such paratopes of binders for CI-M6PR can be described as the residues of the ISVD disclosed herein that contact the epitope residues on the N-terminal domains 1-3 of CI-M6PR.

[0062] In a further particular embodiment, the CI-M6PR specific ISVD specifically binds by contacting a specific paratope of the ISVD, which is, for example, comprised of residues Tyr32, Arg52, Trp53, Ser54, Ser56, Lys57, Ile100, Phe103 and Ser108 as shown in SEQ ID NO:8 (in numerical order, Kabat numbering is not used here), providing the paratope of the ISVD for binding to said epitope as described above. Alternatively, the CI-M6PR specific ISVD specifically binds by contacting a specific paratope of VHH5 or VHH1H52, which upon sequence alignment corresponds to residues 32, 52-57, 100-103, 108 of VHH8.

[0063] In a further alternative embodiment, the protein binder provides a CI-M6PR specific ISVD for internalization, which dissociates gradually from the receptor at pHs present in endosomal compartments, as shown by its pH-dependent binding profile (Callewaert et al., PCT / EP2022 / 054278), such that dissociation occurs similarly to the receptor's natural ligand at pHs around 6-5.5. Thus, the ISVD-based binder provides the conjugate at neutral pH, but dissociates within the endosome (pH 6-5.5), allowing the receptor to cycle back and the ISVD binder to proceed to the lysosome (not recycled to the outer membrane). Such a pH-dependent dissociation profile has been observed, for example, for the VHH7 (SEQ ID NO: 7), VHH1 (SEQ ID NO: 1) and VHH1H11 (SEQ ID NO: 24) ISVDs. Although these VHHs each belong to a different VHH family, they compete for the same binding site on M6PR domains 1-3, and based on co-crystal analysis of VHH7 and VHH1H11 with CI-M6PR domains 1-3, the epitope was determined to be located in N-terminal domain 1. More specifically, the CI-M6PR specific ISVD binding site (also referred to herein as the VHH7 epitope or VHH7 / VHH1H11 epitope or VHH1H11 epitope) can be more specifically described as the ISVD contacting CI-M6PR amino acid residues at positions Lys59, Asn60, Met85, Asp87, Lys89, Ala146, Thr147, and Glu148, as well as Asp118 or Gln119, as shown in SEQ ID NO:23. Epitopes are defined herein as amino acids that are in contact with each other based on integrated analysis, PISA and FastContact analysis at a distance of 4 angstroms or less from the VHH residues as described in Callewaert et al. (PCT / EP2022 / 054278).

[0064] In further specific embodiments, the binding agent comprises an ISVD that specifically binds primarily to domain 1 of CI-M6PR by contacting residues Asp31, Arg33, Asp35, Trp53, Ser54, Ser56, Lys57, Lys96, Asp104 thereof as shown in SEQ ID NO:7 (in numerical order, Kabat numbering not used here), providing a paratope for the ISVD for binding to said epitope as described above. Alternatively, the CI-M6PR specific ISVD specifically binds by contacting with a specific paratope of VHH1 or VHH1H11 that corresponds to residues 31, 33, 35, 53, 54, 56, 57, 96, 104 of VHH7 upon sequence alignment, such as a paratope comprising residues 31-35, 50, 52-57, 96-98 as shown in SEQ ID NO:24.

[0065] In a further aspect, the protein binding agent described herein comprises a CI-M6PR specific ISVD comprising CDR1, CDR2, and CDR3 regions for ISVD binding residues selected from the CDR1, CDR2, and CDR3, respectively, of any of the sequences selected from VHH1, VHH5, VHH7, VHH8, VHH1H11, or VHH1H52 ISVD, wherein the CDR regions are defined according to any one of the annotations known in the art, specifically according to the annotations of Kabat, MacCallum, IMGT, AbM, or Chothia. The determination of the CDR regions can be carried out according to various methods, such as assignment based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745), or according to any of the annotations known as AbM (AbM is an antibody modeling package from Oxford Molecular Ltd. described at http: / / www.bioinf.org.uk / abs / index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196:901-17), Kabat (Kabat et al., 1991; 5th edition, NIH publication 91-3242), or IMGT (LeFranc, 2014; Frontiers in Immunology. 5(22):1-22). The annotation further includes the delineation of CDR and framework regions (FR) in immunoglobulin domain-containing proteins, and thus can be applied to any immunoglobulin protein sequence without undue burden, a method and system known to those skilled in the art.These annotations are slightly different, but each is intended to include the region of the loop that is involved in target binding.The CDR region annotation for each VHH sequence described herein by AbM is provided in Table 12.Alternatively, slightly different CDR annotations known in the art may be applied here to identify the CDR / FR regions of the ISVDs disclosed herein, for example as shown for VHH7 and VHH8 in FIG.

[0066] It should be noted that, as is well known in the art for VH and VHH domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in VH and VHH domains is usually in the range of 110-120, often between 112-115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0067] In another aspect, the protein binding agent provided herein comprises an ISVD that specifically binds to CI-M6PR extracellular N-terminal domains 1 to 3, wherein the ISVD contains a sequence selected from the group of sequences representing VHH1, 5, 7, 8, 1H11 or 1H52 exemplified herein (shown in SEQ ID NOs: 1, 5, 7, 8, 24 and 25, respectively), or a sequence having at least 85%, or at least 90%, or at least 95%, or at least 99% identity thereto, wherein the CDR regions are identical to the respective ISVD sequences, and residue variations are present only in the non-binding residues of the FR regions.

[0068] A further embodiment relates to said protein binding agent comprising a CI-M6PR specific ISVD comprising said CDRs of SEQ ID NO: 1, 5, 7, 8, 24 or 25 annotated according to AbM as defined in Table 12 herein, comprising: -Consensus sequence "xVQLxESGGGLVQ x GGSLxLSCxAx" (SEQ ID NO:78), wherein x at position 1 (x1) is Q, E, or D, x5 is Q or V, x14 is P or A, x19 is R or K, x23 is A, E, T, or V, and x25 is S or A; - an FR2 sequence corresponding to any of the sequences encompassed by the consensus sequence "WxRQxPGKxxExVx" (SEQ ID NO: 79), wherein x at position 2 (x2) is L, F or Y, x5 is A or I, x9 is G, E or Q, x10 is R or I, x12 is G, F or W, and x14 is S or A; - FR3 sequences corresponding to any of the sequences encompassed by the consensus sequence "YxDSxKxRFxxSRDxxKNTxxLxMNSLxxEDTAxxYCxx" (SEQ ID NO: 80), wherein x at position 2 (x2) is A, S, H, or D, x5 is V or A, x7 is G or D, x10 is S, T, or A, x11 is I or V, x15 is D or N, x16 is A, T, or S, x20 is L, I, or V, x21 is Y or N, x23 is R, Q, or Y, x28 is K, Q, or R, x29 is P or T, x34 is V or I, x35 is Y or V, x38 is K, A, or Y, and x39 is A, R, or C; - An FR4 sequence corresponding to any of the sequences encompassed by the consensus sequence "xGQGTxVTVSS" (SEQ ID NO: 81), where x at position 1 (x1) is W or R and x6 is Q or L.

[0069] The "x" residues shown in the consensus FR sequence provide amino acid positions with possible variations without compromising the functionality of the ISVD, the possible differences in identity being provided by the consensus sequence based on the sequences described for VHH1, 5, 7, 8, 1H11 and 1H52, as well as the humanized formats of VHH7 and VHH8 disclosed in SEQ ID NOs: 26-35. Furthermore, further substitutions of those amino acids at the respective positions are also possible without losing effect, since, for example, when considering humanization, amino acids of similar nature / type can be used instead. For example, substitutions can be tolerated between small aliphatic amino acids (I, V, L), or between aromatic amino acids (F, W, Y, H), or between positively charged amino acids (K, R), or between negatively charged amino acids (D or E), or between small polar amino acids (S, T), or between very small neutral amino acids (G, A).

[0070] More particularly, the FR1-4 regions of said CI-M6PR specific ISVD of a protein binding agent of the present invention may be provided by the FR sequences provided in Table 13.

[0071] In a further embodiment, the protein binding agent described herein comprises a CI-M6PR specific ISVD selected from the group of SEQ ID NO: 1, 5, 7, 8, 24 or 25, or a humanized variant of any one of them. The term "humanized variant" of an immunoglobulin single variable domain, such as a domain antibody and a Nanobody® (including a VHH domain), refers to an amino acid sequence of said ISVD that represents the effort of being subjected to humanization, i.e. to increase the degree of sequence identity with the closest human germline sequence. In particular, a humanized immunoglobulin single variable domain, such as a Nanobody® (including a VHH domain), may be an immunoglobulin single variable domain in which there is at least one amino acid residue (in particular at least one framework residue) that is and / or corresponds to a humanization substitution (as further defined herein). Potentially useful humanizing substitutions can be identified by comparing the sequences of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequences of one or more closely related human VH sequences, and then one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein), and the resulting humanized VHH sequence can be tested for affinity to the target, stability, ease and level of expression, and / or other desired properties. In this way, with a limited degree of trial and error, other or even suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled artisan. Also, based on the foregoing, (the framework regions of) immunoglobulin single variable domains, such as Nanobodies® (comprising VHH domains), can be partially humanized or fully humanized. Humanized immunoglobulin single variable domains, particularly Nanobodies, can have several advantages, such as reduced immunogenicity, compared to the corresponding naturally occurring VHH domains.In summary, the humanization substitutions should be selected so that the resulting humanized amino acid sequence of the ISVD and / or VHH still retains the desired properties, such as antigen-binding ability and allosteric modulation ability. The skilled person can select the humanization substitutions or the appropriate combination of humanization substitutions that optimize or achieve the desired or appropriate balance between the desired properties provided by the humanization substitutions on the one hand and the desired properties of the naturally occurring VHH domain on the other hand. Such methods are known to the skilled person. A human consensus sequence can be used as a target sequence for humanization, although other means are known in the art. One alternative includes the skilled person aligning several human germline alleles (such as, but not limited to, the alignment of IGHV3 alleles) and using the alignment to identify residues suitable for humanization in the target sequence. Also, a subset of the human germline alleles most homologous to the target sequence can be aligned as a starting point to identify suitable humanization residues. Alternatively, VHHs are analyzed to identify the closest homologs in human alleles and used for the design of humanized constructs. Humanization techniques applied to camelid VHHs can also be performed by methods involving the replacement of specific amino acids, alone or in combination. The replacements can be selected based on those known from the literature, from known humanization efforts, as well as from human consensus sequences compared to natural VHH sequences, or from the human alleles most similar to the VHH sequence of interest. As can be seen from the data on VHH entropy and VHH variability shown in Tables A-5 to A-8 of WO 08 / 020079, some amino acid residues (i.e. hallmark residues) in the framework regions are more conserved between humans and camelids than others. In general, the invention is not limited in its broadest sense, but any substitutions, deletions or insertions are preferably made at less conserved positions. Also, in general, amino acid substitutions are preferred over amino acid deletions or insertions.For example, the human-like class of camelid single domain antibodies contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensates for this loss of hydrophilicity by another substitution at position 103 that replaces the conserved tryptophan residue present in the VH from the two-chain antibody. Thus, peptides belonging to these two classes show high amino acid sequence homology to the human VH framework regions, from which they can be directly administered to humans without the burden of further humanization, without expecting an undesirable immune response. Indeed, some camelid VHH sequences show high sequence homology to the human VH framework regions, from which they can be directly administered to patients without the additional burden of humanization, without expecting an immune response therefrom. Suitable mutations, in particular substitutions, can be introduced during humanization to generate polypeptides with reduced binding to pre-existing antibodies, for example at at least one of positions 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108 (see, for example, WO 2012 / 175741 and WO 2015 / 173325). The amino acid sequences and / or VHHs of the invention may be suitably humanized at any framework residue(s), such as one or more Hallmark residues (as defined herein) or preferably one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. Depending on the host organism used to express the amino acid sequence, ISVD, VHH or polypeptide of the invention, such deletions and / or substitutions may also be designed to remove one or more sites for post-translational modification (such as one or more glycosylation sites at asparagine replaced with G, A or S; and / or methionine oxidation sites), as is within the ability of the skilled artisan. Alternatively, substitutions or insertions may be designed to introduce one or more sites for attachment of a functional group, for example to allow site-specific pegylation.In some cases, at least one of the typical camelid hallmark residues having hydrophilic properties at positions 37, 44, 45 and / or 47 is replaced (Kabat numbering; see Table A-03 of WO 2008 / 020079). Another example of humanization involves the replacement of residues in FR1 such as at positions 1, 5, 11, 14, 16, and / or 23, and / or 28; residues in FR2 such as at positions 40 and / or 43; residues in FR3 such as at positions 60-64, 73, 74, 75, 76, 78, 79, 81, 82b, 83, 84, 85, 93 and / or 94; and residues in FR4 such as at positions 103, 104, 105, 108 and / or 111 (see Tables A-05-A08 of WO 2008 / 020079; all numbering according to Kabat).

[0072] In certain aspects, the protein binding agents described herein comprise a CI-M6PR-specific ISVD comprising a humanized variant of VHH7 or VHH8, where the retained functionality corresponds to any one of SEQ ID NOs: 26-35 as set forth in Callewaert et al. (PCT / EP2022 / 054278).

[0073] Another embodiment relates to a protein binding agent that is a multispecific agent comprising an ISVD that specifically binds to CI-M6PR domains 1-3 as described herein and a binding agent that specifically binds to an extracellularly accessible target, further comprising a binding agent or moiety that is specifically linked or coupled to either the CI-M6PR specific ISVD or the target specific binding agent, directly or indirectly, to a different epitope and / or a different target. Thus, the additional binding agent or moiety may comprise a binding agent specific for CI-M6PR, but with a different chemical structure than the first binding agent, resulting in a multiparatopic or multispecific binding agent, or the additional binding agent may comprise a binding agent that binds to the same extracellularly accessible target as the binding agent of the fusion protein, but specific for binding to a different epitope on the target, or may bind to a different extracellularly accessible target. Furthermore, the additional binding agent may specifically bind to a different target that can extend the half-life of the fusion protein in a subject, such as, for example, serum albumin protein. Thus, the additional binding agent may comprise an antigen-binding domain and / or may be a functional moiety. If the additional binding agent comprises a binding agent with the same or identical structure or sequence compared to the other building blocks of the fusion protein, i.e., a CI-M6PR-specific and an extracellularly accessible target-specific binding agent, this may provide a multivalent binding agent for any of the respective binding agents, for example, increasing the avidity of binding. Furthermore, the additional binding agent may also include a binding agent with different target specificity or comprise another form of CI-M6PR binding agent that binds to different lysosomal targeting proteins. By coupling several binding agents, which in certain embodiments may all comprise an ISVD, and interacting with different targets, preferably targets present on the cell surface or in the extracellular environment, they are defined as multispecific binding agents. In certain embodiments, the fusion protein comprises more than one VHH disclosed herein for specifically interacting with CI-M6PR and a binding agent for an extracellularly accessible target.Another particular embodiment relates to a fusion protein comprising one binding agent specific for CI-M6PR and a multivalent or multispecific binding agent for an extracellularly accessible target protein of interest. In particular embodiments in which several ISVDs are used as binding agents, for example, a "multispecific" format is formed by linking together two or more immunoglobulin single variable domains, at least one of which has a different specificity.

[0074] Thus, the present invention relates to a bifunctional bispecific agent that targets the CI-M6PR described herein and specifically targets a cell surface or extracellular molecule, i.e., an extracellularly accessible protein (different from the CI-M6PR protein), as a second binding, where such a bispecific agent can increase the degradation of the target compared to the degradation of the cell surface or extracellular molecule in the presence of the CI-M6PR binding agent alone (and thus not coupled to the additional binding agent that specifically binds to the target). The protein binding agent of the present invention is already bispecific in itself, since it binds at least CI-M6PR and another extracellularly accessible protein. Thus, a multispecific binding agent or fusion protein can also be associated with the addition of additional binding agents that can bind to one of the same or additional targets. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, etc. To further illustrate this, any multivalent or multispecific (as defined herein) protein binding agent of the present invention may be suitably directed against two or more different epitopes on the same antigen, for example, epitope 1 on one domain of CI-M6PR and epitope 2 on another domain; or may be directed against two or more different antigens, for example, an antigen against CI-M6PR and an antigen as a half-life extension against serum albumin. One of the most widely used techniques for increasing the half-life and / or reducing immunogenicity of pharmaceutical proteins involves the attachment of a suitable pharmacologically acceptable polymer, such as poly(ethylene glycol) (PEG) or its derivatives (such as methoxypoly(ethylene glycol) or mPEG). Further techniques for increasing the half-life of binding domains may include engineering into bifunctional or bispecific domains (e.g., active antibody fragments against CI-M6PR or active antibody fragments against serum albumin coupled to one or more ISVDs or one ISVD that help extend the half-life), or into fusions of antibody fragments, particularly immunoglobulin single variable domains with peptides (e.g., peptides against serum proteins such as albumin).Coupling to additional moieties results in multispecific binding agents, as further disclosed herein.

[0075] The multivalent or multispecific binding agents of the present invention may also have (or be engineered and / or selected for) increased avidity and / or improved selectivity for the desired CI-M6PR interaction and lysosomal targeting function, and / or for any other desired property or combination of desired properties that may be obtained by the use of such multivalent or multispecific binding agents. For example, the combination of one or more ISVDs that bind to any of the CI-M6PR epitopes and one or more ISVDs that bind to any of the extracellularly accessible target epitopes, as described herein, may result in a multispecific binding agent of the present invention having the potential for cellular uptake or internalization of the complete complex of the protein binding agent and its bound target via CI-M6PR internalization, ultimately resulting in degradation of the target(s) in the lysosome. "Internalization" of an extracellularly accessible target protein, as used herein, means that the target, when bound to a protein binding agent of the present invention (and thus encompasses the CI-M6PR-ISVD), is removed from the cell surface to a greater extent than a control, which may be the same protein binding agent that does not contain the CI-M6PR-ISVD or that contains an alternative ISVD that does not specifically bind to CI-M6PR or other targets for lysosomal uptake; and internalization may also be expressed as voxels / minute (as used herein, an internalization rate of at least 15 voxels / minute, or at least 35, or at least 50, or at least 65, or at least 80, or at least 100, or at least 120 voxels / minute is considered to be "internalized," as determined by live cell imaging methods). "Degradation" or "enhanced degradation" relative to a control, as used herein, means that the amount of protein of the target is reduced when determined for total protein (including the protein fraction retained on the cell surface) or when subcellular fractions or lysates of cells following internalization of the target protein qualitatively show the protein degraded into several fragments (e.g., as determined by Western blot analysis as exemplified herein)."Degradation relative to control" (e.g., untreated or treated with an equivalent protein binding agent lacking the CI-M6PR-specific ISVD of the protein binding agent of the invention) means that protein levels are reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 50%, or more compared to the control (and preferably based on protein levels normalized using a control protein for normalization).

[0076] In a further embodiment, the protein binding agent of the present invention is a multispecific binding agent comprising at least the CI-M6PR specific ISVD described herein and an extracellularly accessible target protein specific binding agent that can be coupled via a linker, spacer.When bound to CI-M6PR, the multispecific binding agent or multivalent ISVD can have an additive or synergistic effect on CI-M6PR internalization activity, or can be used to target cell surface or extracellular molecules and extract or shuffle them from the extracellular or membrane environment to endosomes and lysosomes, or alternatively, can be used to extend their half-life by recycling their targets through the endosomal cycling pathway.The multispecific binding agent of the present invention can be coupled to a functional moiety, a therapeutic (further targeting) moiety, a half-life extension moiety, or a cell-permeable carrier.

[0077] In further particular embodiments, the extracellularly accessible target protein specific binding agent may comprise an antigen binding domain such as an ISVD, a VHH, a Nb, a VHH-Fc fusion, a VHH-Fc-VHH fusion, a knob-into-hole VHH-Fc fusion, or an antibody such as an IgG, or may comprise a small molecule (which may be linked via covalent chemical coupling), or may be a peptide or a peptidomimetic. Further particular embodiments relate to bispecific or multispecific formats comprising the CI-M6PR conjugate based on the ISVD described herein and coupled directly, or indirectly via a spacer or linker, or chemically, to an extracellularly accessible target protein specific and optionally further binding agent. The coupling or fusion of the CI-M6PR specific ISVD with, for example, a VH or VL structure of another ISVD, antibody fragment or antibody type as defined herein, may also occur via a linkage via an Fc tail to produce a bispecific ISVD-Fc antibody as discussed above.

[0078] Thus, certain embodiments envisioned herein encompass those bispecific chimeras in which the ISVD-based binders that specifically interact with the N-terminal portion of CI-M6PR retain their binding to CI-M6PR during its endosomal cycle and have binding affinities that are stable and resistant to dissociation up to pH about 5.5. The anti-CI-M6PR VHHs described herein provide a panel of highly specific and high affinity binders at neutral pH, but with different pH dissociation profiles when lowering the pH (in vitro) to pH 6, 5, 4.5 or 4. Thus, this panel provides a versatile toolbox for exploring bispecifics with different properties of lysosomal degradation and recycling potential depending on the needs of the specific target and application. Furthermore, high affinity (nanomolar to picomolar KD values) of the CI-M6PR binder at neutral pH is required to ensure specific and tight binding to the receptor on the cell surface, while the need for rapid dissociation upon subsequent internalization into endosomes / lysosomes may be desirable to increase the chances of following the same late endosome / lysosome delivery pathway as the native cargo of CI-M6PR. With a view to optimizing binding affinity at specific pH conditions, the skilled artisan knows how to engineer binders such as VHHs using, for example, histidine scanning mutagenesis

[22] , which specifically aims to reduce the binding affinity of the antibody at acidic pH compared to neutral pH. As the imidazole side chain of the histidine residue has a pKa of approximately 6.0, switching its protonation state alters the binding interactions at the interface where it occurs. Briefly, combinatorial phage libraries are obtained with histidines incorporated into the VHH CDRs. This library is then screened by biopanning with binding at pH 7.4 and elution at pH 5.5, followed by BLI to determine the exact binding properties of the resulting VHHs at these pHs.

[0079] Another specific embodiment relates to a protein binding agent comprising a CI-M6PR-specific ISVD described herein and a binding agent for another extracellularly accessible protein fused or coupled by gene fusion and produced by recombinant expression in a host.

[0080] Another aspect of the invention provides a method for detecting the presence, absence or level of CI-M6PR and / or an extracellularly accessible target protein in a sample, comprising contacting the sample with a protein binding agent as described herein and detecting, i.e. quantifying, the presence or absence or level of bound CI-M6PR ISVD or target protein binding agent (optionally a label, conjugate, or multispecific binding agent). A sample as used herein may be a sample isolated from the body, such as a bodily fluid, including inter alia blood, serum, cerebrospinal fluid, or may be an extract, such as a protein extract, cell lysate, etc.

[0081] For the purpose of in vitro or in vivo detection and / or imaging, the protein binding agent or fusion protein of the present invention, comprising CI-M6PR specific ISVD and a binding agent that specifically binds to extracellularly accessible protein as described herein, may further comprise a detection agent (such as a tag or label) in some embodiments.For example, the ISVD, VHH or Nbs as exemplified herein are also tagged.Such tags allow affinity purification and detection of the antibody or active antibody fragment of the present invention.

[0082] Some embodiments include protein binding agents further comprising a label or tag, or more specifically, a fusion protein labeled with a detectable marker. The term detectable label or tag as used herein refers to a detectable label or tag that allows for detection and / or quantification of the fusion protein described herein, and is meant to encompass any label / tag known in the art for these purposes. Particularly preferred are affinity tags such as, but not limited to, chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6xHis or His6), biotin or streptavidin (e.g., Strep Tag®, Strep Tag II® and Twin-Strep Tag®); solubilization tags such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags such as FLAG tags; epitope tags such as V5 tags, myc tags and HA tags; fluorescent proteins (e.g., Fluorescent labels or tags (i.e. fluorochromes / fluorophores) such as GFP, YFP, RFP, etc. and fluorescent dyes (e.g. FITC, TRITC, coumarin and cyanine); luminescent labels or tags such as luciferase, bioluminescent or chemiluminescent compounds (such as luminal, islaminol, theromatic esters, imidazoles, acridinium salts, oxalate esters, dioxetanes or GFP and its analogs); phosphorescent labels; metal chelators; and (other) enzyme labels (e.g. peroxidase, alkaline phosphatase, β-galactosidase, urease or glucose oxidase); radioisotopes. Combinations of any of the above labels or tags are also encompassed. Techniques for producing labeled polypeptides and proteins are well known in the art. The protein binding agents or fusion proteins described herein, comprising a CI-M6PR-specific ISVD of the invention and a binder for an extracellularly accessible target coupled to (or further comprising) a label or tag, allow, for example, immune-based detection of the bound fusion protein.Immuno-based detection is well known in the art and can be achieved by applying a number of approaches. These methods are generally based on the detection of labels or markers such as those mentioned above. For example, see U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. When multiple antibodies are reacted in a single array, each antibody can be labeled with a separate label or tag for simultaneous detection. Further embodiments can include the introduction of one or more detectable labels or other signal generating groups or moieties, or tags, depending on the intended use of the label or tagged fusion protein of the present invention. Other suitable labels will be clear to those skilled in the art and include, for example, moieties that can be detected using NMR or ESR spectroscopy. Such labeled fusion proteins, such as those described herein, may be used, for example, 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 for in vivo imaging purposes, depending on the choice of the particular label.

[0083] Another aspect of the invention relates to pharmaceutical compositions comprising the inventive protein-binding agents or fusion proteins described herein, or comprising the nucleic acid molecules or vectors described herein, and optionally a pharma- ceutically acceptable carrier or diluent or excipient, which can be utilized to achieve a desired pharmacological effect by administration to a patient in need thereof.

[0084] A further aspect relates to said protein binding agents of the present invention, comprising an ISVD-based CI-M6PR specific fusion protein which further recognizes an extracellularly accessible target protein for internalization and degradation, a nucleic acid molecule or vector encoding said protein binding agent or fusion protein, or a pharmaceutical composition comprising the same as described herein for use as a diagnostic.

[0085] In certain embodiments, kits are provided that contain a means for degrading extracellularly accessible target proteins, the kits including protein binding agents described herein, allowing detection or regulation of target protein trafficking in a system that may be an in vitro or in vivo system. It is envisioned that these kits are provided for specific purposes, such as endosome / lysosome labeling, or for inducing target protein degradation in vitro, or for in vivo imaging, or for diagnosis of altered CI-M6PR or target amounts, responses, or effects in a subject. In another embodiment, the kits are provided that contain a means including a nucleic acid molecule, vector, or pharmaceutical composition described herein. The means further provided by the kit depend on the methodology used in the application and the purpose of the kit. For example, detection of a labeled fusion protein as described herein, or a nucleic acid molecule as described herein, may be desired for CI-M6PR or target quantification at the nucleic acid or protein level. For protein-based detection, the kit typically contains a label or a coupled binding agent, such as an ISVD. Similarly, for detection at the nucleic acid level, the kit may contain a label for the nucleic acid, such as a primer or a probe. Additional control agents, antibodies or nucleic acids can also be provided in the kit.Standards for reference or comparison, CI-M6PR or target protein substrates or signal transduction components, reporter genes or proteins, or other means for using the kit can also be included.Of course, the kit can further include pharma- ceutically acceptable excipients, buffers, vehicles or delivery means, instructions, etc.

[0086] A particular aspect of the present invention relates to protein binders, including CI-M6PR binders based on the ISVD described herein and binders that specifically bind to epidermal growth factor receptor (EGFR) extracellularly accessible target proteins located on the cell surface as transmembrane receptor proteins. In addition to the proof-of-concept experiments illustrated herein showing that exogenously added proteins such as GFP can be effectively internalized and degraded in the endosomal / lysosomal machinery, proof-of-concept for the internalization and degradation of transmembrane proteins has been provided by using protein binders in which the EGFR binder is provided by Nb or antibody in combination with a coupled VHH7 or VHH8 CI-M6PR specific ISVD. Targeting EGFR for CI-M6PR mediated internalization and preferably lysosomal degradation thereby provides an alternative approach in the therapeutic treatment of some cancers. The binding agent that targets or specifically binds to EGFR may be envisaged herein as any type of binding agent that can be fused to the CI-M6PR specific ISVD, and thus the EGFR specific binding agent may be an antibody, a small molecule, a peptide, or another antigen binding protein, including an ISVD or a VHH or a Nb. In certain embodiments, the EGFR specific binding agent includes a Nb or a functional (mutant) variant thereof, including, but not limited to, the monovalent 9G8 VHH shown in SEQ ID NO: 12, or a functional homolog thereof having at least 80%, 85%, 90%, 95%, or 97% or 99% identity thereto over the entire length of the monovalent ISVD. A functional homolog means that the binding properties of the ISVD homolog remain very similar or the same, as defined herein.Preferably, the amino acid residues in the CDRs are identical in the functional homologs, except where the mutations do not significantly affect the binding properties and / or where introducing such mutations into the CDRs allows avoiding another obstacle such as glycosylation, as for example in the case of the mutant variant of SEQ ID NO: 12 provided in SEQ ID NO: 17, where N-glycosylation on serine is avoided by a S54A substitution (according to Kabat numbering), or any functional homolog having at least 80%, 85%, 90%, 95%, or 97% or 99% identity thereof over the entire length of the monovalent ISVD. When fused to the CI-M6PR specific VHH, this results in a bispecific fusion protein as exemplified herein in SEQ ID NO: 13, 18, 82 or 84, or a functional homolog having at least 80%, 85%, 90%, 95%, or 97% or 99% identity thereof over the entire length of each monovalent ISVD and / or over the entire length of the fusion protein. Specifically, the EGFR-binding agent fused to the CI-M6PR-specific ISVD may be a multivalent or multispecific EGFR-specific binding agent, more specifically, may comprise SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 85, or a functional homologue having at least 80%, 85%, 90%, 95%, or 97% or 99% identity thereto over the entire length of each monovalent ISVD and / or over the entire length of the fusion protein. In further specific embodiments, the EGFR-specific binding agent is a conventional antibody provided herein as cetuximab, provided by its heavy chain as SEQ ID NO: 87 in combination with a light chain as shown in SEQ ID NO: 86, or, as exemplified herein, the protein binding agent may be provided such that the heavy chain of SEQ ID NO: 87 is fused to the CI-M6PR-specific ISVD described herein (such as provided in SEQ ID NO: 88 or 89), resulting in a multispecific EGFR-specific binding agent, which in combination with the light chain provided by SEQ ID NO: 86, is capable of internalizing and degrading EGFR in a potent manner.Thus, said protein binding agents that specifically target EGFR may be used as medicines, more specifically for use in the treatment of cancer.

[0087] Although specific embodiments, specific configurations, and materials and / or molecules of the products, compositions, methods, uses, samples, and biomarker products according to the present disclosure have been discussed herein, it should be understood that various changes or modifications in form and details may be made without departing from the scope of the present invention. The following examples are provided to better illustrate certain embodiments, and should not be considered as limiting the present application. The present application is limited only by the claims.

[0088] example The examples described below show for the first time the development of nanobody-based LYTACs and provide a proof-of-concept of a novel bispecific / multispecific platform for coupling anti-CI-M6PR VHHs to antigen-binding proteins, such as VHHs or antibodies, against an extracellularly accessible protein of interest to target this protein for internalization and / or lysosomal degradation. The efficacy of internalization of such multispecific fusion proteins is shown herein in a proof-of-concept experiment using GFP and GFP-specific VHHs as extracellular target proteins, and further investigated in the context of transmembrane proteins, such as the relevant disease-causing proteins demonstrated herein using human epidermal growth factor receptor (EGFR) as a target, where binders specifically targeting said receptor were fused to anti-CI-MPR VHHs that we previously isolated after a llama immunization and screening campaign. Since HeLa cells are commonly applied as a general cell-based cancer model, we investigated whether our Nb-based LYTAC system could induce EGFR internalization and / or degradation on those cells.

[0089] Example 1. Production and purification of VHH-based anti-EGFR LYTAC constructs. Anti-CI-M6PR VHHs have been previously generated and characterized in the context of enzyme replacement therapy for lysosomal storage diseases (Callewaert et al., PCT / EP2022 / 054278). Briefly, alpacas were immunized with domains 1-3 of human CI-M6PR and a series of phage pannings were performed according to the method of

[19] to obtain several human / mouse cross-reactive VHHs. The VHHs with the most similar affinities between humans and mice were tested and shown to trigger CI-M6PR-mediated lysosomal uptake. As discussed in PCT / EP2022 / 054278, further characterization revealed that the panel of selected VHHs as disclosed and used herein is suitable for binding and internalization of cell-expressed M6PR (Example 4). The panel could be further grouped into VHHs specific for binding to two epitopes present on the N-terminal M6PR extracellular domain (Example 6). Among these, we demonstrated that incorporation into the initial nanoLYTAC constructs occurs at plasma pH (7.4) (K D VHH 8, which binds with high affinity (approximately 3.35E10-9), was selected in this example. As a negative control for internalization / degradation not mediated by CI-M6PR, a GFP-binding VHH (GBP) was used.

[0090] Since bivalent binding of CI-M6PR has been shown to increase the rate of internalization via receptor dimerization [7], both monovalent and bivalent formats of CI-M6PR of each construct can be analyzed for evaluation of degradation potency and efficacy. To create a bivalent, two CI-M6PR VHHs can be linked, for example, with a standard Gly4 Ser linker.

[0091] In the first example, a monovalent VHH8 is incorporated into a set of anti-EGFR LYTAC constructs together with extracellularly accessible target protein-specific VHHs, more specifically, the target selected herein is EGFR. As described, a VHH (called PMP9G8, hereafter referred to as 9G8; SEQ ID NO: 12) that binds to EGFR [5-6] with monovalent affinity in the low nanomolar range and inhibits EGFR-mediated signaling is used for incorporation into our anti-EGFR nanoLYTACs. Since receptor tyrosine kinases can form homodimers upon activation, bivalent binding of EGFR may improve the apparent affinity and lead to more potent and effective CI-M6PR-mediated internalization. Furthermore, it has also been shown that EGFR dimerization itself can drive receptor internalization and subsequent downregulation by degradation in lysosomes [8]. To account for this, a fusion construct of GBP-VHH connected to two anti-EGFR VHHs in tandem (bivalent format) is envisioned herein.

[0092] The constructs used herein were cloned using a modular cloning platform produced in wild-type Komagataella phaffii (hereafter referred to as Pichia pastoris ) and subsequently purified, after which their EGFR internalization and degradation capabilities were examined in HeLa cells.

[0093] Table 1 summarizes the set of LYTAC constructs that were initially cloned and expressed in P. pastoris. SDS-PAGE analysis of the expression studies showed two distinct bands, one of the expected molecular weight and one corresponding to a larger MW (Figure 1). [Table 1]

[0094] Analysis of endoglycosidase H (EndoH) digestion of the supernatant showed N-glycosylation of the 9G8 anti-EGFR VHH in P. pastoris (Figure 2). Indeed, in the amino acid sequence of 9G8, an NWS sequon was identified in the CDR2 region. Analysis of the existing crystal structure of 9G8 in complex with the EGFR ectodomain revealed that none of the residues from the CDR2 region are involved in antigen binding. In an attempt to remove N-glycosylation, a new set of LYTAC constructs was produced in which 9G8 contains the S54A mutation (Table 1). Expression studies confirmed the removal of glycosylation demonstrated by the absence of additional bands on SDS-PAGE (Figure 3). For each construct, a suitable clone was selected and large-scale expression and purification was performed by benchtop gravity-flow IMAC and desalting (Figure 4).

[0095] Furthermore, to achieve the highest degradation efficiency, it is envisaged to generate a fusion of the CI-M6PR VHH to an anti-EGFR VHH containing a protease-sensitive linker with a cathepsin cleavage site to ensure that the target is released in the late endosome. Thus, a standard flexible 15 amino acid Gly4Ser linker can be used between two identical VHHs.

[0096] Finally, although VHHs have many advantages for therapeutic applications, a downside is the rapid renal clearance upon intravenous administration [9], which is somewhat reduced but still rapid in VHH concatemers. Therefore, improving the pharmacokinetic properties of anti-EGFR nanoLYTACs for intravenous injection in in vivo studies would be predictably beneficial. Therefore, further variants of these nanoLYTAC constructs in which the conjugate is fused to an anti-albumin VHH are also envisioned herein. This is rescued from glomerular filtration as albumin is constantly recycled by neonatal Fc receptors (FcRn) on vascular endothelial cells, resulting in a half-life of 19–21 days in humans

[12] . In addition, this fusion further increases the hydrodynamic radius of the nanoLYTAC constructs.

[0097] Example 2. In vitro assay for evaluation of LYTAC-mediated EGFR internalization efficacy by flow cytometry. The disappearance of the target protein from the cell surface can be quantified by flow cytometry. A decrease in antibody-coupled fluorescent staining intensity signal is detected upon internalization of the target protein.

[0098] The EGFR internalization efficacy of the first set of EGFR-LYTAC constructs after HeLa cell treatment was evaluated by detecting cell surface EGFR using flow cytometry. HeLa cells were left untreated or incubated in duplicate for 24 hours with 5 or 50 nM of constructs 26 (9G8 S54A-VHH8) or 27 (2x9G8 S54A-VHH8) or the corresponding control constructs 28 (9G8 S54A-GBP) or 29 (2x9G8 S54A-GBP). After the incubation period, cells were harvested and specifically stained for EGFR.

[0099] In Figure 5A-B, representative histograms of the fluorescence signal corresponding to cell surface EGFR are shown for untreated HeLa cells and HeLa cells treated with LYTAC constructs (26 and 27) or the corresponding control constructs (28 and 29, respectively). In Figure 5C, a bar graph showing the median fluorescence intensity measured for each condition of the experiment is shown. The results demonstrate that for cells treated with LYTAC constructs 26 and 27, the signal of cell surface EGFR was reduced compared to untreated cells or cells treated with the corresponding control constructs 28 and 29. Furthermore, the bivalent EGFR-binding LYTAC (construct 27) resulted in a lower signal compared to treatment with monovalent EGFR-binding LYTAC (construct 26) at both 5 nM and 50 nM concentrations, with the highest effect for both constructs observed at 50 nM (Figure 5B). The peak of the EGFR signal in cells treated with construct 29 was slightly left-shifted compared to that in untreated cells, indicating a moderate VHH8-independent internalization effect, most likely due to bivalent binding of EGFR.

[0100] Example 3. In vitro assay for assessment of LYTAC-mediated EGFR degradation by Western blot. To quantify which fraction of the target protein is degraded in response to treatment with nanoLYTACs, chemiluminescent Western blot analysis was performed. HeLa cells were incubated with 50 nM of constructs 26 (9G8 S54A-VHH8) or 27 (2x9G8 S54A-VHH8) or the corresponding control constructs 28 (9G8 S54A-GBP) or 29 (2x9G8 S54A-GBP) in OptiMEM for 24 hours (in duplicate). Lysates were obtained and subjected to immunoblotting with anti-EGFR antibodies (Figure 6). As a positive control, cells were incubated with EGF, which is known to induce EGFR degradation. In the described Western blot experiments, we were unable to effectively detect lower levels of total EGFR for cells treated with the nanoLYTAC constructs compared to untreated cells or cells treated with the control constructs. However, the inventors speculate that this may be a cause of the current experimental setup and therefore optimization of conditions and methods is required, as further detailed in Examples 10 and 12.

[0101] Example 4. Anti-CI-M6PR VHH1, VHH5, VHH7 and VHH8 are endocytosed and co-localize with late endosomes and lysosomes. Several experiments were previously performed to demonstrate that a selected panel of M6PR-binding VHHs efficiently internalize and traffic through endosomal compartments to lysosomes, as described in Callewaert et al. (PCT / EP2022 / 054278) and summarized herein.

[0102] Targeting to the endolysosomal compartment was assessed by monitoring the AAF488-VHH signal in live cells over time (3 hours). After LTR incubation and administration of AF488-labeled anti-CI-M6PR VHH, GFP-binding protein (GBP) and recombinant human acid glucosidase alpha (rhGAA; positive control), three fields of view were imaged every 6 minutes. After imaging, we calculated the uptake per cell volume by dividing the sum of the voxel counts of each fluorescent VHH by the sum of the voxel counts per imaged cell at a particular time point.

[0103] Protein uptake relative to cell volume provided the best results for VHH1, -5, -7 and -8. Maximum protein uptake relative to cell volume was observed over a 3-h period with a sigmoidal trend and a mean of 125.5 × 10 4 was observed for VHH7 after an internalization rate of 138.2 × 10 total AF-voxels / min. Similarly, an internalization rate of 138.2 × 10 for VHH1 was calculated by dividing the total number of AF488-positive voxels by time. 4 Compared with VHH7 and -1, the observed intracellular fluorescence of VHH5 was lower and more variable, whereas for VHH8 and rhGAA, the internalization rates were 68.7, 67.3 and 17.8 × 10 4 The profiles of the remaining VHHs (VHH1-11 were analyzed here) were comparable to the negative control (GBP), confirming that they indeed do not bind to cell surface hCI-M6PR.

[0104] The average percentage of VHH colocalization with lysosomes was calculated by taking the voxel number ratio of the intracellular AF488 signal colocalized with LTR and the total intracellular VHH signal. Next to this, the average percentage of the entire endolysomal pool containing a particular VHH or rhGAA was also determined by the voxel number ratio of the VHH signal colocalized with LTR and the total LTR signal. Due to occasional low intracellular AF488 signals and variable percentages, the absolute voxel number of intracellular VHH signals and VHH-LTR colocalization signals were also considered. Primary images after 120 min of incubation are shown in Figure 7.

[0105] After 60 min, the percentage of intralysosomal VHH1, 5 and 7 reaches equilibrium, whereas VHH8 is reaching a plateau at 90 min. LTR-positive voxels in cells treated with VHH1, -8 and rhGAA contained up to 60% internalized protein, whereas the total VHH7-positive LTR-positive pool was near 20% after 3 h. Triangular curves outline the monitored fraction of LTR-stained organelles colocalizing with AF488-VHH or -rhGAA. The total LTR pool positive for AF488 signal was highest for VHH7, between 30-40% after 3 h, and near 15% for VHH1. The fraction of the LTR pool containing VHH was less than 10% for VHH5, VHH8 and rhGAA, and even lower for the other VHHs. Overall, these results clearly demonstrate specific endocytosis and the greatest percentage of lysosomal targeting by anti-CI-M6PR VHH1, 5, 7 and 8 (when compared to the negative control (i.e., GBP); Table 2). The positive control shows only limited lysosomal colocalization. [Table 2]

[0106] Although labeled with differential efficiency, the variation in the endolysosomal content of these four anti-CI-M6PR VHHs upon endocytosis may indicate differential lysosomal delivery or variable cycle pathways of these molecules. This was most striking for VHH7, where we observed a lower VHH7 endolysomal pool compared to the others, but an increased fraction of endolysosomes containing VHH7. To investigate whether these variations in endolysosomal content were the result of true lysosomal delivery, we examined the fraction of AF488-VHHs that colocalized with LAMP1, a lysosomal membrane protein that is increasingly present in mature lysosomes on fixed cells. We did this for VHH7, which was increasingly undergoing endocytosis, and for VHH8, where a lower intracellular fraction but a larger LTR-positive fraction could be observed. After 4 h of incubation on HeLa cells, anti-LAMP1 antibodies were used for staining.

[0107] As shown in Figure 8, the intralysosomal fraction of intracellular VHH7 after 240 min was in the same range (i.e. 19%) as what we observed during live cell imaging experiments after 200 min with LTR (i.e. 20%). However, this is completely opposite for VHH8, as we detected only 2.5-12% intracellular VHH8 colocalization with LAMP1-positive lysosomes compared to 60% with LTR. Next to the intralysosomal fraction, we also calculated the percentage of LAMP1-stained voxels containing VHH (Figure 8A).

[0108] It is noteworthy that while the LAMP1 and LTR colocalized fractions of AF488-VHH7 are similar at 19% and 20%, respectively, VHH8 has a much higher LTR colocalized fraction (i.e., 60%) compared to the LAMP1 colocalized fraction (i.e., up to 12%). Although it is difficult to compare live cell imaging with microscopy after fixation, of course due to the different cell lines used, these experiments may suggest that VHH7 and VHH8 may follow different endolysosomal pathways. VHH7 showed almost equal colocalization with LTR and LAMP1, suggesting increased lysosomal targeting of VHH7. Given the 5-fold higher dissociation rate of VHH7 at pH 6.0 versus pH 7.4, it is plausible that immediate endosomal acidification upon endocytosis allows for rapid dissociation of VHH7, which can then be delivered to lysosomes together with endosomal cargo during maturation. In that case, unbound receptors can be recycled and participate in a new round of binding. The low amount of VHH8 stained with LAMP1 compared to LTR stained organelles can indicate increased colocalization with the previous endolysomal network. However, a higher amount of LAMP1-lysosomes with VHH8 were detected (Figure 8B). Since VHH8 has a dissociation transition between pH 6.0 and pH 5.0, it is plausible that it could remain bound to hCI-M6PR at the early endosomal stage (pH 5.9-6.5) instead of being delivered to lysosomes. The high LAMP1 colocalization of VHH7 on the one hand and the peripheral localization of VHH8 on the other hand can indicate this (Figure 8C).

[0109] It should also be noted that once the VHHs reach the mature lysosomes, they are presumably denatured by lysosomal proteases. What happens to the fluorophore thereafter in terms of localization is unclear. However, it can be assumed that this behavior is similar across the VHHs studied.

[0110] Importantly, throughout the interpretation of the absolute numbers in these microscopy studies, the degree of labeling obtained for all VHHs and recombinant hCI-M6PR D1-D3The first step is to recognize its reduced affinity for VHH7. This is unavoidable and comparable to what is generally expected for these NHS-ester labels, but the absolute number of VHH7 is likely overestimated due to its high labeling efficiency. D1-D3 It is also important to recall that the diverse affinities combined for do not directly correspond to binding to native hCI-M6PR. Furthermore, when we calculated the fraction of colocalized AF488 and LTR or LAMP1 signal, we observed that, together with all other descriptions, low levels of endocytosis together with variability between replicate VHHs can rapidly result in unusually high colocalization percentages. Other VHHs had no evidence of CI-M6PR receptor engagement at the cell surface in previous experiments. However, most showed this low level of highly variable uptake, similar to GBP, and were excluded from the above discussion for these reasons.

[0111] Example 5. Humanized variants of VHH7 and VHH8. Multiple humanized variants of anti-CI-M6PR VHH7 and VHH8 were designed in silico (SEQ ID NOs: 26-29 for VHH7 humanized variants and SEQ ID NOs: 30-35 for VHH8 humanized variants; as disclosed in Callewaert et al. (PCT / EP2022 / 054278)). VHH7hWN and VHH8hWN were produced in HEK293S and purified by IMAC and SEC. Variants VHH7h1-3 and VHH8h1-5 were produced in E. coli and purified by IMAC and desalting.

[0112] For the selection of humanized variants of VHH7 and VHH8, the human CI-M6PR domain 1-3 His 6Biolayer Interferometry (BLI) experiments were performed in which VHHs were biotinylated and coupled to a streptavidin biosensor chip. After loading, during the association phase, the chip was incubated with VHHs serially diluted in kinetic buffer at pH 7.4, and dissociation was performed at pH 7.4, pH 6.5, pH 6.0, pH 5.5 and pH 5.0. All biosensor chips were then regenerated before the analysis of subsequent VHHs. Table 3 summarizes the kinetic parameters retrieved after processing and curve fitting of the BLI measurements. When both association and dissociation were performed at pH 7.4, the obtained affinity constants (K D ), meeting (k on ) and the dissociation rate constant (k off ) was performed according to a 1:1 binding model. For measurements with association at pH 7.4 and dissociation at pH 6.5, 6.0, 5.5 and 5.0, the dissociation rate constants shown are the average of the parameters determined by local curve fitting of the dissociation of 200, 100 and 50 nM VHH. The association-dissociation curves of VHH7 and its humanized variants are shown in Figure 9 and the association-dissociation curves of VHH8 and its humanized variants are shown in Figure 10.

[0113] BLI revealed pH-dependent dissociation of humanized variants VHH7h1, VHH7h2, VHH7h3 and VHH7hWN, with dissociation profiles similar to their non-humanized counterparts VHH7, and dissociation rates that gradually but moderately increased with decreasing pH (Figure 9, Table 3). Furthermore, the human CI-M6PR domain at pH 7.4 1-3 His 6 Their affinity for VHH8 remains largely unchanged upon humanization. Similarly, the pH-dependent dissociation profile of VHH8, whose dissociation rate increases only moderately between pH 7.4 and 6.0, but then shows a rapid surge of nearly an order of magnitude between pH 5.5 and 5.0, remains unchanged for its humanized variants VHH8h1, VHH8h2, VHH8h3 and VHH8hWN (Figure 10, Table 3). Again, the K obtained at pH 7.4 D The values ​​are equivalent for all variants under evaluation. [Table 3]

[0114] Example 6. Multi-angle light scattering and crystallography of VHH-hDom1-3His complex. With a view to identifying the binding sites of the VHHs used herein, a structural analysis of several VHHs in complex with human M6PR domains 1-3 was performed, as previously described by Callewaert et al. (PCT / EP2022 / 054278). Since the three N-terminal domains of CI-M6PR are repeats of CI-M6PR type domains (Pfam PF00878), we determined that the binding sites of the VHHs in hCIMPR are identical to those of the VHHs in hCIMPR. D1-D3 : The molecular weight and oligomeric state of the VHH protein complex were first determined, and the inventors D1-D3 SEC-MALLS elution and fraction samples were analyzed after incubation of VHH with hCI-M6PR and VHH8 in a 1:1 and 1:3 molar fashion. The calculated protein masses corresponded to those expected for VHH and antigen, respectively 17 kDa (±1 kDa) and 51.3 kDa (±0.9 kDa), and 62 kDa (±2 kDa) for the complex, corresponding to equimolar binding of both proteins. When fraction samples were analyzed by non-reducing SDS-PAGE, aggregated or other oligomeric structures could also be detected, but remained limited. Complexation of VHH with antigen proteins was examined after endoglycosidase H digestion, revealing that hCI-M6PR and VHH8 were highly complexed with hCI-M6PR and VHH8, respectively. D1-D3 The present inventors also found that VHH8 and hCI-M6PR D1-D3 Since we found equimolar binding to VHH7 and hCIM6PR, we used equal concentrations of VHH7 and hCIM6PR during the next SEC-MALLS run. D1-D3 was also used. D1-D3Molecular weights of 50 kDa (± 2 kDa) for anti-CI-M6PR VHH7, 15 (± 1 kDa) for anti-CI-M6PR VHH7 and 63.7 (± 0.5 kDa) for the complex were measured. Thus, prior to co-crystallization screening, the (glycosylated) hCI-M6PR complex was determined for both VHH7, VHH8 and VHH1H11 (the latter obtained after a re-panning experiment (see Example 7)). D1-D3 A final preparative SEC run was performed accordingly using After crystallization, the presence of both antigen and VHH was confirmed by SDS-PAGE.

[0115] The first three domains at the N-terminus of CI-M6PR (CI-M6PR D1-D3 ) resembles the previously published conformation. In cocrystals with VHH7 and VHH8, hCI-M6PR D1-D3 Bovine CI-MPR D1-D3 It adopts a trefoil-shaped structure similar to the conformation observed for (pdb 1q25).

[0116] In the co-crystal structure with VHH1H11, the third domain is shifted toward D1 to resemble the conformation present in pdb 6p8i (Figure 14). Although present in the crystallization mixtures of VHH7- and -8-containing complexes, mannose 6-phosphate was not observed in either structure. The N-glycans in the three N-glycosylation sequences (i.e., Asn112, Asn400, and Asn435) could be identified to various degrees from the electron density. In the co-crystal structure with VHH7 and VHH8, clear electron density could be interpreted for the Man3GlcNAc2 or Man4GlcNAc2 containing glycan at Asn112. Only partial core GlcNAc or GlcNAc2 could be interpreted from the electron density at other positions. However, of interest are the crystal contacts observed in these crystal structures between the N-linked glycan on Asn112 of the crystallographically symmetry-related CI-M6PR copy and the M6P binding pocket in D3. More specifically, the α1,3-Man of the oligomannosylated glycan on Asn112 interacts mainly through hydrogen bonds with residues Tyr359, Gln383, Arg426, Glu451 and Tyr456 of CI-M6PR D3 The N-linked glycans on Asn112 and Asn435 of the VHH 1H11 co-crystal structure could be identified as core 1-6 fucosylation.

[0117] The core structure of each domain consists of a flattened β-barrel (Pfam domain CIMR PF00878) containing a five-stranded antiparallel β-sheet (β3-β6), whose strands extend in an orthogonal orientation onto a second five-stranded β-sheet (β8-β11), with the fourth strand inserted between β9 and β11. Each domain is integrated into the CI-M6PR (PDB: 1sy0, 1sz0, 1q25, 6p8i) 17The N-terminus of human domain 2 (residues 161-313) and domain 3 (residues 314-467) should contain four disulfide bonds to be equivalent to the bovine crystal structure of the N-terminal three domains of . The N-terminus of human domain 2 (residues 161-313) and domain 3 (residues 314-467) each contain a linker region composed of a random coil followed by two auxiliary β-strands (β0, β1, and β2) that connect the core flattened β-barrel structure.

[0118] Anti-CI-M6PR VHH7, VHH8 and VHH 1H11 adopt a general immunoglobulin-like fold with neutral and extended twisted CDR3 loops, respectively. D1-D3 The highest resolution crystal structure of a protein complex has been solved to a resolution of 2.2 A (Figure 12A) and was grown at pH 6.5 (Figure 12A). The first protein complex is hCI-M6PR D1 The results reveal VHH7 located on one side packed between two β-sheets of the flattened β-barrel of D1 (Figure 12B). Presenting one side to its antigen, VHH7 interacts through its CDR1,2 but also with residues in CDR3 (Figure 12C). These make contacts with amino acid side chains in intradomain loops A-D of D1 (Figure 12). The complex is near identical in other crystal forms.

[0119] The VHH8 co-crystal structure solved at 2.75 A resolution shows that VHH8 is CI-M6PR hCI-M6PR D2 and hCI-M6PR D3 (Figure 13A). They form a V-shaped surface whose amino acids contact the variable protruding loops of VHH8 (Figure 13B). In general, most of the residues from CDR2 interact with residues in D3, while residues from CDR3 face D2. The contribution of CDR1 is only limited to the overall interaction compared to the other CDRs (Figure 13C).

[0120] VHH7 competing anti-CI-M6PR VHH 1H11 and hCI-M6PR D1-D3The crystal structure of was solved at 2.7A resolution, confirming the results obtained from the mutation screen and competitive BLI. Comparable to VHH7, VHH 1H11 binds hCI-M6PR D1 It faces one side of the flattened β-barrel of the CI-M6PR VHH (Figure 14A-B) and interacts primarily with residues from both the CDR1 and CDR2-bearing β-sheets (Figure 14C). As a general overview, a binding schematic of the lead anti-CI-M6PR VHH is shown in Figure 15.

[0121] PISA

[18] and FastContact

[16] software were examined to determine the D1-D3 The interacting residues in the binding surface of anti-CI-M6PR VHH7, -8 and -1H11 with CI-M6PRD1 were roughly calculated and identified. Since FastContact analysis is biased towards electrostatic interactions, we combined the calculation of desolvation free energy and electrostatic energy calculated in PyMol

[14] with distance measures to approximate the interface residues of anti-CI-M6PR VHH and its antigen. From this information, two very distinct paratopes and epitopes could be delineated for either VHH7, VHH 1H11 and VHH8. The epitope of anti-CI-M6PR VHH7 (Figure 12) consists mainly of amino acids that are part of intradomain loops A, B, C and D of the β-sheet in CI-M6PRD1 (Figure 12). In addition, hydrophobic residues (e.g., Phe143) that constitute the hydrophobic core of the flattened β-barrel contribute to VHH binding. According to current estimates, the critical paratope residues include Arg33, Lys57 and Asp104, which are involved in the hCI-M6PR D1 It interacts with residues Asp87, Glu148 and Lys89, respectively (Table 4, Figure 12D). These calculations allowed us to confirm the similarity of the epitopes of VHH 1H11 and VHH7. In general, the residues predicted to contribute to the interaction were comparable in the epitope and paratope of VHH7 (Table 4). For example, residues Arg33 and Lys57 interact with the epitope of CI-M6PR D1-D3The epitopes of VHH 1H11 were predicted to be highly involved in the binding of VHH 1H11 to hCI-M6PRD2 (Fig. 14D), whereas the residues in CDR3 of VHH 1H11 probably contribute less. Moreover, the predictions here showed high similarity between both epitopes, with Asp87, Lys89 and Glu148 as highly contributing residues (Table 5). The epitopes of anti-CI-M6PR VHH8 are significantly different (Fig. 13). In contrast to VHH7, the interaction of CI-M6PRD1-D3 with VHH8 occurs in the inter- and intra-domain loops of hCI-M6PRD2 and hCI-M6PRD3, but residues within the β-strands of these domains also have an influence (Table 6). As described, the amino acids constituting CDR2 contact D3. Among them, Lys57 of VHH8 is predicted to form electrostatic interactions with Glu409 and Glu433 of D3 (Table 6, Fig. 13D). Strong contacts between D2 and CDR3 were predicted to be Asp102 and Lys191, respectively (Table 6, FIG. 13D).

[0122] The epitope information allows further insight into the (non)cross-reactive binding of VHH7, -8 and 1H11. Despite 75% sequence identity between human domains 1-3 and either Bos taurus or Mus musculus domains 1-3 sequences, the VHH7 and VHH8 interfaces are fairly conserved. In Figure 11, we show that the VHH7 and VHH8 interfaces of hCI-M6PR D1-D3 Each of the specific epitope residues in the orthologous sequences for VHH7 and VHH8 are shown. Considering residues that contribute significantly to the total binding free energy (i.e., ΔG<−1.5 kcal / mol), a higher degree of variation can be observed in VHH7 than in VHH8.

[0123] Tables 4-6, which correspond to information on VHH7, VHH 1H11 and VHH8, respectively, show anti-CI-M6PR VHH and hCI-M6PR VHH by FastContact and PISA. D1-D3The interacting residues and their predicted interaction types are shown as the estimated binding free energies (ΔG) determined by the sum of the calculated electrostatic free energies, desolvation free energies and configurational entropies for the interactions between the residues of . [Table 4] [Table 5] [Table 6]

[0124] Validation of the novelty and specificity of the human M6PR binding site defined herein for VHH7, VHH1H11 and VHH8 required screening a panel of anti-CI-M6PR VHHs developed by LinXis BV (described in Houthoff et al., published as WO2020 / 185069A1) against the extracellular portion of CI-M6PR. Tandem competitive biolayer interferometry of purified alternative anti-CI-M6PR VHHs demonstrated that LinXis VHH 13E8, as well as VHH7 and VHH8 described herein, each bind to the CI-M6PR hDom 1-3 His 6 CI-M6PR hDom 1-3 His 6 No binding to was observed for any additional representative VHHs, thus revealing that no competing binders were identified.

[0125] Example 7. VHH7 or VHH8 Further VHH CDR3 families competing for binding to the human M6PR epitope. Recombinant human CI-M6PR Dom 1-3 His 6The original VHH library from the llama that generated an antigen-specific response upon immunization with CI-M6PR hDom was transformed with CI-M6PR hDom IgG1-specific mAbs, as previously described by Callewaert et al. (PCT / EP2022 / 054278). 1-3 His 6 Fifteen novel VHHs belonging to 12 novel CDR3 groups were identified in these panning efforts.

[0126] To identify anti-CI-M6PR VHHs that bind to epitopes on CI-M6PR that overlap or are identical to VHH7 or VHH8 from different "CDR3" families or VHH families as defined herein, biolayer interferometry (BLI) experiments were performed to identify competitors of VHH7 or VHH8 for the CI-M6PR binding site, and previously characterized VHH1 and VHH5 were also evaluated. Tandem competitive BLI of anti-CI-M6PR VHHs purified from E. coli showed that VHH 1H11 and VHH1 bind to CI-M6PR hDom 1-3 His 6 competes for binding with VHH7 but not with VHH8; and 1H52 and VHH5 bind to CI-M6PR hDom 1-3 His 6 Competed with VHH8 for binding but not with VHH7, whereas 1H21, 1H37, 2H74 and 2H79 competed with CI-M6PR hDom 1-3 His 6 It was revealed that 1H74, 1H44 and 2H60 do not compete with VHH7 or VHH8 for binding to CI-M6PR hDom 1-3 His 6 No saturation binding was obtained (Figures 17 and 18).

[0127] BLI experiments were performed for VHH 1H11 (SEQ ID NO: 24) and VHH 1H52 (SEQ ID NO: 25) in which the human CI-M6PR domain 1-3His6 was biotinylated and coupled to a streptavidin biosensor chip. After loading, during the association phase the chip was incubated with serially diluted VHHs in kinetic buffer at pH 7.4, and dissociation was performed at pH 7.4, pH 6.5, pH 6.0, pH 5.5 and pH 5.0. All biosensor chips were then regenerated before the analysis of subsequent VHHs. Table 7 summarizes the kinetic parameters retrieved after processing and curve fitting of the BLI measurements. The obtained affinity constants (KD), association (kD), and dissociation constants (kD), were calculated using the kinetic constants (KD), when both association and dissociation were performed at pH 7.4. on ) and the dissociation rate constant (k off A global fit was performed according to a 1:1 binding model where p is the binding site and p is the binding site. For measurements with association at pH 7.4 and dissociation at pH 6.5, 6.0, 5.5 and 5.0, the dissociation rate constants shown are the average of the parameters determined by local curve fitting of the dissociation of 200, 100 and 50 nM VHH. The association-dissociation curves of VHH1H11 and VHH1H52 are shown in Figure 19 and Figure 20, respectively.

[0128] Analysis of BLI data was performed using CI-M6PR hDom 1-3 His 6 We reveal that anti-CI-M6PR VHH 1H11, which was demonstrated (in addition to VHH1) to compete with VHH7 for binding via BLI, demonstrates a similar pH-dependent dissociation profile as VHH7 itself (Figure 19). As in the case of VHH7, the value of the dissociation rate constant increases gradually but moderately as the pH is decreased from 7.4 to 5.0 (Table 7). As shown through BLI, the CI-M6PR hDom 1-3 His 6 One of the anti-CI-M6PR VHHs that competed with VHH8 for binding, VHH 1H52 (next to VHH5), also showed a similar pH-dependent dissociation profile to VHH8 (Figure 20). Indeed, there is a rapid increase in the dissociation rate between pH 5.5 and pH 5.0 (Table 7). [Table 7]

[0129] Example 8. Production and purification of VHH-based anti-EGFR nanoLYTAC constructs. Contains anti-EGFR VHH 9G8 S54A and either anti-CI-M6PR VHH VHH7 or VHH8 (G4S) 3 Linker (or further (G4S) 9 The LYTAC construct against EGFR was cloned, coupled at the C-terminus with a (G4S) linker. 3 LYTAC constructs containing two copies of 9G8 S54A, also coupled to a linker, were cloned. As a control for internalization and / or degradation not specifically mediated by CI-M6PR, we designed constructs linking one or two copies of 9G8 S54A to an anti-GFP VHH named "GBP". All these constructs were produced in Pichia pastoris with a C-terminal FLAG3His6 tag, purified by IMAC and desalting, and their expression yields are summarized in Table 8, as well as the composition of the protein constructs. The quality of the protein constructs was checked by SDS-PAGE (Figure 22) and their HeLa cell binding was verified by flow cytometry (data not shown). [Table 8]

[0130] Of the LYTACs containing VHH8 and control constructs, an initial set of proteins were produced early on at small scale (constructs 26-29 described in Examples 1-3), but at the N-terminus potentially contained three additional amino acids related to the cloning methodology that includes the amino acids Arg-Ser-Met (RSM) preceding the sequences provided herein. As described in this example, those constructs were recloned and reproduced in the absence of those additional amino acids, and then their retained in vitro EGFR internalization efficacy was verified by flow cytometry (Example 9). Thus, proteins without the N-terminal additions were used throughout the experiments, further examples are shown, and the results were consistent with those initially observed.

[0131] Example 9. Evaluation of in vitro EGFR internalization efficacy of VHH-based anti-EGFR nanoLYTAC constructs by flow cytometry. The efficacy of VHH-based anti-EGFR nanoLYTAC constructs to reduce the levels of EGFR at the cell surface was evaluated in vitro. For this purpose, HeLa cells were incubated with 50 nM of LYTAC constructs (numbers 34-37, see Table 8) or a control construct in complete medium for 24 h, after which the cells were detached and stained for cell surface EGFR and measured by flow cytometry.

[0132] In Figure 23A, representative histograms of the fluorescent signal corresponding to cell surface EGFR are shown for untreated HeLa cells and HeLa cells treated with 50 ng / ml of recombinant human EGF (rhEGF), LYTAC constructs 34 (9G8 S54A-VHH7), 35 (9G8 S54A-VHH8) or the corresponding control construct 38 (9G8 S54A-GBP). Figure 23B shows representative histograms of untreated HeLa cells and HeLa cells treated with 50 ng / ml of rhEGF, LYTAC constructs 36 (2x 9G8 S54A-VHH7), 37 (2x9G8 S54A-VHH8) or the corresponding control construct 39 (2x9G8 S54A-GBP). In FIG. 23C, a bar graph is shown showing the median fluorescence intensity (MFI) values ​​measured for all conditions in the experiment normalized to the MFI measured for untreated HeLa cells and expressed as a percentage.

[0133] The results of this experiment demonstrate that cells treated with either of the LYTAC constructs that are monovalent for EGFR (constructs 34 and 35) had a significantly reduced signal for cell surface EGFR compared to untreated cells (34: P = 0.001, 35: P = 0.0007) and to cells treated with the corresponding control construct (38) (34: P = 0.00009), 35: P = 0.00005), which also induced a moderate EGFR internalization effect (up to 83% of untreated cells). 9 The monovalent EGFR binding constructs with linkers (constructs 40 and 41) also exhibited their (G4S) 3 We demonstrated efficient induction of EGFR internalization that was comparable to (and slightly more efficient in this experiment than) its counterpart.

[0134] Furthermore, lower signals were detected in HeLa cells treated with either of the bivalent LYTAC constructs against EGFR (constructs 36 and 37) compared to untreated cells (construct 36: P = 0.001, 37: P = 0.0006) and cells treated with the corresponding control construct (39) (construct 36: P = 0.005, 37: P = 0.002). Furthermore, the bivalent EGFR-binding LYTACs (constructs 36 and 37) induced EGFR internalization in HeLa cells more effectively than the monovalent EGFR-binding LYTACs (constructs 34 and 35). Note that the signal corresponding to cell surface EGFR in cells treated with the bivalent control construct (construct 39) was also reduced to 49% of the signal in untreated HeLa cells, an effect expected as a result of bivalent binding of EGFR.

[0135] Example 10. Evaluation of the in vitro EGFR degradation efficacy of VHH-based anti-EGFR nanoLYTAC constructs. To evaluate the efficacy of VHH-based anti-EGFR nanoLYTAC constructs to induce degradation of the target protein, a Western blot assay was optimized. For this purpose, HeLa cells were incubated for 24 h in complete growth medium (as opposed to serum-free optiMEM as detailed in Example 3) with 50 nM of anti-EGFR LYTAC (constructs 34-37) or the corresponding control constructs (38-39), or with 50 ng / ml of rhEGF as a positive control for EGFR degradation. Cell lysates were then obtained in RIPA buffer and equal amounts of protein (measured by BCA assay) were subjected to immunoblotting for fluorescent detection (as opposed to chemiluminescent detection in Example 3) of EGFR and β-tubulin (Figure 24).

[0136] Quantified intensity values ​​corresponding to total EGFR as determined by densitometry demonstrate that there is a consistently lower signal for cells treated with any of the LYTAC constructs (34-37) compared to untreated cells. Furthermore, when normalized to the signal of lysates treated with the monovalent control construct 38 (9G8 S54A-GBP), it is clear that there are consistently lower values ​​for cells treated with any of the monovalent LYTAC constructs (34 and 35), indicating a CI-M6PR-dependent effect and confirming that these full VHH-based anti-EGFR nanoLYTAC constructs can effectively induce degradation of EGFR. We strongly believe that our previous failure to detect lower levels of total EGFR induced by VHH8-containing anti-EGFR nanoLYTAC constructs (26 and 27) (as described in Example 3) is due to both non-optimized cell assay-related and technical conditions.

[0137] Example 11. Evaluation of in vitro inhibition of ligand-induced EGFR activation by VHH-based anti-EGFR nanoLYTAC constructs. To assess whether treatment with the fully VHH-based anti-EGFR nanoLYTAC constructs was effective in inhibiting ligand-induced EGFR activation, a Western blot assay was performed. HeLa cells were treated with 50 nM of nanoLYTAC constructs (34-37) or the corresponding controls (constructs 38-39) or the FDA / EMA approved therapeutic anti-EGFR monoclonal antibody Erbitux® (50 nM or 40 μg / ml) in complete growth medium for 24 h. After the incubation period, cells were stimulated with 50 ng / ml of recombinant human EGF (rhEGF) for 5 min, after which cell lysates were obtained in RIPA buffer and equal amounts of protein were subjected to immunoblotting for fluorescent detection of phosphorylated EGFR (@Tyr1068) (Figure 25).

[0138] The results demonstrate a significant decrease in the levels of phosphorylated EGFR upon treatment with either monovalent EGFR-binding LYTACs (constructs 34 and 35) and bivalent EGFR-binding LYTACs (constructs 36 and 37) compared to stimulated untreated cells and compared to cells treated with their corresponding control constructs (38 and 39, respectively). Interestingly, the strength of this effect is in the same range as treatment with Erbitux, with construct 37 inducing an even stronger inhibitory effect than Erbitux at any of the concentrations tested.

[0139] Example 12. Production of cetuximab-VHH fusions as anti-EGFR nanoLYTAC constructs and in vitro EGFR internalization and degradation efficacy. (G4S) 2 LYTAC constructs against EGFR were designed, consisting of the therapeutic anti-EGFR monoclonal antibody (mAb) cetuximab coupled to either anti-CI-M6PR VHH7 or VHH8 at the C-terminus of the Fc domain using a linker. These cetuximab-based nanoLYTAC constructs were expressed in Chinese Hamster Ovary (CHO) cells and purified from the supernatant through protein A chromatography and SEC. As a control for non-CI-M6PR-mediated EGFR internalization, non-VHH-fused cetuximab was also produced in CHO. The expression yields herein are shown in Table 9 along with a summary of the composition of the protein constructs. Their quality was assessed by SDS-PAGE (Figure 26). [Table 9]

[0140] The efficacy of cetuximab-VHH fusion constructs to reduce the levels of EGFR at the cell surface was evaluated in vitro. For this purpose, HeLa cells were incubated with 5 or 50 nM of LYTAC constructs (Ctx-VHH7 or Ctx-VHH8) or a control construct for 24 h, after which cells were detached and stained for cell surface EGFR and measured by flow cytometry.

[0141] In Figure 27A, representative histograms of the fluorescence signal corresponding to cell surface EGFR are shown for untreated HeLa cells and HeLa cells treated with 50 ng / ml recombinant human EGF (rhEGF), 5 nM LYTAC constructs (Ctx-VHH7 or Ctx-VHH8) or the corresponding control construct (Ctx). In Figure 27B, a bar graph is shown showing the median fluorescence intensity (MFI) values ​​measured for all conditions in the experiment normalized to the MFI measured for untreated HeLa cells and expressed as a percentage.

[0142] The results of this experiment demonstrate that for cells treated with either of the cetuximab-based nanoLYTAC constructs (Ctx-VHH7 and Ctx-VHH8) at 5 nM, the signal of cell surface EGFR was significantly reduced compared to untreated cells (Ctx-VHH7: P = 0.002, Ctx-VHH8: P = 0.002) and cells treated with the corresponding control construct (Ctx) that also induces the expected moderate EGFR internalization effect (Ctx-VHH7: P = 0.002, Ctx-VHH8: P = 0.002). The magnitude of the induction effect does not increase further at higher (50 nM) treatment concentrations.

[0143] Interestingly, the magnitude of EGFR internalization effect in HeLa cells induced by the cetuximab-VHH fusion construct appears to be greater than that induced by the mannose 6 phosphonate (M6Pn) functionalized LYTAC construct described in Banik et al.

[10] and Ahn et al.

[15] , as well as Bertozzi et al. WO2020132100A1. Indeed, when expressed relative to cell surface EGFR levels in untreated HeLa cells, treatment with 50 nM Ctx-VHH7 reduced the levels to 10%, and treatment with 50 nM Ctx-VHH8 reduced the levels to 6%, while 50 nM M6Pn-Ctx construct only reduced EGFR levels to 25-30% (as read from the bar graph shown in Figure 4C by Ahn et al.

[15] ). The inventors believe that the magnitude of effects measured in these flow cytometry experiments can be reliably compared due to the following reasons: (1) both experiments are performed on HeLa cells, (2) the same primary EGFR detection antibody is used in both experiments (EGFR monoclonal antibody 199.12, #MA5-13319, Invitrogen), (3) the LYTAC constructs utilize the same EGFR binding moiety, which is the monoclonal antibody cetuximab, and (4) treatments were performed with the same concentration of each LYTAC construct. Given that a side-by-side comparison was not performed, this difference is significant enough to conclude that the CI-M6PR binding VHHs used to incorporate into the LYTAC constructs disclosed in Callewaert et al. (PCT / EP2022 / 054278) and described herein are more effective at inducing internalization of target proteins in the context of bispecific constructs than M6Pn-ligands.

[0144] To evaluate the efficacy of cetuximab-VHH fusions as nanoLYTAC constructs to induce degradation of EGFR, a Western blot assay was performed. HeLa cells were incubated for 24 hours in complete growth medium with 5 nM Ctx-VHH7 or Ctx-VHH8, or the corresponding non-VHH fusion control Ctx, or 50 ng / ml rhEGF as a positive control for EGFR degradation. After the treatment period, cell lysates were obtained in RIPA buffer, and equal amounts of protein (measured by BCA assay) were subjected to immunoblotting for fluorescent detection of EGFR and β-tubulin (Figure 28).

[0145] Quantified intensity values ​​corresponding to total EGFR determined by densitometry demonstrate that there is a consistently lower signal for cells treated with each LYTAC construct compared to untreated cells. Furthermore, when normalized to the signal for lysates treated with a non-VHH fusion control construct (Ctx), it is clear that there are consistently lower values ​​for cells treated with either of the LYTAC constructs (Ctx-VHH7 and Ctx-VHH8), indicating a CI-M6PR-dependent effect and confirming that the described cetuximab-VHH fusion constructs can effectively induce degradation of EGFR.

[0146] Example 13. Production of VHH-based anti-GFP LYTAC constructs and in vitro GFP internalization and degradation efficacy. To obtain proof of concept that LYTAC constructs based on CI-M6PR VHHs can also effectively induce internalization and degradation of soluble antigens, we conjugated (G4S) to either the anti-CI-M6PR VHHs “VHH7” or “VHH8”. 3LYTAC constructs against GFP were produced containing the anti-GFP VHH "GBP" coupled to the C-terminus with a linker. Additionally, anti-GFP LYTAC constructs were produced coupled to "VHH7" competing VHHs "VHH1" and "VHH 1H11" and to "VHH8" competing VHHs "VHH 5" and "VHH 1H52". As a control for non-CI-M6PR mediated internalization and degradation of GFP, monovalent GBP was also included in the set. These constructs, whose composition is summarized in Table 10, contain the C-terminal FLAG 3 His 6 They were produced in Pichia pastoris with the tag and purified by IMAC and desalting, and their quality was assessed by SDS-PAGE (Figure 29). [Table 10]

[0147] First, we evaluated whether VHH7- and VHH8-containing anti-GFP LYTACs were able to induce cellular internalization and degradation of GFP in vitro. To this end, 50 nM of these LYTAC constructs (43 = GBP-VHH7 and 44 = GBP-VHH8) or the corresponding non-CI-M6PR-VHH fusion control (42 = GBP) were preincubated with 50 nM of recombinant GFP (rGFP) in serum-free OptiMEM (Gibco) for 30 min at room temperature. HeLa cells were then incubated for 24 h with these protein solutions or with a solution containing GFP alone, either in the presence or absence of 200 μM chloroquine, a well-described lysosomotropic compound that inhibits endosomal acidification (and therefore lysosomal degradation). After the incubation period, cell lysates were obtained and equal amounts of protein (measured by BCA assay) were subjected to immunoblotting for fluorescent detection of GFP and β-tubulin as a loading control (Figure 30). The results show that GFP was indeed taken up by cells treated with LYTAC constructs, but also to some extent by cells treated with monovalent GBP and cells incubated only with GFP. However, more important is the appearance of an additional band at a lower molecular weight than expected for full-length GFP, and this is only in the condition treated with LYTAC and not chloroquine. This suggests that GFP is degraded upon internalization only in the presence of GFP-specific LYTAC constructs, and this is in a CI-M6PR and lysosome-dependent manner. These results were also verified in MCF7 cells (Figure 31). Through similar experiments, GFP-LYTAC constructs containing the previously described competitor anti-CI-M6PR VHHs, VHH1 and VHH 1H11, VHH7, and VHH5 and VHH 1H52, VHH8, were evaluated in vitro. However, in this assay, HeLa cells were incubated with 200 nM of each construct in complete growth medium, and due to poor cell conditions, rGFP and chloroquine-treated samples could not be obtained (FIG. 32).As previously established, the low molecular weight bands are degradation products of GFP, and here they were effectively detected for anti-GFP LYTACs containing VHH7, VHH8, VHH1, VHH5 and VHH 1H52, indicating that bispecific constructs with these anti-CI-M6PR VHHs can also effectively and selectively induce degradation of the target antigen. For cells treated with the GBP-VHH 1H11 fusion construct, the degradation products of GFP were unfortunately not detectable in this particular assay.

[0148] In a follow-up experiment, we wanted to evaluate the cellular fate of internalized GFP after treatment washout. Therefore, HeLa cells were treated again for 24 hours with a preincubation solution of 50 nM LYTAC (constructs 43 = GBP-VHH7 and 44 = GBP-VHH8) or monovalent GBP (= 42) and 50 nM rGFP with or without chloroquine, after which the cells were washed with PBS and incubated with fresh complete growth medium for an additional hour. Cell lysates were obtained immediately after the treatment period (+0 hours) as well as after 3 hours (+3 hours) and 7 hours (+7 hours) of additional incubation. As before, the lysates were immunoblotted for the detection of GFP and β-tubulin (Figure 33). Western blot analysis showed the occurrence of a steady-state process in which GFP is exocytosed into the non-GFP-containing medium in the investigated period after treatment washout for all conditions tested, implied by the progressive disappearance of the signal corresponding to GFP. In addition, the pool of recombinant GFP internalized after the initial treatment period is suggested to be further degraded in a CI-M6PR-dependent manner in the post-washout time frame in non-chloroquine LYTAC treatment conditions, as evidenced by the continued detection of lower molecular weight bands.

[0149] material and method cell culture HEK293 suspension cells were cultured in FREX medium (Gibco 14571C) composed of EX-CELL and Freestyle 293 medium (Gibco) supplemented with L-glutamine (Lonza, 2 mM). Expi-Chinese Hamster Ovary (CHO) cells were cultured in ExpiCHO™ Expression Medium (Gibco). HeLa cells were cultured in DMEM (0.1 mM non-essential amino acids (NEAA), 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal calf serum (FCS)) and incubated at 37°C with 5% CO2. MCF7 cells were cultured in DMEM:F12 medium supplemented with FCS (10%) and L-glutamine (2 mM).

[0150] Cloning, production and purification of VHH-based anti-EGFR and anti-GFP nanoLYTAC constructs A modular cloning platform was used to generate expression vectors for Pichia pastoris. Codon-optimized coding sequences were cloned between the promoter and terminator of AOX1, and a FLAG3His6 tag was attached to the C-terminus. The vectors were transformed into competent P. pastoris cells (NCYC2543) by electroporation, and protein was produced by methanol induction

[11] . The clarified supernatant was supplemented with MgCl2 (25 mM), reduced L-Gluthation (100 mg / L, Sigma Aldrich, G4251-1G). After filtration (0.22 μm), the supernatant was loaded onto a HisTrap HP (5 mL) column (GE Healthcare, 17524801), after which bound proteins were washed (5 CV of 20 mM imidazole, 0,5 M NaCl, 20 mM NaH2PO4 / Na2HPO4, pH 7,5) and gradually eluted (10 CV, 400 mM imidazole, 20 mM NaCl, 20 mM NaH2PO4 / Na2HPO4, pH 7,5). Analysis of selected peak fractions was performed by SDS-PAGE (4-20%, Genscript). Fractions containing the protein of interest were pooled and run on a HiLoad 16 / 10 desalting column equilibrated with HBS buffer (50 mM HEPES, 150 mM NaCl, pH 7.5).

[0151] Cloning, production and purification of cetuximab-based anti-EGFR nanoLYTAC constructs Human codon-optimized coding sequences of Ctx-VHH7, Ctx-VHH8 and Ctx containing IgG CH signal peptide were synthetically ordered, incubated with Klenow fragment (3' to 5' exo-) (NEB, M0212L), NEBuffer 2 (NEB), dATP (0.1 mM) for 45 min at 37°C, and cloned using the pcDNA™ 3.3-TOPO™ TA Cloning™ Kit (Thermo Fischer Scientific, K830001) according to the provided protocol. Cloned plasmids were heat-shock transformed (42°C, 90 s) into chemically competent E. coli and sequence verified. For recombinant protein production, the corresponding expression vectors were transfected into Expi-Chinese Hamster Ovary (CHO) suspension cells using the ExpiFectamine™ CHO Transfection Kit (Thermo Scientific). Ten days after transfection, media was collected for purification, and the supernatant was loaded onto a HiTrap MabSelect SuRe (5 mL) column (Cytiva), after which bound proteins were purified with McIlvaine buffer pH 7.2 (0.2 M Na 2 HPO 4 The fractions were washed with 0.1 M citric acid (pH 7.0) and eluted with McIlvaine's buffer pH 3.0. Analysis of selected peak fractions was performed by SDS-PAGE (4–20%, Genscript). Fractions containing the protein of interest were pooled and run on a HiLoad 16 / 600 Superdex 200 pg (Cytiva), and again the eluted fractions were analyzed by SDS-PAGE. Positive fractions were pooled and concentrated in HBS buffer (50 mM HEPES, 150 mM NaCl, pH 7.2).

[0152] Flow cytometry HeLa cells were cultured as described previously, seeded at 100,000 cells / well in 12-well plates, and incubated with various concentrations of LYTAC or control constructs for 24 h. After the incubation period, cells were harvested using cell dissociation buffer (Gibco) and transferred to Eppendorf tubes for final transfer to 96-well V-bottom plates. After harvesting, cells were washed twice with PBS and once with PBS+0.5% BSA, then incubated with anti-EGFR monoclonal antibody (199.12, ThermoFisher, #MA5-13319, 1:40) for 1 h at 4 °C. Cells were washed three times and incubated with secondary anti-mouse IgG PE-AF647 (ThermoFisher, #A-20990, 1:250) for 1 h at 4 °C. After three additional washing steps with PBS+0.5% BSA, cells were resuspended in 100 μl of PBS+0.5% BSA and transferred to tubes for measurement on a BD LSR II flow cytometer.

[0153] Proteolysis analysis by Western blot HeLa and MCF7 cells were cultured as described previously and seeded at 300,000 or 450,000 cells / well, respectively, in 6-well plates. For in vitro GFP degradation assays, 50 or 200 nM of anti-GFP LYTACs were preincubated with equimolar concentrations of recombinant GFP for 30 min at room temperature. Cells were then incubated with these protein solutions with or without the addition of 200 μM chloroquine for 24 h. For in vitro EGFR degradation assays, cells were incubated with 50 nM of anti-EGFR LYTACs for 24 h. For both assays, cell lysates were obtained in 100 μl of RIPA buffer by scraping with a pipette tip, agitating for 1 h at 4 °C, and centrifuging at maximum speed to remove cell debris. Protein concentrations were determined by BCA assay, and equal amounts of protein were mixed with 5 × Laemmli buffer containing DTT and incubated at 98 °C for 10 min. Proteins were separated on precast 4–20% gradient SDS-PAGE gels (GenScript, M00657) and transferred to nitrocellulose membranes by wet blotting. Membranes were blocked for 1 h in 5% skim milk in PBST (or 5% BSA in TBST if phosphorylated EGFR was detected) and incubated overnight at 4°C with anti-EGFR antibody (EGFR rabbit mAb (D38B1), Cell Signaling Technology, #4267S), anti-GFP antibody (GFP rabbit mAb (D5.1), Cell Signaling Technology, #2956) or anti-phospho-EGFR antibody (phospho-EGFR (Tyr1068) (D7A5) rabbit mAb, Cell Signaling Technology, #3777). Three washing steps with PBST (or TBST) for 15 min each were performed, followed by incubation with secondary antibodies.

[0154] For chemiluminescence detection, the membrane was incubated with HRP-conjugated secondary antibody (rabbit IgG from donkey, Cytiva, NA934) for 1 h at room temperature. β-actin was detected with a directly labeled primary antibody (anti-β-actin antibody C4, Santa Cruz, sc-47778 HRP) as a loading control. After another washing step, the membrane was developed with TMB substrate solution (Western Lighting Plus-ECL, Perkin Elmer, NEL103001EA) and imaged on an Amersham Imager 680 (Cytiva).

[0155] For fluorescence detection, the membranes were incubated with mouse anti-β-tubulin antibody (Sigma Aldrich, T4026) for 1 h at room temperature, washed three times, and then incubated with anti-rabbit Dylight800-conjugated secondary antibody (Thermo Scientific) and anti-mouse Dylight680-conjugated secondary antibody (Thermo Scientific). Imaging was performed using an Odyssey Imaging System (LI-COR Biosciences). Densitometric analysis of Western blots was performed using ImageJ.

[0156] Recombinant production of human domain 1-3His6 antigen HEK293 suspension cells were cultured in serum-free EX-CELL (Gibco 14571C-1000ML) and Freestyle 293 medium (Gibco) supplemented with L-glutamine (2 mM) (1:1) at 8% CO with shaking at 125 rpm. 2 The plasmid pTCA-10111 was grown at 37°C in 1000 cc of pcDNA3.3-TOPO-hDom 1-3 His 6 (675 μg) and SV40 large T antigen DNA (1%) were used for transfection of HEK293 suspension cells (300 mL) with polyethylenimine (1:2) (PolyScience, linear, 25 kDa). Supernatants were harvested 3 days after transfection (200 × g, 5') and diluted with MgCl 2(2 mM), reduced L-Gluthation (100 mg / L, Sigma Aldrich, G4251-1G) and 1× cOmplete™ protease inhibitor (Roche, 11697498001). After filtration (0.22 μm), the supernatant was loaded onto a HisTrap HP (5 mL) column (GE Healthcare, 17524801). After washing (5 CV of 20 mM Imidazole, 0.5 M NaCl, 20 mM NaH 2 PO 4 / Na 2 HPO 4 , pH 7.5), the bound proteins were eluted (10 CV, 400 mM imidazole, 20 mM NaCl, 20 mM NaH 2 PO 4 / Na 2 HPO 4 , pH 7.5) and analyzed by SDS-PAGE (4-20%, Genscript). 1-3 His 6 The positive fractions were loaded onto a HiLoad 16 / 600 Superdex 200 pg (GE Healthcare, 28989335), the eluted fractions were analyzed by SDS-PAGE followed by staining with Coomassie B-Blue R250, the positive fractions were pooled and concentrated in MES buffer (50 mM MES, 150 mM NaCl, pH 6.5). mDom1-3His6 was expressed and produced similarly to the human variant, but only purified on a HisTrap (5 mL) column (GE Healthcare, 17524801). The eluted fractions were pooled, concentrated on an Amicon® Ultra-15 Centrifugal Filter Unit (Merck Millipore, UFC901008) and resuspended in MES buffer.

[0157] Production and purification of anti-CI-M6PR VHH Plasmids (1000 ng) were linearized using PmeI (1 U, NEB) and used to transform electrocompetent

[11] Pichia pastoris NRRL-Y-11430 by electroporation. Subsequently, buffered yeast glycerol complex medium (pH 6) was used for inoculation of single clonal transformants and growth for 48 h at 28 °C with shaking at 225 rpm. A buffer switch to buffered yeast complex medium (pH 6) was performed and the culture was grown for another 48 h at 28 °C with shaking at 225 rpm. Every 12 h, the growth culture was spiked with methanol (1%). Finally, the supernatant was collected by centrifugation (1250 rpm, 15') and adjusted to pH 7.

[0158] VHH1, VHH5, VHH6, VHH 1H11 and VHH 1H52 were expressed in E. coli by transforming competent WK6 E. coli cells with the pHEN6c vector containing the VHH open reading frame, Lac operon, PelB secretion signal, ampicillin selection marker and origin of replication. The transformed E. coli cells were inoculated into LB medium containing ampicillin (100 μg / mL) and incubated overnight at 37°C with shaking at 200-250 rpm. One ml of this preculture was added to 330 mL of TB containing ampicillin (100 μg / mL), MgCl2 (2 mM) and glucose (0.1%) and incubated at 37°C with shaking until the OD600 was 0.6-0.9. Once the desired OD600 was reached, expression was induced by the addition of IPTG (Immunosource Cat°102A) (1 mM) and the culture was incubated at 28°C for 16-18 h with shaking. To extract protein, the overnight induced culture was centrifuged at 8000 rpm for 8 min and the cell pellet was resuspended from the 1 L culture in 12 ml of TES by pipetting up and down followed by shaking at 4°C for 1 h. Per 12 ml of TES, 18 ml of TES (diluted 1:4 in MQ) was added and further incubated on ice for another hour with shaking. The whole was centrifuged at 8000 rpm for 30 min at 4°C and the supernatant was used for further purification.

[0159] For all VHHs, the clarified supernatant was diluted with MgCl 2 (2 mM) and supplemented with reduced L-Gluthation (100 mg / L, Sigma Aldrich, G4251-1G). After filtration (0.22 μm), the supernatant was loaded onto a HisTrap HP (5 mL) column (GE Healthcare, 17524801) and then bound proteins were washed (5 CV of 20 mM imidazole, 0.5 M NaCl, 20 mM NaH 2 PO 4 / Na 2 HPO 4, pH 7.5) and slowly eluted (10 CV, 400 mM imidazole, 20 mM NaCl, 20 mM NaH 2 PO 4 / Na 2 HPO 4 , pH 7,5). Analysis of selected peak fractions was performed by SDS-PAGE (4–20%, Genscript). Fractions containing the protein of interest were pooled and run on a HiLoad 16 / 10 desalting column equilibrated with HBS buffer (50 mM HEPES, 150 mM NaCl, pH 7).

[0160] Cloning production and purification of humanized variants of VHH7 and VHH8 IgG C H Signal peptide and His 6Human codon-optimized coding sequences of VHH7hWN and VHH8hWN containing the following were synthetically ordered, incubated with Klenow fragment (3' to 5' exo-) (NEB, M0212L), NEBuffer 2 (NEB), dATP (0.1 mM) for 45 minutes at 37°C, and cloned using the pcDNA™ 3.3-TOPO™ TA Cloning™ Kit (Thermo Fischer Scientific, K830001) according to the provided protocol. Codon-optimized sequences of humanized variants VHH7h1-3 and VHH8h1-5 were cloned into pVDS100 vector using the GenBuilder™ Cloning Kit (GenScript®; Catalog No: L00701) according to the manufacturer's instructions. Cloned plasmids were heat-shock transformed (42°C, 90 seconds) into chemically competent E. coli and sequence confirmed. For recombinant protein production of VHH7hWN and VHH8hWN, the corresponding expression vectors were transfected into HEK293 suspension cells by PEI transfection. The medium was collected for purification 4 days after transfection. VHH7h1-3 and VHH8h1-5 were expressed in E. coli by transforming competent cells with pVDS100 vectors containing the VHH open reading frames. The transformed E. coli cells were inoculated into selective LB medium and incubated overnight at 37°C with shaking at 250 rpm. For autoinduction of protein expression, the preculture was diluted 1:50 in selective TB medium supplemented with glucose and lactose. The culture was incubated for 2 h at 37°C with shaking at 250 rpm, after which the temperature was reduced to 30°C and the culture was incubated for another 26 h. To extract proteins, overnight induced cultures were centrifuged at 4000 rpm for 20 min and cell pellets were resuspended in D-PBS (1 / 12.5 of the expression volume) by pipetting up and down followed by shaking for 1 h at 4° C. The whole was centrifuged at 8500 rpm for 20 min at 4° C. and the supernatant was used for further purification. All supernatants were filtered (0.22 μm) before purification.The supernatants of VHH7hWN and VHH8hWN were loaded onto a HisTrap HP (5 mL) column (GE Healthcare, 17524801) and then bound proteins were washed (5 CV of 20 mM imidazole, 0,5 M NaCl, 20 mM NaH. 2 PO 4 / Na 2 HPO 4 , pH 7.5) and slowly eluted (10 CV, 400 mM imidazole, 20 mM NaCl, 20 mM NaH 2 PO 4 / Na 2 HPO 4 , pH 7.5). VHH positive fractions were loaded onto HiLoad 16 / 600 Superdex 75 pg (GE Healthcare) and eluted fractions were analyzed by SDS-PAGE. Positive fractions were pooled and concentrated in HBS buffer (50 mM HEPES, 150 mM NaCl, pH 7). For VHH7h1-3, VHH8h1-5, IMAC purification was performed on a Janus BioTx system (Perkin Elmer) following standard procedures. Fractions containing the protein of interest were pooled and the buffer was exchanged into PBS before protein concentration.

[0161] Labeling of anti-CI-M6PR VHHs with amine-reactive Alexa Fluor 488 All anti-CI-M6PR VHHs (1 mg) were buffered in HEPES (50 mM), NaCl (150 mM) and NaHCO 3 - (100 mM), diluted at pH 8.3, incubated with 1 mg Alexa Fluor 488 (AF488) NHS ester (Jena Biosciences, APC-002-5) and resuspended in DMSO (1 h at room temperature). Free AF488 NHS ester was then removed using size-exclusion chromatography (HiLoad 16 / 600 Superdex 75 pg, GE Healthcare). Elution fractions were pooled and assessed for the degree of labeling (DOL) and functional binding to hDom1-3His6.

[0162] Microscopic analysis of lysosomal targeting of anti-CI-M6PR VHHs For live cell imaging, 2x10 4 MCF7 cells / well were seeded in OptiMEM medium and incubated the next day with LysoTracker Deep Red DND-99 (50 nM, Thermo Fischer, L12492) at 37 °C, 5% CO 2 The cells were incubated for 30 min in 5% CO and washed with OptiMEM, after which 7.5 μM of AF488-labeled anti-CI-M6PR VHH (in OptiMEM) was incubated on the cells. For all wells (i.e., anti-CI-M6PR VHH), Z-stacks were taken at three different positions every 6 min for a total of 3 h. Z-slices (12) were acquired per position with a step size of 1.5 μm and an XY pixel size of 275 nm × 275 nm. The excitation and emission wavelengths of the fluorescent compounds used were LTR (λ Ex : 633nm and λ Em :665-715nm), AF488(λ Ex : 488 nm and λ Em :520±35nm), Hoechst / DAPI(λ Ex : 405 nm and λ Em :420-470nm).

[0163] HeLa cells were cultured as previously described, with 2.5 × 10 cells in Ham's F-12 medium (supplemented with penicillin and streptomycin), respectively. 4Cells / well were seeded in 8-well chambers (iBidi, 80841). AF488-labeled VHH (5 μM) was incubated on the cells for 4 h and then washed three times with PBS. Cells were fixed in pre-warmed PFA: first in 2% PFA in PBS for 5 min at 37 °C, then with 4% PFA for 10 min at room temperature. Three washes with PBS were performed for 5 min before and after cell permeabilization (0.2% Triton X-100) for 10 min at room temperature. Cells were then blocked for 30 min with normal goat serum diluted (1 / 100) in PBT buffer. Primary mouse anti-LAMP1 monoclonal antibody (Abcam, Ab25630, 1 / 500) was diluted in blocking buffer and incubated overnight at 4 °C. After washing, cells were incubated for 5 min each in PBS and for 2 h at room temperature with secondary goat anti-mouse antibody coupled to DyLight594 (1 / 1,000 in PBT). Cells were washed three times with PBS and then counterstained with DAPI (1 / 1,000 in PBS) for 15 min at 16 °C. Finally, washed and stained cells were stored at 4 °C after mounting in polyvinyl alcohol. Imaging was performed with an LSM880 Airyscan confocal microscope (Zeiss, Jena) used in FAST Airyscan SR mode with a Plan-Apochromat 63x / 1.4 Oil DIC M27 objective. For each VHH, optimal Z-stacks to capture the entire cell volume were taken at three different positions per well for three fluorescent compounds: LAMP1 (λ Ex : 633nm and λ Em :>650nm),AF488(λ Ex :488nm λ Em :495-550nm) and Hoechst / DAPI (λ Ex : 405 nm and λ Em :420-480nm).

[0164] Image Processing and Analysis Images acquired with the Airyscan detector were processed using ZEN software (Zeiss, Jena). Processing included pixel reassignment and default Wiener filtering. Processed images were then imported into Volocity (Quorum Technologies, Ontario) for further analysis. Both images acquired with the LSM880 and Spinning Disk microscopes were analyzed with Volocity software. In both cases, thresholds were determined for the intensity values ​​and size of the segmented objects in the channel of (endo)lysosomal staining and in the channel of labeled VHH. In this way, two populations were created, one containing (endo)lysosomes and one containing labeled VHH. By applying the "intersect" command, we were able to determine the fraction of VHH localized within (endo)lysosomes and the fraction of lysosomes containing VHH. The total volume of the analyzed cells was also measured and corrected. The calculation of the fraction was performed based on the segmented volume, and the volume was expressed in "voxels". A voxel is a 3D version of a pixel and is therefore a unit of volume in an image stack. For live cell imaging results, uptake per cell volume was calculated by dividing the sum of voxel counts for each fluorescent VHH time point by the sum of voxel counts per cell for that time point (representing the cell volume). The percentage of VHH colocalization with lysosomes and the percentage of the total endolysomal pool containing a particular VHH were calculated by taking the voxel count ratio of VHH signal colocalizing with the LTR and total intracellular VHH signal. The percentage of lysosomes bearing VHH was determined by the voxel count ratio of VHH signal colocalizing with the LTR and total LTR signal. The final graph shows the absolute voxel counts of intracellular VHH signal and VHH-LTR colocalization signal.

[0165] Size-exclusion chromatography coupled to multi-angle light scattering. hCI-M6PR D1-D3To estimate the molecular weight and stoichiometry of the and anti-CI-M6PR VHH8 protein complex, we incubated both proteins in a 1:1 and 1:3 molar manner in HBS buffer (50 mM HEPES, 150 mM NaCl, pH 7.5, 0.1 μm filtered) containing sodium azide (0.02%). The total concentrations of both samples were 0.81 and 1.08 mg / ml. After SEC (Superdex 200 increment HR 10 / 30), the eluted proteins were detected at 298 K using an online UV detector (Generic UV), a miniDAWN8 (Wyatt) multi-angle laser light scattering (MALLS) detector and an Optilab refractive index (RI) instrument (Wyatt). The RI increment values ​​(dn / dc values) at 298 K and 658 nm were calculated to determine the protein concentration and molecular weight (dn / dc: 0.1850 ml / g). Elution fractions from 14 to 40 min (0.5 mL / min) were collected for analysis by SDS-PAGE. Data analysis and reporting were performed using ASTRA 7.3.2 software.

[0166] Intact mass analysis of anti-CI-M6PR VHH:receptor complexes Intact proteins were separated on an Ultimate 3000 HPLC system (Thermo Fisher Scientific, Bremen, Germany) online connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientific). Briefly, approximately 4 μg of protein was injected onto a Zorbax Poroshell 300SB-C8 column (5 μm, 300A, 1 × 75 mm IDxL; Agilent Technologies) and separated using a 15 min gradient from 5% to 80% solvent B at a flow rate of 100 μl / min (solvent A: 0.1% formic acid and 0.05% trifluoroacetic acid in water; solvent B: 0.1% formic acid and 0.05% trifluoroacetic acid in acetonitrile). The column temperature was maintained at 60 °C. Eluted proteins were directly sprayed in a mass spectrometer equipped with an ESI source using the following parameters: spray voltage 4.2 kV, surface-induced dissociation 30 V, capillary temperature 325 °C, capillary voltage 35 V and sheath gas flow rate 7 (arbitrary units). The mass spectrometer was operated in MS1 ​​mode using an orbitrap analyzer with a resolution of 100,000 (m / z 400) and a mass range of 600-4000 m / z in profile mode. The obtained MS spectra were deconvoluted with BioPharma Finder 3.0 software (Thermo Fisher Scientific) using the Xtract deconvolution algorithm (isotopically resolved spectra), and the deconvoluted spectra were then automatically annotated using the BioPharma Finder protein sequence manager and protein identification tools.

[0167] Co-crystallization of the VHH-hCI-M6PRD1-D3 complex hCI-M6PR D1-D3 Complexes of hCI-M6PR with either VHH7, -8 or 1H11 were formed and polished by SEC in HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.5). D1-D3and a 1.25 molar excess of either anti-CI-M6PR VHH were injected onto a Superdex 200. Fractions containing the VHH complexes were collected, supplemented with mannose 6-phosphate (1 mM, M3655-100MG, Sigma) and concentrated to 3.5 mg / mL and 7 mg / mL, respectively, using Amicon Ultra-15 protein concentrators (UFC903024, Millipore). The VHH 1H11 complex was concentrated to 3.7 mg / ml without the addition of mannose 6-phosphate. For crystallization of common complexes, nanoliter-scale seated drop vapor diffusion crystallization experiments were set up at 287 K using a commercial sparse matrix crystal screen (Molecular Dimensions, Hampton Research) and a Mosquito crystallization robot (TTP Labtech). Promising hits were further optimized using gradient optimization in 96-wells.

[0168] VHH7:hCI-M6PR D1-D3 Two crystalline forms of were identified: rhombohedral (PGA screen condition C9) which diffracted to 2.2A and was crystallized from 0.3M KBr, 0.1M NaCacodylate pH 6.5, 8% w / v γ-PGA (Na+ form, LM); ; Hu et al. Acta Crystallogr D Biol Crystallogr. 2008;64:957-63), and diffracted to 3.0 A and 0.2 M NH 4 NO 3 , tetragonal crystallized from 0.1M Bis-Tris propane pH 8.5, 18% v / v PEG Smear High (BCS screening condition F6). hCI-M6PR D1-D3 A single crystal form of VHH 8 was identified grown from sodium acetate trihydrate (0.08 M), sodium chloride (0.15 M), Tris (0.1 M), PEG Smear (0.015% v / v), pH 8) (BCS screening condition F3) that diffracted to 2.75 A. VHH 1H11:hCI-M6PR D1-D3Two crystal forms were identified: a poorly diffracting rhombohedral form that crystallized from several conditions of 0.2 M (NH4)2SO4, 0.1 M sodium acetate, 4.6 25 % v / v PEG Smear Broad (BCS screening condition C10), and a tetragonal form that diffracted to 2.7 A that crystallized from several conditions of 0.1 M ammonium sulfate, 0.1 M Tris pH 7.5, 20% w / v PEG 1500 (Proplex screening condition A7).

[0169] Crystals containing the VHH7 and VHH8 complex grown from BCS conditions were cryoprotected in mother liquor supplemented with ZW221 freezing solution (17.5% v / v) consisting of DMSO (40%), ethylene glycol (20%) and glycerol (40%) (Sanchez, et al. Biochemistry 54, no. 21 (2015): 3360-3369). Crystals grown from PGA conditions were cryoprotected in mother liquor supplemented with glycerol (17.5% v / v), and crystals containing the VHH 1H11 complex were cryoprotected in mother liquor supplemented with 17.5% (v / v) ethylene glycol and then vitrified in liquid nitrogen. VHH8-hCI-M6PR D1-D3 Final X-ray diffraction measurements of the crystals were performed at the EMBL P14 beamline (Petra 3 synchrotron, Germany), VHH7-hCI-M6PR D1-D3 Crystallography was performed at the Proxima PX1 beamline (Soleil synchrotron, France) and VHH 1H11-hCI-M6PR D1-D3 Crystallography was performed at ESRF ID30A3. All data sets were derived from individual crystals. Integration and scaling of diffraction data was performed in XDS. Data set statistics are reported in Table 11. Based on the bovine CIMPR structure (PDB:1sz0) and VHH, CIMPR D1D2 , CI-M6PR D3Initial phases were recovered by maximum likelihood based molecular replacement performed in Phaser using Sigma. The structure was iteratively built and refined in Coot, Isolde (Croll, Tristan Ian. 2018. "ISOLDE: A Physically Realistic Environment for Model Building into Low-Resolution Electron-Density Maps." Acta Crystallographica Section D: Structural Biology 74(6):519-30) implemented in ChimeraX and Phenix refine. [Table 11-1] [Table 11-2] [Table 11-3]

[0170] Sequence Listing SEQ ID NOs: 1-11: Amino acid sequences of CI-M6PR-specific VHH1-VHH11. >SEQ ID NO: 12: monovalent EGFR-specific VHH 9G8 (construct 14) >SEQ ID NO: 13: Bispecific 9G8-VHH8 fusion protein (construct 15) >SEQ ID NO: 14: Trivalent bispecific 9G8-9G8-VHH8 fusion protein (construct 16) SEQ ID NO: 15: Bispecific 9G8-GBP fusion protein (construct 17) >SEQ ID NO: 16: Trivalent bispecific 9G8-9G8-GBP fusion protein (construct 18) >SEQ ID NO: 17: monovalent VHH 9G8 S54A (construct 25 / 33) >SEQ ID NO: 18: Bispecific 9G8 S54A-VHH8 fusion protein (construct 26 / 35) >SEQ ID NO: 19: Trivalent bispecific 9G8 S54A-9G8 S54A-VHH8 fusion protein (construct 27 / 37) >SEQ ID NO: 20: Bispecific 9G8 S54A-GBP fusion protein (construct 28 / 38) >SEQ ID NO: 21: Trivalent bispecific 9G8 S54A-9G8 S54A-GBP fusion protein (construct 29 / 39) >SEQ ID NO: 22: FLAG3His6 tag fused C-terminally to the bispecific VHH used in the construct >SEQ ID NO: 23: Amino acid sequence of human cation-independent mannose 6-phosphate receptor precursor [NP_000867.2; 2491aa] >SEQ ID NO: 24: Amino acid sequence of VHH1H11 >SEQ ID NO: 25: Amino acid sequence of VHH1H52 >SEQ ID NO: 26: Amino acid sequence of humanized variant VHH7h1 >SEQ ID NO: 27: Amino acid sequence of humanized variant VHH7h2 >SEQ ID NO: 28: Amino acid sequence of humanized variant VHH7h3 SEQ ID NO: 29: Amino acid sequence of humanized variant VHH7hWN >SEQ ID NO: 30: Amino acid sequence of humanized variant VHH8h1 >SEQ ID NO: 31: Amino acid sequence of humanized variant VHH8h2 >SEQ ID NO: 32: Amino acid sequence of humanized variant VHH8h3 >SEQ ID NO: 33: Amino acid sequence of humanized variant VHH8h4 >SEQ ID NO: 34: Amino acid sequence of humanized variant VHH8h5 SEQ ID NO: 35: Amino acid sequence of humanized variant VHH8hWN [Table 12] [Table 13] SEQ ID NO: 78: FR1 consensus (including humanization) SEQ ID NO: 79: FR2 consensus sequence (including humanization) SEQ ID NO: 80: FR3 consensus sequence (including humanization) SEQ ID NO: 81: FR4 consensus sequence (including humanization) SEQ ID NO: 82: Bispecific 9G8 S54A-VHH7 fusion protein (construct 34) SEQ ID NO: 83: Trivalent bispecific 9G8 S54A-9G8 S54A-VHH7 fusion protein (construct 36) SEQ ID NO: 84: Bispecific 9G8 S54A-VHH7 fusion protein (construct 40) SEQ ID NO: 85: Bispecific 9G8 S54A-VHH8 fusion protein (construct 41) SEQ ID NO: 86: Light chain of cetuximab SEQ ID NO: 87: Heavy chain of cetuximab SEQ ID NO: 88: Heavy chain of cetuximab-VHH7 SEQ ID NO: 89: Heavy chain of cetuximab-VHH8 SEQ ID NO: 90: Monovalent GBP (construct 42) SEQ ID NO: 91: Bispecific GBP-VHH7 fusion protein (construct 43) SEQ ID NO: 92: Bispecific GBP-VHH8 fusion protein (construct 44) SEQ ID NO: 93: Bispecific GBP-VHH1 fusion protein (construct 45) SEQ ID NO: 94: Bispecific GBP-VHH5 fusion protein (construct 46) SEQ ID NO: 95: Bispecific GBP-1H11 fusion protein (construct 47) SEQ ID NO: 96: Bispecific GBP-1H52 fusion protein (construct 48) SEQ ID NO: 97: Amino acid sequence of mouse cation-independent mannose 6-phosphate receptor precursor [NP_034645.2; 2483aa] SEQ ID NO: 98: Amino acid sequence of bovine cation-independent mannose 6-phosphate receptor precursor [NP_776777.1; 2499aa]

[0171] References 1. Hamers-Casterman, C. et al. Nature 363, 446-448 (1993). 2.De Duve C,Pressman BC,Gianetto R,Wattiaux R,Appelmans F:Tissue fractionation studies.6.Intracellular distribution patterns of enzymes in rat-liver tissue.Biochem J 1955,60:604-617. 3.Muyldermans,S.Annu.Rev.Biochem.82,775-797(2013). 4.Schrankel,C.S.,Gokirmak,T.,Lee,C.-W.,Chang,G.&Hamdoun,A.Methods Cell Biol.151,353-376(2019). 5.Laeremans,T.,Haard,H.D.&Hoogenboom,H.R.J.M.(2007)-WO2007042289A2 6.Roovers,R.C.et al.Cancer Immunol.Immunother.56,303-317(2007). 7.York,S.J.et al.J.Biol.Chem.274,1164-1171(1999). 8.Wang,Q.,Villeneuve,G.&Wang,Z.EMBO Rep.6,942-948(2005). 9.Jovcevska,I.&Muyldermans,S.BioDrugs 34,11-26(2020). 10.Banik SM,Pedram K,Wisnovsky S,Ahn G,Riley NM,Bertozzi CR:Lysosome-targeting chimaeras for degradation of extracellular proteins.Nature 2020,584:291-297. 11.Lin-Cereghino J,Wong WW,Xiong S,Giang W,Luong LT,Vu J,Johnson SD,Lin-Cereghino GP:Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris.Biotechniques 2005,38:44-48. 12.Sand,K.M.K.et al.Front.Immunol.5,(2015). 13.Yang J,Zhang Y:I-TASSER server:New development for protein structure and function predictions.Nucleic Acids Research 2015,43:W174-W181. 14.Schrodinger,LLC:The {PyMOL} Molecular Graphics System,Version~1.8.2015. 15.Ahn et al.Nature Chemical Biology,Vol.17;2021;p.937-946 16.Camacho,C.J.&Zhang,C.FastContact:rapid estimate of contact and binding free energies. Bioinforma.Oxf.Engl.21,2534-2536(2005). 17.Olson LJ,Dahms NM,Kim J-JP:The N-terminal Carbohydrate Recognition Site of the Cation-independent Mannose 6-Phosphate Receptor.J Biol Chem 2004,279:34000-34009. 18.E.Krissinel,K.Henrick,J.Mol.Biol.372,774-797(2007). 19.Pardon,et al.A general protocol for the generation of Nanobodies for structural biology.Nature Protocols 2014.9:674-693. 20.Borden,LA,Einstein,R.,Gabel,CA&Maxfield,FRJBiol.Chem.1990.265,8497-8504. 21.Epenetos,AA,Snook,D.,Durbin,H.,Johnson,PM&Taylor-Papadimitriou,J.Cancer Res.1986.46,3183-3191. 22.Schroter,J.et al.mAbs 7,138-151(2014). 23.Pabla,B.,Bissonnette,M.&Konda,VJWorld J.Clin.Oncol.6,133-141(2015). 24.Bockemeyer,J.et al.Eur.J.Cancer Oxf.Engl.1990 48,1466-1475(2012). 25.Tan,J.et al.World J.Gastroenterol.WJG 18,5171-5180(2012). 26.Di Nicolantonio,F.et al.J.Clin.Oncol.Off.J.Am.Soc.Clin.Oncol.26,5705-12(2008). 27.Cunningham,D.et al.N.Engl.J.Med.351,337-345(2004). 28.Arena,S.et al.Clin.Cancer Res.21,2157-2166(2015). 29.Katreddy,RRet al.Oncogenesis 7,5(2018). 30.Weihua,Z.et al.Cancer Cell 13,385-393(2008).

Claims

1. A protein binding agent comprising an immunoglobulin single variable domain (ISVD) that specifically binds to the human cation-independent mannose-6-phosphate receptor (CI-M6PR) on the extracellular N-terminal CI-M6PR domains 1, 2 and / or 3, wherein the ISVD is fused to a binding agent that specifically binds to an extracellularly accessible protein target.

2. 2. The protein binding agent of claim 1, wherein the CI-M6PR-specific ISVD specifically binds to an epitope comprising amino acid residues Lys191, Gly194, Ala195, Tyr196, Leu197, Phe208, Arg219, Gln224, Leu225, Ile297, Lys357, Gly408, Asp409, Asn431, Glu433, and Phe457 set forth in SEQ ID NO:23, or an epitope comprising amino acid residues Lys59, Asn60, Met85, Asp87, Lys89, Ala146, Thr147, and Glu148, and Asp118 or Gln119.

3. 3. The protein binding agent of claim 2, wherein the CI-M6PR-specific ISVD specifically binds to CI-M6PR via a paratope comprising residues 32, 52-57, 100-103, 108 set forth in SEQ ID NO:8, or via a paratope comprising residues 31, 33, 35, 53, 54, 56, 57, 96, 104 set forth in SEQ ID NO:7, or via a paratope comprising residues 31-35, 50, 52-57, 96-98 set forth in SEQ ID NO:

24.

4. 4. The protein binding agent of claim 3, wherein the CI-M6PR-specific ISVD comprises four framework regions (FR) and three complementarity determining regions (CDRs) according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1), and the CDR1, CDR2 and CDR3 regions are selected from the CDR1, CDR2 and CDR3 regions of a sequence selected from the group of sequences of SEQ ID NOs: 1, 5, 7, 8, 24 or 25, wherein the CDR regions are annotated according to Kabat, MacCallum, IMGT, AbM or Chothia.

5. 5. The protein binding agent of claim 4, wherein the CI-M6PR-specific ISVD comprises a CDR1 sequence selected from SEQ ID NOs: 36-41, a CDR2 sequence selected from SEQ ID NOs: 42-47, and a CDR3 sequence selected from SEQ ID NOs: 48-53.

6. 6. The protein binding agent of claim 5, wherein the CI-M6PR-specific ISVD agent comprises an FR1 sequence corresponding to SEQ ID NO: 78, an FR2 sequence corresponding to SEQ ID NO: 79, an FR3 sequence corresponding to SEQ ID NO: 80, and an FR4 sequence corresponding to SEQ ID NO:

81.

7. 2. The protein binding agent of claim 1, wherein the protein binding agent comprises a CI-M6PR specific ISVD comprising a sequence selected from the group of SEQ ID NO: 1, 5, 7, 8, 24 or 25, or sequences having at least 85% amino acid identity thereto and having identical CDRs, or a humanized variant thereof such as any one of SEQ ID NOs: 26-35.

8. 8. The protein binding agent of any one of claims 1 to 7, wherein the CI-M6PR-specific ISVD and the binding agent that specifically binds to an extracellularly accessible protein target are fused by a linker, preferably a short peptide linker such as a glycine-serine linker, or an Fc tail or another moiety.

9. 8. The protein binding agent of any one of claims 1 to 7, wherein the binding agent that specifically binds to an extracellularly accessible protein comprises an antigen-binding protein domain, preferably an ISVD, a VHH-Fc fusion, a VHH-Fc-VHH, or a knob-into-hole VHH-Fc fusion, an antibody such as an IgG.

10. 8. The protein binding agent of any one of claims 1 to 7, wherein the protein binding agent is a multispecific or multivalent binding agent comprising the CI-M6PR-specific ISVD fused to the extracellularly accessible protein-specific binding agent and a further functional moiety such as a therapeutic moiety or another antigen-binding domain or half-life extension.

11. The protein-binding agent of any one of claims 1 to 7, comprising a detectable label or tag.

12. 8. The protein binding agent of any one of claims 1 to 7, wherein the extracellularly accessible protein target is epidermal growth factor receptor (EGFR) and / or the EGFR-specific binding agent comprises SEQ ID NO: 12 or SEQ ID NO: 17, or a homologue thereof having at least 90% identity and identical CDRs, or comprises SEQ ID NOs: 86 and 87.

13. 13. The protein binding agent of claim 12, wherein the protein binding agent comprises a sequence selected from the group of sequences of SEQ ID NOs: 13, 14, 18, 19, 82-85, or a functional homologue thereof having at least 90% identity and identical CDRs, or an antibody formed by a heavy chain-ISVD fusion of SEQ ID NO: 88 or 89 and a light chain of SEQ ID NO:

86.

14. A nucleic acid molecule encoding the protein-binding agent of any one of claims 1 to 7.

15. The protein-binding agent according to any one of claims 1 to 7 for use as a pharmaceutical.

16. 8. The protein-binding agent of any one of claims 1 to 7 for use in the treatment of a disease mediated by an extracellularly accessible protein target specifically bound by the binding agent.

17. The protein-binding agent of any one of claims 1 to 7 for use in the treatment of cancer.

18. The protein-binding agent of any one of claims 1 to 7 for use as a diagnostic or for in vivo imaging.

19. 8. Use of a protein-binding agent according to any one of claims 1 to 7 in a method for removing cell surface molecules and / or for degrading said molecules in lysosomes.

20. 12. Use of the protein-binding agent of claim 11 for in vitro lysosome tracking.

21. Use of the protein binding agent of any one of claims 1 to 7 in drug discovery, structural analysis, or screening assays.

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