Anti-EGFR nanobodies and nanobody constructs, and related products and methods

Abstract

The present invention relates to nanobodies and nanobody constructs capable of recognizing and binding EGFR, and related methods that use thereof, for example methods of diagnosis, treatment and theragnosis that comprise the use of said nanobodies and nanobody constructs.

Description

[0001] ANTI-EGFR NANOBODIES AND NANOBODY CONSTRUCTS, AND RELATED

[0002] PRODUCTS AND METHODS

[0003] TECHNICAL FIELD OF THE INVENTION

[0004] The present invention relates to nanobodies (Nbs) against the Epidermal Growth Factor Receptor (EGFR), nanobody constructs comprising nanobodies of the present invention, compositions comprising said nanobodies and / or nanobody constructs, and methods comprising the use of said nanobodies, nanobody constructs and / or compositions.

[0005] BACKGROUND

[0006] The epidermal growth factor receptor (EGFR) is a member of a family of transmembrane tyrosine kinase receptors that plays a pivotal role in regulating diverse cellular processes such as cell proliferation, survival, migration, and differentiation (Tebbutt, N. et al., 2013). Aberrant activation of EGFR signaling has been implicated in various pathological conditions, particularly cancer (Kumagai S. et al., 2021), making it an attractive target for therapeutic intervention.

[0007] Cetuximab and panitumumab, two well-known EGFR-directed monoclonal antibodies (mAbs), have demonstrated clinical benefits by inhibiting ligand binding, receptor dimerization, and downstream signaling, ultimately leading to cell-cycle arrest and apoptosis (London M. and Gallo E., 2020). However, despite their clinical potential, mAbs are still suboptimal as therapeutic agents because of the slow tumor penetration of a 150-kDa protein and their immunogenicity (Steeland S. et al., 2016).

[0008] Mutations at specific positions within the EGFR ectodomain, such as V441 , S442, R451 , I462, S464, G465, K467, K489, 1491 , and S492, have been identified as drivers of resistance to EGFR antibodies (Tintelnot J. et al., 2019). These mutations, depending on their location relative to the antibody epitopes, can result in decreased antibody binding and subsequent clinical resistance.

[0009] Nanobodies (Nbs), derived from heavy-chain-only antibodies in camelids and sharks, offer a promising avenue for cancer therapy and fighting resistance. With their dimensions, they penetrate deep into tumors and can even cross the blood-brain barrier (Tsitokana M.E. et al., 2023). These nanobodies exhibit high affinity and specificity for target antigens while minimizing off-target accumulation (Muydermans S. et al., 1994). In particular, these small binding moieties, such as the nanobody 7D12, can target specific epitopes overlapping with the EGF-binding site on EGFR (Tintelnot J. et al., 2019; Gainkam L.O. et al., 2008; Schmitz K.R. et al., 2013) When combined with other single-domain antibodies (sdAb) targeting different epitopes, such as 9G8 which binds to the interphase between domain II and III (Schmitz K.R. et al., 2013), the resulting bivalent Nb is more effective in EGFR downregulation (Roovers R.C. et al., 2011 ; Heukers R. et al., 2013). Furthermore, multi-epitope approaches to EGFR targeting, such as the use of Sym004 (combination of futuximab and modotuximab) has been proved to overcome cetuximab resistance (Sanchez-Martin F.J. et al., 2016).

[0010] The emergence of resistance through epitope escape variants and genetic alterations warrants the development of nanobodies targeting different epitopes to provide a wider choice for EGFR-targeted therapy to improve therapeutic outcomes.

[0011] There is therefore a need to provide nanobodies that target EGFR and having an improved affinity for EGFR and that target different epitopes of EGFR as those recognized by nanobodies described in the prior art, for example, 7D12.

[0012] SUMMARY OF THE INVENTION

[0013] In light of this need, the present inventors developed different panning strategies to isolate nanobodies targeted to EGFR from a sdAb library. As a result of these strategies, two potent anti-EGFR Nbs were identified and characterized by ELISA, flow cytometry, microscopy and surface plasmon resonance (SPR). Moreover, these Nbs as well as bivalent Nbs engineered therefrom were tested fortheir effect on proliferation on EGFR- overexpressing cancer cell lines. The sdAb showed extremely high affinity and potent anti-tumor activity in vitro in their bivalent form and were found to be useful for cancer treatment.

[0014] Thus, it is a first aspect of the present invention to provide a nanobody directed against EGFR comprising a FR3 with an amino acid sequence as set forth in SEQ ID NO: 7.

[0015] In an embodiment, the nanobody of the first aspect of the invention, further comprises: a) a CDR1 with an amino acid sequence as set forth in SEQ ID NO: 13 or 14, and b) a CDR2 with an amino acid sequence as set forth in SEQ ID NO: 15 or 16, and c) a CDR3 with an amino acid sequence as set forth in SEQ ID NO: 17 or 18.

[0016] In another embodiment, the nanobody of the first aspect of the invention, further comprises: a) a FR1 with an amino acid sequence as set forth in SEQ ID NO: 1 , and b) a FR2 with an amino acid sequence as set forth in SEQ ID NO: 4, and c) a FR4 with an amino acid sequence as set forth in SEQ ID NO: 10.

[0017] In another embodiment, the nanobody of the first aspect of the invention, comprises a FR3 with an amino acid sequence selected from SEQ ID NO: 8 and 9.

[0018] In an embodiment, the nanobody of the first aspect of the invention comprises a sequence as set forth in SEQ ID NO: 19.

[0019] In another embodiment, the nanobody of the first aspect of the invention comprises a sequence as set forth in SEQ ID NO: 20.

[0020] It is a second aspect of the present invention to provide a nanobody construct comprising a) a nanobody of the first aspect of the invention, and b) at least one other compound comprising i) a pharmaceutical compound, or ii) a reporter compound, or iii) a polypeptide, or iv) a nucleotide-based compound, and c) a linker. wherein the nanobody a) and the at least one other compound b) are linked via the linker c).

[0021] In an embodiment, the nanobody construct of the second aspect of the invention, has a polypeptide comprising at least one nanobody of the first aspect of the invention.

[0022] In an embodiment, the nanobody construct of the second aspect of the invention, comprises two nanobodies of the first aspect of the invention, wherein the nanobodies are either identical or different. In another embodiment, the nanobody construct of the second aspect of the invention , comprises two nanobodies as set forth in SEQ ID NO: 19 or 20, in any combination.

[0023] It is a third aspect of the invention to provide a pharmaceutical composition comprising a nanobody of the first aspect of the invention, or a nanobody of the second aspect of the invention, and a pharmaceutically acceptable excipient selected from diluents, fillers, binders, disintegrants, lubricants, glidants, surfactants, preservatives, antioxidants, pH adjusters, coating agents, flavoring agents, colorants, solvents, emulsifiers, emollients, gelling agents, stabilizers, and or propellants.

[0024] It is a fourth aspect of the invention to provide a method of treatment of a disease that comprises administering a therapeutically effective amount of at least one nanobody of the first aspect of the invention, or a nanobody construct of the second aspect of the invention, or a pharmaceutical composition of the third aspect of the invention to a subject in need thereof.

[0025] In an embodiment, the method of the fourth aspect of the invention, is a method of treatment of a disease that is caused by or related to the biological activity of EGFR.

[0026] In an embodiment, the method of the fourth aspect of the invention further comprises reducing, decelerating, or stopping the proliferation of cells.

[0027] It is a fifth aspect of the invention to provide a method of detecting EGFR in a sample comprising contacting a nanobody of the first aspect of the invention, or a nanobody construct of the second aspect of the invention, with EGFR, wherein said method is performed in vitro.

[0028] In an embodiment, the method of the fifth aspect of the invention comprises detecting the contacting using a technique selected from flow cytometry, immunofluorescence imaging, plate-adsorbed VLP panning, and magnetic bead based panning.

[0029] It is a sixth aspect of the invention to provide a theragnostic method comprising applying the method of the fourth aspect of the present invention , and a method of the fifth aspect of the present invention.

[0030] It is a seventh aspect of the invention to provide a product for use in the treatment, amelioration or prevention of a disease, comprising at least one nanobody of the first aspect of the invention, or at least one nanobody construct of the second aspect of the invention, or a pharmaceutical composition of the third aspect of the invention.

[0031] In an embodiment, the product for use according to the seventh aspect of the invention, used in combination with a method of detection of the fifth aspect of the invention.

[0032] It is as eighth aspect of the invention to provide a use of the nanobody of the first aspect of the invention for directing, aiming or guiding a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell towards an EGFR.

[0033] It is as ninth aspect of the invention to provide a use of the nanobody construct of the second aspect of the invention, for directing, aiming or guiding a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell towards an EGFR.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 shows a schematic representation of the generation and display of the anti- EGFR VHH immune library on E. coli and the methodology adopted for selection of anti- EGFR Nbs.

[0036] Figure 2 shows the results of immunization of Lama glama with EGFR-overexpressing cells and VLPS. The reactivity of pre-immune (day 0: light gray) and immune sera (day 35: black) of llamas assessed by ELISA against purified antigens (Fig. 2A and 2C) or VLPs (Fig. 2B and 2D). A specific response towards EGFR was induced in a Llama (Fig. 2A and 2B), but not in another one (Fig. 2C and 2D).

[0037] Figure 3 shows the results of Colony PCR of clones after each biopanning round. Ten to 14 clones were picked at random to determine the percentage of full size VHHV sequences in each round of biopanning of phage-displayed VHHs. (A) Colony-PCR of the library before panning, where the full-length inserts represent about 90% of the total clones. (B) After the first round of selection and amplification full-length inserts represent between 50-70% of the total. After the second (C) and third (D) round, the percentage of full-length inserts was found to be between 90-100%. Figure 4 shows the expression and purification of Nb. (A) SDS-PAGE of His-tagged purified Nb. (B) SDS-PAGE showing the correct expression of the corresponding bivalent constructs (~30 kDa). (C) Size-exclusion chromatography profile of the IMAC-purified 7D12, 5B1 and 5B12; nanobodies were concentrated on Vivaspin concentrators with 5 kDa molecular weight cut-off (Sartorius Stedim Biotech GmbH, Goettingen, Germany) and loaded onto a superdex S75 (SuperdexTM 75 10 / 300 GL) column in PBS buffer.

[0038] Figure 5 shows 5B1 and 5B12 specific binding to EGFR-expressing cells.

[0039] (A) Expression and purification of Nbs, and an SDS-PAGE of His-tagged purified Nbs.

[0040] (B) Analysis of binding by flow cytometry analysis. One clone from each family was selected for periplasmic Nb expression, purification, and further characterization. Purified Nb were assayed at 500nM by FACS against EGFRhi A431 and EGFRIo MCF7. (C) Specific staining of EGFR-overexpressing HEK293A cells with the selected Nb. Immunofluorescence images of HEK293AhEGFR and HEK293A cells stained with the selected Nb against EGFR. Binding of Nb was detected using anti-His Tag mAb and antimouse IgG (green). Cells were stained with DAPI (blue) and phalloidin (red) to reveal nucleus and F-actin cytoskeleton, respectively.

[0041] Figure 6 shows saturation binding apparent KDs and Bmax values for antibody variants on A431 cells, as determined by flow cytometry. Binding curves of median fluorescence intensity (MFI) versus antibody concentration are shown. Binding at each concentration was normalized to the MFI at 500 nM for each Nb. MFIs were fitted as nonlinear regression curves in GraphPad Prism 8.

[0042] Figure 7 shows the characterization of VHH binding sites. EGFR-specific VHH-phages were tested in competition ELISA using VLPEGFR as antigen. Competition is expressed as percentage of displacement by increasing concentrations of cetuximab

[0043] Figure 8 shows the determination of EGFR Density and EGFR specificity. Cells were labeled with a monovalent anti-EGFR antibody by using a commercially available kit. Labeled cells are shown in grey and non-labeled control cells are shown in black. (A) HEK293A cells. (B) HEK293HEGFR cells. (C) A431 cells. (D) Antibody Binding Capacity (ABC) value, which is equivalent to the number of cell surface receptors per cell.

[0044] Figure 9 shows the Surface plasmon resonance analysis of the interaction between VHH and EGFR at a flow rate of 30 pl / min. SPR sensorgrams showing the interaction of immobilized EGFR and soluble VHHs. (A, B) 5B1 (100-3.125 nM); (C,D) 5B12 (100- 3.125 nM); (E, F) 7D12 (100-3.125 nM).

[0045] Figure 10 shows the Surface plasmon resonance analysis of the interaction between VHH and EGFR at a flow rate of 50 pl / min. SPR sensorgrams showing the interaction of immobilized EGFR and soluble VHHs. (A, B) 5B1 (100-3.125 nM); (C,D) 5B12 (100- 3.125 nM); (E, F) 7D12 (100-3.125 nM).

[0046] Figure 11 shows the Surface plasmon resonance analysis of the interaction between VHH and EGFR. SPR sensorgrams showing the interaction of immobilized EGFR and soluble VHHs at a flow rate of 30 pl / min. (A) 5B1-5B1 (100-0.8 nM); (B) 5B1-5B12 (200- 0.8 nM); (C) 5B12-5B12 (100-0.8 nM); (D) 5B12-5B1 (800-0.8 nM).

[0047] Figure 12 shows how bivalent nanobodies inhibit A431 proliferation. (A) EGF-dependent A431 cells were treated with different equimolar concentrations of the bivalent nanobodies or cetuximab and the inhibition of cell proliferation was measured in triplicates after 6 days by MTS assay. (B) Phosphorylated EGFR status in A431 cells preincubated with serial dilutions of the mono / bivalent nanobodies or cetuximab was determined by Western Blotting after treatment with 170 pM of EGF for 10 min.

[0048] Figure 13 shows the result of proliferation assays performed on glioma stem cell- enriched cell lines of the bivalent Nbs. (A) Effect on 605 cells expressing mutated EGFRVIII. (B) Effect on 608 expressing non-mutated EGFR.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The invention will be described in further detail below, with reference to the accompanying figures and exemplary embodiments below.

[0051] Any technical terminology used herein shall be understood by the common definition utilized in the art and / or by those skilled in the art, unless otherwise explicitly stated or inferred by context.

[0052] As defined herein, Complementarity-Determining Region (CDR) is defined as a hypervariable immunoglobin domain that confines specific antibody binding. As described in the prior art, nanobodies comprise three CDRs. Throughout the present application, CDRs will be numbered according to their position relative to the N-terminal of the amino acid sequence, unless otherwise stated so.

[0053] As defined herein, a Framework Region (FR) is defined as any segment of a nanobody that is not a CDR. FRs are relatively constant, when compared to CDRs, though they can have variations in their sequence. Throughout the present application, FRs will be numbered according to their position relative to the N-terminal of the amino acid sequence, unless otherwise stated so.

[0054] As an example, and without any intent of limiting the scope of the present invention, a nanobody that comprises three CDRs will also contain four FRs, placed in the following order throughout the aminoacidic sequence of the nanobody:

[0055] N-terminal, FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4, C-terminal.

[0056] As clearly shown in this example, FR1 is the framework region comprised from the N- terminal to the CDR1 , FR2 is the framework region comprised between the CDR1 and the CDR2, FR3 is the framework region comprised between the CDR2 and the CDR3, and the FR4 is the framework region comprised between the CDR3 and the C-terminal.

[0057] Throughout the following specification, amino acid sequences may be described by utilizing the amino acid single-letter or the triple-letter code. Whenever a specific location within the sequence presents an amino acid with more than one possible variant, the amino acid in question will be indicated with a parenthesis, with all the possible variants of said amino acid, being each separated with a comma (,). A dash (-) represents that the amino acid is deleted from the sequence. A sequence may contain one or more locations presenting an amino acid with more than one possible variant.

[0058] As an example, and without intent of limiting the scope of the present invention, when describing an amino acid sequence as the following expression

[0059] A(A, C)A(D, E, -)A, it is to be understood that the amino acid in the second position of the sequence can be Alanine (A) or Cysteine (C), while the amino acid in the fourth position of the sequence can be Aspartate (D), Glutamate (E), or a deletion (-). Thus, the amino acid sequence represented by the expression above can be any one of the following: AAADA; AAAEA; AAAA; ACADA; ACAEA; ACAA. As used herein, the term “general” or “generic” when referring to an amino acid sequence, refers to a variant of said amino acid sequence defined in such a way that it comprises all the amino acid sequences contained in a particular list or group, using the aforementioned sequence description rules.

[0060] As used herein, the term “nanobody construct” refers to a molecule in which the structure or function are based on the structure or function of a nanobody. A nanobody construct is hence capable of binding to its specific target or antigen. Accordingly, a nanobody construct of the present invention comprises at least the minimum structural requirements of a nanobody that allows it to target and bind its respective target molecule. This minimum requirement may be defined by the presence of at least three CDRs. Furthermore, the nanobody construct comprises at least one other compound covalently bound to said nanobody that allows it to target and bind its respective target molecule. Examples of said at least one other compound may be, among others, a pharmaceutical compound, a reporter compound, a polypeptide, or a nucleotide-based compound.

[0061] As used herein a “pharmaceutical compound” refers to a molecule that can be administered to a subject in need for the treatment, amelioration or prevention of a disease. Preferably, the pharmaceutical compound is administered in an amount that results in a total or partial treatment, amelioration or prevention of said disease. Preferably, said disease is selected from a proliferative disease, a tumorous disease, a viral disease or an immunological disorder.

[0062] As used herein, the term “subject” refers to a vertebrate animal. These comprise humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic animals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and game birds such as chickens, turkeys, and other gallinaceous birds, ducks, geese, and the like). In an embodiment, the subject may be a mammal, preferably a human. Furthermore, said subject comprises a subject who already suffers a disorder, as well as those in which the disorder is to be prevented.

[0063] As used herein, the term “reporter compound” refers to a molecule that can be used to detect, reveal, measure, or indicate, either directly or indirectly, the presence or activity of a target molecule, biological process, or biochemical reaction. The reporter compound is designed to generate a measurable signal, such as fluorescence, luminescence, color change, radioactive signal, among others, upon interacting with the target molecule, biological process or biochemical reaction.

[0064] The specific nature and application of the reporter compound may vary, and it may be used in combination with various techniques or methodologies known in the art and / or by a person having ordinary skill in the art.

[0065] As used herein, the term “nucleotide-based compound” refers to a compound comprising a nucleotide molecule of DNA or RNA. Said compound may be a mononucleotide or polynucleotide, and may be further covalently bound to another molecules, for example, a lipid, a protein, a plastic or a nanoparticle.

[0066] A first aspect of the present invention relates to a nanobody directed against EGFR comprising a FR3 of SEQ ID NO: 7, more preferably of SEQ ID NO: 8 or 9.

[0067] In an embodiment, the nanobody of the first aspect of the invention further comprises a) a FR1 sequence of SEQ ID NO: 1 , more preferably of SEQ ID NO: 2 o 3, or b) a FR2 sequence as SEQ ID NO: 4, more preferably of SEQ ID NO: 5 or 6, or c) a FR4 sequence of SEQ ID NO: 10, more preferably of SEQ ID NO: 11 or 12.

[0068] In another embodiment, the nanobody of the first aspect of the invention further comprises a) a FR1 sequence of SEQ ID NO: 1 , more preferably of SEQ ID NO: 2 o 3, and b) a FR2 sequence as SEQ ID NO: 4, more preferably of SEQ ID NO: 5 or 6, and c) a FR4 sequence of SEQ ID NO: 10, more preferably of SEQ ID NO: 11 or 12.

[0069] In an embodiment, the nanobody of the first aspect of the present invention further comprises a) a CDR1 sequence of SEQ ID NO: 13 or 14; or b) a CDR2 sequence of SEQ ID NO: 15 or 16; or c) a CDR3 sequence of SEQ ID NO: 17 or 18.

[0070] In an embodiment, the nanobody of the first aspect of the present invention further comprises a) a CDR1 sequence of SEQ ID NO: 13 or 14; and b) a CDR2 sequence of SEQ ID NO: 15 or 16; and c) a CDR3 sequence of SEQ ID NO: 17 or 18.

[0071] In a particularly preferred embodiment, the nanobody of the present invention comprises an amino acid sequence of SEQ ID NO: 19 or 20. More preferably, the nanobody of the present invention consists of an amino acid sequence of SEQ ID NO: 19 or 20.

[0072] Specific embodiments of preferred FRs, CDRs and nanobody sequences are shown in the following Tables 1-8.

[0073] Table 1. List of preferred FR1 sequences of the nanoantibody of the present invention

[0074] Table 2. List of preferred FR2 sequences of the nanoantibody of the present invention

[0075] Table 3. List of preferred FR3 sequences of the nanoantibody of the present invention

[0076] Table 4. List of preferred FR4 sequences of the nanoantibody of the present invention

[0077] Table 5. List of preferred CDR1 sequences of the nanoantibody of the present invention

[0078] Table 6. List of preferred CDR2 sequences of the nanoantibody of the present invention

[0079] Table 7. List of preferred CDR3 sequences of the nanoantibody of the present invention

[0080] Table 8. List of preferred nanobody sequences of the present invention

[0081] Throughout the specification, as well as in the Examples contained herein, the nanoantibodies of the present invention are further characterized by comparing them with an anti-EGFR nanoantibody of the prior art, i.e., 7D12. The following table summarizes the main characteristics of the amino acid sequence of 7D12.

[0082] Table 9. Amino acid sequence and description of the FRs and CDRs of 7D12.

[0083] As one having ordinary skill in the art would appreciate by the description and Examples provided herein, the nanobodies of the present invention show an unexpectedly potent anti-EGFR affinity. This affinity wouldn’t be expected since the nanoantibodies of the present invention show several differences in their amino acid sequence when compared to an EGFR nanobody like 7D12 (see SEQ ID NOs: 21-28), in particular they have several differences in their CDRs (CDR1 : SEQ ID NO 26 vs SEQ ID NOs: 13-14; CDR2: SEQ ID NO 27 vs SEQ ID NOs: 15-16; CDR: SEQ ID NO 28 vs SEQ ID NOs: 17-18).

[0084] Furthermore, the inventors have described that nanoantibodies of the present invention have a stronger affinity for EGFR than that observed in 7D12. Exemplary embodiments of the nanobodies of the invention are shown in the Example 3.5, Table 10. Thus, the nanobodies of the present invention not only show an unexpected affinity for EGFR, as previously described, but even surpass the affinity of nanobodies of the prior art.

[0085] The nanobodies of the present invention are specifically directed to an Epidermal Growth Factor Receptor (EGFR) belonging to any vertebrate including, but without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic animals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild, and game birds such as chickens, turkeys, and other gallinaceous birds, ducks, geese, and the like).

[0086] The nanobodies of the present invention are not limited, constrained nor restrained by a specific antigenic determinant, epitope, domain, part, subunit, or conformation of EGFR that they target (if relevant). However, the nanobodies of the present invention may be designed to target epitopes present or exposed on the surface of a cell.

[0087] In a preferred embodiment of the present invention, and whenever applicable, a nanobody of the present invention may be able to recognize and bind two or more antigenic determinant, epitope, domain, part, subunit, or conformation of EGFR. In that case, said antigenic determinant, epitope, domain, part, subunit, or conformation may be identical or different. As an example, the nanobody of the present invention may recognize EGFR in both the activated and inactivated conformation, may be able to bind to either one of these, or both of them. As an additional example, nanobodies of the invention may be able to bind EGFR bound with its respective ligand, to EGFR in a conformation without its ligand, or both of such conformations.

[0088] Nanobodies of the present invention are also expected to be able to bind to analogs, variants, mutants, alleles, parts and fragments of EGFR that are naturally occurring or synthetic or at least to those analogs, variants, mutants, alleles, parts and fragments of EGFR that contain at least one antigenic epitope. In such case, the nanobodies of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and / or specificity that may be equal or different from the affinity and / or specificity by which the nanobodies of the invention bind to a natural EGFR. It is also included within the scope of the invention that the nanobodies of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of EGFR, but not to others.

[0089] As shown in Example 3.4 and Figure 5B, nanobodies of the present invention may show a high affinity for cells that express low levels of EGFR, for cells that express high levels of EGFR, or for both.

[0090] Thus, the nanobody of the present invention has either a high affinity or low affinity for cells that expresses either high levels or low levels of EGFR. In a preferred embodiment, the nanobody of the present invention has a high or low affinity for cells that express a high level of EGFR, more preferably, the nanobody of the present invention has a high affinity for cells that express a high level of EGFR and a low affinity for cells that express a low level of EGFR.

[0091] In an embodiment, the nanobodies of the present invention have a KD value for EGFR in a flow rate of 30 pl / min that’s lower than 1.7 x 10-8M. Preferably, between 5.0 x 10-9and 1 .5 x 10-8M. More preferably, between 5.0 x 10-9and 9.0 x 10-1° M.

[0092] In an embodiment, the nanobodies of the present invention have a KD value for EGFR in a flow rate of 50 pl / min that’s lower than 3.0 x 10-9M. Preferably, between 1.0 x 10-1° and 2.0 x 10-9M. More preferably, between 1 .4 x 10-1° and 1 .6 x 10-8M. In the context of the present invention, the term "high affinity" when referring to a nanoantibody or antibody, means that the value of the dissociation constant (KD) to a determined target of such antibody or nanoantibody is lower than or equal to 1 .5 x 10-8M at a flow rate of 30 pl / min, or lower than or equal to 2.0 x 10-9M at a flow rate of 50 pl / min.

[0093] In contrast “low affinity”, when referring to a nanoantibody or antibody, means that the value of the dissociation constant (KD) to a determined target of such antibody or nanoantibody is higher than or equal to 1 .7 x 10-8M at a flow rate of 30 pl / min, or higher than or equal to 3.0 x 10-9M at a flow rate of 50 pl / min.

[0094] When referring to a cell, “expressing high levels of EGFR” means to display in the cell surface an amount of EGFR that’s higher than the normal amount of EGFR expected to be found in the cell surface of a particular cell type. One having ordinary skill in the art would be able to determine the normal values of the expression of EGFR for a determined cell type. The expression “expressing low levels of EGFR” means, therefore, that the cell does not “express high levels of EGFR”. These expressions refer only to the relative presence of EGFR in the cell surface in comparison to the normal amount for a particular cell type, and not the absolute amount of EGFR displayed in the cell surface.

[0095] A second aspect of the present invention refers to a nanobody construct comprising a) a nanobody of the first aspect of the present invention, and b) at least one other compound comprising i) a pharmaceutical compound, or ii) a reporter compound, or iii) a polypeptide, or iv) a nucleotide-based compound, and c) a linker, wherein the nanobody a) and the at least one other compound b) are linked via the linker c).

[0096] In an embodiment, the pharmaceutical compound bi) is selected from, but not limited to, monomethyl auristatin E, maleimidocaproyl, valine-citrulline; p-aminocarbamate, tubulysin toxin and variants thereof that causes microtubule destabilization, AZ13599185, emtansine, Adriamycin, paclitaxel, or a photosensitizer, for example, IRDye700DX.

[0097] In an embodiment, the reporter compound bii) may be selected from a radioisotope, a fluorophore, a chromophore, or an enzyme.

[0098] The radioisotope comprises any radioisotope known in the art. Said radioisotope may be detected by any technique for detecting radioisotopes known in the art.

[0099] The fluorophore comprises any fluorophore known in the art. Said fluorophore may be detected by any technique for detecting fluorophores known in the art.

[0100] The chromophore comprises any chromophore known in the art. Said chromophore may be detected by any technique for detecting chromophores known in the art.

[0101] The enzyme comprises any enzyme known in the art. Said enzyme may be detected by any technique for detecting enzymes known in the art.

[0102] In an embodiment, the reporter compound bii) is selected from a fluorescence protein, such as Green Fluorescence protein, Streptavidin scaffolds, FITC, radioisotopes such as 68Ga, 89Zr, 99mTc, 111 In, 225Ac, 1311, 177Lu, 64Cu or any other radioisotope for SPECT, / V-succinimidyl-4-[18F] fluorobenzoate ([18F]-SFB), or any other compound suitable for PET and CT (or micro-CT for animals) imaging known in the art, Infra-Red as Near infra-Red probes such as IRDye800CW, IRDye700DX or IRDye680RD, Alexa Fluor 647, or any other Near infra-Red probe known in the art, lgG1 Fc-fragment coupled with an effector, 177Lu, a y-emission radionuclide, silk fibroin nanoparticles loaded with paclitaxel, quantum dots, preferably, quantum dots suitable for cancer theragnostic, nanoparticles, non-cytotoxic compounds, for example, polyethylene glycol (PEG), an antibiotic for intracellular bacterial infection, any immuno-modulating compound, preferably an anti-inflammatory compound, protein degraders, ligands for cell surface receptor, ligands for proteins and receptors with altered biological activity or expression in cancer, antibody-siRNA conjugates, magnetoliposomes or other MRI imaging probes.

[0103] In a preferred embodiment, the nanobody or nanobody construct of the present invention can be used as a probe for localization of malignant tumors, for example, and without intent to limit the scope of the present invention, bound to a magnetoliposome to serve as contrast agents for MRI serving as imaging probes for localization of malignant tumors.

[0104] In an embodiment, the polypeptide of biii) further comprises a nanoparticle, for example, a metal nanoparticle. More preferably, a magnetic nanoparticle, for example a magnetic bead.

[0105] In an embodiment, the polypeptide of biii) comprises nano-gel, aptamer, N-terminal Gly3 perfluoroaryl macrocyclic, antimicrobial peptides, Cell-Penetrating Peptide (CPP), a polypeptide from a viral-like particle (VLP), a polypeptide from a viral vector, an antibody, and / or a nanobody.

[0106] In a particularly preferred embodiment, the polypeptide of biii) comprises a further nanobody of the first aspect the present invention.

[0107] In an embodiment, the polypeptide of biii) is capable of inducing prophylactic or therapeutical immunity. For example, an HPV tumor antigen, for example, HPV tumor antigen peptide E7, or a CD11 b as expressed on antigen presenting cells. Thus, a preferred embodiment of the present invention comprises an HPV tumor antigen, for example, HPV tumor antigen peptide E7, or a CD11 b as expressed on antigen presenting cells.

[0108] In an embodiment, the nanobody of the present invention can be used in photodynamic therapy, for example, by linking a nanobody of the present invention to a photosensitizer such as IRDye700DX.

[0109] In an embodiment, the nucleotide-based compound of biv) comprises a molecule comprising a nucleotide of DNA or RNA. Said DNA or RNA nucleotide may be a mononucleotide or polynucleotide of DNA or RNA. For example, a mRNA, tRNA, siRNA, microRNA, an expression vector, a probe.

[0110] In an embodiment, the nucleotide-based compound of biv) further comprises a nanoparticle, for example, a metal nanoparticle. More preferably, a magnetic nanoparticle, for example a magnetic bead.

[0111] A nanobody or nanobody construct of the present invention is capable of binding a specific antigen or target thanks to the presence of three CDRs. Furthermore, nanobodies and nanobody constructs of the present invention comprise modified or purified nanobodies, for example, but not limited to, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies. For example, modifying at least one CDR onto a human framework.

[0112] Nanobodies and nanobody constructs of the present invention also comprise other types of modified nanobodies, for example, but not limited to full-length or whole nanobodies and other nanobodies generated by biotechnological or protein engineering methods or processes. Full-length or whole nanobodies comprise monoclonal, recombinant, chimeric, deimmunized, or humanized nanobodies, but may comprise fragments or full- length antibodies. Fragments of antibodies include, but are not limited to, Fv, Fd, Fab, Fab’, F(ab')2, VH, VHH, VL, (s)dAb, or “r IgG” (“half antibody”). Furthermore, nanobody constructs of the present invention comprise modified fragments of antibodies, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, Fab2, Fab3, scFab, scFv-zipper, diabodies, single chain diabodies, tandem diabodies (Tandab’s), tandem di-scFv, tandem tri-scFv, or multibodies, for example, triabodies or tetrabodies, and single domain antibodies such as nanobodies or single variable domain antibodies comprising merely one variable domain, which might be VHH, VL or VH, that specifically bind an antigen or epitope independently of other V regions or domains.

[0113] In an embodiment, nanobodies and nanobodies constructs of the present invention can be modified to obtain a particular preferred characteristic, for example, to prolong their half-life, or to improve their use for imaging or therapeutic application. Strategies include pegylation, multimerization (bivalent, trivalent, tetravalent, pentavalent) and fusion for instance to an Fc region.

[0114] In a preferred embodiment, the nanobody construct of the present invention comprises an antibody, a nanobody, or any other protein or polypeptide capable of targeting, directing or binding a specific epitope.

[0115] Preferably, the nanobody construct of the present invention comprises monovalent, bivalent, trivalent, tetravalent, and polyvalent or multivalent constructs. Accordingly, the present invention comprises monospecific constructs that are capable of binding to a single antigenic structure (for example, an epitope), or bispecific, trispecific, tetraspecific, and polyspecific or multispecific constructs, which are capable of binding to a more than one antigenic structure, e.g. two, three or more, through distinct binding domains. In an embodiment, the nanobody construct of the present invention comprises at least a single polypeptide chain as well as molecules consisting of at least two polypeptide chains. When at least two polypeptide chains are comprised, these may be either identical, for example, homodimers, homotrimers or homo oligomers, or different, for example, heterodimer, heterotrimer or heterooligomer.

[0116] In an embodiment, a nanobody construct of the invention comprises or essentially consists of at least two nanobodies of the invention, such as two, three, or four nanobodies of the invention. Multivalent constructs, as shown in Examples in the present specification can present advantages over proteins or polypeptides comprising a single nanobody of the invention, such as a much improved affinity and / or specificity for EGFR. When at least two nanobodies of the invention are comprised, these may be either identical, for example, homodimers, homotrimers or homo oligomers, or different, for example, heterodimer, heterotrimer or heterooligomer.

[0117] In another embodiment, a polypeptide of the invention comprises at least one nanobody of the invention and at least one other nanobody (i.e. directed against another epitope, antigen, target, protein or polypeptide).

[0118] In another embodiment, a polypeptide of the invention comprises at least one nanobody of the invention. Optionally, the polypeptide of the invention further comprises nanobodies, and at least one other amino acid sequence (such as a protein or polypeptide) which grants the nanobody of the invention and / or the resulting fusion protein of the present invention a desired characteristic or trait.

[0119] In another embodiment, the nanobody construct of the present invention combines two or more of the above embodiments, for example, the nanobody of the present invention may be a trivalent bispecific construct comprising two nanobodies of the invention and one further nanobody, or one or more other amino acid sequences.

[0120] In another embodiment, the nanobody construct of the present invention comprises at least one toxin, preferably, at least one toxin from bacterial or fungal origin. Preferably at least one toxin of bacterial origin, for example, but not limited to, pseudomonal, streptococcal, or escherichial toxins.

[0121] In one preferred embodiment of the invention, the nanobody of the present invention is capable of crossing the blood brain barrier. Such characteristic is obtained by linking one or more Nbs of the invention to one or more acid sequences capable of crossing said barrier.

[0122] The nanobody constructs of the present invention are preferably "in vitro generated nanobody constructs". In other words, the nanobody construct of the present invention may be partially or completely generated by a non-immune cell selection method, e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen. Thus, sequences generated solely by genomic rearrangement in an immune cell in an animal are excluded. A “recombinant nanobody” is an antibody made through the use of recombinant DNA technology or genetic engineering.

[0123] The nanobody of the present invention comprises a linker. Said linker may be selected by a person skilled in the art depending on whether the at least one other compound is a pharmaceutical compound, a reporter compound, or a polypeptide. The specific linker selected, thus, will be dependent on the exact biochemical nature of the at least one other compound.

[0124] In a preferred embodiment, a linker sequence comprises(G4S)x, where X is between 0- 10, more preferably 1 , 2, 3, 4, or 5, more preferably 4, or IEPKI(PQ)XKXKPEP, where X for (PQ)Xis between 0-10, and where X for Kxis between 0-10.

[0125] In a preferred embodiment, the nanobody construct of the present invention comprises a) a first nanobody of the first aspect of the present invention, and b) a second nanobody of the first aspect of the present invention, and c) a linker.

[0126] In a preferred embodiment, the first nanobody and the second nanobody of the nanobody construct each comprise, more preferably consists of, an amino acid sequence selected from any one of SEQ ID NOs: 19 or 20.

[0127] In a preferred embodiment, the nanobody construct comprises a 5B1 nanobody and a 5B12 nanobody, in any combination, and a linker of any one of SEQ ID NOs: 34-37, more preferably, a linker of SEQ ID NO: 34. That is, a nanobody comprising any one of the following dimers: 5B1 -5B1 , 5B1-5B12, 5B12-5B12, 5B12-5B1 , wherein the nanobodies are bound by a linker of SEQ ID NO: 34-37, more preferably a linker of SEQ ID NO: 34. In a preferred embodiment, the nanobody or nanobody construct of the present invention may be incorporated into a cell, preferably incorporated into a cell surface.

[0128] In a preferred embodiment, the nanobody of the first aspect or nanobody construct of the second aspect of the present invention in any of its embodiments can be used to direct, aim or guide a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell towards an EGFR. Preferably towards an EGFR present in the surface of a cell. More preferably, an EGFR present in the surface of a cell that is expressing high levels of EGFR, or the surface of a tumor or cancer cell, or any combination thereof.

[0129] In an embodiment, the nanobody of the first aspect or nanobody construct of the second aspect of the present invention can be used to direct, aim or guide a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell towards an EGFR in an in vitro assay.

[0130] Thanks to the teachings provided herein relating to nanobodies or nanobody constructs of the present invention, a person skilled in the art is now capable of using said nanobodies or nanobody constructs for different purposes. For example, and without intent to limit the scope of the present invention, a person skilled in the art is now capable of linking a nanobody of the present invention with an antibody that doesn’t target EGFR, for the construction of Bispecific T cell engagers, or engineering a cell expressing a nanobody or nanobody construct of the present invention for directing said cell expressing the nanobody or nanobody construct, like CAR-T or CAR-NK cells, to EGFR, or for the engineering of viral-like particles, or viral vectors, in order to direct these towards EGFR. Likewise, a person skilled in the art is now capable of performing better therapeutical or imaging-detection techniques by using the nanobodies or nanobody constructs of the present invention by directing or aiming these towards EGFR.

[0131] The inventors of the present invention, further exemplify, without intent to limit the scope of the present invention, that the nanobody or nanobody construct of the present invention can be bound to a cell, a nucleotide-based compound, such as siRNA, a nanoparticle bonded to a nucleotide-based compound, a viral vector, a viral-like particle. Thanks to the affinity of the nanobody or nanobody construct of the present invention, these different cells, compounds, and particles are directed towards EGFR, or cells that express EGFR, preferably cells that express high levels of EGFR on their surface. The inventors further remark that thanks to the improved affinity of the nanobodies of the present invention, these constructs perform a better engaging, directing, aiming or guiding, than other anti-EGFR nanobodies described in the prior art.

[0132] A third aspect of the present invention refers to a pharmaceutical composition comprising a nanobody of the first aspect or nanobody construct of the second aspect of the present invention.

[0133] In an embodiment, the pharmaceutical composition comprising a nanobody of the first aspect or nanobody construct of the second aspect of the present invention is used in the prevention, treatment or amelioration of a disease. In a preferred embodiment, said disease is selected a proliferative disease, a tumorous disease, a viral disease or an immunological disorder.

[0134] In an embodiment, the pharmaceutical composition comprises two or more different nanobodies or nanobody constructs of the present invention.

[0135] The pharmaceutical composition of the present invention is useful in the treatment, amelioration and / or prevention of a disease in a patient in need thereof.

[0136] The pharmaceutical composition of the present invention may be designed to be administered by specific routes or techniques, with specific frequencies, dosages and regimens, in order to effectively treat a disease. Furthermore, the composition of the present invention encompasses ranges of bioavailability and duration among others.

[0137] In an embodiment, the composition of the present invention comprises a pharmaceutically acceptable excipients selected from diluents, fillers, binders, disintegrants, lubricants, glidants, surfactants, preservatives, antioxidants, pH adjusters, coating agents, flavoring agents, colorants, solvents, emulsifiers, emollients, gelling agents, stabilizers, and or propellants. A person skilled in the art, for example, a physician, will be able to formulate the precise components of the composition according to the administration route or technique and the desired effect to achieve.

[0138] The composition of the present invention may be designed for delivery by any suitable route of administration. Routes of administration include, but are not limited to topical routes, enteral routes, or parenteral routes. In an embodiment, topical routes comprise epicutaneous, inhalational, nasal, ophthalmic, auricular, aural, vaginal, or mucosal routes; enteral routes comprise oral, gastrointestinal, sublingual, sublabial, buccal, or rectal routes; and parenteral routes comprise intracerebroventricular, intravenous, intraarterial, intraosseous, intramuscular, intracerebral, epidural, intrathecal, subcutaneous, intraperitoneal, extra-amniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, transdermal, intranasal, transmucosal, intra-synovial, and intraluminal routes.

[0139] The compositions of the present invention may be administered to a subject in a suitable amount sufficient to totally or partially achieve a desired effect, i.e., treating, ameliorating or preventing a disease. Said amount will depend, among others, on the disease, the administered nanobody or nanobody construct, the therapeutic objectives, the severity of the disease, the patient’s clinical history and response to the therapeutic agent, the route of administration, the patient’s biometrical values (size, body weight, body surface or organ size) and / or condition (the age and general health) of the patient, prior therapy or therapies, and the general state of the patient's own immune system. The administered amount can be adjusted according to the judgment of the attending physician such that it can be administered to the patient once or over a series of administrations, and in order to obtain the optimal therapeutic effect.

[0140] The pharmaceutical composition may be used either as the main therapeutic treatment or in conjunction with other therapies, for example, other anti-cancer treatments. Said other therapies comprise protein-based and non-protein-based drugs. They can be administered concurrently with the composition of the present invention, or they can be administered separately before or after the administration of the composition of the present invention.

[0141] A fourth aspect of the present invention refers to a method of treatment, amelioration or prevention of a disease that comprises administering a therapeutically effective amount of at least one nanobody of the first aspect of the present invention or at least one nanobody construct of the second aspect of the present invention or a pharmaceutical composition of the third aspect of the present invention to a subject in need thereof.

[0142] The term "treatment" refers to either therapeutic use, prophylactic use, or both. The use of the nanobodies or nanobody constructs of the present invention in the treatment of a disease includes its application or administration to the body, tissue, for example, isolated tissue, or cell from a patient who suffers the disease, a symptom of the disease, or a predisposition toward the disease, with the purpose of healing, curing, relieving, ameliorating or improving the disease, the symptom of the disease or the predisposition towards the disease.

[0143] The term “amelioration” as used herein refers to any improvement of the disease state of a patient, by the administration of the composition of the present invention to a subject in need thereof. The improvement may reflect in a deceleration or complete stop of the progression of the disease.

[0144] The term “prevention” as used herein refers to the avoidance of the onset or reoccurrence of a disease in a subject by the administration of the composition of the present invention.

[0145] The term "therapeutically effective amount” as used herein, refers to an amount sufficient to totally or partially achieve a desired effect, i.e., treating, ameliorating or preventing a disease. Said amount will depend, among others, on the disease, the administered nanobody or nanobody construct, the therapeutic objectives, the severity of the disease, the patient’s clinical history and response to the therapeutic agent, the route of administration, the patient’s biometrical values (size, body weight, body surface or organ size) and / or condition (the age and general health) of the patient, prior therapy or therapies, and the general state of the patient's own immune system. The administered amount can be adjusted according to the judgment of the attending physician such that it can be administered to the patient once or over a series of administrations, and in order to obtain the optimal therapeutic effect.

[0146] In an embodiment, the disease to be treated, ameliorated or prevented is a disease caused by or related to cell proliferation. For example, a proliferative disease, a tumorous disease, a viral disease or an immunological disorder.

[0147] In an embodiment, the disease to be treated, ameliorated or prevented is a disease caused by or related to the biological activity of EGFR, for example, EGFR binding to EGF, EGFR transduction signaling, irregular production or expression of EGFR. For example, a proliferative disease, a tumorous disease, a viral disease or an immunological disorder, caused by or related to the biological activity of EGFR, for example, EGFR binding to EGF, EGFR transduction signaling, irregular production or expression of EGFR. In a preferred embodiment, the disease to be treated is a cancer or tumorous disease caused by or related to the biological activity of EGFR. Preferably a cancer or tumorous disease wherein there is overexpression of EGFR.

[0148] In an embodiment, the method of treatment, amelioration or prevention comprises administering two or more nanobodies or nanobody constructs of the present invention in a subject in need thereof. The administration of the two or more nanobodies or nanobody constructs may be done simultaneously or in sequence, in a single or several doses. The person skilled in the art, for example, a physician, would have the knowledge to determine the best administration regime to achieve the desired effect.

[0149] As evidenced by the description and Examples provided herein, the method of treatment, amelioration or prevention of a disease is a result of a reduction, deceleration, or stop of the proliferation of the cells, in particular cells that express high levels of EGFR.

[0150] In an embodiment the method of treatment, amelioration or prevention of the present invention comprises administering at least one nanobody or nanobody construct that has a high affinity for cells that express high levels of EGFR, and a low affinity for cells that express low levels of EGFR.

[0151] In an embodiment method of treatment, amelioration or prevention of the present invention comprises using the nanobody or nanobody construct of the present invention as a directing, aiming or guiding tool for a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell that can be used for treatment, amelioration or prevention of a disease, preferably a proliferative disease, a tumorous disease, a viral disease or an immunological disorder.

[0152] A fifth aspect of the present invention refers to a method of detection of EGFR present in a sample by contacting a nanobody of the first aspect of the present invention or a nanobody construct of the second aspect of the present invention with said EGFR present in a sample.

[0153] A “sample” as used herein, refers to a portion obtained from a subject that may or may not be suffering from a disease. Examples of biological samples include, but are not limited to, complete or partial fractions of tissues or organs, such as tissues or organs such as heart, liver, kidney, lung, arteries, veins, blood or their components, for example plasma, serum, platelets, subpopulations of blood cells, as well as bodily fluids and secretions, for example, saliva, sputum, or any sample retrieved from the mouth and pharynx region.

[0154] The method of detection of the present invention, comprises contacting a sample containing EGFR, preferably irregular levels, for example, high levels of EGFR, with a nanobody of the first aspect of the invention or a nanobody construct of the second aspect of the invention, and detecting the binding of the nanobody of the first aspect of the invention or a nanobody construct of the second aspect of the invention to the EGFR present in the sample by a technique selected from flow cytometry analysis (FACS), immunofluorescence microscopy imaging, plate-adsorbed VLPs, or the use of magnetic- beads associated.

[0155] In an embodiment, the method of detection further comprises comparing the level of binding of the nanobody of the first aspect of the invention or a nanobody construct of the second aspect of the invention to the EGFR present in the sample with the level of binding detected in a control. Said control preferably containing normal level of expression of EGFR.

[0156] In a preferred embodiment, the method of detection is performed in vitro.

[0157] As shown in Examples 2.3, 3.1 and 3.4 of the present invention and Figures 1 , 5B, 5C, and 6, the detection of the binding between the nanobody or nanobody construct of the present invention and EGFR may be performed by different techniques known in the art. For example, by flow cytometry analysis (FACS), immunofluorescence microscopy imaging, plate-adsorbed VLPs, or the use of magnetic-beads associated

[0158] In an embodiment, the method of diagnosis of the present invention can be used for the diagnosis of a disease.

[0159] In an embodiment, the method of diagnosis is for a disease caused by or related to cell proliferation. For example, a proliferative disease, a tumorous disease, a viral disease or an immunological disorder. Preferably, the disease is a cancer or tumorous disease.

[0160] In an embodiment, the method of diagnosis is for a disease caused by or related to the biological activity of EGFR, for example, EGFR binding to EGF, EGFR transduction signaling, irregular production or expression of EGFR. For example, a proliferative disease, a tumorous disease, a viral disease or an immunological disorder, caused by or related to the biological activity of EGFR, for example, EGFR binding to EGF, EGFR transduction signaling, irregular production or expression of EGFR.

[0161] In a preferred embodiment, the disease to be diagnosed is a cancer ortumorous disease caused by or related to the biological activity of EGFR. Preferably a cancer or tumorous disease wherein there is overexpression of EGFR.

[0162] A sixth aspect of the present invention refers to a theragnostic method comprising the method of treatment of the fourth aspect of the invention in any of their embodiments and the method of detection of the fifth aspect of the present invention in any of its embodiments.

[0163] In a preferred embodiment, the theragnostic method comprises a method of diagnosis is for a disease caused by or related to cell proliferation, or to the biological activity of EGFR. More preferably a cancer or tumorous disease caused by or related to the biological activity of EGFR.

[0164] A seventh aspect of the present invention refers to a product for use in the treatment, amelioration or prevention of a disease that comprises administering a therapeutically effective amount of at least one nanobody of the first aspect of the present invention or at least one nanobody construct of the second aspect of the present invention or a pharmaceutical composition of the third aspect of the present invention to a subject in need thereof.

[0165] In an embodiment, the disease to be treated, ameliorated or prevented is a disease caused by or related to cell proliferation. For example, a proliferative disease, a tumorous disease, a viral disease or an immunological disorder.

[0166] In an embodiment, the disease to be treated, ameliorated or prevented is a disease caused by or related to the biological activity of EGFR, for example, EGFR binding to EGF, EGFR transduction signaling, irregular production or expression of EGFR. For example, a proliferative disease, a tumorous disease, a viral disease or an immunological disorder, caused by or related to the biological activity of EGFR, for example, EGFR binding to EGF, EGFR transduction signaling, irregular production or expression of EGFR. In a preferred embodiment, the disease to be treated is a cancer or tumorous disease caused by or related to the biological activity of EGFR. Preferably a cancer or tumorous disease wherein there is overexpression of EGFR.

[0167] In an embodiment, the product for use in the treatment, amelioration or prevention comprises two or more nanobodies or nanobody constructs of the present invention that are meant to be administered in a subject in need thereof. The administration of the two or more nanobodies or nanobody constructs may be done simultaneously or in sequence, in a single or several doses. The person skilled in the art, for example, a physician, would have the knowledge to determine the best administration regime to achieve the desired effect.

[0168] In an embodiment, the product for use of the seventh aspect of the present invention can be used in combination of the method of detection of the fifth aspect of the present invention in any of its embodiments, in order to achieve a product for use in the theragnostic of a disease.

[0169] In a preferred embodiment, the product for use of the seventh aspect of the present invention can be combined with a method of diagnosis for a disease caused by or related to cell proliferation, or to the biological activity of EGFR. More preferably a method of diagnosis a cancer or tumorous disease caused by or related to the biological activity of EGFR.

[0170] All embodiments of the objects, methods, and / or aspects of the present invention resulting from the combination of particular embodiments described herein are to be considered as falling within the scope of the present invention.

[0171] EXAMPLES

[0172] The following examples represent particularly preferred embodiments of the present invention. They are meant to represent exemplary objects and methodologies and are not meant to limit the scope of the present invention in any way.

[0173] Example 1 : Immunization protocol for the production of nanoantibodies of the present invention 1.1 Cell lines

[0174] HEK293A cells were cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1 % penicillin-streptomycin. The his-tagged full-length human EGFR sequence (Cat#HG10001-C-H, Sino Biological, Wayne, PA, USA) was subcloned downstream of a CMV promoter in a pCMV expression plasmid with an hygromycin resistance cassette (Sino Biological, Wayne, PA, USA). HEK293A cells were transfected and individual hygromycin resistant clones were established. An EGFR- expressing HEK293A cell clone (HEK293AhEGFR) was selected for llama immunization following western blot analysis after sample solubilization in 2X Laemmli sample buffer (65.8 mM Tris-HCI, pH 6.8, 2.1 % SDS, 26.3% (w / v) glycerol, 0.01 % bromophenol blue) using Ql-Aexpress anti-His antibodies (Qiagen, Hilden, Germany) and a rabbit monoclonal antibody against EGFR (Cat#10001-R021 , Sino Biological, Wayne, PA, USA). EGFR expression levels on the cellular surface were quantified using the QuantumTM Simply Cellular kit (Bangs Laboratories Inc., Fishers, IN, USA, Cat#814) using anti EGFR-PE as specified by the manufacturer. A431 or MCF7 cells were cultured in DMEM high glucose or DMEM-F12 respectively supplemented with 10% FBS and 1 % penicillin-streptomycin.

[0175] 1.2 The Virus-Like Particle production

[0176] The EGFR virus-like particles (VLPs) were pseudotyped murine leukemia virus (MLV) carrying the full length, membrane integral EGF receptor. VLPs were produced as previously describe by co-transfection of HEK293 cells with pCGP encoding the structural (gag) and (pol) genes of MLV (named p177) and either p2231 , encoding EGFR to generate EGFR-VLPs, or pBSII null plasmid to generate control, ‘naked’ or null- VLPs as previously described [Hoffman TL et al., 2000; Rucker J 2003], Modifications of the previous method included using polyethylenimine (PEI) as the transfection agent in place of calcium-phosphate and testing co-plasmids mixtures to attain the highest EGFR levels on the VLPs, typically at ratios of 3 to 5 receptor per pCGP. VLPs were harvested twice, by collecting the supernatant at 48 h after transfection, by replacing it with fresh DMEM- 5% FBS, then incubating for an additional 24-48 h before a second collection. Both supernatants were filtered through a 0.45 pm filter and concentrated by pelleting through 20% sucrose / HEPES at 140,000 g for 1 h at 4°C. The pellets were re-suspended in 10 mM HEPES pH 7.5 and stored at 4°C. The protein content of VLPs was quantified using the bicinchoninic acid (BOA) titration assay (Cat#23227, Waltham, MA) according to manufacturer’s instructions. The success of the co-transfection of VLPs with EGFR was assessed by dot-blot and the EGFR-VLPs quantification by VLP-ELISA to attain signal saturation. Both methods used anti-CD81 (Cat#MA5-13548, Carlsbad, CA), a HEK293 cell surface marker and anti-EGFR (Cat#10001-R021 , Wayne, PA) antibodies at the recommended dilution as reporters. The plasmids p177, p2231 , and pBSII were a gift from Drs. Willis and Doranz (Integral Molecular, Philadelphia, PA).

[0177] 1.3 Llama immunization

[0178] To induce a humoral immune response directed to EGFR, two llamas were injected subcutaneously with approximately 2 x 107HEK293A-hEGFR cells and boosted twice every two weeks with 1 x 107HEK293A-hEGFR cells. A final boost with 70 U VLPEGFR was administered six weeks after the prime. Preimmune and immune sera were collected at days 0, 35 and 49 and the specific antibodies were monitored by ELISA. Four and seven days after the last antigen injection, two 100 ml blood samples were collected and peripheral blood lymphocytes (PBLs) were purified by density gradient centrifugation on Ficoll-Paque gradients (Amersham Biosciences, Little Chalfont, UK).

[0179] Example 2: Method of selection of the nanoantibodies of the present invention

[0180] 2.1 Phage library construction and selection

[0181] Phage-displayed Nb libraries were constructed from the peripheral blood lymphocytes of the immunized llamas as described in Alzogaray V. et al., 2019. Briefly, mononuclear cells were isolated from 100 ml of blood by Ficoll-Paque (GE Healthcare, Chicago, USA) gradient centrifugation. Total RNA was purified from these cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and cDNA was reverse transcribed. VHH genes were amplified using semi-nested PCR. The amplicons were purified from agarose gels, digested with Sfil and Notl (New England Biolabs, Ipswich, MA, USA) and cloned into the pHEN2 phagemid vector downstream the PelB-leader peptide and upstream the chimeric His6x- Myc epitope tag. Transformation into Escherichia coli XL1-Blue yielded libraries with a size of approximately 107 clones independent transformants and an insert rate of -89% as assessed by colony PCR of 19 clones. Ten clones were used for plasmid DNA preparation (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and sequenced for library diversity analysis (Macrogen, Seoul, Korea). 2.2 Screening of EGFR-specific Nanobodies by phage display technology and phage ELISA identification

[0182] The nanobodies were selected by phage display. Phage particles were rescued from the library using M13K07 helper phage (Life Technologies, Carlsbad, USA) and panned by two alternative methods.

[0183] For panning on plate-adsorbed VLPs, wells of NUNC® MaxiSorp™ microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA, Cat#476635) were coated overnight at 4 °C with 10Opl / well of VLPEGFR or VLPnull (100 ng / well in carbonate buffer pH 9.6). The plates were washed with 0.05% Tween 20-PBS, and then were blocked with 200 pl of 5% skim milk in PBS pH 7.4 for 1 h at 25 °C. After another wash with 0.05% Tween 20- PBS phages from each library were added to the plates and incubated for 1 h at room temperature. After incubation, the plates were washed with 0.05% Tween 20-PBS and bound phage particles were eluted with 100 mM triethylamine (TEA, pH 10.0) and immediately neutralized with 1 M Tris (pH 7.4). The eluted phages were used to infect exponentially growing E. coli TG1 .

[0184] For panning on the soluble purified EGFR ectodomain (ECD) fused to the Fc portion of IgG, EGFR-Fc (Sino Biological, Wayne, PA, USA, Cat#10001-H02H), was captured on Protein G magnetic beads (New England BioLabs, Ipswich, MA, USA, Cat#S1430S). Phages were incubated with 25 pL EGFR-magnetic beads complex at room temperature in a rotator for 2 h to mix them mildly. The magnetic beads were separated by a magnetic rack to wash off the phages which did not bind to the magnetic beads. The magnetic beads were resuspended and eluted with 100 mM TEA (pH 10.0) solution and immediately neutralized with 1 M Tris (pH 7.4). The biopanning protocol was followed as already described for the solid phase strategy.

[0185] The eluted phages were used to infect E. coli TG1 in 30 ml of 2 x YT medium, and the cultures were allowed to grow during the exponential phase until they reached GD600 = 0.5, when the M13K07 helper phages were added to rescue the phages. After infection, samples were incubated ON at 37 °C, 200 rpm. The above steps describe one round of selection, and these rescued phages were used in the next round of selection. After three rounds, the eluted phages were used to infect E. coli TG1 cells. Ninety individual colonies were randomly selected and picked into 96-well plates containing 200 pl of 2 x YT medium overnight. 2.3 Different strategies for selection of EGFR-specific clones from the phage display library produce different outputs

[0186] In order to obtain a cell line with high expression of EGFR, HEK293 cells were transfected with a plasmid expressing EGFR and a stable clone named HEK293HEGFR was selected. EGFR expression in HEK293HEGFR cells was determined to be over 600 times higher than in HEK293A cells (Fig. 8).

[0187] To obtain nanobodies against EGFR, llamas were immunized with HEK293HEGFR cells and Virus-Like Particles (VLPs), and the induction of a specific humoral immune response was followed by testing sera of the animals by ELISA before and after immunization. A specific response towards EGFR was induced only in some llamas (Fig. 2A and 2B), but not in others (Fig. 2C and 2D). The antibodies elicited were also directed towards antigens other than EGFR, as evidenced by the reactivity of immune sera against VLPnuii obtained from HEK293 cells (Fig. 2B)

[0188] The antibody titers for one of the llamas were high enough to qualify for subsequent library construction, which contained approximately 107colony-forming units (CFU), with 88.6% of full-size inserts according to colony PCR.

[0189] For the selection of anti-EGFR nanobodies, two different strategies for the panning were explored:

[0190] (a) on plate-adsorbed VLPs, and

[0191] (b) on the soluble purified EGFR ectodomain (ECD) fused to the Fc portion of IgG.

[0192] The rationale behind both approaches is that panning with VLPs exposes the phages to EGFR epitopes identical to the ones that would be found in cells, while panning with purified EGFR-Fc is devoid of background proteins and thus more specific, therefore lowering the probability of enriching in clones specific for other surface antigens (which would also be present on VLPs, albeit at a lower concentration than EGFR).

[0193] The first selection was performed on plate-adsorbed VLPs, following standard biopanning methods. Screening by VLP-ELISA of 92 random clones from round 3 of this panning, resulted in only 9 clones with modest selectivity ratios (SR) for VLPEGFR / VLPNULL (SR>1 .3) were selected. Sequencing revealed aberrant cloning of the Nb sequence in 7 out of 9 clones, with the majority of them with an insert built from 2 different VHH fragments connected head to tail through the Pstl restriction site. Sequencing of 12 random isolates from the original library revealed each to be unique but two clones contained aberrant sequences, either head to tail tandem VHHS or VHH fragments. These clones do not yield a full functional antibody, even in the case of the head to tail tandems, since the antisense orientation of the upstream fragment generates artificial premature stop codons. Hence the enrichment of such clones is likely the product of a growth advantage of the clone through the amplification steps rather than a specific binding to the target antigen.

[0194] In order to reduce the spurious amplification of dysfunctional clones the inventors decided to tightly control the steps before each round of panning and found that an amplification condition of 4 h at 28°C was the optimal setting to amplify phages enough so that they could be sufficient as an input for next round without over-amplification. Additionally, the inventors monitored the quality of each input / output by colony PCR (SEQ ID NOs: 40-41).

[0195] Sequence analysis showed an in-frame rate of the libraries of 100 % after each round (Fig. 3). Using this strategy, the inventors performed 3 rounds of affinity selection by phage display on purified EGFR-ECD or VLPs. Ninety-two phages from the third round of each selection were individually tested by VLP-ELISA against VLPEGFR for specific binding and VLPNULL for non-specific binding. Eleven clones from the VLP-based panning and 26 clones from the EGFR-ECD panning were positive in the initial screening (i.e., they displayed OD450 >1 .0, with >3-fold higher signal on target VLPs over VLPNULL) These clones were further characterized by EGFR-ECD ELISA, which showed that all clones with a SR<7 that passed the initial criteria had reactivities to EGFR-Fc close to background (corrected OD45o<0.060). From the clones obtained after screening of the VLP-panned output, only 3 out of 11 showed binding to EGFR-ECD. A total of 29 positive colonies with a binding ratio of more than 7 were further sent for sequencing, and 12 different CDR3 sequences were obtained. Finally, the EGFR-specific Nbs were classified into 11 families based on the diversity of their CDR3 amino acid sequences.

[0196] 2.4 Monoclonal phage ELISA

[0197] E. coli bacteria were infected with the eluted phage for 20 minutes at room temperature. Subsequently, the infected cells were spread evenly on LB + ampicillin plates. Following an overnight incubation period at 37°C, colonies of E. coli were chosen at random and inoculated into 200 pl of LB + ampicillin in sterile 96-deep-well plates (Corning, USA). Individual E. coli phage clones were superinfected with M13KO7 at 30°C ON. To perform the phage ELISA, 100 microliters of culture supernatant containing phage particles from the 96-well plates were applied to microtiter plates according to the specified method described in Pardon E. et al., 2014. In order to establish the negative controls, BSA was utilized as an antigen negative control, while M13KO7 helper phage served as the phage negative control.

[0198] 2.5 Expression and purification of single-domain Antibodies (sdAbs).

[0199] VHH DNA sequences (SEQ ID NOs: 38-39) were cloned into the pHEN6 expression vector and the recombinant plasmids were transformed into E. coli WK6. The transformed cells were cultured at 37 °C and 180 rpm in LB medium containing 100 pg / ml ampicillin until they reached the logarithmic growth phase (OD600 = 0.4-0.8). IPTG was added to a final concentration of 1 mM, and the cells were then cultured at 28 °C and 180 rpm ON. The cultures were centrifuged at 10,000 g for 10 min at 4 °C, cells were frozen at -80 °C and thawed at RT, and resuspended in TES buffer (100 mM Tris-HCI pH 8.0, 1 mM EDTA, and 20% sucrose). The supernatant corresponding to the periplasm containing VHHs was collected by centrifugation at 10,000 g for 30 min at 4 °C and dialyzed against equilibration buffer (50mM NaH2PO4, 300mM NaCI, 10 mM imidazole, H2Odd, pH 8) ON at 4 °C using a 3,500 Da cut-off dialysis cellulose membrane (Spectrum™ Spectra / Por™ Dialysis Membrane Tubing, MWCO 3.5KD, ThermoFisher Scientific, Waltham, MA, USA). Monomeric VHHs tagged C-terminally with a 6*His tag were purified from the periplasmic extract of E. coli WK6 cells by immobilized metal affinity chromatography (IMAC) using a His TrapTM HP column (GE Healthcare, Chicago, USA).

[0200] 7D12 is a public domain antibody and its sequence was obtained from Gainkam et al. and synthesized by Genscript (Pistacaway, NJ, USA). 7D12 was cloned into pHEN6 and expressed and purified as the rest of the Nbs.

[0201] Example 3: Nanoantibodies affinity and binding to EGFR

[0202] 3.1 Binding Analysis by Surface Plasmon Resonance (SPR)

[0203] The interaction between the different Nb and EGFR was studied using surface plasmon resonance (SPR) with a BIAcore T100 instrument (Cytiva, Marlborough, MA, USA). EGFR-Fc (the ligand) was diluted in sodium acetate pH 4.5 to a final concentration of 15 pg / ml and immobilized (500 RU) on a CM5 chip surface (Cytiva, Marlborough, MA, USA) through amine coupling, following the manufacturer's instructions. Soluble VHHs were diluted in PBS, pH 7.4 supplemented with 0.005% Tween as the running buffer and injected over the chip surfaces at a flow rate of 50 pl / min for at least 90 sec at 25 °C. The data were analyzed using the BIA evaluation software (Cytiva, Marlborough, MA, USA) considering analyte concentration in the range 100-1.56 nM. The dissociation was monitored for 120 s and glycine pH 3.0 was injected on the surface for 30 s for its regeneration. PBS was not able to wash off the VHHs after interaction with the ligand. The data represent the average of at least two independent assays for VHHs 5B1 , 5B12, and 7D12 under each condition. The dissociation constant (KD) of the system was determined from the kinetic parameters, the association and dissociation rate (kon, koff) by a kinetic analysis using a 1 :1 binding model when it was possible. The bivalent nanobodies did not allow this analysis and the KDs were determined under state steady affinity conditions (SSA method). The standard deviation (SE) of each result was obtained using the BIA evaluation software.

[0204] The dissociation constant (KD) of the system was determine from the kinetic parameters, while the association and dissociation rate {kon, kOff) were determined by a kinetic analysis using a 1 :1 binding model.

[0205] 3.2 Flow cytometric analysis (FACS)

[0206] A431 or MCF7 cells were harvested with Accutase and 5 x 105 cells were incubated with different concentrations of nanobodies in 500 pl of 3% BSA PBS for 2 h at 4 °C in head over tail rotation. Anti-BLS was used as a negative control. The cells were washed three times with 500 pl of 3% BSA PBS at 4 °C to remove unbound antibody. The cells were resuspended and incubated with anti-His-Tag antibody (1 :2000). After incubation, the cells were washed three times with 500 pL of 3% BSA PBS, and resuspended in 500 pL of 3% BSA PBS. The cells were incubated with a Cy5-conjugated mouse anti-His antibody for 30 min at 4 °C in 100 pl of 2% (w / v) BSA in PBS, and were washed three times with 500 pl of 3% BSA PBS to remove unbound antibody. The samples were acquired using a FACS Aria flow cytometer (Beckman Coulter, Brea, CA, USA) and the data analyzed using the FlowJo™ v10 software (BD Biosciences, San Jose, CA).

[0207] 3.3 Immunofluorescence

[0208] HEK293AhEGFR or HEK293A cell lines were grown on a glass coverslip, washed with PBS and fixed with 4% paraformaldehyde for 30 min. The samples were blocked with 10% FBS / PBS for 1 h. Samples were incubated with the VHHs (500nM) ON in PBS / 2.5% FBS, followed by anti-HIS-TAG antibody (Abgent, San Diego, CA, USA) for 1 h, and antimouse Cy5-conjugated secondary antibody (Jackson ImmunoResearch, PA, USA) for 1 h. Cells were washed four times with PBS and mounted on glass slides. All samples were labeled with DAPI (Sig ma-Ald rich, San Luis, USA) and phalloidin (Sigma-Aldrich, San Luis, USA).

[0209] 3.4 Nanobodies 5B1 and 5B12 bind to cell-surface EGFR

[0210] To determine the binding properties of the selected VHHS, one sequence from each of the Nb family was selected for expression of the His-tagged VHH in the periplasm of E. coli strain WK6. Seven unique Nbs were successfully obtained (Fig. 5A, Fig. 4A) and the purified Nbs were tested for their ability to specifically bind EGFR-expressing cells by FACS (Fig. 5B). Two Nbs, 5B1 and 5B12, specifically bind EGFRhiA431 cells, while two additional Nbs, 5A2 and 5F4, could also bind EGFRh' A431 cells albeit with lower affinity. Three of the tested Nbs, 5A1 , 5B4 and 5G1 , did not show differential binding between EGFRhiA431 cells and EGFR10MCF7 cells. Binding of a control Nb (anti-BLS) to EGFRhiA431 cells and of anti-EGFR-Nb (7D12) to EGFR10MCF7 cells was at background levels. On the basis of these results, 5B1 and 5B12 were selected for extensive characterization of their properties. 5B1 , 5B12 and 7D12 preparations were assessed for their aggregation status and the inventors confirmed that they retained a monodisperse SEC peak corresponding to the molecular weight of the VHH monomer (Fig. 4C)

[0211] The selected Nbs were tested for their ability to specifically bind EGFR-expressing cells also by immunofluorescence microscopy (Fig. 5C). Cells were stained either with 5B1 , 5B12, a bivalent Nb against EGFR (b7D12, positive control) or with anti-BLS Nb (negative control). 5B1 and 5B12 stained specifically HEK293AEGFR cells, but not HEK293A cells, which confirms the specific binding of the selected Nbs to EGFR.

[0212] 3.5 Measurement of EGFR binding affinities of 5B1 and 5B12

[0213] Binding of 5B1 and 5B12 to EGFR was further characterized by determining their half maximal effective concentration (EC50) on A431 cells by flow cytometry (Fig. 6). Both nanobodies, as well as the gold standard 7D12, bound to A431 cells with nanomolar affinities, with the novel Nbs displaying an even stronger binding to EGFR overexpressing cells (5B12= 4.7 nM; 5B1 = 76.9 nM; 7D12= 146.8 nM).

[0214] Figure 6 shows saturation binding apparent KDand Bmax values for antibody variants on A431 cells, as determined by flow cytometry. Binding curves of median fluorescence intensity (MFI) versus antibody concentration are shown. Binding at each concentration was normalized to the MFI at 500 nM for each Nb. MFIs were fitted as nonlinear regression curves in GraphPad Prism. These binding properties were confirmed on the purified EGFR ectodomain by Surface Plasmon Resonance (SPR) at a flow rate of 30pl / min. 5B1 and 5B12 KDwere in the same order. However, it is noteworthy that 5B12 exhibited a higher Kon and Koff compared to 5B1 . These differences in the kinetic parameters could potentially confer distinct biological capabilities to the nanobodies. Kinetic parameters of the monovalent VHHS were also determined at a flow rate of 50 pl / min to demonstrate that they were not affected by mass transportation and to minimize re-binding (Table 10, Fig. 10). No significant differences were observed between the kinetic parameters obtained at the two different flow rates.

[0215] In addition, the inventors observed that the nanobodies 5B1 and 5B12 exhibited dissociation constants which were one order of magnitude lower than that of 7D12, indicating a higher affinity of 5B1 and 5B12 towards EGFR compared to 7D12. This represents a clear advantage of the Nbs 5B1 and 5B12 before the Nb 7D12 described in the prior art.

[0216] Furthermore, the higher affinity of 5B1 is due to its long-lived complexes with EGFR compared to 7D12 (Table 10, lower Kofl), while that of 5B12 is due to a 27-fold faster association relative to 7D12 that could favor this Nb rebinding in vitro (Tablel O, higher Kon).

[0217] The inventors point out that the bivalent VHHS did not fit well to the simplest 1 :1 binding model, similar to the other nanobodies under the same conditions. Therefore, alternative models were explored, but the results remain inconclusive and uncertain. Consequently, accurately determining the kinetic parameters, Kon and Koff, and the resulting KD has proven to be challenging. As 5B1-5B1 did not fit a kinetic model, the KDwas calculated using a steady-state (SSA) nonlinear regression method, which allowed for determination of the affinity despite the limitations in obtaining kinetic parameters.

[0218] Table 10. Kinetic parameters of the interaction between EGFR and VHHS determined at a flow of 30pl / min or 50pl / min. Example 4: Bivalent nanobodies of the present invention

[0219] 4.1 Competitive ELISA

[0220] Wells of NUNC® MaxiSorp™ microtiter plates were coated overnight at 4 °C with 100 pL of VLPEGFR. The wells were blocked with 200 pl of PBS containing 5% skim milk for 1 h at RT. Serially diluted cetuximab (0.007pM to 50pM, ErbituxD CETUXIMAB, MERCK) and a fixed amount of VHH phage was added to wells for 1 h at room temperature. The wells were washed 5 times with PBS containing 0.1 % Tween-20, incubated for 1 h with anti-M13 conjugated with Horseradish Peroxidase (HRP), washed 5 times again and then developed with TMB substrate. The absorbance was measured using an ELISA reader at 450 nm. The competitive activity of Nb was expressed as a percentage of displacement of cetuximab ((1- ((OD450 of sample- OD450 of blank) I (OD450 of Nb without cetuximab- OD450 of blank))) / x 100 %).

[0221] 4.2 Synthesis of Bivalent nanobodies of the present invention

[0222] Sequences from selected nanobodies 5B1 and 5B12 (SEQ ID NOs: 38-39) were codon optimized, sequences which were prone to homologous recombination were modified and the genes synthetized by GenScript. The VHH cassettes were subcloned into pHEN6 expression vector, in frame with the pelB leader sequence for secretion into the periplasm. A flexible linker, (G4S)4, was inserted between both VHH cassettes and a His6 tag was cloned on the 3’ end.

[0223] 4.3 5B1 and 5B12 target different epitopes and can be engineered as biparatopic nanobodies

[0224] Competition ELISA was used to gain insight into the epitopes targeted by the two Nbs. Phages expressing the 5B1 , 5B12 or control 7D12 nanobodies were bound to VLPEGFR in the presence of increasing concentrations of Cetuximab, which targets a well-known epitope overlapping the domain III ligand-binding region of EGFR, shared by 7D12. 5B1 phage was competed by Cetuximab in the same way as the control 7D12 phage, evidencing at least a partial overlapping of their epitopes (Fig. 7). On the other hand, 5B12 phage could only be partially displaced by Cetuximab, showing that it binds a different epitope than 5B1 .

[0225] Additionally, to map the epitopes of 5B1 and 5B12, an SPR assay was conducted without surface regeneration. All nanobodies were analyzed at a final concentration of 75 nM. 5B1 was flowed over EGFR at a rate of 30 pl / min for 60 seconds without surface regeneration. When 5B12 was injected at the same concentration, a lower response was observed. Similarly, when the experiment was performed with 5B12 first, followed by 5B1 injection, the lower response was still obtained. These findings would suggest that the epitopes targeted by 5B1 and 5B12, could be partially overlapping or separate but allosteric.

[0226] Figure 7 shows the characterization of VHH binding sites. EGFR-specific VHH-phages were tested in competition ELISA using VLPEGFR as antigen. Competition is expressed as percentage of displacement by increasing concentrations of Cetuximab.

[0227] Example 5: Bivalent nanobodies antitumoral properties

[0228] 5.1 MTT assay

[0229] A431 cells plated at 5,000 cells / well in 96-well plates were incubated with DMEM containing 10% (v / v) FBS for 6 h, followed by incubation with FBS-free DMEM for 24 h. The medium was replaced with the respective Nb or Cetuximab at the appropriate concentration and 170 pM EGF. After incubation for 48 h at 37 °C 5% CO2, cells were treated with MTS (CellTiter 96 Aqueous One Solution Cell Proliferation assay, Promega, Madison, Wl, USA) at 5 mg / ml for 2 h at 37 °C 5% CO2. Optical density of the wells was read at 490 nm on a plate reader (iMark™ Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA). The viability of the cells was calculated as a percentage relative to the control wells treated with media alone (100% viability). All the experiments were performed in triplicate.

[0230] 5.2 Bivalent nanobodies engineered from 5B1 and 5B12 inhibit the proliferation of tumor cells

[0231] The effects of the selected nanobodies on the proliferation of different cell lines were examined using an MTT assay with EGFR-overexpressing A431 cells. Neither 5B1 / 5B12 nor 7D12 showed a significant effect on the proliferation of these cells, unlike Cetuximab which strongly affected cell viability. However, Cetuximab is a complete antibody and it displays two EGFR binding sites, which has already been reported as an important feature for EGFR blocking by antibodies. The inventors construct bivalent nanobodies (bNb) based on 5B1 and 5B12 in order to explore their binding and functional properties. Four bNb (5B1-5B1 , 5B1-5B12, 5B12-5B1 and 5B12- 240 5B12) were built by covalent bonding of two Nb with a flexible peptide linker in a tandem orientation and expressed in E. coli \NKQ (Fig. 4B). The inventors designed two different biparatopic Nbs, 5B1-5B12 and 5B12-5B1 because, even when they are formed by the same “blocks”, which VHH is in the N-terminal or C-terminal affects its properties. bNb were assayed by SPR and exhibited affinities comparable to those observed with the monovalent Nbs (Table 10, Fig. 11)

[0232] Next, the inventors assessed the inhibitory effect of the different Nbs constructs on tumor cell growth. The in vitro studies showed that all bNb had significant anti-proliferative effects on A431 cells, with maximal inhibition rates reaching that of cetuximab, albeit with a weaker inhibition of proliferation at concentrations < 1 nM (Fig. 12A). Of note, the largest inhibition of the bNbs was observed upon localization of 5B1 at the N-terminal domain reaching cetuximab inhibitory effect at 1 nM concentration (Fig. 12A). The different Nb were also assayed for their ability to block EGF-induced EGFR phosphorylation. To assess EGFR phosphorylation status, A431 cells were stimulated with human EGF; cetuximab was used as a positive control to block EGF binding and untreated cell as negative control. In coincidence with the anti-proliferation effects, only bivalent constructs (Biv1 , Biv3 and Biv4) were able to decrease pEGFR levels (Fig. 12B).

[0233] 5.3 Inhibition of glioma stem cell proliferation

[0234] In order to further characterize the novel Nbs, the inventors performed initial studies aiming at determining their capacity to inhibit the in vitro proliferation of the glioma stem cell-enriched cell lines. Interestingly, the biparatopic Nbs 5B1 -5B12 and 5B12-5B1 were able to inhibit the proliferation of G05 cells expressing the mutated EGFRvlll; of note, 5B12-5B1 reached 30% inhibition at 10 nM concentration (Fig. 13A). Surprisingly, none of the bNbs was able to inhibit the in vitro growth of the cell line G08 expressing a nonmutated EGFR (Fig. 13B). This differential effect was also observed with the commercial Ab cetuximab (Fig. 13A and B).

[0235] References

[0236] Alzogaray, V., et al., Characterization of folding-sensitive nanobodies as tools to study the expression and quality of protein particle immunogens. J Biotechnol, 2019. 293: p. 17-23.

[0237] Gainkam, L.O., et al., Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT / micro-CT. J Nucl Med, 2008. 49(5): p. 788-95.

[0238] Heukers, R., et al., Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif. J Cell Sci, 2013. 126(Pt 21): p. 4900-12.

[0239] Hoffman, T.L., et al., A biosensor assay for studying ligand-membrane receptor interactions: binding of antibodies and HIV-1 Envto chemokine receptors. Proc Natl Acad Sci U S A, 2000. 97(21): p. 11215-20.

[0240] Kumagai, S., S. Koyama, and H. Nishikawa, Antitumour immunity regulated by aberrant ERBB family signalling. Nat Rev Cancer, 2021 . 21 (3): p. 181-197.

[0241] London, M. and E. Gallo, Epidermal growth factor receptor (EGFR) involvement in epithelial-derived cancers and its current antibody-based immunotherapies. Cell Biol Int, 2020. 44(6): p. 1267-1282.

[0242] Muyldermans, S., et al., Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng, 1994. 7(9): p. 1129-35.

[0243] Pardon, E., T. Laeremans, S. Triest, S.G. Rasmussen, A. Wohlkonig, A. Ruf, . . . J. Steyaert, A general protocol for the generation of Nanobodies for structural biology Nat Protoc, 2014. 9(3): p. 674-93

[0244] Roovers, R.C., et al., A biparatopic anti-EGFR nanobody efficiently inhibits solid tumour growth. Int J Cancer, 2011. 129(8): p. 2013-24.

[0245] Rucker, J., Optical biosensor assay using retroviral receptor pseudotypes. Methods Mol Biol, 2003. 228: p. 317-28. Sanchez-Martin, F.J., et al., The First-in-class Anti-EGFR Antibody Mixture Sym004 Overcomes Cetuximab Resistance Mediated by EGFR Extracellular Domain Mutations in Colorectal Cancer. Clin Cancer Res, 2016. 22(13): p. 3260-7.

[0246] Schmitz, K.R., et al., Structural evaluation of EGFR inhibition mechanisms for nanobodies / VHH domains. Structure, 2013. 21(7): p. 1214-24.

[0247] Steeland, S., R.E. Vandenbroucke, and C. Libert, Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today, 2016. 21(7): p. 1076-113.

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[0249] Tintelnot, J., et al., Nanobody Targeting of Epidermal Growth Factor Receptor (EGFR) Ectodomain Variants Overcomes Resistance to Therapeutic EGFR Antibodies. Mol Cancer Ther, 2019. 18(4): p. 823-833.

[0250] Tsitokana, M.E., et al., Targeting the Brain with Single-Domain Antibodies: Greater Potential Than Stated So Far? Int J Mol Sci, 2023. 24(3).

[0251] LIST OF SEQUENCES

Claims

CLAIMS1 . A nanobody directed against EGFR comprising a FR3 with an amino acid sequence as set forth in SEQ ID NO: 7.

2. The nanobody according to claim 1 , further comprising: a) a CDR1 with an amino acid sequence as set forth in SEQ ID NO: 13 or 14, and b) a CDR2 with an amino acid sequence as set forth in SEQ ID NO: 15 or 16, and c) a CDR3 with an amino acid sequence as set forth in SEQ ID NO: 17 or 18.

3. The nanobody of any one of claims 1 or 2, further comprising: a) a FR1 with an amino acid sequence as set forth in SEQ ID NO: 1 , and b) a FR2 with an amino acid sequence as set forth in SEQ ID NO: 4, and c) a FR4 with an amino acid sequence as set forth in SEQ ID NO: 10.

4. The nanobody of any one of claims 1-3, comprising a FR3 with an amino acid sequence selected from SEQ ID NO: 8 and 9.

5. The nanobody according to any one of the preceding claims, comprising a sequence as set forth in SEQ ID NO: 19.

6. The nanobody according to any one of the preceding claims, comprising a sequence as set forth in SEQ ID NO: 20.

7. A nanobody construct comprising a) a nanobody according to any one of claims 1 -6, b) at least one other compound comprising i) a pharmaceutical compound, or ii) a reporter compound, or iii) a polypeptide, or iv) a nucleotide-based compound, and c) a linker.wherein the nanobody a) and the at least one other compound b) are linked via the linker c).

8. The nanobody construct according to claim 7, wherein the polypeptide comprises at least one nanobody according to any one of claims 1 -6.

9. The nanobody construct according to claim 8, comprising two nanobodies according to any one of claims 1-6, wherein the nanobodies are either identical or different.

10. The nanobody construct according to claim 9, wherein the nanobodies are as set forth in SEQ ID NO: 19 or 20, in any combination.1 1. A pharmaceutical composition comprising a nanobody according to any one of claims 1-6, or a nanobody construct according to any one of claims 7-10, and a pharmaceutically acceptable excipient selected from diluents, fillers, binders, disintegrants, lubricants, glidants, surfactants, preservatives, antioxidants, pH adjusters, coating agents, flavoring agents, colorants, solvents, emulsifiers, emollients, gelling agents, stabilizers, and propellants.

12. A method of treatment of a disease that comprises administering a therapeutically effective amount of at least one nanobody according to any one of claims 1-6, or a nanobody construct according to any one of claims 7-10, or a pharmaceutical composition according to claim 11 to a subject in need thereof.

13. The method of claim 12, wherein the disease is a disease caused by or related to the biological activity of EGFR.

14. The method of any one of claims 12-13, wherein the method further comprises reducing, decelerating, or stopping the proliferation of cells.

15. A method of detecting EGFR in a sample comprising contacting a nanobody according to any one of claims 1-6, or a nanobody construct according to any one of claims 7-10, with EGFR, wherein said method is performed in vitro.

16. The method according to claim 15, wherein the contacting is detected using a technique selected from flow cytometry, immunofluorescence imaging, plate- adsorbed VLP panning, and magnetic bead based panning.

17. Atheragnostic method comprising applying a method according to claim 12, and a method according to claim 15.

18. A product for use in the treatment, amelioration or prevention of a disease, comprising at least one nanobody of any one of claims 1-6, or at least one nanobody construct of any one of claims 7-10, or a pharmaceutical composition of claim 11 .

19. The product for use according to claim 18, used in combination with a method of detection according to any one of claims 15-16.

20. Use of the nanobody of any one of claims 1 -6 for directing, aiming or guiding a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell towards an EGFR.21 . Use of the nanobody construct of any one of claims 7-10, for directing, aiming or guiding a pharmaceutical compound, reporter compound, polypeptide, nucleotide-based compound, nanoparticle, viral vector, viral-like particle, or cell towards an EGFR.