Cell-type restricted (functional) inhibition of transforming growth factor-beta receptor 2

Abstract

The present disclosure relates to cell-type restricted or -specific inhibition of transforming growth factor-beta receptor 2-mediated responses. In particular there is provided for a conjugate, for example a fusion protein, comprising a first moiety, which is an immunoglobulin chain variable domain which is capable of specifically binding transforming growth factor-beta receptor 2, and at least a second moiety capable of binding a target, for example, expressed on the cell surface of a target cell that expresses transforming growth factor-beta receptor 2, and wherein the first moiety on its own, i.e. in the absence of the second moiety, is unable to inhibit TGFB signaling in such cell. Also provided is for the treatment of a disease or condition with the conjugate.

Description

[0001] Title: Cell-type restricted (functional) inhibition of transforming growth factor-beta receptor 2. GOVERNMENT SUPPORT STATEMENT: This invention was made with government support under GM058670 awarded by the NIH. The government has certain rights in the invention. STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING A Sequence Listing in XML format, entitled P100836US.xml, 135,531 bytes in size, generated on July 16, 2025 and filed herewith, is hereby incorporated by reference in its entirety for its disclosures. FIELD OF THE INVENTION

[0001] This invention pertains in general to cell-type restricted or cell-type specific inhibition of transforming growth factor-beta receptor 2 (TGFBR2) function, in particular, in the treatment of a subject in need thereof.

[0002] In particular, the invention pertains to a conjugate, for example a fusion protein, and use thereof as a medicament. The conjugate comprises a first moiety, which is an immunoglobulin chain variable domain which is capable of specifically binding TGFBR2, in particular capable of specifically binding transforming growth factor-beta receptor 2 extracellular domain, and at least a second moiety capable of binding a target, for example, expressed on the cell surface of a target cell that expresses TGFBR2. The first moiety is characterized by that on its own, i.e. in the absence of the second moiety, it is unable to inhibit Transforming growth factor-β (TGF^; TGFB) signaling in such cell, or only to a limited extent. BACKGROUND OF THE INVENTION

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Transforming growth factor-β (TGFβ; TGFB) is part of a versatile family of cytokines, which includes TGFBs, bone morphogenetic proteins, and activins. These cytokines are crucial regulators of embryonic development, tissue homeostasis, and regeneration. Dysfunction in these pathways is linked to a wide range of conditions, such as cancer, fibrosis, immune diseases, and other pathologies.

[0005] There are three highly structurally related mammalian isoforms of TGFB: TGFB1, TGFB2, and TGFB3. In vitro studies have shown that while all three isoforms produce similar biological effects, they differ in potency depending on the cell-type.

[0006] In the canonical TGFB signaling pathway, TGFB first binds to the low-affinity accessory TGFB type III receptor (TGFBR3), which is abundant on the surface of many cell-types. TGFBR3 presents TGFB to the TGFB receptor complex, which, despite being present at low levels, has a high affinity for TGFB, thereby facilitating signaling. Notably, the TGFB2 isoform particularly relies on TGFBR3 for signaling.

[0007] The TGFB receptor complex is a tetramer consisting of two pairs of transmembrane serine / threonine protein kinases: two TGFB type I receptors (TGFBR1) and two TGFB type II receptors (TGFBR2). When TGFB binds, TGFBR2 transphosphorylates TGFBR1 at specific serine / threonine residues located in the intracellular juxtamembrane region (the GS domain). Following the transduction of the extracellular signal across the plasma membrane, activated TGFBR1 initiates intracellular signaling by phosphorylating SMAD2 / 3. Phosphorylated SMAD2 / 3 dissociates from TGFBR1 and forms a heteromeric complex with SMAD4. This SMAD2 / 3–SMAD4 complex then translocates into the nucleus to activate or repress a wide variety of target genes.

[0008] Beyond the canonical pathway, TGFB also activates several noncanonical signaling pathways downstream of TGFB receptors. For instance, TGFBR1 activates RHO small GTPases and regulates the activity of RHO-associated protein kinase and LIM kinase. This leads to the reorganization of the actin cytoskeleton, affecting cell adhesion, motility, and growth. Additionally, TGFBR2 can directly phosphorylate the cell polarity regulator Partitioning defective protein (PAR)6, which participates in regulating tight junctions and cell migration.

[0009] TGFB signaling has garnered significant interest from cancer researchers due to its extensive roles in regulating cancer cell functions, including cell cycle progression, apoptosis, adhesion, and differentiation. However, TGFB's effects can vary greatly depending on the cell-type and conditions. In normal and premalignant epithelial cells, TGFB typically acts as a tumor suppressor by inhibiting cell proliferation, promoting apoptosis, and maintaining genome stability. Conversely, tumor cells can adapt to or bypass TGFB's suppressive functions and exploit its promotional roles to gain a growth advantage. This includes processes like the epithelial-to-mesenchymal transition (EMT), which facilitates their migration, invasion, intravasation, and extravasation.

[0010] Additionally, TGFB can create a favorable tumor microenvironment (TME) by acting in a paracrine manner to activate cancer-associated fibroblasts (CAFs), promote angiogenesis, produce extracellular matrix (ECM), and suppress anti-tumor immune responses. These actions contribute to cancer progression and metastasis. CAFs are key components of the TME, supporting tumor progression by producing ECM and cytokines, stimulating immune evasion, and promoting angiogenesis.

[0011] Furthermore, TGFB plays a crucial role in enhancing the metastatic capacity of tumor cells by promoting EMT through the upregulation of transcription factors such as SNAIL, via both SMAD- and non-SMAD-dependent pathways.

[0012] Activation of the TGFB signaling pathway can also induce epithelial cancer cells to transition into myofibroblasts through EMT and transform endothelial cells into fibroblast-like cells via endothelial-mesenchymal transition.

[0013] It is therefore not surprising that TGFB signaling has been targeted by pharma and academic researchers in conditions where its dysfunction is implicated. Preclinical results from in vitro cell models and in vivo animal models have shown exciting potential for anti-tumor therapeutics using TGFB-neutralizing antibodies and ligand traps that block the interaction of TGFB with its receptors, as well as selective small- molecule TGFB receptor kinase inhibitors.

[0014] For example, neutralizing antibodies have been designed by pharma and academic researchers to target native TGFB ligands and receptors to block biological activity. Fresolimumab (GC1008), a human antibody that neutralizes TGFB, demonstrated acceptable safety and antitumor activity in phase 1 and phase 2 clinical trials for patients with malignant melanoma, renal carcinoma, glioma, metastatic breast cancer, or relapsed malignant pleural mesothelioma. LY3022859, an antibody that blocks TGFB binding to the ectodomain of TGFBR2, shows antitumor efficacy in several mouse tumor models. However, in a phase 1 clinical trial the maximum tolerated dose for this antibody was not determined due to patients experiencing uncontrolled cytokine release. / pct

[0015] Additionally, small-molecule kinase inhibitors of TGFBR kinases, TGFB ligand traps (TGFBR2 ectodomain-based chimeric fusion proteins designed to prevent TGFB from binding to its receptors), and antisense oligonucleotides are subjects of ongoing research.

[0016] A major challenge is making these pharmacological agents suitable for approved clinical use. Due to TGFB's highly pleiotropic actions, systemic modulation (e.g., inhibition) of TGFB signaling can affect healthy tissues, leading to unwanted side effects and safety concerns.

[0017] Indeed, cancer patients treated with TGFB signaling blockers often experience side effects when TGFB's role in physiological processes to maintain tissue homeostasis is compromised. For instance, some selective TGFBR kinase inhibitors have shown therapeutic effects in cancer patients but caused cardiac toxicity at high doses (hemorrhagic, degenerative, and inflammatory lesions in heart valves) and skin toxicity (eruptive keratoacanthomas, hyperkeratosis, cutaneous squamous-cell carcinomas, and basal cell carcinoma), thus limiting their safe therapeutic window. These adverse effects continue to challenge the clinical application of many anti-TGFB therapies in cancer, fibrosis and other diseases caused by persistent, dysregulated, or overactive TGFB signalling. For an extensive review on TGFB in health and disease, reference is made to Liu et al (2021) (Signal Transduction and Targeted Therapy 6(8) (2021); doi.org / 10.1038 / s41392-020-00436-9).

[0018] In light of this, new products, compositions, methods and uses suitable for use in modulation of TGFB signaling and / or for use in the treatment of conditions characterized by TGFB signaling dysfunction and / or for use in the treatment of conditions that would benefit from modulation of TGFB signaling, for example cancer, would be highly desirable but are not yet readily available. In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods and uses that allow to be suitably used in the aforementioned modulation and / or treatment(s). Accordingly, the technical problem underlying the present invention can at least been seen in the provision of such products, compositions, methods and uses complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below. SUMMARY OF THE INVENTION

[0019] As embodied and broadly described herein, the present invention is directed to the surprising finding that cell-type restricted modulation, preferably inhibition, of TGFB signaling may be provided for by contacting cells with a conjugate according to the invention. The skilled person understands that within the context of the current invention, “inhibition” of TGFB signaling, preferably TGFBR2-mediated TGFB signaling includes functional inhibition of TGFB signaling in a cell, preferably TGFBR2- mediated TGFB signaling in a cell. In embodiments, the conjugate according to the invention is composed of at least two separate moieties, preferably separated by a linker. One of the moieties recognizes TGFBR2 but by itself does not inhibit TGFB- induced TGFBR2-function, where the at least one second moiety recognizes, for example, a cell-type restricted or cell-type specific cell surface protein.

[0020] The present invention can provide for cell-type restricted modulation, in particular cell-type restricted inhibition of TGFBR2-mediated TGFB signaling. In some embodiments the present invention can provide for cell-type specific modulation, in particular cell-type specific inhibition of TGFBR2-mediated TGFB signaling. The skilled person understands that within the context of the current invention both terms (cell- type restricted and cell-type specific) may be used interchangeably expect if from the context it is clear only one of the terms is meant. The conjugate may not inhibit systemic soluble TGFB. In other words, the present invention allows for the inhibition of (cellular) signaling that normally follows from interaction of TGFB with TGFBR2 expressed on the cell surface of a cell-type, and in a way that is cell-type restricted (or -specific). Said otherwise, with the present invention it has become possible to inhibit TGFBR2-mediated TGFB signaling in a first set of cell-types (or cell-type), whereas TGFBR2-mediated TGFB signaling in a second set is not, or only to a limited extent, affected.

[0021] The present invention thus allows for a surprising strategy wherein TGFBR2- mediated TGFB signaling can be inhibited in cell-types wherein such inhibition is desirable, for example in the context of the treatment of a subject in need thereof, without affecting, or only to a limited extent, TGFBR2-mediated TGFB signaling in other cell-types, and wherein it is (indeed) desirable to not inhibit TGFBR2-mediated TGFB signaling. In a non-limiting example, with the invention it has become possible to inhibit TGFBR2-mediated TGFB signaling, in particular cancer cells or cell-types, while at the same time not, or only to a limited extent, influencing TGFBR2-mediated TGFB signaling in other, for example, healthy, cell-type.

[0022] The strategy provided by the invention is based on the unexpected finding that a conjugate can be provided that comprises a first moiety that is capable of binding to TGFBR2 expressed on the cell surface of a cell-type, but that is not able to inhibit TGFBR2-mediated TGFB signaling to a significant degree, for example in the context of treatment of a subject in need thereof, on it’s own. Only when the first moiety is fused to a at least a second moiety, and that is capable of binding to another target, for example another cell-surface receptor, expressed on the cell surface of the (same) cell-type, inhibition (functional inhibition) of TGFBR2-mediated TGFB signaling (or said otherwise: TGFB induced TGFBR2 functioning) occurs. In other words, inhibition of TGFBR2-mediated TGFB signaling in the cell-type is increased by the presence of the second moiety in the conjugate as compared to inhibition of TGFBR2-mediated TGFB signaling by the same in absence of the second moiety in the conjugate. For example, inhibition of TGFBR2-mediated TGFB signaling in the cell-type is increased by the conjugate in comparison to inhibition of TGFBR2-mediated TGFB signaling in the cell-type mediated by only the first moiety (i.e. by only an immunoglobulin chain variable domain, which is capable of specifically binding TGFBR2 according to the invention, and as detailed herein elsewhere).

[0023] The increase in inhibition of TGFBR2-mediated TGFB signaling in the cell-type is preferably many-fold, e.g. more than 2-fold, more than 5-fold, more than 10-fold, more than 50-fold, or even more than 100-fold, although the invention is not in particular limited thereto. As the skilled person understands, any relative and / or absolute increase in the level of inhibition of TGFBR2-mediated TGFB signaling in a cell-type as the consequence of the second moiety in the conjugate according to the invention, and as compared to the same in absence of the second moiety in the conjugate (as explained above) allows for improved cell-type restricted (or -specific) inhibition of TGFBR2-mediated TGFB signaling, in those cells that express on their surface that target that the second moiety in the conjugate of the invention is capable of binding to.

[0024] By way of example, a conjugate according to the invention can be contemplated that comprises a first moiety as defined herein and that comprises a second moiety that is capable of (specifically) binding to a target, for example a cell surface receptor X, that is specifically, or relatively more abundantly, expressed on a cancer cell, as compared to a healthy cell. Such conjugate according to the invention is capable of improved inhibition of TGFBR2-mediated TGFB signaling in the cancer cell as compared to the same in the absence of the second moiety (e.g. the conjugate comprising the first moiety but wherein the second moiety has been removed) and as compared to healthy cells, because of the binding of the second moiety to the target, e.g. cell surface receptor X, specifically or more abundantly expressed on the cell surface of the cancer cells.

[0025] In other words, by combining in the conjugate according to the invention the first moiety as defined herein with a second moiety that is capable of binding to a target that is specifically or more abundantly expressed on the cell surface of a target cell- type of interest, it has now become possible to target TGFBR2-mediated TGFB signaling, more in particular inhibit TGFBR2-mediated TGFB signaling in such target cell-type of interest specifically or at least relatively more specifically as compared to other cell-types that do not or less abundantly express a target that the second moiety is capable of binding to.

[0026] These and other aspects, embodiments and preferences of the invention will be detailed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0028] Figure 1: Immunoprecipitation efficiency of TGFBR2-VHHs was determined by incubating conditioned media containing the various TGFBR2-VHHs (coupled to rabbit Fc) with the lysate from HEK293T cells overexpressing TGFBR2-FLAG or TGFBR2-MYC. The VHHs were pulled down with protein G beads and the co-immunoprecipitated TGFBR2 was visualized by western blot. (A) shows the VHH clones produced in the first round and (B) shows the affinity maturated clones.

[0029] Figure 2: Various VHH clones according to the invention were evaluated for their effect on TGFβ / SMAD signaling using a SMAD3-dependent fluorescent reporter assay (SMAD3 is an essential intracellular effector of TGF-β). (A) HepG2 cells with a stable CAGA-dynGFP reporter (Marvin DL, You L, Bornes L, van Dinther M, Peters N, Dang H, Hakuno SK, Hornsveld M, Kranenburg O, van Rheenen J, Rohling JHT, Chien MP, Ten Dijke P, Ritsma L. Dynamic Visualization of TGF-β / SMAD3 Transcriptional Responses in Single Living Cells. Cancers (Basel).2022 May 19;14(10):2508. doi: 10.3390 / cancers14102508. PMID: 35626109; PMCID: PMC9139966) were pre-incubated for 30 minutes with the conditioned media of the VHHs after which the cells were stimulated with TGFβ (20 pg / ml) for 24h. Images were made and analyzed using the IncuCyte (Sartorius). (B) Similar experiment as in A, but here the signaling was induced by overexpression of TGFBR2 (to non-physiological very high expression levels) in HEK293T cells instead of stimulation of HepG2 cells with exogenously added TGFβ.

[0030] Figure 3: (A) Different CAGA-dynGFP reporter cell lines were used to test the inhibitory effect of TGFBR2-VHH-D10-Y62S coupled to affibodies that recognize epidermal growth factor receptor (zEGFR) (Tolmachev et al. - J Nucl Med - 2009 Feb;50(2):274-83 - doi: 10.2967 / jnumed.108.055525) or zHER2 (Orlova et. al. - Cancer Res - 2006 Apr 15;66(8):4339- 48 - doi: 10.1158 / 0008-5472.CAN-05-3521; wherein the “z” refers to the Z domain, a small protein domain derived from the Staphylococcal protein A. This Z domain serves as the scaffold for an Affibody molecule. Affibody molecules are engineered protein-based binding proteins that can be designed to target specific proteins. In case of zEGFR, its referring to affibody molecule that is engineered to specifically target EGFR.). Assay is the same as way as in Fig 2A, with the only difference being that 1 ng / ml of TGFB was used. Other exemplary embodiments of conjugates according to the invention, including those that for example use TGFBR2-VHH-E03* and TGFBR2-VHH-B03*, will provide comparable results. (B) mRNA expression of EGFR and human epidermal growth factor receptor (HER)2 in the cell lines used in A (data from the Human Protein Atlas website(www.proteinatlas.org, accessed 2024-04- 08)).

[0031] Figure 4: Apparent Kd of various TGFBR2-VHH clones. induced and incubated with different concentrations of TGFBR2-Biotin labelled by a Streptavidin-Alexa-488. The measurement has been done with an incubation of the antigen during 1h30 at 30°C, the assay was done according to Hunter et. al. (Methods Enzymol, 2016-580:21-44. doi: 10.1016 / bs.mie.2016.05.002).

[0032] Figure 5: Schematic examples of conjugates according to the invention, and wherein the first moiety is capable of binding TGFBR2 (as indicated) and the second moiety is capable of binding to EGFR (affibody), or HER2 (affibody).

[0033] Figure 6: Alphafold prediction of TGFBR2-VHH-D10-Y62S binding to extracellular domain of human TGFBR2 (A) as compared to the binding of human TGFB1 (B). Human TGFBR2 extracellular domain is in black, TGFBR2-VHH-D10-Y62S is in dark grey and human TGFB1 is in light grey. Predictions were performed using the alphafold server (https: / / golgi.sandbox.google.com / ). Images were generated using the ChimeraX software (USCF, version 1.8). Other exemplary embodiments of conjugates according to the invention, including those that for example use TGFBR2-VHH-E03* and TGFBR2-VHH-B03*, can provide comparable results.

[0034] Figure 7: Schematic presentation of two conjugate according to the invention, here in particular a TGFBR2xCD4 VHH (TGFBR2-VHH – CD4-VHH) conjugate (being a bispecific antibody)that were used in experiments for which the data in shown in Figure 8 and 9, one without His-tag and one with a C-terminal His-tag (to allow for easier purification). Signal peptide (to direct the secretion of TGFBR2-VHH protein),TGBFR2 nanobody (TGFBR2-VHH, in particular TGFBR2-VHH-D10-Y62S), and CD4- (VHH) and His tag (10xHis) are indicated. A linker, here formed by three alanine residues, that forms a linker between the TGFBR2 VHH (a first moiety according to the invention) and the CD4 VHH (a second moiety according to the invention) is indicated with three A’s.

[0035] Figure 8: A. Analysis of CD4 expression in Jurkat, HBB ALL, MOLT3 and THP1 cells as measured by Western blot analysis of cell lysates probed with a commercially available CD4 specific antibody (Cell signaling CD4 (D2E6M) Rabbit mAb #93518). Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was analyzed to equal protein loading. Whereas HBB ALL, MOLT3 and THP1 express CD4, Jurkat cells are devoid of CD4 expression. B. Analysis of effect of TGFR2VHH fused to CD4 VHH-his on TGF^-induced SMAD3 / 4 dependent transcriptional activation. (y-axis expressed fluorescence of GFP as a measure of CAGA expression, being induced by TGFB / SMAD). Green fluorescent protein (GFP) reporter activity in THP1 cells (which express CD4). When THP1 CAGA-dynGFP cells (referring to cells having a transcriptional reporter for TGF-^ / SMAD signalling, as previously described in Figure 3. CAGA refers to the SMAD3 and SMAD4 DNA binding element.) were challenged with TGF^ in the absence (upper line) or presence of TGFR2 VHH -CD4 VHH-His (middle line), we observed that TGFBR2 VHH -CD4 VHH-His was able to attenuate the TGF^ / SMAD response. TGFBR1 kinase inhibitor (SB-505124; SB; see Persson et al. FEBS Lett. 1998 Aug 28;434(1- 2):83-7. doi: 10.1016 / s0014-5793(98)00954-5. PMID: 9738456.) was taken along as control and blocked the TGF^ / SMAD transcriptional response as expected. C, D and E. Analysis of effect of TGFBR2 VHH -CD4 VHH-His or TGFBR2 VHH -CD4 VHH on TGF^ induced-SMAD2 phosphorylation in Jurkat cells (CD4 negative) HPB ALL, and Molt3 cells (CD4 positive cells). Phosphorylated SMAD2 levels were measured with a specific antibody (as described in Persson et al. FEBS Lett. 1998 Aug 28;434(1-2):83-7. doi: 10.1016 / s0014-5793(98)00954-5. PMID: 9738456). Cells were treated with TGF^ in presence of blank (nothing, except for TGF^, TGFBR1 kinase inhibitor SB-505124 (SB) as positive control) and vehicle control, or two doses of TGFBR2-VHH -CD4 VHH-His or TGFBR2 VHH CD4-VHH . We found that TGFBR2 VHH- CD4-VHH-His or TGFBR2-VHH -CD4 VHH- potently inhibited TGF^ induced phosphorylation of SMAD2 in CD4 positive HBB ALL, MOLT3 and THP1 cells but not in CD4 negative Jurkat cells. GAPDH expression was measured to control for equal loading. Thus, latter results show that TGFBR2 VHH -CD4 VHH inhibits TGFB signaling response selectively in CD4 expressing cells; no inhibitory effect upon TGFBR2 VHH -CD4 VHH challenge on TGFB signaling was observed in CD4 negative cells. Other exemplary embodiments of conjugates according to the invention, including those that for example use TGFBR2-VHH-E03* and TGFBR2-VHH-B03*, will provide comparable results.

[0036] Figure 9: Analysis of effect of TGFBR2 VHH -CD4 VHH-His on TGF^-SMAD2 phosphorylation in primary CD4 T cells. Phosphorylated SMAD2 levels were measured with a specific antibody (see Persson et al. FEBS Lett. 1998 Aug 28;434(1-2):83-7. doi: 10.1016 / s0014-5793(98)00954-5. PMID: 9738456.). Cells were treated with TGF^ in presence of blank (nothing, except for TGF^), vehicle control, TGFBR2-VHH - CD4-VHH-His or TGFBR2-VHH - CD4-VHH-His We found that TGFBR2-VHH - CD4-VHH-His or TGFBR2-VHH - CD4-VHH-His potently inhibits TGF^ induced phosphorylation of SMAD2 in primary CD4 cells. GAPDH expression was measured to control for equal loading. Other exemplary embodiments of conjugates according to the invention, including those that for example use TGFBR2-VHH-E03* and TGFBR2-VHH-B03*, will provide comparable results.

[0037] Figure 10: shows that an affibody to EGFR zEGFR fused to TGFBR2 VHH nanobody is more efficient in inhibiting TGFβ that is exogenously added to the culture medium in A431 cells (that highly express EGFR) as compared to SKOV3 cells (that show very low EGFR expression), and an affibody to HER2 zHER2 fused to TGFBR2 VHH nanobody is more efficient in inhibiting TGFβ that is exogenously added to the culture medium in SKOV3 cells (that highly express HER2) as compared to A431 cells (that show very low HER2 expression). The same experimental set-up as in Figure 3A was used. The results in Figure 10 (using phosphorylated SMAD2 as an assay for TGFB-induced signaling response) are entirely consistent with the results shown in Figure 3A (using TGFB-induced SMAD transcriptional activation as an assay for TGFB-induced signaling response). Both data sets demonstrate the exquisite cell selectivity of affibody fusions to TGFBR2 VHH in inhibiting the TGFB-induced response, and that cell selectivity is determined by which receptor the affibody is directed against. DESCRIPTION Definitions

[0038] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0039] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0040] For purposes of the present invention, the following terms are defined below.

[0041] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a multifunctional conjugate” includes a combination of two or more multifunctional conjugates, and the like. For example, a method for administrating a fusion protein according to the invention includes the administrating of a plurality of fusion proteins (e.g.10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more).

[0042] As used herein, “about” and “approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term “about.”

[0043] As used herein, “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0044] As used herein, "at least a particular value” means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, …, etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, ….-10, -11, etc.

[0045] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps, or components. It also encompasses the more limiting “to consist of”.

[0046] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology, and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0047] As used herein, "exemplary" or “for example” means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations, including those disclosed herein.

[0048] As used herein, the term “antibody” in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including multispecific antibodies, and heteroconjugate antibodies. Within the context of the invention it also includes a single variable domain (e.g., VH ((an immunoglobulin chain variable domain consisting of a VHC domain (Ward et al Nature 1989341:544-546); or VHC), VHH, VL (an immunoglobulin chain variable domain consisting of a VLC domain; of VLC), a domain antibody (dAb)), antigen binding antibody fragments, Fab (a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains), F(ab′)2 (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (consisting of the VHC and CH1 domains), Fv (consisting of the VLC and VHC domains of a single arm of an antibody), disulphide linked Fv, single chain Fv (consisting of VLC and VHC domains joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules), disulphide-linked scFv, diabodies, TANDABS (tandem diabodies), V-NAR (an immunoglobulin chain variable domain consisting of a VHC domain from chondrichthyes IgNAR (Roux et al 1998 Proc Natl Acad Sci USA 95:11804-11809 and Griffiths et al 2013 Antibodies 2:66-81, herein incorporated by reference in their entirety), etc., and modified versions of any of the foregoing. The first moiety and / or the second moiety may also be or comprise an affibody molecule or other antibody mimetics know to the skilled person. As used herein, and antigen- binding fragment (or "'antibody fragment" or "immunoglobulin fragment") refers to a portion of an antibody that specifically binds to its target, for example, to TGFBR2 (in case of the first moiety according to the invention).

[0049] As used herein, "cancer" refers to the physiological condition in mammals that is typically characterized by unregulated cell growth (and invasion and metastasis). The terms "cancer," "neoplasm," and "tumor," are often used interchangeably to describe cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells can be distinguished from non- cancerous cells by techniques known to the skilled person. A cancer cell, as used herein, includes not only primary cancer cells, but also cancer cells derived from such primary cancer cell, including metastasized (secondary) cancer cells, and cell lines derived from cancer cells. Examples include solid tumors and non-solid tumors or blood tumors. Examples of cancers include, without limitation, leukemia, lymphoma, sarcomas, and carcinomas (e.g. colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, melanoma, lymphoma, non-Hodgkin lymphoma, colon cancer, (malignant) melanoma, thyroid cancer, papillary thyroid carcinoma, lung cancer, non-small cell lung carcinoma, and adenocarcinoma of lung). Treatment of a cancer in a subject includes the treatment of a tumor in the subject.

[0050] As used herein, “fibrosis” or “fibrotic disorders” refers to the development of excess fibrous connective tissue in an organ or tissue. It is an underlying manifestation of many disease states. Fibrosis can occur in a variety of tissues or organs. These fibrotic conditions include dermal fibrosis (e.g., associated with scleroderma). Dermal fibrosis is fibrosis that manifests itself in the skin (or dermis). Fibrotic conditions also include non-dermal fibrosis. Non-dermal fibrosis is fibrosis that manifests itself in an organ other than the skin (or dermis). As important example of non-dermal fibrosis is lung (or pulmonary) fibrosis. Lung fibrosis can be associated with interstitial lung disease and diffuse proliferative lung disease. Other examples of non-dermal fibrosis include liver / hepatic fibrosis, ocular fibrosis, fibrosis of the gut, kidney / renal fibrosis, pancreatic fibrosis, vascular fibrosis, cardiac fibrosis, myelofibrosis, and the like.

[0051] Some forms of fibrosis are referred to as interstitial fibrosis, and they include dermal or non-dermal interstitial fibrosis. Examples of fibrotic disorders or fibrosis include pulmonary fibrosis, liver cirrhosis and fibrosis, scleroderma, glomerulonephritis, skin fibrosis, radiation fibrosis, renal fibrosis, proliferative vitreoretinopathy, or uterine fibrosis.

[0052] As used herein the term “fusion protein” refer to any polypeptide, which is not normally found in nature, is a species, in particular a polypeptide in which one or more part of the amino acids sequence are not associated with each other in nature. For example, a fusion protein may comprise a N-terminal part consisting of a first sequence of amino acids and a C-terminal part consisting of a second sequence of amino acids that are not associated with each other in nature and / or are not associated with each other in nature in this order. A fusion protein may for example be obtained from transcription and translation of a fusion gene of nucleic acid. Such fusion gene may be created by joining parts of two different genes / nucleic acid sequences. The sequences may, or may not be separated, by a (flexible) linker.

[0053] As will be understood by the skilled persons, in some embodiments multimers of the first moiety and / or of the second moiety may be used in the conjugate according to the invention. The skilled person is aware of the means and methods to provide therefor.

[0054] As used herein “identity” or “sequence identity” refers to the degree of relatedness between two or more amino acid sequences, or two or more nucleic acid sequences (polynucleotide sequences), as determined by comparing the sequences. The comparison of sequences and determination of sequence identity may be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs available to align two sequences and determine the percent identity between them. The skilled person will appreciate that different algorithms may yield slightly different results.

[0055] Thus, the “percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is the “identities” value, expressed as a percentage, that is calculated by, for example, the basic local alignment tool (BLAST)N algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pairwise BLASTN alignment is performed. Such pairwise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.

[0056] Similarly, the “percent identity” between a query amino acid sequence and a subject amino acid sequence is the “identities” value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pairwise BLASTP alignment is performed. Such pairwise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein.

[0057] The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. Such alterations include at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acids or nucleotides in the query sequence or in one or more contiguous groups within the query sequence. Detailed description

[0058] The invention is defined herein, and in particular in the accompanying claims. Subject-matter, which is not encompassed by the scope of the claims, does not form part of the present claimed invention.

[0059] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment envisaged herein. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are also envisaged herein, and form different embodiments, as would be understood by those in the art.

[0060] It is contemplated that embodiments described herein in relationship to any method, use, or composition can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0061] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0062] As embodied and broadly described herein, the present invention is directed to the surprising finding that cell-type restricted (or -specific) modulation, preferably inhibition, of TGFB signaling may be provided for by contacting cells with a conjugate according to the invention.

[0063] Therefore, according to an embodiment, there is provided for a conjugate comprising. (i) a first moiety, wherein the first moiety is or comprises an immunoglobulin chain variable domain, which is capable of specifically binding transforming growth factor-beta (TGFB) receptor 2 (TGFBR2), and (ii) at least a second moiety, wherein the second moiety is capable of binding a target, and wherein the immunoglobulin chain variable domain of the first moiety is on its own is unable (or only very weakly) to inhibit TGFB signaling (for example, in any cell-type).

[0064] The conjugate according to the invention comprises at least two separate functional parts, herein referred to as a first moiety and a second moiety. Within the conjugate the first moiety is conjugated to the second moiety. In some embodiments the first moiety is directly conjugated to the second moiety. In some embodiments the first moiety is indirectly conjugated to the second moiety, for example in some embodiments the first moiety is conjugated to the second moiety via a connection linker, for example a protein linker. In other embodiments, the first moiety is conjugated to the second moiety via a further moiety, i.e. wherein between the first moiety and the second moiety there is located a third moiety. In some embodiments, more than one first moiety is used, for example one, two, or three first moieties are included in the conjugate according to the invention. In some embodiments, more than one second moiety is used, for example one, two, or three second moieties (each binding to the same target or to different targets) are be included in the conjugate according to the invention.

[0065] In some embodiments, the conjugate is a protein, for example, and preferably, a fusion protein. In some embodiments, the conjugate comprises a protein, for example a fusion protein, as well as a non-protein groups, such as a drug, a fatty acid, a polynucleotide, and / or a sugar. For example, in some embodiments the conjugate comprises a first moiety that is a polypeptide and a second moiety that is a polypeptide; or the conjugate comprises a first moiety that is a polypeptide and a second moiety that is a sugar; or the conjugate comprises a first moiety that is a polypeptide, a second moiety that is a polypeptide, and wherein the conjugate further comprises one or more additional moieties (i.e. functional groups), for example conjugated to the second or first moiety, for example a drug, for example a pharmaceutically acceptable drug used in the treatment of cancer).

[0066] In a preferred embodiment, the conjugate comprises a first moiety that is a polypeptide and at least a second moiety that is a polypeptide, optionally wherein the conjugate comprises one of more additional moieties, for example, wherein the one or more additional moieties provide further functional characteristics to the conjugate (for example, the additional moiety being a drug-moiety, a further antibody moiety, a fluorescent moiety, a radioactive moiety and so on).

[0067] In the conjugate, the first moiety may be conjugated (directly or indirectly) with the second moiety via the C-terminus of the first moiety, but is may also be conjugated (directly or indirectly, i.e. via a linker and / or a further moiety) to the second moiety via the N-terminus of the first moiety.

[0068] The skilled person is aware of conventional techniques to provide for the conjugate according to the invention in any suitable configuration.

[0069] The conjugate comprises a first moiety. The first moiety is an immunoglobulin chain variable domain, which is capable of specifically binding transforming growth factor-beta (TGFB) receptor 2 (TGFBR2). In embodiments, the immunoglobulin chain variable domain, which is capable of specifically binding transforming growth factor- beta (TGFB) receptor 2 (TGFBR2) is not able to inhibit (or only very weakly) TGFB- signaling (TGFB-induced signaling), more in particular TGFBR2-mediated TGFB- signaling (in any cell-type)

[0070] A conventional antibody, also known as immunoglobulin (Ig), is a protein composed of four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The variable domains of the heavy chains are referred to as VHC, while those of the light chains are referred to as VLC. These domains, along with related and derived domains, are collectively known as immunoglobulin chain variable domains.

[0071] The VHC and VLC domains can be further divided into hypervariable regions called "complementarity determining regions" (CDRs), which are interspersed with more conserved regions known as "framework regions" (FRs). The precise definitions of these regions are well-established and in a conventional antibody, each VHC and VLC consists of three CDRs and four FRs, arranged in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino-terminus to the carboxy-terminus.

[0072] The conventional antibody tetramer is formed by two heavy chains and two light chains interconnected by, for example, disulfide bonds. The heavy chains are similarly connected to each other. The constant region of the heavy chain includes three domains: CH1, CH2, and CH3, while the constant region of the light chain comprises one domain, CL. The variable domains of the heavy and light chains serve as binding domains that interact with an antigen.

[0073] The term antibodies include immunoglobulins of types IgA, IgG, IgE, IgD, and IgM (and their subtypes). An exception to the conventional antibody structure is found in the sera of Camelidae which also comprise unique IgG antibodies known as heavy- chain antibodies, which lack the light chain polypeptide and the first constant domain (CH1) and contains a dedicated variable domain called VHH, which associates with its specific antigen (Muyldermans, 2013, "Annu Rev Biochem," 82:775-797; Hamers- Casterman et al., 1993, "Nature," 363(6428):446-448; Muyldermans et al., 1994, "Protein Eng," 7(9):1129-1135).

[0074] The immunoglobulin chain variable domain of the first moiety may be comprised in an antibody or antigen-binding fragment thereof, as will be detailed herein elsewhere.

[0075] The immunoglobulin chain variable domain of the first moiety is capable of specifically binding transforming growth factor-beta receptor 2 (TGFBR2; see e.g., Entrez gene ID 7048 and / or UniProt P37173), preferably mammalian (more preferably human) TGFBR2, primate TGFBR2, or rodent (mice, rat) TGFBR2. In a more preferred embodiment, the immunoglobulin chain variable domain of the first moiety is capable of specifically binding human transforming growth factor-beta receptor 2 (TGFBR2). In another more preferred embodiment, the immunoglobulin chain variable domain of the first moiety is capable of specifically binding transforming growth factor-beta receptor 2 (TGFBR2) expressed in human cells, including human diseased cells such as human cancer cells. The skilled will understand that the immunoglobulin chain variable domain of the first moiety may be cross-reactive for (specifically) binding to TGFBR2 of different species.

[0076] TGFBR2 is a transmembrane serine / threonine kinase forming a TGF-beta- dependent heteromeric complex with the TGF-beta type I serine / threonine kinase receptor, TGFBR1, the non-promiscuous receptor for the TGF-beta cytokines TGFB1, TGFB2 and TGFB3. It transduces the extracellular TGFB1, TGFB2 and TGFB3 signal (also generally referred herein as TGFB) from the cell surface to the cytoplasm (and the nucleus) and thereby regulates a plethora of physiological and pathological processes, including cell cycle arrest in (for example normal and pre-malignant) epithelial and hematopoietic cells, control of mesenchymal cell proliferation and extracellular matrix deposition, cell differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis. The formation of the receptor complex composed of 2 TGFBR1 and 2 TGFBR2 molecules symmetrically bound to the cytokine dimer results in the phosphorylation and activation of TGFBR1 by the constitutively active TGFBR2. Activated TGFBR1 phosphorylates SMAD2 and SMAD3, which dissociate from the receptor and interacts with SMAD4. The SMAD2 and / or SMAD3-SMAD4 complex is subsequently translocated to the nucleus where these heteromeric SMAD complexes modulate the transcription of the TGFB-receptor regulated genes. This constitutes the canonical SMAD-dependent TGFB signaling cascade. TGFBR2 is also involved in non-canonical, SMAD-independent TGF-beta signaling pathways.

[0077] The immunoglobulin chain variable domain of the first moiety preferably comprises at least one complementarity determining region CDR1, at least one complementarity determining region CDR2, and / or at least one complementarity determining region CDR3, for example, as disclosed herein elsewhere. The immunoglobulin chain variable domain of the first moiety preferably comprises at least one framework region FR1, at least one framework region FR2, at least one framework region FR3, and / or at least one framework region FR4, for example, as disclosed herein elsewhere.

[0078] The immunoglobulin chain variable domain of the first moiety is capable of specifically binding transforming growth factor-beta receptor 2. Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding polypeptide can bind. The specificity of an antigen-binding polypeptide is the ability of the antigen-binding polypeptide to recognize a particular antigen as a unique molecular entity and distinguish it from another.

[0079] The immunoglobulin chain variable domain of the first moiety is on its own unable to inhibit TGFB signaling in any cell-type. The term “unable to inhibit TGFB signaling” is to be understood in the context of the current invention, based on the unexpected finding that a conjugate can be provided that comprises a first moiety that alone is capable of binding to TGFBR2 expressed on the cell surface of a cell-type, but that is unable to inhibit TGFBR2-mediated TGFB signaling to a significant degree, for example in the context of treatment of a subject in need thereof. Only in the presence of the at least second moiety that is comprised in the conjugate and that is capable of binding to another target (preferably an extracellular domain of said target), for example another cell-surface receptor, expressed (for example, in a cell-type restricted manner) on the cell surface of the cell-type that also expresses TGFBR2. In other words, the conjugate according to the invention comprises a first moiety (and at least a second moiety), wherein the first moiety is an immunoglobulin chain variable domain, which is capable of specifically binding transforming growth factor-beta receptor 2 (TGFBR2), and wherein inhibition of TGFB signaling is achieved or improved (or dictated) in a cell-type restricted manner by the presence of the at least second moiety in the conjugate (and that is, for example, recognizing a cell surface protein in a particular cell-type restricted of cell-type specific manner or pattern). In other words, inhibition of TGFBR2-mediated TGFB signaling in a particular cell-type by the first moiety is increased if the first moiety is combined with a second moiety in the conjugate that is able to bind to a target in the same cell-type, for example another cell surface receptor. The skilled person is able to measure or determine the “inability (i.e. being unable) to inhibit TGFB signaling, for example, based on the methods disclosed herein, or methods analogous to the methods disclosed herein. Lack of inhibiting TGFB signaling to a significant degree may include no inhibiting at all, or inhibiting to only a limited degree, such as no more than 5 percent or 10 percent in comparison to TGFB signaling that is obtained in the presence of only TGF-beta (and in the absence of the first moiety according to the invention alone, as described above.

[0080] This therefore relates to the potency of the immunoglobulin chain variable domain, which is capable of specifically binding TGFBR2, on it own. Potency is a measure of the activity expressed in terms of the amount required to produce an effect of given intensity. A highly potent agent evokes a greater response at low concentrations compared to an agent of lower potency that evokes a smaller response at low concentrations. Potency is a function of affinity and efficacy. Efficacy refers to the ability to produce a biological response upon binding to a target, for example a cell surface receptor, and the quantitative magnitude of this response. Inhibitory effect on biological response may depend on thresholds. Below certain threshold this is an effect, above threshold there is no effect. Within the context of the invention, the potency of the conjugate of the invention to inhibit TGFBR2 mediate TGFB signaling is higher than that of the same conjugate not comprising the at least second moiety, or for example, than that of the first moiety alone.

[0081] Said otherwise, the conjugate according to the invention provides improved defense to certain restricted cell-types from the effects of TGFB, for example by inhibiting the biological effect of TGFB on the cell-type in which the target, for example, a cell surface receptor, for the second moiety is expressed. In an embodiment of the invention, the conjugate of the invention is thus considered to be a neutralizing conjugate, partially or fully neutralizing TGFBR2 mediate TGFB signaling in a cell-type restricted or -specific manner, whereas the immunoglobulin chain variable domain comprised in the first moiety alone does not show such neutralizing properties (i.e. does not to the same extent has an impact on the cell-types from the effects of TGFB (or TGFBR2-mediated TGFB signaling).

[0082] It is the authors assertion that by including in the conjugate according to the invention at least a second moiety, wherein the second moiety is capable of binding a target, for example to another cell surface receptor expressed on the same cell-type that expressed TGFBR2, the avidity of cell surface binding between the immunoglobulin chain variable domain comprised in the first moiety and TGFBR2 is increased. Avidity is the measure of the strength of binding between an antigen- binding molecule (e.g. a conjugate according to the invention) and the pertinent antigen (e.g. TGFBR2). Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule (e.g. the conjugate according to the invention) and the number of pertinent cell surface binding sites present.

[0083] Indeed, in addition to the first moiety, the conjugate according to the invention comprises at least a second moiety, wherein the second moiety is capable of binding a target. As explained herein elsewhere the second moiety may be of any kind as long as it is able of binding a, for example cell surface, target. Preferably the target of the second moiety is a biological target such as, for example, a protein, a lipid, a sugar, a polypeptide, a polynucleotide. In preferred embodiments the target is a target that is present (or expressed) at or on the cell surface of a cell, in particular of a target cell- type (or cell of interest, for example, a cell-type, wherein TGFBR2-mediated TGFB signaling is to be inhibited).

[0084] It will be understood by the skilled person, the target to which the second moiety capable of binding is preferably not TGFBR2.

[0085] The second moiety itself may be (comprise) an immunoglobulin chain variable domain which is capable of specifically biding to a target, for example in case the target is protein, for example a cell surface protein, for example a cell surface receptor, or a co-receptor, including those described herein elsewhere. However, the second moiety may also be any other moiety that is able of binding a target on a cell, for example present (or expressed) on the cell surface. However, in a preferred embodiment, the second moiety itself comprises an immunoglobulin chain variable domain, which is capable of specifically biding to a target, preferably a target expressed on the cell surface of a target cell-type (or cell of interest and that also expresses, for example expresses TGFBR2), preferably wherein the target is a receptor, for example a cell surface receptor.

[0086] The target, for example cell surface receptor may be a target that is specific for the target cell (or cell of interest) relative to another cell-type or may be a target the expression of which is more abundant on the target cell-type relative to other cell- types.

[0087] In some embodiments it is contemplated that the conjugate according to the invention comprises in addition to the first moiety and the second moiety, additional moieties. It will be understood by the skilled person, specificity of the conjugate according to the invention may be further increased by such conjugate. For example, specificity for a target cell that expresses, in addition to TGFBR2, cell surface receptor X and cell surface protein Y on its cell surface can be provided by a conjugate according to the invention comprising (in addition to the first moiety) a second moiety that (specifically) binds to cell surface receptor X and a further moiety that (specifically) binds to cell surface protein Y (and, for example in comparison to a cell that does not express cell surface protein Y).

[0088] In a preferred embodiment, the second moiety itself comprises an immunoglobulin chain variable domain, which is capable of specifically biding to a target.

[0089] In some embodiments, the second moiety, preferably wherein the second moiety comprises an immunoglobulin chain variable domain which is capable of specifically binding a target, has the potency to modulate the activity of the target, for example to inhibit or to stimulate activity of the target, or to be an antagonist or agonist of said target. However, for the invention this is as such not required (although in some embodiment it is preferred that the second moiety is able of modulating the (biological) activity of the target, for example inhibit or increase activity thereof); for the invention binding of the second moiety, for example specific binding, to its target is sufficient. In some embodiment, the second moiety is capable of modulating (inhibiting or stimulating) signaling that would normally be mediated by the target. For example, in case the target to which the second moiety is capable to bind to would be an epidermal growth factor receptor protein (EGFR), the second moiety may, for example, be an immunoglobulin chain variable domain which is capable of specifically binding to EGFR and thereby capable of inhibiting EGFR-mediated EGF signaling in the cell.

[0090] The immunoglobulin chain variable domain of the first moiety preferably comprises at least one complementarity determining region CDR1, at least one complementarity determining region CDR2, and / or at least one complementarity determining region CDR3, for example, as disclosed herein elsewhere. The immunoglobulin chain variable domain of the first moiety preferably comprises at least one framework region FR1, at least one framework region FR2, at least one framework region FR3, and / or at least one framework region FR4, for example, as disclosed herein elsewhere.

[0091] Within the context of the current invention, the skilled person knows how to provide for a conjugate according to the invention, for example by providing an immunoglobulin chain variable domain, which is capable of specifically binding TGFBR2 but that has low or no potency in inhibiting TGFBR2-mediated TGFB signaling (for example as shown herein), preparing a conjugate comprising in a first moiety said immunoglobulin chain variable domain and further comprising at least a second moiety that is capable of binding a target, for example, expressed on the cell surface of a target cell-type (or cell of interest that also expressed TGFBR2), and, optionally, determining inhibition of TGFBR2 mediate TGFB signaling in the cell-type by the conjugate, optionally in comparison to the provided immunoglobulin chain variable domain.

[0092] The first moiety and / or the second moiety may also be or comprise an affibody molecule or other antibody mimetics know to the skilled person.

[0093] Also provided is for the conjugate according to the invention, wherein the immunoglobulin chain variable domain of the first moiety is an immunoglobulin chain variable domain with an apparent Kd value (to TGFBR2) of between 10 – 5000 pM, preferably between 10 – 2000 pM, for example, between 10 – 1500 pM, between 10 – 100 pM, between 10 – 500 pM, for example between 10 – 250 pM or 10 – 100 pM. In some embodiments the Kd value is at least 10 pM, at least 15 pM, at least 20 pM, or at least 25 pM. The indicated apparent Kd value is the value that is obtained when determining biding of the first moiety on its own, i.e. in the absence of the least second moiety.

[0094] Also provided is for the conjugate according to the invention, wherein the immunoglobulin chain variable domain of the first moiety is an immunoglobulin chain variable domain with an affinity to TGFBR2 that is, for example, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75 fold, at least 100-fold, or at least 150 fold lower than that of TGFB, for example between 5 – 500 fold, between 10 – 400 fold, between 20 – 300 fold, or between 40 – 200 fold lower.

[0095] Affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding molecule (e.g. polypeptide) (Kd), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding molecule (e.g. polypeptide): the lesser the value of the Kd, the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (e.g. polypeptide). Alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1 / Kd. Affinity can be determined by known methods, depending on the specific antigen of interest.

[0096] Suitably, the immunoglobulin chain variable domain of the first moiety will bind with a dissociation constant (Kd) of between 10 – 5000 pM, preferably between 10 – 2000 pM, for example, between 10 – 1500 pM, between 10 – 100 pM, between 10 – 500 pM, for example between 10 – 250 pM or 10 – 100 pM. In some embodiments the Kd value is at least 10 pM, at least 15 pM, at least 20 pM, or at least 25 pM (when determined in absence of the at least one second moiety, i.e. on its own).

[0097] Any Kd value less than 10-6is considered to indicate binding. Specific binding of an antigen-binding molecule to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.

[0098] It was surprisingly found that immunoglobulin chain variable domains of the first moiety capable of specifically binding TGFBR2, preferably TGFBR2 extracellular domain with the indicated affinities may be advantageously used in the conjugate according to the invention.

[0099] In an embodiment of the invention, the immunoglobulin chain variable domains of the first moiety capable of specifically binding TGFBR2 binds to TGFBR2 on a binding-site that at least partially overlaps with the binding-site of TGFB on TGFBR2. The overlap with the binding site may also be by steric hinderance, thereby the immunoglobulin chain variable domains of the first moiety (and or second moiety) hindering interaction of TGFB with the TGBR2.

[0100] Also provided is for a conjugate according to the invention, the conjugate comprising (i) a first moiety, wherein the first moiety is or comprises an immunoglobulin chain variable domain which is capable of specifically binding TGFBR2, and (ii) at least a second moiety, wherein the second moiety is capable of binding a target, and wherein the immunoglobulin chain variable domain fragment of the first moiety comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: -the sequence of CDR1 is selected from SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, SEQ ID NO: 146, and SEQ ID NO: 154. -the sequence of CDR2 is selected from SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, SEQ ID NO: 148, , and SEQ ID NO: 156 and / or -the sequence of CDR3 is selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 150, and , and SEQ ID NO:158.

[0101] Also provided is for a conjugate according to the invention that is a biosimilar to any of the conjugates described herein.

[0102] Also provided is for a conjugate and wherein CDR1, CDR2 and / or CDR3, each comprise one or two amino acid substitutions as compared to any of the sequence of the CDR1, CDR2 and / or CDR3 provided herein. For example, in some embodiments, the CDR1comprised in the immunoglobulin chain variable domain fragment of the first moiety comprises a sequence that is identical to the sequence according to SEQ ID NO:2 wherein 1 or 2 amino acids are substituted. As will be understood by the skilled person, such immunoglobulin chain variable domain fragment of the first moiety would still require it being capable of specifically binding TGFBR2 as disclosed herein.

[0103] In some embodiments, the immunoglobulin chain variable domain fragment of the first moiety comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein:

[0104] -the sequence of CDR1 is selected from SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, SEQ ID NO: 146, and , and SEQ ID NO:154,

[0105] -the sequence of CDR2 is selected from SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, SEQ ID NO: 148, and , and SEQ ID NO: 156, and

[0106] -the sequence of CDR3 is selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 150, and , and SEQ ID NO: 158.

[0107] In some embodiments, the immunoglobulin chain variable domain fragment of the first moiety comprises a CDR1, a CDR2 and a CDR3 with the following sequence combinations (in rows): CDR1 CDR2 CDR3 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 2 4 6 10 12 14 18 20 22 26 28 30 34 36 38 42 44 46 50 52 54 58 60 62 66 68 70 74 76 78 82 84 86 90 92 94 98 100 102 106 108 110 114 116 118 122 124 126 130 132 134 138 140 142 146 148 150 154 156 158

[0108] Also provided is for a conjugate according to the invention and wherein the immunoglobulin chain variable domain fragment of the first moiety comprises a CDR1, a CDR2 and CDR3 as described herein, and wherein the FR1 sequence is selected from SEQ ID Nos: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145 and 153, the FR2 sequence is selected from SEQ ID Nos: 3, 11,19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147 and 155, the FR3 sequence is selected from SEQ ID Nos: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149 and 157, and / or wherein the FR4 sequence is selected from SEQ ID Nos: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151 and 159

[0109] In some embodiments, the immunoglobulin chain variable domain fragment of the first moiety comprises a FR1, a CDR1, a FR2, a CDR2, a FR3, a CDR3 and a FR4 with the following sequence combinations (in rows): FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:) NO:) NO:) NO:) NO:) NO:) NO:) 1 2 3 4 5 6 7 9 10 11 12 13 14 15 17 18 19 20 21 22 23 25 26 27 28 29 30 31 33 34 35 36 37 38 39 41 42 43 44 45 46 47 49 50 51 52 53 54 55 57 58 59 60 61 62 63 65 66 67 68 69 70 71 73 74 75 76 77 78 79 81 82 83 84 85 86 87 89 90 91 92 93 94 95 97 98 99 100 101 102 103 105 106 107 108 109 110 111 113 114 115 116 117 118 119 121 122 123 124 125 126 127 129 130 131 132 133 134 135 137 138 139 140 141 142 143 145 146 147 148 149 150 151 153 154 155 156 157 158 159

[0110] Although the invention is not in particular limited to a conjugate comprising an immunoglobulin chain variable domain fragment of the first moiety having a FR1, CDR1, FR2, CDR2, FR3, CDR3 and / or FR4 as disclosed herein, it was found that in particular a conjugate comprising an immunoglobulin chain variable domain fragment of the first moiety having a FR1, CDR1, FR2, CDR2, FR3, CDR3 and / or FR4 with a sequence as disclosed herein can advantageously be used.

[0111] The first moiety and / or the second moiety may also be or comprise an affibody molecule or other antibody mimetics know to the skilled person.

[0112] Therefor, also provided is a conjugate according to the invention, wherein the immunoglobulin chain variable domain of the first moiety comprises an amino acid sequence having 80% sequence identity to any one of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, SEQ ID NO: 152 and / or SEQ ID NO: 160, preferably wherein the immunoglobulin chain variable domain comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NO: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, SEQ ID NO: 152 and / or SEQ ID NO: 160.

[0113] Preferably, the immunoglobulin chain variable domain comprised in the first moiety comprises any one SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, and / or SEQ ID NO: 143, preferably all. Preferably the immunoglobulin chain variable domain comprised in the first moiety comprises SEQ ID NO: 144. In some embodiments, the immunoglobulin chain variable domain comprised in the first moiety is a biosimilar of the immunoglobulin chain variable domain comprising SEQ ID NO: 144.

[0114] In some embodiments, the immunoglobulin chain variable domain comprises an amino acid sequence having at least 80, 82, 85, 87, 90, 92, 95, 96, 97, 98, or 99% sequence identity to any one of SEQ ID NO: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, SEQ ID NO: 152 and / or SEQ ID NO: 160. In some preferred embodiments, the immunoglobulin chain variable domain comprises an amino acid sequence having at least 80, 82, 85, 87, 90, 92, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 144.

[0115] Also provided is for a conjugate according to the invention, wherein the target that the second moiety is capable of binding is a cell surface receptor, preferably a cell surface receptor that is expressed by a cell-type that expresses TGFBR2, in particular a cell-type of interest, for example, and wherein TGFBR2-mediated TGFB signaling is to be inhibited (in a cell-type restricted manner).

[0116] Also provided is for a conjugate according to the invention, wherein the target that the second moiety is capable of binding is an antigen, for example a neoantigen, preferably an antigen, for example a neoantigen that is expressed by a cell-type that expresses TGFBR2, in particular a cell-type of interest, for example, and wherein TGFBR2-mediated TGFB signaling is to be inhibited (in a cell-type restricted manner).

[0117] Also provided is for a conjugate according to the invention, wherein the target that the second moiety is capable of binding is expressed on the surface of a cell, such as a cell surface receptor, in a cell that expresses TGFBR2. Preferably the target is selected from CD44 (a cell-surface glycoprotein involved in cell–cell interactions, cell adhesion and migration), CD8 (cluster of differentiation 8 (CD8) is a transmembrane glycoprotein that serves as a co-receptor for the T-cell receptor (TCR)), HER2 (human epidermal growth factor receptor 2 is a protein that normally resides in the membranes of cells, and is overexpressed in breast cancer), PLD1 (programmed Cell Death Ligand 1), PD-1 (Programmed Cell Death Protein 1), CTLA4 (Cytotoxic T-lymphocyte associated protein 4), EGFR (Epidermal growth factor receptor), c-MET, CD3, CD4, TCR, CAR (chimeric antigen receptor), Fibroblast activating protein (FAP), fibroblast specific protein (FSP), and / or PDGF-beta receptor.

[0118] As the skilled person understands, in embodiments, the biological activity of target that the second moiety is capable of binding may be stimulated or may be inhibited (for example in case EGFR or HER2 is the target), as desired, upon binding of the second moiety with its target. For example, in some embodiments, the second moiety may be an immunoglobulin chain variable domain which is capable of specifically binding the target and thereby inhibit or stimulate (activate) the target, for example the cell surface receptor. However, in other embodiments the target is not inhibited or stimulated or activated upon binding by the second moiety. In such embodiments, binding of the second moiety to its target is merely aimed at engagement of the conjugate according to the invention with a target cells (for example in case of the target cell being an immune cell, for example expressing CD3, CD4 or CD8 as target for the second moiety (the immune cell may, for example, be a T cell, a NK cell, a dendritic cell, or any other immune cell that elicits immune promoting effects). There are also immune cells that mediate immune suppressive effects such as immune suppressive Tregs, immune suppressive myeloid derived suppressor cells.

[0119] In embodiments wherein the target of the second moiety is expressed on a diseased cell, for example a cancer cell or a cancer-associated fibroblast (CAF), or on an immune cells that mediate immune suppressive effects, preferably the biological activity of the target that the second moiety is capable of binding may be stimulated or may be inhibited, as desired, upon binding of the second moiety with the target.

[0120] As mentioned herein elsewhere, in preferred embodiments, the second moiety is an immunoglobulin chain variable domain, which is capable of specifically binding the target. In such embodiments, preferably the target is a cell surface receptor, preferably a cell surface receptor that is expressed by a cell that expresses TGFBR2.

[0121] In other words, preferably the conjugate according to the invention comprises a first moiety that is or comprises an immunoglobulin chain variable domain, which is capable of specifically binding TGFBR2 and comprises at least a second moiety that is or comprises an immunoglobulin chain variable domain which is capable of specifically binding a target, preferably a target other than TGFBR2. In such embodiments, for example, the first moiety is an antibody as described specifically binding TGFBR2 and the second moiety is an antibody as described specifically binding to a target, preferably wherein the target is selectively expressed on cell types in which the conjugate according to the invention is to inhibit a cell-type specific effect (e.g. mediated via TGFBR2-mediate TGFB signaling and / or cellular responses mediated by the target of the second moiety).

[0122] Therefor, in preferred embodiments, a conjugate according to the invention is provided wherein the immunoglobulin chain variable domain of the first moiety and / or (preferably “and”) the second moiety is an antibody or antibody fragment, preferably selected from a single variable domain, a VH, a VL, a VHH, a dAb, an antigen binding antibody fragment, a Fab, a F(ab′)2, a Fd, a Fv a sCFV, a diabody, a tandem diabody, a nanobody, or a V-NAR.

[0123] Is some embodiments, the immunoglobulin chain variable domain of the first moiety and the immunoglobulin chain variable domain of the second moiety are of the same type (for example, both are a nanobody, or both are a Fab). In other embodiments, the immunoglobulin chain variable domain of the first moiety and the immunoglobulin chain variable domain of the second moiety are of a different type (for example, the immunoglobulin chain variable domain of the first moiety is a nanobody and the immunoglobulin chain variable domain of the second moiety is not, for example is a full IgG antibody).

[0124] As detailed herein elsewhere, in a preferred embodiment, the conjugate is capable of inhibiting TGFB signaling, preferably TGFBR2-mediated TGFB signaling. In such embodiments, TGFB signaling, preferably TGFBR2-mediated TGFB signaling is inhibited upon binding of the first moiety and the second moiety with a target cells (for example, which expresses TGFBR2 and the target of the second moiety of the cell surface).

[0125] The skilled person understand how inhibition of TGFB signaling, preferably TGFBR2 mediated TGFB signaling can be determined, for example, as disclosed herein.

[0126] Also provided is for a conjugate according to the invention and wherein the conjugate comprises at least one further moiety, preferably wherein the further moiety is a therapeutic drug, a toxin, a cytokine, a radionuclide, an enzyme, a ligand, a further immunoglobulin chain variable domain, a stabilizer, a diagnostic molecule, or tag.

[0127] For example, is some embodiments, the conjugate according to the invention comprises a therapeutic drug useful in the treatment of a condition, for example, a tumor. With the conjugate according to the invention the therapeutic drug can be delivered to the target cell.

[0128] In some embodiments, the at least one further embodiment is a stabilizer, for example a stabilizer that improves half-life of the conjugate according to the invention. Is some embodiments, the stabilizer is albumin.

[0129] In some embodiments, the at least one further embodiment is a diagnostic molecule or a tag. For example, the diagnostic molecule or the tag may be used to localize target cells in a tissue.

[0130] In some embodiments, the first moiety, the second moiety, and optionally, the at least one further moiety are linked to each other via a linker, preferably polypeptide linker.

[0131] The skilled person is well-aware of methods and means from providing a conjugate according to the invention, and wherein the first moiety, the second moiety, and optionally, the at least one further moiety are linked to each other via a linker, preferably polypeptide linker (see, for example, Zhang et al (2024) J Med Chem doi: 10.1021 / acs.jmedchem.4c00106.).

[0132] Also provided is for the conjugate according to the invention, wherein the first moiety and the second moiety are, independently, capable of binding to a target expressed on the cell surface of the same cell, preferably wherein the cell is a lymphocyte, a T cell, a CD8+ T cell, a NK cell, a CAR T cell, a CAR NK cell, a tumor cell, a dendritic cell, a monocyte, a macrophage, a neutrophile, a fibroblast, a cancer- associated fibroblast, a macrophage.

[0133] In such embodiment, the cell expresses TGFBR2 and a target of the at least one second moiety and thus allows the conjugate according to the invention to bind with both TGFBR2 and with a target of the at least one second moiety. As explained herein elsewhere, the at least one second moiety thus allows for the selective binding of the conjugate according to the invention with a target cell-type (or cell of interest) that, in addition to TGFBR2, expresses the target to which the second moiety is capable of binding to (in comparison to a cell-type not expressing such target to which the second moiety is capable of binding to). In addition, and as already detailed herein elsewhere, the binding of the conjugate to the target of the second moiety at the same time allows sufficient interaction of the first moiety with the TGFBR2 to allow for inhibition of TGFB signaling, preferably TGFBR2-mediated TGFB signaling. Thus, although the conjugate according to the invention may bind to a TGFBR2 expressed on a cell-type, the presence of the target to which the second moiety is capable of binding to is required for efficient and desirable cell-type restricted (or -specific) inhibition of TGFB signaling, preferably TGFBR2-mediated TGFB signaling.

[0134] The skilled person will understand that selectivity for a particular cell-type (or cell of interest) may be further increased by including in the conjugate according to the invention additional moieties that a capable of binding with a target expressed by said target cell-type (or cell of interest).

[0135] Also provided is for a cell comprising a conjugate according to the invention, preferably wherein the first moiety is bound to TGFBR2 expressed by the cell and / or wherein the second moiety is bound to the target the second moiety is capable of binding expressed by the cell.

[0136] Preferably the cell is cell is a lymphocyte, a T cell, a CD8+ T cell, a NK cell, a CAR T cell, a CAR NK cell, a tumor cell, a dendritic cell, a monocyte, a macrophage, a neutrophil, a fibroblast, a cancer-associated fibroblast, a macrophage. The cell may be a cell in a subject, for example a human subject, for example a patient. The cell may also be a cell that is outside a subject, for example in a test tube. The cell may be in an in vivo tissue or in an in vitro tissue.

[0137] Also provided is for an immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain), which is capable of specifically binding TGFBR2, and wherein the immunoglobulin chain variable domain fragment comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: -the sequence of CDR1 is selected from SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, SEQ ID NO: 146 and SEQ ID NO: 154, -the sequence of CDR2 is selected from SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, SEQ ID NO: 148 and SEQ ID NO: 156, and / or -the sequence of CDR3 is selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 150 and SEQ ID NO: 158.

[0138] Also provided is for an immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain), which is capable of specifically binding TGFBR2, preferably TGFBR2 extracellular domain, according to the invention that is a biosimilar of such immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain).

[0139] Also provided is for an immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) which is capable of specifically binding TGFBR2, preferably TGFBR2 extracellular domain, and wherein CDR1, CDR2 and / or CDR3, each comprise one or two amino acid substitutions as compared to any of the sequence of the CDR1, CDR2 and / or CDR3 provided herein. For example, in some embodiments, the CDR1 comprises a sequence that is identical to the sequence according to SEQ ID NO:10 wherein 1 or 2 amino acids are substituted. As will be understood by the skilled person, such immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) would still require it being capable of specifically binding TGFBR2, preferably TGFBR2 extracellular domain, as disclosed herein.

[0140] In some embodiments, the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: -the sequence of CDR1 is selected from SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, SEQ ID NO: 146 and SEQ ID NO: 154, -the sequence of CDR2 is selected from SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, SEQ ID NO: 148 and SEQ ID NO: 156, and -the sequence of CDR3 is selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 150 and SEQ ID NO: 158.

[0141] In some embodiments, the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises a CDR1, a CDR2 and a CDR3 with the following sequence combinations (in rows): CDR1 CDR2 CDR3 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) 2 4 6 10 12 14 18 20 22 26 28 30 34 36 38 42 44 46 50 52 54 58 60 62 66 68 70 74 76 78 82 84 86 90 92 94 98 100 102 106 108 110 114 116 118 122 124 126 130 132 134 138 140 142 146 148 150 154 156 158

[0142] Also provided is for an immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) according to the invention and wherein the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises a CDR1, a CDR2 and CDR3 as described herein, and wherein the FR1 sequence is selected from SEQ ID Nos: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145 or 153, the FR2 sequence is selected from SEQ ID Nos: 3, 11,19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147 and 155, the FR3 sequence is selected from SEQ ID Nos: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149 and 157, and / or wherein the FR4 sequence is selected from SEQ ID Nos: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143, 151, and 159.

[0143] In some embodiments, the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises a FR1, a CDR1, a FR2, a CDR2, a FR3, a CDR3 and a FR4 with the following sequence combinations (in rows): FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO:) NO:) NO:) NO:) NO:) NO:) NO:) 1 2 3 4 5 6 7 9 10 11 12 13 14 15 17 18 19 20 21 22 23 25 26 27 28 29 30 31 33 34 35 36 37 38 39 41 42 43 44 45 46 47 49 50 51 52 53 54 55 57 58 59 60 61 62 63 65 66 67 68 69 70 71 73 74 75 76 77 78 79 81 82 83 84 85 86 87 89 90 91 92 93 94 95 97 98 99 100 101 102 103 105 106 107 108 109 110 111 113 114 115 116 117 118 119 121 122 123 124 125 126 127 129 130 131 132 133 134 135 137 138 139 140 141 142 143 145 146 147 148 149 150 151 153 154 155 156 157 158 159

[0144] Therefor, also provided is a immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain), wherein the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises an amino acid sequence having 80% sequence identity to any one of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, SEQ ID NO: 152 and / or SEQ ID NO: 160, preferably wherein the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NO: SEQ ID NO: 8 (TGFBR2-VHH- B01), SEQ ID NO: 16 (TGFBR2-VHH-B03), SEQ ID NO: 24 (TGFBR2-VHH-C05), SEQ ID NO: 32 (TGFBR2-VHH-D05) , SEQ ID NO: 40 (TGFBR2-VHH-D09), SEQ ID NO: 48 (TGFBR2-VHH-D10), SEQ ID NO: 56 (TGFBR2-VHH-E05), SEQ ID NO: 64(TGFBR2-VHH-F11), SEQ ID NO: 72 (TGFBR2-VHH-F12), SEQ ID NO: 80 (TGFBR2-VHH-G01), SEQ ID NO: 88 (TGFBR2-VHH-G02), SEQ ID NO: 96 (TGFBR2-VHH-G09), SEQ ID NO: 104 (TGFBR2-VHH-H10), SEQ ID NO: 112 (TGFBR2-VHH-B03*), SEQ ID NO: 120 (TGFBR2-VHH-C09*), SEQ ID NO: 128 (TGFBR2-VHH-C12*), SEQ ID NO: 136 (TGFBR2-VHH-D12*), SEQ ID NO: 144 (TGFBR2-VHH-E03*), SEQ ID NO: 152 (TGFBR2-VHH-H09*), and / or SEQ ID NO: 160 (TGFBR2-VHH-D10-Y62S).

[0145] In some embodiments, the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises an amino acid sequence having at least 80, 82, 85, 87, 90, 92, 95, 96, 97, 98, or 99% sequence identity to any one of SEQ ID NO: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, SEQ ID NO: 152 and / or SEQ ID NO: 160.

[0146] Also provided is for the conjugate according to the invention or the immunoglobulin chain variable domain comprising molecule for use as a medicament, preferably for use in the treatment of cancer, fibrosis, and / or inflammatory disease or in immunotherapy.

[0147] Also provided is for a pharmaceutical composition comprising a conjugate according to the invention or an immunoglobulin chain variable domain comprising molecule according to the invention.

[0148] As will be understood by the skilled person, dosages, and desired concentrations of pharmaceutical compositions of the invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of the skilled person. Animal experiments may provide reliable guidance for the determination of effective doses for human therapy.

[0149] Pharmaceutical compositions according to the invention can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0150] Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0151] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may, for example, comprise between 0.1% and 100% (w / w) active ingredient.

[0152] The agents (e.g. conjugate according to the invention) and (pharmaceutical) compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and / or lymph supply, and / or direct administration to an affected site.

[0153] Also provided is for a method of treatment of a patient in need thereof, wherein the method comprises administration of a conjugate or the immunoglobulin chain variable domain comprising molecule according to the invention to the patient.

[0154] Administration may be any means known to the skilled person, for example as described herein, and preferably in an effective dose.

[0155] Also provided is for a method of preparing a conjugate according to any one of the previous claims wherein the method comprises coupling of the first moiety with the second moiety.

[0156] As explained herein elsewhere, the coupling may be direct coupling (wherein the first moiety and the second moiety are directly connected to each other) or may be direct coupling (for example, wherein the first moiety and the second moiety are connected to each via an intermediate structure, such as a linked or another moiety).

[0157] The skilled person is well aware of methods and means suitable for preparing a conjugate according to the invention, by coupling the first and the second moiety.

[0158] Also provided is for a method of (preferably cell-type restricted or cell-type specific) modulation, preferably inhibition, of TGFB signaling in a cell, preferably TGFBR2-mediated TGFB signaling in a cell, the method comprising -providing a conjugate according to the invention, wherein the second moiety is capable of binding a target that is expressed by the cell (target cell or cell of interest), preferably wherein the target is expressed on the cell surface of the cell, more preferably wherein the target is a cell surface receptor expressed by the cell, -contacting the conjugate with the cell, preferably in the presence of TGFB.

[0159] The method may be performed in vitro or in vivo, for example is a test animal or in a human subject, for example, a patient.

[0160] The skilled person knows how to perform the method of (cell-specific) modulation, preferably inhibition, of TGFB signaling in a cell, preferably TGFBR2- mediated TGFB signaling in a cell. In some embodiment, TGFB is provided in the method, for example in carrying concentrations. In other embodiments, TGFB is naturally present in, for example, the test animal and / or the human subject, in which case it is not needed to provide further TGFB in the method.

[0161] Finally, there is provided for a polynucleotide encoding the conjugate, preferably fusion protein according to the invention or the immunoglobulin chain variable domain comprising molecule according to the invention, or a cell comprising such polynucleotide. The polynucleotide may, for example, be in the form of a vector. A vector is a man-made nucleic acid molecule (polynucleotide) resulting from the use of recombinant DNA technology. These vectors therefore do not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. A vector can be used to deliver the nucleic acid to the interior of a cell (and / or therewith allows translation in the corresponding polypeptide or fusion protein according to the invention). Numerous vectors are known in the art including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds and liposomes. Representative examples of viral vectors include adenoviral vectors, adeno- associated virus vectors, lentivirus vectors and retroviral vectors.

[0162] The cell may be any cell, including prokaryotic cells, eukaryotic cells, cell lines and so on.

[0163] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[0164] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0165] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[0166] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art. EXAMPLES

[0167] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[0168] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

[0169] Experiments were performed using conjugates according to the invention, comprising at least a first moiety according to the invention and a second moiety according to the invention, and results are shown in the figures.

[0170] In Figure 1A the efficiency of different nanobodies fused to Fc domain are tested for the ability to immuno-precipitate TGF-beta type II receptor (TGFBR2), which is provided with an epitope FLAG tag. TGFBR2-FLAG is overexpressed in HEK 293T cells. Input of cell lysate is shown on the right, M2 is an anti-flag antibody and serves as a positive control. The TGFBR2-VHH-D10 is most efficient to immuno-precipitate. No nanobody is negative control and shows now signal.

[0171] Figure 1B shows a similar set up as in Figure 1A. But now the TGF-beta type II receptor is engineered with a MYC epitope tag and is overexpressed in HEK 293T cells. One can see that the affinity matured nanobodies bind more efficiently than the original TGFBR2-VHH-D10 that was used for the affinity maturation. Input of cell lysate is indicated on the left. Positive control with anti-Myc antibody is also shown on the left. No nanobody negative control, no signal is detected.

[0172] The experiment in Figure 2A shows that the TGFBR2 nanobody in Fc format does not inhibit exogenous TGFB induced TGFB signaling as measured with a TGFB / SMAD3 transcriptional reporter construct. SMAD3 is the intracellular effector of TGFB, and SMAD3 upon its phosphorylation forms a heteromeric complex with SMAD4 and mediates a transcriptional response. The so-called CAGA in a SMAD3 / 4 DNA binding element that when multimerized and placed in front of a minimal promotor creates a reporter that can read out TGFB / SMAD3 signaling. Reporter construct is fused to GFP, so the level of GFP signal is a measure for how much SMAD3 is activated by the TGFB receptor complex, in response to TGFB stimulation. Figure 2B shows that when we overexpress TGF-beta type II receptor (TGFBR2) at super high non-physiological expression levels in HEK293T cells we can also activate the CAGA--GFP reporter. This TGFBR2-initiated response can thus occur in the absence of exogenous TGFB. In this way we make the assay highly sensitive and driven by the very high ectopic overexpression of TGBR2. It is TGFBR2 that is targeted by the TGFBR2 nanobody. This assay allowed us to evaluate the inhibitory potency of TGFBR2 nanobody clones; TGFBR2-VHH-B03*-Fc is more efficient than TGFBR2- VHH-B03-Fc, which is more efficient than TGFBR2-VHH-D10-Fc

[0173] Figure 3A shows that an affibody to EGFR zEGFR fused to TGFBR2 nanobody is most efficient in inhibiting TGFB that is exogenously added to the culture medium) in A431 cells as compared to other cell lines. A431 cells highly express the EGFR, and which is not the case in the other cell lines. The affibody HER2 zHER2 to TGFBR2 nanobody is most efficient to inhibit the signaling of exogenous TGFB in SCOV3 cells that highly express HER2 when compared to other cell lines.

[0174] Figure 3B shows the differential expression of EGFR and HER2 in four different cancer cell lines. EGFR is high in A431 and low in other cell lines, and HER2 is high in SVOV3 and lows in other cell lines. This analysis was performed using the human protein atlas (https: / / www.proteinatlas.org / )

[0175] A431 is an epidermal cancer cell line (characteristic for high levels of EGFR expression)., MDA MB231 is a human triple negative breast cancer cell line, Hep2 is a human hepatocellular carcinoma cell line, SVOV3 is a human ovarian carcinoma cell line.

[0176] Figure 4 shows the binding of TGFBR2 nanobodies (at different doses) to TGFBR2 extracellular domain protein. The apparent affinities are indicated.

[0177] In addition, alpha-fold predictions of TGFBR2 extracellular domain with either TGFB (top) and TGFBR2 nanobody (bottom) shows the overlap of binding / contact sites of TGFB and TGFBR2 to TGFBR2 extracellular domain. This is consistent with ability of TGFBR2 nanobody to inhibit, albeit inefficiently the exogenous TGFB binding to TGFBR2 extracellular domain and not being efficient in inhibiting TGFB signaling (see Figure 6).

[0178] Figure 7, 8 and 9 shows results of experiments with conjugates according to the invention, having a first moiety, as disclosed herein, directed to TGFBR2, and a second moiety according to the invention, directed to CD4. Figure 8 A conforms CD4 expression in HBB ALL, MOLT3, and THP1 cells (but not in Jurkat cells) by Western blot. Figure 8B shows that TGFβ-induced SMAD3 / 4-dependent transcriptional GFP reporter activity in THP1 cells was attenuated by the TGFBR2 VHH fused to CD4 VHH with -His epitope tag (TGFBR2-VHH – CD4-CHH-His) (i.e. one of the conjugates shown in Figure 7). Figures 8 C-E show that TGFBR2-VHH - CD4-VHH-His or TGFBR2-VHH - CD4-VHH (as shown in Figure 7), potently inhibited TGFβ-induced SMAD2 phosphorylation in CD4+ cell lines (HBB ALL, MOLT3, THP1) but not in CD4– Jurkat cells. Figure 9 shows that in primary CD4 T cells, both TGFBR2-VHH - CD4- VHH variants shown in Figure 7 inhibited TGF^-induced SMAD2 phosphorylation.

[0179] Figure 10 shows that an affibody to EGFR zEGFR fused to TGFB2 VHH is more efficient in inhibiting signaling of TGF^ that is exogenously added to the culture medium in A431 cells (that highly express EGFR) as compared to SKOV3 cells (that show very low EGFR expression), and an affibody to HER2 zHER2 fused to TGFB2 VHH is more efficient in inhibiting signaling of TGF^ that is exogenously added to the culture medium in SKOV3 cells (that highly express HER2) as compared to A431 cells (that show very low HER2 expression). The experiment in Figure 10 used the same experimental set-up as in Figure 3A.

[0180]

[0181] Experiments with a conjugate according to the invention and wherein the target of the second moiety was CD8, in line with the experiments above, showed in that case the conjugate was successful in inhibiting TGFBR2-mediated TGFB signaling in CD8 expressing cells.

[0182] Material and Methods as used in herein: Reagents

[0183] Recombinant TGF^3 was provided by A. Hinck, University of Pittsburgh, USA. TGF^3 was dissolved in 4 mM HCl / 0.1% ultra pure bovine serum albumin (BSA). Selective small molecule TGFBRI kinase inhibitor SB505124 (1 mM, DMSO, #3263, Tocris, Bristol, UK). MDA-MB-231, HepG2, SKOV3, A431, THP1, and leukemic cell lines were obtained from ATCC. MDA-MB-231, HepG2, SKOV3, and A431 were cultured in DMEM containing 10% fetal bovine serum (FBS) and 100 U / mL penicillin- streptomycin (15140122; Thermo Fisher Scientific, Waltham, MA, USA). THP1 and leukemia cells were cultured in RPMI containing 10% FBS and 100 U / mL penicillin- streptomycin (15140122; Thermo Fisher Scientific, Waltham, MA, USA). Primary CD4 T cells were obtained from Lonza, and cultured, expanded, and activated according to the manufacturer's recommendations. All cell lines were cultured in 37 °C, 5% CO2incubators and routinely tested for the absence of mycoplasma infections. All human cell lines were authenticated using short tandem repeat (STR) profiling. DNA cloning and expression

[0184] All the constructs were made using general cloning methods in the pFUSE vector (Invivogen) from which the Fc was removed. The generated plasmids were transfected in 293 cells using PEI-MAX (polysciences). 24h after transfection the media was changed to DMEM w / o FBS and another 24h later the conditioned media was collected and passed through a 0,22 uM filter. Transcriptional Fluorescent Protein–Based Reporter Assay.

[0185] The CAGA-dynGFP lentiviral vector was used as previously described (Cancers (Basel).2022 May 19;14(10):2508. doi: 10.3390 / cancers14102508. PMID: 35626109; PMCID: PMC9139966). In short, cells containing the CAGA-dynGFP were seeded in 96-well plates. The following day, the cells were stimulated in full media and then placed in the IncuCyte S3 live-cell imaging analysis system (Sartorius). The cells were subsequently imaged every 3 h for a period of 48 h. Fluorescence intensity was analyzed using the IncuCyte software. Western Blotting

[0186] Western blotting was performed as previously described (Cancer Res. 2006 Feb 15;66(4):2202-9. doi: 10.1158 / 0008-5472.CAN-05-3560. PMID: 16489022.). Protein lysates were harvested in Laemmli buffer (0.12 M Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 35 mM β-mercaptoethanol, and bromophenol blue) and boiled for 5 min. Western blotting was performed using standard procedures. Membranes were blocked in 5% non-fat dry milk for 1 h at room temperature and incubated with primary antibody in 2.5% non-fat dry milk overnight at 4 °C. A previously described primary pSMAD2 antibody was used (FEBS Lett. 1998 Aug 28;434(1-2):83-7. doi: 10.1016 / s0014- 5793(98)00954-5. PMID: 9738456). Fluorescently conjugated secondary antibody (1:10,000 in 5% bovine serum albumin (BSA) containing TBST) was used to detect the protein bands by ImageDoc.

Claims

CLAIMS 1. A conjugate comprising (i) a first moiety, wherein the first moiety is or comprises an immunoglobulin chain variable domain which is capable of specifically binding transforming growth factor-beta (TGFB) receptor 2 (TGFBR2), and (ii) at least a second moiety, wherein the second moiety bind a target, and wherein the immunoglobulin chain variable domain of the first moiety is on its own unable to inhibit TGFB signaling.

2. The conjugate according to claim 1, wherein the immunoglobulin chain variable domain of the first moiety is an immunoglobulin chain variable domain with an apparent Kd value of between 10 – 5000 pM, preferably between 10 – 2000 pM, for example, between 10 – 1500 pM, between 10 – 100 pM, between 10 – 500 pM, for example between 10 – 250 pM or 10 – 100 pM.

3. A conjugate comprising (i) a first moiety, wherein the first moiety is or comprises an immunoglobulin chain variable domain which is capable of specifically binding TGFBR2, and (ii) at least a second moiety, wherein the second moiety is capable of binding a target, and wherein the immunoglobulin chain variable domain fragment of the first moiety comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: -the sequence of CDR1 is selected from SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, and SEQ ID NO: 146, -the sequence of CDR2 is selected from SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ IDNO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, and SEQ ID NO: 148, and / or -the sequence of CDR3 is selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, and SEQ ID NO:

150.

4. The conjugate according to claim 3, wherein the immunoglobulin chain variable domain of the first moiety comprises an amino acid sequence having 80% sequence identity to any one of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, and / or SEQ ID NO: 152, preferably wherein the immunoglobulin chain variable domain comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NO: SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, and / or SEQ ID NO:

152.

5. The conjugate according to any one of the previous claims, wherein the target that the second moiety is capable of binding is a cell surface receptor, preferably a cell surface receptor that is expressed by a cell that expresses TGFBR2, and preferably wherein the target is selected from CD44, CD8, HER2, PD-1, CTLA4, EGFR, c-MET, CD3, CD4, TCR, CAR, Fibroblast activating protein (FAP), fibroblast specific protein (FSP), and / or PDGF-beta receptor.

6. The conjugate according to any one of the previous claims, wherein the second moiety is or comprises an immunoglobulin chain variable domain which is capable of specifically binding the target, preferably wherein the target is a cellsurface receptor, preferably a cell surface receptor that is expressed by a cell that expresses TGFBR2.

7. The conjugate according to any one of the previous claims, wherein the immunoglobulin chain variable domain of the first moiety and / or the second moiety is an antibody or antibody fragment, preferably selected from a single variable domain, a VH, a VL, a VHH, a dAb, an antigen binding antibody fragment, a Fab, a F(ab′)2, a Fd, a Fv a sCFV, a diabody, a tandem diabody, a nanobody, or a V-NAR.

8. The conjugate according to any one of the previous claims, wherein the conjugate is capable of inhibiting TGFB signaling.

9. The conjugate according to any one of the previous claims, wherein the conjugate comprises at least one further moiety, preferably wherein the further moiety is a therapeutic drug, a toxin, a cytokine, a radionuclide, an enzyme, a ligand, a further immunoglobulin chain variable domain, a stabilizer, a diagnostic molecule, or tag.

10. The conjugate according to any one of the previous claims, wherein the first moiety, the second moiety, and optionally, the at least one further moiety are linked to each other via a linker, preferably polypeptide linker.

11. The conjugate according to any one of the previous claims, wherein the first moiety and the second moiety are, independently, capable of binding to a target expressed on the cell surface of the same cell, preferably wherein the cell is a lymphocyte, a T cell, a CD8+ T cell, a NK cell, a CAR T cell, a CAR NK cell, a tumor cell, a dendritic cell, a monocyte, a macrophage, a neutrophil, a fibroblast, a cancer-associated fibroblast, a macrophage.

12. A cell comprising a conjugate according to any one of the previous claims, preferably wherein the first moiety is bound to TGFBR2 expressed by the celland / or wherein the second moiety is bound to the target the second moiety is capable of binding expressed by the cell.

13. An immunoglobulin chain variable domain comprising molecule which is capable of specifically binding TGFBR2, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein: -the sequence of CDR1 is selected from SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 42, SEQ ID NO: 50, SEQ ID NO: 58, SEQ ID NO: 66, SEQ ID NO: 74, SEQ ID NO: 82, SEQ ID NO: 90, SEQ ID NO: 98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, and SEQ ID NO: 146, -the sequence of CDR2 is selected from SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 36, SEQ ID NO: 44, SEQ ID NO: 52, SEQ ID NO: 60, SEQ ID NO: 68, SEQ ID NO: 76, SEQ ID NO: 84, SEQ ID NO: 92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, and SEQ ID NO: 148, and / or -the sequence of CDR3 is selected from SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 46, SEQ ID NO: 54, SEQ ID NO: 62, SEQ ID NO: 70, SEQ ID NO: 78, SEQ ID NO: 86, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, and SEQ ID NO:

150.

14. The immunoglobulin chain variable domain comprising molecule according to claim 13, wherein the immunoglobulin chain variable domain comprises an amino acid sequence having 80% sequence identity to any one SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, and / or SEQ ID NO: 152, preferably wherein the immunoglobulin chain variable domain (or a molecule comprising such immunoglobulin chain variable domain) comprises an amino acid sequence having 100% sequence identity to any one of SEQ ID NO: SEQ ID NO: 8, SEQ IDNO: 16, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40, SEQ ID NO: 48, SEQ ID NO: 56, SEQ ID NO: 64, SEQ ID NO: 72, SEQ ID NO: 80, SEQ ID NO: 88, SEQ ID NO: 96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, and / or SEQ ID NO:

152.

15. The conjugate according to any one of the previous claims or the immunoglobulin chain variable domain comprising molecule for use as a medicament, preferably for use in the treatment of cancer, fibrosis, an (auto)immune disease and / or inflammatory disease or in immunotherapy.

16. A method of treatment of a patient in need thereof, wherein the method comprises administration of a conjugate or the immunoglobulin chain variable domain comprising molecule according to any one of the previous claims to the patient.

17. A method of preparing a conjugate according to any one of the previous claims wherein the method comprises coupling of the first moiety with the second moiety.

18. A method of modulation of TGFB signaling in a cell, preferably in a cell-type restricted manner, the method comprising (i) providing a conjugate according to any one of the previous claims, wherein the second moiety is capable of binding a target that is expressed by the cell, preferably wherein the target is expressed on the cell surface of the cell or is a cell surface receptor, (ii) contacting the conjugate with the cell, preferably in the presence of TGFB.

19. A polynucleotide encoding the conjugate, preferably fusion protein or the immunoglobulin chain variable domain comprising molecule according to any one of the previous claims, or a cell comprising the polynucleotide.

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