Targeted PD-L1 decomposition

Abstract

The present invention relates to binding polypeptides comprising a first binding domain that binds to a transmembrane E3 ligase and a second binding domain that binds to a disease-related polypeptide, wherein the first binding domain comprises furin domain 1 and / or furin domain 2 of R-spondin (RSPO), and the second binding domain is deficient in wnt signaling activity and / or bone morphogenetic protein (BMP) signal inhibitory activity, as well as related polynucleotides, host cells, pharmaceutical uses, and methods.

Description

[Technical Field]

[0001] The present invention relates to binding polypeptides comprising furin domain 1 and / or furin domain 2 of R-spondin (RSPO), which are deficient in wnt signaling activity and / or bone morphogenetic protein (BMP) signal inhibitory activity, and which comprise a first binding domain that binds to a transmembrane E3 ligase and a second binding domain that binds to a disease-related polypeptide, as well as polynucleotides, host cells, medical uses, and methods related thereto. [Background technology]

[0002] Targeted protein degradation (TPD) is a field that has rapidly advanced, expanding the scope of therapeutic targets to include previously undruggable proteins and overcoming drug resistance (see, for example, Sun et al., Signal Transduct Target Ther 4:64 doi:10.1038 / s41392-019-0101-6 (2019)). Unlike traditional small molecule or antibody-based modalities that inhibit the function of a protein of interest (POI), TPD induces the degradation of targeted proteins. Among the various TPD platforms, proteolysis-targeting chimeric molecules (PROTACs) have been successfully applied to the degradation of various POIs implicated in multiple diseases, including cancer, infectious diseases, and neurodegenerative disorders (He et al., Front. Cell Dev. Biol 9:685106, doi:10.3389 / fcell.2021.685106 (2021)).

[0003] ZNRF3 / RNF43 is a single-pass transmembrane E3 ligase with a well-structured extracellular domain and a functional intracellular RING domain (Zebisch et al., Nature comm 4:2787, doi:10.1038 / ncomms3787 (2013)). Its ligands, R-spondins (RSPOs), are a family of four secreted stem cell growth factors important in various biological processes, from development to cancer (de Lau et al., Genome Biology 13:242 (2012); de Lau et al., Genes Dev 28:305, doi:10.1101 / gad.235473.113(2014)). Recent studies have revealed that among the four RSPOs, RSPO2 and RSPO3 are bifunctional ligands that activate WNT signaling and inhibit BMP signaling. (Lee et al., Nature comm 11: 5570, doi:10.1038 / s41467-020-19373-w (2020); Sun et al., Cell rep 36: 109559, doi:10.1016 / j.celrep.2021.109559 (2021)). RSPO2 and RSPO3 accomplish this by using ZNRF3 / RNF43 to target different substrates.

[0004] ZNRF3 and RNF43 ubiquitinate the WNT receptors Frizzled and LRP6, leading to their internalization and subsequent lysosomal degradation (Hao et al., Nature 485:195, doi:10.1038 / nature11019 (2012); Koo et al., Nature 488:665, doi:10.1038 / nature11308 (2012)). In addition to activating WNT signaling, RSPO2 and RSPO3 also inhibit BMP signaling. Notably, the WNT agonist function of R-spondins has been linked to tumorigenesis (ter Stege et al. (2021), Oncogene 40(47):6469), limiting their therapeutic utility.

[0005] The programmed death-ligand 1 (PD-L1) protein, also known as CD274, is a 40-kDa transmembrane protein known to bind to the PD-1 receptor on activated T cells and has been implicated in regulating CD8+ T cell proliferation. Accordingly, PD-L1 antagonists have been proposed to inhibit the interaction between PD-L1 and PD-1, thereby promoting the activation of tumor-directed T cells in cancer therapy; for example, atezolizumab is a therapeutic monoclonal antibody that targets PD-L1. Summary of the Invention

[0006] In light of the above, there remains a need for improved cancer treatments, particularly by activating and / or maintaining an immune response against cancer cells. The technical problem underlying the present invention can be seen as the provision of means and methods that meet the aforementioned need. This technical problem is solved by the embodiments characterized in the claims and herein below.

[0007] Accordingly, the present invention relates to binding polypeptides comprising a first binding domain that binds to a transmembrane E3 ligase and a second binding domain that binds to a disease-associated polypeptide, preferably programmed death-ligand 1 (PD-L1). [Brief explanation of the drawings]

[0008] [Figure 1a] RSPO chimeras bridge ZNRF3 / RNF43 and PD-L1. a. Top: Domain structure of the indicated proteins. Bottom: Structure prediction from RSPO2 (PDB 4ufr) and PD-1 HAC (PDB 5ius). R2FA is the RSPO2 furin domain with the LGR-binding-defective F109A mutation. PD1HAC is a high-affinity consensus (HAC) mutant of PD-1 with enhanced binding to PD-L1. SP is signal peptide; FU is furin domain; GS is glycine-serine linker; TSP1 is thrombospondin domain 1. [Figure 1b-d]RSPO chimeras bridge ZNRF3 / RNF43 and PD-L1. b. ELISA binding assay. c-e. ELISA assays as in (b) relating to the direct interaction of RSPO chimeras with (c) PD-L1ECD, (d) ZNRF3ECD, and (e) RNF43ECD. f. ELISA-based ternary complex assay. g-h. ELISA-based ternary complex assays as in (f). R2PD1 bridges the interaction of PD-L1ECD with (g) ZNRF3ECD and (h) RNF43ECD. [Figure 1e-h] Continuation of Figure 1b-d. [Figure 2-1] RSPO chimeras promote the degradation of overexpressed PD-L1. a. PD-L1 degradation assay. b–e, g. Immunoblot analysis of the indicated proteins in 293T cells. Cells were transfected as indicated and analyzed after 24 hours of incubation with conditioned medium containing the indicated proteins (b, d, g) or the indicated purified proteins (c, e). Control conditioned medium and mock-purified proteins were used as controls. Normalized ratios of PD-L1 and ERK are shown in (c). f. Immunofluorescence staining of the indicated proteins in 293T cells. Cells were transfected with PD-L1-GFP and ZNRF3-HA for 2 days. After an additional 5 hours of incubation with equal amounts of the indicated purified proteins, cells were fixed and stained for GFP and the lysosomal marker LAMP1. Notably, colocalization of PD-L1 and LAMP1 (arrows) was observed upon R2PD1 treatment. Scale bar = 10 μm. In (c), 0.2, 0.8, 2.2, 5.4, and 13.6 nM of purified R2PD1 protein were used, and in (e), 5.4 nM of purified R2PD1 protein was used. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 3-1]RSPO chimeras promote the degradation of endogenous PD-L1 in cancer cells. a-e, h, j-l, o-p, r, s. Immunoblot analysis of the indicated proteins in MEL624 and COLO-800 cells. (a, j) Cells were transfected with the indicated siRNAs for 3 days. (be, kl, p, rs) Cells were analyzed after incubation with the indicated proteins or conditioned medium for 24 hours or the indicated times. (h) Cells were treated with 333 U IFN-γ for 24 hours, incubated with purified proteins for an additional 24 hours, and then harvested for analysis. (o) Untreated cells were analyzed. Mock purified protein or control conditioned medium served as controls. Normalized PD-L1 to TfR ratios are shown below. f-g, m. Flow cytometry analysis of cell surface PD-L1 in MEL624 cells (f-g) and COLO-800 cells (m). Quantitative data are shown in (g, m). i, n, q. qRT-PCR analysis of the expression of the indicated genes in MEL624 and COLO-800 cells. (i, n) Cells were analyzed after 24 hours of incubation with the indicated purified proteins. (q) Untreated cells were analyzed. (b) The conditioned medium used in (c) was used at 0.027, 0.081, 0.27, 0.81, and 2.7 nM of purified protein. (d) 0.24 nM of purified protein was used. (e–g, i) 1 nM of purified protein was used. (h) 0.027, 0.081, 0.27, 0.81, and 2.7 nM of purified protein was used. (k) 0.081, 0.27, 0.81, and 2.7 nM of purified protein was used. (l–n) 0.81 nM of protein was used. (p) 0.7 nM of purified protein was used. (r) 0.07 and 0.24 nM purified protein or anti-PD-L1 antibody (atezolizumab) were used, and (s) 0.07 and 0.7 nM purified protein or anti-PD-L1 antibody (atezolizumab) were used. Mock purified protein and isotype antibody control were used as controls. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Figure 3-4]Continued from Figure 3-3. [Figure 4-1] PD-L1 degradation by RSPO chimeras is independent of WNT / β-cat signaling. a-d. Immunoblot analysis of the indicated proteins in MEL624 (a-c) and COLO-800 (d) cells. Cells were analyzed after incubation with the indicated proteins for 12 hours (a) or 24 hours (b-d). (c-d) Cells were transfected with the indicated siRNA for 2 days before incubation with purified proteins. e. qRT-PCR analysis of Axin2 expression in COLO-800 cells. Cells were analyzed after incubation with purified proteins in the presence of WNT3A for 24 hours. f. TOPflash assay using HEK293T cells. Cells were analyzed after incubation with the indicated proteins for 12 hours. DKK1 protein at 10, 50, and 200 ng ml-1 was used in (a-b). 0.24 nM purified R2PD1 protein was used in (b). Purified R2PD1 was used at 0.24 nM (c), 0.73 nM (d), and 0.8 nM (e). In (f), RSPO2 or R2PD1 was used at 0.12, 0.36, 1.08, and 3.24 nM. [Figure 4-2] Continued from Figure 4-1. [Figure 5-1]PD-L1 degradation by RSPO chimeras requires ZNRF3 / RNF43. ag. PD-L1 degradation assay. PD-L1 degradation by R2PD1 in the presence of the competitor soluble RNF43ECDFc (a-b) or after ZNRF3 / RNF43 knockdown (cg). b, d, f, h-i. Immunoblot analysis of the indicated proteins in MEL624 (b, d), COLO-800 (f), and A375 (h-i) cells. Cells were analyzed after 24 hours of incubation with the indicated proteins. (d, f) Cells were transfected with the indicated siRNA for 2 days before incubation with purified proteins. e, g. Flow cytometry analysis of cell surface PD-L1 in MEL624 cells (e) and COLO-800 cells (g). Values ​​were normalized to the corresponding control group. RNF43ECDFc was used at 50, 150, 500, and 1,500 ng ml-1 in (b). Purified R2PD1 was used at 0.27 nM (b), 0.14 nM (d), 1 nM (e), 0.8 nM (f), and 2.5 nM (g). Purified R2PD1 was used at 0.073, 0.24, 0.73, and 2.44 nM in (h–i). [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 6-1](a) Flow cytometric quantification of activated cytotoxic CD107a+ T cells. HEK293T cells were transfected with plasmids encoding the MART-1 minigene, PD-L1, and ZNRF3 / RNF43 for 2 days. T cells were transfected with the indicated TCR and PD-1 mRNA for 1 day. After incubation with the indicated purified proteins, HEK293T cells were mixed with T cells for 5 hours. After 5 hours of coculture, cells were harvested, and activated cytotoxic CD107a+ CD8+ T cells were quantified. Purified R2PD1 was used at concentrations of 0.4, 1.1, 3.3, 10, and 30 nM. n = single sample. Representative data from two independent experiments are shown. (b) Flow cytometric quantification of cytotoxic TNFα+ T cells. T cells were transfected with mRNA expressing the TCR and PD-1 for 1 day. MEL624 cells were incubated overnight with the indicated proteins. After an additional 5 hours of co-culture, MEL624 cells and T cells were analyzed by flow cytometry. Equal numbers of CD8+ T cells were analyzed from each combination. 0.14, 0.41, 1.23, 3.7, and 11.1 nM of purified R2PD1 or PD-L1 antibody atezolizumab were used. n = single sample. Representative data from three independent experiments with the same conclusion are shown. (c-d) Real-time cell proliferation assay. MEL624 cells were pretreated with IFNγ for 2 days and then mixed with T cells in the presence of the indicated proteins. T cells were transfected with mRNA expressing TCR and PD-1 for 1 day. MEL624 cell proliferation was monitored using the XCELLigence System. n = three independent experimental samples. Data were normalized to the time point at which T cells were added and presented as the mean. For the last time point, the standard error of the mean (SEM) is shown. For (c), ****P<0.0001 from one-way ANOVA. For (d), ****P<0.0001 by two-tailed paired t-test. In (c), 0.41, 3.7, and 11.1 nM purified R2PD1 were used. In (d), 0.41 and 3.7 nM purified R2PD1 or 3.7 and 11.1 nM PD-L1 antibody atezolizumab were used. [Figure 6-2] Continued from Figure 6-1. DETAILED DESCRIPTION OF THE INVENTION

[0009] In general, terms used herein are to be given their ordinary and accustomed meanings to those skilled in the art, and unless otherwise specified, are not limited to any special or modified meanings as necessary. When used hereinafter, the terms "have," "comprise," or "include," or any grammatical variations thereof, are used in a non-exclusive sense. Thus, these terms can refer to both a situation in which no additional features are present in the entity described in this context, in addition to the features introduced by these terms, as well as a situation in which one or more additional features are present. As an example, the expressions "A has B," "A comprises B," and "A includes B" can refer to both a situation in which no other elements are present in A besides B (i.e., a situation in which A consists entirely of B), and a situation in which, in addition to B, one or more additional elements are present in entity A, such as element C, elements C and D, or even other elements. It will also be appreciated by those skilled in the art that the terms "comprising a" and "comprising an" are preferably equivalent to "comprising one or more," i.e., "comprising at least one." Thus, unless otherwise specified, a reference to one item of a plurality preferably refers to at least one such item, more preferably to a plurality of such items; thus, for example, reference to specifying a "cell" refers to reference to specifying at least one cell, preferably to a plurality of cells.

[0010] Furthermore, as used hereinafter, the terms "preferably," "more preferably," "most preferably," "particularly," "more particularly," "particularly," "more particularly," or similar terms may be used in conjunction with any feature without further limiting the possibilities. Therefore, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The present invention can be practiced using alternative features, as will be recognized by those skilled in the art. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any limitations on further embodiments of the invention, without any limitations on the scope of the invention, and without any limitations on the possibility of combining the feature so introduced with other optional or non-optional features of the invention.

[0011] The methods specified herein below are preferably in vitro methods. The steps of the methods can in principle be performed in any order deemed appropriate by a person skilled in the art, but are preferably performed in the order indicated; also, one or more, preferably all, of the steps may be assisted or performed by automated devices. Furthermore, the methods may comprise further steps in addition to those explicitly mentioned above.

[0012] As used herein, the term "standard conditions" refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., preferably a temperature of 25°C and an absolute pressure of 100 kPa, unless otherwise specified; and preferably, standard conditions also include a pH of 7. Furthermore, unless otherwise specified, the term "about" refers to the stated value with a technical precision generally accepted in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5% of the stated value. Furthermore, the term "essentially" indicates the absence of error that would affect the stated result or use, i.e., potential errors would not produce a specified result with an error of more than ±20%, more preferably ±10%, and most preferably ±5%. Thus, "consisting essentially of" means including the specified component but excluding other components, excluding materials present as impurities, unavoidable materials present as a result of the process used to provide the component, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "consisting essentially of" may include known and acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a set of ingredients contains less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of non-specific ingredients.

[0013] The term "binding" is understood by those skilled in the art; preferably, the term refers to the dissociation constant K D at most 10 -6 mol / l, more preferably at most 10 -7 mol / l, more preferably at most 10 -8 mol / l, most preferably at most 10 -9The term "specific binding" refers to the interaction of two molecules that is 100 mol / l. The term "specific binding" is also understood by those skilled in the art. Preferably, specific binding refers to binding of a binding polypeptide identified elsewhere herein with at least 10-fold, preferably at least 100-fold, and more preferably at least 1000-fold higher affinity for its cognate binding partner than for any non-cognate binding partner. Thus, the dissociation constant (K) of the binding polypeptide / non-cognate binding partner complex is D ) is preferably at least 10 -6 mol / l, more preferably at least 10 -5 mol / l, most preferably at least 10 -4 It is in mol / l.

[0014] The degree of identity (e.g., expressed as "% identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the sequence fragment in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the sequences compared for optimal alignment. The percentage is calculated by determining the number of positions where identical residues occur in both sequences, preferably over the entire length of the polynucleotide or polypeptide, to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), computer implementations of these algorithms (e.g., BLAST, GAP, BESTFIT, PASTA, or TFASTA), or by visual inspection. Once two sequences have been identified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment and thus the degree of identity. Preferably, default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used. More preferably, the Basic Local Alignment Search Tool (BLAST) implementation is used, using default parameter values ​​for alignment. In the context of biological sequences referred to herein, the term "essentially identical" indicates a percent identity of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%. Of course, the term essentially identical includes 100% identity. The foregoing also applies mutatis mutandis to the term "essentially complementary."

[0015] The term "fragment" of a biological macromolecule, preferably a polynucleotide or polypeptide, is used herein in a broad sense to refer to any subpart, preferably a subdomain, of the respective biological macromolecule comprising the indicated sequence, structure, and / or function. Thus, the term encompasses not only subparts generated by actual fragmentation of the biological macromolecule, but also subparts derived from the respective biological macromolecule by theoretical methods, e.g., in silico. Thus, as used herein, fragments of immunoglobulins can refer not only to Fc or Fab fragments, but also, for example, to single-chain antibodies, bispecific antibodies, and nanobodies.

[0016] Unless otherwise indicated herein, the identified compounds, particularly polynucleotides and polypeptides, may be included in larger structures, e.g., covalently or non-covalently linked to additional sequences, carrier molecules, retardants, and other excipients. In particular, the identified polypeptides may be included in fusion polypeptides containing additional peptides, which may be useful, for example, as tags for purification and / or detection, as linkers, or to extend the in vivo half-life of the compound. The term "detectable tag" refers to a series of amino acids added or introduced into a fusion polypeptide; preferably, the tag is added to the C-terminus or N-terminus of the fusion polypeptide. The series of amino acids preferably allows the polypeptide to be detected by an antibody that specifically recognizes the tag; or preferably allows the polypeptide to form a functional higher-order structure, such as a chelator; or preferably allows visualization, as in the case of a fluorescent tag. Preferred detectable tags are Myc tags, FLAG tags, 6-His tags, HA tags, GST tags, or fluorescent protein tags, such as GFP tags. All of these tags are well known in the art. Other additional peptides preferably included in the fusion polypeptide include additional amino acids or other modifications that can function as mediators of secretion, mediators of blood-brain barrier crossing, cell-penetrating peptides, and / or immunostimulatory agents. Additional polypeptides or peptides to which the polypeptide can be fused are signal sequences and / or transport sequences, such as the IL-2 signal sequence, and linker sequences.

[0017] As used herein, the term "polypeptide" refers to a molecule comprising several, generally at least 20, amino acids covalently linked to each other by peptide bonds. Molecules consisting of fewer than 20 amino acids covalently linked by peptide bonds are generally considered "peptides." Preferably, a polypeptide comprises 50 to 1,000 amino acids, more preferably 100 to 1,000, even more preferably 200 to 500, and most preferably 250 to 400 amino acids. A polypeptide may also be a complex of two or more amino acid chains, i.e., a multimer, e.g., a dimer, trimer, etc. In such cases, the complex of two or more amino acid chains may be referred to as a "polypeptide oligomer" or "protein complex." Preferably, the complex of two or more amino acid chains is a heteromultimer, more preferably a heterodimer, preferably comprising at least one first binding domain and at least one second binding domain in a non-covalent complex. A polypeptide may also comprise additional non-peptide structures, such as at least one glycosylation, lipid conjugation, etc. More preferably, the designated polypeptide, particularly the binding polypeptide, comprises all of the indicated structural components contained in one continuous, covalently linked peptide chain; thus, the polypeptide, particularly the binding polypeptide, preferably is or is comprised in a fusion polypeptide. Unless otherwise noted, reference herein to a particular polypeptide preferably includes variants of the polypeptide.

[0018] As used herein, the term "polypeptide variant" refers to any chemical molecule that contains at least one polypeptide identified herein and has the indicated biological activity, but differs in structure from said specific polypeptide. Preferably, a polypeptide variant includes a polypeptide having a contiguous amino acid sequence that corresponds at least 50%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% to the amino acid sequence of a specifically identified polypeptide, in particular SEQ ID NO: 7. Furthermore, it should be understood that a polypeptide variant referred to in accordance with the present invention has an amino acid sequence that differs by at least one amino acid substitution, deletion, and / or addition, and that the amino acid sequence of the variant is preferably still at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% identical to the amino acid sequence of a specific polypeptide, in particular SEQ ID NO: 7 or 9. In view of the above, the percent identity values ​​indicated herein below are preferably lower limit values ​​of percent identity, and the required percent identity can be higher, e.g., having the values ​​mentioned above. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art, as described herein above. The polypeptide variants referred to above may be allelic variants or other species-specific homologs, paralogs, or orthologs. Furthermore, the polypeptide variants referred to herein include fragments of a particular polypeptide or polypeptide variants of the aforementioned types, so long as these fragments and / or variants have the specified biological activity. Such fragments may also be, for example, degradation products or splice variants of the polypeptide, or those derived therefrom. Also included are variants that differ by post-translational modifications, such as phosphorylation, glycosylation, ubiquitination, sumoylation, myristylation, etc., by the inclusion of unnatural amino acids, and / or by being peptidomimetics.The above applies mutatis mutandis and independently to the domains of the polypeptides described herein. That is, the first binding domain and the second binding domain may be variant domains that still have the indicated activity, in particular binding activity, as specified herein below. Thus, the first binding domain may be, for example, a variant of a polypeptide comprising the furin1 and furin2 domains of R-spondin, and may comprise, for example, an amino acid sequence at least 80% identical to SEQ ID NO:2, and the second binding domain may be a polypeptide variant of the extracellular IgV-like domain of the PD-1 polypeptide, and may comprise, for example, an amino acid sequence at least 85% identical to SEQ ID NO:3.

[0019] As used herein, the term "polynucleotide" refers to a linear or circular nucleic acid molecule. The polynucleotides of the present invention are preferably provided as isolated polynucleotides (i.e., isolated from their natural environment) or in genetically modified form, preferably containing at least one heterologous sequence. The term encompasses single-stranded and double-stranded polynucleotides. Furthermore, it also encompasses naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or chemically modified polynucleotides, including artificially modified derivatives, such as biotinylated polynucleotides and locked nucleic acids. Polynucleotides of the present invention have the activity of encoding a binding polypeptide or at least one binding domain thereof, as identified herein. Methods for testing whether a given polynucleotide has the aforementioned biological activity are known in the art and are described herein below. Unless otherwise specified, reference herein to a particular polynucleotide preferably includes polynucleotide variants.

[0020] As used herein, the term "polynucleotide variant" refers to a variant of a polynucleotide referred to herein, including a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition, and / or deletion, and which possesses the biological activity identified for the specific polynucleotide. Preferably, the polynucleotide variant is an ortholog, paralog, or other homolog of the specific polynucleotide. Also preferably, the polynucleotide variant is or is derived from a non-naturally occurring allele of the specific polynucleotide. Polynucleotide variants also encompass polynucleotides comprising a nucleic acid sequence capable of hybridizing to the aforementioned specific polynucleotide under stringent hybridization conditions. These stringent conditions are known to those skilled in the art and are described in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Those skilled in the art will know how to determine the necessary hybridization conditions by consulting textbooks such as those mentioned above or elsewhere herein. Alternatively, polynucleotide variants can be obtained by PCR-based methods such as mixed oligonucleotide primer-based amplification of DNA. Furthermore, variants include polynucleotides comprising nucleic acid sequences that are at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% identical to the specifically designated nucleic acid sequences. Also encompassed are polynucleotides comprising nucleic acid sequences that encode amino acid sequences that are at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% identical to the specifically designated amino acid sequences.The percent identity value is preferably calculated over the entire amino acid or nucleic acid sequence region as specified herein above. The polynucleotides of the present invention consist of, consist essentially of, or comprise the aforementioned nucleic acid sequences. Thus, they may also contain additional nucleic acid sequences. Specifically, the polynucleotides of the present invention may encode fusion proteins, in which one partner of the fusion protein is a polypeptide encoded by the above-mentioned nucleic acid sequence. The polynucleotides of the present invention may also be included in expression constructs and / or vectors.

[0021] As used herein, the term "expression construct" refers to a heterologous polynucleotide comprising the aforementioned polynucleotide and nucleic acid sequences necessary for the expression of the polynucleotide. Generally, such additional nucleic acid sequences, preferably heterologous to the polynucleotide encoding the binding polypeptide or at least one binding domain thereof, may be promoter sequences, regulatory sequences, and / or transcription termination sequences, such as terminators. Expression of a polynucleotide involves transcription of the polynucleotide into RNA. Regulatory elements ensuring expression in cells, particularly eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably include regulatory sequences ensuring transcription initiation and, optionally, a polyA signal ensuring transcription termination and stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers. Regulatory elements enabling expression in prokaryotic host cells include, for example, the lac, trp, or tac promoters of E. coli. Examples of regulatory elements enabling expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV, SV40, or RSV promoters (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin introns in mammalian and other animal cells. Furthermore, inducible expression control sequences can be used in the expression constructs encompassed by the present invention. Such inducible constructs can include tet or lac operator sequences, or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to elements responsible for transcription initiation, such regulatory elements can also include transcription termination signals downstream of the polynucleotide, such as the SV40 polyA site or tk polyA site. Preferably, the expression construct is a eukaryotic expression construct, i.e., an expression construct containing all elements necessary for expression, preferably inducible expression, in eukaryotic host cells. However, the expression construct can also be a bacterial expression construct for producing the binding polypeptide in a bacterial cell.

[0022] The term "vector" preferably encompasses phage, plasmid, viral, or retroviral vectors, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Furthermore, the term also relates to targeting constructs that allow random or site-specific integration of the targeting construct into genomic DNA. Such targeting constructs preferably contain DNA of sufficient length for homologous or heterologous recombination, as described in detail below. Vectors containing polynucleotides of the present invention preferably further contain a selectable marker for propagation and / or selection in a host. Vectors can be incorporated into host cells by various techniques well known in the art. For example, plasmid vectors can be introduced in precipitates, such as calcium phosphate precipitates or rubidium chloride precipitates, or in complexes with charged lipids, or in carbon-based clusters, such as fullerenes. Alternatively, plasmid vectors can be introduced by heat shock or electroporation. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before application to host cells. Viral vectors, particularly retroviral vectors, can be replication-competent or replication-deficient. In the latter case, viral propagation generally occurs only in complementary host / cells. More preferably, in the vectors of the present invention, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells or isolated fractions thereof. That is, preferably, the polynucleotide is contained in an expression vector. In this context, suitable expression vectors are Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (InVitrogene), or pSPORT1 (GIBCO BRL), which are known in the art. Preferably, the vector is an expression vector, gene transfer and / or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, and adeno-associated viruses can be used to deliver polynucleotides or vectors to target cell populations.Recombinant viral vectors can be constructed using methods well known to those skilled in the art; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY, and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0023] In view of the above, those skilled in the art will understand that reference herein to a gene or its gene product includes polynucleotide variants of the gene and its transcription products, i.e., particularly alleles, homologs, and mutants of the gene, and transcript variants, such as splice variants and RNA editing variants of RNA gene products. More preferably, reference to a gene relates to the gene and its naturally occurring alleles. Reference to a polypeptide gene product also includes polypeptide variants identified herein above, particularly isoforms and / or mutant proteins having the indicated degree of sequence identity and possessing the indicated biological activity. For genes or gene products referenced by Genbank accession number or similar designation, the respective sequences are incorporated herein by reference. Unless otherwise indicated, database entries refer to the status of each database entry on the date prior to the filing date of this application.

[0024] The term "R-spondin" is known to those skilled in the art to be related to a family of tectal plate-specific spondins, and is reviewed, for example, by de Lau et al., Genome Biology 13:242 (2012). Thus, the R-spondin may be one of R-spondins 1 to 4, and is preferably one of human R-spondins 1 to 4. Preferably, the R-spondin is R-spondin 2 or R-spondin 3. The term "R-spondin2" is known to those skilled in the art. Human R-spondin2 polypeptide has several isoforms, and for example, the amino acid sequence of isoform 1 precursor is provided as Genbank accession number NP_848660.3, SEQ ID NO: 12. Therefore, the term "R-spondin2" as used herein preferably relates to said human R-spondin2 or a polypeptide having an amino acid sequence at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% identical to the amino acid sequence of said human R-spondin2. Preferably, R-spondin2 is human R-spondin2 or a homolog thereof, preferably a vertebrate homolog, more preferably a mammalian homolog. R-spondin2 homologs are known, for example, from the HomoloGene database entry 18235; furthermore, R-spondin2 homologs of other species can be identified by sequence comparison, in particular by determining the degree of identity as described elsewhere herein. Thus, preferably, R-spondin2 is human, chimpanzee, rhesus monkey, rat, mouse, cow, dog, chicken, zebrafish or xenopus R-spondin2, more preferably human R-spondin2. The term "R-spondin3" is also known to those skilled in the art. The amino acid sequence of human R-spondin3 precursor is available, for example, as Genbank accession number NP_116173.2, SEQ ID NO: 2.Therefore, the term "R-spondin3" as used herein preferably relates to said human R-spondin3 or a polypeptide having an amino acid sequence at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to the amino acid sequence of said human R-spondin3. Preferably, R-spondin3 is human R-spondin3 or its homolog, preferably a vertebrate homolog, more preferably a mammalian homolog. R-spondin3 homologs are known, for example, from the HomoloGene database entry 12484; furthermore, R-spondin3 homologs of other species can be identified by sequence comparison, in particular by determining the degree of identity as described elsewhere herein. Thus, preferably, R-spondin3 is human, chimpanzee, rhesus monkey, rat, mouse, cow, chicken, zebrafish or xenopus R-spondin3, more preferably human R-spondin3.

[0025] As used herein, the term "binding domain" refers to a substructure of a binding polypeptide having an indicated biological binding activity; preferably, the binding is specific. Preferably, the binding domain comprises, and more preferably consists of, a contiguous amino acid stretch having the indicated binding activity. Preferably, the binding domain has only the indicated binding activity. That is, it preferably has no biological activities other than those specifically identified, although such additional biological activities may be present in the protein from which the binding domain is derived. Thus, preferably, the binding domain, particularly the first binding domain, more preferably both binding domains, and most preferably the binding polypeptide, does not have wnt signaling activity and / or bone morphogenetic protein (BMP) signaling inhibitory activity, and more preferably does not have signaling activity. As specified herein, the binding polypeptide comprises at least a first binding domain and a second binding domain, wherein the first binding domain has the biological activity of binding to a transmembrane E3 ligase; and the second binding domain has the biological activity of binding to a disease-related polypeptide.

[0026] The first binding domain has biological activity of binding to a transmembrane E3 ligase. The term "transmembrane E3 ligase" is understood by those skilled in the art to refer to a transmembrane member of the E3 ligase family (EC 2.3.2.27). Preferably, the transmembrane E3 ligase is a RING-finger E3 ligase. Preferably, the transmembrane E3 ligase is expressed by a target cell. Preferably, the transmembrane E3 ligase is ZNRF3 E3 ligase, preferably human ZNRF3 E3 ligase, more preferably having the amino acid sequence of Genbank Accession No. NP_001193927.1, SEQ ID NO: 10, or one of its isoforms; also preferably, the transmembrane E3 ligase is RNF43 E3 ligase, preferably human RNF43 E3 ligase, more preferably having the amino acid sequence of Genbank Accession No. NP_060233.3, SEQ ID NO: 11, or one of its isoforms. Polypeptides having the activity of binding to transmembrane E3 ligases are known in the art, in particular the R-spondins specified herein above. Thus, preferably, the first binding domain comprises a portion of R-spondin having the aforementioned activity, preferably R-spondin2. Thus, the first binding domain preferably comprises furin domain 1 of R-spondin (RSPO) and / or furin domain 2 without Wnt activation activity. Preferably, furin domain 1 corresponds to amino acids 37-84 of SEQ ID NO: 12 or corresponding amino acids in other spondin polypeptides, and / or furin domain 2 corresponds to amino acids 90-134 of SEQ ID NO: 12 or corresponding amino acids in other R-spondin polypeptides. More preferably, furin domain 2 comprises a substitution of amino acid F109 with a non-identical amino acid, preferably an F109A amino acid substitution. In this case, amino acid position 109 is the position bearing this number in human R-spondin2 specified herein above, or the corresponding amino acid position in one of the other R-spondins.Other amino acid exchanges in R-spondin and / or furin domains that abolish wnt signaling are known in the art, and those skilled in the art know how to implement such amino acid exchanges in the context of the first binding domain; therefore, a further preferred amino acid exchange with the aforementioned effect is the exchange of amino acid F105 with a non-identical amino acid, preferably the F105A amino acid exchange, where amino acid position 105 is the position with this number in human R-spondin2 specified hereinabove, or the corresponding amino acid position in one of the other R-spondins. Preferably, the first binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1. More preferably, the first binding domain comprises the furin domain 1 and the furin domain 2 of RSPO2. Thus, more preferably, the first binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 2. More preferably, the first binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, more preferably consists of an amino acid sequence at least 80% identical to SEQ ID NO: 2. Thus, the first binding domain preferably lacks the TSP1 domain; also preferably, the first binding domain consists of said furin domain 1 and / or said furin domain 2, preferably comprising said F109A amino acid exchange.

[0027] As used herein, the term "subject" relates to vertebrates, preferably mammals, and in particular to livestock, companion animals, and laboratory animals. More preferably, the subject is a human. Preferably, the subject has been diagnosed with and / or is at risk of developing a disease as identified herein below. More preferably, the subject has been diagnosed with cancer and / or is at risk of developing recurrence and / or metastasis.

[0028] The term "disease" is used broadly herein and preferably relates to any pathological condition in a subject that is known or believed to be in need of treatment, and more preferably treatable by a binding polypeptide as specified herein. Thus, preferably, the disease is a pathological condition caused or exacerbated by (over)expression of at least one disease-associated polypeptide as specified elsewhere herein. Thus, the disease may be caused or exacerbated, in particular, by overexpression of a polypeptide in unphysiological amounts and / or by expression of a mutant protein of the polypeptide of interest, which may, for example, have increased activity, new activity, and / or inhibit physiological regulation. Preferably, the disease may be caused or exacerbated by a malfunction of the immune system, e.g., cancer or an autoimmune disease. Preferably, the disease is caused or exacerbated by overexpression of at least one immune checkpoint inhibitor, preferably PD-L1. More preferably, the disease is cancer, even more preferably a PD-L1-expressing cancer.

[0029] As used herein, the term "cancer" relates to a disease in animals, including humans, characterized by the uncontrolled growth of somatic cells ("cancer cells"). This uncontrolled growth may involve the invasion and destruction of surrounding tissues (infiltration) and, in some cases, the spread of cancer cells to other parts of the body (metastasis). Preferably, the term cancer also includes recurrence (relapse) of cancer. Thus, preferably, the cancer is a solid cancer, metastasis, or recurrence thereof. Also preferably, the cancer is a non-solid cancer, particularly leukemia, especially recurrent or advanced leukemia. Preferably, the cancer is acute myeloid leukemia (AML), acute lymphocytic leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, basal cell carcinoma, bile duct cancer, bladder cancer, brainstem glioma, breast cancer, Burkitt's lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, Endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular malignant melanoma, Kaposi's sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, Oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, central nervous system The cancer is selected from the group consisting of primary systemic lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, testicular cancer, pharyngeal cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. More preferably, the cancer is melanoma.

[0030] The second binding domain has the biological activity of binding to a disease-associated polypeptide, preferably the extracellular domain thereof, preferably programmed death-ligand 1 (PD-L1), preferably the extracellular domain thereof.

[0031] The term "disease-associated polypeptide" is understood by those skilled in the art to include any polypeptide causally involved in the pathogenesis of a disease; thus, a disease-associated polypeptide is preferably known to be overexpressed in a disease and / or to be a mutant protein of a polypeptide normally present in a subject, and preferably, this overexpression or expression of the mutant protein is causally associated with the disease. Therefore, a decrease in the amount of a disease-associated polypeptide is preferably known to at least cause an improvement in the disease, more preferably to cause an improvement in the disease. A disease-associated polypeptide is preferably membrane-associated and preferably binds to the cytoplasmic membrane of a target cell. Therefore, a disease-associated polypeptide preferably comprises at least one transmembrane domain, i.e., is preferably a transmembrane polypeptide. Also preferably, a disease-associated polypeptide comprises at least one extracellular domain. However, a disease-associated polypeptide can also be a non-transmembrane polypeptide; preferably, in this case, the disease-associated polypeptide is a membrane-associated polypeptide, particularly a lipid-anchored polypeptide, or an extracellular, preferably soluble polypeptide, non-covalently bound to a membrane, particularly the cytoplasmic membrane. Non-covalent binding of soluble polypeptides to membranes is mediated by protein-protein interactions, protein-lipid interactions, protein-polysaccharide interactions, metal ions, etc. Preferably, the disease-associated polypeptide is a regulatory polypeptide that contributes to (i) regulating cell proliferation, (ii) regulating immune responses, particularly cellular immune responses, and / or (iii) regulating intercellular communication. Thus, the disease-associated polypeptide may in particular be a T-cell regulatory polypeptide or an immune checkpoint polypeptide. In particular, the disease-associated polypeptide may be PD-L1.

[0032] Preferably, the disease-associated polypeptide is programmed cell death ligand (PD-L1). The terms "programmed cell death ligand 1" and "PD-L1" refer to a group of polypeptides known by this designation, which may also be referred to as "CD274" or "B7-H1". Preferably, PD-L1 is expressed on target cells. Preferably, PD-L1 is human PD-L1, and more preferably has the amino acid sequence of Genbank Accession No. NP_054862.1, SEQ ID NO: 13, or one of its isoforms.

[0033] Polypeptides and domains thereof having binding activity to disease-associated polypeptides are known in the art and can be selected by those skilled in the art based on the disease-associated polypeptide to be targeted. Thus, in particular, the second binding domain can be a binding domain of an interaction partner of the disease-associated polypeptide, preferably a naturally occurring one. Thus, when the disease-associated polypeptide is a receptor, the second binding domain can comprise, for example, the binding domain of the receptor's cognate ligand or a derivative thereof having the binding activity. The second binding domain can also comprise an aptamer, a spiegelmer, a designed ankyrin repeat (DARPIN) domain, a Kunitz-type domain, an antibody, particularly a single-chain antibody or a single-domain antibody (see Hey et al. (2005), Trends Biotechnol. 23:514), or a derivative thereof having the aforementioned binding activity.

[0034] Polypeptides having binding activity to PD-L1 are known in the art and include, in particular, programmed cell death protein 1 (PD-1). The terms "programmed cell death protein 1" and "PD-1" refer to a member of a family of T cell regulators known by this designation to those skilled in the art, which may also be referred to as "CD279." Preferably, the PD-1 is human PD-1, and more preferably comprises the amino acid sequence set forth in GenBank Accession No. NP_005009.2, SEQ ID NO: 14, or the amino acid sequence of one of its isoforms. PD-1 comprises an extracellular IgV-like domain that binds to the extracellular domain of PD-L1; therefore, the second binding domain preferably comprises the extracellular IgV-like domain of the PD-1 polypeptide, more preferably a high-affinity variant thereof. Thus, the second binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 3 or 4. Also preferably, the second binding domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 5. Thus, if the second binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 3 or 4, preferably it also comprises an amino acid sequence at least 75% identical to SEQ ID NO: 5. More preferably, the second binding domain consists of one of the foregoing amino acid sequences.

[0035] As used herein, the term "binding polypeptide" relates to a polypeptide having the structural elements and biological activity or activities identified above. Thus, a binding polypeptide comprises at least a first binding domain having the biological activity of binding to a transmembrane E3 ligase; and a second binding domain having the biological activity of binding to a disease-associated polypeptide. Thus, a binding polypeptide has the biological activities of binding to a transmembrane E3 ligase and a disease-associated polypeptide. Preferably, the binding activity interconnects the transmembrane E3 ligase and the disease-associated polypeptide at the cytoplasmic membrane of a target cell via the binding polypeptide, and preferably causes the disease-associated polypeptide to be degraded by the cell.

[0036] In view of the above description of the present specification, the binding polypeptide preferably has only the binding activity indicated, i.e., preferably does not have any biological activity other than that specifically indicated, although such additional biological activity may be present in the protein from which the binding polypeptide is derived. Thus, the binding polypeptide preferably does not have Wnt signaling activity and / or bone morphogenetic protein (BMP) signal inhibitory activity, more preferably does not have signal modulation activity, and preferably does not have signaling activity. Also, the binding polypeptide preferably does not have additional sequences from the protein from which it is derived. Thus, the binding polypeptide preferably does not contain the transmembrane domain and / or intracellular domain of PD-1; also, the binding polypeptide preferably does not contain the active TSP-1 domain of R-spondin, i.e., when the binding polypeptide contains a TSP-1 domain, it is preferably a signaling-inactive polypeptide variant. More preferably, the binding polypeptide preferably does not contain the active TSP-1 domain of R-spondin, and more preferably does not contain the amino acid sequence of at least 7 amino acids downstream of amino acid 143 of SEQ ID NO: 12.

[0037] Nevertheless, the binding polypeptide may comprise, in addition to the amino acid sequence specifically shown, for example, a tag or flag sequence, a signal peptide, e.g., one having the amino acid sequence of SEQ ID NO: 8, or at least one linker sequence. Further amino acid sequences or other structural elements may be included to provide additional binding activity, improved serum stability, tissue targeting, etc. Preferably, however, the binding polypeptide does not comprise a transmembrane domain and / or an intracellular domain. Thus, the binding polypeptide is preferably a non-transmembrane integral polypeptide, more preferably a soluble polypeptide.

[0038] Preferably, the binding polypeptide is a complex of two or more amino acid chains, i.e., preferably a multimer, e.g., a dimer, trimer, etc.; in such cases, the complex of two or more amino acid chains may also be referred to as a "binding polypeptide oligomer" or a "binding polypeptide complex." Preferably, the complex of two or more amino acid chains is a heteromultimer, more preferably a heterodimer, preferably comprising at least one first binding domain and at least one second binding domain in a non-covalent complex. Methods for providing suitable complexes are known in the art, for example, using suitable ligand-receptor pairs, antigen-antibody reactions, etc. Exemplarily, the interaction of biotin with streptavidin or an interaction pair derived therefrom may be used. Preferably, a colicin / immunoaffinity pair is used for complex formation. As will be understood by those skilled in the art, when the binding polypeptide is a binding polypeptide complex, the complex can be formed in vitro, for example, by mixing a first binding domain covalently bound to a first partner of an affinity pair with a second binding domain bound to a second partner of the affinity pair; however, the complex can also be formed in vivo, for example, by administering a first binding domain covalently bound to a first partner of an affinity pair and further administering a second binding domain bound to a second partner of the affinity pair. As will be understood by those skilled in the art, it is also possible to use two or more second binding domains with one first binding domain, for example, by using a first binding domain covalently bound to a first partner of an affinity pair with multiple second binding domains each bound to a second partner of the affinity pair; in this way, two or more disease-related polypeptides can be targeted.

[0039] More preferably, in the binding polypeptide, the first binding domain and the second binding domain are directly or indirectly covalently linked. More preferably, the first binding domain and the second binding domain are contained in a common polypeptide, i.e., preferably, the first binding domain and the second binding domain jointly form a fusion polypeptide. Preferably, the first binding domain and the second binding domain are connected via a linker, i.e., preferably, a linker is interposed between the first binding domain and the second binding domain. The terms "linker," "linker sequence," and "linker peptide" are generally known to those skilled in the art. Those skilled in the art are familiar with how to select an appropriate linker peptide. Preferably, the linker contains 1 to 20, more preferably 5 to 10, amino acids, preferably independently selected from the group consisting of glycine (G), proline (P), and serine (S). A particularly preferred linker peptide comprises the amino acid sequence shown as SEQ ID NO: 6. Preferably, the first binding domain is located N-terminal to the second binding domain in the binding polypeptide. Thus, when a linker is present, the order of structural elements in the binding polypeptide is preferably N-terminus, first binding domain, linker, second binding domain, C-terminus. As will be understood by those skilled in the art, when additional structural elements are present in the binding polypeptide, they are preferably attached to the N-terminus and / or C-terminus of the aforementioned preferred structures. Additionally, additional structural elements may be added with or without an intervening linker, which may or may not be the same as the linker intervening between the first and second binding domains.

[0040] In accordance with the above, the binding polypeptide preferably comprises an amino acid sequence at least 80% identical to SEQ ID NO: 7 or 9. More preferably, the binding polypeptide consists essentially of an amino acid sequence at least 80% identical to SEQ ID NO: 7 or 9, and most preferably, the binding polypeptide consists of an amino acid sequence at least 80% identical to SEQ ID NO: 7 or 9.

[0041] Preferably, the binding polypeptide is contained in a substance composition containing additional components; thus, the binding polypeptide can be contained in a solution, particularly an aqueous solution, which may further contain, for example, at least one buffer, at least one salt, and / or any other components deemed appropriate by those skilled in the art. Preferably, the composition is a pharmaceutical composition, which preferably further comprises a pharmaceutically acceptable carrier. The terms "medicament" and "pharmaceutical composition" are used essentially interchangeably herein and are generally known to those skilled in the art. As referred to herein, this term preferably relates to any composition containing an active agent, designated as a pharmaceutically active compound, and optionally one or more excipients. The pharmaceutically active compound may be present in liquid or dry, e.g., lyophilized, form. It is understood that the form and characteristics of a pharmaceutically acceptable excipient, e.g., a carrier or diluent, are determined by the amount of active ingredient with which it is incorporated, the route of administration, and other well-known variables. An excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient of the formulation. Excipients employed include solids, gels, or liquids. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Examples of liquid carriers include phosphate buffered saline, saline, Ringer's solution, dextrose solution, Hank's solution, syrup, oil, water, emulsions, various wetting agents, and the like. Similarly, the carrier or diluent can include a time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. Suitable carriers include those listed above and others known in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The excipient is selected so as not to affect the biological activity of the formulation. However, the excipient may be selected to improve uptake of the active agent into cells, particularly cancer cells.

[0042] Medicaments may be administered by any route deemed appropriate, e.g., by a physician, preferably in a therapeutically effective amount. A therapeutically effective amount refers to that amount of active compound that prevents, ameliorates, or treats symptoms associated with a disease or condition referred to herein. The therapeutic efficacy and toxicity of a drug can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the ED50 (the dose therapeutically effective in 50% of the population) and the LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. The dosing regimen is determined by the attending physician and clinical factors. As is well known in the medical field, the dosage administered to a single patient can depend on many factors, including the type and severity of the disease, the patient's size and age, the specific formulation of the drug administered, sex, the time and route of administration, general health, and other drugs administered concomitantly. The medicaments referred to herein are preferably administered at least once, e.g., as a bolus. However, the medicament may be administered more than once, preferably at least twice, for example, after a defined time period, permanently, or periodically. Progress can be monitored by periodic evaluation. The recommended dosage may be indicated in the prescriber's or user's instructions to anticipate dosage adjustments depending on the intended recipient. The medicament according to the present invention may contain additional active agents in addition to the aforementioned active agents. Preferably, the pharmaceutically active compound according to the present invention is to be applied together with at least one additional agent, and therefore may be formulated with at least one additional agent as a medicament. More preferably, in the case of cancer treatment, the at least one additional active agent is a chemotherapeutic agent or an immunotherapeutic agent, such as a T cell or immune checkpoint modulator. It is also understood that the formulation of the pharmaceutical composition is preferably carried out under GMP-standardized conditions, etc., to ensure the quality, pharmaceutical safety, and efficacy of the medicament.

[0043] Advantageously, research underlying the present invention has revealed that the binding polypeptides described in these Examples induce degradation of PD-L1, a model target polypeptide, in target cells, thereby not only alleviating PD-1-mediated immune checkpoint blockade but also eliminating one of the factors mediating it, thereby improving therapeutic efficacy. The binding polypeptides were found to target and cause degradation of the highly important immune checkpoint protein PD-L1. Importantly, PD-L1 degradation occurred in the picomolar to low nanomolar range, demonstrating the high potency of these binding polypeptides. Notably, the exemplary transmembrane E3 ligases used in the Examples, ZNRF3 and RNF43, exhibit widespread to ubiquitous expression across many tissues and cell lines (see, e.g., www.ncbi.nlm.nih.gov / gene / 84133 and -54894). Thus, the present disclosure provides an R-spondin binding domain that (i) is appropriately mutated to disable WNT and BMP signaling, (ii) binds to an appropriate second binding polypeptide, and (iii) retains biological activity to bind to E3 ligase with high affinity, be internalized, and degraded. Thus, the present study provides a generally applicable signaling-disabled R-spondin-derived first binding domain for proximity-based proteolytic therapy.

[0044] The above definitions apply mutatis mutandis below. The additional definitions and explanations further below also apply mutatis mutandis to all embodiments described herein.

[0045] The present invention further relates to polynucleotides encoding the binding polypeptides according to the invention.

[0046] The polynucleotides of the present invention are described herein above; as described above, the polynucleotides may be configured in expression constructs and / or vectors, i.e., preferably in expression vectors, all of which are also described herein above.

[0047] The present invention also relates to host cells comprising a binding polypeptide according to the invention and / or a polynucleotide according to the invention.

[0048] The term "cell" as used herein is understood by those skilled in the art to refer to the smallest structural unit in biology that has the primary capability of self-replication. Accordingly, this term includes archaea, prokaryotes, and eukaryotes. The term "host cell" refers to a cell that can transiently, preferably stably, maintain the polynucleotides or vectors described herein and / or express the binding polypeptides from the expression constructs or expression vectors described herein; thus, the host cell can be, for example, a bacterial cell, particularly an E. coli cell; or a eukaryotic cell, such as a subject's cell to be treated with the binding polypeptide. The term "target cell" refers to a cell known or believed to express at least one transmembrane E3 ligase and a disease-related polypeptide; preferably, the target cell is a cancer cell. Preferably, the target cell is a subject's cell or a cultured cell derived therefrom.

[0049] The present invention also relates to a device comprising a binding polypeptide according to the invention and / or a polynucleotide according to the invention.

[0050] As used herein, the term "device" refers to a system of means comprising at least the described means, preferably operatively linked to each other and / or to further means so as to allow administration of the compound or composition of the present invention. Preferred means for administering binding polypeptides are well known in the art and have been described herein above. How the means are linked for operation depends on the type of means included in the device and the type of administration envisaged. Preferably, in such cases, the means are included in a single device. The device may accordingly comprise a delivery unit for administering the binding polypeptide and, optionally, a storage unit for storing the binding polypeptide until administration. However, it is also envisaged that the means of the present invention may appear as separate devices in such embodiments, preferably packaged together as a kit. Those skilled in the art will understand how to link the means without further explanation. Preferred devices are those that can be applied without the special knowledge of a specialist. In a preferred embodiment, the device is a syringe containing the binding polypeptide, more preferably with an injection needle. More preferably, the device is an intravenous infusion (IV) device comprising the binding polypeptide. Also preferably, the device is a tube or endoscopic device comprising a pharmaceutical preparation for flow to the administration site, e.g., the heart, or further comprises a needle for local application of the compound or composition, e.g., application to a tumor.

[0051] The present invention also relates to a kit comprising a binding polypeptide according to the invention and / or a polynucleotide according to the invention, and optionally means for administration.

[0052] The term "kit" as used herein refers to a collection of the aforementioned compounds, means, or reagents, which may or may not be packaged together. The components of the kit may be configured in separate vials (i.e., as a kit of separate parts) or may be provided in a single vial, e.g., as a composition as specified hereinabove. In view of the above, the kit may, for example, comprise (i) a first binding domain bound to a first partner of an affinity pair and (ii) at least one second binding domain bound to a second partner of the affinity pair, either in separate vials or as a preformed complex. In one embodiment, the kit housing allows for transfer, particularly general transfer, of the components of the kit; therefore, the housing may particularly be a transportable container containing all specified components. Furthermore, it is understood that the kit of the present invention may be used to practice the methods mentioned hereinabove. It is preferably envisioned that all components are provided in a ready-to-use manner for practicing the methods mentioned hereinabove. Furthermore, the kit preferably includes instructions for practicing the method. The instructions may be provided as instruction manuals in paper or electronic form. For example, the instructions may include instructions for interpreting results obtained when using the kit to perform the aforementioned method. Preferably, the kit is adapted for use in the method of the invention, and more preferably is adapted to include all reagents necessary to perform said method.

[0053] The present invention also relates to a method for killing cancer cells, comprising contacting said cancer cells with a binding polypeptide according to the invention and / or a polynucleotide according to the invention.

[0054] The method for killing cancer cells of the present invention is preferably an in vitro method. However, the method can also be performed in vivo, for example, as part of a method for treating and / or preventing cancer as specified herein below. The method may include steps in addition to those specified above. For example, further steps may involve, for example, identifying cancer cells, determining the expression of at least one transmembrane E3 ligase and / or disease-related polypeptide on the cells, etc. Furthermore, one or more of the steps may be performed or assisted by an automated device. Thus, the method can be performed, in particular, on cells expressing a disease-related polypeptide, preferably target cells. Also preferably, the method is performed, in particular, on cells overexpressing at least one transmembrane E3 ligase, in particular ZNRF3 and / or RNF43, preferably target cells; therefore, preferably, the method further comprises contacting the cancer cells with an agent that provides a transmembrane E3 ligase.

[0055] The term "agent that provides a transmembrane E3 ligase" includes each agent that, when contacted with a cell, preferably a target cell, causes at least one transmembrane E3 ligase to be present in the cell in an increased amount compared to a cell not contacted with the agent. Thus, the agent that provides a transmembrane E3 ligase can be a transmembrane E3 ligase polypeptide, preferably contained in a liposome that can fuse with the cytoplasmic membrane of the cell. More preferably, the agent that provides a transmembrane E3 ligase is a polynucleotide encoding the transmembrane E3 ligase, more preferably an expression construct for the transmembrane E3 ligase.

[0056] The present invention also relates to a binding polypeptide according to the invention and / or a polynucleotide according to the invention for use in medicine; and to the use of a binding polypeptide according to the invention and / or a polynucleotide according to the invention for the manufacture of a medicament.

[0057] Furthermore, the present invention relates to a binding polypeptide according to the invention and / or a polynucleotide according to the invention for use in the treatment and / or prevention of cancer; and to the use of a binding polypeptide according to the invention and / or a polynucleotide according to the invention for the manufacture of a medicament for the treatment and / or prevention of cancer.

[0058] The terms "treat" and "treatment" refer to the amelioration of a disease or disorder referred to herein or its associated symptoms to a significant extent, and as used herein, this term includes the prevention of the worsening of the disease, disorder, or its associated symptoms. As used herein, the treatment may also include the complete restoration of health associated with the disease or disorder referred to herein. It should be understood that treatment as used herein may not be effective in all subjects treated. However, the term preferably requires that a statistically significant portion of subjects suffering from the disease or disorder referred to herein can be successfully treated. Whether a statistically significant portion is present can be determined by those skilled in the art using various well-known statistical evaluation tools, such as determining a confidence interval, determining a p-value, Student's t-test, Mann-Whitney test, etc., without further elaboration. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment should be effective in at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a given cohort or population of subjects. Preferably, treating comprises inhibiting the growth of, and more preferably killing, cancer cells. Preferably, treating cancer comprises reducing the tumor and / or cancer cell burden in the subject. Preferably, treating cancer comprises increasing an immune response, more preferably an increased cellular immune response, against cancer cells of the cancer compared to an untreated control group. As will be appreciated by those skilled in the art, the effectiveness of, for example, cancer treatment depends on various factors, including, for example, the stage and type of cancer. Also preferably, the treatment of cancer further comprises at least one of the administration of an agent that provides a transmembrane E3 ligase, chemotherapy, immunotherapy including administration of T cells, preferably CAR T cells and / or recombinant T cell receptor T cells, surgery, and radiation therapy.

[0059] The terms "prevent" and "prevention" refer to maintaining the health of a subject with respect to a disease or disorder referred to herein for a certain period of time. It is understood that the period may depend on the amount of pharmaceutical compound administered and the individual factors of the subject discussed elsewhere herein. It should be understood that prevention may not be effective in all subjects treated with a binding polypeptide. However, the term preferably requires that a statistically significant portion of a cohort or population of subjects is effectively prevented from contracting a disease or disorder referred to herein or its associated symptoms. Preferably, a cohort or population of subjects that would normally, i.e., would develop a disease or disorder referred to herein in the absence of the preventive measures according to the present invention, is envisioned in this context. Whether a portion is statistically significant can be determined by one of ordinary skill in the art using various well-known statistical evaluation tools described elsewhere herein without further elaboration. In the context of cancer treatment, prevention particularly relates to preventing the occurrence of cancer, preventing the occurrence of metastasis, and / or preventing recurrence.

[0060] The present invention also provides a method for treating and / or preventing cancer in a subject, comprising: (a) administering to said subject a binding polypeptide according to the invention and / or a polynucleotide according to the invention, and (b) thereby treating and / or preventing cancer in said subject. The present invention relates to a method comprising:

[0061] Said therapeutic methods are preferably in vivo methods and may include steps in addition to those specifically mentioned, which may, for example, be related to identifying a subject in need of cancer treatment or at risk of developing cancer, or may be related to further therapeutic steps as identified herein above.

[0062] The present invention also relates to a method for identifying whether a subject suffering from cancer is susceptible to treatment with a binding polypeptide according to the invention and / or a polynucleotide according to the invention, comprising the steps of: (A) determining the expression of a disease-associated polypeptide or a surrogate marker thereof in cancer cells of the subject; and (B) identifying the subject as susceptible to the treatment based on the determination in step (A). The present invention relates to a method comprising:

[0063] The method for identifying a subject is preferably an in vitro method and may include steps in addition to the specifically indicated steps. Further steps may, for example, involve providing a sample, e.g., a cancer sample, for step (A) and / or treating the subject as specified herein above, particularly if the subject is determined to be susceptible to said treatment. Whether a subject is susceptible to said treatment can be determined by a person skilled in the art based on the indicated determination; that is, preferably, if it is determined that the subject's cancer cells express a disease-associated polypeptide or a surrogate marker indicative of disease-associated polypeptide expression, the subject is preferably considered to be susceptible to said treatment.

[0064] In step (A), the expression of a disease-associated polypeptide can be determined by methods known to those skilled in the art, particularly immunohistochemistry, using a biopsy sample, as shown in this example. However, instead of or in addition to the disease-associated polypeptide, a surrogate marker can also be determined. The term "surrogate marker" refers to a biological molecule that is not identical to a disease-associated polypeptide but indicates its expression in cells. Such surrogate markers are known in the art and include, in particular, mRNA encoding a disease-associated polypeptide. Step (A) can include additional determinations; preferably, the expression of ZNRF3 and / or RNF43 is further determined, and the subject is determined to be susceptible to the treatment based on the expression of ZNRF3 and / or RNF43. Preferably, the subject is determined to be susceptible to the treatment if the subject's cancer cells express ZNRF3 and / or RNF43, and more preferably, if ZNRF3 and / or RNF43 are determined not to contain a loss-of-function mutation.

[0065] The present invention also relates to the use, preferably in vitro, of a binding polypeptide according to the invention and / or a polynucleotide according to the invention to cause degradation of a disease-associated polypeptide in a host cell and / or to kill a host cell expressing a disease-associated polypeptide.

[0066] In view of the above, the following embodiments are particularly contemplated: Embodiment 1: A binding polypeptide comprising a first binding domain that binds to a transmembrane E3 ligase; and a second binding domain that binds to a disease-associated polypeptide, preferably programmed cell death-ligand 1 (PD-L1).

[0067] Embodiment 2: The binding polypeptide of embodiment 1, wherein the first binding domain comprises a furin domain 1 of R-spondin (RSPO) and / or a furin domain 2 that does not have wnt-activating activity.

[0068] Embodiment 3: A binding polypeptide according to embodiment 1 or 2, wherein said furin domain 2 comprises an exchange of amino acid F109 with a non-identical amino acid, preferably an F109A amino acid exchange.

[0069] Embodiment 4: The binding polypeptide of any of embodiments 1 to 3, wherein the first binding domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:1. Embodiment 5: The binding polypeptide of any one of embodiments 1 to 4, wherein the first binding domain comprises furin domain 1 and furin domain 2 of RSPO2.

[0070] Embodiment 6: The binding polypeptide of any of embodiments 1 to 5, wherein the first binding domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2. Embodiment 7: The binding polypeptide of any of embodiments 1 to 6, wherein the first binding domain lacks wnt signaling activity and / or bone morphogenetic protein (BMP) signal inhibitory activity.

[0071] Embodiment 8: A binding polypeptide according to any of embodiments 1 to 7, wherein the first binding domain lacks a TSP1 domain, and preferably the first binding domain consists of the furin domain 1 and / or the furin domain 2.

[0072] Embodiment 9: The binding polypeptide of any one of Embodiments 1 to 8, wherein the second binding domain binds to the extracellular domain of PD-L1. Embodiment 10: The binding polypeptide of any one of embodiments 1 to 9, wherein the second binding domain binds to the extracellular domain of the disease-related polypeptide.

[0073] Embodiment 11: The binding polypeptide of any of embodiments 1 to 10, wherein the disease-associated polypeptide is PD-L1 and the second domain comprises the extracellular IgV-like domain of a programmed death-1 (PD-1) polypeptide. Embodiment 12: A binding polypeptide according to any one of embodiments 1 to 11, wherein the second binding domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or 4.

[0074] Embodiment 13: The binding polypeptide of any of embodiments 1 to 12, wherein the second binding domain comprises an amino acid sequence that is at least 75% identical to SEQ ID NO:5. Embodiment 14: A binding polypeptide according to any one of embodiments 1 to 13, wherein the first binding domain and the second binding domain are covalently linked.

[0075] Embodiment 15: A binding polypeptide according to any of embodiments 1 to 14, wherein the first binding domain and the second binding domain together form a fusion polypeptide.

[0076] Embodiment 16: The binding polypeptide of embodiment 15, wherein the first binding domain and the second binding domain are linked via a linker, preferably a GS linker, more preferably a linker comprising the amino acid sequence of SEQ ID NO: 6.

[0077] Embodiment 17: A binding polypeptide according to any one of embodiments 1 to 16, wherein the binding polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:7. Embodiment 18: A binding polypeptide according to any one of embodiments 1 to 17, wherein said binding polypeptide consists of an amino acid sequence at least 80% identical to SEQ ID NO:7.

[0078] Embodiment 19: The binding polypeptide of any of embodiments 1 to 18, wherein the disease-related polypeptide is a transmembrane polypeptide comprising at least one extracellular domain. Embodiment 20: A binding polypeptide according to any one of claims 1 to 19, wherein the disease-related polypeptide is a T cell regulatory polypeptide or an immune checkpoint polypeptide.

[0079] Embodiment 21: The binding polypeptide of any one of claims 1 to 20, wherein the disease-associated polypeptide is PD-L1. Embodiment 22: A polynucleotide encoding a binding polypeptide according to any one of embodiments 1 to 21.

[0080] Embodiment 23: A host cell comprising a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22. Embodiment 24: A device comprising a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22.

[0081] Embodiment 25: A kit comprising a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22, and optionally an administration means. Embodiment 26: A method of killing cancer cells, comprising contacting the cancer cells with a binding polypeptide of any of embodiments 1 to 21 and / or a polynucleotide of embodiment 22.

[0082] Embodiment 27: The method of embodiment 26, wherein said method is an in vitro method. Embodiment 28: The method of embodiment 26 or 27, wherein the cancer cells are PD-L1-overexpressing cancer cells.

[0083] Embodiment 29: A binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22 for use in medicine. Embodiment 30: Use of a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22 for the manufacture of a medicament.

[0084] Embodiment 31: A binding polypeptide according to any of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22 for use in the treatment and / or prevention of cancer.

[0085] Embodiment 32: A binding polypeptide and / or polynucleotide for use according to embodiment 31, wherein said treatment and / or prevention further comprises the administration of an agent that provides a transmembrane E3 ligase.

[0086] Embodiment 33: A binding polypeptide and / or polynucleotide for use according to embodiment 31 or 32, wherein said treatment and / or prevention further comprises the application of at least one of immunotherapy, surgery, radiotherapy, and chemotherapy.

[0087] Embodiment 34: Binding polypeptide and / or polynucleotide for use according to any of embodiments 31 to 33, wherein said immunotherapy comprises the administration of T cells, preferably CAR T cells and / or recombinant T cell receptor T cells.

[0088] Embodiment 35: A binding polypeptide and / or polynucleotide for use according to any of embodiments 31 to 34, wherein the cancer is melanoma.

[0089] Embodiment 36: Use of a binding polypeptide according to any of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22 for the manufacture of a medicament for treating and / or preventing cancer.

[0090] Embodiment 37: A method for treating and / or preventing a disease in a subject, comprising the steps of: (a) administering to a subject a binding polypeptide of any one of embodiments 1 to 22 and / or a polynucleotide of embodiment 22; and (b) thereby treating and / or preventing cancer in said subject. A method including

[0091] Embodiment 38: A method for identifying a subject suffering from cancer susceptible to treatment with a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22, comprising the steps of: (A) determining expression of a disease-associated polypeptide, preferably PD-L1, or a surrogate marker thereof, in cancer cells of said subject; and (B) identifying the subject as susceptible to the treatment based on the determination in step (A). A method comprising:

[0092] Embodiment 39: The method of embodiment 38, wherein the subject is identified as being susceptible to said treatment if it is determined in step (a) that said cancer cells express said disease-associated polypeptide, preferably PD-L1, preferably overexpress PD-L1.

[0093] Embodiment 40: Use of a binding polypeptide of any of Embodiments 1 to 21 and / or a polynucleotide of Embodiment 22 to cause degradation of a disease-related polypeptide, preferably PD-L1, in a host cell and / or to kill a host cell expressing a disease-related polypeptide, preferably PD-L1. Embodiment 41: The subject matter of any of embodiments 1-40, wherein the transmembrane E3 ligase is ZNRF3 E3 ligase and / or RNF43 E3 ligase.

[0094] All documents cited herein are incorporated by reference in their entirety and with respect to the disclosure content specifically mentioned herein.

[0095] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. [Example]

[0096] 1. Method 1.1 Cell lines and their growth conditions HEK293T, MEL624, and A375 cells were maintained in DMEM High Glucose (Gibco 11960) supplemented with 10% FBS (Capricorn FBS-12A), 1% penicillin-streptomycin (Sigma P0781), and 2 mM L-glutamine (Sigma G7513). A375 wild-type and ZNRF3 / RNF43 double knockout cells were kindly provided by the Bryja laboratory and have been validated in a previous study (Radaszkiewicz et al., Elife 10, doi:10.7554 / eLife.65759 (2021)). COLO-800 cells were maintained in RPMI (Gibco 21875) supplemented with 10% FBS, 1% penicillin-streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate (Sigma S8636). All cell lines were cultured at 37°C in a humidity-controlled incubator with 5% CO2. Mycoplasma contamination was negative for all cell lines used.

[0097] The primary human T cell line was derived from an ex vivo expansion culture of tumor-infiltrating lymphocytes obtained from a pancreatic cancer sample (T222, human, male, pancreatic ductal adenocarcinoma) as previously described (Poschke et al., (2020), Clin. Cancer Res. 26:4289). To ensure future availability, cells were repeatedly expanded using the rapid expansion protocol outlined in the same publication and cryopreserved. Three days prior to electroporation, cells were thawed and plated at 3 x 10 in 24-well plates containing X-vivo 15 (Lonza BE02-053Q) supplemented with 2% HSA (CSL Behring 01468366) and 300 IU / mL human IL-2 (Clinigen Healthcare 17152.00.00). 6 Cells were rested at a concentration of 0.1% cells / mL. Cells were maintained in a humidity-controlled incubator at 37°C and 5% CO2. Mycoplasma contamination tests were negative for all cell lines used.

[0098] 1.2 Construct The human RSPO2 wild-type construct was C-terminally tagged with Flag in the pCS2+ vector, as previously demonstrated (Sun et al., Cell rep 36:109559, doi:10.1016 / j.celrep.2021.109559 (2021)). R2, which contains a phenylalanine-alanine mutation at position 109 within the FU2 domain. FA was obtained by mutagenic PCR. ECD was cloned into the pCS2+ vector by inserting its N-terminus into the end of the IgV-like domain of human PD-1. HAC ) (Maute et al., Proc Natl Acad Sci USA 112:E6506-6514, doi:10.1073 / pnas.1519623112 (2015)) was generated by Gibson assembly using synthetic oligonucleotides. The human PD-L1 extracellular domain was inserted into the alkaline phosphatase (AP)-pCS2+ vector, and the AP fusion protein (PD-L1 ECD R2 FA and PD1 HAC The FU1 / 2 domain fragment of the construct was used to assemble the RSPO chimera R2 by Gibson assembly. FA PD1 HAC The resulting fragment was inserted into the pCS2+ vector. A flexible 10-amino acid glycine-serine linker (GSGSGGSGSG, SEQ ID NO: 6) was inserted between the FU2 domain of RSPO2 and the PD1 domain to promote autonomous folding of the domains. HAC Human ZNRF3-HA, ZNRF3 ΔRINGThe -myc and RNF43-Flag constructs have been previously described (Chang et al., Elife 9, doi:10.7554 / eLife.51248(2020); Kim et al., Elife 10, doi:10.7554 / eLife.70885(2021)). Human PD-L1-GFP, pEGFP-N1 / PD-L1, was a gift from Mien-Chie Hung (Addgene plasmid # 121478; RRID: Addgene_121478). The sequences of the constructs generated in this study were confirmed by individual DNA sequencing.

[0099] 1.3 Cell transfection siRNAs and plasmids were transfected using DharmaFECT 1 transfection reagent (Dharmacon T-2001) and X-tremeGENE9 DNA transfection reagent (Roche 06365809001), respectively, according to the manufacturer's protocols.

[0100] 1.4 Synthesis of conditioned medium and protein purification HEK 293T cells were seeded on a 10 cm culture dish and transfected with RSPO2-Flag, R2 FA -Flag, PD1-Flag, PD1 HAC -Flag, R2PD1-Flag and PD-L1 ECDAP was transiently transfected. After 24 hours, the medium was replaced with fresh DMEM containing 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin, and cells were harvested daily for the next 3 days. The conditioned medium was verified and quantified by immunoblotting. Medium containing equimolar amounts of protein was used for subsequent analyses. WNT3A-conditioned medium was produced in L cells as previously described (Kazanskaya et al., Dev Cel 7:525-534 (2004)). For protein purification, the conditioned medium was incubated overnight with anti-Flag antibody-conjugated agarose beads (Sigma A2220). After washing with ice-cold PBS, the protein bound to the beads was eluted with 200 mM glycine (pH 2.6) and neutralized with an equal volume of 1 M Tris (pH 8.0). The eluate was dialyzed against at least a 1000-fold excess of ice-cold PBS. Protein concentrations were quantified by Coomassie staining using BSA standards (Sigma P0914).

[0101] 1.5 In vitro binding assay In a high-binding 96-well plate (Greiner M5811), 2 μg ml -1 Recombinant human PD-L1 ECD Fc (Peprotech 310-35), ZNRF3 ECD Fc (R&D systems 7994-RF-025) or RNF43 ECD Fc (R&D systems 7964-RN-050) protein was coated overnight at 4°C in bicarbonate coating buffer (50 mM NaHCO3, pH 9.6). Coated wells were washed three times with TBST (TBS, 0.1% Tween-20) and blocked with 5% BSA in TBST for 1 hour at room temperature. After overnight incubation with Flag-tagged proteins, wells were washed three times with TBST. Proteins bound to the wells were detected with a peroxidase-conjugated anti-Flag antibody (Sigma A8592) and analyzed by QuantaBlu. TM Fluorogenic Peroxidase Substrate Kit (Thermo ScientificTM In the ternary complex formation assay, PD-L1 was used instead of HRP-anti-Flag antibody. ECD AP was added to the wells. After overnight incubation, AP signals were detected using chemiluminescent AquaSpark AP substrate (Serva 42593.01). Data are presented as the mean and SD of biological replicates.

[0102] 1.6 PD-L1 degradation assay HEK 293T cells were seeded in cell culture plates and transfected with the indicated plasmids. 24 hours after transfection, cells were treated with either conditioned medium or the indicated purified proteins. After a further 24 hours of incubation, cells were harvested for immunoblotting. Cancer cells were seeded in cell culture plates and treated as indicated. After 24 hours of incubation, or at the indicated times, cells were harvested for immunoblotting. For the IFNγ-stimulated degradation assay, MEL624 cells were preincubated with 333 U IFNγ (ImmunoTools 11343536) for 24 hours. PD-L1 antibodies used in the assay Atezolizumab and matched isotype controls were obtained from the Offringa lab, DKFZ, Germany.

[0103] 1.7 Immunoblot Cultured cells were harvested and lysed in ice-cold RIPA buffer supplemented with cComplete Protease Inhibitor Cocktail (Roche 11697498001). The lysate was mixed with Laemmli buffer containing β-mercaptoethanol and boiled at 70°C for 10 minutes to prepare SDS-PAGE samples. Saponin buffer was used to detect cytoplasmic β-catenin. Immunoblot images were acquired using a SuperSignal West pico ECL (ThermoFisher 34580) on an LAS-3000 system (Fujifilm). Blot quantification was performed using ImageJ software.

[0104] 1.8 Immunofluorescence (IF) Cells were seeded onto cell culture plates with glass coverslips. 24 hours after transfection with the indicated plasmids, cells were treated with purified proteins for the indicated periods. After fixation with 4% PFA for 10 minutes, cells were stained with primary antibodies (1:250) overnight at 4°C and then with fluorescently labeled secondary antibodies (1:250) for 2 hours at room temperature. Images were captured using an LSM 700 (Zeiss) and analyzed with ImageJ. Approximately 100 cells were analyzed per group. The following primary antibodies were used for staining: chicken anti-GFP antibody (Millipore AB16901) and mouse anti-LAMP1 antibody (Cell Signaling Technology 15665).

[0105] 1.9 Quantitative Real-Time PCR Cultured cells were lysed in Macherey-Nagel RA1 buffer containing 1% β-mercaptoethanol, and total RNA was isolated using a NucleoSpin RNA isolation kit (Macherey-Nagel 740955). Reverse transcription and PCR amplification were performed as previously described (Sun et al., Cell rep 36: 109559, doi:10.1016 / j.celrep.2021.109559 (2021)). The primers used for AXIN2 were forward 5'-CCACACCCTTCTCCAATCC-3' (SEQ ID NO: 15) and reverse 5'-TGCCAGTTTCTTTGGCTCTT-3' (SEQ ID NO: 16). The primers used for PD-L1 were forward 5'-CCTACTGGCATTTGCTGAACG-3' (SEQ ID NO: 17) and reverse 5'-AGACAATTAGTGCAGCCAGGT-3' (SEQ ID NO: 18). The primers used for ZNRF3 were: forward 5'-TGTGCCATCTGTCTGGAGAA-3' (SEQ ID NO: 21) and reverse 5'-TTCCTGTGAAACCGGTGAGT-3' (SEQ ID NO: 22). The primers used for RNF43 were: forward 5'-GTTTGCTGGTGTTGCTGAAA-3' (SEQ ID NO: 23) and reverse 5'-TGGCATTGCACAGGTACAG-3' (SEQ ID NO: 24). The primers used for GAPDH were: forward 5'-AGCCACATCGCTCAGACAC-3' (SEQ ID NO: 19) and reverse 5'-GCCCAATACGACCAAATCC-3' (SEQ ID NO: 20). The graph shows relative gene expression to GAPDH. Data are presented as mean values ​​with SD from multiple experimental replicates.

[0106] 1.10 TOPflash luciferase reporter assay TOPflash luciferase assays were performed as previously described (Berger et al., EMBO rep 18:712-725, doi:10.15252 / embr.201643585 (2017)). Data are presented as the mean and SD of biological replicates.

[0107] 1.11 Quantification and statistical analysis Statistical analysis was performed using PRISM7 software using unpaired t-test or one-way ANOVA test. Not significant (ns) means p>0.05, * means p<0.05, ** means p<0.01, *** means p<0.001, and **** means p<0.0001.

[0108] 1.12 Flow cytometry Cells were harvested non-enzymatically, pelleted, and resuspended in ice-cold blocking buffer (PBS supplemented with 1% BSA and 0.1% NaN3). After blocking with an Fc receptor binding inhibitor (eBioscience 14916173), cells were stained with an anti-PD-L1 antibody (CST 86744) and then incubated with a fluorochrome-conjugated secondary antibody. Cells stained with secondary antibody alone served as a control. Dead cells were excluded by counterstaining with propidium iodide. FACS samples were analyzed using a FACSCanto or LSRFortessa (BD Biosciences), and data were processed using FlowJo software. Rabbit anti-PD-L1 antibody (CST 86744) was used to measure cell surface PD-L1.

[0109] For T cell activation assays, intracellular and extracellular staining was performed using the following antibodies: anti-human CD107a (BioLegend 328608), anti-human TNFa (BioLegend 502915), anti-mouse TRBC (BioLegend 109230), anti-human CD3 (BioLegend 317328), anti-human CD4 (BioLegend 344614), anti-human CD8 (BioLegend 300920), and LIVE / DEAD. TMFixable Aqua Dead Cell Stain Kit (Thermofisher L34966). PD-1 antibody (BioLegend 329906) was used to measure surface PD-1 expression. Data were acquired on a Fortessa cytometer (BD Biosciences) and analyzed using Flowjo v10.3 software (BD Biosciences).

[0110] 1.13 Co-culture assay for assessing T cell activation Co-culture assays were performed to examine the ability of TCR-transfected T cells to recognize tumors and other target contexts. To monitor tumor recognition, CD107a and TNFα levels were measured. 50,000 effector cells (T cells) and 200,000 target cells were incubated in a U-bottom 96-well plate at 37°C for 5 hours. To detect TNF and CD107a signaling in response to T cell activation, commercially available GolgiPlug (BD Bioscience 555028) and GolgiStop (BD Bioscience 554715) were used at a 1:000 dilution. When human tumor cells (MEL624 and COLO-800) were used as targets, they were pretreated with commercially available human IFN-β at 333 U ml-1 for 48 hours. TCR reactivity was assessed by flow cytometry after 5 hours of incubation of effector and target cells. The percentage of activated T cells was measured by upregulation of CD107a or TNFα in viable CD8+ T cells. Transfected effector cells were assayed against medium to determine background reactivity. The results of this assay were subtracted to check the reactivity of effector cells.

[0111] 1.14 Cytotoxicity Assay The effects of R2PD1 chimeras on cell proliferation and cytotoxicity were assessed using the CellTiter-Glo assay (Promega) according to the manufacturer's instructions. MEL624 and COLO-800 cells were seeded at a density of 500 cells per well in 96-well microplates, with three wells per group. After overnight incubation, R2PD1 chimeras were added to the cells in growth medium, and potential cytotoxicity was measured 72 hours later. CellTiter-Glo reagent (100 μl) was added to each well, and luminescence was recorded using a Spark Multimode Microplate Reader (Tecan). Relative luminescence was determined by normalizing the fold change to the mean luminescence of wells with control medium on the same plate. Protein-untreated cells served as a control, and background luminescence was measured in wells containing medium without cells.

[0112] 1.15 xCELLigence-based tumor killing assay For the tumor killing assay, a 96-well microplate (Agilent 5232376001) with a gold microelectrode cell sensor integrated into the bottom of each well was used with the xCELLigence system (Agilent). The basal impedance of the medium was measured before tumor cell addition. 20,000 tumor cells were seeded into each well, and when the tumor cells reached a cell index of 1, an equal number of effector cells were added. The cell index was measured every minute for the first 30 minutes after effector cell addition and every 5 minutes thereafter. After the initiation of co-culture, target cell proliferation was monitored for 4–5 days. Each condition had three independent replicates, which were monitored in parallel. The normalized cell index was calculated by dividing the cell index value at each time point by the cell index at the time of effector cell addition. RTCA Software Pro (version 2.3.0) was used for analysis.

[0113] 2.Results 2.1 Design and validation of bispecific RSPO chimeras To generate a bispecific chimera capable of simultaneously binding to ZNRF3 / RNF43 and PD-L1, we engineered RSPO2, which has the highest binding affinity to ZNRF3 (Park et al., J. Biol. Chem. 2018, 293 (25), 9759-9769) (Fig. 1a). To prevent unwanted activation of WNT signaling, we engineered an LGR-binding-deficient mutant (R2 FA To achieve this, we mutated phenylalanine 109 in the FU2 domain of RSPO2 (FA mutation). To obtain the PD-L1 binding unit, we cloned the extracellular IgV-like domain of PD-1 (PD1) and generated its high-affinity binding mutant (Maute et al., Proc Natl Acad Sci USA 2015, 112 (47), E6506-6514). HAC To obtain the final bispecific reagent, RSPO2 FU1 / 2 FA The PD1 domain is connected via a flexible glycine-serine linker. HAC We fused the module to the TSP1 domain of RSPO2, which is dispensable for ZNRF3 / RNF43 binding, to generate the bispecific RSPO2 chimera R2PD1. Replacing the TSP1 domain of RSPO2 also abolished its inhibition of BMP signaling.

[0114] Recombinant PD1 HAC , R2 FA The R2PD1 protein was readily produced and secreted into the medium of transfected HEK293T cells. As expected, in cell surface binding assays, R2PD1 bound to LGR4 much less strongly than the chimera without the FA mutation. To examine whether R2PD1 retained its binding activity to its interacting partners, we performed a solid-phase ELISA assay (Fig. 1b). PD1 bound weakly to immobilized PD-L1, whereas PD1 HAC The HAC mutations strongly bound to PD-L1 (Fig. 1c), confirming that they exhibited higher affinity for PD-L1. Importantly, R2PD1 and PD1 bind to immobilized PD-L1. HAC showed similarly strong binding (Fig. 1c). FAR2PD1 bound to immobilized ZNRF3 and RNF43 in a similar manner (Fig. 1d-e). Thus, R2PD1 maintains tight binding to both PD-L1 and ZNRF3 / RNF43 targets.

[0115] A prerequisite for E3 ligase-mediated protein degradation is the formation of an E3 ligase-target complex. To examine whether R2PD1 bridges the interaction between ZNRF3 / RNF43 and PD-L1, we performed an ELISA assay using immobilized ZNRF3 and RNF43 (Fig. 1f). As expected, PD-L1 bound to ZNRF3 (Fig. 1g) and RNF43 (Fig. 1h) only in the presence of R2PD1, but not to R2PD1. FA and isolated PD1 HAC The RSPO chimera R2PD1 effectively bridges the interaction between PD-L1 and ZNRF3 / RNF43.

[0116] 2.2 RSPO chimeras promote degradation of overexpressed PD-L1 in 293T cells To investigate whether RSPO chimeras induce PD-L1 degradation, we performed a degradation assay using HEK293T cells transfected with human PD-L1, with or without ZNRF3 / RNF43 (Figure 2a). Cotransfection of ZNRF3 or RNF43 did not affect PD-L1 levels. However, the addition of recombinant R2PD1 effectively reduced the amount of overexpressed PD-L1 only in cells cotransfected with ZNRF3 or RNF43 (Figure 2b). PD-L1 degradation reached a plateau after incubation with 5.4 nM purified recombinant protein, with a maximum 60% reduction in total protein detected by immunoblot (Figure 2c).

[0117] It was then hypothesized that simultaneous binding of PD-L1 and ZNRF3 / RNF43 is required for PD-L1 degradation. FA or PD1 HACNo reduction in PD-L1 levels was detected in cells incubated with either R2PD1 or R2PD1 (Figure 2d-e). This finding was further confirmed by immunostaining, where R2PD1 abolished cell surface staining of PD-L1 and instead induced colocalization with the lysosomal marker LAMP1 (Figure 2f). FA and PD1 HAC This result is consistent with previous findings that ZNRF3 / RNF43 uses the lysosomal pathway for targeted protein degradation.

[0118] To investigate the importance of E3 ligase activity coordinated with PD-L1 degradation, we investigated the effect of a dominant-negative variant of ZNRF3 lacking the intracellular RING domain (ZNRF3 ΔRING ) was transfected into 293T cells. R2PD1 efficiently reduced the total PD-L1 protein in a dose-dependent manner in cells expressing full-length ZNRF3, but not in cells expressing ZNRF3. ΔRING Transfection with RSPO chimeras abolished PD-L1 degradation (Fig. 2g). We conclude that RSPO chimeras induce degradation of overexpressed PD-L1 in 293T cells in the presence of overexpressed ZNRF3 / RNF43.

[0119] 2.3 RSPO chimeras promote degradation of endogenous PD-L1 in cancer cells To examine whether the RSPO chimera induces degradation of endogenous PD-L1, we focused on cancer cell lines expressing PD-L1. PD-L1 small interfering RNA (siRNA) knockdown in the human melanoma cell line MEL624 reduced PD-L1 protein levels, confirming the specificity of the antibody and the cells' expression of PD-L1 (Figure 3a). Addition of R2PD1-conditioned medium reduced PD-L1 levels by approximately 90% (Figure 3b). PD-L1 reduction plateaued at as little as 0.27 nM R2PD1 protein (Figure 3c). PD-L1 reduction by R2PD1 was readily detected within 6 h (Figure 3d). Furthermore, PD-L1 reduction was observed only with R2PD1, not with R2PD1. FA and PD1 HACThis was not observed with R2PD1 (Figure 3e), confirming the need for dual specificity. Flow cytometry analysis confirmed that R2PD1 induced cell surface removal of PD-L1 (Figure 3f-g).

[0120] PD-L1 expression is regulated by various cytokines present in the tumor microenvironment, particularly interferon-gamma (IFNγ). We investigated whether R2PD1 could degrade IFNγ-induced PD-L1. As expected, IFNγ increased PD-L1 protein levels, and co-culture with R2PD1 reduced them (Figure 3h).

[0121] To exclude the possibility that the decrease in PD-L1 was due to its transcriptional misregulation rather than induced proteolysis, we monitored PD-L1 mRNA in MEL624 cells. qRT-PCR analysis revealed that no significant changes in PD-L1 mRNA were observed in cells incubated with the effector proteins studied (Figure 3i).

[0122] We confirmed this finding using another melanoma cell line, COLO-800, which expresses PD-L1 and can be downregulated by siRNA transfection (Figure 3j). Again, the R2PD1 chimera reduced both the cell surface and total protein levels of PD-L1, although less completely than in MEL624 cells (Figures 3k-m). Again, no significant changes in PD-L1 mRNA were detected by qRT-PCR analysis (Figure 3n). Despite similar PD-L1 protein levels in MEL624 and COLO-800 cells (Figure 3o), the reduction of PD-L1 in COLO-800 cells was only evident after 24 h of incubation with the R2PD1 chimera (Figure 3p), which was slower than that in MEL624 cells (Figure 3d). We speculated that the lower efficiency and different kinetics of PD-L1 degradation by R2PD1 might be due to the different expression levels of ZNRF3 / RNF43. Indeed, qRT-PCR analysis revealed that the expression levels of ZNRF3 / RNF43 were significantly higher in MEL624 cells than in COLO-800 cells (Fig. 3q).

[0123] Atezolizumab is a therapeutic monoclonal antibody that targets PD-L1 and promotes tumor-directed T cell activation by inhibiting its interaction with PD-1. Here, we compared the effects of the RSPO chimera and atezolizumab on PD-L1 protein abundance. The R2PD1 chimera again reduced PD-L1 in both MEL624 and COLO-800 cells, whereas the addition of atezolizumab did not alter PD-L1 protein abundance (Figure 3r-s). Together, these data demonstrate that the R2PD1 chimera induces degradation of endogenous PD-L1 protein in cancer cells.

[0124] 2.4 PD-L1 degradation by RSPO chimeras is independent of WNT / β-cat signaling RSPO is a potent WNT signaling agonist and BMP signaling agonist. These signaling activities may cause unwanted side effects when applying RSPO chimeric proteins. Therefore, the R2PD1 protein used here lacks the TSP1 domain, completely eliminating the BMP inhibitory activity, and contains F in the FU2 domain. 109 The presence of the A(FA) mutation should inhibit LGR binding and therefore WNT activation. To rule out the possibility that the furin domain of R2PD1 retains residual WNT / β-cat signaling activity, we performed PD-L1 degradation assays in the presence of WNT inhibitors. The R2PD1 chimera degraded PD-L1 in both the presence of the potent WNT signaling inhibitor DKK1 (Figure 4a-b) and β-catenin siRNA knockdown (Figure 4c-d). Furthermore, unlike wild-type RSPO2, the chimeric protein did not induce expression of the WNT target gene AXIN2 or affect WNT reporter (TOPflash) activity (Figure 4e-f). These findings confirm that the RSPO chimera abolishes WNT signaling and that PD-L1 degradation is independent of WNT / β-cat signaling.

[0125] 2.5 PD-L1 degradation by RSPO chimeras requires ZNRF3 / RNF43 We demonstrated that the bispecific chimeric R2PD1 induces PD-L1 degradation. To investigate whether PD-L1 degradation occurs via ZNRF3 / RNF43, the R2PD1 chimeric protein was co-transfected with the soluble RNF43 extracellular domain (RNF43) prior to addition to cells. ECD Fc) (Figure 5a). ECD Since Fc directly binds to R2PD1 (Fig. 1e), the rationale is that if the chimera acts specifically, it should abolish the effect of R2PD1. ECDR2PD1 preincubated with Fc rescued PD-L1 from degradation (Figure 5b). Furthermore, while knockdown of either ZNRF3 or RNF43 alone with siRNA was insufficient to block R2PD1-mediated PD-L1 degradation, dual knockdown successfully blocked degradation in both MEL624 cells (Figure 5c-e) and COLO-800 cells (Figure 5f-g), indicating that this chimera is involved in both E3 ligases. To confirm these findings, we utilized the ZNRF3 / RNF43 double mutant A375 melanoma cell line (dKO). R2PD1 reduced PD-L1 in control A375 cells but failed to reduce PD-L1 in dKO cells (Figure 5h-i). We conclude that in melanoma cells, the RSPO chimera engages ZNRF3 and RNF43 for PD-L1 degradation.

[0126] 2.6 R2PD1 chimeras reactivate cytotoxic T cells and suppress tumor cell proliferation To confirm that targeting PD-L1 with the R2PD1 chimera promotes cytotoxic T cell activation, we performed T cell activation assays in which primary T cells were cocultured with target cells and tumor cell killing assays. Primary T cells were transfected with human DMF5 T cell receptor (TCR) mRNA to recognize target cells displaying the matching MART-1 melanoma antigen. Furthermore, PD-1 mRNA was co-transfected into the T cells to enhance PD-L1 / PD-1 signaling.

[0127] First, HEK 293T cells were transfected with PD-L1 and MART-1 minigenes to target T cells and then cocultured with T cells transfected with the corresponding TCR. Expression of CD107a or TNFα was monitored as a marker of cytotoxic T cell activation. When the transfected cells were cocultured, approximately 60% of CD8+ T cells were CD107a-positive. Coexpression of PD-L1 / PD-1 in HEK293T cells and T cells, respectively, reduced the percentage of activated T cells by approximately 80%. Addition of the R2PD1 chimera to these HEK293T-restricted T cells dose-dependently restored T cell activation, as monitored by CD107a expression (Figure 6(a)).

[0128] Using this experimental system, we next replaced the engineered HEK293T cells with MEL624 cells, which endogenously express MART-1, as T cell targets. When MEL624 cells were cocultured with T cells, the R2PD1 chimera activated T cells in a dose-dependent manner (Figure 6(b)). R2PD1 was found to be significantly more potent than atezolizumab, a clinically used checkpoint inhibitor that is a humanized IgG1 monoclonal antibody targeting PD-L1, at the same concentration (Figure 6(b)). The R2PD1 response reached saturation at low nM concentrations, where atezolizumab was still ineffective. Even at the highest dose employed, R2PD1 was 2.5-fold more potent than atezolizumab (Figure 6(b)).

[0129] Furthermore, the R2PD1 chimera inhibited MEL624 cell proliferation when cocultured with T cells (Figure 6(c)), but not without T cells. In this tumor-killing assay, R2PD1 was also more potent than atezolizumab at equivalent concentrations (3.7 nM), suppressing tumor cell proliferation by 30% versus 7% (Figure 6(d)). Note that while overall growth inhibition is modest in such short-term in vitro assays, the full tumor-suppressing potential of checkpoint inhibitors is exerted over a long period in vivo. We conclude that the R2PD1 chimera functions as a checkpoint inhibitor in vitro, reactivating T cells, and suppressing tumor cell proliferation more potently than the established checkpoint inhibitor atezolizumab.

[0130] Prior art documents Berger et al., EMBO rep 18:712-725, doi:10.15252 / embr.201643585 (2017) Chang et al., Elife 9, doi:10.7554 / eLife.51248 (2020); de Lau et al., Genome Biology 13:242 (2012) de Lau et al., Genes Dev 28:305, doi:10.1101 / gad.235473.113 (2014) Hao et al., Nature 485:195, doi:10.1038 / nature11019 (2012) He et al., Front. Cell Dev. Biol 9:685106, doi:10.3389 / fcell.2021.685106 (2021) Hey et al. (2005), Trends Biotechnol. 23:514 Kazanskaya et al., Dev Cel 7:525-534 (2004) Kim et al., Elife 10, doi:10.7554 / eLife.70885 (2021) Koo et al., Nature 488:665, doi:10.1038 / nature11308 (2012) Lau et al., Genome Biology 13:242 (2012) Lee et al., Nature comm 11: 5570, doi:10.1038 / s41467-020-19373-w (2020) Maute et al., Proc Natl Acad Sci U S A 112:E6506-6514, doi:10.1073 / pnas.1519623112 (2015) Park et al., J. Biol. Chem. 2018, 293 (25), 9759-9769) Poschke et al. (2020), Clin. Cancer Res. 26:4289 Radaszkiewicz et al., Elife 10, doi:10.7554 / eLife.65759 (2021) Sun et al, Signal Transduct Target Ther 4:64 doi:10.1038 / s41392-019-0101-6 (2019) Sun et al., Cell rep 36: 109559, doi:10.1016 / j.celrep.2021.109559 (2021) ter Stege et al. (2021), Oncogene 40(47):6469 Zebisch et al., Nature comm 4:2787, doi:10.1038 / ncomms3787 (2013)

Claims

1. A binding polypeptide comprising a first binding domain that binds to a transmembrane E3 ligase and a second binding domain that binds to a disease-related polypeptide, wherein the first binding domain comprises furin domain 1 and / or furin domain 2 of R-spondin (RSPO), and the second binding domain lacks wnt signaling activity and / or bone morphogenetic protein (BMP) signal inhibitory activity.

2. The binding polypeptide of claim 1 , wherein the first binding domain comprises furin domain 1 and furin domain 2 of RSPO2.

3. 3. The binding polypeptide of claim 2, wherein the furin domain 2 comprises an exchange of amino acid F109 with a non-identical amino acid, preferably an F109A amino acid exchange.

4. 4. The binding polypeptide of claim 1, wherein the first binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:

2.

5. The binding polypeptide of any one of claims 1 to 4, wherein the first binding domain lacks the TSP1 domain.

6. 6. A binding polypeptide according to any one of claims 1 to 5, wherein the first binding domain consists of the furin domain 1 and / or the furin domain 2.

7. 7. The binding polypeptide of any one of claims 1 to 6, wherein the disease-related polypeptide is a T-cell regulatory polypeptide or an immune checkpoint polypeptide.

8. 8. The binding polypeptide of any one of claims 1 to 7, wherein the disease-related polypeptide is Programmed Death-Ligand 1 (PD-L1) and the second binding domain comprises the extracellular IgV-like domain of a Programmed Death-1 (PD-1) polypeptide.

9. 9. A binding polypeptide according to any one of claims 1 to 8, wherein the second binding domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 3 or 4, preferably the second binding domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:

5.

10. 10. A binding polypeptide according to any one of claims 1 to 9, wherein the first binding domain and the second binding domain together form a fusion polypeptide, preferably wherein the first binding domain and the second binding domain are linked via a linker, preferably a GS linker, more preferably a linker comprising the amino acid sequence of SEQ ID NO:

6.

11. 11. The binding polypeptide of any one of claims 1 to 10, wherein the binding polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

7.

12. A polynucleotide encoding a binding polypeptide according to any one of claims 1 to 11.

13. A host cell comprising a binding polypeptide according to any one of claims 1 to 11 and / or a polynucleotide according to claim 12.

14. 13. An in vitro method of killing cancer cells comprising contacting cancer cells with a binding polypeptide according to any one of claims 1 to 11 and / or a polynucleotide according to claim 12.

15. A binding polypeptide according to any one of claims 1 to 11 and / or a polynucleotide according to claim 12 for use in medicine.

16. 13. A binding polypeptide according to any one of claims 1 to 11 and / or a polynucleotide according to claim 12 for use in the treatment and / or prevention of cancer, preferably wherein the cancer is melanoma.

17. 13. A method for identifying a subject suffering from cancer as being susceptible to treatment with a binding polypeptide according to any one of claims 1 to 11 and / or a polynucleotide according to claim 12, comprising: (A) determining PD-L1 expression or a surrogate marker thereof in cancer cells of the subject; and (B) identifying the subject as susceptible to the treatment based on the determination in step (A). A method comprising:

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