Targeted PD-L1 degradation

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 the furin domain 1 and / or furin domain 2 of R-pondin (RSPO) wherein the first binding domain lacks wnt signaling activity and / or lacks bone morphogenetic protein (BMP) signaling inhibitory activity; and to polynucleotides, host cells, medical uses and methods related thereto.

Description

Technical Field

[0001] The present invention relates to 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, wherein the first binding domain comprises furin domain 1 and / or furin domain 2 of R-spondin (RSPO), wherein the first binding domain lacks wnt signaling activity and / or lacks bone morphogenetic protein (BMP) signal inhibition activity; and to polynucleotides, host cells, medical uses and methods related thereto. Background Art

[0002] Targeted protein degradation (TPD) is a rapidly developing field that has broadened the scope of therapeutic targets to include historically undruggable proteins and overcome drug resistance (see, e.g., Sun et al., Signal Transduct Target Ther 4:64 doi:10.1038 / s41392-019-0101-6 (2019)). Unlike the inhibition of the function of the target protein (POI) by traditional small molecule drugs and antibody-based models, TPD induces the degradation of the targeted protein. Among the various TPD platforms, protein degradation targeting chimeras (PROTACs) have been successfully applied to the degradation of various POIs involved in a variety of diseases, including cancer, infectious diseases, and neurodegenerative diseases (He et al., Front. Cell Dev. Biol 9:685106, doi:10.3389 / fcell.2021.685106 (2021)).

[0003] ZNRF3 / RNF43 are single-spanning E3 ligases with a well-structured extracellular domain and a functional intracellular RING domain (Zebisch et al., Nature comm 4:2787, doi:10.1038 / ncomms3787 (2013)). Their ligands, R-spondin (RSPO), are a family of four secreted stem cell growth factors that are crucial in a variety of 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 shown 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)). They achieve this function by targeting different substrates using ZNRF3 / RNF43.

[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 WNT signaling activation, RSPO2 and RSPO3 also inhibit BMP signaling. Notably, the WNT agonist function of R-spondin has been implicated in tumorigenesis (ter Stege et al. (2021), Oncogene 40(47):6469), which limits their therapeutic applicability.

[0005] The programmed death ligand 1 (PD-L1) protein, also known as CD274, is a 40 kDa transmembrane protein that is known to bind to the PD-1 receptor found on activated T cells and has been proposed to be involved in controlling CD8+ T cell proliferation. Therefore, PD-L1 antagonists have been proposed to block the interaction of PD-L1 with PD-1, thereby promoting tumor-directed T cell activation in cancer treatment; for example, atezolizumab is a therapeutic monoclonal antibody that targets PD-L1. Summary of the invention

[0006] In view of the above, there is still a need to improve the treatment of cancer, in particular by activating and / or maintaining an immune response to cancer cells. The technical problem of the present invention can be seen as providing a device and a method for meeting the above needs. The technical problem is solved by the embodiments characterized in the claims and below.

[0007] Therefore, the present invention relates to 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 to programmed death-ligand 1 (PD-L1).

[0008] In general, the terms used herein will be given the common and customary meanings of ordinary technicians in the field, unless otherwise specified, without limitation to special or customized meanings. As used below, the terms "having", "including" or "comprising" or any arbitrary grammatical variants thereof are used in a non-exclusive manner. Therefore, these terms can refer to the situation where there are no other features in the entity described in the context of this article except the features introduced by these terms, and can also refer to the situation where one or more other features are present. As an example, the expressions "A has B", "A contains B" and "A includes B" can all refer to the situation where there are no other elements in A except B (i.e., A is composed of B alone and exclusively), and can also refer to the situation where there are one or more other elements in entity A except B, such as element C, element C and D or even other elements. In addition, as understood by those skilled in the art, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e., equivalent to "comprising at least one". Thus, unless stated otherwise, a statement relating to one of a plurality of items preferably relates to at least one such item, more preferably a plurality of such items; thus, for example, identifying a "cell" relates to identifying at least one cell, preferably to identifying a plurality of cells.

[0009] In addition, as used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in combination with optional features without limiting further possibilities. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As will be appreciated by those skilled in the art, the present invention can be implemented by using alternative features. Similarly, the features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any limitation on other embodiments of the invention, without any limitation on the scope of the invention, and without any limitation on the possibility of combining the features introduced in this manner with other optional or non-optional features of the invention.

[0010] The methods detailed herein below are preferably in vitro methods. In principle, the method steps may be performed in any arbitrary order deemed suitable by the skilled person, but are preferably performed in the order indicated; likewise, one or more (preferably all) of the steps may be assisted or performed by automated equipment. Furthermore, the method may comprise steps other than those explicitly mentioned above.

[0011] As used herein, if not otherwise specified, 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; preferably, standard conditions include a pH value of 7. In addition, if not otherwise indicated, the term "about" relates to an indicated value with a technical accuracy generally accepted in the relevant field, preferably to an indicated value of ±20%, more preferably ±10%, and most preferably ±5%. In addition, the term "substantially" means that there is no deviation that has an impact on the indicated result or use, i.e., the potential deviation will not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially consisting of..." means including the specified components but not including other components, which do not include materials present as impurities, unavoidable materials present due to the process for providing the components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase "substantially consisting of..." includes any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of one group of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, most preferably less than 0.1% by weight of the non-specified component or components.

[0012] The term "combination" is understood by the skilled person; preferably, the term relates to a combination of at most 10 -6 mol / l, more preferably at most 10-7 mol / l, even more preferably at most 10 -8 mol / l, most preferably up to 10 -9 Dissociation constant K in mol / l D The term "specific binding" is also understood by those skilled in the art. Preferably, specific binding involves binding, as described elsewhere herein, wherein the affinity of the binding polypeptide for its cognate binding partner is at least 10 times, preferably at least 100 times, more preferably at least 1000 times higher than the affinity for any non-cognate binding partner. Thus, the dissociation constant (K) of any 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 mol / l.

[0013] 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, wherein the sequence fragments in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the sequences compared to which the optimal alignment is obtained. The percentage is calculated by determining the number of positions where the same residues occur in the two sequences (preferably over the entire length of the polynucleotide or polypeptide) to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. The optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the similarity search method of Pearson and Lipman (1988), by computerized implementations of these algorithms (e.g., BLAST, GAP, BESTFIT, PASTA or TFASTA) or by visual inspection. In view of having determined two sequences for comparison, preferably GAP and BESTFIT are used to determine their optimal alignment, thereby determining the degree of identity. Preferably, the default value of 5.00 for the gap weight and the default value of 0.30 for the gap weight length are used. More preferably, the comparison is performed using the basic local alignment search tool (BLAST) implementation and default parameter values. In the context of the biological sequences mentioned herein, the term "substantially identical" means at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99% identity % value. It is understood that the term "substantially identical" includes 100% identity. The foregoing applies to the term "substantially complementary" with necessary changes.

[0014] The term "fragment" of a biomacromolecule (preferably a polynucleotide or polypeptide) is used in a broad sense to refer to any sub-portion (preferably a subdomain) of each biomacromolecule comprising the sequence, structure and / or function shown. Thus, the term includes sub-portions resulting from actual fragmentation of the biomacromolecule, but also sub-portions derived from the corresponding biomacromolecule in an abstract manner (e.g., on a computer). Thus, as used herein, Fc or Fab fragments, but also, for example, single-chain antibodies, bispecific antibodies and nanobodies can be referred to as fragments of immunoglobulins.

[0015] Unless otherwise specifically indicated herein, the indicated compounds, particularly polynucleotides and polypeptides, may be included in a larger structure, for example, may be covalently or non-covalently linked to other sequences, carrier molecules, blockers and other excipients. In particular, the indicated polypeptide may be included in a fusion polypeptide comprising other peptides, which may be used as, for example, tags for purification and / or detection, as linkers, or for extending the in vivo half-life of the compound. The term "detectable label" refers to an amino acid stretch (a stretch of amino acids) added to or introduced into a fusion polypeptide; preferably, the label is added to the C-terminus or N-terminus of the fusion polypeptide. The amino acid stretch preferably allows the polypeptide to be detected by an antibody that specifically recognizes the label; or preferably allows the formation of a functional conformation, such as a chelator; or preferably allows visualization, such as in the case of a fluorescent label. Preferred detectable labels are Myc tags, FLAG tags, 6-His tags, HA tags, GST tags or fluorescent protein tags, such as GFP tags. These tags are all well known in the art. Other further peptides preferably included in the fusion polypeptide contain other amino acids or other modifications, which can be used as secretion mediators, as mediators of blood-brain barrier passage, as cell penetrating peptides and / or as immunostimulants. Additional polypeptides or peptides that can be fused to the polypeptide are signal sequences and / or transport sequences, such as the IL-2 signal sequence and linker sequences.

[0016] As used herein, the term "polypeptide" refers to a molecule comprising a plurality of (typically at least 20) amino acids covalently linked by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are generally considered "peptides". Preferably, the polypeptide comprises 50 to 1000, more preferably 100 to 1000, more preferably 200 to 500, and most preferably 250 to 400 amino acids. The polypeptide may be a complex of more than one amino acid chain, i.e., a multimer, such as a dimer, a trimer, etc.; in this case, the complex of more than one amino acid chain may also be referred to as a "polypeptide oligomer" or a "protein complex". Preferably, the complex of more than one amino acid chain 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. In addition, the polypeptide may comprise additional non-peptide structures, such as at least one glycosylation, lipid conjugation, etc. More preferably, the indicated polypeptide, in particular a binding polypeptide, comprises all the structural components indicated contained in one continuous covalent peptide chain, and therefore, the polypeptide, in particular a binding polypeptide, is preferably or is contained in a fusion polypeptide. Unless otherwise specifically indicated, references herein to a particular polypeptide preferably include polypeptide variants.

[0017] As used herein, the term "polypeptide variant" relates to any chemical molecule comprising at least one polypeptide as indicated herein, which has the indicated biological activity, but is structurally different from the specific polypeptide. Preferably, the polypeptide variant comprises a polypeptide having a continuous amino acid sequence corresponding to at least 50%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% of the polypeptide indicated specifically (particularly the amino acid sequence of SEQ ID NO: 7). In addition, it should be understood that the polypeptide variant mentioned according to the present invention should have an amino acid sequence that differs due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant still has preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity with the amino acid sequence of the specific polypeptide (particularly SEQ ID NO: 7 or 9). In view of the above, the identity % values ​​indicated herein below are preferably lower limits of the identity % and the desired identity % may be higher, for example having the above values. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and as described above. The polypeptide variants mentioned above may be allelic variants or any other species-specific homologues, paralogues or orthologues. In addition, the polypeptide variants mentioned herein include fragments of a particular polypeptide or polypeptide variants of the aforementioned type, as long as these fragments and / or variants have the biological activity as indicated. These fragments may be or may be derived from, for example, degradation products or splice variants of the polypeptide. This also includes variants that differ due to post-translational modifications (e.g., phosphorylation, glycosylation, ubiquitination, sumoylation or myristylation), which include non-natural amino acids and / or are peptide mimetics. The foregoing applies independently, mutatis mutandis, to the domains of the polypeptides described herein, i.e., the first binding domain and the second binding domain may be variant domains, still having the indicated activity, particularly the binding activity, as described below. Thus, the first binding domain may, for example, be a variant of a polypeptide comprising furin domain 1 and furin domain 2 of R-spondin, for example, may comprise an amino acid sequence having at least 80% identity with SEQ ID NO:2, and the second binding domain may be a polypeptide variant of the extracellular IgV-like domain of a PD-1 polypeptide, for example, may comprise an amino acid sequence having at least 85% identity with SEQ ID NO:3.

[0018] As used herein, the term "polynucleotide" refers to a linear or circular nucleic acid molecule. The polynucleotides of the present invention should preferably be provided as isolated polynucleotides (i.e., isolated from their natural background) or in a genetically modified form, preferably comprising at least one heterologous sequence. The term includes single-stranded polynucleotides and double-stranded polynucleotides. In addition, the term also includes chemically modified polynucleotides, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides or artificially modified derivatives, such as biotinylated polynucleotides, locked nucleic acids, etc. The polynucleotides of the present invention have the activity of encoding a binding polypeptide as described herein or at least one binding domain thereof. Methods for testing whether a given polynucleotide has the aforementioned biological activity are known in the art and are described below. Unless otherwise specifically indicated, reference to a particular polynucleotide herein preferably includes polynucleotide variants.

[0019] As used herein, the term "polynucleotide variant" refers to a variant of a polynucleotide comprising a nucleic acid sequence as mentioned herein, characterized in that the sequence can be derived from the above-mentioned specific nucleic acid sequence by at least one nucleotide substitution, addition and / or deletion, wherein the polynucleotide variant should have the biological activity as described for the specific polynucleotide. Preferably, the polynucleotide variant is an ortholog, a paralog or another 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 include polynucleotides comprising a nucleic acid sequence that can preferably hybridize with the above-mentioned specific polynucleotide under stringent hybridization conditions. These stringent conditions are known to the skilled person and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 63.1-63.6. The skilled person knows how to determine the desired hybridization conditions by reference to textbooks (such as the above-mentioned textbooks or textbooks indicated elsewhere herein). Alternatively, polynucleotide variants can be obtained by PCR-based techniques (e.g., DNA amplification based on mixed oligonucleotide primers). In addition, variants include polynucleotides comprising nucleic acid sequences, the nucleic acid sequences having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99% identity with the nucleic acid sequences of specific indications. In addition, polynucleotides are also included, which include nucleic acid sequences encoding amino acid sequences, the amino acid sequences having at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99% identity with the amino acid sequences of specific indications. Preferably, the identity percentage value is calculated over the entire amino acid or nucleic acid sequence region, preferably as described above. The polynucleotides of the present invention are composed of, substantially composed of or comprise the above-mentioned nucleic acid sequences. Therefore, they may also comprise other nucleic acid sequences. Specifically, the polynucleotides of the present invention may encode fusion proteins, wherein a partner of the fusion protein is a polypeptide encoded by the above-mentioned nucleic acid sequences. In addition, the polynucleotides may be included in expression constructs and / or vectors.

[0020] As used herein, the term "expression construct" refers to a heterologous polynucleotide comprising the aforementioned polynucleotide and a nucleic acid sequence required for expressing the polynucleotide. Typically, this other nucleic acid sequence (which is preferably heterologous to a polynucleotide encoding a binding polypeptide or at least one of its binding domains) can be a promoter sequence, a regulatory sequence and / or a transcription termination sequence, such as a terminator. The expression of the polynucleotide includes transcription of the polynucleotide into RNA. Regulatory elements that ensure expression in cells (particularly eukaryotic cells, preferably mammalian cells) are well known in the art. They preferably include regulatory sequences that ensure transcription initiation and optionally poly-A signals that ensure transcription termination and transcript stability. Additional regulatory elements can include transcription and translation enhancers. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, lac, trp or tac promoters in Escherichia coli, and examples of regulatory elements that allow expression in eukaryotic host cells are CMV-promoters, SV40-promoters, RSV-promoters (Rous sarcoma virus), CMV-enhancers, SV40-enhancers or globin introns in yeast AOX1 or GAL1 promoters or mammals and other animal cells. In addition, inducible expression control sequences can be used in the expression constructs encompassed by the present invention. Such inducible constructs can comprise tet or lac operator sequences or sequences induced by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to the elements responsible for initiating transcription, such regulatory elements can also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site downstream of the polynucleotide. Preferably, in eukaryotic host cells, the expression construct is a eukaryotic expression construct, i.e. an expression construct comprising all elements required for expression (preferably induced expression). However, the expression construct can also be a bacterial expression construct for producing a binding polypeptide in a bacterial cell.

[0021] The term "vector" preferably includes bacteriophage, plasmid, viral vector or retroviral vector and artificial chromosome, such as bacterial or yeast artificial chromosome. In addition, the term also relates to a targeting construct, which allows the targeting construct to be randomly or site-specifically integrated into genomic DNA. Such a target construct preferably comprises a DNA of sufficient length for homologous recombination or heterologous recombination as described in detail below. The vector comprising the polynucleotide of the present invention preferably also comprises a selective marker for propagation and / or selection in a host. The vector can be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced into a precipitate (such as a calcium phosphate precipitate or a rubidium chloride precipitate), or into a complex with a charged lipid, or into a carbon-based cluster (such as fullerene). Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line before being applied to a host cell. Viral vectors, particularly retroviral vectors, may have replication ability or replication defects. In the latter case, viral proliferation usually occurs only in a complementary host / cell. More preferably, in the vector of the present invention, the polynucleotide is operably linked to an expression control sequence that allows expression in a prokaryotic or eukaryotic cell or an isolated fraction thereof, i.e. preferably, the polynucleotide is contained in an expression vector. In this case, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (InVitrogene) or pSPORT1 (GIBCO BRL). Preferably, the vector is an expression vector, a gene transfer and / or a targeting vector. Expression vectors derived from viruses (e.g., retroviruses, vaccinia viruses or adeno-associated viruses) can be used to deliver polynucleotides or vectors to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; 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).

[0022] In view of the above, it is understood by those skilled in the art that reference to a gene or its gene product herein includes polynucleotide variants of a gene and one or more of its transcription products, i.e., in particular alleles, homologs and mutants of a gene, and, for example, transcript variants, such as splice variants and RNA editing variants of an RNA gene product. More preferably, reference to a gene relates to a gene and its naturally occurring alleles. In addition, reference to a polypeptide gene product includes polypeptide variants as described above, in particular isoforms and / or mutant proteins having a specified degree of sequence identity and having a specified biological activity. For those genes or gene products referred to by a Genbank accession number or similar designation, the corresponding sequences are included in this specification by reference. Unless otherwise indicated, database entries refer to the status of the corresponding database entry on the day before the filing date of the present application.

[0023] The term "R-spondin" is known to the skilled person as relating to the roof plate-specific spondin family, for example as reviewed in de Lau et al., Genome Biology 13:242 (2012). Thus, the R-spondin may be one of R-spondin 1 to 4, preferably one of human R-spondin 1 to 4. Preferably, the R-spondin is R-spondin 2 or R-spondin 3. The term "R-spondin 2" is known to the skilled person. Human R-spondin 2 polypeptides have several isoforms, for example the amino acid sequence of the isoform 1 precursor is provided as Genbank accession number NP_848660.3, SEQ ID NO:12. Thus, as used herein, the term "R-spondin 2" preferably relates to the aforementioned human R-spondin 2, or to a polypeptide having an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99% identical to the amino acid sequence of said human R-spondin 2. Preferably, R-spondin 2 is human R-spondin 2 or a homologue thereof, preferably a vertebrate homologue, more preferably a mammalian homologue. Homologues of R-spondin 2 are known, for example from the HomoloGene database entry 18235; in addition, homologues of R-spondin 2 in other species can be identified by sequence comparison, in particular by determining the degree of identity as described elsewhere herein. Thus, R-spondin 2 is preferably human, chimpanzee, rhesus monkey, rat, mouse, cow, dog, chicken, zebrafish or R-spondin 2 from Xenopus tropicalis, more preferably human R-spondin 2. The term "R-spondin 3" is also known to the person skilled in the art. The amino acid sequence of the human R-spondin 3 precursor is available, for example, as Genbank accession number NP_116173.2, SEQ ID NO: 2. Therefore, as used herein, the term "R-spondin 3" preferably relates to the aforementioned human R-spondin 3, or to a polypeptide having an amino acid sequence that is at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, most preferably at least 95% identical to the amino acid sequence of said human R-spondin 3. Preferably, R-spondin 3 is human R-spondin 3 or a homologue thereof, preferably a vertebrate homologue, more preferably a mammalian homologue.Homologues of R-spondin 3 are known, for example from the HomoloGene database entry 12484; in addition, homologues of R-spondin 3 in other species can be identified by sequence comparison, in particular by determining the degree of identity as described elsewhere herein. Thus, R-spondin 3 is preferably human, chimpanzee, rhesus monkey, rat, mouse, cow, chicken, zebrafish or R-spondin 3 from Xenopus tropicalis, more preferably human R-spondin 3.

[0024] As used herein, the term "binding domain" relates to a substructure of a binding polypeptide having an indicated biological binding activity; preferably, the binding is a specific binding. Preferably, the binding domain comprises, more preferably consists of, a continuous amino acid chain having an indicated binding activity. Preferably, the binding domain has only the indicated binding activity, i.e. preferably has no biological activity other than those specifically indicated, although such additional biological activity may already be present in the protein from which the binding domain is derived. Thus, preferably the binding domain, in particular the first binding domain, more preferably both the first binding domain and the second binding domain, most preferably the binding polypeptide, has no wnd signaling activity and / or has no bone morphogenetic protein (BMP) signaling inhibitory activity, more preferably has no signaling activity. As described herein, the binding polypeptide comprises at least a first binding domain and a second binding domain, wherein the first binding domain has a biological activity of binding to a transmembrane E3 ligase; and the second binding domain has a biological activity of binding to a disease-associated polypeptide.

[0025] The first binding domain has a biological activity of binding a transmembrane E3 ligase. The term "transmembrane E3 ligase" is understood by a skilled person 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 a ZNRF3 E3 ligase, preferably a human ZNRF3 E3 ligase (more preferably having Genbank accession number NP_001193927.1, an amino acid sequence of SEQ ID NO: 10) or one of its isoforms; also preferably, the transmembrane E3 ligase is a RNF43 E3 ligase, preferably a human RNF43 E3 ligase (more preferably having Genbank accession number NP_060233.3, an amino acid sequence of SEQ ID NO: 11) or one of its isoforms. Polypeptides having the activity of binding to transmembrane E3 ligases are known in the art and include in particular R-spondin as described above. Thus, preferably, the first binding domain comprises a sub-portion of R-spondin, preferably R-spondin 2, having the above-mentioned activity. Thus, the first binding domain preferably comprises furin domain 1 and / or furin domain 2 of R-spondin (RSPO), wherein the furin domain 2 does not have wnt-activation activity. Preferably, the furin domain 1 corresponds to amino acids 37 to 84 of SEQ ID NO: 12 or the corresponding amino acids of other spondin polypeptides, and / or the furin domain 2 corresponds to amino acids 90 to 134 of SEQ ID NO: 12 or the corresponding amino acids of other r-spondin polypeptides. More preferably, the furin domain 2 comprises an exchange of amino acid F109 with a non-identical amino acid, preferably an F109A amino acid exchange, wherein amino acid position 109 is the position with this number in human R-spondin 2 as described above, or the corresponding amino acid position in one of the other R-spondins. Other amino acid exchanges that eliminate wnt signaling in R-spondin and / or furin domains are known in the art, and the skilled person knows how to practice such amino acid exchanges in the context of the first binding domain; therefore, another preferred amino acid exchange with the above effect is an exchange of amino acid F105 with a non-identical amino acid, preferably an F105A amino acid exchange, wherein amino acid position 105 is the position with this number in human R-spondin 2 as described above, or the corresponding amino acid position in one of the other R-spondins. Preferably, the first binding domain comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1.More preferably, the first binding domain comprises furin domain 1 and furin domain 2 of RSPO2. Therefore, the first binding domain more preferably comprises an amino acid sequence having at least 80% identity with SEQ ID NO:2. More preferably, the first binding domain consists of an amino acid sequence having at least 80% identity with SEQ ID NO:1, more preferably at least 80% identity with SEQ ID NO:2. Therefore, the first binding domain preferably lacks the TSP1 domain; also preferably, the first binding domain consists of the furin domain 1 and / or the furin domain 2, wherein the furin domain 2 preferably comprises the aforementioned F109A amino acid exchange.

[0026] The term "subject" as referred to herein relates to a vertebrate, preferably a mammal, in particular a livestock, companion animal or laboratory animal. More preferably, the subject is a human. Preferably, the subject has been diagnosed as having a disease as described below and / or has been diagnosed as being at risk of developing a disease. More preferably, the subject has been diagnosed as having cancer and / or has been diagnosed as having a risk of recurrence and / or metastasis.

[0027] The term "disease" is used in a broad sense herein and preferably relates to any pathological state of a subject, preferably requiring treatment, and more preferably known or suspected to be treatable by a binding polypeptide as described herein. Thus, the disease is preferably a pathological state caused or aggravated by the expression (overexpression) of at least one disease-related polypeptide, as described elsewhere herein. Thus, the disease may be caused or aggravated in particular by overexpression of a polypeptide at non-physiological levels and / or by expression of a mutant protein of a polypeptide of the subject, which mutant protein may, for example, have increased activity, neoactivty and / or may inhibit physiological regulation. Preferably, the disease is caused or aggravated by an immune system dysfunction (eg, cancer or an autoimmune disease). Preferably, the disease is caused or aggravated by overexpression of at least one immune checkpoint inhibitor (preferably PD-L1). More preferably, the disease is cancer, and more preferably a cancer expressing PD-L1.

[0028] As used herein, the term "cancer" relates to a disease in animals, including humans, characterized by the uncontrolled growth of a group of somatic cells ("cancer cells"). This uncontrolled growth may be accompanied by invasion and destruction of surrounding tissues (infiltration) and the possible spread of cancer cells to other parts of the body (metastasis). Preferably, the term cancer also includes the recurrence of cancer (recurrence). Thus, preferably, the cancer is a solid cancer, a metastasis or a recurrence thereof. Also preferably, the cancer is a non-solid cancer, in particular a leukemia, in particular a relapsed or advanced leukemia. Preferably, the cancer is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem 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, hypothalamus and optic plexus. Pathway glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, medulloblastoma, medullary epithelioma, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral 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, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sézary syndrome syndrome), small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, waldenström macroglobulinemia and wilms tumor. More preferably, the cancer is melanoma.

[0029] The second binding domain has a biological activity of binding to a disease-associated polypeptide (preferably an extracellular domain thereof), preferably has an activity of binding to programmed death-ligand 1 (PD-L1) (preferably an extracellular domain thereof).

[0030] The term "disease-associated polypeptide" is understood by the skilled person to include any and all polypeptides that are causally related to the etiology of the disease; thus, the disease-associated polypeptide is preferably known to be overexpressed in the disease and / or is known to be a mutant protein of a polypeptide normally present in a subject, wherein preferably the overexpression or expression of the mutant protein is causally related to the disease. Thus, reducing the amount of the disease-associated polypeptide preferably causes, more preferably is known to cause, at least an improvement in the disease. The disease-associated polypeptide is preferably membrane-bound, preferably bound to the cytoplasmic membrane of the target cell. Thus, the disease-associated polypeptide preferably comprises at least one transmembrane domain, i.e., preferably a transmembrane polypeptide. Also preferably, the disease-associated polypeptide comprises at least one extracellular domain. However, the disease-associated polypeptide may also be a non-transmembrane polypeptide; preferably, in this case, the disease-associated polypeptide is a membrane-bound polypeptide, in particular a lipid-anchored polypeptide, or an extracellular (preferably soluble) polypeptide that is non-covalently bound to a membrane (in particular a cytoplasmic membrane). The non-covalent binding of soluble polypeptides to the membrane may be mediated by protein-protein interactions, protein-lipid interactions, protein-glycan interactions, metal ions, etc. Preferably, the disease-associated polypeptide is a regulatory polypeptide that helps regulate (i) cell proliferation, (ii) immune response, in particular cellular immune response, and / or (iii) intercellular communication. Therefore, the disease-associated polypeptide may be in particular a T cell regulator polypeptide or an immune checkpoint polypeptide. In particular, the disease-associated polypeptide may be PD-L1.

[0031] Preferably, the disease-associated polypeptide is programmed death-ligand 1 (PD-L1). The terms "programmed death-ligand 1" and "PD-L1" relate to the group of polypeptides known under this name, which may also be referred to as "CD274" or "B7-H1". Preferably, PD-L1 is expressed by the target cell. Preferably, PD-L1 is human PD-L1 (more preferably having the amino acid sequence of Genbank accession number NP_054862.1, SEQID NO: 13) or one of its isoforms.

[0032] Polypeptides and domains thereof having activity in combination with disease-related polypeptides are known in the art and are selected by technicians based on the disease-related polypeptides intended to be targeted. Therefore, the second binding domain may be a binding domain of a (preferably naturally occurring) interaction partner of a disease-related polypeptide in particular. Therefore, in the case where the disease-related polypeptide is a receptor, the second binding domain may, for example, comprise a cognate ligand of the receptor or a binding domain of a derivative thereof having the above-mentioned binding activity. In addition, the second binding domain may comprise an aptamer, a spiegelmer, a designed ankyrin repeat (darpin) domain, a Kunitz-type domain, an antibody, in particular a single-chain antibody or a single-domain antibody (see Hey et al., (2005), Trends Biotechnol. 23:514) or a derivative thereof having the above-mentioned binding activity.

[0033] Polypeptides having the activity of binding to PD-L1 are known in the art, and in particular include programmed cell death protein 1 (PD-1). The terms "programmed cell death protein 1" and "PD-1" relate to members of the family of T cell regulators known to those skilled in the art under this name, which may also be referred to as "CD279". Preferably, PD-1 is human PD-1 (more preferably comprising the amino acid sequence as shown in Genbank accession number NP_005009.2, SEQ ID NO: 14) or 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 an extracellular IgV-like domain of a PD-1 polypeptide, more preferably a high affinity variant thereof. Therefore, the second binding domain comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3 or 4. Also preferably, the second binding domain comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 5. Thus, where the second binding domain comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3 or 4, it preferably also comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 5. More preferably, the second binding domain consists of one of the aforementioned amino acid sequences.

[0034] As used herein, the term "binding polypeptide" relates to a polypeptide having a structural element and a specified biological activity or multiple biological activities as described above. Therefore, the binding polypeptide comprises at least a first binding domain having a biological activity of binding to a transmembrane E3 ligase; and a second binding domain having a biological activity of binding to a disease-associated polypeptide. Therefore, the binding polypeptide has a biological activity of binding to a transmembrane E3 ligase and binding to a disease-associated polypeptide. Preferably, the binding activity causes the transmembrane E3 ligase and the disease-associated polypeptide to become interconnected at the cytoplasmic membrane of the target cell through the binding polypeptide, and preferably causes the disease-associated polypeptide to be degraded by the cell.

[0035] In view of the above description, the binding polypeptide preferably has only the indicated binding activity, i.e. preferably has no biological activity other than those specifically indicated, although such additional biological activity may already be present in the protein from which the binding polypeptide is derived. Thus, the binding polypeptide preferably has no wnt signaling activity and / or has no bone morphogenetic protein (BMP) signaling inhibitory activity, more preferably has no signaling modulating activity, preferably has no signaling activity. It is also preferred that the binding polypeptide does not contain other sequences of the protein or proteins from which it is derived. Thus, the binding polypeptide preferably does not comprise the transmembrane domain and / or intracellular domain of PD-1; in addition, the binding polypeptide preferably does not comprise the active TSP-1 domain of R-spondin, i.e. in the case where the binding polypeptide comprises the TSP-1 domain, the binding polypeptide is preferably a signaling inactive polypeptide variant. More preferably, the binding polypeptide preferably does not comprise the active TSP-1 domain of R-spondin, more preferably does not comprise any amino acid sequence of at least 7 amino acids in length downstream of amino acid 143 of SEQ ID NO: 12.

[0036] Nevertheless, the binding polypeptide may comprise amino acid sequences other than those specifically indicated, such as a tag or marker sequence, a signal peptide (e.g., an amino acid sequence having SEQ ID NO: 8), or at least one linker sequence. Other amino acid sequences or other structural elements may be included to provide further binding activity, improved serum stability, tissue targeting, etc. However, preferably, the binding polypeptide does not comprise a transmembrane domain and / or an intracellular domain. Therefore, the binding polypeptide is preferably a non-membrane-integrated polypeptide, more preferably a soluble polypeptide.

[0037] Preferably, the binding polypeptide is a complex of more than one amino acid chain, i.e. preferably a multimer, such as a dimer, trimer, etc.; in this case, the complex of more than one amino acid chain may also be referred to as a "binding polypeptide oligomer" or a "binding polypeptide complex". Preferably, the complex of more than one amino acid chain 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, using, for example, suitable ligand-receptor pairs, antibody-antigen interactions, etc. On an exemplary basis, 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 appreciated by those skilled in the art, in the case where the binding polypeptide is a binding polypeptide complex, the complex can be formed in vitro, for example by mixing a first binding domain covalently coupled to a first partner of an affinity pair with a second binding domain coupled 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 coupled to a first partner of an affinity pair, and further administering a second binding domain coupled to a second partner of the affinity pair. As will also be appreciated by those skilled in the art, it is also possible to use more than one second binding domain and one first binding domain, for example by using a first binding domain covalently coupled to a first partner of an affinity pair and a plurality of second binding domains, each of which is coupled to a second partner of the affinity pair; in this way, more than one disease-associated polypeptide can be targeted.

[0038] 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 together form a fusion polypeptide. Preferably, the first binding domain and the second binding domain are connected via a linker, i.e. preferably, the linker is between the first binding domain and the second binding domain. The terms "linker", "linker sequence" and "linker peptide" are in principle known to the skilled person. The skilled person knows how to select a suitable linker peptide. Preferably, the linker comprises 1 to 20, more preferably 5 to 10 amino acids, which are preferably independently selected from the group consisting of glycine (G), proline (P) and serine (S). A particularly preferred linker peptide comprises an amino acid sequence as shown in SEQ ID NO: 6. Preferably, the first binding domain is located at the N-terminus of the second binding domain in the binding polypeptide; therefore, in the presence of a linker, the order of the structural elements in the binding polypeptide is preferably N-terminus, first binding domain, linker, second binding domain, C-terminus. As will be appreciated by those skilled in the art, where additional structural elements are present in the binding polypeptide, they are preferably attached to the N-terminus and / or C-terminus of the preferred structures described above. In addition, additional structural elements may be added with or without an intervening linker, wherein the optional linker may be the same or different from the linker inserted into the first and second binding domains.

[0039] According to the above, the binding polypeptide preferably comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 7 or 9. More preferably, the binding polypeptide consists essentially of an amino acid sequence having at least 80% identity with SEQ ID NO: 7 or 9, and most preferably, the binding polypeptide consists of an amino acid sequence having at least 80% identity with SEQ ID NO: 7 or 9.

[0040] Preferably, the binding polypeptide is contained in a material composition comprising other components; thus, the binding polypeptide may be contained in a solution, in particular an aqueous solution, which may additionally contain, for example, at least one buffer, at least one salt and / or one or more other components deemed suitable by the skilled person. Preferably, the composition is a pharmaceutical composition, which preferably also comprises a pharmaceutically acceptable carrier. The terms "drug" and "pharmaceutical composition" are used interchangeably herein and are known in principle to the skilled person. As mentioned herein, the term preferably relates to any material composition comprising one or more specified active agents as one or more pharmaceutically active compounds and optionally one or more excipients. The one or more pharmaceutically active compounds may be present in liquid or dry (e.g., lyophilized) form. It should be understood that the form and properties of a pharmaceutically acceptable excipient (e.g., a carrier or diluent) depend on the amount of active ingredient combined therewith, the route of administration and other well-known variables. One or more excipients must be acceptable in the sense of being compatible with the other ingredients of the formulation and harmless to its receptor. The excipients used may include solids, gels or liquids. The present invention relates to the invention of the present invention.Exemplary solid carriers are lactose, terra alba, sucrose, talcum, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, etc.Exemplary liquid carriers are phosphate buffered saline solution, physiological saline, Ringer's solution, dextrose solution, Hank's solution, syrup, oil, water, emulsion, various types of wetting agents, etc.Similarly, the carrier or diluent can include slow-release materials well known in the art, such as single glyceryl monostearate or glyceryl distearate or glyceryl monostearate or glyceryl distearate with wax.The suitable carrier includes those mentioned above and other carriers well known in the art, referring to, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.Select one or more excipients, so as not to affect the biological activity of the combination.However, excipients can also be selected to improve the uptake of the active agent into cells (particularly cancer cells).

[0041] The drug can be administered by any route deemed appropriate, such as by a physician, preferably with a therapeutically effective dose. A therapeutically effective dose refers to the amount of an active compound that prevents, improves or cures the symptoms of a disease or condition mentioned in this specification. The therapeutic efficacy and toxicity of a drug can be determined by standard drug procedures in cell culture or experimental animals, such as ED50 (dose effective for treatment in 50% of the population) and LD50 (dose lethal to 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage of any one patient may depend on many factors, including the type and severity of the disease, the patient's body shape, age, the specific formulation of the drug to be administered, sex, time and route of administration, general health, and other drugs administered simultaneously. The drug mentioned herein is preferably administered at least once, such as as a bolus. However, the drug can be administered more than once, and preferably at least twice, such as permanently or regularly administered after a limited time window. Progress can be monitored by regular assessment. The dosage recommendation may be indicated in the prescription or instructions for use so that the dosage adjustment can be made in advance according to the receptor under consideration. The drug according to the present invention may contain other active agents in addition to the one or more active agents mentioned above. Preferably, the pharmaceutically active compound according to the present invention is used together with at least one other drug, and therefore can be formulated as a drug together with the at least one other drug. More preferably, in the case of cancer treatment, the at least one other active agent is a chemotherapeutic agent or an immunotherapeutic agent, such as a T cell or immune checkpoint regulator. In addition, it should be understood that the preparation of the pharmaceutical composition is preferably carried out under GMP standardized conditions, etc., to ensure the quality, drug safety and effectiveness of the drug.

[0042] Advantageously, it was found in the work of the present invention that the binding polypeptides described in the examples induce degradation of PD-L1, a model target polypeptide on target cells, thereby not only alleviating PD-1-mediated immune checkpoint blockade, but also removing one of the factors that mediate it, thereby improving therapeutic efficacy. It was found that the binding polypeptides target the highly relevant immune checkpoint protein PD-L1 and cause its degradation. Importantly, PD-L1 degradation occurs in the pico-nanomolar to low-nanomolar range, indicating that the binding polypeptides are highly effective. It is worth noting that the exemplary transmembrane E3 ligases ZNRF3 and RNF43 used in the examples show widespread to ubiquitous expression in many tissues and cell lines (see, for example, www.ncbi.nlm.nih.gov / gene / 84133 and -54894). Therefore, the present specification provides an R-spondin binding domain that (i) inactivates WNT- and BMP signaling by introducing appropriate mutations; (ii) is coupled to a suitable second binding polypeptide, and (iii) retains the biological activity of binding to the E3 ligase with high affinity and internalizing and degrading. Thus, the current work provides the first generally applicable, signaling-inactive R-spondin-derived binding domain for proximity-based protein degradation therapeutics.

[0043] The above definitions apply mutatis mutandis to the following. Other definitions and explanations given further below also apply mutatis mutandis to all embodiments described in this specification.

[0044] The present invention also relates to polynucleotides encoding the binding polypeptides according to the present invention.

[0045] The polynucleotides of the invention have been described above; as indicated, said polynucleotides may be comprised in an expression construct and / or a vector, ie preferably in an expression vector, all of which are also as described above.

[0046] The present invention further relates to a host cell comprising a binding polypeptide according to the invention and / or a polynucleotide according to the invention.

[0047] As used herein, the term "cell" is understood by the skilled person to refer to the smallest structural unit in biology that has the primary ability to replicate itself. Thus, the term includes archaeal cells, prokaryotic cells and eukaryotic cells. The term "host cell" refers to a cell capable of transiently (preferably stably) maintaining a polynucleotide or vector as described herein and / or expressing a binding polypeptide from an expression construct or expression vector as described herein; thus the host cell may be, for example, a bacterial cell, in particular an E. coli cell; or it may be a eukaryotic cell, such as a cell of a subject treated with a binding polypeptide. The term "target cell" refers to a cell known or suspected to express at least one transmembrane E3 ligase and a disease-associated polypeptide; preferably, the target cell is a cancer cell. Preferably, the target cell is a cell of a subject or a cultured cell derived therefrom.

[0048] The present invention further relates to a device comprising a binding polypeptide according to the invention and / or a polynucleotide according to the invention.

[0049] As used herein, the term "device" relates to a system of devices comprising at least the described devices, preferably operably connected to each other, and / or also to other devices that allow the administration of the compounds or compositions of the invention. Preferred devices for administering binding polypeptides are well known in the art and described above. How the devices are operatively connected will depend on the type of devices included in the device and the kind of administration envisaged. Preferably, in this case, the device is comprised by a single device. The device may therefore include a delivery unit for administering the binding polypeptide and optionally a storage unit for storing the binding polypeptide until administration. However, it is also contemplated that the device of the present invention may be presented as a separate device in such an embodiment, and preferably packaged together as a kit. A person skilled in the art will recognize how to connect the devices without further explanation. Preferred devices are those that can be applied without specific knowledge of a skilled person. In a preferred embodiment, the device is a syringe containing a binding polypeptide, more preferably with a needle. More preferably, the device is an intravenous infusion (IV) device containing a binding polypeptide. Also preferably, the device is a tube or endoscopic device that contains the formulation for flushing the site of administration (eg, the heart), or also contains a needle for local administration of the compound or composition (eg, to a tumor).

[0050] The present invention further relates to a kit comprising a binding polypeptide according to the invention and / or a polynucleotide according to the invention and optionally an administration device.

[0051] As used herein, the term "kit" refers to a collection of the above-mentioned compounds, devices or reagents, which may or may not be packaged together. The components of the kit may be contained in separate vials (i.e., a kit as separate parts), or may be provided in a single vial, for example as a composition as described above. In view of the above, the kit may, for example, contain (i) a first binding domain coupled to a first partner of an affinity pair and (ii) at least one second binding domain coupled to a second partner of an affinity pair, for example in separate vials, or as a preformed complex. The housing of the kit in one embodiment allows translocation of the compounds of the kit, in particular common translocation; therefore, the housing may in particular be a portable container containing all the specified components. In addition, it should be understood that the kit of the present invention can be used to implement the above-mentioned method. It is preferably envisaged that all components are provided in a ready-to-use manner for implementing the above-mentioned method. In addition, the kit preferably contains instructions for implementing the method. These instructions may be provided by a user manual in paper or electronic form. For example, the manual may contain instructions for explaining the results obtained when the above-mentioned method is performed using the kit. Preferably, the kit is suitable for use in the methods of the present invention, more preferably suitable for containing all reagents required to perform one or more of the methods.

[0052] The present invention also relates to a method of killing cancer cells, comprising contacting said cancer cells with a binding polypeptide according to the present invention and / or a polynucleotide according to the present invention.

[0053] 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 described below. The method may include steps in addition to those steps explicitly mentioned above. For example, additional steps may involve, for example, identifying cancer cells (for determining the expression of at least one transmembrane E3 ligase and / or disease-related polypeptide on the cells), etc. In addition, one or more of the steps may be performed or assisted by automated equipment. Therefore, the method can be implemented in particular on cells (preferably target cells) expressing disease-related polypeptides. It is also preferred that the method is implemented on cells (preferably target cells) that overexpress at least one transmembrane E3 ligase (particularly ZNRF3 and / or RNF43); therefore, preferably, the method further comprises contacting the cancer cells with an agent that provides a transmembrane E3 ligase.

[0054] The term "agent providing a transmembrane E3 ligase" includes every agent which, when contacted with a cell (preferably a target cell), results in the presence of at least one transmembrane E3 ligase in the cell, the amount of which is increased compared to a cell not contacted with the agent. Thus, the agent providing a transmembrane E3 ligase may 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 providing a transmembrane E3 ligase is a polynucleotide encoding a transmembrane E3 ligase, more preferably an expression construct for a transmembrane E3 ligase.

[0055] The present invention also relates to a binding polypeptide according to the invention and / or a polynucleotide according to the invention for medical use; and to a binding polypeptide according to the invention and / or a polynucleotide according to the invention for the manufacture of a medicament.

[0056] In addition, the present invention relates to a binding polypeptide according to the present invention and / or a polynucleotide according to the present invention for use in treating and / or preventing cancer; and to the use of a binding polypeptide according to the present invention and / or a polynucleotide according to the present invention in the preparation of a medicament for treating and / or preventing cancer.

[0057] The terms "treating" and "treatment" refer to significantly improving the diseases or conditions mentioned herein or the symptoms associated therewith; as used herein, the terms include preventing the deterioration of diseases, conditions or symptoms associated therewith. The treatment as used herein may also include full recovery of health for the diseases or conditions mentioned herein. It should be understood that, as the terms are used herein, treatment may not be effective in all subjects to be treated. However, the term should require that, preferably, a statistically significant portion of the subjects suffering from the diseases or conditions mentioned herein can be successfully treated. Without further explanation, a person skilled in the art can use various well-known statistical evaluation tools (e.g., determination of confidence intervals, determination of p values, Student's t-test, Mann-Whitney test, etc.) to effortlessly determine whether a portion is statistically significant. 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 is effective for at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the subjects in a given cohort or population. Preferably, the treatment comprises inhibiting the proliferation of cancer cells, more preferably killing cancer cells. Preferably, treating cancer reduces the tumor and / or cancer cell load in the subject. Preferably, treating cancer comprises increasing the immune response to cancer cells of the cancer, more preferably comprising increasing the cellular immune response, preferably compared to an untreated control. As will be appreciated by those skilled in the art, the effectiveness of a treatment (e.g., treatment of cancer) depends on a variety of factors, including, for example, the stage of cancer and the type of cancer. Also preferably, cancer treatment further comprises administering at least one of an agent providing a transmembrane E3 ligase, chemotherapy, immunotherapy, surgery and radiotherapy, wherein immunotherapy preferably comprises administering T cells, preferably CAR T cells and / or recombinant T cell receptor T cells.

[0058] The terms "preventing" and "prevention" refer to maintaining health in a subject relative to a disease or condition mentioned herein for a certain period of time. It is understood that the time period may depend on the amount of the drug compound that has been administered and the individual factors of the subject discussed elsewhere in this specification. It should be understood that prevention may not be effective in all subjects treated with the binding polypeptide. However, the term requires that, preferably, a statistically significant portion of subjects in a cohort or population is effectively prevented from suffering from a disease or condition mentioned herein or its associated symptoms. Preferably, in this case, a cohort or population of subjects is envisioned, which would normally (i.e., without the preventive measures according to the present invention) develop a disease or condition as mentioned herein. Without further explanation, a person skilled in the art can easily determine whether a portion is statistically significant using various well-known statistical evaluation tools discussed elsewhere in this specification. In the case of cancer treatment, prevention particularly relates to preventing cancer development, preventing metastasis formation and / or preventing recurrence.

[0059] Furthermore, the present invention relates to a method for treating and / or preventing cancer in a subject, said method comprising

[0060] (a) administering to the subject a binding polypeptide according to the invention and / or a polynucleotide according to the invention, and

[0061] (b) thereby treating and / or preventing cancer in said subject.

[0062] The above-mentioned treatment methods are preferably in vivo methods and may include steps other than those specifically mentioned. The additional steps may, for example, involve identifying a subject in need of cancer treatment or at risk of developing cancer, or may involve other treatment steps as described above.

[0063] The present invention also relates to a method of identifying a subject suffering from cancer as being susceptible to treatment with a binding polypeptide according to the present invention and / or a polynucleotide according to the present invention, the method comprising

[0064] (A) determining the expression of a disease-associated polypeptide or a surrogate marker thereof in cancer cells of the subject; and

[0065] (B) identifying the subject as being sensitive to the treatment based on the determination in step (A).

[0066] The method for identifying a subject is preferably an in vitro method and may include steps other than those specifically indicated. Other steps may, for example, involve providing a sample (e.g., a cancer sample) for step (A), and / or involve treating the subject as described above, particularly in the case where the subject is determined to be sensitive to the treatment. Whether the subject is sensitive to the treatment can be confirmed by a skilled person based on an indicated assay; i.e., preferably, a subject is preferably considered to be sensitive to the treatment if it is determined that the subject's cancer cells express a disease-associated polypeptide or a surrogate marker indicating expression of a disease-associated polypeptide.

[0067] In step (A), the expression of the disease-associated polypeptide may be determined according to methods known to those skilled in the art, and as shown in the examples herein, in particular by immunohistochemistry, for example on a biopsy sample. However, instead of or in addition to the disease-associated polypeptide, a surrogate marker may also be determined, the term "surrogate marker" relating to any biological molecule that is different from the disease-associated polypeptide but indicates its expression in a cell. Such surrogate markers are known in the art and in particular include mRNA encoding a disease-associated polypeptide. Step (A) may comprise an additional determination; preferably, the expression of ZNRF3 and / or RNF43 is further determined, and the subject is additionally determined to be sensitive to the treatment based on the expression of ZNRF3 and / or RNF43. Preferably, the subject is considered to be sensitive to the treatment if it is determined that the cancer cells of the subject express ZNRF3 and / or RNF43, and more preferably, ZNRF3 and / or RNF43 do not contain a loss-of-function mutation.

[0068] Furthermore, the present invention relates to the use of a binding polypeptide according to the present invention and / or a polynucleotide according to the present invention for causing degradation of a disease-associated polypeptide in a host cell and / or for killing a host cell expressing a disease-associated polypeptide, wherein the use is preferably an in vitro use.

[0069] In view of the above, the following implementation plans are specifically envisaged:

[0070] 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 to programmed death-ligand 1 (PD-L1).

[0071] Embodiment 2: The binding polypeptide of embodiment 1, wherein the first binding domain comprises furin domain 1 and / or furin domain 2 of R-spondin (RSPO), wherein the furin domain 2 does not have wnt-activation activity.

[0072] Embodiment 3: The binding polypeptide of embodiment 1 or 2, wherein the furin domain 2 comprises an exchange of amino acid F109 with a non-identical amino acid, preferably comprises an exchange of amino acid F109A.

[0073] Embodiment 4: The binding polypeptide of any one 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.

[0074] 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.

[0075] Embodiment 6: The binding polypeptide of any one 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.

[0076] Embodiment 7: The binding polypeptide of any one of Embodiments 1 to 6, wherein the first binding domain lacks wnt signaling activity and / or lacks bone morphogenetic protein (BMP) signaling inhibition activity.

[0077] Embodiment 8: The binding polypeptide of any one of embodiments 1 to 7, wherein the first binding domain lacks the TSP1 domain, preferably wherein the first binding domain consists of the furin domain 1 and / or the furin domain 2.

[0078] 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.

[0079] 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-associated polypeptide.

[0080] Embodiment 11: The binding polypeptide of any one of Embodiments 1 to 10, wherein the disease-associated polypeptide is PD-L1, and wherein the second domain comprises an extracellular IgV-like domain of a programmed death-1 (PD-1) polypeptide.

[0081] Embodiment 12: The binding polypeptide of 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.

[0082] Embodiment 13: The binding polypeptide of any one 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.

[0083] Embodiment 14: The binding polypeptide of any one of Embodiments 1 to 13, wherein the first binding domain and the second binding domain are covalently linked.

[0084] Embodiment 15: The binding polypeptide of any one of Embodiments 1 to 14, wherein the first binding domain and the second binding domain together form a fusion polypeptide.

[0085] Embodiment 16: The binding polypeptide of embodiment 15, wherein the first binding domain and the second binding domain are connected by a linker, and the linker is preferably a GS linker, more preferably comprises the amino acid sequence of SEQ ID NO:6.

[0086] Embodiment 17: The binding polypeptide of 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.

[0087] Embodiment 18: The binding polypeptide of any one of Embodiments 1 to 17, wherein the binding polypeptide consists of an amino acid sequence that is at least 80% identical to SEQ ID NO:7.

[0088] Embodiment 19: The binding polypeptide of any one of Embodiments 1 to 18, wherein the disease-associated polypeptide is a transmembrane polypeptide comprising at least one extracellular domain.

[0089] Embodiment 20: The binding polypeptide of any one of claims 1 to 19, wherein the disease-associated polypeptide is a T cell regulator polypeptide or an immune checkpoint polypeptide.

[0090] Embodiment 21: The binding polypeptide of any one of claims 1 to 20, wherein the disease-associated polypeptide is PD-L1.

[0091] Embodiment 22: A polynucleotide encoding a binding polypeptide according to any one of embodiments 1 to 21.

[0092] Embodiment 23: A host cell comprising the binding polypeptide according to any one of embodiments 1 to 21 and / or the polynucleotide according to embodiment 22.

[0093] 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.

[0094] 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 device.

[0095] Embodiment 26: A method for killing cancer cells, comprising contacting the cancer cells with a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22.

[0096] Embodiment 27: The method described in Embodiment 26, wherein the method is an in vitro method.

[0097] Embodiment 28: The method of Embodiment 26 or 27, wherein the cancer cell is a cancer cell that overexpresses PD-L1.

[0098] 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.

[0099] Embodiment 30: The binding polypeptide of any one of embodiments 1 to 21 and / or the polynucleotide of embodiment 22, for use in the manufacture of a medicament.

[0100] Embodiment 31: A binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22 for use in treating and / or preventing cancer.

[0101] Embodiment 32: The binding polypeptide and / or polynucleotide for use according to embodiment 31, wherein the treatment and / or prevention further comprises administering an agent that provides a transmembrane E3 ligase.

[0102] Embodiment 33: The binding polypeptide and / or polynucleotide for use according to Embodiment 31 or 32, wherein the treatment and / or prevention further comprises administering at least one of immunotherapy, surgery, radiotherapy and chemotherapy.

[0103] Embodiment 34: The binding polypeptide and / or polynucleotide for use according to any one of Embodiments 31 to 33, wherein the immunotherapy comprises administration of T cells, preferably CAR T cells and / or recombinant T cell receptor T cells.

[0104] Embodiment 35: The binding polypeptide and / or polynucleotide for use according to any one of Embodiments 31 to 34, wherein the cancer is melanoma.

[0105] Embodiment 36: Use of the binding polypeptide of any one of Embodiments 1 to 21 and / or the polynucleotide of Embodiment 22 in the preparation of a medicament for treating and / or preventing cancer.

[0106] Embodiment 37: A method for treating and / or preventing a disease in a subject, the method comprising:

[0107] (a) administering to the subject a binding polypeptide according to any one of embodiments 1 to 22 and / or a polynucleotide according to embodiment 22, and

[0108] (b) thereby treating and / or preventing cancer in said subject.

[0109] Embodiment 38: A method of identifying a subject with cancer that is sensitive to treatment with a binding polypeptide according to any one of embodiments 1 to 21 and / or a polynucleotide according to embodiment 22, the method comprising:

[0110] (A) determining the expression of a disease-associated polypeptide (preferably PD-L1 expression) or the expression of a surrogate marker thereof in cancer cells of the subject; and

[0111] (B) identifying the subject as being sensitive to the treatment based on the determination in step (A).

[0112] Embodiment 39: The method of Embodiment 38, wherein in step (a), when it is determined that the cancer cells express the disease-associated polypeptide (preferably PD-L1), preferably overexpress PD-L1, the subject is identified as being sensitive to the treatment.

[0113] Embodiment 40: Use of the binding polypeptide of any one of embodiments 1 to 21 and / or the polynucleotide of embodiment 22 for causing degradation of a disease-associated polypeptide (preferably PD-L1) in a host cell and / or for killing a host cell expressing a disease-associated polypeptide (preferably expressing PD-L1).

[0114] Embodiment 41: The subject matter of any one of Embodiments 1 to 40, wherein the transmembrane E3 ligase is a ZNRF3 E3 ligase and / or a RNF43 E3 ligase.

[0115] All references cited in this specification are incorporated by reference into their entire disclosures as well as the disclosures specifically mentioned in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 . RSPO chimeras bridge ZNRF3 / RNF43 and PD-L1. a. Top, domain structures of the indicated proteins. Bottom, structure predictions from RSPO2 (PDB 4ufr) and PD-1 HAC (PDB 5ius). R2 FA , with LGR binding defective F 109A Mutated RSPO2 furin domain. PD1 HAC b. ELISA binding assay. ce. ELISA assays as described in (b) for RSPO chimeras with (c) PD-L1. ECD (d)ZNRF3 ECD 、(e)RNF43 ECD Direct interaction between R2PD1 and PD-L1. f. ELISA-based ternary complex analysis. gh. ELISA-based ternary complex analysis, as described in (f). ECD With (g) ZNRF3 ECD and (h)RNF43 ECD The interaction between them.

[0117] Figure 2 RSPO chimeras promote degradation of overexpressed PD-L1. a. Analysis of PD-L1 degradation. b, g. Immunoblot analysis of the indicated proteins in 293T cells. Cells were transfected as indicated and analyzed after 24 h 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 between PD-L1 and ERK are indicated in (c). f. Immunofluorescence staining of 293T cells for the indicated proteins. Cells were transfected with PD-L1-GFP and ZNRF3-HA for 2 days. After incubation with equal amounts of the indicated purified proteins for an additional 5 h, cells were fixed and stained for GFP and the lysosomal marker LAMP1. Note the colocalization of PD-L1 with LAMP1 upon treatment with R2PD1 (arrows). Scale bar, 10 μm. 0.2, 0.8, 2.2, 5.4 and 13.6 nM purified R2PD1 protein were used in (c). 5.4 nM purified R2PD1 protein was used in (e).

[0118] Figure 3RSPO chimeras promote degradation of endogenous PD-L1 in cancer cells. ae, h, jl, op, 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 h or the indicated hours. (h) Cells were treated with 333U IFN-γ for 24 h and harvested for analysis after incubation with purified proteins for another 24 h. (o) Untreated cells were analyzed. Mock purified proteins or control conditioned medium were used as controls. The normalized ratios between PD-L1 and TfR are shown below. fg, m. Flow cytometric analysis of cell surface PD-L1 in MEL624 (fg) and COLO-800 (m) cells. Quantification is 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 h of incubation with the indicated purified proteins. (q) Untreated cells were analyzed. Indicated conditioned media were used in (b). 0.027, 0.081, 0.27, 0.81, and 2.7 nM purified proteins were used in (c). 0.24 nM purified proteins were used in (d). 1 nM purified proteins were used in (eg, i). 0.027, 0.081, 0.27, 0.81, and 2.7 nM purified proteins were used in (h). 0.081, 0.27, 0.81, and 2.7 nM purified proteins were used in (k). 0.81 nM protein was used in (ln). 0.7 nM purified proteins were used in (p). (r) 0.07 and 0.24 nM purified protein or anti-PD-L1 antibody (atezolizumab), (s) 0.07 and 0.7 nM purified protein or anti-PD-L1 antibody (atezolizumab). Mock purified protein and isotype antibody control were used as controls.

[0119] Figure 4. PD-L1 degradation by RSPO chimeras is independent of WNT / β-cat signaling. ad. Immunoblot analysis of the indicated proteins in MEL624 (ac) and COLO-800 (d) cells. Cells were analyzed after 12 h (a) or 24 h (bd) incubation with the indicated proteins. Cells were transfected with the indicated siRNAs for 2 days and then incubated with purified proteins (cd). e. qRT-PCR analysis of Axin2 expression in COLO-800 cells. Cells were analyzed after 24 h incubation with purified proteins in the presence of WNT3A. f. TOPflash analysis with HEK293T cells. Cells were analyzed after 12 h incubation with the indicated proteins. 10, 50, and 200 ng ml used in (ab). -1 DKK1 protein. 0.24 nM purified R2PD1 protein was used in (b). Purified R2PD1 was used with 0.24 nM (c), 0.73 nM (d), 0.8 nM (e). 0.12, 0.36, 1.08, and 3.24 nM RRSPO2 or R2PD1 were used in (f).

[0120] Figure 5 . PD-L1 degradation by RSPO chimeras requires ZNRF3 / RNF43. ag. PD-L1 degradation assay. In the presence of competing soluble RNF43 ECD PD-L1 degradation by R2PD1 in the presence of Fc (ab) or after ZNRF3 / RNF43 knockdown (cg). b, d, f, hi. Immunoblot analysis of the indicated proteins in MEL624 (b, d), COLO-800 (f), and A375 (hi) cells. Cells were analyzed after 24 h of incubation with the indicated proteins. Cells were transfected with the indicated siRNA for 2 days and then incubated with purified proteins (d, f). e, g. Flow cytometric analysis of cell surface PD-L1 in MEL624 (e) and COLO-800 (g) cells. Values ​​were normalized to the corresponding control groups. 50, 150, 500, and 1500 ng ml were used in (b). -1 RNF43 ECD Fc. R2PD1 purified using 0.27 nM (b), 0.14 nM (d), 1 nM (e), 0.8 nM (f), 2.5 nM (g). R2PD1 purified using 0.073, 0.24, 0.73, and 2.44 nM (hi).

[0121] Figure 6: (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 mRNAs for TCR and PD-1 for 1 day. HEK293T cells were mixed with T cells for 5 h after incubation with the indicated purified proteins. After 5 h of co-incubation, cells were harvested and activated cytotoxic CD107a+CD8+ T cells were quantified. 0.4, 1.1, 3.3, 10, and 30 nM purified R2PD1 was used. 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 mRNAs expressing TCR and PD-1 for 1 day. MEL624 cells were incubated with the indicated proteins overnight. After an additional 5 h of co-incubation, 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 purified R2PD1 or PD-L1 antibody atezolizumab were used. n = single sample. Representative data from three independent experiments with the same conclusions are shown. (cd) Real-time cell growth analysis. MEL624 cells were pretreated with IFNγ for 2 days and 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. The growth of MEL624 cells was monitored using the XCELLigence system. n = 3 experimentally independent samples. Data were normalized to the time point of T cell addition and shown as mean values. Standard error of the mean (SEM) is indicated for the last time point. ****P<0.0001 from a one-way ANOVA test for (c). ****P<0.0001 from two-tailed paired t-test for (d). 0.41, 3.7, and 11.1 nM purified R2PD1 was used in (c). 0.41 and 3.7 nM purified R2PD1 or 3.7 and 11.1 nM PD-L1 antibody atezolizumab was used in (d). DETAILED DESCRIPTION

[0122] The following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way.

[0123] Example 1: Method

[0124] 1.1 Cell lines and growth conditions

[0125] HEK 293T, 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 lab and validated in previous studies (Radaszkiewicz et al., ELife 10, doi:10.7554 / eLife.65759(2021)). COLO-800 cells were maintained in RPMI (Gibco 21875) with 10% FBS, 1% penicillin-streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate (Sigma S8636). All cell lines were cultured in a humidity controlled incubator at 37°C and 5% CO2. Mycoplasma contamination was negative in all cell lines used.

[0126] The primary human T cell line was derived from an ex vivo expanded culture of tumor infiltrating lymphocytes obtained from a pancreatic cancer sample (T222, human, male, pancreatic ductal adenocarcinoma), which has been previously described (Poschke et al. (2020) Clin. Cancer Res. 26:4289). To ensure future availability, cells were repeatedly expanded and cryopreserved using the rapid expansion protocol outlined in the same publication. Three days prior to electroporation, cells were thawed and cultured at a density of 3x10 6 10 cells / mL were plated 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). Cells were maintained in a humidity-controlled incubator at 37°C and 5% CO2. Mycoplasma contamination was negative in all cell lines used.

[0127] 1.2 Construct

[0128] The wild-type construct of human RSPO2 was tagged with Flag at the C-terminus in the pCS2+ vector, which was verified in a previous study (Sun et al., Cell rep 36:109559, doi:10.1016 / j.celrep.2021.109559 (2021)). R2 with a phenylalanine-to-alanine mutation at position 109 within the FU2 domain was obtained by mutagenesis PCR. FAPD1 was cloned by inserting the N-terminus into the end of the IgV-like domain of human PD-1 in the pCS2+ vector ECD High-affinity consensus mutants of PD-1 (PD1 HAC ) (Maute et al., Proc Natl Acad Sci USA 112: E6506-6514, doi: 10.1073 / pnas.1519623112 (2015)). The human PD-L1 extracellular domain was inserted into the alkaline phosphatase (AP)-pCS2+ vector and expressed as an AP fusion protein (PD-L1 ECD AP). RSPO chimera R2 FA PD1 HAC It is used from R2 FA and PD1 HAC The fragments of the FU1 / 2 domain of the construct were obtained by Gibson assembly and inserted into the pCS2+ vector. HAC A flexible 10-amino acid glycine-serine linker (GSGSGGSGSG, SEQ ID NO: 6) was inserted between the two domains to promote autonomous folding of these domains. ΔRING -myc and RNF43-Flag constructs (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 were kindly provided by Mien-Chie Hung (Addgene plasmid #121478; RRID:Addgene_121478). The sequences of the constructs generated in this study were confirmed by separate DNA sequencing.

[0129] 1.3 Cell transfection

[0130] siRNA 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.

[0131] 1.4 Conditioned medium generation and protein purification

[0132] HEK 293T cells were seeded into 10 cm culture dishes and expressed with RSPO2-Flag, R2 FA-Flag, PD1-Flag, PD1 HAC -Flag, R2PD1-Flag, and PD-L1 ECD AP transient transfection. After 24 hours, the medium was replaced with fresh DMEM containing 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin and harvested every day for the next three days. The conditioned medium was verified and quantified by immunoblotting. The medium containing equimolar amounts of protein was used in subsequent analyses. WNT3A conditioned medium was prepared in L-cells as described previously (Kazanskaya et al., Dev Cel 7:525-534 (2004)). For protein purification, the conditioned medium was incubated overnight with agarose beads conjugated to anti-Flag antibody (Sigma A2220). After washing with ice-cold PBS, the protein bound to the beads was eluted with 200mM glycine (pH 2.6) and neutralized with an equal volume of 1M Tris (pH 8.0). The eluate was dialyzed with at least a 1000-fold excess volume of ice-cold PBS. Protein concentration was quantified by Coomassie staining using BSA standards (Sigma P0914).

[0133] 1.5 In vitro binding assay

[0134] High-binding 96-well plates (Greiner M5811) were coated with 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 The coated wells were coated with Fc (R&D Systems 7964-RN-050) protein overnight. The 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 incubation with Flag-tagged protein overnight, the wells were washed three times with TBST. Protein bound to the wells was detected with peroxidase-conjugated anti-Flag antibody (Sigma A8592) and quantified with QuantaBlu™ Fluorescent Peroxidase Substrate Kit (Thermo Scientific™ 15169). For the ternary complex formation assay, PD-L1 ECD AP (instead of HRP-anti-Flag antibody) was added to the wells. After overnight incubation, AP signal was detected with chemiluminescent AquaSpark AP substrate (Serva 42593.01). Data are shown as the mean of biological replicates with SD.

[0135] 1.6 PD-L1 degradation analysis

[0136] HEK 293T cells were seeded into cell culture plates and transfected with the indicated plasmids. 24 h after transfection, cells were treated with conditioned medium or purified proteins as indicated. After an additional 24 h of incubation, cells were harvested for immunoblotting. Cancer cells were seeded into cell culture plates and treated as indicated. After 24 h of incubation or the indicated times, cells were harvested for immunoblotting. For degradation analysis in the presence of IFNγ stimulation, MEL624 cells were pre-incubated with 333U IFNγ (ImmunoTools11343536) for 24 h. The PD-L1 antibody atezolizumab and matched isotype controls used in the analysis were obtained from Offringa Laboratories, DKFZ, Germany.

[0137] 1.7 Immunoblotting

[0138] Cultured cells were harvested and lysed in ice-cold RIPA buffer with cOmplete protease inhibitor cocktail (Roche 11697498001). The lysate was mixed with Laemmli buffer containing β-mercaptoethanol and boiled at 70°C for 10 min to prepare SDS-PAGE samples. For cytoplasmic β-catenin detection, saponin buffer was used. Immunoblot images were acquired with SuperSignal West pico ECL (ThermoFisher 34580) using a LAS-3000 system (FujiFilm). Quantification of blots was performed using ImageJ software.

[0139] 1.8 Immunofluorescence (IF)

[0140] Cells were seeded onto cell culture plates with glass coverslips with inserts. 24 h after transfection with the indicated plasmids, cells were treated with purified proteins for the indicated times. After fixation with 4% PFA for 10 min, cells were stained with primary antibodies (1:250) overnight at 4 °C and with fluorophore-conjugated secondary antibodies (1:250) for 2 h at room temperature. Images were acquired using LSM 700 (Zeiss) and analyzed with ImageJ. Approximately 100 cells were analyzed for each group. The following primary antibodies were used for staining: chicken anti-GFP (Millipore AB16901), mouse anti-LAMP1 (Cell Signaling Technology 15665).

[0141] 1.9 Quantitative real-time PCR

[0142] Cultured cells were lysed in Macherey-Nagel RA1 buffer containing 1% β-mercaptoethanol, and total RNA was isolated using the NucleoSpin RNA Isolation Kit (Macherey-Nagel 740955). Reverse transcription and PCR amplification were performed as described previously (Sun et al., Cell rep 36:109559, doi:10.1016 / j.celrep.2021.109559(2021)). Primers for AXIN2 were: forward 5'-CCACACCCTTCTCCAATCC-3' (SEQ ID NO: 15) and reverse 5'-TGCCAGTTTCTTTGGCTCTT-3' (SEQ ID NO: 16). Primers for PD-L1 were: forward 5'-CCTACTGGCATTTGCTGAACG-3' (SEQ ID NO: 17) and reverse 5'-AGACAATTAGTGCAGCCAGGT-3' (SEQ ID NO: 18). Primers for ZNRF3 were: forward 5'-TGTGCCATCTGTCTGGAGAA-3' (SEQ ID NO:21) and reverse 5'-TTCCTGTGAAACCGGTGAGT-3' (SEQ ID NO:22). Primers for RNF43 were: forward 5'-GTTTGCTGGTGTTGCTGAAA-3' (SEQ ID NO:23) and reverse 5'-TGGCATTGCACAGGTACAG-3' (SEQ ID NO:24). Primers for GAPDH were: forward 5'-AGCCACATCGCTCAGACAC-3' (SEQ ID NO:19) and reverse 5'-GCCCAATACGACCAAATCC-3' (SEQ ID NO:20). Graphs show gene expression relative to GAPDH. Data are shown as means with SD from multiple experimental replicates.

[0143] 1.10 TOPflash luciferase reporter gene assay

[0144] TOPflash luciferase assay was performed as previously described (Berger et al., EMBO rep 18:712-725, doi:10.15252 / embr.201643585(2017)). Data are shown as means of biological replicates with SD.

[0145] 1.11 Quantitative and statistical analysis

[0146] Statistical analysis was performed using unpaired t-test or one-way ANOVA test with PRISM7 software. Not significant (ns), p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0147] 1.12 Flow cytometry

[0148] Cells were harvested non-enzymatically, pelleted and resuspended in ice-cold blocking buffer (PBS supplemented with 1% BSA and 0.1% NaN3). After blocking cells with Fc receptor binding inhibitor (eBioscience 14916173), cells were stained with anti-PD-L1 antibody (CST 86744) and then incubated with fluorescent dye-labeled secondary antibodies. Staining with secondary antibody alone was used as a control. Dead cells were excluded by counterstaining with propidium iodide. FACS samples were analyzed with FACSCanto or LSRFortessa (BD Biosciences), and data were processed with FlowJo software. Rabbit anti-PD-L1 (CST 86744) antibody was used for cell surface PD-L1 measurement.

[0149] For T cell activation analysis, the following antibodies were used for intracellular and extracellular staining: 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™ Fixable Aqueous (Aqua) Dead Cell Stain Kit (Thermofisher L34966). PD-1 antibody (BioLegend 329906) was used for surface PD-1 expression measurement. Data were acquired on a Fortessa cytometer (BD Biosciences) and analyzed using Flowjo v10.3 software (BD Biosciences).

[0150] 1.13 Co-culture assays for assessing T cell activation

[0151] Co-culture analysis was performed to examine the recognition ability of TCR-transfected T cells against tumor or other target environments. To monitor tumor recognition, CD107a and TNFa levels were measured. U-bottom 96-well plates with 50,000 effector cells (T cells) and 200,000 target cells were incubated at 37°C for 5 hours. To accumulate TNF and CD107a signals in response to T cell activation, commercially available GolgiPlug (BD bioscience 555028) and GolgiStop (BDbioscience 554715) were used at a ratio of 1:000. When human tumor cells (MEL624 and COLO-800) were used as target cells, they were pretreated with commercially available human IFN- at 333U ml-1 for 48 hours. After incubation of effector and target cells for 5 hours, TCR reactivity was assessed by flow cytometry. The percentage of activated T cells was measured by upregulation of CD107a or TNFa on live CD8+ T cells. Media from transfected effector cells were assayed to determine background reactivity. Results from the assay were subtracted to examine effector cell reactivity.

[0152] 1.14 Cytotoxicity assay

[0153] The effects of R2PD1 chimeras on cell proliferation and cytotoxicity were assessed using the CellTiter-Glo assay (Promega) following the manufacturer’s instructions. MEL624 and COLO-800 cells were seeded in 96-well microplates at a density of 500 cells / well in three wells per group. After overnight incubation, R2PD1 chimeras were added to the cells in growth medium and potential cytotoxicity was measured 72 h 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 obtained by normalizing it to the fold change relative to the mean luminescence of wells with control medium on the same plate. Protein-untreated cells were used as controls and background luminescence was measured in wells containing medium without cells.

[0154] 1.15 Tumor killing analysis based on xCELLigence

[0155] For tumor killing analysis, a 96-well microplate (Agilent 5232376001) with a gold microelectrode cell sensor built into the bottom of each well was used with the xCELLigence system (Agilent). The basal impedance of the culture medium was measured before adding tumor cells. Each well was seeded with 200,00 tumor cells, and an equal number of effector cells were added once the tumor cells reached a cell index of 1. The cell index was measured every minute for the first 30 minutes after the addition of effector cells and every five minutes thereafter. Target cell growth was monitored for 4-5 days after the start of co-culture. Each condition had three independent replicates that 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.

[0156] 2. Results

[0157] 2.1 Design and validation of bispecific RSPO chimeras

[0158] We modified RSPO2, which has the highest binding affinity for ZNRF3 (Park et al., J. Biol. Chem. 2018, 293(25), 9759-9769), to generate a bispecific chimera with simultaneous binding capacity for ZNRF3 / RNF43 and PD-L1 ( Figure 1 a). To prevent unwanted activation of WNT signaling, we mutated phenylalanine 109 of RSPO2 within the FU2 domain (FA mutation) to obtain an LGR-binding defective mutant (R2 FA To obtain PD-L1 binding units, we cloned the extracellular IgV-like domain of PD-1 (PD1) and generated its high-affinity binding variant (Maute et al., Proc Natl Acad Sci USA 2015, 112 (47), E6506-6514). HAC To obtain the final bispecific reagent, we linked RSPO2 FU1 / 2 via a flexible glycine-serine linker. FA Domain structure and PD1 HAC The modules were fused, effectively replacing the TSP1 domain of RSPO2, which is dispensable for ZNRF3 / RNF43 binding, to generate the bispecific RSPO2 chimera R2PD1. Replacing the TSP1 domain in RSPO2 also abolished BMP signaling inhibition.

[0159] Recombinant PD1 HAC , R2 FAand R2PD1 proteins were readily produced and secreted into the culture medium of transfected HEK293T cells. Expectedly, R2PD1 bound LGR4 much weaker than the chimera without the FA mutation in the cell surface binding assay. To test whether R2PD1 retains binding activity to its interacting partners, we performed a solid phase-based ELISA assay ( Figure 1 b). For fixed PD-L1, PD1 binds weakly, but PD1 HAC Strong binding ( Figure 1 c), confirming that the HAC mutant has a higher affinity for PD-L1. Importantly, R2PD1 and PD1 HAC Showed equally strong binding to immobilized PD-L1 ( Figure 1 c). In addition, R2PD1 binds immobilized ZNRF3 and RNF43, similar to R2 FA ( Figure 1 de). Thus, R2PD1 maintains a tight binding toward its two targets, PD-L1 and ZNRF3 / RNF43.

[0160] A prerequisite for E3 ligase-mediated protein degradation is the formation of an E3 ligase-target complex. To test whether R2PD1 bridges the interaction between ZNRF3 / RNF43 and PD-L1, we employed an ELISA assay using immobilized ZNRF3 and RNF43 ( Figure 1 f). As expected, only in the presence of R2PD1, but not in the presence of R2 FA or isolated PD1 HAC In the presence of protein, PD-L1 binds to ZNRF3 ( Figure 1 g) and RNF43 ( Figure 1 h). We conclude that the RSPO chimera R2PD1 effectively bridges the interaction between PD-L1 and ZNRF3 / RNF43.

[0161] 2.2 RSPO chimeras promote degradation of 293T cells overexpressing PD-L1

[0162] To test whether RSPO chimeras induce PD-L1 degradation, we performed degradation assays using HEK293T cells transfected with human PD-L1 with or without ZNRF3 / RNF43 ( Figure 2 a). Co-transfection of ZNRF3 or RNF43 had no effect on PD-L1 levels. However, addition of recombinant R2PD1 effectively reduced the levels of overexpressed PD-L1, but only when ZNRF3 or RNF43 was co-transfected ( Figure 2b). After incubation with 5.4 nM purified recombinant protein, the degradation of PD-L1 reached a plateau, with a maximum reduction of 60% of total protein detected in immunoblotting ( Figure 2 c).

[0163] Next, we hypothesized that PD-L1 degradation requires simultaneous binding to PD-L1 and ZNRF3 / RNF43. FA or PD1 HAC No decrease in PD-L1 levels was detected in cells incubated with Figure 2 This finding was further confirmed by IF, where R2PD1 abolished PD-L1 cell surface staining while inducing colocalization with the lysosomal marker LAMP1 ( Figure 2 f). Use R2 FA or PD1 HAC This result is consistent with previous findings that ZNRF3 / RNF43 employs the lysosomal pathway for targeted protein degradation.

[0164] To test the importance of E3 ligase activity in coordinating PD-L1 degradation, we used a dominant-negative variant of ZNRF3 lacking the intracellular RING domain (ZNRF3 ΔRING R2PD1 effectively reduced total PD-L1 protein levels in a dose-dependent manner in cells expressing full-length ZNRF3, whereas PD-L1 degradation was not observed in cells expressing ZNRF3. ΔRING Eliminated after transfection ( Figure 2 g). Overall, we conclude that RSPO chimeras cause degradation of overexpressed PD-L1 in 293T cells in the presence of overexpressed ZNRF3 / RNF43.

[0165] 2.3 RSPO chimeras promote degradation of endogenous PD-L1 in cancer cells

[0166] To investigate whether the RSPO chimera induces degradation of endogenous PD-L1, we turned to cancer cell lines with PD-L1 expression. Knockdown of PD-L1 by small interfering RNA (siRNA) in the human melanoma cell line MEL624 reduced PD-L1 protein levels, confirming antibody specificity and that the cells express PD-L1 ( Figure 3 a). Addition of R2PD1-conditioned medium reduced PD-L1 levels by approximately 90% ( Figure 3 b). Using as little as 0.27nM R2PD1 protein reduced PD-L1 to a plateau ( Figure 3 c). PD-L1 reduction by R2PD1 was readily detected within 6 hours ( Figure 3d). In addition, only when using R2PD1, not when using R2 FA or PD1 HAC ( Figure 3 e), a reduction in PD-L1 was observed, confirming the requirement for bispecificity. Flow cytometric analysis confirmed that R2PD1 induced cell surface removal of PD-L1 ( Figure 3 fg).

[0167] The expression of PD-L1 is regulated by various cytokines present in the tumor microenvironment, especially interferon-γ (IFNγ). We asked whether R2PD1 could degrade PD-L1 induced by IFNγ. Expectedly, IFNγ increased PD-L1 protein levels, and co-incubation with R2PD1 reduced PD-L1 protein levels ( Figure 3 h).

[0168] To exclude that the reduction of PD-L1 was due to its transcriptional dysregulation rather than induced protein degradation, we monitored PD-L1 mRNA in MEL624 cells. In this study, qRT-PCR analysis showed no significant changes in PD-L1 mRNA in cells incubated with effector proteins ( Figure 3 i).

[0169] We confirmed our findings using another melanoma cell line, COLO-800, in which PD-L1 is expressed and can be knocked down by siRNA transfection ( Figure 3 j). Again, R2PD1 chimeras reduced both cell surface PD-L1 and total PD-L1 protein, although not as completely as in MEL624 cells ( Figure 3 Again, no significant changes in PD-L1 mRNA were detected by qRT-PCR analysis ( Figure 3 n). Although the protein levels of PD-L1 in MEL624 and COLO-800 cells were comparable ( Figure 3 o), but the reduction of PD-L1 in COLO-800 was only evident after 24 h of incubation with R2PD1 chimeras ( Figure 3 p), later than in MEL624 cells ( Figure 3 d). We speculate that the lower efficiency and unique kinetics of PD-L1 degradation by R2PD1 are due to the different expression levels of ZNRF3 / RNF43. Indeed, qRT-PCR analysis showed that the expression of ZNRF3 / RNF43 in MEL624 cells was significantly higher than that in COLO-800 cells ( Figure 3 q).

[0170] Atezolizumab is a therapeutic monoclonal antibody that targets PD-L1 and blocks its interaction with PD-1, thereby promoting tumor-directed T-cell activation. Here, we compared the effects of RSPO chimeras and atezolizumab on PD-L1 protein levels. R2PD1 chimeras again reduced PD-L1 in both MEL624 and COLO-800 cells, whereas the addition of atezolizumab did not alter PD-L1 protein levels ( Figure 3 Collectively, these data support that the R2PD1 chimera causes degradation of endogenous PD-L1 protein in cancer cells.

[0171] PD-L1 degradation by RSPO chimeras is independent of WNT / β-cat signaling

[0172] RSPO is a potent WNT signaling agonist and BMP signaling antagonist. Since these signaling activities may lead to unwanted side effects when RSPO chimeric proteins are applied, the R2PD1 protein used here lacks its TSP1 domain, which completely abolishes its BMP inhibition, and it contains the F2 domain within the FU2 domain. 109 A(FA) mutation, which should abolish LGR-binding and thus should prevent WNT activation. To exclude that the R2PD1 furin domain retains residual WNT / β-cat signaling activity, we performed PD-L1 degradation assays in the presence of WNT inhibitors. In the presence of the potent WNT signaling inhibitor DKK1 ( Figure 4 ab) and degradation of PD-L1 upon β-catenin siRNA knockdown ( Figure 4 cd), R2PD1 chimeras degraded PD-L1. In addition, unlike wild-type RSPO2, the chimeric protein could not induce expression of the WNT-targeted gene AXIN2 or affect the activity of the WNT reporter gene (TOPflash) ( Figure 4 ef). These findings confirm that RSPO chimeras are WNT signaling-inactive and that PD-L1 degradation is independent of WNT / β-cat signaling.

[0173] 2.5 PD-L1 degradation by RSPO chimera requires ZNRF3 / RNF43

[0174] We have shown that the bispecific chimera R2PD1 causes degradation of PD-L1. To address whether PD-L1 degradation occurs through ZNRF3 / RNF43, we incubated the R2PD1 chimeric protein with the soluble extracellular domain of RNF43 (RNF43 ECD Fc) and then added to cells ( Figure 5 a). The basic principle is that, due to RNF43ECD Fc directly binds to R2PD1 ( Figure 1 e) As long as the chimera functions specifically, it should nullify the effects of R2PD1. ECD Fc-preincubated R2PD1 rescued PD-L1 from degradation ( Figure 5 b). Furthermore, although single siRNA knockdown of ZNRF3 or RNF43 was insufficient to block PD-L1 degradation by R2PD1, double knockdown successfully prevented the degradation of PD-L1 by MEL624 ( Figure 5 ce) and COLO-800 cells ( Figure 5 fg), indicating that the chimera engages both E3 ligases. To confirm these findings, we utilized the ZNRF3 / RNF43 double mutant A375 melanoma cell line (dKO). While R2PD1 reduced PD-L1 in control A375 cells, it failed to reduce PD-L1 in dKO cells ( Figure 5 hi). We conclude that in melanoma cells, RSPO chimeras bind ZNRF3 and RNF43 to degrade PD-L1.

[0175] 2.6 R2PD1 chimeras reactivate cytotoxic T cells and inhibit tumor cell proliferation

[0176] To verify that targeting PD-L1 by R2PD1 chimera enhanced cytotoxic T cell activation, we applied T cell activation assays and tumor cell killing assays by co-culturing primary T cells with target cells. Primary T cells were transfected with human DMF5 T cell receptor (TCR) mRNA to enable recognition of target cells presenting the matching MART-1 melanoma antigen. In addition, to enhance PD-L1 / PD-1 signaling, PD-1 mRNA was co-transfected into T cells.

[0177] We initially engineered HEK 293T cells as T cell targets by using PD-L1 and MART-1 minigenes and co-cultured them with T cells transfected with matching TCRs. As markers of cytotoxic T cell activation, we monitored the expression of CD107a or TNFa. After co-incubation of transfected cells, approximately 60% of CD8+ T cells were CD107a-positive. Co-expression of PD-L1 / PD-1 in HEK293T cells and T cells, respectively, reduced the percentage of activated T cells by approximately 80%. When the R2PD1 chimera was added to such HEK293T-restricted T cells, it restored T cell activation in a dose-dependent manner, as monitored by CD107a expression ( Figure 6 (a)).

[0178] Using this experimental protocol, we next replaced the T cell target engineered HEK293T cells with MEL624 cells that endogenously express MART-1. After co-culture of MEL624 and T cells, R2PD1 chimeras activated T cells in a dose-dependent manner ( Figure 6 (b)). When compared with equal concentrations of the clinically used checkpoint inhibitor atezolizumab (a humanized IgG1 monoclonal antibody targeting PD-L1), we found that R2PD1 was considerably more effective ( Figure 6 (b) The R2PD1 response saturates at low nM concentrations, while atezolizumab shows no effect, and at the highest dose employed, R2PD1 is still 2.5-fold more potent than atezolizumab ( Figure 6 (b)).

[0179] Furthermore, R2PD1 chimeras reduced the growth of MEL624 cells when co-cultured with T cells ( Figure 6 (c) , but did not reduce T cell growth. Also in this tumor killing assay, at equivalent concentrations (3.7 nM), R2PD1 was more effective than atezolizumab, reducing tumor cell growth by 30% (R2PD1) vs 7% (atezolizumab) ( Figure 6 (d)). Note that while overall growth inhibition was modest in these short-term in vitro assays, the full tumor-restricting potential of checkpoint inhibitors is revealed over extended periods of time in vivo. We conclude that the R2PD1 chimera functions as a checkpoint inhibitor in vitro, where it is more effective at reactivating T cells and restricting tumor cell growth than the established checkpoint inhibitor atezolizumab.

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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-associated polypeptide, wherein the first binding domain comprises furin domain 1 and / or furin domain 2 of R-spondin (RSPO), wherein the first binding domain lacks wnt signaling activity and / or lacks bone morphogenetic protein (BMP) signaling inhibition activity.

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

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 comprises an exchange of F109A amino acid.

4. The binding polypeptide of any one of claims 1-3, wherein the first binding domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

2.

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

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

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

8. The binding polypeptide of any one of claims 1-7, wherein the disease-associated polypeptide is programmed death-ligand 1 (PD-L1), and wherein the second binding domain comprises an extracellular IgV-like domain of a programmed death-1 (PD-1) polypeptide.

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

5.

10. The binding polypeptide of any one of claims 1-9, 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 connected by a linker, and the linker is preferably a GS linker, more preferably comprising the amino acid sequence of SEQ ID NO:

6. E 11. The binding polypeptide of any one of E 1-10, 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. An in vitro method for killing cancer cells, comprising contacting said 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. 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. A method for identifying a subject with cancer who is susceptible to treatment with a binding polypeptide according to any one of claims 1 to 11 and / or a polynucleotide according to claim 12, the method comprising (A) determining the expression of PD-L1 or a surrogate marker thereof in cancer cells of the subject; and (B) identifying the subject as being sensitive to the treatment based on the determination in step (A).

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