Screening method for effective combinations of target-E3 ligases

Abstract

The present invention relates to a method for identifying an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein, where the combination is effective if the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein upon forced dimerization of the membrane-bound protein, preferably by ubiquitination of the membrane-bound protein. The method of the present invention includes the step of exposing a cell to a heterobifunctional molecule, the heterobifunctional molecule comprising a first binding domain capable of specifically binding to the extracellular portion of the transmembrane E3 ubiquitin ligase and a second binding domain capable of specifically binding to the extracellular portion of the membrane-bound protein. The method further includes the step of determining the reduction in the surface level of the membrane-bound protein. Furthermore, the present invention relates to a heterobifunctional molecule that targets an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein.

Description

[Technical Field]

[0001] This invention relates to the field of molecular and cellular biology, particularly to the fields of targeted molecular therapy and cancer cell biology. The invention relates to a method for screening effective combinations of target membrane-bound proteins and E3 ubiquitin ligases, and for generating heterobifunctional molecules that simultaneously target these discovered effective combinations. Therefore, the invention relates to the use of heterobifunctional molecules that can simultaneously bind to transmembrane ubiquitin ligases and membrane-bound proteins in order to mediate the internalization of membrane-bound proteins. [Background technology]

[0002] Cells communicate with their surroundings through the activation of receptors embedded in the cell membrane, which capture external chemical signals and initiate intracellular signaling cascades to drive cellular responses. Receptor availability on the cell surface is a critical determinant of signal specificity and sensitivity, and misregulation of these events is often associated with the development or progression of diseases, including, but not limited to, cancer, autoimmune diseases, neurological disorders, and inflammatory disorders, as well as treatment resistance.

[0003] For example, the mutagenesis, activation, or overexpression of receptors (e.g., EGFR, ERBB2, PDGFR, TGFβR, IGFR1, GHR, FZD, LRP6) is a major and widely recognized cancer-promoting mechanism in numerous tissues. The dependence of cancer cells on abnormal receptor activity has spurred the development of various neutralizing antibodies and small molecule inhibitors. However, successfully neutralizing receptor activity requires the creation of potent binders that reach sufficient plasma concentrations to demonstrate high efficacy without inducing toxicity, which can be difficult in the case of non-covalent interacting substances. Furthermore, compensatory receptor stabilization or upregulation is a major pathway for resistance.

[0004] Post-translational modification of the cytosolic region of membrane-bound receptors with ubiquitin leads to rapid removal of the receptor from the cell surface via induced endocytosis. The internalized receptor may then be subjected to lysosomal degradation. In healthy stem cells, high levels of Wnt signaling induce the expression of two homologous membrane-bound ubiquitin ligases, RNF43 and ZNRF3, which are known to mediate the ubiquitination and removal of Frizzled (FZD), a Wnt receptor, from the cell surface (Koo et al, Nature 2012, 488(7413):665-9). Therefore, this negative feedback loop helps modulate the sensitivity of stem cells to Wnt by controlling the effective number of Frizzled (FZD) receptors on the cell surface. The activity of RNF43 / ZNRF3 against FZD is neutralized in the stem cell niche by the LGR4 / 5 receptor and the secreted protein R-spongin (RSPO), which forms a complex with RNF43 / ZNRF3 (Hao et al, Nature 2012, 485(7397):195-200). This trimer, the RSPO-LGR4 / 5-RNF43 / ZNRF3 complex, is then removed from the cell surface, resulting in stabilization of FZD receptor expression and an increase in Wnt signaling levels. Wnt signaling is often misregulated in cancer. Such cancers show increased expression of Wnt target genes, including RNF43 and ZNRF3.

[0005] E3 ubiquitin ligases recruit ubiquitin-conjugated E2 enzymes that carry ubiquitin to the protein substrate, thereby assisting in or directly catalyzing the transfer of ubiquitin to the protein substrate.

[0006] It is known that transmembrane ubiquitin E3 ligase-mediated receptor ubiquitination results in endocytosis and subsequent degradation of the ubiquitinated substrate. It is known in the art that such degradation preferably occurs within lysosomes. Lysosomal degradation requires ligation of monoubiquitin, multiubiquitin, Lys11, Lys29, Lys48, or Lys63-linked polyubiquitin chains to membrane-bound receptors. This, in contrast to the activity of cytosolic ubiquitin ligases, primarily utilizes the proteasomal degradation pathway, namely the proteasomal degradation pathway via the coupling of Lys11, Lys29, or Lys48-linked polyubiquitin chains to cytosolic target proteins.

[0007] Therefore, transmembrane E3 ubiquitin ligases may interact with different members of the E2 enzyme family to selectively target membrane-bound substrates. The ubiquitinated substrates can be taken up and subsequently degraded, preferably via lysosomal degradation.

[0008] There remains a strong need in the art to effectively target and inhibit the activity of membrane-bound receptors, particularly those involved in the development or progression of disease. For example, there is a particularly strong need in the art to effectively target and inhibit the activity of membrane-bound receptors involved in the development or progression of cancer, autoimmune diseases, neurological disorders, rare diseases, and inflammatory disorders, as well as treatment resistance. [Overview of the Initiative]

[0009] The present invention can be summarized in the following embodiments. Embodiment 1. A method for identifying effective combinations of transmembrane E3 ubiquitin ligase and membrane-bound proteins, wherein a combination is effective when the transmembrane E3 ubiquitin ligase, preferably by ubiquitination of the membrane-bound protein, can co-bind to a heterobifunctional molecule, thereby reducing the surface level of the membrane-bound protein, and this method involves the following steps: a) providing cells expressing a transmembrane E3 ubiquitin ligase and a membrane-bound protein on the cell surface; b) exposing the cells to a heterobifunctional molecule, wherein the heterobifunctional molecule i) a first binding domain capable of specifically binding to the extracellular portion of the transmembrane E3 ubiquitin ligase, and ii) a second binding domain capable of specifically binding to the extracellular portion of the membrane-bound protein ; and c) determining the surface level of the membrane-bound protein of the cells ; and a decrease in the surface level of the membrane-bound protein indicates that the combination is an effective combination, and this decrease is preferably a decrease compared to the surface level of the membrane-bound protein of the cells before step b).

[0010] Embodiment 2. The method according to embodiment 1, wherein the membrane-bound protein is a transmembrane protein.

[0011] Embodiment 3. The method according to embodiment 1 or 2, wherein the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130 and RNF128.

[0012] Embodiment 4. - The transmembrane E3 ubiquitin ligase contains a first extracellular non-native epitope tag, and the first binding domain of the heterobifunctional molecule binds to the first non-native epitope tag ; and - The membrane-bound protein contains a second extracellular non-native epitope tag, and the second binding domain of the heterobifunctional molecule binds to the second non-native epitope tag The method according to any one of the preceding embodiments, which is one of them.

[0013] Embodiment 5. The method according to Embodiment 4, wherein the first and second non-natural epitope tags are different tags.

[0014] Embodiment 6. The method according to Embodiment 4 or 5, wherein the first non-natural epitope tag is at least one of an alpha tag and an E6 tag, and / or the second non-natural epitope tag is at least one of an alpha tag and an E6 tag.

[0015] Embodiment 7. At least one of the first and second non-natural epitope tags is a transmembrane E3 ubiquitin ligase and a membrane-bound protein, respectively. i) N-terminus; ii) C-terminus; and / or iii) Extracellular loop region A method according to any one of embodiments 4 to 6, wherein the method is located in at least one of the above.

[0016] Embodiment 8. The method according to any one of the above embodiments, wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody.

[0017] Embodiment 9. The method according to Embodiment 8, wherein the first binding domain of the heterobifunctional molecule is anti-alpha VHH and the second binding domain is anti-E6 VHH, or the first binding domain of the heterobifunctional molecule is anti-E6 VHH and the second binding domain is anti-alpha VHH.

[0018] Embodiment 10. The method according to any one of the above embodiments, wherein the membrane-bound protein comprises a third non-natural epitope tag and / or the transmembrane ubiquitin E3 ligase comprises a fourth non-natural epitope tag, preferably the third and / or fourth epitope tags being at least one of His-tag, FLAG-tag and myc-tag.

[0019] Embodiment 11. The method according to any one of the above embodiments, wherein the cell surface level of the membrane-bound protein in step c) is determined by detecting the protein on the cell surface, preferably by immunofluorescence.

[0020] Embodiment 12. The method according to any one of the above embodiments, wherein the combination is effective when the cell surface level of the membrane-bound protein is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least about 95% compared to the cell surface level of the membrane-bound protein before step b), preferably by at least about 60%, 70%, 80%, 90%, or at least about 95% compared to the cell surface level of the membrane-bound protein before step b).

[0021] Embodiment 13. In step a), first and second cells are provided, - The first cell expresses the first transmembrane E3 ubiquitin ligase and the first membrane-bound protein on its cell surface; and - The second cell expresses the second transmembrane E3 ubiquitin ligase and the first membrane-bound protein on its cell surface. The first and second transmembrane E3 ubiquitin ligases are different ligases containing the same first extracellular non-native epitope tag; In step b), the first and second cells are exposed to the heterobifunctional molecule, and the heterobifunctional molecule i) A first binding domain capable of specifically binding to a first non-natural epitope tag; ii) The extracellular portion of the membrane-bound protein, preferably a second binding domain capable of specifically binding to a second non-natural epitope tag, Includes, The method according to any one of Embodiments 4 to 11, wherein in step c), the surface levels of membrane-bound proteins in the first and second cells are determined, and the combination is valid if the cell surface level of membrane-bound proteins in the first cell is reduced by at least about 10%, 20%, 30%, 40%, 50%, or at least about 60% compared to the cell surface level of membrane-bound proteins in the second cell after step b).

[0022] Embodiment 14. A third, fourth, or further cell is provided, each expressing a third, fourth, or further transmembrane E3 ubiquitin ligase and a first membrane-bound protein on its cell surface, respectively. Transmembrane E3 ubiquitin ligase is a different ligase that contains the same first extracellular non-natural epitope tag. The combination is effective if the cell surface level of membrane-bound proteins in the first cell decreases by at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least approximately 95% compared to the cell surface level of membrane-bound proteins in the second, third, fourth, and subsequent cells after step b). The method of Embodiment 13, wherein the method is carried out in a multiplexed manner.

[0023] Embodiment 15. The method according to any one of the above embodiments, wherein the decrease in the surface level of membrane-bound proteins is determined by a decrease in the total amount of membrane-bound proteins in the cells, preferably measured by microscopy, biochemical analysis and / or FACS.

[0024] Embodiment 16. The method according to any one of the above embodiments, wherein the cells provided in step a) overexpress, and if necessary, permanently overexpress, at least one of the transmembrane E3 ubiquitin ligase and membrane-bound proteins.

[0025] Embodiment 17. The method according to any one of the above embodiments, wherein the cells prepared in step a) express transmembrane E3 ubiquitin ligase and membrane-bound proteins at endogenous levels.

[0026] Embodiment 18. The method of Embodiment 17, wherein in the cells prepared in step a), the genomic sequence encoding the transmembrane E3 ubiquitin ligase is modified to incorporate sequences encoding a first and optionally a fourth non-natural epitope tag.

[0027] Embodiment 19. The method according to Embodiment 17 or 18, wherein in the cells prepared in step a), the genomic sequence encoding a membrane-bound protein is modified to incorporate a second, and optionally third, non-natural epitope tag encoding sequence.

[0028] Embodiment 20. The method according to any one of the above embodiments, wherein the heterobifunctional molecule comprises a peptide linker between a first binding domain and a second binding domain, preferably the peptide linker is (GGGGS)n, where n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and preferably n is 3 or 5.

[0029] Embodiment 21. A heterobifunctional molecule comprising a first and a second binding domain, i) The first binding domain can specifically bind to transmembrane E3 ubiquitin ligase; and ii) The second binding domain can specifically bind to membrane-bound proteins, A heterobifunctional molecule in which a transmembrane E3 ligase and a membrane-bound protein are an effective combination, as determined in the method of any one of Embodiments 1 to 20.

[0030] Embodiment 22. The heterobifunctional molecule according to Embodiment 21, wherein the molecule binds to the extracellular portion of a transmembrane E3 ubiquitin ligase and the extracellular portion of a membrane-bound protein.

[0031] Embodiment 23. A heterobifunctional molecule according to Embodiment 21 or 22, wherein the membrane-bound protein is a receptor, preferably a receptor involved in at least one of cancer, autoimmune diseases, neurological disorders, and inflammatory disorders.

[0032] Embodiment 24. A heterobifunctional molecule according to any one of Embodiments 21 to 23, wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody.

[0033] Example 25. A heterobifunctional molecule according to any one of Embodiments 21 to 24, for use as a pharmaceutical. [Brief explanation of the drawing]

[0034] [Figure 1] This is a schematic diagram of an exemplary embodiment of the present invention. The heterobifunctional molecule of the present invention simultaneously binds to a transmembrane E3 ubiquitin ligase and a transmembrane protein. As a result, the transmembrane protein is ubiquitinated, internalized, and degraded. [Figure 2] This figure shows the functional evaluation of the A / C dimerizer. HEK293T cells were transfected with RNF43-FKBP and TβRII-Flag-FRB and treated overnight with the A / C dimerizer or an equal volume of 100% ethanol. The TβRII construct was immunoprecipitated from the cell lysates using Flag-M2 beads. The IP samples and whole cell lysates were separated by SDS-page, blotted, and stained for Flag and RNF43 to detect binding between the two constructs. [Figure 3]This figure shows how forced dimerization of RNF43 and TβRII induces relocalization of both proteins to perinuclear lysosomes. (a) Confocal image of HEK293T cells transfected with TβRII-Flag-FRB. (B) Confocal image of HEK293T cells transfected with RNF43-FKBP and TβRII-Flag-FRB. Cells were treated overnight with A / C dimerizer or an equal volume of 100% ethanol. TβRII and RNF43 were visualized by Flag and RNF43 staining, respectively. (C) Confocal image of HEK293T cells transfected with CD63-GFP, RNF43-FKBP, and TβRII-Flag-FRB. Cells were treated overnight with A / C dimerizer, and TβRII-Flag-FRB was visualized by Flag staining. Arrows indicate perinuclear lysosomes. [Figure 4] This figure shows the degradation of TβRII by forced dimerization of RNF43 and TβRII. HEK293T cells were transfected with RNF43-FKBP and TβRII-Flag-FRB and treated overnight with an A / C dimerizer or an equal volume of 100% ethanol. Cell lysates were separated by SDS-page, blotted, and stained for Flag and RNF43 to visualize protein levels. [Figure 5] This figure shows that VHH-mediated dimerization of RNF43 or RNF167 with the receptor TβRII or EGFR induces receptor internalization and co-clustering in the perinuclear region. Cells were treated with 100 nM bi-VHH for 5 hours before fixation. E3 ligases were visualized with Myc staining and receptors with Flag staining. Confocal images of HEK293 T cells transfected with (A) E6-Flag-TβRII and Alpha-Myc-RNF43, (B) E6-Flag-TβRII and Alpha-Myc-RNF167, (C) E6-Flag-EGFR and Alpha-Myc-RNF43, and (D) E6-Flag-EGFR and Alpha-Myc-RNF167. Arrows indicate co-clustering of E3 ligases and receptors in the perinuclear region. [Figure 6]This figure shows that bifunctional VHH treatment promotes the internalization of transmembrane receptors from the cell surface mediated by membrane-bound E3 ligases. HEK293T cells were transfected with one of the E3 ligases RNF43, RNF128, RNF130, or RNF167 and the receptors CTLA-4, FLT-3, PD-1, or PD-L1. Cells were either left untreated or treated overnight with 50 nM bi-VHH before fixation. Receptors present on the cell surface were visualized by flag staining of impermeable cells. (A) Confocal images of HEK293T cells transfected with E6-Flag-CTLA-4 and Alpha-Myc-RNF43 or Alpha-Myc-RNF167, (B) E6-Flag-FLT-3 and Myc-RNF43, Alpha-Myc-RNF128 or Alpha-Myc-RNF167, (C) E6-Flag-PD-1 and Alpha-Myc-RNF43, Alpha-Myc-RNF128, Alpha-Myc-RNF130 or Alpha-Myc-RNF167, and (D) E6-Flag-PD-L1 and Alpha-Myc-RNF43, Alpha-Myc-RNF128, Alpha-Myc-RNF130 or Alpha-Myc-RNF167. [Figure 7] Bifunctional VHH treatment promotes the internalization of the type III multispan protein CMTM6 from the cell surface mediated by membrane-bound E3 ligases. (A) Schematic diagram of the Snorkel tag for detecting surface expression of multispan proteins. (B) HEK293T cells were transfected with one of the E3 ligases RNF43, RNF128, RNF130, or RNF167, and the multispan receptor CMTM6. Cells were left untreated or treated overnight with 50 nM bi-VHH before fixation. Receptors present on the cell surface were visualized by Flag staining of impermeable cells. Confocal images of HEK293T cells transfected with E6-Flag-Snorkel-CMTM6 and Alpha-Myc-RNF43, Alpha-Myc-RNF128, Alpha-Myc-RNF130, or Alpha-Myc-RNF167 are shown. [Figure 8-1]This figure examines the effect of bi-VHH on E3 ligase and target combinations at the endogenous level. (A) Strategies for generating endogenously tagged proteins. [Figure 8-2] (B) A diagram showing the effect of bi-VHH on E3 ligase and target combinations at the endogenous level. (A) A schematic diagram of the bi-VHH approach to target cell surface removal using an endogenously tagged version of the E3 ligase and target combination. [Modes for carrying out the invention]

[0035] definition Various terms relating to the methods, compositions, formulations, uses, and other embodiments of the present invention are used throughout this specification and the claims. Unless otherwise indicated, such terms shall be given the common meaning in the art to which the invention pertains. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein. Any methods and materials similar or equivalent to those described herein may be used in the test implementation of the present invention, but preferred materials and methods are described herein.

[0036] The methods for carrying out the prior art used in the methods of the present invention will be apparent to those skilled in the art. The practices of the prior art in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields are well known to those skilled in the art and are discussed, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987, and periodic updates; and in the series Methods in Enzymology, Academic Press, San Diego.

[0037] "A," "an," and "the": These singular terms include plural references unless the context explicitly indicates otherwise. Therefore, the indefinite article "a" or "an" usually means "at least one." Thus, for example, a reference to "cells" includes combinations of two or more cells.

[0038] "Approximately" and "about": When these terms refer to measurable values ​​such as quantities and durations, they mean that such variation should be appropriate for the disclosed method, and should include variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value. In addition, quantities, ratios, and other numerical values ​​may be presented in range format as herein. Such range formats should be used concisely for convenience and should be understood flexibly to include numerical values ​​explicitly designated as limits of the range, but also to include all individual numerical values ​​or subranges contained within that range, as individual numerical values ​​and subranges are explicitly designated. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly stated limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, as well as subranges such as about 10 to about 50, about 20 to about 100.

[0039] "and / or": The term "and / or" refers to a situation in which one or more of the listed conditions may occur, either alone, in combination with at least one of the listed conditions, or with up to all of the listed conditions.

[0040] "Includes": This term is to be interpreted as comprehensive, open-ended, and non-exclusive. Specifically, this term and its variations mean including the identified features, steps, or components. These terms should not be interpreted as excluding the presence of other features, steps, or components.

[0041] “Exemplary”: This term means “to serve as an example, case, or illustration” and should not be construed as excluding other configurations disclosed herein.

[0042] The term "hetero-bifunctional molecule" is defined herein as a molecule comprising two different functional binding domains. In particular, the hetero-bifunctional molecule of the present invention has a first functional binding domain for binding to a transmembrane E3 ubiquitin ligase and another second functional binding domain for binding to a second molecule. As the name "hetero-bifunctional" already indicates, the second functional binding domain binds to a second molecule, and the second molecule is not the same molecule that can bind to the first functional binding domain, i.e., it is not the same transmembrane E3 ubiquitin ligase. Preferably, the second functional binding domain does not bind to a transmembrane E3 ubiquitin ligase.

[0043] The terms “protein” or “polypeptide” refer to a molecule consisting of a chain of amino acids without referring to a specific mode of action, size, three-dimensional structure, or origin. Thus, a “fragment” or “part” of a protein may still be referred to as a “protein.” A protein as defined herein and used in any manner defined herein may be an isolated protein. “Isolated protein” is used to refer to a protein that no longer exists in nature (e.g., in vitro or in recombinant bacterial or plant host cells). Preferably, a protein contains more than 50 amino acid residues.

[0044] In this specification, the term "protein molecule" is understood to mean a molecule containing a short chain of amino acid monomers linked by peptide (amide) bonds. A short chain of amino acid monomers contains two or more amino acid residues. Preferably, the amino acid chain has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues. Preferably, there are 100 or fewer amino acid residues. Preferably, there are 50 or fewer amino acid residues in the protein molecule. Preferably, the protein molecule has about 2 to 100, 3 to 50, 4 to 40, or 5 to 30, or 6 to 20 amino acid residues. Preferably, the protein molecule has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues. If necessary, the protein molecule may include one or more further organic moieties, such as linkages, to form a cyclic protein molecule.

[0045] An "aptamer" is preferably a nucleic acid molecule having a specific nucleotide sequence. An aptamer can contain any appropriate number of nucleotides. An aptamer can contain RNA or DNA, or both ribonucleotide residues and deoxyribonucleotide residues. An aptamer can be single-stranded, double-stranded, or contain double-stranded or triple-stranded regions. Furthermore, an aptamer can contain chemically modified residues, for example, to improve its stability.

[0046] Aptamers are typically about 10 to 300 nucleotides long. More commonly, aptamers are about 30 to 100 nucleotides long.

[0047] An aptamer for a given target (i.e., a transmembrane E3 ubiquitin ligase or further transmembrane protein) includes a nucleic acid that can be identified from a candidate mixture of nucleic acids by using a method comprising: (a) contacting a candidate mixture with the target, wherein nucleic acids having increased affinity for the target compared to other nucleic acids in the candidate mixture can be separated from the remainder of the candidate mixture; (b) separating the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to produce a nucleic acid-enriched mixture, thereby identifying the aptamer of the target molecule.

[0048] While affinity interactions are recognized as a matter of degree, in this context, the "specific binding affinity" of an aptamer to its target means that the aptamer binds to that target with a much higher degree of affinity than it would to other non-target components in the mixture or sample.

[0049] Aptamers have a specific binding region that allows them to form a complex with an intended target molecule in an environment where other substances in the same environment do not complex with nucleic acids. Binding specificity can be defined by comparing the comparative dissociation constant (Kd) of the aptamer with respect to its ligand to the dissociation constant of the aptamer with respect to other materials in the environment or generally unrelated molecules. Typically, the Kd of the aptamer with respect to its ligand is at least about 10 times smaller than the Kd of the aptamer with unrelated or accompanying materials in the environment. More preferably, the Kd is at least about 50 times smaller, more preferably at least about 100 times smaller, and most preferably at least about 200 times smaller.

[0050] In one embodiment, the aptamer that binds to the transmembrane protein has a dissociation constant (K) of 1 mM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. d ) has. In one embodiment, the anti-transmembrane protein antibody binds to an epitope that is conserved between different species.

[0051] In one embodiment, the aptamer that binds to the transmembrane E3 ubiquitin ligase has a dissociation constant (K) of 1 mM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. d ) has. In one embodiment, the anti-transmembrane protein antibody binds to an epitope conserved across different species.

[0052] The term "antibody" is used in its broadest sense, specifically to cover, as long as it exhibits the desired biological and / or immunological activity, for example, monoclonal antibodies, e.g., agonists and antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies, single-chain antibodies and functional fragments of antibodies, e.g., Fab, Fab', F(ab')2 and Fv fragments, diabodies, triabodies, single-domain antibodies (sdAb), heavy-chain antibodies, and nanobodies.

[0053] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" in this specification. Antibodies may be human and / or humanized.

[0054] The term "anti-transmembrane E3 ubiquitin ligase antibody" specifically covers, insofar as it exhibits the desired biological and / or immunological activity, for example, single anti-transmembrane E3 ubiquitin ligase monoclonal antibodies, e.g., agonists and antagonists, preferably agonists, neutralizing antibodies, full-length or intact monoclonal antibodies, polyclonal antibodies, naked antibodies, multivalent antibodies, single-chain anti-transmembrane E3 ubiquitin ligase antibodies and fragments of anti-transmembrane E3 ubiquitin ligase antibodies, e.g., Fab, Fab', F(ab')2 and Fv fragments, diabodies, triabodies, single-domain antibodies (sdAb), heavy-chain antibodies and nanobodies. Preferred antibodies may be nanobodies. Preferably, anti-transmembrane E3 ubiquitin ligase antibodies specifically bind to E3 ubiquitin ligases as defined below herein.

[0055] The term "anti-transmembrane protein antibody" specifically covers, insofar as it exhibits the desired biological and / or immunological activity, for example, single anti-transmembrane protein monoclonal antibodies, e.g., agonists and antagonists, preferably antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies, polyclonal antibodies, naked antibodies, multivalent antibodies, single-chain anti-transmembrane protein antibodies and fragments of anti-transmembrane protein antibodies, e.g., Fab, Fab', F(ab')2 and Fv fragments, diabodies, triabodies, single-domain antibodies (sdAb), heavy-chain antibodies and nanobodies. Preferred antibodies may be nanobodies. Preferably, anti-transmembrane protein antibodies specifically bind to transmembrane proteins as defined below herein.

[0056] The term "anti-transmembrane E3 ubiquitin ligase antibody" or "antibody that binds to transmembrane E3 ubiquitin ligase" refers to an antibody that can bind to transmembrane E3 ubiquitin ligase with sufficient affinity so that the antibody is useful as the first binding domain of a heterobifunctional molecule as defined herein. Preferably, the degree of binding of the anti-transmembrane E3 ubiquitin ligase antibody to unrelated proteins is less than about 10% of the binding of the antibody to transmembrane E3 ubiquitin ligase, as measured, for example, by radioimmunoassay (RIA) or ELISA. In some embodiments, the antibody that binds to transmembrane E3 ubiquitin ligase has a dissociation constant (K) of 1 mM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. d ) has. In one embodiment, the anti-transmembrane E3 ubiquitin ligase antibody binds to an epitope conserved across different species.

[0057] The term "anti-transmembrane protein antibody" or "antibody that binds to a transmembrane protein" refers to an antibody that can bind to a specific or selected transmembrane protein with sufficient affinity so that the antibody is useful as a second binding domain of a heterobifunctional molecule as defined herein. Preferably, the degree of binding of the anti-transmembrane protein antibody to an unrelated protein is less than about 10% of the binding of the antibody to the transmembrane protein, as measured, for example, by radioimmunoassay (RIA) or ELISA. In some embodiments, the antibody that binds to the transmembrane protein has a dissociation constant (K) of 1 mM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. d ) has. In one embodiment, the anti-transmembrane protein antibody binds to an epitope conserved across different species.

[0058] An antibody that "binds" to the target antigen, i.e., a transmembrane E3 ubiquitin ligase or further transmembrane protein of interest, is an antibody that binds to the antigen with sufficient affinity so that the antibody is useful as the first or second binding domain of a heterobifunctional molecule as defined herein.

[0059] Antibodies acting as either the first or second binding domain in a heterobifunctional molecule can be basic quadrivalent antibodies. Such basic quadrivalent antibody units are preferably heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H) (IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide called a J chain, and thus contain 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 basic quadrivalent units along with the J chain).

[0060] In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H chain and L chain also have regularly spaced intrachain disulfide cross-links. Each H chain has a variable domain (V H ) at the N-terminus, followed by three constant domains (C H ) for each of the α and γ chains, and four C H domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) at the N-terminus, followed by a constant domain (C L ) at the other terminus. V L aligns with V H , and C L aligns with the first constant domain (C H 1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of V H and V L together forms a single antigen-binding site. For the structure and properties of antibodies of different classes, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0061] L chains from any vertebrate species can be assigned to one of two clearly distinct types called κ and λ based on the amino acid sequence of the constant domain. Depending on the amino acid sequence of the constant domain (C H ) of the heavy chain, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes have C HBased on relatively small differences in sequence and function, they are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0062] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain is called "V H It can be called "V". The variable domain of the light chain is "V L These domains can be referred to as "antigen-binding sites." These domains are generally the most variable parts of an antibody and contain the antigen-binding site.

[0063] The term "variable" refers to the significant sequence differences between antibodies in a particular segment of the variable domain. The V domain mediates antigen binding and defines the specificity of a particular antibody to a specific antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable domain. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs), separated by shorter, extremely variable regions called "hypervariable regions" (HVRs), each 9-12 amino acids long. The variable domains of natural heavy and light chains each contain four FRs, primarily employing a β-sheet structure, connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions within each chain are held together in close proximity by the fiber-retaining domain (FR), and together with hypervariable regions from other chains, they contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domain does not directly participate in antibody binding to the antigen, but exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity (ADCC).

[0064] "Undamaged" antibodies have an antigen-binding site and C L and at least heavy chain constant domain, C H 1. C H 2 and C H The antibody contains 3. The constant domain may be the natural sequence constant domain (e.g., the human natural sequence constant domain) or an amino acid sequence variant thereof.

[0065] An "antibody fragment" is a portion of an intact antibody, preferably including at least the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; triabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment includes the antigen-binding site of the intact antibody and therefore retains the ability to bind to an antigen.

[0066] The term "nanobody" is well known in the relevant technical field. A nanobody is a V of an antibody consisting only of the heavy chain. H An antibody fragment containing or consisting of an H domain. The terms “nanobody,” “single-domain antibody,” and “single-domain antibody fragment” can be used interchangeably herein. A single-domain antibody fragment has a single monomeric variable antibody domain, preferably with a molecular weight of about 12-15 kDa. Nanobodies, like whole antibodies, can selectively bind to specific antigens. Nanobodies may be derived from dromedary camels, camels, llamas, alpacas, or sharks. Preferred nanobodies are derived from camelids, preferably from llamas.

[0067] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, and the remaining "Fc" fragment, a name reflecting its ability to readily crystallize. Each Fab fragment consists of the entire L chain, along with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0068] Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and can still crosslink the antigen. The Fab' fragment contains one or more cysteine ​​from the antibody hinge region. H It differs from the Fab fragment by having a few additional residues at the carboxyl terminus of one domain. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments having a hinged cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0069] The Fc fragment contains the carboxyl-terminal portions of both H chains, held together by a disulfide. The effector function of the antibody is determined by the sequence of the Fc region, which is also the portion recognized by the Fc receptor (FcR) found in certain types of cells.

[0070] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain and one light chain variable domain, which are tightly and non-covalently associated. In the single-stranded Fv (scFv) species, the one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that in the double-stranded Fv species. From the folding of these two domains, six hypervariable loops (three from the H chain and three from the L chain) are generated, which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, but with lower affinity than the entire binding site.

[0071] "Single-stranded Fv" is also abbreviated as "sFv" or "scFv," and refers to V connected to a single polypeptide chain. H and V L An antibody fragment containing an antibody domain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, enabling the sFv to form a desired structure for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0072] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigen site, in contrast to polyclonal antibody preparations which contain different antibodies directed to different determinants (epitopes). Monoclonal antibodies have the advantage of being able to be synthesized without contamination by other antibodies. The modifying phrase “monoclonal” should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies useful as the first or second binding domain in the heterobifunctional molecule of the present invention can be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or can be produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0073] The monoclonal antibodies described herein include “chimeric” antibodies, as well as fragments of such antibodies, insofar as they exhibit the desired biological activity, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody originating from another species or belonging to another antibody class or subclass (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies of interest herein include “primatized” antibodies containing variable domain antigen-binding sequences and human constant region sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.).

[0074] The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing the minimal sequence derived from the non-human antibody. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced with residues from the hypervariable region (donor antibody) of a non-human species such as mouse, rat, rabbit, or non-human primate, possessing the desired antibody specificity, affinity, and capabilities. In some examples, a small number of framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody typically contains two variable domains, with all or substantially all of the hypervariable loop corresponding to that of the non-human immunoglobulin, and all or substantially all of the FR being from the human immunoglobulin sequence. The humanized antibody also, if necessary, contains at least a portion of the immunoglobulin constant region (Fc), typically from that of the human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also, see the following overview articles and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

[0075] The terms “hypervariable region” and “HVR,” as used herein, refer to regions of antibody variable domains that are hypervariable in sequence and / or form structurally defined loops involved in antigen binding. Generally, antibodies contain six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Numerous hypervariable region contours have been used and are incorporated herein. The hypervariable region is generally derived from amino acid residues of the "complementarity-determining region" or "CDR" (for example, approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the VL, and approximately residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the VH, according to the Kabat numbering system); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from the "hypervariable loop" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, and 26-32 (H1), 52-56 (H2), and 95-101 (H3) in VH, when numbered according to the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and / or those residues from the "hypervariable loop" / CDR (e.g., residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in VL, when numbered according to the IMGT numbering system, and 27-38 (H1), 56-65 (H2), and 105-120 (H3) in VH; Lefranc, MP et al. Nucl. Acids Includes Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000).If necessary, the antibody, when numbered according to Honneger, A. and Plunkthun, AJ (Mol. Biol. 309:657-670 (2001)), has symmetrical insertions at one or more of the following locations in VL: 28, 36 (L1), 63, 74-75 (L2), and 123 (L3), and in VH: 28, 36 (H1), 63, 74-75 (H2), and 123 (H3). The hypervariable region / CDR of the antibody of the present invention is preferably defined and numbered according to the IMGT numbering system.

[0076] A “framework” or “FR” residue is a variable domain residue other than a hypervariable region residue as defined herein.

[0077] A "blocking" antibody or "antagonist" antibody inhibits or reduces the biological activity of the antigen to which it binds. A preferred blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

[0078] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of the polypeptide of interest.

[0079] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to endogenous binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). d) can be expressed as follows. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art and any of them can be used for the purposes of the present invention. Specific exemplary embodiments are described below.

[0080] "K d " or "K d The value can be measured by a surface plasmon resonance assay using BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip with approximately 10-50 response units (RUs). Briefly, the carboxymethylated dextran biosensor chip (CM5, BIAcore Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions for use. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate at pH 4.8 and injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurements, two-fold series dilutions of antibody or Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween 20 are injected at a flow rate of approximately 25 μl / min at 25°C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K) was calculated using a simple one-to-one Langmuir coupled model (BIAcore evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. d ) to k off / k onThe calculation was performed as a ratio. For example, see Chen, Y., et al., (1999) J. Mol Biol 293:865-881. The on rate was 10 by the above surface plasmon resonance assay. 6 M -1 S -1 If it exceeds this, the on-rate can be determined by measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band passthrough) at 25°C in the presence of increased antigen concentrations using a spectrophotometer such as an Aviv Instruments spectrophotometer with stop-flow or an 8000 series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred red cuvette.

[0081] Furthermore, the present invention also refers to "on-rate," "meeting speed," "meeting rate," or "k on As mentioned above, this can be determined using the same surface plasmon resonance technique described above with BIAcore(trademark)-2000 or BIAcore(trademark)-3000 (BIAcore, Inc., Piscataway, NJ).

[0082] Preferably, the antibody intended for use as a first or second binding domain in a heterobifunctional molecule does not significantly cross-react with other proteins.

[0083] The terms "antigen-binding protein" and "binding domain" in the heterobifunctional molecules of the present invention can be used interchangeably in this specification.

[0084] The term “epitope” refers to a portion of a molecule that is bound by the first or second binding domain of the heterobifunctional molecule of the present invention, respectively. The term includes any determinant that can specifically bind to an antigen-binding protein, for example, to the first or second domain of the heterobifunctional molecule as defined herein. Epitopes can be continuous or discontinuous (for example, in a polypeptide, amino acid residues that are not continuous with each other in the polypeptide sequence but are in the context of the molecule are bound by the antigen-binding protein). Epitopes are preferably located on a transmembrane E3 ubiquitin ligase as defined herein, or on a further transmembrane protein of interest as defined herein.

[0085] Epitope determinants may include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl, sulfonyl, or sulfate groups, and may possess specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on the target antigen in complex mixtures of proteins and / or macromolecules.

[0086] The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain may differ, the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226, or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly manipulating the nucleic acid encoding the antibody heavy chain.

[0087] The term "antibody containing an Fc region" refers to an antibody that contains an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody purification or by recombinant manipulation of the nucleic acid encoding the antibody. Therefore, the heterobifunctional molecule containing an antibody having an Fc region according to the present invention may include antibodies having K447 or antibodies from which K447 has been removed.

[0088] "Amino acid sequence": This refers to the order of amino acid residues in a protein, or within a protein. In other words, any order of amino acids in a protein can be called an amino acid sequence.

[0089] "Nucleotide sequence": This refers to the sequence of nucleotides in a nucleic acid, or within a nucleic acid. In other words, any sequence of nucleotides in a nucleic acid can be called a nucleotide sequence.

[0090] The terms “homology,” “sequence identity,” and others are used interchangeably herein. Sequence identity is defined herein as the association between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing sequences. In the art, “identity” also, as there may be, the degree of sequence association between amino acid sequences or nucleic acid sequences, as determined by matching strings of such sequences. The “similarity” between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of the second polypeptide.

[0091] The term “complementarity” is defined herein as the sequence identity of a nucleotide sequence to a fully complementary chain (e.g., a second or reverse chain). For example, a 100% complementary (or fully complementary) sequence is understood herein to have 100% sequence identity with a complementary chain, and for example, an 80% complementary sequence is understood herein to have 80% sequence identity with a (fully) complementary chain.

[0092] "Identity" and "similarity" can be readily calculated by known methods. "Sequence identity" and "sequence similarity" can be determined by aligning two peptide sequences or two nucleotide sequences using global or local alignment algorithms, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Sequences can then be said to be "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (as defined below) (for example, when optimally aligned by the program GAP or BESTFIT using default parameters). GAP uses the Needleman and Wunsch global alignment algorithms to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. Global alignment is appropriately used to determine sequence identity when two sequences have similar lengths. Generally, GAP default parameters are used, with a gap generation penalty of 50 (nucleotides) / 8 (proteins) and a gap elongation penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and sequence identity percentage scores can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open-source software such as EmbossWIN version 2.10.0's "needle" program (using the global Needleman-Wunsch algorithm) or "water" program (using the local Smith-Waterman algorithm), using the same parameters as GAP described above, or using default settings (for both "needle" and "water," and for both protein and DNA alignments, the default gap-open penalty is 10.0 and the default gap-expanding penalty is 0.5; the default scoring matrix is ​​Blosum62 for protein and DNAFull for DNA). Local alignment, such as that using the Smith-Waterman algorithm, is preferred when sequences have substantially different full lengths.

[0093] Alternatively, percentage similarity or identity can be determined by searching public databases using algorithms such as FASTA and BLAST. Therefore, the nucleic acid and protein sequences of the present invention can further be used as "query sequences" for searching public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) from Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. A BLAST protein search can be performed using the BLASTx program, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecule of the present invention. For comparative purposes, gapped alignment can be obtained using Gapped BLAST, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., BLASTx and BLASTn) can be used. Please refer to the National Center for Biotechnology Information website at http: / / www.ncbi.nlm.nih.gov / .

[0094] As used herein, the terms “prevent,” “prevent,” and “prevention” mean the prevention or reduction of the recurrence, onset, onset or progression of a disease, preferably as defined herein below, or the prevention or reduction of the severity and / or duration of a disease or one or more of its symptoms.

[0095] As used herein, the terms “treatment” and “therapy” may mean any protocol, method, and / or agent that can be used in the prevention, treatment, management, or improvement of a disease, preferably a disease as defined herein below, or one or more symptoms thereof.

[0096] As used herein, the terms “to treat,” “to treat,” and “treatment” mean reducing or improving the progression, severity, and / or duration of a disease, preferably as defined herein below, and / or reducing or improving one or more symptoms of the disease.

[0097] As used herein, the term “effective dose” means an amount of a treatment, e.g., a prophylactic or therapeutic agent, preferably a heterobifunctional molecule as defined herein, that is sufficient to reduce the severity and / or duration of a disease, improve one or more of its symptoms, prevent the progression of the disease, or cause regression of the disease, or to prevent the onset, recurrence, initiation or progression of the disease or one or more of its symptoms, or to enhance or improve the prophylactic and / or therapeutic effect of another treatment (e.g., another therapeutic agent). Preferably, the disease is a disease as defined herein below.

[0098] This invention relates to a concept of employing a heterobifunctional molecule for the targeted internalization and subsequent degradation of membrane-bound proteins. The heterobifunctional molecule of this invention can simultaneously bind to a transmembrane ubiquitin ligase and a membrane-bound protein such as a cancer-promoting receptor. Induced proximity of the ubiquitin ligase to the desired target transmembrane protein (i.e., "forced dimerization") results in ubiquitination of the target, followed by removal from the cell surface and subsequent degradation. For example, this can result in impaired proliferation of cancer cells. A schematic diagram of an exemplary embodiment of this invention is provided in Figure 1.

[0099] The advantages of this approach include at least the following: i) The heterobifunctional molecules of the present invention enable increased potency by requiring only a substoichiometric amount of the molecule compared to the target molecule, compared to conventional "occupancy-based" therapies. ii) The required specific binding of two proteins, namely transmembrane E3 ubiquitin ligase and membrane-bound protein, also reduces potential off-target toxicity. Preferably, a ubiquitin ligase that localizes to the cell membrane and shows increased expression in cancer cells is employed. iii) Targeted protein degradation results in long-lasting pharmacodynamic effects because it requires time to synthesize a sufficient amount of new transmembrane protein. iv) Heterobifunctional molecules bind to extracellular protein portions and therefore do not need to pass through the cell membrane. v) Cancer cells are known to richly express several types of transmembrane E3 ubiquitin ligases, such as RNF43 and ZNRF3, in cancer cells with self-regenerative properties. In this example, four alleles produce proteins that exhibit ubiquitination activity, reducing the potential for mutation-induced inactivation and resistance.

[0100] The inventors further discovered that not all membrane-bound proteins can be effectively targeted by any transmembrane E3 ubiquitin ligase; that is, bringing a membrane-bound protein into proximity with a transmembrane E3 ubiquitin ligase does not necessarily result in cell surface removal of the membrane-bound protein. Therefore, for the development of effective heterobifunctional molecules, a screening method should be employed to determine combinations that, when placed near a transmembrane E3 ubiquitin ligase, result in effective internalization of the membrane-bound protein.

[0101] The inventors have discovered an efficient method for screening effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins, such as combinations in which induced proximity ("forced dimerization") of the transmembrane ubiquitin E3 ligase and membrane-bound protein results in cell surface removal of the membrane-bound protein. Using this direct method, effective heterobifunctional molecules can be constructed to target effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins.

[0102] A particular advantage of the method described herein is that it allows for the screening of different combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins using a single heterobifunctional molecule, for example, by using the same first epitope for all transmembrane E3 ubiquitin ligases and the same second epitope for all membrane-bound proteins. This provides an objective method for determining effective combinations without having to consider any variability that may exist between different heterobifunctional molecules, such as variable binding affinity.

[0103] Accordingly, in one embodiment, the present invention relates to a heterobifunctional molecule comprising a first and a second binding domain. The first binding domain can specifically bind to a transmembrane E3 ubiquitin ligase, and the second binding domain can bind to a specific membrane-bound protein. The combination of transmembrane E3 ubiquitin ligase and membrane-bound protein is preferably identified using the screening method described herein.

[0104] The simultaneous binding of a transmembrane E3 ubiquitin ligase to a membrane-bound protein brings these two molecules into close proximity. As a result, the transmembrane E3 ubiquitin ligase can subsequently ubiquitize the membrane-bound protein.

[0105] Therefore, preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein results in ubiquitination of the membrane-bound protein.

[0106] It is known that ubiquitination leads to the degradation of ubiquitinated proteins. Therefore, more preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein leads to the degradation of the membrane-bound protein.

[0107] The simultaneous binding of a transmembrane E3 ubiquitin ligase and a membrane-bound protein brings these two molecules into close proximity. As a result, the membrane-bound protein can be internalized and, preferably, subsequently degraded.

[0108] Screening method Prior to the production of heterobifunctional molecules that bind to natural epitopes, such as heterobifunctional molecules as defined herein for use in the treatment of diseases, it is possible to first identify effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins. Preferably, a combination is considered effective if the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein, preferably by ubiquitination of the membrane-bound protein, when the E3 ligase and membrane-bound protein are in close proximity to each other, i.e., preferably when forced dimerization of the transmembrane E3 ubiquitin ligase and membrane-bound protein is present. Therefore, preferably, a combination is considered effective if the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein, preferably by ubiquitination of the membrane-bound protein, during forced dimerization of the transmembrane E3 ubiquitin ligase and membrane-bound protein. Preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are brought into proximity by simultaneous binding to the heterobifunctional molecule as defined herein. Therefore, preferably, the combination is considered effective if the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein when the transmembrane E3 ubiquitin ligase and the membrane-bound protein heterobifunctional molecule are simultaneously bound to the heterobifunctional molecule, preferably as defined herein.

[0109] The inventors have developed a method for effectively screening suitable combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins, such as combinations that can be effectively targeted by heterobifunctional molecules as defined herein.

[0110] Therefore, in one aspect, the present invention relates to a method for identifying effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins, wherein the combination is effective if the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein. Preferably, the combination is effective if the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein by ubiquitination of the membrane-bound protein, preferably subsequently by internalization of the ubiquitinated membrane-bound protein. The internalized ubiquitinated membrane-bound protein can then be degraded, preferably by lysosomes. Preferably, the method is a) A step of preparing cells that express transmembrane E3 ubiquitin ligase and membrane-bound proteins on their cell surface; b) A step of exposing cells to a heterobifunctional molecule, wherein the heterobifunctional molecule is i) A first binding domain capable of specifically binding to the extracellular portion of transmembrane E3 ubiquitin ligase; and ii) A second binding domain that can specifically bind to the extracellular portion of membrane-bound proteins. Steps including; c) A step to determine the surface level of membrane-bound proteins in cells. The decrease in the surface level of the membrane-bound protein, including the above, indicates that the combination is an effective combination. Preferably, the decrease is a decrease compared to the surface level of the membrane-bound protein of the cell prior to step b). Preferably, the decrease in the protein level is a decrease compared to the protein level of the membrane-bound protein of the same or similar cell that has not been exposed to the heterobifunctional molecule, for example, a decrease compared to the protein level of the membrane-bound protein of the cell provided in step a) of the method of the present invention.

[0111] The present invention further relates to a method for reducing the surface level of membrane-bound proteins in cells. The method is preferably, a) The step of preparing cells that express transmembrane E3 ubiquitin ligase and membrane-bound proteins on their cell surface; and b) Exposing cells to heterobifunctional molecules as defined herein. It includes. Hetero-bifunctional molecules are i) A first binding domain capable of specifically binding to the extracellular portion of transmembrane E3 ubiquitin ligase; and ii) A second binding domain that can specifically bind to the extracellular portion of membrane-bound proteins. Includes.

[0112] The method preferably further comprises step c) determining the surface level of the cell's membrane-bound protein. The decrease is preferably a decrease compared to the surface level of the cell's membrane-bound protein prior to step b). Preferably, the decrease in protein level is a decrease compared to the protein level of the membrane-bound protein of the same or similar cell that has not been exposed to the heterobifunctional molecule, for example, a decrease compared to the protein level of the cell's membrane-bound protein provided in step a) of the method of the present invention.

[0113] This method is preferably an ex vivo method, and preferably an in vitro method.

[0114] Step a) Prepare the cells In step a) of the method of the present invention, cells are prepared. Any cells suitable for the expression of transmembrane E3 ubiquitin ligase and membrane-bound proteins may be used in the method of the present invention. The cells preferably express transmembrane E3 ubiquitin ligase and membrane-bound proteins on their cell surface. Preferably, the cells are immortalized cells, preferably cell lines, preferably cancer cell lines. The cells may be bacterial, yeast, plant, or animal cells. Preferably, the cells are animal cells. Preferred animal cells are vertebrate cells, preferably rodent or primate cells, preferably mouse or human cells. The cells may be cell cultures, cell lines, biopsies, and organoids, or parts thereof. Preferably, the cells are patient-derived tissue, preferably part of cultured patient-derived tissue, or derived therefrom. The cells may be part of a biopsy or organoid, preferably a tumor organoid, or derived therefrom. The biopsy may be an excision biopsy, an incision biopsy, or a core biopsy. The organoid is preferably a cancer organoid. The organoids are preferably patient-derived organoids, and more preferably tumor organoids.

[0115] Preferred cells are human cells, preferably at least one of cancer cells, immune cells, and nerve cells. Preferred cells are human cell lines, preferably human cancer cell lines, human immune cell lines, and / or human nerve cell lines. The cell line may be an immortalized cell line. Preferably, the cells are HEK293T cells.

[0116] The provided cells may express membrane-bound proteins and / or transmembrane E3 ubiquitin ligases at endogenous levels, or may be modified to induce or increase the expression of membrane-bound proteins and / or transmembrane E3 ubiquitin ligases. Furthermore, or alternatively, cells may be modified to express transmembrane E3 ubiquitin ligases containing one or more non-natural epitope tags as defined herein, and / or membrane-bound proteins containing one or more non-natural epitope tags as defined herein. The transmembrane E3 ubiquitin ligase containing (non-natural) epitopes and the membrane-bound proteins containing (non-natural) epitopes are preferably expressed in the same cell.

[0117] The provided cells, preferably cell lines, may express at least one of wild-type or "native" transmembrane E3 ubiquitin ligase and wild-type membrane-bound protein. At least one of wild-type transmembrane E3 ubiquitin ligase and wild-type membrane-bound protein may be overexpressed intracellularly. Cells may transiently overexpress at least one of wild-type transmembrane E3 ubiquitin ligase and wild-type membrane-bound protein. If necessary, at least one of wild-type transmembrane E3 ubiquitin ligase and wild-type membrane-bound protein may be constitutively overexpressed intracellularly.

[0118] Alternatively, or further, the provided cells, preferably cell lines, may express at least one of the engineered transmembrane E3 ubiquitin ligase and engineered membrane-bound proteins. The engineered transmembrane E3 ubiquitin ligase includes a first, and optionally a fourth, non-natural epitope tag as defined herein. The engineered membrane-bound proteins include a second, and optionally a third, non-natural epitope tag as defined herein. The provided cells may transiently overexpress at least one of the engineered transmembrane E3 ubiquitin ligase and engineered membrane-bound proteins. If necessary, the cells may constitutively overexpress at least one of the engineered transmembrane E3 ubiquitin ligase and engineered membrane-bound proteins.

[0119] The expression of at least one of an engineered transmembrane E3 ubiquitin ligase and an engineered membrane-bound protein, and constitutive expression as needed, can be achieved by any conventional means known to those skilled in the art. For example, constitutive expression can be achieved, for example, by incorporating an expression cassette expressing at least one of the (engineered) transmembrane E3 ubiquitin ligase and the (engineered) membrane-bound protein into the cellular genome.

[0120] The expression of transmembrane E3 ubiquitin ligases (including, if necessary, non-natural epitopes) may be regulated by their native promoters or by non-natural promoters, such as, but not limited to, constitutively active promoters. The expression of membrane-bound proteins (including, if necessary, non-natural epitopes) may be regulated by their native promoters or by non-natural promoters, such as, but not limited to, constitutively active promoters.

[0121] Sequences encoding at least one of the following (including, optionally, non-natural epitopes) – a transmembrane E3 ubiquitin ligase and / or membrane-bound protein (including, optionally, non-natural epitopes) – can be introduced into the provided cells for transient or permanent expression. Optionally, the encoding sequence(s) are contained within an expression cassette introduced into the cells. The expression cassette preferably further comprises one or more elements that control the expression of the transmembrane E3 ubiquitin ligase and / or the expression of the membrane-bound protein. Preferred expression elements are natural or non-natural promoters. The expression cassette may also be part of an expression vector. Preferred expression vectors are naked DNA, a DNA complex, or a viral vector. Preferred naked DNA is a linear or circular nucleic acid molecule, e.g., a plasmid. A plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted by standard molecular cloning techniques, for example. A DNA complex can be a DNA molecule bound to any carrier suitable for delivering DNA into cells. Preferred carriers are selected from the group consisting of lipoplexes, liposomes, polymerosomes, polyplexes, viral vectors, dendrimers, inorganic nanoparticles, viromosomes, and transcellular peptides.

[0122] The provided cells may be modified to express, at an endogenous level, a transmembrane E3 ubiquitin ligase containing a non-natural epitope tag(s) and a membrane-bound protein containing a non-natural epitope tag(s). As a non-limiting example, sequences encoding a first, and optionally a fourth, non-natural epitope tag, may be incorporated into the genomic sequence of the provided cells. Furthermore, sequences encoding a second, and optionally a third, non-natural epitope tag, may be incorporated into the genomic sequence of the provided cells.

[0123] Therefore, the genomic sequence of the provided cells encoding a transmembrane E3 ubiquitin ligase can be modified to incorporate sequences encoding a first, and optionally a fourth, non-natural epitope tag. The modified genomic sequence preferably encodes and expresses a transmembrane E3 ubiquitin ligase containing the first, and optionally a fourth, non-natural epitope tag as defined herein. Preferably, in the provided cells, the genomic sequence encoding a membrane-bound protein can be modified to incorporate sequences encoding a second, and optionally a third, non-natural epitope tag. The modified genomic sequence preferably encodes and expresses a membrane-bound protein containing the second, and optionally a third, non-natural epitope tag as defined herein.

[0124] Methods for targeted genome modification to incorporate sequences encoding first, second, and optionally third and fourth non-natural epitope tags are well known to those skilled in the art and include, but are not limited to, site-specific endonucleases that generate double-strand breaks at genomic locations for incorporating sequences encoding first, and optionally fourth non-natural epitope tags, or sequences encoding second, and optionally third non-natural epitope tags. A preferred site-specific nuclease is the CRISPR-Cas system. Preferably, the generated double-strand breaks are specific to a single location in the genome. Alternatively, double-strand breaks may be generated at two or more genomic locations, at least one of which is located within or near a sequence encoding a transmembrane E3 ubiquitin ligase, or within or near a sequence encoding a membrane-bound protein, incorporating sequences encoding the first or second tag, and optionally third and / or fourth tags, respectively.

[0125] Those skilled in the art will readily understand that additional sequences, such as selection cassettes for selecting modified cells, can be incorporated into the genome, though not limited to these. Such selection cassettes are preferably incorporated into intergenetic regions or intronic regions, preferably into introns of transmembrane ubiquitin E3 ligases and / or introns of membrane-bound proteins.

[0126] Therefore, the first, and optionally fourth, non-natural epitope tags can be introduced into the cellular genome by the step of introducing into the cell an oligonucleotide or donor plasmid containing the sequences encoding the first, and optionally fourth tags, i) a site-directed nuclease that generates a double-strand break in or near the sequence encoding the transmembrane E3 ubiquitin ligase, and ii) the sequences encoding the first, and optionally fourth tags. The double-strand break is preferably located such that the mature transmembrane E3 ubiquitin ligase contains the first, and optionally fourth tags. Preferably, the double-strand break is located such that the first, and optionally fourth tags are located between the signal peptide and the mature transmembrane ubiquitin E3 ligase. Preferably, the double-strand break is located such that the first, and optionally fourth tags are located extracellularly. Preferably, the double-strand break is located such that at least one of the first, and optionally fourth tags is located at or near the N-terminus of the mature transmembrane ubiquitin E3 ligase. Alternatively, or further, at least one of the first and, optionally, the fourth tag may be located at or near the C-terminus of the mature transmembrane ubiquitin E3 ligase. Alternatively, or further, at least one of the first and, optionally, the fourth tag may be located in the extracellular loop region of the mature transmembrane ubiquitin E3 ligase.

[0127] Cells expressing a transmembrane ubiquitin ligase containing a first, and optionally a fourth, non-natural epitope tag, can be used in the screening method defined herein. In this embodiment, the heterobifunctional molecule may include a first binding domain for specifically binding to a first non-natural epitope tag, and a second binding domain for binding to a natural epitope present in the wild-type transmembrane protein.

[0128] Preferably, in the same cell, a second, and optionally third, non-native epitope tag can be introduced into the cellular genome by the step of introducing into the cell i) a site-specific nuclease that generates a double-strand break in or near the sequence encoding the membrane-bound protein, and ii) an oligonucleotide or donor plasmid containing the sequence encoding the second, and optionally third, tag. Preferably, the double-strand break is located such that the second, and optionally third, tag is located extracellularly. Preferably, the double-strand break is located such that the mature membrane-bound protein includes the second, and optionally third, tag at its N-terminus. Alternatively, or further, at least one of the second, and optionally third, tags may be located at or near the C-terminus of the membrane-bound protein. Alternatively, or further, at least one of the second, and optionally third, tags may be located in the extracellular loop region of the membrane-bound protein.

[0129] The oligonucleotide or donor plasmid preferably contains sequences for promoting homologous sequence-dependent repair.

[0130] Alternatively, or furthermore, the first, second, and optionally third and fourth, non-natural epitope tags can be introduced into the genome using CRISPR-Cas prime editing technology.

[0131] If necessary, CRISPR technologies such as the CRISPR technology described above can be used to generate suitable controls for methods defined herein, including, but not limited to, the production of transmembrane E3 ubiquitin ligases lacking functional ligase domains.

[0132] Step b) Exposure of cells to heterobifunctional molecules Step b) of exposing cells to the heterobifunctional molecule is preferably under conditions that allow the heterobifunctional molecule to bind simultaneously with the transmembrane E3 ubiquitin ligase and the transmembrane protein. Such conditions are well known to those skilled in the art. As a non-limiting example, the heterobifunctional molecule can be added directly to the cell culture medium.

[0133] The concentration of the heterobifunctional molecule used in step b) of the method of the present invention may vary. For example, the concentration may depend on the epitopes present in the heterobifunctional molecule and / or the transmembrane E3 ubiquitin ligase and / or the membrane-bound protein. The concentration of the heterobifunctional molecule can be experimentally determined using standard techniques. Preferably, the concentration of the heterobifunctional molecule exposed to the cells is about 0.1 nM to 1000 nM, about 0.5 nM to 500 nM, about 5 nM to 100 nM, about 20 nM to 80 nM, or about 40 nM to 60 nM. The concentration of the heterobifunctional molecule is preferably about 50 nM. The heterobifunctional molecule described herein can co-binding to the transmembrane E3 ubiquitin ligase and the membrane-bound protein. The transmembrane E3 ubiquitin ligase is preferably the transmembrane E3 ubiquitin ligase described herein.

[0134] The membrane-bound protein is preferably a transmembrane protein. The transmembrane protein may be at least one of type I, type II, and type III transmembrane proteins. The transmembrane protein may be a so-called "multispan" protein. The transmembrane protein may be any transmembrane protein described herein.

[0135] The duration of exposure of cells to heterobifunctional molecules is preferably at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, or 24 hours, or preferably at least 1, 2, 3, 4, 5, 6, or 7 days.

[0136] At least one of the transmembrane E3 ubiquitin ligase and membrane-bound protein may be a wild-type protein, for example, a protein naturally encoded in the genome and optionally present (expressed) in the provided cell. The transmembrane E3 ubiquitin ligase and membrane-bound protein are preferably expressed in the same cell. Optionally, at least one of the wild-type proteins is overexpressed in the provided cell. Thus, optionally, the wild-type transmembrane E3 ubiquitin ligase for use in the method of the present invention has induced or increased expression in the provided cell. Optionally, the wild-type membrane-bound protein for use in the method of the present invention has induced or increased expression in the provided cell. The heterobifunctional molecule for use in the method of the present invention preferably comprises a first binding domain capable of binding to a (natural) epitope present in the wild-type transmembrane E3 ubiquitin ligase and / or a second binding domain capable of binding to a (natural) epitope present in the wild-type membrane-bound protein, preferably the wild-type transmembrane protein.

[0137] Instead, at least one of the transmembrane E3 ubiquitin ligase and membrane-bound proteins is not a wild-type protein. Preferably, the transmembrane E3 ubiquitin ligase contains a first non-natural epitope tag. Preferably, the first non-natural epitope tag is located in the extracellular portion of the ubiquitin ligase. Therefore, preferably, the first non-natural epitope tag is exposed on the cell surface of the provided cell. Preferably, the first non-natural epitope tag is i) The N-terminus of transmembrane E3 ubiquitin ligase; ii) The C-terminus of transmembrane E3 ubiquitin ligase; and / or ii) Extracellular loop region of transmembrane E3 ubiquitin ligase It is located in or near that location.

[0138] If the transmembrane E3 ubiquitin ligase contains a first non-natural epitope tag, the heterobifunctional molecule preferably contains a first binding domain that selectively binds to the first non-natural epitope tag.

[0139] Preferably, the membrane-bound protein contains a second non-natural epitope tag. Preferably, the second non-natural epitope tag is located in the extracellular portion of the membrane-bound protein. Therefore, preferably, the second non-natural epitope tag is exposed on the cell surface of the provided cell.

[0140] Preferably, the second non-natural epitope tag is i) The N-terminus of a membrane-bound protein; ii) The C-terminus of membrane-bound proteins; and / or iii) Extracellular loop region of membrane-bound proteins It is located in or near that location.

[0141] If the membrane-bound protein contains a second non-natural epitope tag, the heterobifunctional molecule preferably contains a second binding domain that selectively binds to the second non-natural epitope tag.

[0142] In a preferred method of the present invention, cells are exposed to a heterobifunctional molecule, and the heterobifunctional molecule is i) A first binding domain capable of specifically binding to a first non-natural epitope tag located in the extracellular portion of a transmembrane E3 ubiquitin ligase; and ii) A second binding domain capable of specifically binding to a second non-native epitope tag located in the extracellular portion of a membrane-bound protein. Includes.

[0143] "Non-natural epitope tag" is understood herein as an epitope not typically present in wild-type, naturally occurring proteins. The terms "epitope" and "epitope tag" may be used interchangeably herein.

[0144] The first non-natural epitope tag is preferably located in the extracellular portion of the transmembrane E3 ubiquitin ligase. If the transmembrane E3 ubiquitin ligase includes an extracellular N-terminal portion, the first epitope, preferably the first non-natural epitope, is preferably located at or near the original N-terminus of the transmembrane E3 ubiquitin ligase. Optionally, additional amino acid residues are located between the original N-terminus and the first epitope. Optionally, about 10 to 100, 5 to 50, or about 1 to 10 amino acid residues are located between the original N-terminus of the transmembrane E3 ubiquitin ligase and the first, optionally non-natural, epitope. These amino acid residues may be native to the transmembrane E3 ubiquitin ligase, or the transmembrane E3 ubiquitin ligase may be further elongated by these additional amino acid residues.

[0145] If the transmembrane E3 ubiquitin ligase includes an extracellular C-terminal portion, the first epitope, preferably the first non-native epitope, is preferably located at or near the original C-terminus of the transmembrane E3 ubiquitin ligase. Optionally, additional amino acid residues are located between the original C-terminus and the first epitope. Optionally, there may be about 10–100, 5–50, or about 1–10 amino acid residues between the original C-terminus of the transmembrane E3 ubiquitin ligase and the first, optionally non-native, epitope. These amino acid residues may be native to the transmembrane E3 ubiquitin ligase, or the transmembrane E3 ubiquitin ligase may be further elongated by these additional amino acid residues.

[0146] Whether or not the transmembrane E3 ubiquitin ligase contains an extracellular portion at the N-terminus and / or C-terminus, the first, optionally non-native, epitope tag may be located in, or further than, the extracellular loop region of the transmembrane E3 ubiquitin ligase.

[0147] Alternatively, or further, the first non-natural epitope tag may be extended by one or more amino acid residues located adjacent to the intracellular portion of the transmembrane E3 ubiquitin ligase, for example, located at or near the original intracellular N-terminus and / or C-terminus, but not limited to these. In this embodiment, the non-natural epitope tag may be extended by a transmembrane domain, which causes the extracellular expression of the tag, preferably a peptide-tag or protein-tag as defined herein. Non-limiting examples of such extended tags are described in International Publication No. 2012 / 116076 and Brown et al (PLoS One, 2013 Sep 2;8(9):e73255 ("Snorkel Tag")) incorporated herein by reference. The transmembrane domain extending the non-natural epitope tag preferably has an arrangement such as that shown in Figure 1 of Brown et al (opposite). The Snorkel tag preferably has the sequence shown in Figure 1 of Brown et al (cited above).

[0148] The second non-natural epitope tag is preferably located in the extracellular portion of the membrane-bound protein. If the membrane-bound protein includes an extracellular N-terminal portion, the second epitope, preferably the second non-natural epitope, is preferably located at or near the original N-terminus of the membrane-bound protein. Optionally, additional amino acid residues are located between the original N-terminus and the second epitope. Optionally, about 10 to 100, 5 to 50, or about 1 to 10 amino acid residues are located between the original N-terminus of the membrane-bound protein and the second, optionally non-natural epitope. These amino acid residues may be native to the membrane-bound protein, or the membrane-bound protein may be further elongated by these additional amino acid residues.

[0149] If the membrane-bound protein includes an extracellular C-terminal portion, the second epitope, preferably a second non-native epitope, is preferably located at or near the original C-terminus of the membrane-bound protein. Optionally, additional amino acid residues are located between the original C-terminus and the second epitope. Optionally, there may be about 10–100, 5–50, or about 1–10 amino acid residues between the original C-terminus of the membrane-bound protein and the second, optionally non-native, epitope. These amino acid residues may be native to the membrane-bound protein, or the membrane-bound protein may be further elongated by these additional amino acid residues.

[0150] Regardless of whether the membrane-bound protein includes an extracellular portion at the N-terminus and / or C-terminus, a second, optionally non-native epitope tag may be located in, or further than, the extracellular loop region of the membrane-bound protein.

[0151] Alternatively, or further, the second non-natural epitope tag may be extended by one or more amino acid residues located adjacent to the intracellular portion of the membrane-bound protein, for example, located at or near the original intracellular N-terminus and / or C-terminus. In this embodiment, the non-natural epitope tag may be extended by a transmembrane domain, which causes the extracellular expression of the tag, preferably a peptide-tag or protein-tag as defined herein. Non-limiting examples of such extended tags are described in International Publication 2012 / 116076 and Brown et al (PLoS One, 2013 Sep 2;8(9):e73255 ("Snorkel Tag")) incorporated herein by reference. The transmembrane domain extending the tag preferably has the sequence shown in Figure 1 of Brown et al (op.). The Snorkel tag preferably has the sequence shown in Figure 1 of Brown et al (op.).

[0152] The incorporation of non-natural epitope tags into transmembrane E3 ubiquitin ligases and membrane-bound proteins, respectively, can be carried out using any conventional molecular biological techniques known in the art. The first and second epitope tags can be any suitable tags. The epitope tags may be linear or structural epitopes. The tags are preferably peptide tags or protein tags. Preferably, the tags are short amino acid sequences. The length of the first and / or second non-natural epitope tags is preferably about 2–50, 3–40, 4–30, 5–20, or 8–15 amino acid residues. Preferably, the tags are amino acid sequences against which an antibody or antibody fragment, preferably a nanobody, can be induced using any conventional means known to those skilled in the art. The non-natural epitope tags may be publicly available tags or newly discovered sequences. The first and second epitope tags may be the same tag or different tags. Preferably, the first and second epitope tags are different tags.

[0153] The first and / or second non-natural epitope tag may be a peptide tag selected from the group consisting of Alpha tag, E6 tag, V5 tag, VSV tag, Avi tag, C- tag, calmodulin tag, polyglutamate tag, polyarginine tag, E- tag, FLAG- tag, HA- tag, His- tag, Myc- tag, NE- tag, Rho1D4- tag, S- tag, SBP- tag, Sof tag 1, Spot- tag, Strep- tag, T7- tag, TC tag, Ty tag, and Xpress tag.

[0154] The first and / or second non-natural epitope tag may be a protein tag selected from the group consisting of GFP tag (green fluorescent protein), RFP tag (red fluorescent protein), YFP tag (yellow fluorescent protein), BFP tag (blue fluorescent protein), BCCP tag (biotin carboxyl carrier protein), glutathione-S-transferase tag, Halo tag, SNAP tag, CLIP tag, HUH tag, maltose-binding protein tag, Nus tag, thioredoxin tag, Fc tag, carbohydrate recognition domain (CRD), and CRDSAT tag.

[0155] A first and / or second non-natural epitope tag may be extended by one or more amino acid residues that induce extracellular expression of the tag. The portion that induces extracellular expression is preferably a transmembrane domain, preferably a transmembrane domain (TMD), preferably the TMD shown in Figure 1 of Brown et al. (cited above). The transmembrane domain can result in extracellular expression of a protein-tag or peptide-tag as defined herein.

[0156] A transmembrane domain can result in the extracellular expression of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more protein-tags or peptide-tags as defined herein. Preferably, a transmembrane domain can result in the extracellular expression of at least one of the myc-tag, FLAG-tag, Alpha-tag, and E6-tag. Preferably, a transmembrane domain can result in the extracellular expression of at least the E6-tag and FLAG-tag. Alternatively, or further, a transmembrane domain can result in the extracellular expression of at least the alpha-tag and myc-tag.

[0157] Non-natural epitope tags may be selected from the group consisting of Alpha tags, E6 tags, myc tags, FLAG tags, His tags, V5 tags, VSV tags, GFP tags, and RFP tags.

[0158] The first epitope tag may preferably be the Alpha tag described by Goetzke et al (2019, Nature Communications, 10(1), 1-12). Alternatively, the first epitope tag may be the UBC6e tag (E6 tag) described by Ling et al. (2019, Molecular Immunology, 114(July), 513-523). The second epitope tag may preferably be the Alpha tag described by Goetzke et al (cited above). Alternatively, the second epitope tag may be the UBC6e tag (E6 tag) described by Ling et al. (cited above). Preferably, the first tag may be an Alpha tag and the second tag may be an E6 tag. Alternatively, the first tag may be an E6 tag and the second tag may be an Alpha tag.

[0159] Any suitable combination of an epitope tag and a corresponding antibody or antibody fragment that recognizes the epitope may be used in the manner defined herein. Preferred antibody fragments are nanobodies. Thus, any suitable combination of an epitope and a corresponding nanobody that recognizes the epitope can be used in the manner defined herein.

[0160] A person skilled in the art can select a suitable combination of epitope-antibody or antibody fragment. For example, a person skilled in the art can select a suitable combination of epitope-antibody or antibody fragment that is known in the art. Alternatively, or further, the combination of epitope-antibody or antibody fragment may be a newly discovered combination and can be used in the manner defined herein.

[0161] The antibody or antibody fragment may be any suitable antibody or antibody fragment that specifically binds to the first or second epitope tag. A preferred antibody fragment is a nanobody. Preferably, the heterobifunctional molecule for use in the method of the present invention is a bispecific antibody, preferably a bispecific nanobody.

[0162] Preferably, when the first epitope tag is an Alpha tag and the second epitope tag is an E6 tag, the first binding domain of the heterobifunctional molecule may contain anti-Alpha VHH, and the second binding domain may contain anti-E6 VHH. Preferably, when the first epitope tag is an E6 tag and the second epitope tag is an Alpha tag, the first binding domain of the heterobifunctional molecule may contain anti-E6 VHH (Ling et al, cited above), and the second binding domain may contain anti-Alpha VHH (Goetzke et al, cited above). Therefore, preferred epitope tag-binding domain combinations are: i) Alpha tags - anti-Alpha VHH (see, for example, Goetzke et al (cited above)); and ii) E6 tag - Anti-E6 VHH (see, for example, Ling et al.) It is at least one of the following.

[0163] A preferred Alpha tag has at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 96. A preferred E6 tag has at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 97. A preferred anti-Alpha VHH has at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 98. The CDR3 sequence of a preferred anti-E6 VHH has at least approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequence number 99.

[0164] The membrane-bound protein may contain a third non-natural epitope tag. The third non-natural epitope tag may be used to determine the protein level of the membrane-bound protein, preferably to determine at least one of the following: the protein cell surface level, the total protein level, and the intracellular level of the membrane-bound protein. The third non-natural epitope tag is preferably located in the extracellular portion of the membrane-bound protein. Therefore, preferably, the third non-natural epitope tag is exposed on the cell surface of the provided cell. Preferably, the third non-natural epitope tag is i) The N-terminus of a membrane-bound protein; ii) The C-terminus of membrane-bound proteins; and / or iii) Extracellular loop region of membrane-bound proteins It is located in or near that location.

[0165] The location of the third non-natural epitope tag may be as shown above for the second epitope tag. The third non-natural epitope tag may be located at the N-terminus and / or C-terminus of the second (non-natural) epitope tag.

[0166] The transmembrane E3 ubiquitin ligase may contain a fourth non-natural epitope tag. The fourth non-natural epitope tag can be used to determine the protein level of the transmembrane E3 ubiquitin ligase, preferably at least one of the following: the protein cell surface level, the total protein level, and the intracellular level of the transmembrane E3 ubiquitin ligase. The fourth non-natural epitope tag is preferably located in the extracellular portion of the transmembrane E3 ubiquitin ligase. Therefore, preferably, the fourth non-natural epitope tag is exposed on the cell surface of the provided cell. Preferably, the fourth non-natural epitope tag is, i) The N-terminus of transmembrane E3 ubiquitin ligase; ii) The C-terminus of transmembrane E3 ubiquitin ligase; and / or ii) Extracellular loop region of transmembrane E3 ubiquitin ligase It is located in or near that location.

[0167] The location of the fourth non-natural epitope tag may be as shown above for the first epitope tag. The fourth non-natural epitope tag may be located at the N-terminus and / or C-terminus of the first (non-natural) epitope tag.

[0168] Therefore, in one embodiment, the transmembrane E3 ubiquitin ligase may contain first and fourth non-natural epitope tags, and the membrane-bound protein may contain second and third non-natural epitope tags. The third and / or fourth non-natural epitope tags may be any conventional tags known to those skilled in the art for protein detection, such as peptide tags or protein tags.

[0169] If necessary, peptide tags are selected from the group consisting of Alpha tags, E6 tags, V5 tags, VSV tags, Avi tags, C- tags, calmodulin tags, polyglutamate tags, polyarginine tags, E- tags, FLAG- tags, HA- tags, His- tags, Myc- tags, NE- tags, Rho1D4- tags, S- tags, SBP- tags, Sof tags 1, Spot- tags, Strep- tags, T7- tags, TC tags, Ty tags, and Xpress tags. If necessary, protein tags are selected from the group consisting of GFP tags (green fluorescent protein), RFP tags (red fluorescent protein), YFP tags (yellow fluorescent protein), BFP tags (blue fluorescent protein), BCCP tags (biotin carboxyl carrier protein), glutathione-S-transferase tags, Halo tags, SNAP tags, CLIP tags, HUH tags, maltose-binding protein tags, Nus tags, thioredoxin tags, Fc tags, carbohydrate recognition domains (CRDs), and CRDSAT tags.

[0170] A third non-natural epitope tag may be selected from the group consisting of myc-tag, his-tag, FLAG-tag, V5-tag, VSV-tag, HA-tag, GFP, and RFP. A fourth non-natural epitope tag may be selected from the group consisting of myc-tag, his-tag, FLAG-tag, V5-tag, VSV-tag, HA-tag, GFP, and RFP.

[0171] The preferred combinations of the first and fourth tags are myc- tag and Alpha tag, myc- tag and E6 tag, FLAG tag and Alpha tag, or FLAG tag and E6 tag. The preferred combinations of the second and third tags are Flag tag and E6 tag, FLAG tag and Alpha tag, myc tag and E6 tag, or myc tag and Alpha tag.

[0172] A third and / or fourth non-natural epitope tag may further include a portion that causes extracellular expression of the tag. The portion that causes extracellular expression is preferably a transmembrane domain, preferably a transmembrane domain (TMD), preferably a TMD as shown in Figure 1 of Brown et al. (cited above). The tag expressed extracellularly is preferably a peptide-tag or protein-tag as defined herein.

[0173] Step c): Determining the surface level of membrane-bound proteins The level or amount of membrane-bound proteins may be determined using any conventional means known to those skilled in the art. Such means include, but are not limited to, microscopy, Western blotting, quantitative immunofluorescence and / or quantitative Western blotting as needed, cell surface biotinylation, FACS analysis, labeling of membrane-bound proteins with cell-impermeable fluorescent probes (e.g., SNAP labeling), and quantitative mass spectrometry.

[0174] The absolute amount or level of membrane-bound proteins can be determined, for example, by directly comparing the levels of membrane-bound proteins before and after exposure to heterobifunctional molecules, or by determining the fluorescence intensity on the cell surface before and after exposure, for example, by fluorescence microscopy or FACS analysis.

[0175] For microscopic analysis, membrane-bound proteins localized on the surface can be labeled using fluorescent labels for membrane-bound proteins, preferably in impermeable cells. The total number of cells can be determined, for example, by staining the cell nuclei with DAPI or a similar dye. The average fluorescence intensity values ​​from multiple images taken at the same magnification, laser power, optical settings, and exposure time can be analyzed in ImageJ or a similar analysis program. Each image is preferably normalized to the number of stained nuclei, for example, to its own DAPI value, and the resulting value represents the relative amount of membrane-bound proteins on the surface per cell.

[0176] Alternatively, or even further, a dual labeling of membrane-bound proteins can be used by first using a first epitope tag to fluorescently label surface-localized membrane-bound proteins in impermeable cells, and then using a second epitope tag to fluorescently label total membrane-bound proteins in permeable cells. For microscopic analysis, fluorescence intensity values ​​from multiple images taken at the same magnification, laser power, optical settings, and exposure time can be analyzed in ImageJ or a similar analysis program. Background levels from untransfected cells are preferably subtracted for each image, and intensity values ​​are preferably averaged over each condition. The ratio of the average fluorescence intensity of surface-localized proteins to the average fluorescence intensity of total (membrane-bound) proteins is a measure of the relative amount of membrane-bound proteins on the surface (e.g., Stueber et al, ACS Chem Biol, 2019, 14(6):1154-1163).

[0177] Alternatively, or furthermore, for analyses using flow cytometry, dead cells and non-cellular components are preferably excluded by forward and side scattering plots, as well as based on negative DNA staining. Subsequently, doublet cells are preferably excluded using forward and side scattering region versus height. The fluorescence signal can be quantified based on a set threshold, for example, using FACSdiva, FlowJo, or a similar analysis program, based on the fluorescence intensity determined for untransfected cells. The ratio of the mean fluorescence intensity of surface-localized proteins to the mean fluorescence intensity of total (membrane-bound) proteins is a measure of the relative amount of membrane-bound proteins on the surface (Stueber et al., op. cit.).

[0178] Alternatively, or even further, the surface level of membrane-bound proteins can be determined based on the mature versus immature form of the protein. Membrane-bound proteins may undergo complex glycosylation and further post-translational modifications during biosynthesis, resulting in different molecular weights (MWs) when visualized, for example by Western blotting, with higher molecular weights (MWs) representing the mature form present on the cell surface. The average pixel intensity of the bands representing mature and immature membrane-bound proteins can be determined by Western blotting analysis software such as ImageQuant (Zeiss), ImageJ, or similar programs. The ratio of the intensity of mature form to the intensity of the total amount of membrane-bound protein is a measure of the relative amount of membrane-bound protein on the surface (e.g., described by Koo et al 2021, Nature, 2012;488(7413):665-9).

[0179] Alternatively, or even further, the relative levels or amounts of membrane-bound proteins can be determined, for example, by comparing them to household protein levels or total cellular protein levels before and after exposure using the Western blot analysis described above.

[0180] Alternatively, or in addition to the above, the surface level of membrane-bound proteins can be determined by surface biotinylation. For this purpose, surface proteins are labeled with cell-impermeable biotin, and then all surface proteins are isolated by streptavidin pull-down. Subsequently, the pool of total protein and surface-localized proteins is analyzed, for example, using Western blotting. The ratio of the intensity of membrane-bound proteins in the surface pool to the intensity of membrane-bound proteins in the total protein pool, determined by analytical software such as ImageQuant (Zeiss), ImageJ, or a similar program, is a measure of the relative amount of membrane-bound proteins on the surface (Dubey et al, Elife, 2020;9:e54469; Hao et al, Nature 2012; 485(7397): 195-200).

[0181] Alternatively, or even further, the reduction in the protein level of a membrane-bound protein can be determined by comparing different combinations of membrane-bound proteins and transmembrane ubiquitin E3 ligases. As a non-limiting example, the protein level determined after forced dimerization of a membrane-bound protein with a first transmembrane ubiquitin E3 ligase can be compared to the protein level determined after forced dimerization of the same membrane-bound protein with a second transmembrane ubiquitin E3 ligase. Such a method allows for the determination of the most effective combination of membrane-bound proteins and transmembrane ubiquitin E3 ligases, for example, the transmembrane ubiquitin E3 ligase that causes the most potent reduction in the surface level of the membrane-bound protein during forced dimerization.

[0182] Transmembrane ubiquitin E3 ligases that cause the most potent reduction in the surface level of membrane-bound proteins during forced dimerization are preferably those that reduce the surface level by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to transmembrane ubiquitin E3 ligases that cause the weakest reduction in the surface level of membrane-bound proteins during forced dimerization or do not cause any reduction at all.

[0183] The method of the present invention can be carried out directly in a multiplexed manner. As a non-limiting example, a first cell can be brought into contact with a first heterobifunctional molecule, and a second cell can be brought into contact with a second heterobifunctional molecule. The first and second cells are preferably physically separated, for example, by maintaining them in separate wells. The first and second cells may have the same genetic background. The first and second cells may be the same cell.

[0184] A first cell can be exposed to a first heterobifunctional molecule, and a second cell can be exposed to a second heterobifunctional molecule. The first and second heterobifunctional molecules may differ in their first binding domain and / or their second binding domain.

[0185] The first and second heterobifunctional molecules may differ in their first and second binding domains.

[0186] The first and second heterobifunctional molecules may differ in their first binding domains, but not in their second binding domains. The first binding domain of the first heterobifunctional molecule may selectively bind to a native or unnatural epitope tag located on the first transmembrane E3 ubiquitin ligase. The first binding domain of the second heterobifunctional molecule may selectively bind to a native or unnatural epitope tag located on the second transmembrane E3 ubiquitin ligase. The second binding domains of the first and second heterobifunctional molecules may selectively bind to a native or unnatural epitope tag located on a membrane-bound protein. This allows for a simple approach to determine the transmembrane ubiquitin E3 ligase that causes the most potent, i.e., most effective reduction at the surface level of the membrane-bound protein during forced dimerization.

[0187] The first and second heterobifunctional molecules may differ in their second binding domains, but not in their first binding domains. The second binding domain of the first heterobifunctional molecule may selectively bind to a native or unnatural epitope tag located on a membrane-bound protein. The second binding domain of the second heterobifunctional molecule may selectively bind to a native or unnatural epitope tag located on a second membrane-bound protein. The first binding domains of the first and second heterobifunctional molecules preferably bind to a native or unnatural epitope tag located on a transmembrane E3 ubiquitin ligase. This allows for a direct approach to determining the membrane-bound protein that is effectively reduced during forced dimerization by a certain transmembrane E3 ubiquitin ligase.

[0188] Instead, the first and second heterobifunctional molecules are identical in their first and second binding domains, but the first cell expresses a first transmembrane ubiquitin E3 ligase containing a non-natural epitope tag to which the first binding domain of the heterobifunctional molecule can bind, and the second cell expresses a second transmembrane ubiquitin E3 ligase containing the same non-natural epitope tag. The first and second cells preferably express membrane-bound proteins containing a native or non-natural epitope tag to which the second binding domain of the heterobifunctional molecule can bind. The first and second transmembrane ubiquitin E3 ligases are preferably two different transmembrane ubiquitin E3 ligases. The first transmembrane ubiquitin E3 ligase is preferably selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128. The second transmembrane ubiquitin E3 ligase is preferably selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128.

[0189] Instead, the first and second heterobifunctional molecules are identical in their first and second binding domains, but the first cell expresses a first membrane-bound protein containing a non-native epitope tag to which the second binding domain of the heterobifunctional molecule can bind, and the second cell expresses a second membrane-bound protein containing the same non-native epitope tag. The first and second cells preferably express a transmembrane E3 ubiquitin ligase containing a native or non-native epitope tag to which the first binding domain of the heterobifunctional molecule can bind. The first and second membrane-bound proteins are preferably two different membrane-bound proteins.

[0190] Those skilled in the art will readily understand that the first and second cells can be easily extended to a third, fourth, fifth cell, and so on. Similarly, the first and second heterobifunctional molecules can be easily extended to a third, fourth, fifth heterobifunctional molecule, and so on.

[0191] Preferably, the method of the present invention includes step a) preparing first and second cells, - The first cell expresses the first transmembrane E3 ubiquitin ligase and the first membrane-bound protein on its cell surface; and - The second cell expresses the second transmembrane E3 ubiquitin ligase and the first membrane-bound protein on its cell surface. The first and second transmembrane E3 ubiquitin ligases are different ligases containing the same first extracellular non-native epitope tag; In step b), the first and second cells are exposed to the heterobifunctional molecule, and the heterobifunctional molecule is i) A first binding domain capable of specifically binding to a first non-natural epitope tag; and ii) A second binding domain that can specifically bind to the extracellular portion of a membrane-bound protein, preferably to a second non-natural epitope tag. Includes, In step c), the surface levels of membrane-bound proteins in the first and second cells are determined. The combination is preferably effective if the cell surface level of membrane-bound proteins in the first cell is reduced by at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% compared to the cell surface level of membrane-bound proteins in the second cell after step b). An effective combination is the combination of the first transmembrane E3 ubiquitin ligase and the membrane-bound protein.

[0192] When simultaneously bound to a heterobifunctional molecule, one combination of transmembrane E3 ubiquitin ligase and membrane-bound protein preferably results in a more potent reduction in the surface level of the membrane-bound protein than a combination of another transmembrane E3 ubiquitin ligase and the (identical) membrane-bound protein. This more effective combination is shown herein as the combination in “first cell”. However, those skilled in the art will readily understand that this method concerns differences between cells. For example, it is equally reasonable that a combination is effective if the cell surface level of membrane-bound protein in second cell is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% compared to the cell surface level of membrane-bound protein in first cell after step b). In this scenario, the second cell therefore contains the effective combination.

[0193] Preferably, a third, fourth, or further cell is provided, each expressing a third, fourth, or further transmembrane E3 ubiquitin ligase and a first membrane-bound protein on its cell surface, wherein the transmembrane E3 ubiquitin ligases are different ligases containing the same first extracellular non-natural epitope tag, and the combination is effective when the cell surface level of the membrane-bound protein in the first cell is reduced by at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% compared to the cell surface level of the membrane-bound protein in the second, third, fourth, and further cells after step b). Preferably, the method is carried out in a multiplexed manner.

[0194] Therefore, the most effective combination can be determined using the method of the present invention. The most effective combination is preferably a combination of transmembrane E3 ubiquitin ligase and membrane-bound protein, which results in the most potent reduction of the cell surface level of the membrane-bound protein upon co-binding to the heterobifunctional molecule. Preferably, the most effective combination is one that results in a reduction of at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least about 95% of the cell surface level of the membrane-bound protein compared to all other tested combinations. The most effective combination of a particular membrane-bound protein is preferably a transmembrane E3 ubiquitin ligase that most efficiently mediates the removal of this membrane-bound protein from the cell surface compared to other tested transmembrane E3 ubiquitin ligases upon co-binding of the transmembrane E3 ubiquitin ligase and membrane-bound protein to the heterobifunctional molecule.

[0195] Furthermore, those skilled in the art will readily understand that the methods detailed herein are not limited by these embodiments and that variations constitute equivalent parts of the present invention.

[0196] The protein levels of membrane-bound proteins can be determined before and after exposure to heterobifunctional molecules as defined herein.

[0197] The protein levels of membrane-bound proteins can be determined after exposing cells to heterobifunctional molecules for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, or 24 hours, or preferably at least 1, 2, 3, 4, 5, 6, or 7 days.

[0198] Preferably, the cell surface level of membrane-bound proteins is reduced compared to the cell surface level of membrane-bound proteins in the same cells that have not been exposed to the heterobifunctional molecule, for example, the cells provided in step a) of the method of the present invention.

[0199] The terms “(protein) level” and “(protein) quantity” may be used interchangeably herein. Preferably, the cell surface level is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% compared to the cell surface level of membrane-bound proteins prior to step b) of the method of the present invention. herein, a 100% reduction is understood to indicate that the transmembrane protein is no longer detectable on the cell surface. The reduction in the cell surface protein level may be determined by directly determining the level, or quantity, of the protein remaining on the cell surface after exposure to the heterobifunctional molecule. A preferred method for detecting the level of membrane-bound proteins at the cell surface level is immunofluorescence, preferably quantitative immunofluorescence.

[0200] Transmembrane ubiquitin E3 ligases preferably ubiquitinate membrane-bound proteins. Therefore, instead or further, a decrease in the surface level of membrane-bound proteins can be determined by determining the level of ubiquitination of the membrane-bound proteins. An increase in ubiquitination levels preferably correlates inversely with the cell surface level of membrane-bound proteins. Methods for determining the ability of a transmembrane E3 ubiquitin ligase to ubiquitinate membrane-bound proteins include, but are not limited to, immunoprecipitation of membrane-bound proteins via a third epitope tag and determination of the bound ubiquitin molecule using an anti-ubiquitin antibody. Alternatively or further, tagged, preferably His-tagged, ubiquitin can be co-expressed with membrane-bound proteins, and the level of ubiquitination can be determined using an anti-tagged antibody, preferably an anti-His-tagged antibody. Alternatively, or instead, proteomics approaches can be used to identify and quantify ubiquitin chains (as outlined in Fulzele and Bennett 2018 Methods Mol Biol).

[0201] Ubiquitination preferably results in the internalization and degradation of ubiquitinated membrane-bound proteins. Therefore, instead, or even further, the reduction in the surface level of membrane-bound proteins may be determined by determining the total protein level, or amount, of the membrane-bound proteins in the cell after exposure to the heterobifunctional molecule, for example, after step b). The total protein level of the cell is preferably reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% compared to the total protein level of the membrane-bound proteins in the cell before step b) of the method of the present invention. Hereinafter, a 100% reduction is understood to indicate that the transmembrane protein is no longer detectable in the cell. Methods for determining total protein levels are well known and may include, for example, standard biochemical analysis and FACS.

[0202] Alternatively, or further, a decrease in the cell surface level of membrane-bound proteins may be determined by determining an increase in the intracellular localization of membrane-bound proteins, preferably an increase in the endosomal localization of membrane-bound proteins. Ubiquitination of membrane-bound proteins preferably results in internalization of membrane-bound proteins. Internalized proteins may then be degraded, preferably in lysosomes. To determine the increase in the intracellular protein localization of a protein, the method may include a step of inhibiting lysosomal turnover by treating cells with bafilomycin A1, for example, but not limited to, prior to the step of determining the increase in the intracellular localization of membrane-bound proteins. Preferably, the intracellular localization of membrane-bound proteins increases by at least about 1.5, 2, 3, 4, 5, 6 or more times compared to the intracellular localization of membrane-bound proteins prior to step b) of the method of the present invention.

[0203] Alternatively, or further, a reporter assay and / or downstream signaling readout can be used to determine a decrease in cell surface levels of membrane-bound proteins. Such reporter assays and / or downstream signaling readouts are known in the art and readily available for use in the methods of the present invention.

[0204] Alternatively, or furthermore, the co-localization of membrane-bound proteins and one or more lysosomal markers can be determined using standard techniques. The level of co-localization is preferably inversely correlated with the cell surface level of the membrane-bound proteins.

[0205] The method defined herein may also be considered a method for selecting a combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein. Preferably, the method defined herein is a method for selecting an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein. Preferably, the combination is effective when, in close proximity, the transmembrane E3 ubiquitin ligase can ubiquitinize the membrane-bound protein. Ubiquitination of the membrane-bound protein preferably results in the internalization of the protein. Preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are in close proximity by simultaneous binding to a heterobifunctional molecule as defined herein. The methods of the present invention as defined herein can also be considered as methods for determining the efficiency or efficacy of heterobifunctional molecules, preferably heterobifunctional molecules as defined herein, for reducing the surface level of membrane-bound proteins in cells. The methods preferably include steps outlined above. Preferably, the methods include a) A step of preparing cells that express transmembrane E3 ubiquitin ligase and membrane-bound proteins on their cell surface; and b) Exposing cells to a heterobifunctional molecule, preferably a heterobifunctional molecule as defined herein. Includes.

[0206] The method preferably further comprises step c) determining the surface level of membrane-bound proteins in the cell. The decrease is preferably a decrease compared to the surface level of membrane-bound proteins in the cell prior to step b). The methods defined herein are also, - A method for selecting an effective combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein, preferably a transmembrane protein, wherein the combination is selected when the protein level of the transmembrane protein decreases after step c); - A method for screening effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins, preferably transmembrane proteins; - A method for producing a heterobifunctional molecule as defined herein, wherein the heterobifunctional molecule selectively binds to a selected combination of a transmembrane E3 ubiquitin ligase and a membrane-bound protein, preferably a transmembrane protein; - A method for determining the ability of a transmembrane E3 ubiquitin ligase to ubiquitinate membrane-bound proteins, wherein if the protein level of the transmembrane protein decreases after step c), the transmembrane E3 ligase can ubiquitinate the membrane-bound protein; - Methods for targeting membrane-bound proteins for degradation by heterobifunctional molecules; - A method for determining the ubiquitination of a membrane-bound protein, wherein a decrease in the surface level of the membrane-bound protein indicates ubiquitination of the membrane-bound protein, and the decrease is preferably a decrease compared to the surface level of the membrane-bound protein of the cell prior to step b), It can be considered to be at least one of these.

[0207] As described above in this specification, the methods defined herein can be used to produce effective heterobifunctional molecules, such as effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins, for example, heterobifunctional molecules targeting these molecules. Accordingly, the present invention also relates to heterobifunctional molecules, preferably heterobifunctional molecules as defined herein, wherein the transmembrane E3 ubiquitin ligases and membrane-bound proteins to which the heterobifunctional molecules selectively bind are selected using the methods defined above herein, preferably the selection methods. The method preferably comprises the following steps: a) A step of preparing cells that express transmembrane E3 ubiquitin ligase and membrane-bound proteins on their cell surface, - Transmembrane E3 ubiquitin ligase contains a first non-natural epitope tag in its extracellular portion; - Membrane-bound proteins include a second non-natural epitope tag in their extracellular portion, and; b) A step of exposing cells to a heterobifunctional molecule, wherein the heterobifunctional molecule is - A first binding domain capable of specifically binding to a first non-natural epitope tag; and - A second binding domain capable of binding to a second non-natural epitope tag. Steps including; c) A step of determining the surface level of membrane-bound proteins in cells; d) A step of selecting transmembrane E3 ubiquitin ligases and transmembrane proteins in which the surface level of the membrane-bound proteins has decreased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about 100%, wherein the decrease is a decrease compared to the surface level of the membrane-bound proteins of the cell prior to step b), and Includes.

[0208] In one embodiment, the present invention relates to a transmembrane E3 ubiquitin ligase comprising a first and optionally fourth non-natural epitope tag as defined herein.

[0209] In another embodiment, the present invention relates to membrane-bound proteins comprising a second, and optionally third, non-natural epitope tag as defined herein.

[0210] In one aspect, the present invention is - Transmembrane E3 ubiquitin ligases comprising a first and optionally fourth non-natural epitope tag as defined herein; and - Membrane-bound proteins comprising a second, and optionally a third, non-natural epitope tag as defined herein, It relates to the combination of these. In one embodiment, the present invention relates to a host cell expressing a transmembrane E3 ubiquitin ligase containing a first, and optionally a fourth, non-natural epitope tag as defined herein. The host cell may preferably further express a membrane-bound protein containing a second, and optionally a third, non-natural epitope tag as defined herein. A preferred host cell of the present invention is the cell provided in step a) of the method of the present invention.

[0211] Transmembrane E3 ubiquitin ligase that binds to the first binding domain The present invention further relates to heterobifunctional molecules for use in the methods of the present invention. Furthermore, the present invention relates to heterobifunctional molecules that target effective combinations of transmembrane E3 ubiquitin ligases and membrane-bound proteins, preferably as specified by the methods of the present invention.

[0212] The heterobifunctional molecule of the present invention comprises a first binding domain that can specifically bind to the extracellular portion of a transmembrane E3 ubiquitin ligase, and a second binding domain that can specifically bind to the extracellular portion of a membrane-bound protein.

[0213] The first binding domain of the heterobifunctional molecule can specifically bind to a transmembrane E3 ubiquitin ligase. Preferably, the transmembrane E3 ubiquitin ligase contains a natural epitope to which the heterobifunctional molecule, as defined herein, can specifically bind when the heterobifunctional molecule is used as a pharmaceutical. Alternatively, the transmembrane E3 ubiquitin ligase may be engineered to contain a non-natural epitope, preferably a non-natural epitope as defined herein. The non-natural epitope can preferably specifically bind to the heterobifunctional molecule when the heterobifunctional molecule is used in the method of the present invention, preferably in a selected method of the present invention.

[0214] Transmembrane E3 ubiquitin ligases can mediate the ubiquitination and endocytosis of membrane-bound proteins, that is, they can mediate the ubiquitination and endocytosis of proteins bound by the second binding domain of heterobifunctional molecules as defined herein.

[0215] Ubiquitination and endocytosis of the substrate preferably remove the substrate from the cell surface. The internalized substrate can then be degraded. Therefore, preferably, transmembrane E3 ubiquitin ligases can mediate ubiquitination, cell surface removal, and degradation of membrane-bound proteins, i.e., they can mediate ubiquitination, cell surface removal, and degradation of proteins bound by the second binding domain of a heterobifunctional molecule as defined herein.

[0216] Therefore, preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein leads to the internalization of the membrane-bound protein, thereby removing the membrane-bound protein from the cell surface.

[0217] Preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein results in the internalization and degradation of the membrane-bound protein. Therefore, preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are expressed in the same cell. If necessary, at least one of the transmembrane E3 ubiquitin ligase and the membrane-bound protein may be overexpressed in the cell.

[0218] Ubiquitination and degradation can be evaluated using any suitable method known in the art. As a non-limiting example, ubiquitination and degradation can be evaluated as described in Koo et al, Nature (2012) (cited above), which is incorporated herein by reference.

[0219] Substrate proteins are selected for ubiquitin-mediated modification of lysine residues via interaction with an E3 ligase protein that recruits a ubiquitin-charged E2 enzyme (Clague MJ and Urbe S (2010), Cell; 43(5):682-5). This can result in the transfer of a single ubiquitin molecule to the substrate (monoubiquitination) or the coupling of further ubiquitin molecules to the previous ubiquitin molecule via, for example, a lysine residue present in the previous ubiquitin molecule, thereby forming a chain. The seven lysine residues of ubiquitin offer the formation of different isopeptide chain links with different three-dimensional structures, all of which are present in eukaryotic cells (Xu et al. (2009), Cell 137, 133-145). The specific combination of E2 and E3 enzymes recruited to the substrate determines the type of chain linkage.

[0220] In particular, lysosomal degradation sometimes requires substrate proteins with different ubiquitination patterns than proteasomal degradation. For example, substrates labeled with lysine 48 (Lys48)-linked polyubiquitin chains often result in proteasomal targets. Alternatively, substrates labeled with monoubiquitin, multiubiquitin, Lys11, Lys29, Lys48-linked, or Lys63-linked polyubiquitin are directed to lysosomes.

[0221] The degradation mediated by transmembrane E3 ubiquitin ligase can be at least one of lysosomal degradation and proteasomal degradation. Preferably, the degradation mediated by transmembrane E3 ubiquitin ligase is at least lysosomal degradation.

[0222] Therefore, preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein results in the internalization of the membrane-bound protein. Preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein results in the internalization of the membrane-bound protein, as well as at least one of proteasomal and lysosomal degradation. Preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein results in the internalization and lysosomal degradation of the membrane-bound protein.

[0223] Preferably, the transmembrane E3 ubiquitin ligase ubiquitinates membrane-bound proteins with monoubiquitin, multiubiquitin, Lys11, Lys29, Lys48, or Lys63-linked polyubiquitin chains. Preferably, the transmembrane E3 ubiquitin ligase polyubiquitinates membrane-bound proteins with monoubiquitin, multiubiquitin, or Lys63-linked polyubiquitin chains. Preferably, the transmembrane E3 ubiquitin ligase polyubiquitinates membrane-bound proteins with at least one of Lys11, Lys29, Lys48, and Lys63-linked polyubiquitin chains. Preferably, the transmembrane E3 ubiquitin ligase polyubiquitinates membrane-bound proteins with Lys63-linked polyubiquitin chains.

[0224] Many transmembrane E3 ubiquitin ligases exhibit tissue-specific expression or overexpression in one or more cancer types. Therefore, preferably, transmembrane E3 ubiquitin ligases capable of binding to the heterobifunctional molecules defined herein are those expressed in selective tissues. As an unspecified example, transmembrane E3 ubiquitin ligases RNF43 and ZNRF3 are selectively expressed in adult stem cell populations of multiple tissues, including the intestine, but are not limited to these examples. Even more unspecified examples include transmembrane E3 ubiquitin ligases MARCH1 and MARCH9, which show increased expression in immune cells.

[0225] Preferably, the transmembrane E3 ubiquitin ligase is expressed only in selective tissues, such as cancerous tissue, but is not limited to these.

[0226] Alternatively, or furthermore, transmembrane E3 ubiquitin ligases that can bind to heterobifunctional molecules as defined herein are transmembrane E3 ubiquitin ligases that exhibit expression, preferably overexpression, in one or more types of cancer.

[0227] Preferably, the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128. Preferably, the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF145, RNFT1, RNF130, and RNF128. Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43, RNF167, RNF128, and RNF130. Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43, RNF128, and RNF167. Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43 and RNF128.

[0228] Transmembrane E3 ubiquitin ligases can be overexpressed. As a non-limiting example, it is known in the art that RNF43, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF167, RNF130, and RNF128 show increased expression in cancer.

[0229] In one embodiment, the heterobifunctional molecule comprises a first and a second binding domain, i) The first binding domain can specifically bind to transmembrane E3 ubiquitin ligase, which is expressed in cancer tissue, preferably selectively or overexpressed; ii) The second binding domain can specifically bind to membrane-bound proteins, and the membrane-bound proteins are known to be involved in or are expected to be involved in the aforementioned cancer tissue. Simultaneous binding of a transmembrane E3 ubiquitin ligase and a hetero-bifunctional molecule to a membrane-bound protein preferably results in ubiquitination and internalization of the transmembrane protein.

[0230] Preferably, the transmembrane E3 ubiquitin ligase and the membrane-bound protein are expressed in the same cell, preferably in the same cancerous cell.

[0231] Preferably, both the E3 ubiquitin ligase and the membrane-bound protein are expressed in cancerous cells selected from the group consisting of lung cancer, colorectal cancer, hepatocellular carcinoma, osteosarcoma, pancreatic cancer, gastric cancer, liver cancer, skin cancer, breast cancer, bladder cancer, ovarian cancer, esophageal cancer, thyroid cancer, cervical cancer, glioblastoma, squamous cell carcinoma, prostate cancer (gene expression atlas) and intestinal cancer and / or their metastases.

[0232] Preferably, the membrane-bound protein is a transmembrane protein.

[0233] In one embodiment, the hetero-bifunctional molecule comprises a first and a second binding domain, i) the first binding domain can specifically bind to a transmembrane E3 ubiquitin ligase, which is expressed in immune cells, preferably selectively expressed or overexpressed; ii) the second binding domain can specifically bind to a membrane-bound protein, which is expressed in the same immune cells; Simultaneous binding of a transmembrane E3 ubiquitin ligase and a hetero-bifunctional molecule to a membrane-bound protein preferably results in ubiquitination and internalization of the membrane-bound protein.

[0234] Preferably, the membrane-bound protein is a transmembrane protein.

[0235] In one embodiment, the hetero-bifunctional molecule comprises a first and a second binding domain, i) The first binding domain can specifically bind to a transmembrane E3 ubiquitin ligase, which is expressed, preferably selectively expressed, or overexpressed in nerve cells; and ii) The second binding domain can specifically bind to a membrane-bound protein, which is expressed in the same nerve cell, and the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the membrane-bound protein preferably results in ubiquitination and internalization of the membrane-bound protein.

[0236] Preferably, the membrane-bound protein is a transmembrane protein.

[0237] Preferably, the transmembrane E3 ubiquitin ligase has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29.

[0238] Preferably, the transmembrane E3 ubiquitin ligase is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30.

[0239] Preferably, the transmembrane E3 ubiquitin ligase is at least one of RNF43 and ZNRF3. The proteins RNF43 and ZNRF3 preferably have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 1 and 3, respectively. Preferably, the RNF43 and ZNRF3 proteins are encoded by sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 2 and 4, respectively.

[0240] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF43. Preferably, the RNF43 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 1. Preferably, the RNF43 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 2.

[0241] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is ZNRF3. Preferably, the ZNRF3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 3. Preferably, the ZNRF3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 4.

[0242] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF13. Preferably, the RNF13 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 5. Preferably, the RNF13 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6.

[0243] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is AMFR. Preferably, the AMFR protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7 (or SEQ ID NO: 51). Preferably, the AMFR protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8 (or SEQ ID NO: 52).

[0244] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH1. Preferably, the MARCH1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. Preferably, the MARCH1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10.

[0245] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH4. Preferably, the MARCH4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. Preferably, the MARCH4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0246] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH2. Preferably, the MARCH2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 13. Preferably, the MARCH2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 14.

[0247] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH8. Preferably, the MARCH8 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 15. Preferably, the MARCH8 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16.

[0248] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH9. Preferably, the MARCH9 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 17. Preferably, the MARCH9 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 18.

[0249] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF149. Preferably, the RNF149 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. Preferably, the RNF149 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.

[0250] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF145. Preferably, the RNF145 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 21. Preferably, the RNF145 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22.

[0251] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNFT1. Preferably, the RNFT1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23. Preferably, the RNFT1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 24.

[0252] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF167. Preferably, the RNF167 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25. Preferably, the RNF167 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26.

[0253] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF130. Preferably, the RNF130 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27. Preferably, the RNF130 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 28.

[0254] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF128. Preferably, the RNF128 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 29. Preferably, the RNF128 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 30.

[0255] Protein that binds to the second binding domain As detailed herein, the heterobifunctional molecule of the present invention has a first binding domain that can bind to a transmembrane E3 ubiquitin ligase, preferably the transmembrane E3 ubiquitin ligase defined above herein.

[0256] The heterobifunctional molecule of the present invention further comprises a second binding domain, which can bind to a membrane-bound protein. Preferably, the membrane-bound protein preferably comprises a native epitope to which the heterobifunctional molecule defined herein can specifically bind when the heterobifunctional molecule is used as a medicament. Alternatively, the membrane-bound protein can be engineered to comprise a non-native epitope, preferably a non-native epitope defined herein. The non-native epitope preferably allows the heterobifunctional molecule to specifically bind when the heterobifunctional molecule is used in the methods of the present invention, preferably in the selection methods of the present invention.

[0257] Preferably, the protein that can bind to the second binding domain of the heterobifunctional molecule is a protein that is at least partially exposed to the outside of the cell. The protein can be attached to the cell membrane from one side or can span across the entire membrane, i.e., is a transmembrane protein. Preferably, the second binding domain can specifically bind to a transmembrane protein.

[0258] As the term "hetero-bifunctional" already suggests, a membrane-bound protein capable of binding to a second binding domain is different from a transmembrane E3 ubiquitin ligase capable of binding to a first binding domain. Preferably, the second binding domain does not bind specifically and / or effectively to the transmembrane E3 ubiquitin ligase.

[0259] Preferably, the membrane-bound protein is a transmembrane protein, and preferably at least one of a nutrient transporter, an ion channel, and a cell surface receptor. Preferably, the second binding domain of the heterobifunctional molecule can specifically bind to the transmembrane receptor. Preferably, the receptor is at least one of an ion channel-linked receptor, an enzyme-linked receptor, a G protein-linked receptor, and an Fc receptor. The second binding domain of the heterobifunctional molecule may bind to the monomeric and / or dimerized form of the receptor. Furthermore, or alternatively, the second binding domain may bind to the inactive and / or active conformation of the receptor.

[0260] Membrane-bound proteins may be associated with or involved in the onset, progression, or severity of disease. Membrane-bound proteins may be known or expected to be involved in cancer, autoimmune diseases, inflammatory diseases, neurological disorders, rare diseases, infectious diseases, and / or genetic diseases.

[0261] Preferably, the membrane-bound protein is not at least one of LGR4, LGR5, and LGR6.

[0262] In preferred embodiments, membrane-bound proteins are known to or expected to be involved in neurological disorders. In preferred embodiments, membrane-bound proteins are known to or expected to be involved in rare diseases.

[0263] In preferred embodiments, membrane-bound proteins are known to or expected to be involved in diseases selected from the group consisting of Charcot-Marie-Tooth disease (CMT), Gaucher disease (GD), anti-Mag peripheral neuropathy, CD38-related neurodegenerative pathology, myostatin-related neuromuscular disease, demyelinating disease, MS, ALS, and Guillain-Barré (GB). CD38-related neurodegenerative pathology may be at least one of ALS, MS, PD, and AD. Myostatin-related neuromuscular disease may be at least one of Duchenne disease and cachexia.

[0264] In preferred embodiments, membrane-bound proteins, preferably membrane-bound receptors, are known to or expected to be involved in cancer. “Cancer-involved receptors” are understood herein to be membrane-bound receptors that may directly or indirectly influence cancer malignancy.

[0265] In one embodiment, a membrane-bound receptor involved in cancer may be a receptor that, upon activation or increased activity, induces or enhances the malignant characteristics of a cell. For example, but not limited to, activation of a membrane-bound receptor may affect at least one of the following: stem cell properties, differentiation ability, metabolism, viability, proliferation, and immune evasion ability of a cell. As used herein, the activity of a receptor includes, but not limited to, a receptor having one or more activating mutations, and / or a receptor having increased expression and / or increased availability of a receptor ligand, and / or a receptor having decreased turnover, and is stabilized, for example, on the cell membrane.

[0266] Furthermore, or alternatively, membrane-bound receptors known or expected to be involved in cancer may include, for example, receptors present on immune cells and / or stromal cells. As a non-limiting example, inhibiting receptors present on immune cells may result in the activation of immune cells to target tumor cells, and inhibiting receptors present on stromal cells may result in a reduction of tumor angiogenesis.

[0267] Therefore, it is understood herein that receptors involved in cancer may be membrane-bound receptors present on tumor cells, and / or membrane-bound receptors present on cells that have a direct or indirect effect on tumor cells.

[0268] The phrase "receptors associated with or involved in cancer" includes, but is not limited to, proliferative disorders such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndromes, or preleukemia.

[0269] In one embodiment, the cancer associated with the activation or increased activity of membrane-bound receptors described herein is a hematological cancer. In one embodiment, the cancer associated with the activation or increased activity of membrane-bound receptors described herein is a solid tumor. Further diseases associated with the activation or increased activity of membrane-bound receptors described herein include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with the activation of membrane-bound receptors described herein. Non-cancer-related indications associated with the activation or increased activity of membrane-bound receptors described herein include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation.

[0270] Preferably, the membrane-bound receptor to which the second domain of the heterobifunctional molecule can bind is a cancer-related receptor, and preferably the receptor has increased activity, e.g., increased downstream signaling. The downstream signaling is preferably increased compared to otherwise identical cells that do not have activation or increased activity of the membrane-bound receptor. The increased activity may be due to, but is not limited to, mutagenic activation of the receptor, upregulation of the receptor, increased stabilization of the receptor, and / or increased availability of the receptor ligand.

[0271] The receptor may be involved in a specific type of cancer. Alternatively, the receptor may be involved in many different types of cancer. For example, the receptor may be involved in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of cancer. Alternatively, or even further, the receptor may be involved in cancer angiogenesis.

[0272] The receptor may be involved in solid tumors or hematological cancers. The receptor may be involved in solid tumors. Preferably, solid tumors include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine cancer, thyroid cancer. The following are selected from the group consisting of: cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors in childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, axial spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, and metastatic lesions of the above cancers.

[0273] The receptor may be involved in solid tumors or hematological malignancies. Preferably, the hematological cancer is selected from one or more of the following: chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prelymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

[0274] Preferably, the membrane-bound protein to which the second binding domain of the heterobifunctional molecule can bind is associated with cancers selected from the group consisting of colorectal cancer, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, prostate cancer, lung cancer, melanoma, leukemia, pancreatic cancer, and bladder cancer.

[0275] Preferably, a membrane-bound protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in colorectal cancer. Preferably, the membrane-bound protein is at least one of EGFR, IGF1R, MET, LRP6, WLS, and ERBB2.

[0276] Preferably, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is activated or has increased activity in hepatocellular carcinoma. Preferably, the membrane-bound protein is LRP6.

[0277] Preferably, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is activated or has increased activity in colorectal cancer. Preferably, the membrane-bound protein is LRP6 or WLS.

[0278] Preferably, a transmembrane protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in breast cancer. Preferably, the membrane-bound protein is WLS, EGFR, or ERBB2.

[0279] Preferably, a transmembrane protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in esophageal cancer. Preferably, the membrane-bound protein is ERBB2 or VEGFR2.

[0280] Preferably, a transmembrane protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in gastric cancer. Preferably, the membrane-bound protein is WLS, ERBB2, or VEGFR2.

[0281] Preferably, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is activated or has increased activity in leukemia. Preferably, the membrane-bound protein is FLT3.

[0282] Preferably, a transmembrane protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in melanoma. Preferably, the membrane-bound protein is KIT.

[0283] Preferably, a transmembrane protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in non-small cell lung cancer. Preferably, the membrane-bound protein is EGFR or MET.

[0284] Preferably, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is activated or has increased activity in ovarian cancer. Preferably, the membrane-bound protein is EGFR.

[0285] Preferably, a transmembrane protein capable of binding to the second binding domain of a heterobifunctional molecule is activated or has increased activity in pancreatic cancer. Preferably, the membrane-bound protein is LRP6 or EGFR.

[0286] Preferably, membrane-bound proteins capable of binding to the second binding domain of the heterobifunctional molecule are selected from the group consisting of TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6, CMTM4, WLS, SLC7A5, and SLC16A7.

[0287] Preferably, the membrane-bound protein capable of binding to the second binding domain of the heterobifunctional molecule has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 84, 86, 88, 90, 92, 94, 100, 102, and 104.

[0288] Preferably, the membrane-bound protein capable of binding to the second binding domain of the heterobifunctional molecule is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 85, 87, 89, 91, 93, 95, 101, 103, and 105.

[0289] In preferred embodiments, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is TGFβR1 or TGFβR2.

[0290] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is TGFβR1. Preferably, the TGFβR1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31. Preferably, the TGFβR1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 32.

[0291] In a preferred embodiment, the transmembrane protein that can be bound by the second binding domain of the heterobifunctional molecule is TGFβR2. Preferably, the TGFβR2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 33. Preferably, the TGFβR2 protein is encoded by a sequence that has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 34.

[0292] Preferably, the second binding domain of the heterobifunctional molecule can specifically bind to TGFβR2, and the first binding domain can specifically bind to RNF167.

[0293] In a preferred embodiment, the transmembrane protein to which the second binding domain of the heterobifunctional molecule can bind is EGFR. Preferably, the EGFR protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 35. Preferably, the EGFR protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 36.

[0294] Preferably, the second binding domain of the heterobifunctional molecule can specifically bind to EGFR, and the first binding domain can specifically bind to RNF167.

[0295] In preferred embodiments, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is ERBB2 or ERBB3.

[0296] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is ERBB2. Preferably, the ERBB2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 37. Preferably, the ERBB2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 38.

[0297] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is ERBB3. Preferably, the ERBB3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 39. Preferably, the ERBB3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 40.

[0298] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is IGF1R. Preferably, the IGF1R protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 41. Preferably, the IGF1R protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42.

[0299] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is MET. Preferably, the MET protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 43. Preferably, the MET protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 44.

[0300] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is VEGFR2. Preferably, the VEGFR2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 45. Preferably, the VEGFR2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 46.

[0301] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is KIT. Preferably, the KIT protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 47. Preferably, the KIT protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 48.

[0302] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FLT3. Preferably, the FLT3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 49. Preferably, the FLT3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 50.

[0303] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is PDGFRA. Preferably, the PDGFRA protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 53. Preferably, the PDGFRA protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 54.

[0304] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is PDGFRB. Preferably, the PDGFRB protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 55. Preferably, the PDGFRB protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 56.

[0305] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10.

[0306] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD1. Preferably, the FZD1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 57. Preferably, the FZD1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 58.

[0307] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD2. Preferably, the FZD2 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 59. Preferably, the FZD2 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 60.

[0308] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD3. Preferably, the FZD3 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 61. Preferably, the FZD3 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 62.

[0309] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD4. Preferably, the FZD4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 63. Preferably, the FZD4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 64.

[0310] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD5. Preferably, the FZD5 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 65. Preferably, the FZD5 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 66.

[0311] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD6. Preferably, the FZD6 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 67. Preferably, the FZD6 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 68.

[0312] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD7. Preferably, the FZD7 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 69. Preferably, the FZD7 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 70.

[0313] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD8. Preferably, the FZD8 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 71. Preferably, the FZD8 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 72.

[0314] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD9. Preferably, the FZD9 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 73. Preferably, the FZD9 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 74.

[0315] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is FZD10. Preferably, the FZD10 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 75. Preferably, the FZD10 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 76.

[0316] In preferred embodiments, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is LRP5 or LRP6.

[0317] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is LRP5. Preferably, the LRP5 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 77. Preferably, the LRP5 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 78.

[0318] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is LRP6. Preferably, the LRP6 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 79. Preferably, the LRP6 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 80.

[0319] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is a growth hormone receptor (GHR). Preferably, the GHR protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 84. Preferably, the GHR protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 85.

[0320] In preferred embodiments, the transmembrane protein functions as an immune checkpoint inhibitor. Preferably, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is selected from the group consisting of PD-1, PD-L1, CTLA4, CMTM6, CMTM4, and WLS.

[0321] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is PD-1. Preferably, the PD-1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 86. Preferably, the PD-1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 87.

[0322] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is PD-L1. Preferably, the PD-L1 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 88. Preferably, the PD1L1 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 89.

[0323] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is CTLA4. Preferably, the CTLA4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 90. Preferably, the CTLA4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 91.

[0324] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is CMTM6. Preferably, the CMTM6 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 92. Preferably, the CMTM6 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 93.

[0325] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is CMTM4. Preferably, the CMTM4 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 94. Preferably, the CMTM4 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 95.

[0326] In a preferred embodiment, the transmembrane protein capable of binding to the second binding domain of the heterobifunctional molecule is WLS / GPR177. Preferably, the WLS protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 100. Preferably, the WLS protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 101.

[0327] In a preferred embodiment, the transmembrane protein to which the second binding domain of the heterobifunctional molecule can bind is SLC7A5. Preferably, the SLC7A5 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 102. Preferably, the SLC7A5 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 103.

[0328] In a preferred embodiment, the transmembrane protein to which the second binding domain of the heterobifunctional molecule can bind is SLC16A7(MCT2). Preferably, the SLC16A7 protein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 104. Preferably, the SLC16A7 protein is encoded by a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 105.

[0329] Preferably, the second binding domain of the heterobifunctional molecule binds to the extracellular portion of the membrane-bound protein. Therefore, preferably, the heterobifunctional molecule does not need to pass through the cell membrane to bind to the membrane-bound protein.

[0330] Preferably, the first and second binding domains of the heterobifunctional molecule bind to the extracellular portions of the transmembrane E3 ubiquitin ligase and the membrane-bound protein, respectively. Preferably, the heterobifunctional molecule binds extracellularly.

[0331] First binding domain The heterobifunctional molecule of the present invention comprises at least a first binding domain and a second binding domain. The first binding domain can specifically bind to a transmembrane E3 ubiquitin ligase. Preferably, the first binding domain can specifically bind to a transmembrane E3 ubiquitin ligase as defined in the section "Protein to which the first binding domain binds" above. The first binding domain of the heterobifunctional molecule may selectively bind to a native epitope of the transmembrane E3 ubiquitin ligase, preferably when the heterobifunctional molecule is used pharmaceutically. Alternatively, the first binding domain of the heterobifunctional molecule may selectively bind to a non-native epitope manipulated within the transmembrane E3 ubiquitin ligase, preferably when the heterobifunctional molecule is used in the method of the present invention, preferably in a selected method of the present invention.

[0332] The first binding domain of the heterobifunctional molecule may be any domain capable of specifically binding to the transmembrane E3 ubiquitin ligase. Preferably, the first binding domain of the heterobifunctional molecule binds to the extracellular portion of the transmembrane E3 ubiquitin ligase.

[0333] Those skilled in the art will understand, for example, methods for generating the first binding domain of the heterobifunctional molecule of the present invention by screening compound libraries, immunotherapy studies, and / or hybridoma techniques for generating antibodies or functional fragments thereof. Preferred functional antibody fragments are nanobodies. Details of these techniques are described, for example, by (Antibodies: A Laboratory Manual, Harlow et al., Cold Spring Harbor Publications, p. 726, 1988), or by (Campbell, AM "Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands, 1984), or by (St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Details of the generation of VHH / nanobodies against natural epitopes are described, for example, by Pardon et al, Nature Protocols 2014, which is incorporated herein by reference.

[0334] In a preferred embodiment, the molecule capable of binding to the transmembrane E3 ubiquitin ligase is an antibody. Therefore, preferably, the antibody may function as the first binding domain in the heterobifunctional molecule of the present invention.

[0335] Preferably, the antibody is an antibody fragment. Preferably, the antibody fragment is a nanobody. Therefore, in a preferred embodiment, the molecule that can bind to the transmembrane E3 ubiquitin ligase is a nanobody. Therefore, preferably, the nanobody may function as the first binding domain in the heterobifunctional molecule of the present invention.

[0336] In a preferred embodiment, the first binding domain is an organic small molecule.

[0337] In a preferred embodiment, the first binding domain is an aptamer.

[0338] In a preferred embodiment, the first binding domain is a protein molecule. The protein molecule may be cyclized, and therefore, preferably, the protein molecule is a cyclic peptide. The peptide may be cyclized by a direct covalent bond between two amino acid residues or by using a crosslinking moiety. Such crosslinking moieties are well known in the art and include, for example, those described in International Publication No. 2012 / 057624, incorporated herein by reference. The protein molecule may be a protein molecule conventionally known in the art.

[0339] Accordingly, the present invention extends to molecules known in the art for specific binding to transmembrane E3 ubiquitin ligases, which can function as the first binding domain of the heterobifunctional molecule of the present invention. Such known molecules include, but are not limited to, at least one of known antibodies, protein molecules, aptamers, or known small organic molecules. Preferably, antibodies, protein molecules, aptamers, or small organic molecules are known in the art for binding to the extracellular portion of transmembrane E3 ubiquitin ligases.

[0340] Antibodies that bind to transmembrane E3 ubiquitin ligases are known in the art, and it would not be difficult for those skilled in the art to obtain such antibodies. Any known antibody that can specifically bind to transmembrane E3 ubiquitin ligases, preferably specifically to the extracellular portion of transmembrane E3 ubiquitin ligases, would be suitable for use as the first binding domain in the heterobifunctional molecule of the present invention.

[0341] A preferred known molecule capable of binding to transmembrane E3 ubiquitin ligase is a nanobody. Therefore, preferably, a nanobody may function as the first binding domain in the heterobifunctional molecule of the present invention.

[0342] In a preferred embodiment, the first binding domain is a native ligand of a transmembrane E3 ubiquitin ligase, or a functional fragment thereof, i.e., a fragment of a native ligand that is capable of binding to a transmembrane E3 ubiquitin ligase.

[0343] As a non-limiting example, the natural ligands for RNF43 and ZNRF3 are Rspondin(RSPO)-1, -2, -3, and -4. Therefore, in one embodiment, the heterobifunctional molecule comprises a first binding domain capable of binding to RNF43, the first binding domain being selected from the group consisting of Rspondin 1, Rspondin 2, Rspondin 3, and Rspondin 4 or their functional fragments. Alternatively, the heterobifunctional molecule comprises a first binding domain capable of binding to ZNRF3, the first binding domain being selected from the group consisting of Rspondin 1, Rspondin 2, Rspondin 3, and Rspondin 4 or their functional fragments.

[0344] Second binding domain The heterobifunctional molecule of the present invention comprises at least a first binding domain and a second binding domain. The second domain can specifically bind to a membrane-bound protein, preferably a transmembrane protein. Preferably, the second binding domain can specifically bind to a membrane-bound protein as defined in the section "Protein to which the second binding domain binds" above. Preferably, the second binding domain of the heterobifunctional molecule can selectively bind to a native epitope of a membrane-bound protein when the heterobifunctional molecule is used pharmaceutically. Alternatively, preferably, the second binding domain of the heterobifunctional molecule can selectively bind to a non-native epitope manipulated within a membrane-bound protein when the heterobifunctional molecule is used in the method of the present invention, preferably in a selected method of the present invention.

[0345] The second binding domain of the heterobifunctional molecule may be any domain capable of specifically binding to a membrane-bound protein, preferably a transmembrane protein. Preferably, the second binding domain of the heterobifunctional molecule binds to the extracellular portion of the membrane-bound protein.

[0346] The second binding domain can be an antibody, peptide, aptamer, or small organic molecule.

[0347] Those skilled in the art will understand, for example, methods for generating the second binding domain of the heterobifunctional molecule of the present invention by screening compound libraries, immunotherapy studies, and / or hybridoma techniques for generating antibodies or functional fragments thereof. Preferred functional antibody fragments are nanobodies. Details of these techniques are described, for example, by (Antibodies: A Laboratory Manual, Harlow et al., Cold Spring Harbor Publications, p. 726, 1988), or by (Campbell, AM "Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands, 1984), or by (St. Groth et al., J. Immunol. Methods 35:1-21, 1980).

[0348] In preferred embodiments, the molecule capable of binding to a membrane-bound protein is an antibody. Therefore, preferably, the antibody may function as a second binding domain in the heterobifunctional molecule of the present invention.

[0349] Preferably, the antibody is an antibody fragment. Preferably, the antibody fragment is a nanobody. Therefore, in a preferred embodiment, the molecule that can bind to a membrane-bound protein is a nanobody. Therefore, preferably, the nanobody may function as a second binding domain in the heterobifunctional molecule of the present invention.

[0350] In a preferred embodiment, the first binding domain is an organic small molecule.

[0351] In a preferred embodiment, the first binding domain is an aptamer.

[0352] In a preferred embodiment, the second binding domain is a protein molecule. The protein molecule may be cyclized, and therefore, preferably, the protein molecule is a cyclic peptide. The peptide may be cyclized by a direct covalent bond between two amino acid residues or by using a crosslinking moiety. Such crosslinking moieties are well known in the art and include, for example, those described in International Publication No. 2012 / 057624, incorporated herein by reference. The protein molecule may be a protein molecule conventionally known in the art.

[0353] Therefore, the present invention extends to molecules known in the art for specifically binding to membrane-bound proteins, preferably membrane-bound proteins as defined herein. Such molecules can function as a second binding domain of the heterobifunctional molecule of the present invention.

[0354] Such known molecules include, but are not limited to, at least one of known antibodies, protein molecules, aptamers, or known small organic molecules. Preferably, antibodies, protein molecules, aptamers, or small organic molecules that bind to the extracellular portion of membrane-bound proteins as defined herein are known in the art.

[0355] Antibodies that bind to membrane-bound proteins, preferably transmembrane proteins as defined herein, are known in the art, and it would not be difficult for those skilled in the art to obtain such antibodies. Any known antibody that can specifically bind to a membrane-bound protein as defined herein, preferably specifically to the extracellular portion of a membrane-bound protein as defined herein, would be suitable for use as a second binding domain in the heterobifunctional molecule of the present invention.

[0356] A preferred known molecule capable of binding to membrane-bound proteins is a nanobody. Therefore, preferably, a nanobody may function as a second binding domain in the heterobifunctional molecule of the present invention.

[0357] In preferred embodiments, the second binding domain is a native ligand for a membrane-bound protein, preferably a transmembrane protein as defined herein. Preferably, the native ligand is an antagonist of the transmembrane protein.

[0358] Heterobifunctional molecules The first and second binding domains can each specifically bind to transmembrane proteins or membrane-bound proteins, i.e., target proteins.

[0359] Specific binding is understood herein as the degree of binding of a domain of a “non-target” protein to a particular target protein being less than approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the domain’s binding to that particular target protein, as determined by fluorescence-activated cell sorting (FACS) analysis, ELISA, or radioimmunoprecipitation (RIA). With respect to the binding of a domain to a target protein, the terms “specifically binding” or “specifically binding” to an epitope on a particular polypeptide target or to a particular polypeptide or to be “specific” mean binding that is measurably different from nonspecific interactions. Specific binding can be measured, for example, by comparing the binding of a target protein to the binding of a control protein, which is generally a similarly structured protein that does not possess binding activity. For example, specific binding can be determined by competition with a control protein similar to the target, such as an excess of unlabeled targets. In this case, specific binding is indicated if the binding of a labeled target to a probe is competitively inhibited by an excess of unlabeled targets.

[0360] As used herein, the terms “specifically binding” or “specifically binding” to a particular polypeptide or epitope on a particular polypeptide target means, for example, at least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 This can be represented by a binding domain having a Kd (which may be determined as described above) for M or a higher target. In one embodiment, the term “specific binding” refers to binding where the binding domain binds to a specific polypeptide or epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0361] The simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a membrane-bound protein results in ubiquitination and degradation of the transmembrane protein. Preferably, the membrane-bound protein is a transmembrane protein. Therefore, preferably, the simultaneous binding of a heterobifunctional molecule to a transmembrane E3 ubiquitin ligase and a transmembrane protein results in ubiquitination and degradation of the transmembrane protein. Preferably, the degradation is at least one of proteasomal degradation and lysosomal degradation. Preferably, the degradation is lysosomal degradation.

[0362] Hetero-bifunctional molecules as defined herein may, therefore, knock down or knock out the presence of membrane-bound proteins on the cell membrane by targeting transmembrane E3 ubiquitin ligases, i.e., bringing them closer to the membrane-bound proteins. In other words, the steady-state level of membrane-bound proteins is reduced.

[0363] Steady-state levels may be defined herein as the amount of protein present per cell. Compared to reference cells, steady-state levels of membrane-bound proteins may decrease by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or by about 100%, meaning that membrane-bound proteins are completely absent due to the binding of heterobifunctional molecules.

[0364] In preferred embodiments, the heterobifunctional molecule is a bispecific antibody. Bispecific antibodies are described, for example, in Wu X and Demarest SJ, Methods. (2019) 154:3-9. Therefore, preferably, the first binding domain is an antibody that can specifically bind to a transmembrane E3 ubiquitin ligase, preferably the transmembrane E3 ubiquitin ligase defined in the section "Transmembrane E3 ubiquitin ligase to which the first binding domain binds". Preferably, the second binding domain is also an antibody that can specifically bind to a membrane-bound protein, preferably a transmembrane protein, preferably the transmembrane protein defined in the section "Protein to which the second binding domain binds". The two antibodies (i.e., the first and second binding domains) can bind directly to each other, or a linker can be present between the two antibodies, preferably a linker as defined herein.

[0365] In preferred embodiments, the heterobifunctional molecule is a bispecific nanobody. Bispecific nanobodies are disclosed, for example, in International Publication No. 2015 / 044386 and Conrath et al. (Camel Single-domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs, JBC, 2001). Preferably, the first binding domain is a nanobody that can specifically bind to a transmembrane E3 ubiquitin ligase, preferably the transmembrane E3 ubiquitin ligase defined in the section "Transmembrane E3 ubiquitin ligase to which the first binding domain binds". Preferably, the second binding domain is also a nanobody that can specifically bind to a membrane-bound protein, preferably a transmembrane protein, preferably the transmembrane protein defined in the section "Protein to which the second binding domain binds". The two nanobodies (i.e., the first and second binding domains) can be directly bound to each other, or a linker can be present between the two nanobodies, preferably a linker as defined herein.

[0366] In preferred embodiments, the heterobifunctional molecule is a bicyclic peptide. Preferably, the first binding domain is a cyclic peptide that can specifically bind to a transmembrane E3 ubiquitin ligase, preferably the transmembrane E3 ubiquitin ligase defined in the section "Transmembrane E3 ubiquitin ligase to which the first binding domain binds." Preferably, the second binding domain is also a cyclic peptide that can specifically bind to a membrane-bound protein, preferably a transmembrane protein, preferably the transmembrane protein defined in the section "Protein to which the second binding domain binds." The two cyclic peptides (i.e., the first and second binding domains) can bind directly to each other, for example, by using the same crosslinking region, or a linker can be present between the two cyclic peptides, preferably a linker as defined herein.

[0367] The heterobifunctional molecule may, if necessary, include a moiety that increases molecular stability. Such moieties may include, but are not limited to, a binding domain for specific binding of albumin.

[0368] The heterobifunctional molecule may optionally include a tag for purification or detection of the heterobifunctional molecule, preferably a peptide-tag or protein-tag as defined herein. Preferred purification tags are His-tags or Avi-tags. A preferred detection tag is a V5-tag. Such heterobifunctional molecules are particularly useful for use in the (screening) method of the present invention.

[0369] The heterobifunctional molecules of the present invention may include a linker between the first binding domain and the second binding domain. The linker may be any suitable linker known in the art. Preferably, the linker is a Gly-Ser sequence. Those skilled in the art know how to select the linker depending on the first and second binding domains. The linker may be a very flexible linker of the form (GGGGS)n, (GGS)n, and (G)n, for example, a more rigid linker of the form (EAAAK)n, (SPKKKRKVEAS)n (SEQ ID NO: 81), or (SGSETPGTSESATPES)n (SEQ ID NO: 82), or (KSGSETPGTSESATPES)n (SEQ ID NO: 83), or any variant thereof, where n is preferably 1 to 15, preferably 1 to 7, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0370] The linker is preferably 2 to 250 amino acids, 2 to 30 amino acids, 3 to 23 amino acids, or 3 to 18 amino acids in length.

[0371] The linker may be a cleavable linker, for example, by introducing a 3C protease cleavage site into the linker. Such a linker is particularly useful for use in the (screening) method of the present invention because it provides control over the fact that forced dimerization should be disabled, for example by cleaving the linker.

[0372] therapeutic use The heterobifunctional molecules defined herein can be used to reduce the level of any selected membrane-bound protein by the simultaneous binding of a transmembrane ubiquitin E3 ligase to a selected membrane-bound protein. The combination of the transmembrane ubiquitin ligase and the selected membrane-bound protein is preferably an effective combination and is preferably selected using the selection method of the present invention.

[0373] In one embodiment, the heterobifunctional molecules as defined herein are for use as pharmaceuticals. The heterobifunctional molecules for pharmaceutical use preferably bind to native epitopes present on transmembrane ubiquitin E3 ligases and to native epitopes present on selected membrane-bound proteins. The medical uses described herein are formulated as heterobifunctional molecules for use as pharmaceuticals for the treatment of the aforementioned diseases(s) by administration of an effective amount of the heterobifunctional molecule, but can also be similarly formulated as a method for treating the aforementioned diseases(s) using the heterobifunctional molecules as defined herein, comprising the steps of administering an effective amount of the heterobifunctional molecule to a subject, administering the heterobifunctional molecule as defined herein for use in the preparation of a pharmaceutical for treating the aforementioned diseases(s), wherein the heterobifunctional molecule is administered in an effective amount, and using the heterobifunctional molecule as defined herein for the treatment of the specified diseases(s) by administration of an effective amount. All such medical uses are envisioned by the present invention.

[0374] Those skilled in the art will understand that an increase in the activity of any membrane-bound protein involved in the onset, severity, or duration of a disease may be a suitable target for a heterobifunctional molecule as defined herein. Therefore, the heterobifunctional molecule is not limited to any specific membrane-bound protein or any specific disease. Preferably, the disease is characterized by an increase in the activity of a membrane-bound protein, preferably a receptor, and the increase in the activity of the membrane-bound receptor preferably affects or defines the onset, severity, or duration of the disease.

[0375] As a non-limiting example, heterobifunctional molecules can be used to treat at least one of the following conditions: cancer, dementia, heart disease, neurological disorders, rare diseases, and infections.

[0376] It is well known in the art that increased activity of membrane-bound receptors plays a crucial role, for example, in the onset, severity, or duration of cancer. Therefore, in one embodiment, heterobifunctional molecules are used to treat, prevent, alleviate, or suppress cancer-related symptoms.

[0377] Preferably, cancer is cancer as defined in the “protein to which the second binding domain binds” section of this specification.

[0378] Preferably, the cancer is a solid tumor or a hematological cancer. Alternatively, or even more preferably, the receptor may be involved in cancer angiogenesis.

[0379] Preferably, the solid tumor is a solid tumor as defined in the section "Protein to which the second binding domain binds" above in this specification.

[0380] Preferably, hematological cancer is a hematological cancer as defined in the section "Proteins to which the second binding domain binds" above in this specification.

[0381] In one embodiment, the present invention relates to a composition comprising a heterobifunctional molecule as defined herein. The heterobifunctional molecule preferably binds to an effective combination of a transmembrane ubiquitin E3 ligase and a membrane-bound protein, selected using a preferred method of the present invention. The composition may be suitable for use in cell culture, preferably animal cell culture, more preferably mammalian cell culture. Furthermore, or alternatively, the composition is preferably a pharmaceutical composition or a cosmetic composition.

[0382] The composition may contain one type of heterobifunctional molecule, or it may contain at least two different heterobifunctional molecules to knock down or knock out the presence of two or more different membrane-bound proteins. The composition may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of heterobifunctional molecules.

[0383] Compositions containing the heterobifunctional molecules described above can be prepared in various other media, such as pharmaceutical or cosmetic compositions, or as human or animal foods, including medical foods and nutritional supplements.

[0384] "Medical foods" are products intended for specific dietary management of diseases or conditions that have unique nutritional requirements. For example, medical foods include, but are not limited to, vitamin and mineral preparations delivered through a feeding tube (known as enteral administration).

[0385] "Dietary supplements" means products intended to supplement a human diet, typically provided in the form of formulations such as pills, capsules, and tablets. For example, but not limited to, dietary supplements may contain one or more of the following ingredients: vitamins, minerals, herbs, herbal medicines; amino acids, dietary substances intended to supplement a diet by increasing total dietary intake; and concentrates, metabolites, ingredients, extracts, or combinations thereof of any of the aforementioned. Dietary supplements may be incorporated into foods, including but not limited to food bars, beverages, powders, cereals, prepared foods, food additives, and candies.

[0386] Therefore, the composition in question may be compounded with other ingestible, physiologically acceptable materials, including but not limited to food. Furthermore, or alternatively, the compositions for use described herein may be administered orally in combination with (separate) administrations of food.

[0387] The compositions may be administered alone or in combination with other pharmaceutical or cosmetic agents, and can be combined with physiologically acceptable carriers. In particular, the heterobifunctional molecules described herein can be formulated as pharmaceutical or cosmetic compositions by formulating them with pharmaceutically or physiologically acceptable excipients, carriers, and additives such as vehicles.

[0388] Appropriate pharmaceutically or physiologically acceptable excipients, carriers, and vehicles include processing agents and drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-p-cyclodextrin, polyvinylpyrrolidinone, low-melting-point waxes, ion-exchange resins, and any combination of two or more of these. For other pharmaceutically acceptable excipients, see "Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1991), and "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, Philadelphia, 20th edition (2003), 21 st edition (2005) and 22 nd This is described in edition (2012), which is incorporated herein by reference.

[0389] The pharmaceutical or cosmetic composition comprising the heterobifunctional molecule for use described in the present invention may be in any form suitable for the intended method of administration, for example, a solution, suspension, or emulsion. In preferred embodiments, the heterobifunctional molecule is administered in solid or liquid form.

[0390] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the heterobifunctional molecule may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also contain additional substances other than the inert diluent, such as lubricants, such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. Tablets and pills can be further prepared using enteric coatings.

[0391] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or saline. Such compositions may also contain wetting agents, emulsifiers and suspending agents, adjuvants such as cyclodextrins, as well as sweeteners, flavorings, and fragrances.

[0392] Liquid carriers are typically used in the preparation of solutions, suspensions, and emulsions. In preferred embodiments, liquid carriers / liquid dosage forms intended for use in the implementation of the present invention include, for example, water, physiological saline, pharmaceutically acceptable organic solvents, pharmaceutically acceptable oils or fats, and mixtures of two or more thereof. In preferred embodiments, the heterobifunctional molecules of the present invention as defined herein are miscible with an aqueous solution before administration. The aqueous solution should be suitable for administration, and such aqueous solutions are well known in the art. It is further known in the art that the suitability of the aqueous solution for administration may depend on the route of administration.

[0393] In a preferred embodiment, the aqueous solution is an isotonic aqueous solution. The isotonic aqueous solution is preferably nearly (or perfectly) isotonic with respect to plasma. In a further preferred embodiment, the isotonic aqueous solution is physiological saline.

[0394] The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickeners, viscosity modifiers, stabilizers, and flavoring agents. Preferred flavoring agents are sweeteners such as monosaccharides and / or disaccharides. Suitable organic solvents include, for example, monohydric alcohols such as ethanol and polyhydric alcohols such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, and cottonseed oil.

[0395] For parenteral administration, the carrier may be an oily ester such as ethyl oleate or isopropyl myristate. The composition for use in the present invention may be in the form of fine particles, microcapsules, liposome capsules, or any combination of two or more thereof.

[0396] Time-release, sustained-release, or controlled-release delivery systems may utilize diffusion-controlled matrix systems or erodible systems, as described, for example, in Lee, "Diffusion-Controlled Matrix Systems", pp. 155-198 and Ron and Langer, "Erodible Systems", pp. 199-224, in "Treatise on Controlled Drug Delivery", A. Kydonieus Ed., Marcel Dekker, Inc., New York 1992. The matrix may be a biodegradable material that can be spontaneously degraded in situ and in vivo by hydrolysis or enzymatic cleavage, for example, by proteases. The delivery system may be, for example, a naturally occurring or synthetic polymer or copolymer, e.g., in the form of a hydrogel. Exemplary polymers having cleavable bonds include polyesters, polyorthoesters, polyanhydrides, polysaccharides, poly(phosphoesters), polyamides, polyurethanes, poly(imide carbonates), and poly(phosphazenes).

[0397] The heterobifunctional molecules of the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally obtained from phospholipids or other lipid substances. Liposomes are formed by monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipids that can form liposomes can be used. In addition to the heterobifunctional molecules defined herein, the compositions of the present invention in liposome form may include stabilizers, preservatives, excipients, etc. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholine (lecithin). Methods for forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NY, p. 33 et seq (1976).

[0398] A pharmaceutical or cosmetic composition may include a unit dose formulation, where the unit dose is a dose sufficient to have a therapeutic or inhibitory effect on the disorder or condition as defined herein, and / or an effective amount to reduce or knock out the expression of a membrane-bound protein. The unit dose may be a single dose sufficient to have a therapeutic or inhibitory effect on the disorder or condition as defined herein and / or an effective amount to reduce the expression of a target membrane-bound protein. Alternatively, the unit dose may be a dose administered periodically during the course of treatment or suppression of the disorder or condition as defined herein. During the course of treatment, the concentration of the composition may be monitored to ensure that the desired level is maintained.

[0399] Heterobifunctional molecules or compositions comprising heterobifunctional molecules as defined herein may be administered enterally, orally, parenterally, sublingually, by inhalation (e.g., as a mist or spray), rectally, or topically, and may preferably be administered in a dosage unit formulation comprising a conventional non-toxic pharmaceutically or physiologically acceptable carrier, adjuvant, and vehicle, as desired. For example, preferred modes of administration include oral, subcutaneous, percutaneous, transmucosal, iontophoresis, intravenous, intra-arterial, intramuscular, intraperitoneal, intranasal (e.g., via nasal mucosa), subdural, rectal, gastrointestinal, etc., and direct administration to specific or affected organs or tissues, such as cancerous tissue. For administration to the central nervous system, spinal administration, epidural administration, ventricular administration, etc., may be used. Topical administration may involve the use of percutaneous administration such as percutaneous patches or iontophoresis devices. As used herein, the term parenteral includes subcutaneous injection, intravenous injection, intramuscular injection, intrastitial injection, or infusion techniques.

[0400] The heterobifunctional molecules can be mixed with pharmaceutically acceptable carriers, adjuvants, and vehicles suitable for the desired route of administration. The heterobifunctional molecules of the present invention may be administered by supplementation via a gastric tube or percutaneous tube.

[0401] In preferred embodiments, the present invention relates to heterobifunctional molecules as defined above for use in the treatment, prevention, or suppression of cancer-related symptoms by administration of an effective total daily dose.

[0402] The dosage form for oral administration may be a solid oral dosage form. The class of solid oral dosage forms mainly consists of tablets and capsules, but other forms are also known in the art and may be equally suitable. When used as a solid oral dosage form, the heterobifunctional molecules as defined herein may be administered, for example, in the form of an immediate-release tablet (or capsule, etc.) or a sustained-release tablet (or capsule, etc.). As will be apparent to those skilled in the art, any suitable immediate-release or sustained-release solid dosage form can be used in the context of the present invention.

[0403] The heterobifunctional molecules described for use as described herein may be administered in solid form, liquid form, aerosol form, or in tablet, pill, powder mixture, capsule, granule, injection, cream, solution, suppository, enema, colon lavage, emulsion, dispersion, food premix, and other suitable forms. The heterobifunctional molecules of c may also be administered in liposomal formulations. The compounds may also be administered as prodrugs, where the prodrug is converted into a form that is therapeutically effective in the treated subject. Additional methods of administration are known in the art.

[0404] Injectable preparations, such as sterile aqueous or oily suspensions for injection, can be formulated according to known techniques using appropriate dispersants or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in propylene glycol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, and isotonic sodium chloride solutions. Furthermore, sterile fixative oils have conventionally been employed as solvents or suspension media. For this purpose, any brand of fixative oil, including synthetic mono or diglycerides, may be employed. Additionally, fatty acids such as oleic acid are used in the preparation of injectable preparations.

[0405] Suppositories for rectal administration of heterobifunctional molecules can be prepared by mixing the heterobifunctional molecule with a suitable non-irritating excipient, such as cocoa butter or polyethylene glycol, which is solid at room temperature but liquid at rectal temperature, and therefore dissolves in the rectum to release the heterobifunctional molecule.

[0406] The heterobifunctional molecules for use described herein may be administered as single active pharmaceutical (or cosmetic) agents, but they may also be used in combination with one or more other agents used to treat or suppress a disease or disorder. Representative agents useful in combination with the heterobifunctional molecules of the present invention for treating, preventing or suppressing symptoms associated with a disease or disorder include, but are not limited to, coenzyme Q, vitamin E, idebenone, MitoQ, EPI-743, vitamin K and its analogues, naphthoquinone and its derivatives, other vitamins, and antioxidant compounds.

[0407] When additional activators are used in combination with the heterobifunctional molecules of the present invention, the additional activators may generally be administered in therapeutic doses as shown in the Physicians' Desk Reference (PDR) 53rd Edition (1999), incorporated herein by reference, or in therapeutically useful doses known to those skilled in the art. The heterobifunctional molecules of the present invention and other therapeutically active agents or agents may be administered at the recommended maximum clinical dose or at lower doses. The dose levels of the active compounds in the compositions of the present invention may be varied to obtain the desired therapeutic response, depending on the route of administration, the severity of the disease, and the patient's response. When administered in combination with other therapeutic agents, the therapeutic agents may be formulated as separate compositions administered simultaneously or at different times, or the therapeutic agents may be administered as a single composition.

[0408] Manufacturing of heterobifunctional molecules In one embodiment, the present invention relates to a method for producing the heterobifunctional molecule of the present invention, wherein the method is - Preferably, the step of selecting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein using the method of the present invention; - A step of constructing a first binding domain that can specifically bind to transmembrane E3 ubiquitin ligase; - A step of constructing a second binding domain that can specifically bind to membrane-bound proteins; and - A step of coupling a first binding domain to a second binding domain, wherein the coupling is either a direct coupling or a coupling via a linker, preferably a linker as defined herein. Includes.

[0409] It is understood herein that the steps of constructing the first and second binding domains may be carried out using any conventional means in the art. As a non-limiting example, at least one of the first and second binding domains may be a binding domain conventionally known in the art, for example, an antibody known in the art for specific binding to transmembrane E3 ubiquitin ligases or an antibody known in the art for specific binding to membrane-bound proteins.

[0410] Alternatively, at least one of the first and second binding domains is a de novo binding domain, such as, but not limited to, an antibody or nanobody binding domain discovered through immunological studies. The step of selecting a transmembrane E3 ubiquitin ligase and a membrane-bound protein may be achieved by incorporating a first non-native epitope tag into the transmembrane E3 ubiquitin ligase and a second non-native epitope tag into the membrane-bound protein. When expressed intracellularly, the first and second epitope tags are preferably presented in their respective extracellular portions, i.e., extracellularly. A heterobifunctional molecule having a first binding domain capable of binding to the first epitope tag and a second binding domain capable of binding to the second epitope tag may then be used to evaluate its ability to target the transmembrane E3 ubiquitin ligase to the membrane-bound protein. After forcing the E3 ubiquitin ligase and the membrane-bound protein to interact using the heterobifunctional molecule, efficacy may be evaluated by determining the extent to which the transmembrane E3 ubiquitin ligase can ubiquitinize and internalize the membrane-bound protein. Furthermore, or alternatively, the ability of this selection system may be assessed by determining the extent to which the cell surface level and / or total protein level of the membrane-bound protein is reduced after forcing the interaction of the E3 ubiquitin ligase with the membrane-bound protein using this selection system. Alternatively, the step of selecting the transmembrane E3 ubiquitin ligase with the membrane-bound protein may also be considered a method for reducing the surface level of the membrane-bound protein in a cell, as detailed below herein.

[0411] After selecting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein, a heterobifunctional molecule may be constructed containing a first binding domain that can specifically bind to the extracellular portion of the (natural) transmembrane E3 ubiquitin ligase and a second binding domain that can specifically bind to the extracellular portion of the (natural) membrane-bound protein. [Examples]

[0412] [Example 1] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units / mL penicillin, and 100 μg / mL streptomycin (Invitrogen). Cells were cultured at 37°C in 5% CO2. Transfection was performed using FuGENE 6 (Promega) according to the manufacturer's protocol for microscopic observation, and using PEI for biochemical examination. A / C Heterodimerizer (Takara Bio, #635056) was administered at 1 μM overnight at 37°C, with the control condition being treatment with an equal volume of 100% ethanol. TGFβ was administered at 1.5 ng / mL for 45 minutes.

[0413] Constructs and antibodies TGF-βII type serine / threonine kinase receptor (TβRII)-Flag-FKBP and -Flag-FRB were provided by Peter ten Dijke (LUMC, Leiden). RNF43-FKBP and -FRB were prepared by adding FKBP to the C-suffix of human RNF43 using Q5 High-Fidelity 2× Master Mix (NEB). 36V Alternatively, it was obtained by inserting the FRB coding sequence. All constructs were sequence-validated. CD63-GFP was donated by J. Klumperman (UMCU, Utrecht). The following primary antibodies were used for immunoblotting (IB), immunofluorescence (IF), and immunoprecipitation (IP): rabbit anti-FLAG (Sigma-Aldrich), rat anti-HA (Roche), mouse anti-FLAG (M2; Sigma-Aldrich), mouse anti-Actin (MP Biomedicals), and rabbit anti-RNF43 (Sigma-Aldrich). Primary antibodies were diluted according to the manufacturer's instructions for use. Secondary antibodies used for IB or IF were obtained from either Rockland or Invitrogen at a dilution of 1:8000 or 1:300, respectively.

[0414] Immunofluorescence and confocal microscopy HEK293T cells were grown on laminin (Sigma)-coated glass coverslips in 24-well plates. After overnight transfection, cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS). Cells were blocked at room temperature (RT) for 30 minutes in a buffer containing 2% BSA and 0.1% saponin in PBS. Subsequently, cells were incubated with primary and secondary antibodies in blocking buffer at room temperature for 1 hour. Cells were mounted on Prolong Diamond (Life Technologies) and images were acquired using an LSM700 confocal microscope. Images were analyzed and processed in ImageJ.

[0415] Immunoprecipitation and Western blotting After transfection, cells were grown in a 10 cm dish until 80% confluence. After washing the cells with PBS, they were scraped off and lysed in a cell lysis buffer containing 100 mM NaCl, 50 mM Tris pH 7.5, 0.25% Triton X-100, 10% glycerol, 50 mM NaF, 10 mM Na3VO4, 10 μM leupeptin, 10 μM aprotinin, and 1 mM PMSF. The lysate was clarified by centrifugation at 16,000 × g at 4°C for 15 minutes. The lysate was placed in SDS sample buffer and heated at 37°C for 1 hour. For immunoprecipitation, the lysate was incubated with 25 μl of pre-bound Flag-M2 beads (Sigma) and incubated overnight at 4°C. After washing, the beads were eluted in sample buffer and heated at 37°C for 1 hour. After SDS-PAGE, the proteins were transferred to Immobilon-FL PVDF membranes (Milipore) by Western blotting. After blocking with Odyssey blocking buffer (LI-COR), the proteins were labeled with specified primary antibodies and detected using an Amersham Typhoon Biomolecular Imager (GE Health Care) with goat anti-mouse / rabbit Alexa 680 (Invitrogen), donkey anti-rat Alexa 680 (Invitrogen), or goat anti-mouse / rabbit IRDye 800 (Rockland).

[0416] Results and Discussion Forced dimerization of RNF43 and TGF-βII-type serine / threonine kinase receptor (TβRII) induces lysosomal localization and degradation of TβRII. To demonstrate the proof of concept of redirecting transmembrane E3 ligases to target selected cell surface proteins for internalization and lysosomal degradation, we used an FKBP / FRB dimerization system. Either an FKBP domain or an FRB domain was fused to the C-terminus of both TβRII and RNF43. When co-expressed in HEK293T cells, these proteins did not interact (Figure 2). However, upon addition of an A / C dimerizer, co-immunoprecipitation of RNF43 and TβRII was induced (Figure 2). The dimerizer itself did not inhibit the stability of TβRII or RNF43 (Figure 2, whole cell lysates). Next, we investigated whether forced interaction between RNF43 and TβRII altered the intracellular localization of TβRII. In the absence of a dimerizing agent, TβRII was primarily localized to the cell membrane, both in the absence of RNF43 (Figure 3A) and in the co-expression of RNF43 (Figure 3B). However, upon addition of a dimerizing agent, both TβRII and RNF43 were strongly relocalized to perinuclear vesicles positive for the lysosomal marker CD63 (Figure 3C). These findings suggest that forced dimerization of RNF43 and TβRII induces increased levels of TβRII into lysosomes.

[0417] To determine whether the amount of functional TβRII decreased as a result of enhanced lysosomal localization of TβRII, protein levels were analyzed using Western blotting. While RNF43 expression itself did not affect TβRII stability, the amount of TβRII protein was clearly reduced when dimerization between RNF43 and TβRII was induced (Figure 4). These results together indicate that forced dimerization of the normally unrelated transmembrane receptor TβRII with the transmembrane E3 ligase RNF43 targets TβRII for lysosomal degradation.

[0418] [Example 2] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units / mL penicillin, and 100 μg / mL streptomycin (Invitrogen). Cells were cultured at 37°C and 5% CO2. Transfection was performed using FuGENE 6 (Promega) according to the manufacturer's protocol.

[0419] Constructs and antibodies E6-Flag-TGF-βII-type serine / threonine kinase receptor (TβRII) and Epidermal Growth Factor Receptor (EGFR), as well as Alpha-myc-RNF43 and RNF167, were obtained by subcloning using Q5 High-Fidelity 2× Master Mix (NEB). All constructs were sequenced. For immunofluorescence (IF), the following primary antibodies were used: rabbit anti-Flag (Sigma-Aldrich) and mouse anti-Myc (hybridoma 9E10). Primary antibodies were diluted according to the manufacturer's instructions. Secondary antibodies used for IF were used at a 1:300 ratio (Life Technologies).

[0420] Immunofluorescence and confocal microscopy HEK293T cells were grown on laminin (Sigma-Aldrich) coated glass coverslips in 24-well plates. After overnight transfection, cells were incubated with 20 nM Bafilomycin A1 (Sigma-Aldrich) for 1 hour before and during a 5-hour treatment with 100 nM bi-VHH (VHH Alpha-(G4S)3-VHH E6). After treatment, cells were washed twice with warmed medium and fixed with 4% formaldehyde in 0.05 M phosphate buffer pH 7.4. Cells were blocked at room temperature (RT) for 30 minutes in a buffer containing 2% BSA and 0.1% saponin in PBS. Subsequently, cells were incubated in the blocking buffer with either a primary antibody against Flag or Myc at room temperature for 1 hour, followed by incubation with a secondary antibody at room temperature for 1 hour. Cells were mounted on Prolong Diamond (Life Technologies) and images were acquired using an LSM700 confocal microscope. The images were analyzed and processed using ImageJ.

[0421] Results and Discussion RNF43 and RNF167 induce the removal of TβRII and EGFR from the cell surface by forced dimerization using bispecific VHH. To further confirm the functionality of the heterobifunctional molecule of the present invention, the selected receptor was targeted with an E3 ligase via VHH-mediated dimerization in the extracellular domain. For this purpose, epitope tags were fused to the extracellular domains of both the target (E6 tag) and the E3 ligase (Alpha tag). These epitope tags were selected to be recognized by VHH (Goetzke et al., 2019, Nature Communications, 10(1), 1-12; Ling et al., 2019, Molecular Immunology, 114(July), 513-523), and a bispecific VHH (bi-VHH) for these two epitopes was created, enabling VHH-mediated dimerization. In addition, to determine the changes in protein localization, the Myc epitope tag was incorporated into the E3 ligase and the Flag epitope tag into the receptor. When co-expressed in HEK293T cells, none of the receptors co-localized with any of the E3 ligases. The E3 ligases were mainly localized in intracellular compartments, while the receptors were mainly localized on the cell membrane (data not shown). However, after 5 hours of treatment with bi-VHH, both RNF43 and RNF167 induced the removal of TβRII and EGFR from the cell membrane. Furthermore, in bafilomycin-treated cells, which inhibit lysosome turnover, the internalized proteins co-clustered in the perinuclear region, indicating that E3 ligases and their targets accumulated in late endosome / lysosome structures (Figure 5A-D). These findings suggest that heterobifunctional molecules such as bi-VHH can be used to intentionally dimerize transmembrane E3 ligases with selected transmembrane receptors, thereby inducing receptor removal from the cell surface.

[0422] [Example 3] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units / mL penicillin, and 100 μg / mL streptomycin (Invitrogen). Cells were cultured at 37°C and 5% CO2. Transfection was performed using FuGENE 6 (Promega) or Effectene (Qiagen) according to the manufacturer's protocol.

[0423] Constructs and antibodies E6-Flag-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), receptor tyrosine protein kinase FLT3 (FLT-3), programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1), as well as Alpha-myc-RNF43, RNF128, RNF130, and RNF167 were obtained by subcloning using Q5 High-Fidelity 2× Master Mix (NEB). All constructs were sequenced. For immunofluorescence (IF), the following primary antibodies were used: rabbit anti-Flag or mouse anti-Flag (Sigma-Aldrich). Primary antibodies were diluted according to the manufacturer's instructions. Secondary antibodies used for IF were used at a 1:300 ratio (Life Technologies).

[0424] Immunofluorescence and confocal microscopy HEK293T cells were grown on laminin (Sigma-Aldrich) coated glass coverslips in 24-well plates. Six hours after transfection, cells were incubated overnight with 50 nM bi-VHH (VHH Alpha-(G4S)3-VHH E6). After treatment, cells were washed twice with warmed medium and fixed with 4% formaldehyde in 0.05 M phosphate buffer pH 7.4. Cells were blocked at room temperature (RT) for 30 minutes with a buffer containing 2% BSA in PBS. Subsequently, cells were incubated in blocking buffer with primary antibody against Flag at room temperature for 1 hour, and then with secondary antibody at room temperature for 1 hour. Cells were mounted on Prolong Diamond (Life Technologies) and images were acquired using an LSM700 confocal microscope with a 5x objective lens or an EVOS-M5000 microscope with a 20x objective lens. Images were analyzed and processed in ImageJ.

[0425] Results and Discussion Surface removal of the target becomes possible through forced dimerization using bispecific VHH, which is achieved through a specific combination of E3 ligase and the target. To screen for further candidate E3 ligase-receptor combinations, we constructed Alpha-Myc-RNF128, Alpha-Myc-RNF130, E6-Flag-CTLA-4, E6-Flag-FLT-3, E6-Flag-PD-1, and E6-Flag-PD-L1 in addition to previously constructed constructs. When co-expressed in HEK293T cells, all of CTLA-4, FLT-3, PD-1, and PD-L1 localized to the cell surface. After overnight treatment with bi-VHH, the following E3-target combinations resulted in removal of the surface targets: CTLA-4 and RNF167; FLT-3 and RNF43, RNF128, or RNF167; PD-1 and RNF128, RNF130, or RNF167; and PD-L1 and RNF43, RNF128, or RNF130 (Figures 6A-D). These findings expand the scope of use for heterobifunctional molecules like bi-VHH to intentionally dimerize various transmembrane E3 ligases with selected transmembrane receptors, thereby inducing the removal of these receptors from the cell surface. Furthermore, these findings highlight that not all combinations are necessarily effective.

[0426] [Example 4] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were cultured in RPMI (Invitrogen) supplemented with 10% fetal bovine serum (GE Healthcare), 2 mM UltraGlutamine (Lonza), 100 units / mL penicillin, and 100 μg / mL streptomycin (Invitrogen). Cells were cultured at 37°C and 5% CO2. Transfection was performed using FuGENE 6 (Promega) or Effectene (Qiagen) according to the manufacturer's protocol.

[0427] Constructs and antibodies E6-Flag-Snorkel CKLF-like Marvell transmembrane domain-containing family protein 6 (CMTM6) was obtained by subcloning using Q5 High-Fidelity 2× Master Mix (NEB). The constructs were sequenced. Immunofluorescence (IF) was performed using a mouse anti-Flag (Sigma-Aldrich) primary antibody. The primary antibody was diluted according to the manufacturer's instructions. The secondary antibody used for IF was used at a 1:300 ratio (Life Technologies).

[0428] Immunofluorescence and confocal microscopy HEK293T cells were grown on laminin (Sigma-Aldrich) coated glass coverslips in 24-well plates. Six hours after transfection, cells were incubated overnight with 50 nM bi-VHH (VHH Alpha-(GGGGS)3-VHH E6). Post-treatment, cells were washed twice with warm medium and fixed with 4% formaldehyde in 0.05 M phosphate buffer at pH 7.4. Cells were blocked at room temperature (RT) for 30 minutes with a buffer containing 2% BSA in PBS. Subsequently, cells were incubated with a primary antibody against Flag at room temperature for 1 hour, followed by incubation with a secondary antibody in blocking buffer at room temperature for 1 hour. Cells were mounted on Prolong Diamond (Life Technologies) and images were acquired using an LSM700 confocal microscope with a 5x objective lens. Images were analyzed and processed in ImageJ.

[0429] Results and Discussion RNF43 and RNF128 induce cell surface removal of the multispan receptor CMTM6 during forced dimerization using bispecific VHH. To expand the applicability of the heterobifunctional molecule of the present invention to type II or type III transmembrane proteins, the Snorkel tag was used. Type II and type III transmembrane proteins are located intracellularly at their N-terminus, or both their N-terminus and C-terminus, respectively. To detect their surface expression, the Snorkel tag can be added to the intracellular N-terminal or C-terminal region, enabling protein tagging and extracellular detection without disrupting their structure or intracellular localization. The Snorkel tag consists of a transmembrane domain (TMD) surrounded by a linker region and two epitope tags. Depending on the multispan protein of interest, the inventors incorporated the Alpha-Myc tag for multispan E3 ligases or the E6-Flag tag for multispan targets (Figure 7A). As an example, the inventors generated E6-Flag-Snorkel-CMTM6 in addition to previously generated constructs. When co-expressed in HEK293T cells, CMTM6 localized to the cell surface. In overnight treatment with bi-VHH, RNF43, and to a lower degree RNF128, were able to have CMTM6 removed from the surface when treated with bi-VHH (Figure 7B).

[0430] These findings expand the range of heterobifunctional molecules, such as bi-VHH, that can be used to intentionally dimerize multispan receptors like CMTM6 with various transmembrane E3 ligases, thereby inducing the removal of these receptors from the cell surface. Furthermore, these findings reiterate the need to screen for effective E3 ligase-target combinations, as not all combinations are effective.

[0431] [Example 5] To verify the promising combinations obtained from the above screening in physiological conditions, C Using RISPR / Cas9 technology, we express endogenously tagged E3 ligases and targets. Prepare a cancer cell line. The inventors use guide RNA and Alpha-Myc or E It is used in combination with 6-Flag tagged donor DNA, and within the E3 ligase or target Between the signal peptide (SP) at the gene locus and the coding sequence of the first mature amino acid This facilitates the insertion of these tags (Figure 7A). Using these cell lines, bi- Removal of endogenous targets from the cell surface by forced dimerization with VHH, using microscopy and F Evaluate using either ACS or Western blotting (Figure 7B). Examples of the inventions of this application include the following: [1] A method for identifying effective combinations of transmembrane E3 ubiquitin ligase and membrane-bound proteins, wherein the combination is effective when the transmembrane E3 ubiquitin ligase can reduce the surface level of the membrane-bound protein, preferably by ubiquitination of the membrane-bound protein, upon co-binding of the transmembrane E3 ubiquitin ligase to a heterobifunctional molecule, and the method is a) A step of providing cells that express the transmembrane E3 ubiquitin ligase and the membrane-bound protein on their cell surface; b) The cells are exposed to the heterobifunctional molecule in the step, the heterobifunctional molecule is i) A first binding domain capable of specifically binding to the extracellular portion of the transmembrane E3 ubiquitin ligase; and ii) A second binding domain that can specifically bind to the extracellular portion of the membrane-bound protein. Steps including; c) A step of determining the surface level of the membrane-bound protein in the cell. A method comprising the following, wherein a decrease in the surface level of the membrane-bound protein indicates that the combination is an effective combination, and the decrease is preferably a decrease compared to the surface level of the membrane-bound protein in the cell prior to step b). [2] The method according to [1] above, wherein the membrane-bound protein is a transmembrane protein. [3] The method according to [1] or [2] above, wherein the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128. [4] - The transmembrane E3 ubiquitin ligase comprises a first extracellular non-natural epitope tag, and the first binding domain of the heterobifunctional molecule binds to the first non-natural epitope tag; and - The membrane-bound protein includes a second extracellular non-natural epitope tag, and the second binding domain of the heterobifunctional molecule binds to the second non-natural epitope tag. The method described in any one of the above [1] to [3], wherein at least one of the above is the method described in [1] to [3]. [5] The method according to [4] above, wherein the first and second non-natural epitope tags are different tags. [6] The method according to [4] or [5] above, wherein the first non-natural epitope tag is at least one of an alpha tag and an E6 tag, and / or the second non-natural epitope tag is at least one of an alpha tag and an E6 tag. [7] At least one of the first and second non-natural epitope tags is the transmembrane E3 ubiquitin ligase and the membrane-bound protein, respectively. i) N-terminus; ii) C-terminus; and / or iii) Extracellular loop region The method described in any one of the above [4] to [6], wherein the method is located in at least one of the above. [8] The method according to any one of the above [1] to [7], wherein the heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody. [9] The method according to [8], wherein the first binding domain of the heterobifunctional molecule is anti-Alpha VHH and the second binding domain is anti-E6 VHH, or the first binding domain of the heterobifunctional molecule is anti-E6 VHH and the second binding domain is anti-Alpha VHH.

[10] The method according to any one of the above [1] to [9], wherein the membrane-bound protein comprises a third non-natural epitope tag and / or the transmembrane ubiquitin E3 ligase comprises a fourth non-natural epitope tag, preferably the third and / or fourth epitope tag being at least one of His-tag, FLAG-tag, and myc-tag.

[11] The method according to any one of the above [1] to

[10] , wherein the cell surface level of the membrane-bound protein in step c) is determined by detecting the protein on the cell surface, preferably by immunofluorescence.

[12] The method according to any one of the above [1] to

[11] , wherein the combination is effective when the cell surface level of the membrane-bound protein is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least about 95% compared to the cell surface level of the membrane-bound protein before step b), preferably by at least about 60%, 70%, 80%, 90%, or at least about 95% compared to the cell surface level of the membrane-bound protein before step b).

[13] In step a), the first and second cells are provided, - The first cell expresses a first transmembrane E3 ubiquitin ligase and a first membrane-bound protein on its cell surface; and - The second cell expresses a second transmembrane E3 ubiquitin ligase and a first membrane-bound protein on its cell surface. The first and second transmembrane E3 ubiquitin ligases are different ligases containing the same first extracellular non-natural epitope tag; In step b), the first and second cells are exposed to a heterobifunctional molecule, and the heterobifunctional molecule i) a first binding domain capable of specifically binding to the first extracellular non-natural epitope tag; ii) The extracellular portion of the membrane-bound protein, preferably a second binding domain capable of specifically binding to the second non-natural epitope tag, Includes, The method according to any one of the above [4] to

[11] , wherein in step c), the surface levels of the membrane-bound protein in the first and second cells are determined, and the combination is valid if the cell surface level of the membrane-bound protein in the first cell is reduced by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least about 95% compared to the cell surface level of the membrane-bound protein in the second cell after step b).

[14] A third, fourth, or further cell is provided, expressing a third, fourth, or further transmembrane E3 ubiquitin ligase and the first membrane-bound protein, respectively, on the cell surface. The transmembrane E3 ubiquitin ligase is a different ligase containing the same first extracellular non-natural epitope tag. The combination is effective if the cell surface level of the membrane-bound protein in the first cell decreases by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least about 95% compared to the cell surface level of the membrane-bound protein in the second, third, fourth, and further cells after step b). Preferably, the method according to

[13] above, wherein the method is carried out in a multiplexed manner.

[15] The method according to any one of the above [1] to

[14] , wherein the reduction in the surface level of the membrane-bound protein is determined by a reduction in the total amount of the membrane-bound protein in the cell, preferably by microscopic examination, biochemical analysis and / or FACS.

[16] The method according to any one of the above [1] to

[15] , wherein the cells provided in step a) overexpress, and if necessary permanently overexpress, at least one of the transmembrane E3 ubiquitin ligase and the membrane-bound protein.

[17] The method according to any one of the above [1] to

[16] , wherein the cells provided in step a) express the transmembrane E3 ubiquitin ligase and the membrane-bound protein at endogenous levels.

[18] The method according to

[17] , wherein in the cells provided in step a), the genomic sequence encoding the transmembrane E3 ubiquitin ligase is modified to incorporate the first, and optionally fourth, sequences encoding non-natural epitope tags.

[19] The method according to

[17] or

[18] , wherein in the cells provided in step a), the genomic sequence encoding the membrane-bound protein is modified to incorporate the second, and optionally third, non-natural epitope tag sequences.

[20] The method according to any one of the above [1] to

[19] , wherein the heterobifunctional molecule comprises a peptide linker between the first binding domain and the second binding domain, preferably the peptide linker is (GGGGS)n, where n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, and preferably n is 3 or 5.

[21] A heterobifunctional molecule comprising a first and a second binding domain, i) The first binding domain can specifically bind to transmembrane E3 ubiquitin ligase; and ii) The second binding domain can specifically bind to transmembrane proteins, A heterobifunctional molecule in which the transmembrane E3 ligase and the membrane-bound protein are an effective combination, as determined by the method described in any one of the above [1] to

[20] .

[22] The heterobifunctional molecule described in

[21] above, which binds to the extracellular portion of the transmembrane E3 ubiquitin ligase and the extracellular portion of the membrane-bound protein.

[23] The heterobifunctional molecule according to

[21] or

[22] , wherein the transmembrane protein is a receptor, preferably a receptor involved in at least one of cancer, autoimmune diseases, neurological disorders and inflammatory disorders.

[24] A heterobifunctional molecule according to any one of the above

[21] to

[23] , which is a bispecific antibody, preferably a bispecific nanobody.

[25] A heterobifunctional molecule as described in any one of the above

[21] to

[24] for use as a pharmaceutical.

Claims

1. A method for identifying effective combinations of transmembrane E3 ubiquitin ligase and membrane-bound proteins, wherein the combination is effective when the transmembrane E3 ubiquitin ligase, upon simultaneous binding to a heterobifunctional molecule, can reduce the surface level of the membrane-bound protein by ubiquitinating the protein, and the method is as follows: a) Providing cells that express the transmembrane E3 ubiquitin ligase and the membrane-bound protein on their cell surface; b) The cells are exposed to the heterobifunctional molecule in the step of exposing them to the heterobifunctional molecule, i) A first binding domain capable of specifically binding to the extracellular portion of the transmembrane E3 ubiquitin ligase; and ii) A second binding domain that can specifically bind to the extracellular portion of the membrane-bound protein. Steps including; c) A step of determining the surface level of the membrane-bound protein in the cell. A method comprising the following, wherein a decrease in the surface level of the membrane-bound protein indicates that the combination is an effective combination, and the decrease is at least 10% compared to the surface level of the membrane-bound protein in the cell prior to step b).

2. The method according to claim 1, wherein the membrane-bound protein is a transmembrane protein.

3. The method according to claim 1 or 2, wherein the transmembrane E3 ubiquitin ligase is selected from the group consisting of RNF43, RNF167, ZnRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF130, and RNF128.

4. - The transmembrane E3 ubiquitin ligase comprises a first extracellular non-natural epitope tag, and the first binding domain of the heterobifunctional molecule binds to the first non-natural epitope tag; and - The membrane-bound protein contains a second extracellular non-natural epitope tag, and the second binding domain of the heterobifunctional molecule binds to the second non-natural epitope tag. The method according to any one of claims 1 to 3, wherein at least one of the following is the method according to claim 1 to 3.

5. The method according to claim 4, wherein the first and second non-natural epitope tags are different tags.

6. The method according to claim 4 or 5, wherein the first non-natural epitope tag is at least one of an alpha tag and an E6 tag, and / or the second non-natural epitope tag is at least one of an alpha tag and an E6 tag.

7. At least one of the first and second non-natural epitope tags is a transmembrane E3 ubiquitin ligase and a membrane-bound protein, respectively. i) N-terminus; ii) C-terminus; and / or iii) Extracellular loop region The method according to any one of claims 4 to 6, wherein the method is located in at least one of the following:

8. The method according to any one of claims 1 to 7, wherein the heterobifunctional molecule is a bispecific antibody containing an Fc region.

9. The method according to claim 8, wherein the first binding domain and the second binding domain are domains of a heavy chain antibody.

10. The method according to claim 9, wherein the bispecific antibody is a bispecific nanobody.

11. The method according to claim 10, wherein the first binding domain of the heterobifunctional molecule is anti-Alpha VHH and the second binding domain is anti-E6 VHH, or the first binding domain of the heterobifunctional molecule is anti-E6 VHH and the second binding domain is anti-Alpha VHH.

12. The method according to any one of claims 1 to 9, wherein the membrane-bound protein comprises a third non-natural epitope tag, and / or the transmembrane ubiquitin E3 ligase comprises a fourth non-natural epitope tag.

13. The method according to claim 12, wherein the third and / or fourth epitope tag is at least one of His-tag, FLAG-tag, and myc-tag.

14. The method according to any one of claims 1 to 10, wherein the cell surface level of the membrane-bound protein in step c) is determined by detecting the protein on the cell surface.

15. In step a), first and second cells are provided. - The first cell expresses a first transmembrane E3 ubiquitin ligase and a first membrane-bound protein on its cell surface; and - The second cell expresses the second transmembrane E3 ubiquitin ligase and the first membrane-bound protein on its cell surface. The first and second transmembrane E3 ubiquitin ligases are different ligases containing the same first extracellular non-natural epitope tag; In step b), the first and second cells are exposed to a heterobifunctional molecule, and the heterobifunctional molecule i) a first binding domain capable of specifically binding to the first extracellular non-natural epitope tag; ii) A second binding domain that can specifically bind to the extracellular portion of the membrane-bound protein and Includes, The method according to any one of claims 4 to 14, wherein in step c), the surface levels of the membrane-bound protein in the first and second cells are determined, and the combination is effective if the cell surface level of the membrane-bound protein in the first cell is reduced by at least about 10% compared to the cell surface level of the membrane-bound protein in the second cell after step b).

16. The method according to claim 15, wherein the second binding domain is specifically capable of binding to the second non-natural epitope tag.

17. A third, fourth, or further cell is provided, each expressing a third, fourth, or further transmembrane E3 ubiquitin ligase and the first membrane-bound protein on its cell surface. The transmembrane E3 ubiquitin ligase is a different ligase containing the same first extracellular non-natural epitope tag. The combination is effective if the cell surface level of the membrane-bound protein in the first cell is reduced by at least about 10% compared to the cell surface level of the membrane-bound protein in the second, third, fourth, and further cells after step b), The method according to claim 15 or 16, wherein the above method is carried out in a multiplexed manner.

18. The method according to any one of claims 1 to 14, wherein the decrease in the surface level of the membrane-bound protein is determined by a decrease in the total amount of the membrane-bound protein in the cell.

19. The method according to any one of claims 1 to 18, wherein the cells provided in step a) overexpress, and if necessary permanently overexpress, at least one of the transmembrane E3 ubiquitin ligase and the membrane-bound protein.

20. The method according to any one of claims 1 to 19, wherein the cells provided in step a) express the transmembrane E3 ubiquitin ligase and the membrane-bound protein at endogenous levels.

21. The method according to claim 20, wherein in the cells provided in step a), the genomic sequence encoding the transmembrane E3 ubiquitin ligase is modified to incorporate the first, and optionally fourth, non-natural epitope tag sequences.

22. The method according to claim 20 or 21, wherein in the cells provided in step a), the genomic sequence encoding the membrane-bound protein is modified to incorporate the second, and optionally third, sequences encoding the non-natural epitope tag.

23. The method according to any one of claims 1 to 19, wherein the heterobifunctional molecule includes a peptide linker between the first binding domain and the second binding domain.

24. The method according to claim 23, wherein the peptide linker is (GGGGS)n, where n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

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