Reverse turboid

Abstract

Described herein are fusion protein(s), e.g., fusion protein(s) comprising: a protein of interest described herein, e.g., a protein that is to be degraded, a variant thereof, or an enzymatically active portion thereof, genetically fused to a Proximity Labeling Enzyme described herein, e.g., such as a promiscuous Proximity Labeling Enzyme. Also described are polynucleotide sequence(s) encoding the fusion protein(s), vector(s) comprising the polynucleotide sequence(s), and cells comprising the vector(s). Also described herein are methods of using the fusion protein(s), polynucleotide sequence(s), vector(s), and cell(s).

Description

[0001] Monte Rosa Therapeutics AG

[0002] F&R Ref.: 52271 -0033W01 PCT Application

[0003] REVERSE TURBOID CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No.

[0005] 63 / 745,975, filed on January 16, 2025. The entire contents of the foregoing are incorporated herein by reference.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named “52271-0033W01_SL_ST26.XML.” The XML file, created on January 14, 2026, is 9,899 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0008] TECHNICAL FIELD

[0009] Described herein are fusion protein(s), e.g., fusion protein(s) comprising: i) a protein of interest, and ii) a Proximity Labeling Enzyme, e.g., a promiscuous biotinylation enzyme. Also described are polynucleotide sequence(s) encoding the fusion protein(s), vector(s) comprising the polynucleotide sequence(s), and cells comprising the vector(s). Also described herein are methods of using the fusion protein(s), polynucleotide sequence(s), vector(s), and cell(s).

[0010] BACKGROUND

[0011] The ubiquitin proteasome system can be manipulated, e.g., with various small molecules to trigger interaction, e.g., targeted degradation of specific proteins of interest. While many E3 ligase substrate receptors are known, there is a need for methods for identifying and / or validating new E3 ligase substrate receptors.

[0012] SUMMARY

[0013] Provided herein are systems for detecting or validating modulator-dependent proximitybased interactions between a protein of interest and an E3 ligase substrate receptor, the system comprising: a) cell(s) expressing one or more fusion proteins, each fusion protein comprising the protein and a proximity labeling enzyme; and b) an E3 ligase binding modulator.Monte Rosa Therapeutics AG

[0014] F&R Ref.: 52271 -0033W01 PCT Application

[0015] Also described herein are methods for detecting the interaction between a protein of interest and one or more E3 ligase substrate receptor(s), the method comprises: a) providing (i) cell(s) expressing a fusion protein comprising the protein and a proximity labeling enzyme; and (ii) optionally, an E3 ligase binding modulator; b) incubating the cell(s) and, optionally, the modulator under conditions effective for the proximity labeling enzyme to label protein(s) in the proximity of the fusion protein; and c) determining the presence and / or amount of labeled E3 ligase(s), thereby detecting the interaction of an E3 ligase and the protein.

[0016] Also described are methods for identifying E3 ligase substrate receptors(s) that interact with a protein of interest in a modulator-dependent manner or not, the method comprising:

[0017] I) a) providing i) first cell(s) one or more fusion protein(s) each comprising the protein and a proximity labeling enzyme; and ii) an E3 ligase binding modulator; b) incubating the cell(s) and modulator under conditions effective for the proximity labeling enzyme to label the one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein(s); and c) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s);

[0018] II) a) providing i) second cell(s) expressing the fusion protein(s); and ii) a negative control for the modulator; b) incubating the second cell(s) and negative control under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein(s); and c) detecting the presence and / or amount of labeled E3 ligase substrate receptors(s);

[0019] III) comparing the presence and / or amount of labeled E3 ligase substrate receptors(s), from step I to those in step II; and

[0020] IV) identifying one or more E3 ligase substrate receptors(s) that interact with the protein in a modulator-dependent manner based on the step III comparison.

[0021] Described herein are also methods for validating a predicted modulator-dependent interaction between one or more E3 ligase substrate receptor(s) and a protein of interest, the method comprising:

[0022] I) a) providing i) first cell(s) expressing a fusion protein comprising the protein and a proximity labeling enzyme; and ii) an E3 ligase binding modulator; b) incubating the first cell(s) and modulator under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein; and c) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s);

[0023] II) a) providing i) second cell(s) expressing the fusion protein; and ii) a negative control for the modulator; b) incubating the second cell(s) and negative control under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptor(s)Monte Rosa Therapeutics AG

[0024] F&R Ref.: 52271 -0033W01 PCT Application

[0025] in the proximity of the fusion protein; and c) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s);

[0026] III) comparing the presence and / or amount of labeled E3 ligase substrate receptor(s), from step I with those from step II; and

[0027] IV) validating the predicted modulator-dependent interaction between one or more E3 ligase substrate receptor(s) and the protein or not based on the comparing of step III.

[0028] In some embodiments of the methods above, the negative control for the modulator is DMSO.

[0029] In some embodiments of the methods above, the conditions effective for the proximity labeling enzyme to label protein(s) in the proximity of the fusion protein comprise incubating in a composition comprising a substrate for the proximity labeling enzyme. In some embodiments, the substrate for the proximity labeling enzyme is biotin.

[0030] In some embodiments of the methods above, the incubation is carried out in the presence of a 26S proteasome inhibitor. In some embodiments, the 26S proteasome inhibitor is selected from the group consisting of bortezomib, ixazomib, carfilzomib, MG-132, MG-115, oprozomib, marizomib, MLN9708, and combinations thereof.

[0031] In some embodiments of the methods above, the detecting the presence and / or amount of labeled protein(s) comprises quantitative mass spectrometry and / or Western Blot analysis.

[0032] In some embodiments of the methods above, the the protein is identified as having a modulator-dependent interaction with an E3 ligase substrate receptor, or vice-versa, when the amount of E3 ligase substrate receptor that is labeled after incubation with the modulator is greater than the amount of E3 ligase substrate receptor that is labeled after incubation under the same conditions with a negative control for the modulator.

[0033] In some embodiments of any one of the above-described methods or system, the proximity labeling enzyme is a promiscuous biotinylation enzyme. In some embodiments, the promiscuous biotinylation enzyme is selected from TurboID, BioID, BioID2, miniTurbo, AirlD, BASU, microID, ultralD, HRP, APX, APEX, APEX2 and variations thereof.

[0034] In some embodiments of any one of the above-described methods or system, the one or more of the fusion protein(s) further comprises a linker between the protein and the proximity labeling enzyme. In some embodiments, the linker(s) are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long.

[0035] In some embodiments of any one of the above-described methods or system, the one or more of the fusion protein(s) further comprises a self-cleaving peptide, optionally T2A and / or aMonte Rosa Therapeutics AG

[0036] F&R Ref.: 52271 -0033W01 PCT Application

[0037] detection label, optionally a fluorescent protein, optionally green fluorescent protein (GFP), optionally eGFP.

[0038] In some embodiments of any one of the above-described methods, the modulator is a molecular glue degrader.

[0039] In some embodiments of any one of the above-described methods or system, the cell is selected from the group consisting of HEK293T cells, CAL51 cells, HCT116 cells, MCF7 cells, SKMEL28 cells, THP1 cells, U937 cells, and combinations thereof.

[0040] Also described herein are cells of any one of the above-described methods or system. Also described herein are fusion proteins of any one of the above-described methods or system.

[0041] Also described herein are nucleic acid sequences encoding the fusion proteins of any one of the above-described methods or system. Also described are vectors comprising the nucleic acids. In some embodiments, described herein are cells comprising the vectors.

[0042] Also described herein are protein complexes comprising the fusion protein and the protein of the system or methods as described above. In some embodiments, described herein are cells comprising the protein complex.

[0043] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.

[0044] Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0045] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0046] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount ofMonte Rosa Therapeutics AG

[0047] F&R Ref.: 52271 -0033W01 PCT Application

[0048] something present in addition to determining whether it is present or absent depending on the context.

[0049] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0051] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0052] DESCRIPTION OF DRAWINGS

[0053] Figure 1. A schematic showing the methods described herein for identifying and / or validating interactions between a protein of interest and an E3 ligase substrate receptor(s).

[0054] Figure 2A. A western blot showing that a fusion protein comprised of TurboID and MEK1 was able to interact with the E3 ligase substrate receptor, CRBN, in the presence of the molecular glue degrader (MGD), Compound A.

[0055] Figure 2B. A western blot showing that a fusion protein comprised of TurboID and Protein of Interest 1 (the FRB domain of mTOR) was able to interact with the E3 ligase substrate receptor, CRBN, in the presence of the MGDs, Compound B or Compound C.

[0056] Figure 2C. A western blot showing that a fusion protein comprised of TurboID and Protein of Interest 2 was able to interact with the E3 ligase substrate receptor, CRBN, in the presence of the MGD, Compound D.

[0057] Figure 2D. A TMT proteomics volcano plot showing that a fusion protein comprised of TurboID and CCNE1 was able to interact with the E3 ligase substrate receptor, CRBN, in the presence of the MGDs, Compounds F and G, but not in the presence of Compound E.Monte Rosa Therapeutics AG

[0058] F&R Ref.: 52271 -0033W01 PCT Application DETAILED DESCRIPTION

[0059] The study of protein interactions requires high sensitivity. Described herein are methods for determining protein interactions in the presence / absence of molecules, wherein the interactions are detected based on the association of a Proximity Labeling Enzyme and an E3 ligase substrate receptor, resulting in the labeling of the latter.

[0060] FUSION PROTEIN(S)

[0061] The fusion protein(s) described herein comprise a protein of interest described herein, e.g., a protein that is to be degraded, a variant thereof, or an enzymatically active portion thereof, genetically fused to a Proximity Labeling Enzyme described herein, e.g., such as a promiscuous Proximity Labeling Enzyme.

[0062] As used herein, an “enzymatically active portion” of a protein of interest is one that retains the ability to function in its normal way.

[0063] In some embodiments, the fusion protein comprises, from N-terminal to C-terminal: (a) a Proximity Labeling Enzyme described herein, e.g., such as a promiscuous Proximity Labeling Enzyme; and (b) protein of interest described herein, e.g., a protein that is to be degraded, a variant thereof, or an enzymatically active portion thereof. In some embodiments, the protein of interest does not comprise a leading methionine (M).

[0064] In some embodiments, the fusion protein comprises, from N-terminal to C-terminal: (a) a protein of interest described herein, e.g., a protein that is to be degraded, a variant thereof, or an enzymatically active portion thereof; and (b) a Proximity Labeling Enzyme described herein, e.g., such as a promiscuous Proximity Labeling Enzyme. In some embodiments, the Proximity Labeling Enzyme does not comprise a leading methionine (M).

[0065] In some embodiments, the fusion protein comprises a linker (e.g., a flexible linker) between the protein of interest and the Proximity Labeling Enzyme. In some embodiments, the linker is from 1 to 20 amino acids long, e.g., in some embodiments the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the linker comprises GSG, or variations thereof (e.g., SGGGGSGGGGSGGGGS).

[0066] In some embodiments, the fusion protein comprises or consists of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the fusion protein comprises or consists of an amino acid sequence at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.Monte Rosa Therapeutics AG

[0067] F&R Ref.: 52271 -0033W01 PCT Application

[0068] Proteins of Interests

[0069] The methods described herein are useful for identifying and validating E3 ligases, that selectively interact with a known protein of interest in the presence of a molecule. Identification and validation of the E3 ligase involves the use of fusion proteins that comprise a protein of interest.

[0070] In some embodiments, the protein of interest (also referred to herein as an E3 ligase binding target) is a protein comprising a structural feature on its surface that mediates its recruitment and degradation by an E3 ligase complex (i.e., a degron).

[0071] In some embodiments, the protein of interest is a protein comprising an E3 ligase-accessible loop, e.g., a cereblon-accessible loop, e.g., a G-loop.

[0072] In some embodiments, the protein of interest is a protein listed in Table 1 or a variant, derivative, ortholog, or homolog thereof.

[0073] Table 1. Examples of Proteins of Interest

[0074]

[0075] Monte Rosa Therapeutics AG

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[0103] Monte Rosa Therapeutics AG

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[0169] Monte Rosa Therapeutics AG

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[0192]

[0193] Proximity Labeling Enzymes

[0194] The fusion proteins as described herein also comprise a Proximity Labeling Enzyme. Proximity Labeling Enzyme(s) (PLEs), upon addition of a small-molecule substrate, such as biotin, initiate covalent tagging of endogenous proteins within a few nanometers of the promiscuous enzyme. PLEs are described, e.g., in Branon et al., “Efficient Proximity Labeling in Living Cells and Organisms with TurboID,” Nature Biotechnology (2018) doi:10.1038 / nbt.4201.

[0195] Promiscuous Biotinylation Enzymes

[0196] In some cases, the proximity labeling enzyme is a promiscuous biotinylation enzyme. Bifunctional ligase / repressor BirA, e.g., E. coli BirA acts both as a biotin— [acetyl-CoA-carboxylase] ligase and a biotin-operon repressor. In the presence of ATP, BirA activates biotin to form the BirA-biotinyl-5'-adenylate (BirA-bio-5'-AMP or holoBirA) complex. HoloBirA can either transfer the biotinyl moiety to the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase, or bind to the biotin operator site and inhibit transcription of the operon. The wild type E. coli BirA biotinylates only a single cellular protein. See, e.g., Choi-Rhee et al., “Promiscuous Protein Biotinylation by Escherichia coli biotin protein ligase,” Protein Science 13(11):3043-50 (2004).

[0197] Wild-type E. coli BirA has the amino acid sequence of SEQ ID NO: 1 :

[0198] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGY SLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGR RGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPND LYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDR NTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQG ALLLEQDGIIKPWMGGEISLRSAEK.

[0199] In some embodiments, the proximity labeling enzyme is a promiscuous biotin ligase, e.g., a mutant of E. coli BirA that attaches biotin to more proteins than does the wild-type BirA, preferably a large number of cellular proteins, preferably in vivo, e.g., as described in Branon etMonte Rosa Therapeutics AG

[0200] F&R Ref.: 52271 -0033W01 PCT Application

[0201] al., “Efficient Proximity Labeling in Living Cells and Organisms with TurboID,” Nature Biotechnology (2018) doi:10.1038 / nbt.420L

[0202] In some embodiments, the promiscuous biotin ligase is TurboID of SEQ ID NO:2: MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGY SLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGS RGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPND LYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDR NTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGA LLLEQDGVIKPWMGGEISLRSAEK. See Branon et al., “Efficient Proximity Labeling in Living Cells and Organisms with TurboID,” Nature Biotechnology (2018) doi:10.1038 / nbt.420L In some embodiments, the promiscuous biotin ligase comprises or consists of SEQ ID NO:2. In some embodiments, the promiscuous biotin ligase comprises or consists of an amino acid sequence at least 80%, e.g., at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:2Error! Reference source not found..

[0203] In some embodiments, the promiscuous biotin ligase is AirlD of SEQ ID NO:3:

[0204] MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGY CLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGS RGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAETLQKLGADGVRVKWPN DLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDR NQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQG ALLLEQDGVIKPWIGGEISLRSA. See Kido et al., “AirlD, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions,” Elife 9:e54983 (2020). In some embodiments, the promiscuous biotin ligase comprises or consists of SEQ ID NO:3. In some embodiments, the promiscuous biotin ligase comprises or consists of an amino acid sequence at least 80%, e.g., at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:3.

[0205] EXPRESSION SYSTEMS

[0206] To use the fusion proteins described herein, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the fusion protein can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and / or expression.

[0207] Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the fusion protein. The nucleic acid encoding the fusion protein can also be cloned into an expression vector, for administrationMonte Rosa Therapeutics AG

[0208] F&R Ref.: 52271 -0033W01 PCT Application

[0209] to a plant cell, fungal cell, bacterial cell, protozoan cell, or animal cell, preferably a mammalian cell or a human cell.

[0210] Thus, described herein are nucleic acid(s) encoding the fusion protein(s) described herein, vectors comprising the nucleic acid(s), and cells comprising the vector(s).

[0211] In some embodiments, the vector is a lentivirus vector. See, e.g., Milone et al., “Clinical Use of Lentiviral Vectors,” Leukemia 32: 1529-41 (2018). In some embodiments, the vector is a retrovirus vector. In some embodiments, the vector is a gamma retroviral vector. In some embodiments, the vector is a non-viral vector, e.g., a piggyback non-viral vector (PB transposon, see, e.g., Wu et al., “piggyback is a Flexible and Highly Active Transposon as Compared to Sleeping Beauty, Tol2, and Mosl in Mammalian Cells,” PNAS 103(41): 15008-13 (2006)), a sleeping beauty non-viral vector (SB transposon, see, e.g., Hudecek et al., “Going Non-Viral: the Sleeping Beauty Transposon System Breaks on Through to the Clinical Side,” Critical Reviews in Biochemistry and Molecular Biology 52(4):355-380 (2017)), or an mRNA vector.

[0212] To obtain expression, a sequence encoding a fusion protein is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, ALaboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

[0213] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of SV40, CMV, UBC, EFl A, PGK, and CAGG.

[0214] In some embodiments, the promoter is an inducible promoter. See, e.g., Kallunki et al., “How to Choose the Right Inducible Gene Expression System for Mammalian Studies?” Cells 8:796 (2019).

[0215] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. Atypical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the fusion protein and any signals required, e.g., for efficient polyadenylation of the transcript,Monte Rosa Therapeutics AG

[0216] F&R Ref.: 52271 -0033W01 PCT Application

[0217] transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

[0218] The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the fusion protein, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc.

[0219] Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0220] Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

[0221] Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the fusion protein.

[0222] In some embodiments, the cell(s) are stably transduced, transformed, or transfected. In some embodiments, the cell(s) are transiently transduced, transformed, or transfected.

[0223] In some embodiments, the cell(s) are selected from the group consisting of HEK293T cells, CAL51 cells, HCT116 cells, MCF7 cells, SKMEL28 cells, THP1 cells, U937 cells, and combinations thereof.

[0224] In some cases, the cell(s) are adherent. In some cases, the cell(s) are non-adherent.Monte Rosa Therapeutics AG

[0225] F&R Ref.: 52271 -0033W01 PCT Application

[0226] In some embodiments, the cell(s) are cancer cells. In some embodiments, the cell(s) are selected from the group consisting of NIHOVCAR3, HL60, CACO2, HEL, HEL9217, M0N0MAC6, LS513, A101D, C2BBE1, NCIH2077, 253 J, HCC827, ONCODG1, HS294T, NCIH1581, SLR21, SKBR3, T24, MCF7, MHHCALL2, NCIH1693, PATU8988S, PATU8988T, OPM2, CH157MN, 253JBV, GOS3, KPL1, HCC827GR5, PCM, PANC0213, MHHCALL3, NCIH1819, PLB985, NCIH1650, U343, SI 17, EHEB, SKNMC, U118MG, RDES, PANC0203, HS895T, MDAMB134VI, MV411, ACHN, GCIY, TOV112D, HEKTE, NCIH929, TE617T, A673, KARPAS299, HT1080, D283MED, DOHH2, OPM 1, ML1, SUPB15, PANC1005, HH, RERFLCMS, HS616T, SALE, OCIAML5, HCC4006, HS683, RECI, HS611T, 697, HS706T, MEG01, GRANTA519, KU812, U87MG, NCO2, MJ, MHHNB11, TE125T, BDCM, GDM1, G292CLONEA141B1, HS281T, MUTZ3, T3M4, ACCMESO1, SKES1, HS172T, NCIH684, PC3, OV56, NCIH2452, PANC0504, HPAFII, D341, G401, ZR751, GAMG, SIMA, RH41, KE37, GMS10, CAOV4, LOUCY, ALLSIL, JVM2, CAPAN2, KP3, NCIH3255, NCCSTCK140, HCC1187, SIGM5, OCIAML2, SU8686, VCAP, OAW28, EFM192A, HUPT3, HS863T, CHP212, NCIH2405, SUPT11, COV434, OCILY19, TO175T, KG1C, SLR20, LN319, NCIH1341, NALM19, HS229T, JH0S2, HS729, HS274T, HS940T, CHP126, 8MGBA, CFPAC1, PANC0327, PFEIFFER, SNU308, CAL29, HCC2429, RERFGC1B, SKLMS1, THP1, T47D, HS578T, SKNSH, HCC2935, JM1, M059K, NCIH2052, HS888T, SW1990, MHHCALL4, A4FUK, OCILY3, OSRC2, BT12, CORL105, GA10, SW579, PANCI, HS751T, KASUMI6, KE97, N0M01, RD, PRECLH, VMRCRCZ, TM87, JHUEM3, CAL62, TE159T, LOUNH91, NCIH660, HS766T, NCIH1618, HS839T, SCC9, SNU869, L363, HS343T, HS737T, NCIH2444, CORL311, SCC25, RCC10RGB, HDMYZ, BHT101, MFE280, KARPAS620, HS934T, SET2, HCC1599, TALL1, EOL1, HS255T, NMCG1, A204, COLO320, NH6, LP1, PK59, C8166, DETROIT562, U178, SNU1079, CADOES1, DAOY, CAL120, HUPT4, HS675T, LN382, JHESOAD1, JHH6, PL21, A375, MINO, SNU398, ASPC1, HCC1937, HS819T, ECC12, SUPM2, KPNYN, BICR31, HS822T, HS742T, KALS1, U251MG, DEL, CAKI2, PANC0403, SW1417, JHOM1, SCC4, HUGIN, HS600T, JK1, RT4, DANG, DKMG, BL41, SLR23, OCUM1, AU565, CL11, KMRC20, NCIH2887, LSI 034, COLO201, SCC15, LMSU, COV318, CORL279, DU4475, KELLY, SKNAS, RERFLCAI, UOK101, KASUMI1, CALU6, KP4, SNU213, HDLM2, SNU245, AM38, HP AC, SUDHL10, SLR24, SF539, HS852T, HS834T, HCC38, HCC1419, COV362, EWS502, SNU840, KP2, NCIH1755, A1207, HS840T, TOLEDO, SNU1033, NUDUL1, BT549, SNU466, NCIH209, OV90, NCIH841, KLE, NB4, EM2, OUMS23, NCIH889, NCIH2029, HNT34, SLR25, LAMA84, SNU1077, SNU5, WM115, ECGH0,Monte Rosa Therapeutics AG

[0227] F&R Ref.: 52271 -0033W01 PCT Application

[0228] HS688AT, PK1, EFO21, SKLU1, IMR32, NCIH2122, SKNBE2, KMRC3, HCC2108, KARPAS422, SNU886, TUHR14TKB, TE10, MPP89, PSN1, M0LM6, HT144, 42MGBA, JHOC5, SNU620, JURLMK1, NCIH1395, LN215, CCFSTTG1, EFM19, ISTMES2, YAPC, JHOM2B, DB, MSTO211H, 0CIAML3, NCIH3122, SR786, HCC461, HS870T, SKNFI, CL14, NCIH522, SNU668, KPNRTBM1, JVM3, QGP1, RPMI7951, HCC1500, COLO678, MKN1, HCC1428, TE15, CAPAN1, NCIH82, MKN45, JEKO1, NCIH69, MG63, NCIH508, SKHEP1, M0LM13, SKMM2, U2OS, SUDHL4, SKNDZ, NCIH226, SNU1105, M0LM16, SNU626, RL, P12ICHIKAWA, SKM1, HCC1143, G402, SF295, SNU478, NCIH647, NCIH1781, KMS12BM, T84, CORL24, OE33, SW780, SKRC20, KG1, TF1, NUDHL1, H4, LUDLU1, MHHES1, CALU3, HLF, NCIH2081, NCIH520, J82, TEN, RI1, NCIH2196, SKCO1, COLO800, BL70, NCIH747, K029AX, MEC1, U937, SNU685, TE5, OVSAHO, SAOS2, 769P, SNU1197, HS739T, NCIH1944, BICR6, NCIH838, PANC0813, SW1353, KMS28BM, SNU449, SW837, SNU475, SKMEL3, TC71, UACC62, KMS20, NCIN87, UO31, A704, TYKNU, NCIH1694, BV173, CAKI1, NCIH1915, EFE184, 0CIMY7, SW1088, LU65, MEI, CA46, SH4, RERFLCSQ1, OVKATE, LU99, KNS60, KPNSI9S, NCIH2228, NCIH1666, MESSA, MELHO, NCIH2085, TE8, MOLP2, HCC95, LN428, BCPAP, CAL54, CJM, TUHR10TKB, SNU8, SNU1196, NALM1, NCIH460, CAS1, SKMEL1, SNU216, HCC56, PK45H, YH13, SW1463, LI7, HSC2, RT112, HUH1, JHH4, MALME3M, SNU387, KNS81, HUH7, NCIH2170, RERFLCKJ, SNU182, VMRCRCW, GSU, KU1919, F36P, TE11, SW1116, SF767, NCIH716, MUTZ5, SNU423, OELE, TUHR4TKB, NCIH1792, KO52, EW8, SNU46, LS123, TCCPAN2, BICR16, SNB75, RKN, NCIH146, KE39, CORL88, HUT78, NCIH1299, CALU1, INA6, SNU1272, NCIH1092, HCC33, CAL78, SNU410, CAL33, PEER, 59M, NCIH2030, UMUC3, NCIH1184, KURAMOCHI, NCIH2171, HS821T, OVISE, ABC1, T173, DMS114, RS5, SNU61, NCIH2004RT, WSUDLCL2, BXPC3, BT20, SNU761, HUTU80, HS618T, HS606T, KMS34, HEYA8, SNU489, OE21, VMCUB1, HSC4, HT1197, BHY, SNU1076, IGR39, K562, HT29, SQ1, UACC893, A498, SIHA, AML193, A172, NCIH1836, ECC10, TDOTT, HCC78, EBC1, KHM1B, RCM1, SW1710, ST486, UACC812, ISTMES1, YKG1, T98G, G361, MDAMB436, FUOV1, HCC364, KMS27, JHH2, HCC1171, UACC257, C32, SNU16, COLO741, MCI 16, JHOS4, EPLC272H, NCIH1876, NCIH1975, KMS26, NCIH1437, NCIH2073, LN235, TM31, BC3C, DMS153, LN229, LCLC97TM1, TTC709, KMS21BM, PATU8902, SLR26, MIAPACA2, M07E, BEN, KY01, TE6, PECAPJ34CLONEC12, KYM1, COV644, SF126, NCIH2227, SUDHL6, HUT102, HOS, RVH421, SKMEL28, HS746T, OVCAR4, SNU1041, PECAPJ15, JHH1, MDAMB157, KNS42, SNU201, HCC1806, HEP3B217, U266B1, LCLC103H, NCIH596, IOMMLEE, YD8, KS1,Monte Rosa Therapeutics AG

[0229] F&R Ref.: 52271 -0033W01 PCT Application

[0230] HS944T, FU97, LN340, SNU119, RPMI8402, KYSE520, NCIH441, NCIH211, SKMEL31, CMK, HMEL, HDQP1, COLO829, JL1, OVMANA, TE1, NCIH28, 7860, IGR37, SW620, SUIT2, JJN3, RAJI, SF268, SUDHL8, A2780, KMS18, SCLC21H, SUDHL5, WM1799, CORL23, OVTOKO, SUDHL1, SKMES1, NCIH1355, HCC44, HCC70, SW900, SBC5, HUH6, IALM, LN443, NUGC4, NCIH1734, LN464, SW1573, MKN7, 0E19, SW948, A549, SNU1066, SNU503, KMRC1, L33, SNU878, CH, 0V7, RH18, HCC2814, HCC2157, SNU899, KYSE180, TE9, CORL47, 0VCAR8, A3KAW, DMS53, HCC1395, NCIH2882, RMUGS, L1236, DMS79, OAW42, LC1F, EKVX, P3HR1, SNU283, KMRC2, NCIH854, JIMT1, HCC1833, CA0V3, KMS11, SNU1214, TT2609C02, COLO68ON, NCIH2291, RMGI, TCCSUP, HMC18, SNUC1, YD10B, HT1376, HCC202, TE14, NCIH2066, KASUMI2, NCIH1963, SKMEL5, HCC2279, PECAPJ41CLONED2, NCIH1838, JHH5, PECAPJ49, SNU601, NCIH1385, GB1, HEPG2, A253, UBLC1, DM3, CORL95, NCIH1623, M0LP8, GSS, NCIH1703, SJSA1, DMS273, LOXIMVI, 0CIM1, NCIH196, JMSU1, L428, HCC2218, Gil, A427, MKN74, MDAMB175VII, LNZ308, NUGC2, YD38, MM1S, SH10TC, WM983B, NCIH1648, NCIH526, MDAMB231, LK2, P31FUJ, BICR56, TE441T, KIJK, RERFLCAD2, NCIH727, ONS76, KYSE30, HSC3, PC9, NCIH1105, NCIH2023, SEM, CAMA1, KYSE70, NCIH2126, DAUDI, LXF289, A2058, NCIH810, SHP77, RERFLCAD1, BFTC909, KATOIII, BICR22, M0LT13, MCAS, HLFA, CL40, HS695T, NCIH446, HS936T, BFTC905, COLO668, NB1, COLO679, L540, SNU738, HUH28, KYSE410, SKMEL30, SK0V3, COLO783, T3M10, HS939T, KMH2, NCIH524, RPMI8226, BT483, LN18, SW403, EJM, SKMEL24, KYSE140, KYSE510, HOP92, CAL12T, WM793, ZR7530, HUNS1, NCIH1436, HEC50B, CAL27, RH30, UMUC1, GCT, YD15, NCIH322, AM01, SCABER, HCC366, NCIH2087, SW480, HARA, DMS454, NCIH1373, FADU, HGC27, JHH7, MDAMB468, HS698T, MORCPR, NCIH1435, NCIH661, 0CIMY5, KYSE150, CAL51, CAL851, KNS62, HCC1954, NCIH358, HOP62, KMBC2, DBTRG05MG, COLO684, KYSE450, NCIH1048, CHAG0K1, HCC1195, NCIH1568, NCIH1930, NCIH510, HCC515, KYSE270, RS411, NCIH2347, MDAMB415, EB1, HCC15, MFE296, AGS, MELJUSO, IGR1, SW1783, MDAMB435S, T0V21G, NCIH2009, SF172, NCIH1793, KMM1, SW1271, HCC1438, NCIH1563, NCIH1651, NCIH1869, CL34, 647V, FTC238, SNU719, WM88, NCIH23, HCC1359, CAL148, FTC133, NCIH2106, 5637, ES2, SNU349, SNU520, JHUEM2, MDAMB453, NUGC3, NCIH2286, ESSI, HT, IPC298, NCIH1573, TE4, M0LT16, IM95, CMLT1, NCIH1339, RCHACV, BCP1, NCIH2172, DV90, HT55, BT474, JHUEM1, NCIH2110, HCC1569, HMCB, SNU1, SNU324, MDAMB361, MDST8, EFO27, PF382, NALM6, SKUT1, AN3CA, HEC1B, HPBALL, RKO, NAMALWA, NCIH650, HEC265, 0VK18, 2313287, TGBC11TKB, LOVO, NCIH2342,Monte Rosa Therapeutics AG

[0231] F&R Ref.: 52271 -0033W01 PCT Application

[0232] MDAPCA2B, SUPT1, HEC1A, SNU407, 22RV1, LS180, SW48, SNUC4, REH, ISHIKAWAHERAKLIO02ER, OC314, CCK81, M0LT3, RL952, IGR0V1, SNUC2A, COLO792, KM12, SNUC5, HCT116, HEC151, 639V, SNGM, HCC2450, HUCCT1, LNCAPCLONEFGC, EN, DU145, NCIH1155, DND41, GP2D, KCL22, HEC6, LS411N, HT115, MEWO, MFE319, SNU175, HEC108, SNU81, BICR18, JHUEM7, HEC59, JURKAT, HEC251, HCT15, CW2, SNU1040, 1321N1, 143B, 451LU, A673STAG2KO16, A673STAG2KO45, A673STAG2NT14, A673STAG2NT23, ACCS, AZ521, BECKER, BGC823, BJHTERT, BT16, C3A, CBAGPN, CGTHW1, CHL1, CHLA06ATRT, CHLA10, CHLA218, CHLA266, CHLA32, CHLA57, CHLA9, CHLA99, CMK115, CMK86, COGE352, COLO205, COLO699, COLO704, COLO775, COLO818, COLO849, CORL51, COV504, CPCN, CW9019, D384, D425, D458, D556, DERL2, DL, DL40, DLD1, DOV13, EB2, EVSAT, EWS834, F5, FEPD, GLC82, GRM, NCIH292, HCC1588, HCC1897, HCC2998, HCT8, HELA, HK2, HLC1, HLE, HN, HRT18, HS571T, HS604T, HTK, JR, KARPAS384, KCIM0H1, KD, KHYG, KLM1, KOPN8, KP1N, KP1NL, KPMRTRY, L82, LC1SQSF, M059J, MAC2A, MEC2, MKL1, MKL2, MOGGCCM, MOGGUVW, MOLT4, MON, M0N0MAC1, MOTN1, MSDASH1, MTA, MYLA, NCIH187, NCIH1993, NCIH2141, NHAHTDD, NKL, OC315, OC316, OCILYIO, OCILY12, OCILY132, OUMS27, OVCAR5, PCM6, CCLFPEDS0001T, CCLFPEDS0003T, PETA, PL45, R256, R262, RCC4, RPMI6666, RT11284, SCMCRM2, SF8657, SHSY5Y, SJRH30, SKMEL2, SKNEP1, SKPNDW, SKRC31, SMSCTR, SMZ1, SNB19, SNUC2B, STM9101, SUMB002, SUPHD1, TC32, TTC466, TIG3TD, TK10, TTC1240, TTC549, TTC642, U138MG, UMRC2, UMRC6NEO, UPCISCC090, UPCISCC152, UPCISCC154, UT7, UW228, VMRCLCD, VMRCLCP, WM2664, YMB1, 127399, FUJI, SW982, SYO1, YAMATO, BIN67, SCCOHT1, SCS214, CHLA258, TC106, COGAR359, Y79, CHLA15, COGN278, COGN305, NB1643, 8305C, 8505C, HA1E, PLCPRF5, TT, CME1, A431, ANGMCSS, BICR10, BICR78, C33A, C4I, C4II, CASKI, CHP134, COLO794, COV413A, DOTC24510, GIMEN, GP5D, H103, H157, HTCC3, JOPACA1, LAN2, LAN6, MB1, MCF10A, MDAMB330, ME180, MS751, NCIH1770, NCIH2135, NCIH345, NCIH847, NGP, NMB, OACM51, OCIC5X, OCIP5X, OV17R, PAI, PACADD119, PACADD135, PACADD137, PACADD159, PACADD161, PACADD165, PACADD188, PWR1E, RO82W1, RPMI2650, SCLC22H, SUM102PT, SUM1315MO2, SUM149PT, SUM159PT, SUM185PE, SUM190PT, SUM229PE, SUM44PE, SUM52PE, SUM225CWN, SW156, SW626, SW954, SW13, SW756, TOM, UMUC13, UMUC14, UMUC16, UMUC4, UMUC5, UMUC10, UMUC11, UMUC6, UMUC7, UMUC9, UMC11, UWB1289, VP229, WERIRB1, WPE1NA22, TC138, TC205, CCLFPEDS0008T, 921, A388, ASH3, BLUE1, BOKU, BONNA12, BPH1,Monte Rosa Therapeutics AG

[0233] F&R Ref.: 52271 -0033W01 PCT Application

[0234] CIO, C125PM, C75, C80, C84, C99, CI, CII, CORL32, CORL321, EGI1, EMTOKA, ESO26, ESO51, FARAGE, FLO1, H357, H376, H413, HCA1, HCC1008, HCS2, HCSC1, HEC1, HEC116, HEMCSS, HG3, HKA1, HMY1, HSC1, HSC5, HT3, HU09, IHH4, JAR, JEG3, JMURTK2, KKU100, KKU213, KML1, KMLS1, KMS28PE, KON, KOSC2CL343, KYAE1, LS, LU135, MCC13, MCC142, MCC26, MEL202, MEROM, MERO25, MERO41, MERO48A, MERO82, MERO83, MERO84, MERO95, MM127, MM370, MM383, MM386, MM415, MM426, M0LM1, MUTZ8, NCCIT, NCIH1417, NCIH64, NH12, NO10, NO11, NOZ, NP2, NP3, NP5, NP8, OCIAML4, OCILY18, OCILY7, OCIM2, OCUG1, ONDA7, ONDA8, ONDA9, OSC19, OSC20, P4E6, PEA1, PEO1, PEO4, PGA1, RAMOS, RCK8, ROS50, SAT, SCC3, SEKI, SHI1, SHMAC4, SHMAC5, SISO, SKGI, SKGII, SKGT2, SKGT4, SKN, SKNO1, SNU638, SUSA, TASK1, TFK1, TGW, U2904, U698M, UH01, UMRC3, UMRC7, UPCISCC026, UPCISCC040, UPCISCC074, UPCISCC116, UPCISCC131, VAESBJ, VAL, VMRCMELG, WSUNHL, PFSK1, HS860T, CAL72, GOTO, OCIC4P, SEMK2, HB1119, HSB2, CCRFCEM, RH28, RMS13, RC2, RHJT, TTC442, RH36, RH4, CCLFPEDS0018T, SNU1544, RH18DM, LPS6, LPS27, 93T449, 94T778, 95T1000, LPS141, LPS853, LPS510, LPS067, OS252, C396, MFM223, COLO824, SW527, 184B5, ICC10, ICC106, ICC108, ICC12, ICC137, ICC15, ICC2, ICC3, ICC4, ICC5, ICC6, ICC8, ICC9, G415, HKGZCC, KMCH1, RBE, SG231, SSP25, TGBC1TKB, TGBC52TKB, TKKK, YSCCC, JHC7, MUGCHOR1, UMCHOR1, MSI, CCLP1, CCSW1, GB2, NZOV9, NALM16, ECC2, 9505BIK, A375SKINCJ1, A375SKINCJ2, A375SKINCJ3, UACC62SKINCJ1, SKMEL19, MP46, MEL285, MEL290, 0MM1, OMM25, HOKUG, SKGIIIA, PMFKO14, TGBC18TKB, ECC4, TT1TKB, HHUA, HOUAI, SAS, HSKTC, PK8, HMVII, HOTHC, T3M5, CA922, HSQ89, HO1U1, HTMMT, LU134A, LU139, ATN1, P30OHK, SLVL, HSSCH2, NOS1, HSOS1, LU165, NB69, HSSYII, 201T, BB65RCC, CAL39, CHSA0011, CHSA0108, CHSA8926, CORL303, CP50MELB, CP66MEL, CP67MEL, CS1, DJM1, EMCBAC1, EMCBAC2, ESI, ES3, ES4, ES5, ES6, ES7, ES8, EW1, EW11, EW12, EW13, EW16, EW18, EW22, EW24, EW3, EW7, GAK, GMEL, GT3TKB, H2369, H2373, H2461, H2591, H2595, H2722, H2731, H2795, H2803, H2804, H2810, H2818, H2869, H290, H513, HA7RCC, HEY, HSC39, HUO3N1, ISTMEL1, ISTSL1, ISTSL2, JHOS3, K2, K5, KGN, LB1047RCC, LB2241RCC, LB2518MEL, LB373MELD, LB647SCLC, LB996RCC, LC1SQ, LC2AD, LU99A, M14, MCIXC, MKN28, MMACSF, MRKNU1, MZ1PC, MZ2MEL, MZ7MEL, NCC010, NCC021, NCIH1304, NCIH1688, NCIH250, NCIH322M, NCIH378, NCIH720, NCIH740, NCIH748, NCIH835, NY, OCUBM, OMC1, OVCA420, OVCA433, OVMIU, PC3JPC3, PL18, PL4, RCCAB, RCCER, RCCFG2, RCCJF, RCCJW, RCCMF, RERFLCFM, RH1, RXF393, SBC1,Monte Rosa Therapeutics AG

[0235] F&R Ref.: 52271 -0033W01 PCT Application

[0236] SBC3, SCH, SN12C, SW962, TCYIK, TMK1, UCH2, WM1552C, WM278, WM35, YMB1E, ALLPO, ARH77, BALLI, BB30HNC, BB49HNC, BC1, BC3, BE13, BE2M17, CESS, COLO320HSR, CROAP2, CTB1, CTV1, D245MG, D247MG, D263MG, D336MG, D392MG, D423MG, D502MG, D542MG, D566MG, DG75, DIFI, DOK, DSH1, EB3, ETK1, GRST, H3118, H9, HAL01, HC1, HCE4, HO INI, HS445, HS633T, IM9, IMR5, JHU011, JHU022, JHU029, JIYOYEP2003, JSC1, KARPAS1106P, KARPAS231, KARPAS45, KINGS1, KMOE2, KNS81FD, KOSC2, KPNYS, KY821, KYSE220, KYSE50, LB771HNC, LB831BLC, LC41, LN405, LNZTA3WT4, MCCAR, MFHINO, MHHPREB1, ML2, MLMA, MN60, MYM12, NBTU110, NB10, NB12, NB13, NB14, NB17, NB5, NB6, NB7, NCIH128, NCIH630, NEC8, NK92MI, NKM1, NTERA2CLD1, OACP4C, P32ISH, PCI15A, PCI30, PCI38, PCI4B, PCI6A, QIMRWIL, RAMOS2G64C10, RF48, RPMI8866, SKMG1, SKN3, STS0421, SUDHL16, SUPB8, SW684, SW872, TE12, TGBC24TKB, TK, TUR, WIL2NS, YT, 184A1, 600MPE, HBL100, HCC2185, HCC2688, HCC3153, LY2, MACLS2, MCF12A, MX1, SKBR5, SKBR7, ZR75B, GISTT1, HCC827GR, SS1A, UCH1, HCET, JHU028, M980513, MOT, NBSUSSR, BB30PBL, BB49EBV, BB65EBV, CAR1, CP50EBV, CP66EBV, DIPG007, GBM001, HA7EBV, L542, LB1047EBV, LB2241EBV, LB2518EBV, LB373EBV, LB647PBL, LB771PBL, LB831EBV, LB996EBV, MZ1B, MZ7B, NCIBL128, NCIBL1395, NCIBL1437, NCIBL1770, NCIBL2009, NCIBL2052, NCIBL2087, NCIBL209, NCIBL2122, NCIBL2126, NCIBL2171, HCC1187BL, HCC1599BL, HCC1937BL, LS1034PBL, HCC38BL, HCC1143BL, J82EBV, COLO829BL, HCC2157BL, HCC1395BL, HCC2218BL, HCC1954BL, M00921, M1203273, MET2B, ACN, MC1010, UDSCC2, SCI, CROAP3, GEO, HUH6CLONE5, SARC9371, KMHDASH2, CCLFUPGI0005T, HT144SKINFV1, HT144SKINFV3, HT144SKINFV2, RVH421SKINFV1, HAP1, WM3211, WM4235, M040416, and M140325.

[0237] In some embodiments, the fusion protein includes a nuclear localization domain which provides for the protein to be translocated to the nucleus. Several nuclear localization sequences (NLS) are known, and any suitable NLS can be used. For example, many NLSs have a plurality of basic amino acids, referred to as a bipartite basic repeats (reviewed in Garcia-Bustos et al, 1991, Biochim. Biophys. Acta, 1071:83-101). An NLS containing bipartite basic repeats can be placed in any portion of chimeric protein and results in the chimeric protein being localized inside the nucleus. In preferred embodiments a nuclear localization domain is incorporated into the final fusion protein, as the ultimate functions of the fusion proteins described herein will typically require the proteins to be localized in the nucleus. However, it may not be necessary to add a separate nuclear localization domain in cases where the DBD domain itself, or another functional domain within the final chimeric protein, has intrinsic nuclear translocation function.Monte Rosa Therapeutics AG

[0238] F&R Ref.: 52271 -0033W01 PCT Application VARIANTS

[0239] In some embodiments, the fusion protein(s) or components thereof described herein, or the polynucleotides encoding the fusion protein(s) or components thereof described herein, are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., as described herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent.

[0240] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity" is equivalent to nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0241] Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7);

[0242] "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher PlusTM, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for target proteins or nucleic acids, the length of comparison can be any length, up to and including full length of the target (e.g., 5%,Monte Rosa Therapeutics AG

[0243] F&R Ref.: 52271 -0033W01 PCT Application

[0244] 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence.

[0245] For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences is accomplished using Smith Waterman Alignment with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0246] Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0247] E3 LIGASE BINDING MODULATORS

[0248] The methods described herein are useful, for example, for identifying compound (e.g., drug)-dependent proximity interactions. In some embodiments, the methods are used to validate and / or identify targets that selectively interact with an E3 ligase in the presence of a compound, e.g., an E3 ligase binding modulator, e.g., a cereblon binding modulator. In some instances, the E3 ligase binding modulator is a molecular glue degrader (MGD). A molecular glue degrader is a non-heterobifunctional small molecule that induces interaction between a E3 ligase substrate receptor and a protein of interest resulting in degradation of the protein of interest.

[0249] E3 ligase binding modulators, e.g., E3 ligase substrate receptor binding modulators, e.g., cereblon binding modulators, are described, for example, in WO2021 / 069705 and WO2021 / 053555, which are hereby incorporated by reference in their entirety.

[0250] In some embodiments, the E3 ligase binding modulator, e.g., E3 ligase substrate receptor binding modulator, e.g., cereblon binding modulator, is a compound shown in Tables 5 and 6, below, or pharmaceutically acceptable salts thereof, or a stereoisomers thereof.

[0251] Table 2. Examples of E3 Ligase Binding Modulators

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[0386] METHODS OF USING THE FUSION PROTEINS, VECTORS, AND EXPRESSION SYSTEMS

[0387] The fusion proteins, vectors, and expression systems described herein, in various combinations are useful, for example, for identifying and / or validating an E3 ligase.

[0388] FIG. 1 shows a schematic of the methods described herein for identification and / or validation of an E3 ligase.

[0389] Specifically, described herein are methods for identifying and / or validating an E3 ligase substrate receptors(s) that interact with a protein of interest in a modulator-dependent manner or not. The methods described herein comprise: I) a) providing i) first cell(s) one or more fusion protein(s) each comprising the protein and a proximity labeling enzyme; and ii) an E3 ligase binding modulator; b) incubating the cell(s) and modulator under conditions effective for the proximity labeling enzyme to label the one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein(s); and c) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s); II) a) providing i) second cell(s) expressing the fusion protein(s); and ii) a negative control for the modulator; b) incubating the second cell(s) and negative control under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein(s); and c) detecting the presence and / or amount of labeled E3 ligase substrate receptors(s); III) comparing the presence and / or amount of labeled E3 ligase substrate receptors(s), from step I to those in step II; and IV) identifying one or more E3 ligase substrate receptors(s) that interact with the protein in a modulator-dependent manner based on the step III comparison.

[0390] Suitable fusion proteins and components thereof, targets, and cells / expression systems are described herein.

[0391] In some embodiments, the incubation composition comprises a cell culture medium. Suitable cell culture media are known and described in the art. See, e.g., Yang et al., “Culture Conditions and Types of Growth Media for Mammalian cells,” Intech dx.doi.org / 10.5772 / 52301.

[0392] In some embodiments, e.g., when the proximity labeling enzyme is a promiscuous biotinylation enzyme (e.g., TurboID, BioID, BioID2, miniTurbo, AirlD, BASU, microID, ultralD, HRP, APX, APEX, APEX2 and variations thereof).

[0393] In some embodiments, suitable substrates for the proximity labeling enzyme can be biotin (5-[(3aS,4S,6aR)-2-oxo-l,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid), a derivative of biotin (e.g., biotin-phenol), or a modified biotin. In some embodiments, the substrate is present in or added to the incubation composition. In some embodiments, the substrate is biotin and the amount of biotin in the composition, e.g., at the beginning of theMonte Rosa Therapeutics AG

[0394] F&R Ref.: 52271 -0033W01 PCT Application

[0395] incubation or at the time the biotin is added to the composition, is from or from about 0.01 to or to about 0.10 mM. In some embodiments, the amount of biotin in the composition, e.g., at the beginning of the incubation, is or is about 0.05 mM.

[0396] In some embodiments, the cell(s) are incubated in the incubation medium without the substrate for the proximity labeling enzyme for a length of time before adding the substrate for the proximity labeling enzyme to the composition. In some embodiments, the cell(s) are incubated for from or from about 5 minutes to or to about 10 hours before adding the substrate for the proximity labeling enzyme to the composition. In some embodiments, the cell(s) are incubated in a buffer (e.g., PBS or any suitable buffer) without the substrate for the proximity labeling enzyme for a length of time before adding the substrate for the proximity labeling enzyme to the composition. In some embodiments, the cell(s) are incubated for from or from about 30 seconds to or to about 5 minutes in a suitable buffer before adding the substrate for the proximity labeling enzyme to the composition.

[0397] In some embodiments, a 26S proteasome inhibitor is present in or added to the incubation composition. In some embodiments, the 26S proteasome inhibitor is selected from the group consisting of bortezomib (([(17?)-3-methyl-l-[[(25)-3-phenyl-2-(pyrazine-2-carbonylamino)propanoyl]amino]butyl]boronic acid)), ixazomib ([(lR)-l-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]boronic acid), carfilzomib ((2S)-4-methyl-N-[(2S)-l-[[(2S)-4-methyl-l-[(2R)-2-methyloxiran-2-yl]-l-oxopentan-2-yl]amino]-l-oxo-3-phenylpropan-2-yl] -2- [ [(2 S)-2- [(2-morpholin-4-ylacetyl)amino] -4-phenylbutanoyl]amino]pentanamide), MG-132 (benzyl 7V-[(25)-4-methyl-l-[[(25)-4-methyl-l-[[(25)-4-methyl-l-oxopentan-2-yl]amino]-l-oxopentan-2-yl]amino]-l-oxopentan-2-yl]carbamate), MG-115 (benzyl N-[(2S)-4-methyl-l-[[(2S)-4-methyl-l-oxo-l-[[(2S)-l-oxopentan-2-yl]amino]pentan-2-yl]amino]-l-oxopentan-2-yl]carbamate), Proteasome Inhibitor I (tert-butyl (45)-5-[[(25)-l-[[(25)-4-methyl-l-oxopentan-2-yl]amino]-l-oxopropan-2-yl]amino]-4-[[(25)-3-methyl-2-(phenylmethoxycarbonylamino)pentanoyl]amino]-5-oxopentanoate), oprozomib (7V-[(25)-3-methoxy-l-[[(25)-3-methoxy-l-[[(25)-l-[(27?)-2-methyloxiran-2-yl]-l-oxo-3-phenylpropan-2-yl]amino]-l-oxopropan-2-yl]amino]-l-oxopropan-2-yl]-2-methyl-l,3-thiazole-5-carboxamide), marizomib ((U?,47?,55)-4-(2-chloroethyl)-l-[(5)-[(15)-cyclohex-2-en-l-yl]-hydroxymethyl]-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3, 7-dione), MLN9708 (4-(carboxymethyl)-2-[(U?)-l-[[2-[(2,5-dichlorobenzoyl)amino]acetyl]amino]-3-methylbutyl]-6-oxo-l,3,2-dioxaborinane-4-carboxylic acid), and combinations thereof. In some embodiments, the amount of proteasome inhibitor in the incubation composition, e.g., at the beginning of the incubation or at the time it is added to the composition, is from or from about 0.02 pM to or toMonte Rosa Therapeutics AG

[0398] F&R Ref.: 52271 -0033W01 PCT Application

[0399] about 2.0 pM. In some embodiments, the amount of proteasome inhibitor in the incubation composition, e.g., at the beginning of the incubation or at the time it is added to the composition is or is about 0.2 pM.

[0400] In some embodiments, a modulator, as described herein, is present in or added to the incubation composition. In some embodiments, the modulator is provided as part of a composition comprising DMSO. In some embodiments, the amount of modulator in the composition, e.g., at the beginning of the incubation or at the time it is added to the composition, is from or from about 1 to or to about 50 mM. In some embodiments, the amount of modulator in the composition, e.g., at the beginning of the incubation or at the time it is added to the composition, is or is about 10 mM.

[0401] In some embodiments, the cell(s) are incubated in the incubation medium without the substrate for the proximity labeling enzyme before adding the modulator to the composition. In some embodiments, the cell(s) are incubated for from or from about 5 minutes to or to about 10 hours before adding the modulator to the composition.

[0402] In some embodiments, the proximity labeling enzyme substrate and the modulator are added to the composition at the same time or about the same time.

[0403] Detecting the presence and / or amount of labeled protein(s) (e.g., labeled E3 ligase substrate receptor(s)), can be carried out by any suitable means, which are known in the art.

[0404] In some embodiments of any of the methods described herein, e.g., in particular for identifying and / or validating an E3 ligase, the fusion protein(s) may be tagged, e.g., in a manner that is not dependent on the proximity labeling enzyme. In some embodiments, the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose-binding protein (MBP), chitin binding protein, a streptavidin tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys, FLAG-tag (e.g., DYKDDDDK (SEQ ID NO:4)), a biotin tag, and combinations thereof.

[0405] In some embodiments, detecting the presence and / or amount of labeled protein(s), comprises a step of selectively isolating the labeled protein(s), e.g., by affinity chromatography.

[0406] In some embodiments of any of the methods described herein, detecting the presence and / or amount of labeled protein(s), comprises an immunoprecipitation step. In some embodiments, e.g., when the proximity labeling enzyme is a promiscuous biotinylation enzyme, immunoprecipitation comprises an antibody, avidin, neutravidin, streptavidin, rhizavidin, or any variations of these. For example, the immunoprecipitation comprises streptavidin based immunoprecipitation, e.g., streptavidin bead based immunoprecipitation.Monte Rosa Therapeutics AG

[0407] F&R Ref.: 52271 -0033W01 PCT Application

[0408] In some embodiments of any of the methods described herein, the first and / or second fusion proteins can be conjugated to a fluorophore (thereby allowing for the detecting the presence and / or amount of labeled protein(s) based on the quantification of fluorescence signal) or with an enzyme which can produce a detectable light signal e.g. a luciferase or peroxidase (e.g. HRP) in presence of its corresponding substrate(s).

[0409] In some embodiments of any of the methods described herein, the cells are harvested and pelleted, e.g., prior to detecting the presence and / or amount of protein(s), e.g., prior to immunoprecipitation.

[0410] In some embodiments of any of the methods described herein, immunoprecipitation comprises incubating the cell(s), e.g., the harvested cell pellet, in a lysis buffer. In some embodiments, the lysis buffer is a urea buffer. In some embodiments, the lysis buffer comprises a protease inhibitor. In some embodiments, the protease inhibitor is selected from the group consisting of AEBSF, Bestatin, E-64, Pepstatin A, Phosphoramidon, Leupeptin, Aprotinin, 1,10-Phenanthroline, and combinations thereof. Following lysis, the labeled protein(s) can be harvested, e.g., with streptavidin beads, and analyzed, for example, but Western Blot and / or Mass spectrometry, e.g., quantitative mass spectrometry.

[0411] In some embodiments of any of the methods described herein, an E3 ligase is validated and / or identified, if after incubation with the modulator, e.g., as described herein, the amount of labeled proteins is greater than the amount of protein that is labeled after incubation under the same conditions except without a modulator (e.g., with DMSO as a negative control). In some embodiments, the log2 fold change of the labeled proteins when incubated with the modulator versus the control (e.g., DMSO) is at least 0.5, at least 1, at least 1.5, at least 2, or at least 3. In some embodiments, the p-value of detecting a given log2 fold change across sample conditions is 0.1 or less, e.g., 0.05 or less, e.g., 0.001 or less, e.g., 0.0001, 0.00001, 0.000001, 0.0000001, 0.00000001, 0.000000001 or less.

[0412] EXAMPLES

[0413] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0414] Example 1: TurboID Fusion Proteins

[0415] Fusion proteins comprising TurboID and MEK1 (SEQ ID NO: 5; shown below), Protein of Interest 1 (mTOR(FRB); SEQ ID NO: 6, shown below), CCNE1 (SEQ ID NO: 7, shown below), or Protein of Interest 2 were generated.Monte Rosa Therapeutics AG

[0416] F&R Ref.: 52271 -0033W01 PCT Application

[0417] SEQ ID NO: 5 (TurboID-MEKl):

[0418] MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGY SLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGS RGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPND LYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDR NTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGA LLLEQDGVIKPWMGGEISLRSAEKSGGGGSGGGGSGGGGSPKKKPTPIQLNPAPDGSAV NGTSSAETNLEALQKKLEELELDEQQRKRLEAFLTQKQKVGELKDDDFEKISELGAGNG GVVFKVSHKPSGLVMARKLIHLEIKPAIRNQIIRELQVLHECNSPYIVGFYGAFYSDGEISI CMEHMDGGSLDQVLKKAGRIPEQILGKVSIAVIKGLTYLREKHKIMHRDVKPSNILVNS RGEIKLCDFGVSGQLIDSMANSFVGTRSYMSPERLQGTHYSVQSDIWSMGLSLVEMAV GRYPIPPPDAKELELMFGCQVEGDAAETPPRPRTPGRPLSSYGMDSRPPMAIFELLDYIV NEPPPKLPSGVFSLEFQDFVNKCLIKNPAERADLKQLMVHAFIKRSDAEEVDFAGWLCS TIGLNQPSTPTHAAGV

[0419] SEQ ID NO: 6 (TurboID-mTOR(FRB)):

[0420] (MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGY SLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGS RGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVKWPND LYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDR NTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGA LLLEQDGVIKPWMGGEISLRSAEKSGGGGSGGGGSGGGGSNTLVQQAMMVSEELIRVA ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRD LMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQLPQLTSLELQYVSPKLLMCR DLELAVPGTYDP)

[0421] SEQ ID NO: 7 (TurboID-CCNEl):

[0422] (MPRERRERDAKERDTMKEDGGAEFSARSRKRKANVTVFLQDPDEEMAKIDRTARDQC GSQPWDNNAVCADPCSLIPTPDKEDDDRVYPNSTCKPRIIAPSRGSPLPVLSWANREEV WKIMLNKEKTYLRDQHFLEQHPLLQPKMRAILLDWLMEVCEVYKLHRETFYLAQDFFD RYMATQENVVKTLLQLIGISSLFIAAKLEEIYPPKLHQFAYVTDGACSGDEILTMELMIM KALKWRLSPLTIVSWLNVYMQVAYLNDLHEVLLPQYPQQIFIQIAELLDLCVLDVDCLE FP YGIL AAS ALYHF S S SELMQK VSGYQWCDIENC VKWMVPF AM VIRETGS SKLKHFRG VADEDAHNIQTHRDSLDLLDKARAKKAMLSEQNRASPLPSGLLTPPQSGKKQSSGPEMMonte Rosa Therapeutics AG

[0423] F&R Ref.: 52271 -0033W01 PCT Application

[0424] AGSGMKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVP GKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQA GRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGADKVRVK WPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGI NLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGID KQGALLLEQDGVIKPWMGGEISLRSAEK)

[0425] CAL51 cells were transduced with a lentiviral vector expressing one of the fusion proteins noted above. The cells were cultured in DMEM medium with 10% FBS and vector expression was selected with 2ug / mL puromycin treatment for two days.

[0426] Molecular glue degrader (MGD) compounds (CP) were dissolved in DMSO to a concentration of lOmM. Both lOuM compound or DMSO (as a control), 2uM bortezomib and 50uM Biotin were added to the cultures for 6 hours. Cells were harvested on ice by washing with PBS, centrifuging, discarding the supernatant, re-suspending the pellet in ImL cold PBS IX, and centrifuging again. The pellets were stored at -80°C. Urea buffer (2M urea + lOmM Tris HC1 pH8) was prepared, filtered, and stored at 4°C. A solution of lOmL lysis buffer (e.g., NP-40 or RIP A) with plus lOOpL each of Protease inhibitor cocktail 1 (Sigma, #P8340), Phosphatase inhibitor cocktail 2 (Sigma, #P5726), and Phosphatase inhibitor cocktail 3 (Sigma, #P0044), and IpL 0.2pM Bortezomib was prepared fresh and kept on ice.

[0427] The harvested cell pellet was resuspended in ImL lysis buffer, vortexed, sonicated, and kept on ice for 20min. The lysate was centrifuged at max speed for lOmin at 4°C and the supernatant was transferred in a new 1.5mL Eppendorf tube and put on ice.

[0428] Immunoprecipitation was carried out using magnetic beads in a 1.5mL Eppendorf tube. To prepare the magnetic beads, lysis buffer was added to the beads and mixed before applying magnetic force to separate the beads from the supernatant. The supernatant was removed and discarded. This process was repeated. Then, the cells were prepared for either Western Blot or Mass Spectrometry analysis, as follows.

[0429] For Western Blot, 900pL of the cell lysate (protein concentration ~0.5 mg / mL) was added to 40pL of beads and incubated for 4h at 4°C on a rotating device. 50pL of the cell lysate was put back into a tube with 15pL 4X Laemmli Sample Buffer and 1.4pL dithiothreitol (DTT) (stock solution at IM to give a final concentration of 20mM) and then heated 10 min at 95°C. The beads were collected with the magnetic stand, and the unbound sample was removed and saved for analysis. 50uL of the unbound sample was put in an Eppendorf tube with 15uL 4X Laemmli Sample Buffer and 1.4uL DTT (stock solution at IM to have a final concentration of 20mM) and then heated lOmin at 95°C. The beads were first washed twice with ImL RIPAMonte Rosa Therapeutics AG

[0430] F&R Ref.: 52271 -0033W01 PCT Application

[0431] buffer, then with ImL urea buffer, and finally once with ImL lysis buffer. 50uL of lysis buffer solution (with protease inhibitors) was added to the beads to elute the samples, with 20uL 4X Laemmli Sample Buffer, 1.6uL DTT (20mM) and 16uL biotin (lOmM). The sample was vortexed to mix and heated at 95°C for 15min, and then stored at -80°C ready to use for Western Blot analysis.

[0432] For Mass Spectrometry, 900pL of the cell lysate (protein concentration around Img / mL) was added to 50pL of beads and incubated for 4h at 4°C on a rotating device. The beads were collected with the magnetic stand, and the unbound sample was removed and saved for analysis. The beads were washed twice with ImL RIP A buffer, then three times with ImL urea buffer, and finally with ImL IX PBS. The supernatant was removed and the sample with the beads was frozen at -80°C.

[0433] As shown in FIGs. 2A-2D, interaction between each fusion protein and CRBN was observed in the presence of a MGD.

[0434] OTHER EMBODIMENTS

[0435] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Monte Rosa Therapeutics AGF&R Ref.: 52271 -0033W01 PCT Application WHAT IS CLAIMED IS:

1. A system for detecting or validating modulator-dependent proximity -based interactions between a protein of interest and an E3 ligase substrate receptor, the system comprising:a) cell(s) expressing one or more fusion proteins, each fusion protein comprising the protein and a proximity labeling enzyme; andb) an E3 ligase binding modulator.

2. A method for detecting the interaction between a protein of interest and one or more E3 ligase substrate receptor(s), the method comprises:a) providing (i) cell(s) expressing a fusion protein comprising the protein and a proximity labeling enzyme; and (ii) optionally, an E3 ligase binding modulator;b) incubating the cell(s) and, optionally, the modulator under conditions effective for the proximity labeling enzyme to label protein(s) in the proximity of the fusion protein; andc) determining the presence and / or amount of labeled E3 ligase(s), thereby detecting the interaction of an E3 ligase and the protein.

3. A method for identifying E3 ligase substrate receptors(s) that interact with a protein of interest in a modulator-dependent manner or not, the method comprising:I)a) providing i) first cell(s) one or more fusion protein(s) each comprising the protein and a proximity labeling enzyme; and ii) an E3 ligase binding modulator;b) incubating the cell(s) and modulator under conditions effective for the proximity labeling enzyme to label the one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein(s); andc) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s); II)a) providing i) second cell(s) expressing the fusion protein(s); and ii) a negative control for the modulator;b) incubating the second cell(s) and negative control under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein(s); and78Monte Rosa Therapeutics AGF&R Ref.: 52271 -0033W01 PCT Applicationc) detecting the presence and / or amount of labeled E3 ligase substrate receptors(s); III) comparing the presence and / or amount of labeled E3 ligase substrate receptors(s), from step I to those in step II; andIV) identifying one or more E3 ligase substrate receptors(s) that interact with the protein in a modulator-dependent manner based on the step III comparison.

4. A method for validating a predicted modulator-dependent interaction between one or more E3 ligase substrate receptor(s) and a protein of interest, the method comprising:I)a) providing i) first cell(s) expressing a fusion protein comprising the protein and a proximity labeling enzyme; and ii) an E3 ligase binding modulator;b) incubating the first cell(s) and modulator under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptors(s) in the proximity of the fusion protein; andc) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s); II)a) providing i) second cell(s) expressing the fusion protein; and ii) a negative control for the modulator;b) incubating the second cell(s) and negative control under conditions effective for the proximity labeling enzyme to label one or more E3 ligase substrate receptor(s) in the proximity of the fusion protein; andc) detecting the presence and / or amount of labeled E3 ligase substrate receptor(s); III) comparing the presence and / or amount of labeled E3 ligase substrate receptor(s), from step I with those from step II; andIV) validating the predicted modulator-dependent interaction between one or more E3 ligase substrate receptor(s) and the protein or not based on the comparing of step III.

5. The method of claim 3 or 4, wherein the negative control for the modulator is DMSO.

6. The method of any one of claims 2-5, wherein conditions effective for the proximity labeling enzyme to label protein(s) in the proximity of the fusion protein comprise incubating in a composition comprising a substrate for the proximity labeling enzyme.79Monte Rosa Therapeutics AGF&R Ref.: 52271 -0033W01 PCT Application7. The method of claim 6, wherein the substrate for the proximity labeling enzyme is biotin.

8. The method of any one of claims 2-7, wherein incubation is carried out in the presence of a 26 S proteasome inhibitor.

9. The method of claim 8, wherein the 26S proteasome inhibitor is selected from the group consisting of bortezomib, ixazomib, carfilzomib, MG-132, MG-115, oprozomib, marizomib, MLN9708, and combinations thereof.

10. The method of any one of claims 2-9, wherein detecting the presence and / or amount of labeled protein(s) comprises quantitative mass spectrometry and / or Western Blot analysis.

11. The method of any one of claims 2-10, wherein the protein is identified as having a modulator-dependent interaction with an E3 ligase substrate receptor, or vice-versa, when the amount of E3 ligase substrate receptor that is labeled after incubation with the modulator is greater than the amount of E3 ligase substrate receptor that is labeled after incubation under the same conditions with a negative control for the modulator.

12. The method or system of any of the preceding claims, wherein the proximity labeling enzyme is a promiscuous biotinylation enzyme.

13. The method or system of claim 12, wherein the promiscuous biotinylation enzyme is selected from TurboID, BioID, BioID2, miniTurbo, AirlD, BASU, microID, ultralD, HRP, APX, APEX, APEX2 and variations thereof.

14. The method or system of any one of the preceding claims, wherein one or more of the fusion protein(s) further comprises a linker between the protein and the proximity labeling enzyme.

15. The method or system of claim 14, wherein the linker(s) are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long.80Monte Rosa Therapeutics AGF&R Ref.: 52271 -0033W01 PCT Application16. The method or system of any of the preceding claims, wherein one or more of the fusion protein(s) further comprises a self-cleaving peptide, optionally T2A and / or a detection label, optionally a fluorescent protein, optionally green fluorescent protein (GFP), optionally eGFP17. The method of any one of the preceding claims, wherein the modulator is a molecular glue degrader.

18. The method or system of any one of the preceding claims, wherein the cell is selected from the group consisting of HEK293T cells, CAL51 cells, HCT116 cells, MCF7 cells, SKMEL28 cells, THP1 cells, U937 cells, and combinations thereof.

19. A cell of the system or method of any of the preceding claims.

20. A fusion protein of the system or method of any of the preceding claims.

21. A nucleic acid sequence encoding the fusion protein of the system or method of any of the preceding claims.

22. A vector comprising the nucleic acid sequence of claim 21.

23. A cell comprising the vector of claim 22.

24. A protein complex comprising the fusion protein and the protein of the system or method of any of the preceding claims.

25. A cell comprising the protein complex of claim 24.81

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