Membrane ubiquitin ligases that target protein degradation

Abstract

This invention provides a heterobifunctional molecule that can simultaneously bind to transmembrane ubiquitin ligases and transmembrane proteins, thereby mediating the internalization of transmembrane proteins. [Solution] The present invention relates to a heterobifunctional molecule comprising a first and a second binding domain, wherein i) the first binding domain can specifically bind to a transmembrane E3 ubiquitin ligase; and ii) the second binding domain can specifically bind to a transmembrane protein, and the simultaneous binding of the heterobifunctional molecule to the transmembrane E3 ubiquitin ligase and the transmembrane protein results in ubiquitination and internalization of the transmembrane protein. The present invention further relates to a heterobifunctional molecule for use in the treatment of a disease, preferably at least one of cancer, autoimmune disease, inflammatory disease, infectious disease, and genetic disease.

Description

[Technical Field]

[0001] Field of Invention This invention relates to the field of molecular and cellular biology, particularly to the field of cancer cell biology. This invention relates to membrane It simultaneously binds to transmembrane ubiquitin ligases and transmembrane proteins, thereby binding to transmembrane proteins. This relates to the use of heterobifunctional molecules that can mediate internalization. [Background technology]

[0002] background Cells capture external chemical signals and initiate intracellular signal transduction cascades to respond to cellular signals. The response is driven by the activation of receptors embedded in the cell membrane, which communicate with the surrounding environment of the cell. The availability of receptors on the cell surface is a crucial factor in determining signal specificity and sensitivity. These are determinants, and misregulation of these events is often linked to cancer development and treatment resistance. It is.

[0003] In fact, receptor mutations, activation, or overexpression are major in many tissues. These are widely recognized cancer-promoting mechanisms (e.g., EGFR, ERBB2, PDG) FR, TGFβR, IGFR1, GHR, FZD, LRP6). Abnormal receptor activity in cancer cells. Sex dependence has led to the development of various neutralizing antibodies and small molecule inhibitors. However, To successfully neutralize receptor activity, it is necessary to demonstrate high efficacy without inducing toxicity. The creation of a strong binder that can reach sufficient plasma concentrations is necessary, and non-covalent interacting substances In some cases, this may prove difficult. Furthermore, stabilization or elevation of compensatory receptors Resistance is the primary pathway for tolerance.

[0004] When the cytosolic region of the membrane-bound receptor is post-translationally modified with ubiquitin, induced endo cytosis is induced, leading to rapid removal of the receptor from the cell surface. The internalized receptor may then be subjected to lysosomal degradation. In healthy stem cells, high-level Wnt sig naling induces the expression of two homologous membrane-bound ubiquitin ligases, RNF43 and ZNRF3, which are known to mediate ubiquitination and removal of Frizzled (FZD), the receptor for Wnt, from the cell surface (Koo et al, Nature 2012, 48 8(7413):665-9). Thus, this negative feedback loop helps regulate 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 towards FZD is neutralized in the stem cell niche by the LGR4 / 5 receptor and the secreted protein R-spondin (RSPO), which forms a complex with RNF43 / ZNRF3 (Hao et al, Nature 2012, 485(7397):195-200). Next, this trimeric RSPO-LGR4 / 5-RNF43 / ZNR F3 complex is removed from the cell surface, resulting in stabilization of FZD receptor expression and an increase in the level of Wnt signaling. Wnt signaling is often misregulated in cancer. Such cancers show increased expression of Wnt target genes, including RNF43 and ZNRF3.

[0005] An E3 ubiquitin ligase loads ubiquitin onto a protein substrate and recruits an enzyme that helps or directly catalyzes the transfer of ubiquitin to the protein substrate or directly catalyzes the conjugation of E2 ubiquitin.

[0006] Ubiquitination of receptors mediated by transmembrane ubiquitin E3 ligase is endothelial It is known to cause itosis and subsequent degradation of ubiquitinated substrates. It is known in the art that such degradation preferably occurs within lysosomes. Lysosomal degradation involves monoubiquitin, multiubiquitin, Lys48, or L Ligation of ys63-linked polyubiquitin chains to membrane-bound receptors is required. This primarily utilizes the proteasome degradation pathway, namely Lys48-linked polyubiquitin The coupling of the chain to cytosolic target proteins primarily facilitates proteasome degradation. This is in contrast to the activity of cytosolic ubiquitin ligases that utilize this pathway.

[0007] Therefore, transmembrane E3 ubiquitin ligases are different members of the E2 enzyme family. It may interact with and selectively target membrane-bound substrates. Ubiquitinated groups The substance can be internalized and then degraded via lysosomal degradation.

[0008] Effectively targeting transmembrane receptors, particularly those involved in the onset or progression of disease. Therefore, inhibiting its activity remains strongly needed in the field of technology. Effectively targeting transmembrane receptors involved in the development of this condition and inhibiting their activity is It is especially strongly needed in the field of technology. [Overview of the project]

[0009] Summary of the Invention The present invention can be summarized in the following embodiments. Embodiment 1. A heterobifunctional molecule comprising a first and a second binding domain, i) The first binding domain specifically binds to transmembrane E3 ubiquitin ligase. Can; ii) The second binding domain can specifically bind to transmembrane proteins, The simultaneous binding of heterobifunctional molecules to E3 ubiquitin ligases and transmembrane proteins is Preferably, this brings about ubiquitination and internalization of transmembrane proteins. A heterobifunctional molecule.

[0010] Embodiment 2. The molecule is the extracellular component of the transmembrane E3 ubiquitin ligase and the transmembrane protein A heterobifunctional molecule according to Embodiment 1 that binds to the extracellular portion of a substance.

[0011] Embodiment 3. Simultaneous binding of molecules to transmembrane E3 ubiquitin ligase and transmembrane protein The combination results in the degradation of transmembrane proteins, preferably lysosomal degradation, as in Embodiment 1. The heterobifunctional molecule described in 2.

[0012] Embodiment 4. Transmembrane E3 ubiquitin ligase is monoubiquitin, multiubiquitin, Transmembrane proteins are ubiquitously linked to polyubiquitin chains with Lys48 or Lys63 ligation. A heterobifunctional molecule described in any one of the above embodiments, which is chinified.

[0013] Embodiment 5. The transmembrane protein is a receptor, preferably a receptor involved in cancer. , a heterobifunctional molecule as described in any one of the embodiments described above.

[0014] Embodiment 6. Transmembrane E3 ubiquitin ligase is RNF43, RNF167, ZNRF 3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8 , MARCH9, RNF149, RNF145, RNFT1, RNF130 and RNF Heterodifunctional according to any one of the embodiments described above, selected from the group consisting of 128. sex molecule.

[0015] Embodiment 7. Transmembrane proteins include TGFβR1, TGFβR2, EGFR, and ERBB. 2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA , PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD 6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD - Selected from the group consisting of L1, CTLA4, CMTM6, CMTM4, and WLS. A heterobifunctional molecule as described in any one of the embodiments described above.

[0016] Embodiment 8. The molecule includes a linker between the first binding domain and the second binding domain. , a heterobifunctional molecule as described in any one of the embodiments described above.

[0017] Embodiment 9. At least one of the first domain and the second domain (at lea The first domain and the second domain are formed by an organic small molecule or protein molecule. The heterobifunctional molecule described in any one of the embodiments described above.

[0018] Embodiment 10. The heterobifunctional molecule is a bicyclic peptide, as described in Embodiment 9. Ronifunctional molecule.

[0019] Embodiment 11. At least one of the first domain and the second domain is an antibody The functional fragment is preferably a nanobody, as described above. A heterobifunctional molecule described in any one of the states.

[0020] Embodiment 12. The heterobifunctional molecule is a bispecific antibody, preferably a bispecific nano The body is the heterobifunctional molecule described in Embodiment 11.

[0021] Embodiment 13. At least one of the first domain and the second domain is an apter A heterobifunctional molecule as described in any one of the embodiments described above.

[0022] Embodiment 14. The method described in any one of the above embodiments for use as a pharmaceutical product. A terobifunctional molecule.

[0023] Embodiment 15. The embodiment described in any one of the above embodiments for use in the treatment of cancer. A heterobifunctional molecule, the cancer is preferably colorectal cancer, ovarian cancer, breast cancer, From esophageal cancer, stomach cancer, prostate cancer, lung cancer, melanoma, leukemia, pancreatic cancer, and bladder cancer A heterobifunctional molecule selected from the following group.

[0024] Embodiment 16. A method for identifying transmembrane proteins as targets for cancer treatment. Therefore, the method is i) Expose cells to one or more members of a library of heterobifunctional molecules. The step involves a heterobifunctional molecule having a first binding domain and a second binding domain. Includes n, The first binding domain can bind to transmembrane E3 ubiquitin ligase, and the second domain The main part is a step containing a scrambled sequence; ii) Viability and differentiation capacity of exposed cells ), at least one of stem cell properties and proliferation capacity Another step to decide; iii) Of the viability, differentiation ability, stem cell properties and proliferation ability of the exposed cells, less Each of the following is considered: at least one of the following: viability of control cells, differentiation ability, stem cell properties, and proliferative capacity. A step to compare with one; iv) Compared to control cells, exposed cells have higher viability, differentiation potential, stem cell properties, and proliferation. Identifying heterobifunctional molecules that reduce or increase at least one of the capabilities Up and; v) A membrane to which the heterobifunctional molecules identified in step iv) can bind. Steps to identify transemission proteins A method that includes [this].

[0025] Embodiment 17. The heterobifunctional molecule is a bispecific antibody, preferably a bispecific nano The method according to embodiment 16, which is the body.

[0026] Example 18. Cells are obtained from patient-derived tissue, preferably a portion of cultured patient-derived tissue. The cells are either present or derived therefrom, preferably from a biopsy or organoid, preferably from a biopsy or organoid. Embodiments 16 or 17 are part of or derived from a tumor organoid. Method of description.

[0027] Embodiment 19. A method for reducing the surface level of membrane-bound proteins in cells. The method is, a) Express transmembrane E3 ubiquitin ligase and membrane-bound proteins on the cell surface. The steps include preparing the cells; b) A step of exposing cells to a heterobifunctional molecule, wherein the heterobifunctional molecule is i) A transmembrane E3 ubiquitin ligase that can specifically bind to the extracellular portion. 1 binding domain; and ii) A second binding agent that can specifically bind to the extracellular portion of membrane-bound proteins. main Steps including; c) If necessary, a step to determine the surface level of membrane-bound proteins in the cell. and The decrease at the surface level is due to the reduction in the surface level of the cell membrane-bound proteins prior to step b). A method that shows a decrease compared to [the previous method].

[0028] Embodiment 20. The method described in Embodiment 19, wherein the membrane-bound protein is a transmembrane protein. Law.

[0029] Embodiment 21. - Transmembrane E3 ubiquitin ligase contains a first non-natural epitope tag in its extracellular portion. Furthermore, the first binding domain of the heterobifunctional molecule binds to the first unnatural epitope tag. ru; as well as - The membrane-bound protein contains a second non-natural epitope tag in its extracellular portion, and is heterozygous. The second binding domain of the functional molecule binds to the second non-natural epitope tag. The method according to embodiment 19 or 20, which is at least one of the following.

[0030] Embodiment 22. Embodiment in which the first and second non-natural epitope tags are different tags. The method described in 21.

[0031] Embodiment 23. The first non-natural epitope tag is one of the alpha tag and E6 tag. At least one, and / or a second non-natural epitope tag, is the alpha tag. The method according to embodiment 21 or 22, wherein the tag is at least one of the E6 tags.

[0032] Embodiment 24. The first and second non-natural epitope tags are transmembrane E3 ubiquitin ligators. The N-terminus of the enzyme and membrane-bound protein, as in Embodiments 19-23 The method described in one of the following options.

[0033] Embodiment 25. The heterobifunctional molecule is a bispecific antibody, preferably a bispecific nano The method according to any one of embodiments 19 to 24, which is the body.

[0034] Embodiment 26. The first binding domain of the heterobifunctional molecule is anti-alpha-VHH, The second binding domain is anti-E6 VHH, or the first binding domain of a heterobifunctional molecule The main binding domain is anti-E6 VHH, and the second binding domain is anti-alpha VHH; in practice... The method described in condition 25.

[0035] Embodiment 27. The membrane-bound protein contains a third non-natural epitope tag, and / or The transmembrane ubiquitin E3 ligase contains a fourth non-natural epitope tag, preferably the third. and / or a fourth epitope tag is His-tag, FLAG-tag and myc-tag The method according to any one of embodiments 19 to 26, wherein at least one of the Gs.

[0036] Embodiment 28. The cell surface level of membrane-bound proteins in step c) is the cell surface The above protein is detected, preferably determined by immunofluorescence, and the implementation is carried out. The method described in any one of forms 19 to 27.

[0037] Embodiment 29. The cell surface level of membrane-bound proteins is prior to step b), Compared to the cell surface level of protein, at least about 10%, 20%, 30%, 40%, A reduction of 50% or at least about 60% as described in any one of embodiments 19 to 28. method.

[0038] Embodiment 30. A decrease in the surface level of membrane-bound proteins leads to a decrease in the intracellular membrane-bound proteins. The decrease in total amount is preferably determined by biochemical analysis, as in Embodiments 19-29. Either one of the methods.

[0039] Embodiment 31. The cells prepared in step a) are transmembrane E3 ubiquitin rigger Overexpress ze and membrane-bound proteins, and permanently overexpress them as needed. The method according to any one of embodiments 19 to 30.

[0040] Embodiment 32. The cells prepared in step a) are transmembrane E3 ubiquitin rigger Any one of Embodiments 19 to 30 expresses ze and membrane-bound proteins at an endogenous level. The method used.

[0041] Embodiment 33. In the cells prepared in step a), transmembrane E3 ubiquitin The genome sequence encoding the ligase is the first, and optionally the fourth, non-natural epithet The method according to embodiment 32, which has been modified to incorporate an array that codes for a puttag.

[0042] Embodiment 34. In the cells prepared in step a), the membrane-bound protein is The genome sequence being coded codes for a second, and optionally a third, non-natural epitope tag. The method according to embodiments 32 and 33, which has been modified to incorporate a sequence.

[0043] Embodiment 35. A heterobifunctional molecule between the first binding domain and the second binding domain It contains a peptide linker, preferably the peptide linker is (GGGGS)n, and n n is preferably 1, 2, 3, 4, 5, 6 or 7, and n is preferably 3 or 5. or the method according to any one of embodiments 19 to 34.

[0044] Embodiment 36. Transmembrane E3 ubiquitin ligase and membrane-bound protein, a) Express transmembrane E3 ubiquitin ligase and membrane-bound proteins on the cell surface. This is a step in which cells are prepared, - Transmembrane E3 ubiquitin ligase contains a first non-natural epitope tag in its extracellular portion. fruit; - The membrane-bound protein contains a second non-natural epitope tag in its extracellular portion, step and; b) A step of exposing cells to a heterobifunctional molecule, wherein the heterobifunctional molecule is - A first binding domain that can specifically bind 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) The surface level of membrane-bound proteins is at least approximately 10%, 20%, 30%, and 40%. Transmembrane E 3. A step of selecting ubiquitin ligases and membrane-bound proteins, wherein the reduction is Step b) is a decrease compared to the surface level of the cell membrane-bound protein, Top A heterogeneous Bifunctional molecule.

[0045] Definition Various terms related to the methods, compositions, formulations, uses and other aspects of the present invention are used throughout this specification and the claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the relevant art to which the invention pertains. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein . Any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention , but the preferred materials and methods are described herein .

[0046] Methods for practicing the prior art used in the methods of the present invention will be apparent to those skilled in the art . 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 considered, for example, in the following references: Sambrook et al., Molecular Cloning. A Laboratory Manua l, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987, and periodic updates; and the series Methods in Enzymo logy, Academic Press, San Diego.

[0047] "a", "an", and "the": these singular forms Terms include multiple references unless the content explicitly indicates otherwise. The definite article "a" or "an" usually means "at least one." Therefore, for example, a reference to "cell" includes combinations of two or more cells.

[0048] "Approximately" and "about": These terms refer to measurable values ​​such as quantity and duration. If such a variation is suitable for performing the disclosed method, then the specified value or ±20% or ±10%, civilization ±5%, further civilization ±1%, and further This more preferably means including a variation of ±0.1%. In addition, quantity, ratio, and Other values ​​may be presented in range form in this specification. - The matte is used concisely for convenience and includes a number explicitly specified as the limit of the range. It should be understood flexibly, but also, individual numbers and subranges are not explicitly specified. To ensure that it includes all individual numbers or subranges contained within that range, It should be understood. For example, a ratio in the range of approximately 1 to approximately 200 is an explicit representation of approximately 1 and approximately 200. It should be understood that this includes the limits described, as well as approximately 2, approximately 3, and approximately 4, etc. It is understood that this includes the individual ratios, as well as sub-ranges such as approximately 10 to approximately 50, and approximately 20 to approximately 100. It should be done.

[0049] "and / or": The term "and / or" means that one or more of the listed cases are single In Germany, or in combination with at least one of the cases described, the most of the cases described. This refers to a situation that can occur in conjunction with all other factors.

[0050] "Including": This term is comprehensive and open-ended, and not exclusive. It is interpreted as follows: Specifically, this term and its variations are used to describe identified features, steps or It means that it includes components. These terms refer to other features, steps, or components. It should not be interpreted as excluding its existence.

[0051] "Exemplary": This term means "to serve as an example, case, or illustration," and is intended to be illustrative. This should not be interpreted as excluding other components disclosed in the details.

[0052] In this specification, the term "hetero-bifunctional molecule" refers to two different functionally binding domains. It is defined as a molecule containing. In particular, the heterobifunctional molecule of the present invention is transmembrane E3 ubiquitous. A first functional binding domain for binding to tin ligase, and a second domain for binding to the molecule It has another second functional binding domain. The term "heterobifunctional" has already been indicated. Thus, the second functional binding domain binds to the second molecule, and the second molecule binds to the first functional domain. They are not the same molecule that can bind to the potential-binding domain, i.e., the same transmembrane molecule. It is not an E3 ubiquitin ligase. Preferably, the second functional binding domain is a transmembrane E 3. Does not bind to ubiquitin ligase.

[0053] The terms "protein" or "polypeptide" refer to a specific type of protein with a specific mode of action, size, and three-dimensional structure. It refers to a molecule consisting of a chain of amino acids, without referring to its composition or origin. Therefore, tan A “fragment” or “part” of a protein may still be referred to as a “protein.” Proteins defined as such and used in any manner defined herein are isolated. It may be an isolated protein. An "isolated protein" is an example of a protein that no longer exists in nature. For example, referring to proteins that are not present in in vitro or recombinant bacteria or plant host cells. It is used for this purpose. Preferably, the protein contains more than 50 amino acid residues.

[0054] In this specification, the term "protein molecule" refers to molecules linked by peptide (amide) bonds. It is understood as a molecule containing short chains of amino acid monomers. Short chains of amino acid monomers consist of two or more molecules. It contains the amino acid residues shown above. Preferably, the amino acid chain has at least 2, 3, 4, 5, 6 It has 7, 8, 9, 10, 11, 12, 13, 14, and 15 amino acid residues. There are 100 or fewer amino acid residues. Preferably, there are 50 in the protein molecule. The following amino acid residues are present. Preferably, the protein molecule has about 2 to 100, 3 to 5 It has 0, 4-40, 5-30, or 6-20 amino acid residues. Preferably The protein molecules are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 It has 4 or 15 amino acid residues. If necessary, the protein molecule is a cyclic protein. To generate a ligature molecule, one or more further organic parts such as linkages, but not limited to these. Includes minutes.

[0055] "Aptamer" is preferably a nucleic acid molecule having a specific nucleotide sequence. A ptamer can contain any appropriate number of nucleotides. A aptamer is RNA. or containing DNA, or ribonucleotide residues and deoxyribonucleotide residues It can contain both. The aptamer can be single-stranded, double-stranded, or double-stranded. It can contain a triple-stranded region. Furthermore, the aptamer can improve its stability, for example. To achieve this, it may contain chemically modified residues.

[0056] Aptamers are typically about 10 to 300 nucleotides in length. In this case, the aptamer is approximately 30 to 100 nucleotides in length.

[0057] A predetermined target (i.e., transmembrane E3 ubiquitin ligase or further transmembrane protein) The aptamer for the substance is (a) a step of bringing the candidate mixture into contact with the target, Nucleic acids that have increased affinity for the target compared to other nucleic acids in the comix are selected from the candidate mixture. (b) A step in which the increased affinity nucleic acid can be separated from the remainder of the candidate mixture. (c) a step of separating the nucleic acids; and (c) amplifying the increased affinity nucleic acids to enrich the mixture of nucleic acids. Using a method that includes the step of generating a substance, thereby identifying the aptamer of the target molecule The method includes nucleic acids that can be identified from a candidate mixture of nucleic acids.

[0058] While affinity interactions are recognized as a matter of degree, in this context, their target The "specific binding affinity" of an aptamer to a mixture is determined by whether the aptamer is generally found in the mixture or It binds to its target with a much higher affinity than it binds to other non-target components in the sample. It means to do something.

[0059] Aptamers are intended targets in environments where other substances in the same environment do not complex with nucleic acids. It has a specific binding region that can form a complex with target molecules. The specificity of the binding is its The comparative dissociation constant (Kd) of the aptamer relative to the ligand is compared to other materials in the environment or the general context. It can be defined by comparing it with the dissociation constant of the aptamer for molecules unrelated to it. Typically, the Kd of the aptamer with respect to its ligand is unrelated to the material in the environment or attached to it. It is at least about 10 times smaller than the Kd of the aptamer having the accompanying material. Alternatively, Kd is at least about 50 times smaller, and more preferably at least about 100 times smaller. Most preferably, it is at least about 200 times smaller.

[0060] In one embodiment, the aptamer that binds to the transmembrane protein is less than 1 mM and 100 nucleotides. Dissociation constants (K) less than or equal to M, less than or equal to 10 nM, less than or equal to 1 nM, or less than or equal to 0.1 nM d ) has In one embodiment, an anti-transmembrane protein antibody is used in an epitope conserved across different species. Combine.

[0061] In one embodiment, the aptamer that binds to transmembrane E3 ubiquitin ligase is 1 mM or less. Below, dissociation constants (K) of 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 is conserved across different species. It binds to an epitope.

[0062] The term "antibody" is used in its broadest sense, specifically to refer to a desired biological and / or Insofar as it exhibits immunological activity, for example, monoclonal antibodies, for example, agonists and an Tagonists, neutralizing antibodies, full-length or intact monoclonal antibodies, polyclonal antibodies, poly Valuable 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 It covers the body, the nanobody.

[0063] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" in this specification. Antibodies can be human and / or humanized.

[0064] The term "anti-transmembrane E3 ubiquitin ligase antibody" specifically refers to the desired biological and / or insofar as it exhibits immunological activity, for example, a monochromatic anti-transmembrane E3 ubiquitin ligase monochromator Ronal antibodies, such as agonists and antagonists, preferably agonists and neutralizing antibodies Body, full-length or intact monoclonal antibody, polyclonal antibody, naked antibody, polyvalent antibody, Single-chain anti-transmembrane E3 ubiquitin ligase antibody and anti-transmembrane E3 ubiquitin ligase antibody Fragments of, for example, Fab, Fab', F(ab')2 and Fv fragments, diabody, tri It covers abodies, single-domain antibodies (sdAbs), heavy chain antibodies, and nanobodies. The antibody may be a nanobody. Preferably, an anti-transmembrane E3 ubiquitin ligase antibody. It specifically binds to E3 ubiquitin ligase as defined below in this specification.

[0065] The term "anti-transmembrane protein antibody" specifically refers to a desired biological and / or immunological antibody. As long as it exhibits scientific activity, for example, a single anti-transmembrane protein monoclonal antibody, such as Ago Nist and antagonist, preferably antagonist, neutralizing antibody, full length or intact Monoclonal antibody, polyclonal antibody, naked antibody, multivalent antibody, single-chain anti-transmembrane protein Fragments of cytoplasmic antibodies and anti-transmembrane protein antibodies, e.g., Fab, Fab', F(ab') 2 and Fv fragments, diabody, triabody, single-domain antibody (sdAb), heavy chain Covers antibodies and nanobodies. Preferred antibodies may be nanobodies. Preferably Anti-transmembrane protein antibodies are specifically targeted at transmembrane proteins as defined below in this specification. To combine in an unusual way.

[0066] The term "anti-transmembrane E3 ubiquitin ligase antibody" or "transmembrane E3 ubiquitin ligase" "An antibody that binds to" means an antibody that binds to the first heterobifunctional molecule as defined herein. To be useful as a binding domain, it binds to transmembrane E3 ubiquitin ligases with sufficient affinity. This refers to an antibody that can bind to an unrelated protein, preferably an antitransmembrane E3 antibody. The degree of binding of ubiquitin ligase antibodies can be determined, for example, by radioimmunoassay (RIA) or When measured by ELISA, the binding of antibodies to transmembrane E3 ubiquitin ligase is measured. It is less than 10% of the total. In one embodiment, an antibody that binds to transmembrane E3 ubiquitin ligase The body contains substances with concentrations of 1 mM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. The dissociation constant (K d ) has. In one embodiment, an anti-transmembrane E3 ubiquitin ligase antibody It binds to epitopes that are conserved across different species.

[0067] The term "anti-transmembrane protein antibody" or "antibody that binds to a transmembrane protein" refers to an anti-transmembrane protein antibody. The body is useful as the second binding domain of a heterobifunctional molecule as defined herein. As such, binding to specific or selected transmembrane proteins with sufficient affinity is possible. This refers to antibodies that can be produced. Preferably, anti-transmembrane protein antibodies against unrelated proteins. The degree of binding can be measured, for example, by radioimmunoassay (RIA) or ELISA. In this case, the binding of antibodies to transmembrane proteins is less than approximately 10%. Antibodies that bind to transmembrane proteins have concentrations of 1 mM or less, 100 nM or less, and 10 nM or less. Dissociation constant (K) of 1 nM or less, or 0.1 nM or less. d ) has. In one embodiment, anti Transmembrane protein antibodies bind to epitopes that are conserved across different species.

[0068] The target antigen, i.e., transmembrane E3 ubiquitin ligase or further transmembrane antigen of the target. Antibodies that "bind" to proteins are antibodies that are heterobifunctional molecules as defined herein. Sufficient to be useful as the first or second binding domain, respectively. It is an antibody that binds to the aforementioned antigen with high affinity.

[0069] It can be used as the first or second binding domain in a heterobifunctional molecule. The antibody used may be a basic four-chain antibody. Such basic four-chain antibody units are preferably It is a heterotetramer sugar composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies are proteins, and they consist of five groups along with an additional polypeptide called the J chain. It consists of a basic heterotetramer unit and therefore contains 10 antigen-binding sites, while, Secretory IgA antibodies polymerize to form multivalent aggregates containing 2 to 5 basic 4-chain units along with the J chain. (It can form a body.)

[0070] In the case of IgG, a 4-chain unit is generally about 150,000 daltons. Each L chain is one The H chain is linked by a covalent disulfide bond, while the two H chains are connected by a covalent disulfide bond. Depending on the isotype, they are linked to each other by one or more disulfide bonds. Each H chain and The L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain at its N-terminus. In (VH ) and subsequently has three constant domains (C H ) for the μ and ε isotypes, and has four C H domains. Each L chain has a variable domain (V L ) at the N-terminus and subsequently has a constant domain (C L ) at the other terminus. V L aligns with V H , and C L aligns with the first constant domain of the heavy chain (C H 1). Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains. The pairing of V H with 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.

[0071] L chains from any vertebrate species can be assigned to one of two clearly distinct types called kappa and lambda based on the amino acid sequence of the constant domain. Based on the amino acid sequence of the constant domain of the heavy chain (C H ), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, each having a heavy chain designated α, δ, ε, γ, and μ, respectively. The γ and α classes have C domains HRelatively small in array and function Based on the differences, they are further divided into subclasses; for example, humans are classified into the following subclasses: Ig It expresses G1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0072] The "variable region" or "variable domain" of an antibody refers to the amino terminus of the heavy or light chain of the antibody. Refers to a domain. The variable domain of the heavy chain is "V H It can be called "". The variable domain of the light chain is " V L These domains can be referred to as "the most variable parts of an antibody." It contains an antigen-binding site.

[0073] The term "variable" refers to the significant difference in sequence between different antibodies in a segment containing a variable domain. This refers to the V domain, which mediates antigen binding and determines the specificity of a particular antibody against a particular antigen. It is defined. However, across the 110-amino acid span of the variable domain, the variability is uniform. It is not distributed in one area. Instead, the V region is called the Framework Region (FR), which is 15 It consists of relatively unchanging stretches of ~30 amino acids, with each being a "highly variable region" of 9~12 amino acids in length. It is divided into extremely variable, shorter regions called "HVRs". The variable domains of the chain and light chain each contain four FRs and primarily employ a β-sheet structure, 3 Connected by two highly variable regions, these connect the β-sheet structure, and in some cases... It forms loops that form part of the chain. The hypervariable regions in each chain are brought together in close proximity by FR. It is held there and, together with the hypervariable region from other chains, contributes to the formation of the antigen-binding site of the antibody. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public He See Health Service, National Institutes of Health, Bethesda, MD. (1991). The constant domain is not directly involved in the binding of antibodies to antigens, but antibody-dependent cells It exhibits various effector functions, such as the involvement of antibodies in toxicity (ADCC).

[0074] "Intact" antibodies have an antigen-binding site and C L and at least Also heavy chain constant domain, C H 1. C H 2 and C H This antibody contains 3. The constant domain is, Natural sequence constant domains (e.g., human natural sequence constant domains) or their amino acid combinations It can be a column variant.

[0075] "Antibody fragment" is a part of an intact antibody, preferably at least the antigen-bound and / or containing a variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragment; diabody; triabody; linear antibody (US Patent No. 5,641,870) Specification, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] See reference; 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 the antigen It retains the ability to bind.

[0076] The term "nanobody" is well known in the relevant technical field. Nanobody is a molecule consisting only of heavy chains. V of antibody H An antibody fragment containing or consisting of an H domain. Preferred nanobody. It can be induced from the camelid family, preferably from the llama family.

[0077] The papain digestion of antibodies involves two identical antigen-binding fragments called "Fab" fragments, and The remaining "Fc" fragments are produced, a name that reflects their ability to easily crystallize. The fragment consists of the entire light chain along with the variable region domain of the heavy chain (VH), and one heavy chain (CH1 It consists of the first constant domain of ). Each Fab fragment is monovalent with respect to antigen binding, that is, It has a single antigen-binding site.

[0078] Pepsin treatment of antibodies produces a single large F(ab')2 fragment, which is a bivalent antigen-binding fragment. It closely corresponds to the two active disulfide bond Fab fragments and still crosslinks the antigen. This can be done. The Fab' fragment contains one or more cysteine ​​from the antibody hinge region. H 1 By having a few additional residues at the carboxyl terminus of the domain, it differs from the Fab fragment. Yes. Fab'-SH has a cysteine ​​residue (or more) in its constant domain that can contain a free thiol group. This is the name used herein for Fab' which possesses the original F(ab')2 antibody fragment. Subsequently, a pair of Fab' fragments containing hinge cysteine ​​was generated between them. Antibody cleavage Other chemical couplings of these two are also known.

[0079] The Fc fragment consists of the carboxyl terminal portion of both H chains held together by a disulfide. This includes the effector function of the antibody, which is determined by the sequence of the Fc region, and this region also This is a region recognized by Fc receptors (FcRs) found in certain types of cells.

[0080] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. The fragment consists of one heavy chain and one light chain variable region, which are firmly and non-covalently associated. It consists of a main dimer. In the single-stranded Fv(scFv) species, it has one heavy chain and one The light chain variable domain is a "dimer" in which the light chain and heavy chain are similar to those in the double-stranded Fv species. They can associate in a structure, covalently linked by a flexible peptide linker. It can be formed. From the folding of these two domains, amino acids for antigen binding Six hypervariable loops (H chain and) contribute to the residues and confer antigen-binding specificity to the antibody. Three loops are generated from each of the L chains. However, a single variable domain (or anti Even half of the Fv (containing only three CDRs specific to the source) has the ability to recognize and bind to the antigen. It possesses some force, but its affinity is lower than that of the entire binding site.

[0081] "Single-stranded Fv" is also abbreviated as "sFv" or "scFv," but it refers to a single polyparticle. V connected to the butyl chain H and V L It is an antibody fragment containing an antibody domain. Preferably, The sFv polypeptide allows sFv to form the desired structure for antigen binding. It further includes a polypeptide linker between the VH and VL domains. For an overview of sFv, see , Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg an See d. Moore eds., Springer-Verlag, New York, pp. 269–315 (1994).

[0082] The term "monoclonal antibody," as used herein, refers to a substantially homogeneous antibody. This refers to antibodies obtained from a population; that is, individual antibodies, including those from a population, can exist in small amounts. They are identical except for possible naturally occurring mutations. Monoclonal antibodies are highly specific. Polyclonal antibodies containing different antibodies directed toward different determinants (epitopes) In contrast to body preparations, monoclonal antibodies are directed to a single antigen site. It has the advantage of being able to be synthesized without contamination by antibodies. Modifier "monoc "Nal" should be interpreted as requiring the production of antibodies by any specific method. No. For example, the first or second binding domain in the heterobifunctional molecule of the present invention The first useful monoclonal antibody was identified by Kohler et al., Nature, 256:495 (1975). They can be prepared by the hybridoma methodology described, or by bacteria, eukaryotes, etc. It can be produced using recombinant DNA methods in the cells of living animals or plants (e.g.) For example, see U.S. Patent No. 4,816,567. "Monoclonal antibody" "Also, for example, Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Phage antibody library using the technique described in J. Mol. Biol., 222:581-597 (1991) - It can be isolated from.

[0083] The monoclonal antibodies described herein, insofar as they exhibit the desired biological activity, have heavy chains and / or The light chain portion is derived from a specific species or from a specific antibody class or subclass. The sequence is identical or homologous to the corresponding sequence in the antibody to which it belongs, while the rest of the chain(s) is different. The corresponding antibody in an antibody derived from the species or belonging to another antibody class or subclass. "Chimera" antibodies that are identical or homologous to the sequence, as well as fragments of such antibodies (US) National Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci See USA, 81:6851-6855 (1984). The chimeric antibody of interest in this specification is Variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.), It also includes a "primate-like" antibody containing a human constant region sequence.

[0084] The "humanized" form of non-human (e.g., rodent) antibodies is the minimal sequence derived from the non-human antibody. It is a chimeric antibody containing human immunoglobulin ( The cypient antibody is one in which residues derived from the hypervariable region of the recipient antibody contribute to the desired antibody specificity. Non-human species such as mice, rats, rabbits, or non-human primates that have affinity and ability It is replaced with residues derived from the hypervariable region of the (donor antibody). In some cases, human immunoglobulins A small number of framework region (FR) residues in brin are replaced with corresponding non-human residues. Furthermore, the humanized antibody contains residues not found in the recipient antibody or donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies The body typically contains two variable domains and all or substantially all of the hypervariable loop. This corresponds to non-human immunoglobulins, and all or substantially all of FR are human immunoglobulins. It is a globulin sequence. Humanized antibodies also, if necessary, in the immunoglobulin constant region. This includes at least a portion of the region (Fc), typically that of human immunoglobulins. For example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323- See 329 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following overview article and the references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1:105-115 (1998); Harris, Bioch em. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

[0085] The terms "hypervariable region" and "HVR" as used herein refer to hypervariable in the array. Antibodies that are and / or form structurally defined loops involved in antigen binding This refers to the region of the variable domain. Generally, antibodies have six hypervariable regions; three in VH (H1, H2, H3), and three (L1, L2, L3) in the VL. Numerous rings of hypervariable regions. The term "column" is used and is included herein. The hypervariable region is generally referred to as the "complementarity determination region." amino acid residues derived from the "region" or "CDR" (for example, according to the Kabat numbering system) If numbered accordingly, approximately 24-34 (L1) and 50-56 (L2) residues in the VL are approximately 89-97 (L3), and approximately 31-35 (H1) and 50-65 (H2) during VH. ) and 95-102(H3); Kabat et al., Sequences of Proteins of Immunologic al Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda sda, Md. (1991)) and / or those residues derived from the "hypervariable loop" (e.g., Ch When numbered according to the Othia numbering system, residues 24-34 in VL (L1 ), 50-56 (L2) and 89-97 (L3), as well as 26-32 (H1) in VH. ), 52-56(H2) and 95-101(H3); Chothia and Lesk, J. Mol. Biol . 196:901-917 (1987)); and / or those residues from "hypervariable loops" / CDRs (For example, if numbered according to the IMGT numbering system, residues 27-3 in VL) 8 (L1), 56-65 (L2), and 105-120 (L3), as well as 27 in VH -38 (H1), 56-65 (H2), and 105-120 (H3); Lefranc, MP e t al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:21 Includes 9-221 (2000)). If necessary, antibodies are included. Honneger, A. and Plunkthun, AJ (M If numbered according to ol. Biol. 309:657-670 (2001), the following points apply: 28 in VL, 36 (L1), 63, 74-75 (L2), and 123 (L3), as well as 28 in VH. , one or more of 36(H1), 63, 74-75(H2), and 123(H3) It has symmetric insertion. The hypervariable region / CDR of the antibody of the present invention is in the IMGT numbering system. Therefore, it is preferable that they be defined and numbered.

[0086] A “framework” or “FR” residue is a hypervariable region residue as defined herein. These are variable domain residues other than the base group.

[0087] "Blocking" antibodies or "antagonist" antibodies are antibodies that bind to the biological activity of the antigen they inhibit. It inhibits or reduces sexual activity. Preferred blocking antibody or antagonist Antibodies substantially or completely inhibit the biological activity of antigens.

[0088] When used herein, "agonist antibody" refers to the functional activity of the target polypeptide. It is an antibody that mimics at least one of the following.

[0089] "Binding affinity" generally refers to the relationship between a single binding site of a molecule (e.g., an antibody) and its binding partners. This refers to the sum of the strengths of non-covalent interactions between toners (e.g., antigens). Unless otherwise specified... To the extent that it is used herein, “binding affinity” means the members of a binding pair (for example, This refers to the intrinsic binding affinity that reflects the 1:1 interaction between the antibody and the antigen. The affinity for -toner Y is generally determined by the dissociation constant (K d It can be expressed as ). Affinity is , including those described herein, by general methods known in the art This can be measured. Low affinity antibodies generally bind slowly to antigens and easily Although they tend to dissociate, high-affinity antibodies generally bind to antigens faster and for longer periods. There is a tendency to continue this. 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 The following is a description of the details.

[0090] "Kd " or "K d The "value" is approximately 10-50 response units (RU) of immobilized antigen CM5 chip. Using a pump, at 25°C, use BIAcore(trademark)-2000 or BIAcore(trademark)- Surface plastics using 3000 (BIAcore, Inc., Piscataway, NJ) This can be measured using a Zumon resonance assay. Simply put, Ruboxymethylated Dextran Biosensor Chip (CM5, BIAcore Inc.) .) In accordance with the supplier's instructions for use, N-ethyl-N'-(3-dimethylaminopropyl (Lu)-Carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) It is activated by 5 μg / ml (approximately 0.2) of the antigen in 10 mM sodium acetate at pH 4.8. After diluting to μM, inject at a flow rate of 5 μl / min, and the coupled protein will be approximately 10 A response unit (RU) is achieved. After antigen injection, 1M ethanolamine is injected, and no reaction occurs. Block the group. For kinetic measurements, use an antibody or Fab (0.78 nM~500 nM) The 2x series dilutions of ) were subjected to a flow rate of approximately 25 μl / min at 25°C to obtain 0.05% Tween 20 Inject into PBS (PBST) containing [the substance]. Association rate (k on ) and dissociation rate (k off )of, By simultaneously matching the association and dissociation sensorgrams, a simple one-to-one Langmui can be created. The calculation was performed using the r-coupled model (BIAcore evaluation software version 3.2). . Equilibrium dissociation constant (K d ) to k off / k on It was calculated as a ratio. For example, Chen, Y., et al. See l., (1999) J. Mol Biol 293:865-881. Surface plasmon resonance assay described above. The on-rate is 10 6 M -1 S -1 If it exceeds this, the on-rate will have a stop flow. Equipped with a spectrophotometer (Aviv Instruments) or a stirred red cuvette. The 8000 series SLM-Aminco spectrophotometer (ThermoSpectroni c) When measured with a spectrometer such as the above, in the presence of increased antigen concentrations, in PBS, pH 7.2 Changes in fluorescence emission intensity of a 20nM anti-antigen antibody (Fab form) at 25°C (excitation = 295nm) By using fluorescence quenching technology to measure emission (340nm, 16nm band passthrough) It is possible to make a decision.

[0091] Furthermore, the present invention is also known as "on-rate" or "meeting speed" or "meeting rate" or " k on " is, as mentioned above, BIAcore(trademark)-2000 or BIAcore( Trademark)-3000 (BIAcore, Inc., Piscataway, NJ) This can be determined using the same surface plasmon resonance technique described above.

[0092] Preferably, it is used as a first or second binding domain in a heterobifunctional molecule. The antibody does not significantly cross-react with other proteins.

[0093] The terms "antigen-binding protein" and "binding domain" in the heterobifunctional molecule of the present invention are These can be used interchangeably in this specification.

[0094] The term "epitope" refers to the first or second binding domain of the heterobifunctional molecule of the present invention. These are parts of the molecules that are bound by each of them. This term is specific to antigen-binding proteins. They can be coupled in a specific manner, for example, the heterobifunctional components defined below in this specification. It includes any determinant that can specifically bind to the first or second domain of the child. Pitopes can be continuous or discontinuous (for example, in polypeptides, polyp In a ptide sequence, amino acid residues that are not consecutive to each other but are within the molecular context are antigens. (Bound by binding proteins). The epitope is preferably defined herein. On the transmembrane E3 ubiquitin ligase that is intended, or for the purpose as defined herein It is located on further transmembrane proteins.

[0095] Epitope determinants include amino acids, sugar side chains, phosphoryl, sulfonyl, or sulfate groups. It may contain chemically active surface groupings of the molecule, and has a unique three-dimensional structure. It may have sex and / or specific charge properties. Generally, it is specific to a particular target antigen. Antibodies target epitopes on target antigens in complex mixtures of proteins and / or macromolecules. Prioritize recognition.

[0096] The term "Fc region" as used herein includes both the natural sequence Fc region and the variant Fc region. , used to define the C-terminal region of immunoglobulin heavy chains. The boundary of the Fc region can vary, but the human IgG heavy chain Fc region is usually Cys226 It is defined as an amino acid residue at the position, or an extension from Pro230 to its carboxyl terminus. It is understood that the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) is, for example If, during the production or purification of antibodies, or if the nucleic acid encoding the heavy chain of an antibody is recombinantly manipulated... It can be removed by doing so.

[0097] The term "antibody containing an Fc region" refers to an antibody that contains an Fc region. n (residue 447 according to the EU numbering system) is, for example, during antibody purification, or in the antibody The nucleic acid encoding it can be removed by recombinant manipulation. The heterobifunctional molecule containing an antibody having an Fc region according to the present invention has K447 or Alternatively, it may include an antibody from which K447 has been removed.

[0098] "Amino acid sequence": This refers to the order of amino acid residues in a protein, or within a protein. This refers to the sequence of amino acids in a protein. In other words, any sequence of amino acids in a protein can be called an amino acid sequence. .

[0099] "Nucleotide sequence": This refers to the sequence of nucleotides in a nucleic acid, or within a nucleic acid. In other words, any order of nucleotides in a nucleic acid can be called a nucleotide sequence.

[0100] The terms "homology" and "sequence identity" are used interchangeably in this specification. Identity, as determined herein by comparison of sequences, is defined as two or more A Between amino acid (polypeptide or protein) sequences or between two or more nucleic acids (polynucleotides) (d) Defined as the association between sequences. In the art, "identity" is also, in some cases Accordingly, if determined by matching between strings of such sequences, the amino acid sequences This refers to the degree of sequence association between nucleic acid sequences. The "similarity" between two amino acid sequences is, The amino acid sequence of one polypeptide and its conserved amino acid substitutions are used to create a second polypeptide. It is determined by comparing it with the sequence of CHIDO.

[0101] The term "complementarity" as used herein refers to a chain that is completely complementary (e.g., a second chain or a reverse chain). It is defined as sequence identity of nucleotide sequences. For example, 100% complementary ( Sequences that are (or are completely complementary) are, in this specification, considered to have 100% sequence identity with complementary strands. It is understood that such sequences have, for example, in this specification, 80% complementary sequences are (completely) complementary It is understood to have 80% sequence identity with the chain.

[0102] "Identity" and "similarity" can be easily calculated by known methods. Sequence identity and sequence similarity depend on the lengths of the two sequences, and can be determined globally or locally. Using a precise alignment algorithm, two peptide sequences or two nucleotides This can be determined by aligning the array. Arrays of similar length are preferably , a global alignment algorithm that optimally aligns the sequence over its entire length (for example For example, they are aligned using Needleman Wunsch, and are arranged in substantially different lengths. The columns are preferably aligned using a local alignment algorithm (e.g., SmithWatts). The array is then sorted using (for example, using default parameters). (When optimally aligned using the GAP or BESTFIT program) If a minimum percentage of oneness (as defined below) is shared, then "substantially" They can be described as "essentially identical" or "essentially similar." GAP is Needleman and Using the Wunsch global alignment algorithm, over the entire length (full length) This sorts two arrays, maximizes the number of matches, and minimizes the number of gaps. Global Alignment is appropriately used to determine sequence identity when two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotide) / 8 (protein) and a gap extension penalty = 3 (nucleotide) / 2 (protein). For nucleotides, the default score matrix used is nwsgapdna, and for proteins , the default score matrix is Blosum62 (Henikoff & Henikof f, 1992, PNAS 89, 915-919). The scores for sequence alignment and percentage sequence identity are obtained 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 the EmbossWIN version 2.10.0 program "needle" (which uses the global Needleman Wun sch algorithm) or "water" (which uses the local Smith Wa terman algorithm), or with default settings (for both "needle" and "water", and for both protein and DNA alignment, the default gap open penalty is 1 0.0 and the default gap extension penalty is 0.5; the default score matrix is Blosum62 for proteins and DNAFull for DNA). For both protein and DNA alignment, the default gap open penalty is 10.0 and the default gap extension penalty is 0.5; the default score matrix is Blosum62 for proteins and DNAFull for DNA. It can be determined using (which is ll). If the sequences have substantially different total lengths, Smi Local alignment methods, such as those using the Waterman algorithm, are preferred. It seems so.

[0103] Instead, percentage similarity or identity is expressed using terms like FASTA, BLAST, etc. This can be determined by using Gorhythm to search public databases. Therefore, the nucleic acid and protein sequences of the present invention can be further used, for example, with other families. To identify the leader or related sequence, a search is performed against public databases. It can be used as a "query sequence". Such searches are described by Altschul, et al. (1990) BLASTn and BLASTx programs in J. Mol. Biol. 215:403-10 (bar This can be done using John 2.0). BLAST nucleotide search is the nucleus of the present invention. To obtain a nucleotide sequence homologous to the acid molecule, use the NBLAST program, score = 10. It can be performed with 0 and word length = 12. BLAST protein search is the protein of the present invention To obtain an amino acid sequence homologous to the molecule, use the BLASTx program, score = 50. This can be done with a word length of 3. To obtain a gapped alignment for comparison purposes. In addition, Ga as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402 Gapped BLAST can be used. BLAST and Gapped BLAST When using ST programs, each program (for example, BLASTx and B) You can use the default parameters of LASTn. http: / / www.ncbi.nlm. Please refer to the National Center for Biotechnology Information website at nih.gov / .

[0104] As used herein, the terms “prevent,” “prevent,” and “prevention” are used with respect to the same meaning. This refers to the recurrence, onset, or onset of a disease, preferably a disease as defined herein below. This is for the prevention or reduction of progression, or the severity of the disease or one or more of its symptoms and / or This refers to the prevention or reduction of the period.

[0105] As used herein, the terms “treatment” and “therapy” mean disease, preferably the present disease. In this document, prevention, treatment, and management of the diseases, or one or more of their symptoms, as defined below. This refers to any protocol(s) or method(s) that can be used in the improvement process. It can refer to a name / or drug(s).

[0106] As used herein, the terms “to treat,” “to treat,” and “treatment” mean the same as “treatment.” , disease, preferably the progression, severity, and / or the disease as defined herein below. This refers to a reduction or improvement in duration, and / or a reduction in one or more symptoms of a disease. We will improve it.

[0107] As used herein, the term “effective dose” means the severity and / or duration of the disease. It reduces the symptoms, improves one or more of the symptoms, prevents the progression of the disease, or slows the regression of the disease. Is it sufficient to cause, or is it the onset, recurrence, or commencement of the disease or one or more of its symptoms? Or it may lead to prevention of progression, or the preventive effect of another treatment (e.g., another therapeutic agent) and / or treatments that are sufficient to enhance or improve the therapeutic effect, such as prophylactic agents or Refers to the amount of a therapeutic agent, preferably a heterobifunctional molecule as defined herein. Preferably the disease is a disease as defined herein below. **BRIEF DESCRIPTION OF THE INVENTION**

[0108] **DETAILED DESCRIPTION** The present invention relates to the concept of the present invention employing a hetero bifunctional molecule for the targeted internalization and subsequent degradation of membrane-bound proteins. The heterobifunctional molecule of the present invention can simultaneously bind to a transmembrane ubiquitin ligase and a membrane-bound protein such as a cancer-promoting receptor. The induced proximity of the ubiquitin ligase to the desired target transmembrane protein results in targeted ubiquitination, followed by removal from the cell surface and subsequent degradation. For example as a result, cancer cell proliferation is impaired. A schematic diagram of an exemplary embodiment of the present invention is provided in FIG. 1.

[0109] The advantages of this approach include at least the following: i) The heterobifunctional molecule of the present invention enables an increase in potency by requiring only sub-stoichiometric amounts of the molecule compared to the target molecule when compared to conventional "occupancy-based" therapies. ii) The requirement for specific binding of two proteins, namely a transmembrane E3 ubiquitin ligase and a transmembrane protein, also reduces potential off-target toxicity. Preferably, a ubiquitin ligase that localizes to the cell membrane and exhibits increased expression in cancer cells is employed. iii) Target protein degradation results in a long-term pharmacodynamic effect because sufficient time is required to synthesize a new transmembrane protein. ​​​​​​​​iv) Heterobifunctional molecules bind to extracellular protein moieties and therefore pass through the cell membrane. There's no need to waste time on it. v) Cancer cells possess self-regenerative properties, and RNF43 and ZNRF3 It is known that it richly expresses several types of transmembrane E3 ubiquitin ligases, such as Yes. In this example, four alleles produce proteins that exhibit ubiquitination activity. It reduces the possibility of inactivation and resistance due to mutations.

[0110] Furthermore, the inventors have found that the effective interaction between transmembrane E3 ubiquitin ligase and membrane-bound proteins is The combination, for example, the induced proximity of transmembrane ubiquitin E3 ligase and membrane-bound proteins Effectiveness of screening for combinations that result in cell surface removal of membrane-bound proteins A rational method was discovered. Using this simple method, effective heterobifunctional molecules were constructed. Therefore, targeting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein It is possible.

[0111] Therefore, in the first aspect, the present invention relates to a heterogeneous molecule comprising first and second binding domains. Regarding a bifunctional molecule. The first binding domain specifically binds to transmembrane E3 ubiquitin ligase. It can bind, and the second binding domain can bind to specific membrane-bound proteins. can.

[0112] The simultaneous binding of transmembrane E3 ubiquitin ligase and membrane-bound proteins involves these two molecules They are brought into close proximity to each other. As a result, the transmembrane E3 ubiquitin ligase then proceeds to the membrane It can ubiquitinate binding proteins.

[0113] Therefore, preferably, transmembrane E3 ubiquitin ligase and hete to membrane-bound proteins The simultaneous binding of rhodifunctional molecules leads to ubiquitination of membrane-bound proteins.

[0114] It is well known that ubiquitination leads to the degradation of ubiquitinated proteins. However Furthermore, preferably, transmembrane E3 ubiquitin ligase and hete to membrane-bound proteins The simultaneous binding of difunctional molecules leads to the degradation of membrane-bound proteins.

[0115] The simultaneous binding of transmembrane E3 ubiquitin ligase and membrane-bound proteins involves these two molecules They are brought into close proximity to each other. As a result, membrane-bound proteins are internalized, preferably so It can be subsequently broken down.

[0116] Transmembrane E3 ubiquitin ligase bound to the first binding domain The first binding domain of the heterobifunctional molecule is specific to transmembrane E3 ubiquitin ligase. It enables target binding of membrane-bound proteins. Transmembrane E3 ubiquitin ligase enables target binding of membrane-bound proteins. It can mediate transformation and endocytosis, that is, heterogeneous as defined herein. Ubiquitination of proteins bound by the second binding domain of a rhinofunctional molecule It can also mediate endocytosis.

[0117] Ubiquitination and endocytosis of the substrate preferably remove the substrate from the cell surface. Remove. The internalized substrate can then be degraded. Therefore, preferably, transmembrane E3 Ubiquitin ligases mediate ubiquitination, cell surface removal, and membrane-bound protein degradation. It can intervene, that is, the second binding domain of a heterobifunctional molecule as defined herein. It can mediate the ubiquitination, cell surface removal, and degradation of proteins bound by this compound. ru.

[0118] Therefore, preferably, transmembrane E3 ubiquitin ligase and hete to membrane-bound proteins The simultaneous binding of difunctional molecules leads to the internalization of membrane-bound proteins, thereby the membrane Removes the binding protein from the cell surface.

[0119] Preferably, the heterobifunctional component of the transmembrane E3 ubiquitin ligase and membrane-bound protein. The simultaneous binding of these proteins leads to the internalization and degradation of membrane-bound proteins. Therefore, it is preferable Transmembrane E3 ubiquitin ligase and membrane-bound proteins are expressed in the same cell. If necessary, a small number of transmembrane E3 ubiquitin ligases and membrane-bound proteins may be used. At least one of these compounds may be overexpressed in the cell.

[0120] Ubiquitination and degradation are carried out using any suitable method known in the art. It can be evaluated by... As a non-limiting example, ubiquitination and degradation can be evaluated by... As described in Koo et al, Nature (2012) (cited above), which is incorporated herein, the evaluation is performed. It is possible.

[0121] The substrate protein is an E3 ligase protein that recruits a ubiquitin-charged E2 enzyme. Selected for ubiquitin-mediated modification of lysine residues through interaction with (Clague M J and Urbe S (2010), Cell; 43(5):682-5). This is the process of transferring a single ubiquitin molecule to a substrate. Movement (monoubiquitination), or, for example, lysine residues present in the previous ubiquitin molecule. This leads to further coupling of ubiquitin molecules to the previous ubiquitin molecule, It can form chains. The seven lysines of ubiquitin have different three-dimensional structures. It provides the formation of isopeptide chain links, all of which are present in eukaryotic cells (Xu et al.). (2009), Cell 137, 133-145). A specific combination of E2 and E3 enzymes recruited to the substrate, Determine the type of chain linkage.

[0122] In particular, lysosomal degradation is based on a different ubiquitination pattern than proteasomal degradation. It may require high-quality proteins. For example, lysine-48 (Lys48) linked polyubitus. Substrates labeled with chitin chains often result in proteasome targets. Instead, mono Ubiquitin, multiubiquitin, Lys48-linked or Lys63-linked polyubiquitin A substrate labeled with either of these labels is directed towards the lysosome.

[0123] Transmembrane E3 ubiquitin ligase-mediated degradation is lysosomal degradation and pro- It may be at least one of the theasome degradation processes. Preferably, transmembrane E3 ubiquitin Ligase-mediated degradation is at least lysosomal degradation.

[0124] Therefore, preferably, transmembrane E3 ubiquitin ligase and hete to membrane-bound proteins The simultaneous binding of difunctional molecules leads to the internalization of membrane-bound proteins. Preferably, transmembrane binding. The simultaneous binding of heterobifunctional molecules to E3 ubiquitin ligase and membrane-bound proteins is a membrane-bound process. Internalization of binding proteins, and less than proteasome and lysosomal degradation. It also brings about one of the following: preferably, transmembrane E3 ubiquitin ligase and membrane-bound proteins. The simultaneous binding of heterobifunctional molecules facilitates the internalization and lysosomal degradation of membrane-bound proteins. To bring about.

[0125] Preferably, the transmembrane E3 ubiquitin ligase converts transmembrane proteins into monoubiquitin, Multiubiquitin, Lys63-linked polyubiquitin, or Lys48-linked polyubiquitin Ubiquitination occurs in the transmembrane chain. Preferably, the transmembrane E3 ubiquitin ligase is used to ubiquitinate the transmembrane protein. The substance is formed using monoubiquitin, multiubiquitin, or Lys63-linked polyubiquitin chains. It ubiquitinates. Preferably, the transmembrane E3 ubiquitin ligase is a transmembrane protein. Poly Ubiquitination occurs. Preferably, the transmembrane E3 ubiquitin ligase converts the transmembrane protein into ubiquitin. Polyubiquitination occurs with Lys63-linked polyubiquitin chains.

[0126] Many transmembrane E3 ubiquitin ligases exhibit tissue-specific expression or one or more of them. It shows overexpression in cancer types. Therefore, preferably, as defined herein Transmembrane E3 ubiquitin ligases that can bind to heterobifunctional molecules are selected. It is a transmembrane E3 ubiquitin ligase expressed in the target tissue. As a non-limiting example, Transmembrane E3 ubiquitin ligases RNF43 and ZNRF3 are used in, but are not limited to, the intestines, etc. It is selectively expressed in adult stem cell populations of multiple tissues. Further examples include, but are not limited to, Furthermore, transmembrane E3 ubiquitin ligases MARCH1 and MARCH9 are used by immune cells to detect odors. This results in increased expression.

[0127] Preferably, the transmembrane E3 ubiquitin ligase is selected from cancerous tissue and other tissues, but is not limited to this selection. It is expressed only in the target tissue.

[0128] Alternatively, or even further, they bind to heterobifunctional molecules as defined herein. Transmembrane E3 ubiquitin ligases that can be expressed in one or more types of cancer Preferably, it is a transmembrane E3 ubiquitin ligase that exhibits overexpression.

[0129] Preferably, the transmembrane E3 ubiquitin ligase is RNF43, RNF167, ZNRF 3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8 , MARCH9, RNF149, RNF145, RNFT1, RNF130 and RNF Selected from the group consisting of 128. Preferably, the transmembrane E3 ubiquitin ligase is RN F43, RNF167, ZNRF3, RNF13, AMFR, MARCH1, MARCH 2, MARCH4, MARCH8, MARCH9, RNF145, RNFT1, RNF1 Selected from the group consisting of 30 and RNF128. Preferably, transmembrane E3 ubiquitin. Ligase is one of the following: RNF43, RNF167, RNF128, and RNF130 At most one. Preferably, the transmembrane E3 ubiquitin ligase is RNF43 and R It is at least one of NF167.

[0130] Transmembrane E3 ubiquitin ligases can be overexpressed. A non-limiting example is RNF43. , ZNRF3, RNF13, AMFR, MARCH1, MARCH2, MARCH4, M ARCH8, MARCH9, RNF149, RNF145, RNFT1, RNF167, RNF130 and RNF128 show increased expression in cancer, according to this technology. It is publicly known in the field.

[0131] Non-restrictive examples: - RNF43 is used in lung cancer and colorectal cancer (EMBL-EBI gene expression atlas) ) is overexpressed in; - ZNRF3 is found in hepatocellular carcinoma and (metastatic) colorectal cancer (gene expression atlas). It is overexpressed in [location]; - RNF13 is used in osteosarcoma (gene expression atlas) and pancreatic cancer (Zhang Q, et al, It is overexpressed in Cell Res. 2009;19(3):348-57); AMFR is not limited to lung cancer, stomach cancer, colorectal cancer, liver cancer, and skin cancer. It is overexpressed in many cancers, including breast cancer and bladder cancer, and its elevated expression is These cancers were found to be associated with poor prognosis and metastasis (Chiu CG et al.) al, Expert Rev Anticancer Ther. 2008;8(2):207-17; B. Huang and A. Raz, Cell Res 1995; 5(2):221-234; Gene Expression Atlas); - MARCH1 is overexpressed in breast and ovarian cancer (Meng Y et al, Oncol Rep. 2016; 36(5): 2463-2470; Gene Expression Atlas); - MARCH2 is highly expressed in many different tumor types (human protein Atlas of the material; - MARCH4 is overexpressed in esophageal cancer and thyroid cancer (gene expression truss); - MARCH8 is overexpressed in esophageal cancer and lung cancer (Singh S et al, C ancer Cell Int. 2017;17:116; Fan J et al, Oncotarget. 2017; 8(64): 108238-108248 ); - MARCH9 is overexpressed in lung cancer (Fan J, op. cit.); - RNF149 is overexpressed in osteosarcoma (gene expression atlas); - RNF145 is overexpressed in osteosarcoma (gene expression atlas); - RNFT1 is overexpressed in glioblastoma (gene expression atlas); - RNF167 is overexpressed in squamous cell carcinoma (gene expression atlas); - RNF130 is overexpressed in esophageal cancer and prostate cancer (gene expression atlas). To be revealed; and - RNF128 is overexpressed in intestinal cancer (gene expression atlas).

[0132] Furthermore, gene amplification is used in many cancers (cBioPortal), and in breast cancer. It is commonly found for RNFT1 and RNF13. Gene amplification is one Generally, this leads to an increase in protein expression.

[0133] Furthermore, some members of the MARCH family are MARCH1 and MARCH Immune cells including H9 (Wang X et al, Semin Cancer Biol. 2008; 18(6): 441-450) It is expressed at a high level.

[0134] In one embodiment, the heterobifunctional molecule comprises first and second binding domains, i) The first binding domain specifically binds to transmembrane E3 ubiquitin ligase. Transmembrane E3 ubiquitin ligase is expressed in cancer tissue, preferably selectively. It is expressed or overexpressed in; ii) The second binding domain can specifically bind to transmembrane proteins, The protein is known to be involved in the aforementioned cancer tissue, or is expected to be involved. It was thought, Simultaneous application of heterobifunctional molecules to transmembrane E3 ubiquitin ligases and transmembrane proteins The binding preferably results in ubiquitination and internalization of the transmembrane protein.

[0135] Preferably, the transmembrane E3 ubiquitin ligase and the transmembrane protein are in the same cell. It is expressed, preferably within the same cancer cells.

[0136] Preferably, both E3 ubiquitin ligase and transmembrane protein are used in lung cancer, colorectal cancer. Cancer, hepatocellular carcinoma, osteosarcoma, pancreatic cancer, stomach cancer, liver cancer, skin cancer, breast cancer, bladder cancer Ovarian cancer, esophageal cancer, thyroid cancer, cervical cancer, glioblastoma, squamous cell carcinoma, prostate cancer Select from the group consisting of (gene expression atlas) and colon cancer and / or their metastases. It is expressed in cancer cells.

[0137] In one embodiment, the heterobifunctional molecule comprises first and second binding domains, i) The first binding domain specifically binds to transmembrane E3 ubiquitin ligase. Transmembrane E3 ubiquitin ligase can be expressed in immune cells, preferably selectively. It is expressed or overexpressed in; ii) The second binding domain can specifically bind to transmembrane proteins, The same protein is expressed in the same immune cells; Simultaneous application of heterobifunctional molecules to transmembrane E3 ubiquitin ligases and transmembrane proteins The binding preferably results in ubiquitination and internalization of the transmembrane protein.

[0138] Preferably, the transmembrane E3 ubiquitin ligase is SEQ ID NOs: 1, 3, 5, 7, 9, 11, Sequences selected from the group consisting of 13, 15, 17, and 19, and at least about 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 It has % or 100% sequence identity.

[0139] Preferably, the transmembrane E3 ubiquitin ligase is SEQ ID NOs: 2, 4, 6, 8, 10, 12 Sequences selected from the group consisting of 14, 16, 18, and 20, and at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, It is coded by an array that has 99% or 100% array identity.

[0140] Preferably, the transmembrane E3 ubiquitin ligase is one of RNF43 and ZNRF3. There is at least one. The proteins RNF43 and ZnRF3 are preferably each At least approximately 60%, 65%, 70%, 75%, 80%, and 85% of sequence numbers 1 and 3. , having sequence identity of 90%, 95%, 96%, 97%, 98%, 99%, or 100% Preferably, the RNF43 and ZNRF3 proteins are SEQ ID NO: 2 and 4 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, Co They'll be killed.

[0141] In preferred embodiments, the transmembrane E3 ubiquitin ligase is RNF43. Preferably The RNF43 protein is at least approximately 60%, 65%, 70%, and 75% of SEQ ID NO: 1. %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It has sequence identity. Preferably, the RNF43 protein has sequence identity with at least SEQ ID NO: 2. Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% It is encoded by sequences having 98%, 99%, or 100% sequence identity. Alternatively, the first binding domain of the heterodifunctional molecule binds to RNF43, and the heterodifunctional The second binding domain of the sex molecule is the sex of the protein bound to the second binding domain. In the context, it binds to the membrane-bound protein as defined below in this specification. Preferably, Transmembrane proteins that can bind to the second binding domain of a telobifunctional molecule are T GFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, F ZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, F ZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6 and Selected from the group consisting of CMTM4 and WLS. Preferably, heterobifunctional components. Transmembrane proteins that can bind to the second binding domain of this protein include TGFβR2, E The group is selected from GFR, FLT3 and FZD7, and PD-L1.

[0142] Preferably, the heterobifunctional molecules as defined herein are specific to RNF43. A first binding domain that can bind to TGFβR2, and a domain that specifically binds to TGFβR2. It includes a second binding domain that enables this.

[0143] Preferably, the heterobifunctional molecules as defined herein are specific to RNF43. A first binding domain that can bind to, and a domain that specifically binds to EGFR. It includes a second binding domain.

[0144] Preferably, the heterobifunctional molecules as defined herein are specific to RNF43. A first binding domain that can bind to, and a domain that specifically binds to FLT3. It includes a second binding domain.

[0145] Preferably, the heterobifunctional molecules as defined herein are specific to RNF43. A first binding domain that can bind to, and a domain that specifically binds to FZD7. It includes a second binding domain.

[0146] Preferably, the heterobifunctional molecules as defined herein are specific to RNF43. A first binding domain capable of binding to PD-L1, and specific binding to PD-L1. It includes a second binding domain that enables this.

[0147] In preferred embodiments, the transmembrane E3 ubiquitin ligase is ZNRF3. Preferably The ZNRF3 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 3. %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It has sequence identity. Preferably, the ZNRF3 protein has sequence identity with at least SEQ ID NO: 4. Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% It is encoded by sequences having 98%, 99%, or 100% sequence identity. Alternatively, the first binding domain of the heterobifunctional molecule binds to ZNRF3, and the heterobifunctional The second binding domain of the sex molecule is the sex of the protein bound to the second binding domain. In the context, it binds to the membrane-bound protein as defined below in this specification. Preferably, Transmembrane proteins that can bind to the second binding domain of a telobifunctional molecule are T GFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, F ZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, F ZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6, CM The group is selected from TM4 and WLS.

[0148] In preferred embodiments, the transmembrane E3 ubiquitin ligase is RNF13. Preferably The RNF13 protein is at least approximately 60%, 65%, 70%, and 75% of SEQ ID NO: 5. %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It has sequence identity. Preferably, the RNF13 protein has sequence identity with at least SEQ ID NO: 6. Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% It is encoded by sequences having 98%, 99%, or 100% sequence identity. Alternatively, the first binding domain of the heterobifunctional molecule binds to RNF13, and the heterobifunctional The second binding domain of the sex molecule is the sex of the protein bound to the second binding domain. In the context, it binds to the membrane-bound protein as defined below in this specification. Preferably, Transmembrane proteins that can bind to the second binding domain of a telobifunctional molecule are T GFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, F ZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, F ZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6, CM The group is selected from TM4 and WLS.

[0149] In preferred embodiments, the transmembrane E3 ubiquitin ligase is AMFR. Preferably The AMFR protein is at least approximately 60%, 65% of the SEQ ID NO: 7 or SEQ ID NO: 51. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% Or it has 100% sequence identity. Preferably, the AMFR protein is sequence number 8 Or sequence number 52 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, It has sequence identity of 90%, 95%, 96%, 97%, 98%, 99%, or 100%. Encoded by the sequence. Preferably, the first binding domain of the heterobifunctional molecule is A The second binding domain of the heterobifunctional molecule binds to the MFR, and the second binding domain is " In the section "bound proteins," the membrane-bound proteins defined herein below It binds to the substance. Preferably, it binds to the second binding domain of the heterobifunctional molecule. The transmembrane proteins that can be produced are TGFβR1, TGFβR2, EGFR, ERBB2, and ER BB3, IGF1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDG FRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZ D7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, The group is selected from CTLA4, CMTM6, CMTM4, and WLS.

[0150] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH1. Furthermore, the MARCH1 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 9. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It has 0% sequence identity. Preferably, the MARCH1 protein is small compared to sequence number 10. Even without it, approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 96%. Encoded by sequences having 97%, 98%, 99%, or 100% sequence identity. Preferably, the first binding domain of the heterobifunctional molecule binds to MARCH1, The second binding domain of the telobifunctional molecule is "protein bound to the second binding domain." In the section of this specification, it binds to the membrane-bound protein defined below. Alternatively, a transmembrane protein that can bind to the second binding domain of a heterobifunctional molecule The components are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R , MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, F ZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, F ZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMT The group is selected from M6, CMTM4, and WLS.

[0151] In preferred embodiments, the transmembrane E3 ubiquitin ligase is MARCH4. Furthermore, the MARCH4 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 11. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the MARCH4 protein is sequence number 12 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to MARCH4. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM The group is selected from TM6, CMTM4, and WLS.

[0152] In preferred embodiments, the transmembrane E3 ubiquitin ligase is MARCH2. Furthermore, the MARCH2 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 13. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the MARCH2 protein is sequence number 14 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to MARCH2. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM The group is selected from TM6, CMTM4, and WLS.

[0153] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH8. Furthermore, the MARCH8 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 15. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the MARCH8 protein is sequence number 16 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to MARCH8. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM The group is selected from TM6, CMTM4, and WLS.

[0154] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is MARCH9. Furthermore, the MARCH9 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 17. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the MARCH9 protein is sequence number 18 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to MARCH9. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM The group is selected from TM6, CMTM4, and WLS.

[0155] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF149. Furthermore, the RNF149 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 19. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the RNF149 protein is sequence number 20 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to RNF149. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM The group is selected from TM6, CMTM4, and WLS.

[0156] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF145. Furthermore, the RNF145 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 21. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the RNF145 protein is sequence number 22 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to RNF145. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM The group is selected from TM6, CMTM4, and WLS.

[0157] In preferred embodiments, the transmembrane E3 ubiquitin ligase is RNFT1. Preferably The RNFT1 protein is at least approximately 60%, 65%, 70%, and 7% of the SEQ ID NO: 23. 5%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It has % sequence identity. Preferably, the RNFT1 protein has less than 24 SEQ ID NO: 24. Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 It is encoded by sequences with 7%, 98%, 99%, or 100% sequence identity. Preferably, the first binding domain of the heterobifunctional molecule binds to RNFT1, and the heterobi The second binding domain of the functional molecule is the "protein bound to the second binding domain". In the action, it binds to a membrane-bound protein as defined below in this specification. Preferably Transmembrane proteins that can bind to the second binding domain of heterobifunctional molecules are , TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, IGF1R, ME T, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1 , FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 , FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CMTM6, The group is selected from CMTM4 and WLS.

[0158] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF167. Furthermore, the RNF167 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 25. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the RNF167 protein is sequence number 26 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to RNF167. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM Selected from the group consisting of TM6, CMTM4, and WLS. Preferably, heterodifunctional. Transmembrane proteins that can bind to the second binding domain of sex molecules include TGFβR2. The group is selected from EGFR, FLT3, PD-1, and CTLA4.

[0159] Preferably, the heterobifunctional molecule as defined herein is specific to RNF167. A first binding domain that can bind specifically, and a domain that specifically binds to TGFβR2. It includes a second binding domain that can perform this action.

[0160] Preferably, the heterobifunctional molecule as defined herein is specific to RNF167. A first binding domain that can bind specifically, and which binds specifically to EGFR. It includes a second binding domain that enables this.

[0161] Preferably, the heterobifunctional molecule as defined herein is specific to RNF167. A first binding domain that can bind specifically, and that binds specifically to FLT3. It includes a second binding domain that enables this.

[0162] Preferably, the heterobifunctional molecule as defined herein is specific to RNF167. A first binding domain that can bind specifically, and which specifically binds to PD-1. It includes a second binding domain that enables this.

[0163] Preferably, the heterobifunctional molecule as defined herein is specific to RNF167. A first binding domain that can bind specifically, and a domain that specifically binds to CTLA4. It includes a second binding domain that enables this.

[0164] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF130. Furthermore, the RNF130 protein is at least approximately 60%, 65%, and 70% of the protein in Sequence ID No. 27. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the RNF130 protein is sequence number 28 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to RNF130. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM Selected from the group consisting of TM6, CMTM4, and WLS. Preferably, heterodifunctional. Transmembrane proteins that can bind to the second binding domain of sex molecules include PD-1 and It is at least one of PD-L1, preferably as defined herein. The heterobifunctional molecule is a first binding domain that can specifically bind to RNF130. It includes a second binding domain that can specifically bind to PD-1. In this specification, heterobifunctional molecules are specifically bound to RNF130. A first binding domain that can bind, and which can specifically bind to PD-L1. It includes a second binding domain.

[0165] In a preferred embodiment, the transmembrane E3 ubiquitin ligase is RNF128. Furthermore, the RNF128 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 29. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 1 It has 00% sequence identity. Preferably, the RNF128 protein is sequence number 30 and At least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% Encoded by arrays having %, 97%, 98%, 99%, or 100% array identity. Preferably, the first binding domain of the heterobifunctional molecule binds to RNF128. The second binding domain of a heterobifunctional molecule is "the protein bound to the second binding domain In the section "Quality," it binds to the membrane-bound protein defined below in this specification. Mashiku is a transmembrane tangent that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IGF1. R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, CM Selected from the group consisting of TM6, CMTM4, and WLS. Preferably, heterodifunctional. Transmembrane proteins that can bind to the second binding domain of sex molecules include FLT3 and P The group is selected from D-1 and PD-L1.

[0166] Preferably, the heterobifunctional molecule as defined herein is specific to RNF128. A first binding domain that can bind specifically, and that binds specifically to FLT3. It includes a second binding domain that enables this.

[0167] Preferably, the heterobifunctional molecule as defined herein is specific to RNF128. A first binding domain that can bind specifically, and which specifically binds to PD-1. It includes a second binding domain that enables this.

[0168] Preferably, the heterobifunctional molecule as defined herein is specific to RNF128. A first binding domain that can bind specifically, and a domain that specifically binds to PD-L1. It includes a second binding domain that enables this.

[0169] Preferably, the first binding domain of the heterobifunctional molecule is a transmembrane E3 ubiquitin ligator. - It binds to the extracellular portion of the enzyme. Therefore, preferably, the heterobifunctional molecule transmembrane It does not need to pass through the cell membrane to bind to E3 ubiquitin ligase.

[0170] Protein bound to the second binding domain As detailed herein, the heterobifunctional molecules of the present invention are transmembrane E3 ubiquitous. Tin ligase, preferably transmembrane E3 ubiquitin ligase as defined herein above. It has a first binding domain that can bind to gauze.

[0171] The heterobifunctional molecule of the present invention further comprises a second binding domain, and the second binding domain It can bind to membrane-bound proteins.

[0172] Preferably, a protein that can bind to the second binding domain of the heterobifunctional molecule Proteins are proteins that are exposed to the outside of the cell, at least partially. It can attach to the cell membrane from one side, or it can extend across the entire membrane. In other words, it is a transmembrane protein. Preferably, the second binding domain is a transmembrane protein. It can specifically bind to proteins.

[0173] As the term "heterobifunctionality" already suggests, it can bind to a second binding domain. The transmembrane protein is a transmembrane E3 ubiquitin ligase that can bind to the first binding domain. This is different. Preferably, the second binding domain is specific to transmembrane E3 ubiquitin ligase. They do not bind directly and / or effectively.

[0174] Preferably, the membrane-bound protein is a transmembrane protein, preferably a cell surface receptor. Therefore, preferably, the second binding domain of the heterobifunctional molecule is a transmembrane receptor. It can specifically bind to it. Preferably, the receptor is an ion channel-coupled receptor. It is at least one of the following: enzyme-linked receptors, G protein-linked receptors, and Fc receptors. ru.

[0175] The second binding domain of the heterobifunctional molecule binds to the monomeric and / or dimeric forms of the receptor. It can bind to the embodied form. Furthermore, or alternatively, the second binding domain inactivates the receptor. It can bind to sexual and / or active conformations.

[0176] Membrane-bound proteins may be associated with or involved in the onset, progression, or severity of disease. Membrane-bound proteins are involved in cancer, autoimmune diseases, inflammatory diseases, infectious diseases, and / or It may be known or expected that it is involved in a genetic disorder.

[0177] Preferably, the membrane-bound protein is one of LGR4, LGR5, and LGR6. They are not the same.

[0178] In preferred embodiments, transmembrane receptors are known or predicted to be involved in cancer. It may be thought of as such. In this specification, "receptors involved in cancer" refers to receptors directly involved in malignant tumors of cancer. It is understood as a transmembrane receptor that can exert influence indirectly or indirectly.

[0179] In one embodiment, transmembrane receptors involved in cancer, upon activation or increased activity, It may be a receptor that induces or enhances malignant characteristics in cells. For example, but is not limited to membranes. Activation of transcatheter receptors affects one of the following aspects of a cell's stem cell nature: differentiation ability, viability, and proliferation ability. It may affect at least one of them. When used herein, the receptor activity is not limited to However, receptors with one or more activating mutations, and / or increased receptor ligands Receptors with increased expression and / or increased availability, and / or decreased It contains receptors that have metabolic turnover and are stabilized, for example, on the cell membrane.

[0180] Furthermore, or alternatively, transmembrane cytology known or expected to be involved in cancer The receptor may be, for example, a receptor present on immune cells and / or stromal cells. As a limited example, inhibiting receptors present on immune cells can target tumor cells. This can lead to the activation of immune cells, and inhibition of receptors present on stromal cells This can lead to a reduction in tumor angiogenesis.

[0181] Therefore, receptors involved in cancer include transmembrane receptors present on tumor cells, and / or transmembrane receptors present on cells that have a direct or indirect effect on tumor cells. It is understood in this specification that this is possible.

[0182] The phrase "receptors associated with or involved in cancer" is not limited to cancer, but also includes or proliferative disorders such as malignant tumors, or myelodysplasia, myelodysplastic syndrome, or preleukemia This includes precancerous conditions such as those listed above.

[0183] In one embodiment, the activation or increased activity of the transmembrane receptor described herein relates to The related cancers are hematological cancers. In one embodiment, the transmembrane receptor described herein Cancers associated with the activation or increased activity of the body are solid tumors. Further diseases related to the activation or increased activity of transmembrane receptors, but not limited to these. However, as described herein, for example, atypical and / or non-classical cancers, malignant tumors, and pre- This includes cancerous conditions or proliferative disorders associated with transmembrane receptor activation. Non-cancer-related indications associated with the activation or increased activity of transmembrane receptors listed include: However, it is not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies) This includes (and asthma) and transplantation.

[0184] Preferably, the receptor to which the second domain of the heterobifunctional molecule can bind is involved in cancer. The receptor is one that increases the activity of, for example, the downstream signal It has transmembrane signaling. Downstream signaling preferably involves activation or increase of transmembrane receptors. The activity increases compared to other identical cells that do not possess the activity. The increased activity is limited. However, receptor mutations and activation, receptor upregulation, increased receptor stabilization, and / or This may be due to the increased availability of receptor ligands.

[0185] The receptor may be involved in a specific type of cancer. Alternatively, the receptor may be involved in many different types of cancer. The receptor may be involved in many types of cancer. (different cancer types). For example, the receptors are at least 1, 2, 3, 4, 5, 6, 7. It may be involved in 8, 9, 10 or more types of cancer. Alternatively, or further The receptor may also be involved in cancer angiogenesis.

[0186] The receptor may be involved in solid tumors or hematological malignancies. It may also be possible. Preferably, the solid tumors are 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, brain cancer Cancers of the neck, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, and anal cancer. Stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Vulvar carcinoma, Hodgkin's disease, non-Hodgkin lymphoma, endocrine cancer, thyroid cancer, parathyroid gland Adenocarcinoma, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in childhood, Bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma , epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, The group is selected from the group consisting of the aforementioned cancer and metastatic lesions.

[0187] The receptor may be involved in solid tumors or hematological malignancies. Preferably, hematological malignancies are Chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B-cell leukemia lymphocytic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic bone marrow B-cell prolymphocytic leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, - Kitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia Small cell or large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, man Toll cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes Non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasia One or more of the following: biological, Waldenstrom macroglobulinemia, or preleukemia. Selected from.

[0188] Preferably, a transmembrane to which the second binding domain of the heterobifunctional molecule can bind. Proteins are used in colorectal cancer, ovarian cancer, breast cancer, esophageal cancer, stomach cancer, prostate cancer, and lung cancer. This relates to cancer selected from the group consisting of melanoma, leukemia, pancreatic cancer, and bladder cancer. .

[0189] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in colorectal cancer. Alternatively, transmembrane proteins include EGFR, IGF1R, MET, and ERBB2. There is at least one.

[0190] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in breast cancer. Preferably The transmembrane proteins are EGFR or ERBB2.

[0191] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in esophageal cancer. Preferably The transmembrane protein is either ERBB2 or VEGFR2.

[0192] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in gastric cancer. Preferably The transmembrane protein is either ERBB2 or VEGFR2.

[0193] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in leukemia. Preferably The transmembrane protein is FLT3.

[0194] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in melanoma. Preferably The transmembrane protein is KIT.

[0195] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in non-small cell lung cancer. In other words, the transmembrane protein is either EGFR or MET.

[0196] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in ovarian cancer. Preferably The transmembrane protein is EGFR.

[0197] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The protein is activated or has increased activity in pancreatic cancer. Preferably The transmembrane protein is EGFR.

[0198] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The proteins are TGFβR1, TGFβR2, EGFR, ERBB2, ERBB3, and IG. F1R, MET, VEGFR2, KIT, FLT3, PDGFRA, PDGFRB, GH R, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, FZD10, LRP5, LRP6, PD-1, PD-L1, CTLA4, The group is selected from CMTM6, CMTM4, and WLS.

[0199] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The proteins are: 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, Sequences selected from the group consisting of 79, 84, 86, 88, 90, 92, and 94 and fewer Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 It has 7%, 98%, 99%, or 100% sequence identity.

[0200] Preferably, a transmembrane molecule that can bind to the second binding domain of a heterobifunctional molecule. The proteins are sequence numbers 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50. , 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, Sequences selected from the group consisting of 80, 85, 87, 89, 91, 93, and 95 and fewer Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 It is encoded by sequences with 7%, 98%, 99%, or 100% sequence identity.

[0201] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane proteins that can be used are TGFβR1 or TGFβR2.

[0202] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is TGFβR1. Preferably, the TGFβR1 protein is , Sequence ID 31 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90% It has sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%. In addition, the TGFβR1 protein is at least approximately 60%, 65%, and 7% of the TGFβR1 protein, as shown in SEQ ID NO: 32. 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and It is coded by an array that has 100% array identity.

[0203] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is TGFβR2. Preferably, the TGFβR2 protein is , Sequence ID 33 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90% It has sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%. Furthermore, the TGFβR2 protein is at least approximately 60%, 65%, and 7% of the TGFβR2 protein, according to SEQ ID NO: 34. 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and It is encoded by a sequence having 100% sequence identity. Preferably, heterobifunctional. The second binding domain of the sex molecule can specifically bind to TGFβR2, and the first binding domain The binding domain specifically binds to at least one of RNF43 and RNF167. This is possible. Preferably, the second binding domain of the heterobifunctional molecule is TGFβR It can specifically bind to 2, and the first binding domain specifically binds to RNF43. It is possible.

[0204] Preferably, the second binding domain of the heterobifunctional molecule binds specifically to TGFβR2. The first binding domain can bind to RNF167 specifically. ru.

[0205] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is EGFR. Preferably, the EGFR protein has a sequence number No. 35 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably EGFR protein is at least approximately 60%, 65%, 70%, and 75% of SEQ ID NO: 36. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is encoded by a sequence having sequence identity. Preferably, the second of a heterobifunctional molecule. The binding domain can specifically bind to EGFR, and the first binding domain is R It can specifically bind to at least one of NF43 and RNF167.

[0206] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to EGFR. The first binding domain can specifically bind to RNF167.

[0207] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane proteins that can be used are ERBB2 or ERBB3.

[0208] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein that can be used is ERBB2. Preferably, the ERBB2 protein is distributed Column number 37 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the ERBB2 protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 38. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0209] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein that can be used is ERBB3. Preferably, the ERBB3 protein is distributed Column number 39 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the ERBB3 protein is at least approximately 60%, 65%, and 70% of the SEQ ID NO: 40. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0210] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is IGF1R. Preferably, the IGF1R protein is distributed Column number 41 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the IGF1R protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 42. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0211] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is MET. Preferably, the MET protein is SEQ ID NO: 4 3 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, It has sequence identity of 96%, 97%, 98%, 99%, or 100%. Preferably, M The ET protein is at least approximately 60%, 65%, 70%, 75%, and 80% of SEQ ID NO: 44. The same array as %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has uniformity.

[0212] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is VEGFR2. Preferably, the VEGFR2 protein is , Sequence ID 45 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90% It has sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%. More specifically, the VEGFR2 protein is at least approximately 60%, 65%, and 7% of the SEQ ID NO: 46. 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and It is coded by an array that has 100% array identity.

[0213] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is KIT. Preferably, the KIT protein is SEQ ID NO: 4 7 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, It has sequence identity of 96%, 97%, 98%, 99%, or 100%. Preferably, K IT proteins are at least approximately 60%, 65%, 70%, 75%, and 80% of sequence number 48. The same array as %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has uniformity.

[0214] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FLT3. Preferably, the FLT3 protein has a sequence number No. 49 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FLT3 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 50. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is encoded by a sequence having sequence identity. Preferably, the second of a heterobifunctional molecule. The binding domain can specifically bind to FLT3, and the first binding domain is R It specifically binds to at least one of NF43, RNF167, and RNF128. It is possible.

[0215] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to FLT3. The first binding domain can specifically bind to RNF43.

[0216] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to FLT3. The first binding domain can specifically bind to RNF167.

[0217] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to FLT3. The first binding domain can specifically bind to RNF128.

[0218] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is PDGFRA. Preferably, the PDGFRA protein is , Sequence ID 53 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90% It has sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%. More specifically, the PDGFRA protein is at least approximately 60%, 65%, and 7% of the protein in sequence number 54. 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and It is coded by an array that has 100% array identity.

[0219] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is PDGFRB. Preferably, the PDGFRB protein is , SEQ ID NO: 55 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90% It has sequence identity of %, 95%, 96%, 97%, 98%, 99%, or 100%. More specifically, the PDGFRB protein is at least approximately 60%, 65%, and 7% of the SEQ ID NO: 56. 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and It is coded by an array that has 100% array identity.

[0220] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane proteins that can be broken are FZD1, FZD2, FZD3, FZD4, FZD5, FZD The group is selected from 6, FZD7, FZD8, FZD9, and FZD10.

[0221] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD1. Preferably, the FZD1 protein has a sequence number Number 57 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD1 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 58. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0222] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD2. Preferably, the FZD2 protein has a sequence number Number 59 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD2 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 60. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0223] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD3. Preferably, the FZD3 protein has a sequence number Number 61 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD3 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 62. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0224] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD4. Preferably, the FZD4 protein has a sequence number Number 63 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD4 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 64. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0225] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD5. Preferably, the FZD5 protein has a sequence number Number 65 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD5 protein is at least approximately 60%, 65%, 70%, and 75% of the sequence number 66. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0226] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD6. Preferably, the FZD6 protein has a sequence number Number 67 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD6 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 68. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0227] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD7. Preferably, the FZD7 protein has a sequence number Number 69 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD7 protein is at least approximately 60%, 65%, 70%, and 75% of the sequence number 70. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is encoded by a sequence having sequence identity. Preferably, the second of a heterobifunctional molecule. The binding domain can specifically bind to FZD7, and the first binding domain is R It can specifically bind to NF43.

[0228] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD8. Preferably, the FZD8 protein has a sequence number Number 71 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD8 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 72. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0229] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD9. Preferably, the FZD9 protein has a sequence number Number 73 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The FZD9 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 74. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0230] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is FZD10. Preferably, the FZD10 protein is distributed Column number 75 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the FZD10 protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 76. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0231] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane proteins that can be used are LRP5 or LRP6.

[0232] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is LRP5. Preferably, the LRP5 protein has a sequence number Number 77 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably LRP5 protein is at least approximately 60%, 65%, 70%, and 75% of SEQ ID NO: 78. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0233] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is LRP6. Preferably, the LRP6 protein has a sequence number Number 79 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of %, 96%, 97%, 98%, 99%, or 100%. Preferably The LRP6 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 80. , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It is coded by an array that has array identity.

[0234] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is the growth hormone receptor (GHR). Preferably, GHR The protein is at least 60%, 65%, 70%, 75%, 80%, and 8% of the sample in sequence number 84. Sequence identity of 5%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% It has. Preferably, the GHR protein is at least about 60%, 65% of SEQ ID NO: 85. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% Alternatively, it is coded by an array that has 100% array identity.

[0235] In a preferred embodiment, the transmembrane protein functions as an immune checkpoint inhibitor. Preferably, a membrane capable of binding to the second binding domain of the heterobifunctional molecule. The transpermeable proteins are PD-1, PD-L1, CTLA4, CMTM6, CMTM4 and Selected from the group consisting of WLS.

[0236] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is PD-1. Preferably, the PD-1 protein is SEQ ID NO: 86 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% It has sequence identity of 96%, 97%, 98%, 99%, or 100%. Preferably, The PD-1 protein is at least approximately 60%, 65%, 70%, and 75% of the SEQ ID NO: 87. Distribution of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Encoded by a sequence having column identity. Preferably, the second of the heterobifunctional molecules The binding domain can specifically bind to PD-1, and the first binding domain is RNF1 Transmembrane E3 ubiquitin selected from the group consisting of 67, RNF128, and RNF130. It can specifically bind to ligases.

[0237] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-1. The first binding domain can specifically bind to RNF167.

[0238] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-1. The first binding domain can specifically bind to RNF128.

[0239] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-1. The first binding domain can specifically bind to RNF130.

[0240] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is PD-L1. Preferably, the PD-L1 protein is distributed Column number 88 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the PD1L1 protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 89. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0241] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-L1. The first binding domain is RNF43, RNF128 and RNF130 It can specifically bind to transmembrane E3 ubiquitin ligases selected from the group consisting of the following: ru.

[0242] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-L1. The first binding domain can specifically bind to RNF43.

[0243] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-L1. The first binding domain can specifically bind to RNF128. .

[0244] Preferably, the second binding domain of the heterobifunctional molecule specifically binds to PD-L1. The first binding domain can specifically bind to RNF130. .

[0245] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein that can be used is CTLA4. Preferably, the CTLA4 protein is distributed Column number 90 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the CTLA4 protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 91. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 Encoded by a sequence having 0% sequence identity. Preferably, a heterobifunctional molecule. The second binding domain can specifically bind to CTLA4, and the first binding domain This substance can specifically bind to RNF167.

[0246] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein that can be used is CMTM6. Preferably, the CMTM6 protein is distributed Column number 92 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the CMTM6 protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 93. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0247] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is CMTM4. Preferably, the CMTM4 protein is distributed Column number 94 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, It has 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. Furthermore, the CMTM4 protein is at least approximately 60%, 65%, and 70% of the protein in sequence number 95. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 10 It is coded by an array with 0% array identity.

[0248] In preferred embodiments, it can bind to the second binding domain of the heterobifunctional molecule. The transmembrane protein is WLS / GPR177. Preferably, WLS protein This is at least approximately 60%, 65%, 70%, 75%, 80%, and 85% of sequence number 100. It has sequence identity of 90%, 95%, 96%, 97%, 98%, 99%, or 100%. Preferably, the WLS protein is at least about 60%, 65%, and 7% of SEQ ID NO: 101. 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and It is coded by an array that has 100% array identity.

[0249] Preferably, the second binding domain of the heterobifunctional molecule is extracellular to the membrane-bound protein. It binds to a portion. Therefore, preferably, the heterobifunctional molecule binds to the membrane-bound protein and It does not need to pass through the cell membrane to bind.

[0250] Preferably, the first and second binding domains of the heterobifunctional molecule are transmembrane E3 ubiquitous. It binds to the extracellular portion of the chitin ligase and the transmembrane protein, respectively. Heterobifunctional molecules bind extracellularly.

[0251] First binding domain The heterobifunctional molecule of the present invention comprises at least a first binding domain and a second binding domain It contains . The first binding domain specifically binds to transmembrane E3 ubiquitin ligase. This is possible. Preferably, the first binding domain is the one to which the first binding domain binds. It specifically binds to the transmembrane E3 ubiquitin ligase as defined in the section on "proteins". can.

[0252] The first binding domain of the heterobifunctional molecule is specific to transmembrane E3 ubiquitin ligase. It can be any domain that can bind to it. Preferably, a heterobifunctional molecule The first binding domain binds to the extracellular portion of transmembrane E3 ubiquitin ligase.

[0253] Those skilled in the art can, for example, screen compound libraries, conduct immunotherapy studies, and / or This invention relates to the heterogeneity of the present invention, which is achieved by hybridoma technology that generates antibodies or functional fragments thereof. To understand how to generate the first binding domain of a bifunctional molecule. Preferred functional antibody fragment. These are nanobodies. Details of these technologies can be found, for example, (Antibodies: A Laboratory Man). This is described in Harlow et al., Cold Spring Harbor Publications, p. 726, 1988. (Campbell, AM "Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands, (1984) or (St. Groth et al., J. Immunol. Methods 35:1-21, 1980) Therefore, it is described. Details of VHH / nanobody generation in relation to natural epitopes are, for example, This is described in Pardon et al, Nature Protocols 2014, and is referenced in this specification. It will be incorporated into the book.

[0254] In a preferred embodiment, molecules that can bind to transmembrane E3 ubiquitin ligase are , an antibody. Therefore, preferably, the antibody is in the heterobifunctional molecule of the present invention. It may function as the first binding domain.

[0255] Preferably, the antibody is an antibody fragment. Preferably, the antibody fragment is a nanobody. Therefore, in a preferred embodiment, it can bind to transmembrane E3 ubiquitin ligase. The molecules that can be used are nanobodies. Therefore, preferably, the nanobodies are the heterogeneous molecules of the present invention. It may function as the first binding domain in rhinofunctional molecules.

[0256] In a preferred embodiment, the first binding domain is an organic small molecule.

[0257] In a preferred embodiment, the first binding domain is an aptamer.

[0258] In a preferred embodiment, the first binding domain is a protein molecule. The offspring may be cyclic, and therefore, preferably, the protein molecule is a cyclic peptide. Peptides are formed by direct covalent bonds between two amino acid residues, or by cross-linking. The portion may be annularized by using a part. Such a bridging portion is in the art. This is well known, for example, International Publication No. 2012 / 0, which is incorporated herein by reference. Examples include, but are not limited to, the cross-linked portions described in patent no. 57624. Protein-based The molecule may be a protein molecule that is conventionally known in this field.

[0259] Therefore, the present invention relates to the technology for specifically binding to transmembrane E3 ubiquitin ligase. This extends to molecules known in the field, and this molecule is the first component of the heterobifunctional molecule of the present invention. It can function as a synthesizing domain. Known molecules that can do this include known antibodies. This includes at least one protein molecule, aptamer, or known small organic molecule, This is not limited to these. Preferably, antibodies, protein molecules, aptamers, or small organic molecules. It is known in the art to bind to the extracellular portion of transmembrane E3 ubiquitin ligase. Yes, they are.

[0260] Antibodies that bind to transmembrane E3 ubiquitin ligases are known in the art and are accessible to those skilled in the art. If such antibodies exist, obtaining them would not be difficult. Transmembrane E3 ubiquitin A gauze that can specifically bind to the extracellular portion, preferably a transmembrane E3 ubiquitin ligase. Any known antibody that can specifically bind is the first in the heterobifunctional molecule of the present invention. It would be suitable for use as a binding domain.

[0261] Preferred known molecules that can bind to transmembrane E3 ubiquitin ligases are nanobo Therefore, preferably, the nanobody is located in the heterobifunctional molecule of the present invention. It may function as the first binding domain.

[0262] In a preferred embodiment, the first binding domain is the natural binding domain of the transmembrane E3 ubiquitin ligase. The ligand, or its functional fragment, can bind to transmembrane E3 ubiquitin ligases. It is a fragment of a natural ligand in its current state.

[0263] As a non-limiting example, natural ligands for RNF43 and ZNRF3 include Rspo These are ndin(RSPO)-1, -2, -3, and -4. Therefore, in one embodiment, The heterobifunctional molecule contains a first binding domain that can bind to RNF43. The first binding domain is Rspondin 1, Rspondin 2, Rspondin Selected from the group consisting of 3 and Rspondin 4 or its functional fragments. In addition, the heterobifunctional molecule has a first binding domain that can bind to ZNRF3. Including, the first binding domain is Rspondin 1, Rspondin 2, Rspon Selected from the group consisting of din 3 and Rspondin 4 or their functional fragments. .

[0264] Second binding domain The heterobifunctional molecule of the present invention comprises at least a first binding domain and a second binding domain It contains the second domain, which is specific to membrane-bound proteins, preferably transmembrane proteins. They can bind to each other. Preferably, the second binding domain is the "second binding domain" It specifically binds to transmembrane proteins as defined in the section "proteins to which the protein binds". can.

[0265] The second binding domain of the heterobifunctional molecule is a membrane-bound protein, preferably a transmembrane membrane. It can be any domain that can specifically bind to a protein. Preferably, The second binding domain of the telobifunctional molecule binds to the extracellular portion of membrane-bound proteins.

[0266] The second binding domain can be an antibody, peptide, aptamer, or small organic molecule.

[0267] Those skilled in the art can, for example, screen compound libraries, conduct immunotherapy studies, and / or This invention relates to the heterogeneity of the present invention, which is achieved by hybridoma technology that generates antibodies or functional fragments thereof. To understand how to generate a second binding domain of a bifunctional molecule. Preferred functional antibody fragment. These are nanobodies. Details of these technologies can be found, for example, (Antibodies: A Laboratory Man). This is described in Harlow et al., Cold Spring Harbor Publications, p. 726, 1988. (Campbell, AM "Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands, (1984) or (St. Groth et al., J. Immunol. Methods 35:1-21, 1980) Therefore, it is recorded.

[0268] In a preferred embodiment, the molecule that can bind to the membrane-bound protein is an antibody. Therefore, preferably, the antibody is the second binding domain in the heterobifunctional molecule of the present invention. It may function as an input.

[0269] 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 membrane-bound protein is It is a nanobody. Therefore, preferably, the nanobody is the heterobifunctional component of the present invention. It may function as a second binding domain in the offspring.

[0270] In a preferred embodiment, the first binding domain is an organic small molecule.

[0271] In a preferred embodiment, the first binding domain is an aptamer.

[0272] In a preferred embodiment, the second binding domain is a protein molecule. The offspring may be cyclic, and therefore, preferably, the protein molecule is a cyclic peptide. Peptides are formed by direct covalent bonds between two amino acid residues, or by cross-linking. The portion may be annularized by using a part. Such a bridging portion is in the art. This is well known, for example, International Publication No. 2012 / 0, which is incorporated herein by reference. Examples include, but are not limited to, the cross-linked portions described in patent no. 57624. Protein-based The molecule may be a protein molecule that is conventionally known in this field.

[0273] Therefore, the present invention relates to membrane-bound proteins, preferably membrane-bound proteins as defined herein. To specifically bind to composite proteins, it extends to molecules known in this field. Such molecules can function as the second binding domain of the heterobifunctional molecule of the present invention. ru.

[0274] Such known molecules include known antibodies, protein molecules, aptamers, or known At least one small organic molecule is an example, but is not limited to these. Preferably, an anti Body, protein molecules, aptamers, or small organic molecules are membrane-bound proteins as defined herein. It is known in this art to bind to the extracellular portion of nitrates.

[0275] It binds to membrane-bound proteins, preferably transmembrane proteins as defined herein. Antibodies are known in the art, and it would be difficult for those skilled in the art to obtain such antibodies. It should not be difficult. Specific binding to transmembrane proteins as defined herein is possible. It can, preferably, specifically bind to the extracellular portion of a transmembrane protein as defined herein. Any known antibody that can perform the second binding in the heterobifunctional molecule of the present invention It would be suitable for use as a domain name.

[0276] A preferred known molecule that can bind to transmembrane proteins is a nanobody. Therefore, preferably, the nanobody is the second bond in the heterobifunctional molecule of the present invention. It may function as a combined domain.

[0277] In preferred embodiments, the second binding domain is a native ligand for a transmembrane protein, The ligand is a transmembrane protein as defined herein. Preferably, the natural ligand is It is a transmembrane protein antagonist.

[0278] Heterobifunctional molecules The first and second binding domains are transmembrane proteins or membrane binding domains, respectively. It can specifically bind to proteins, i.e., target proteins.

[0279] Specific binding, as used herein, refers to the degree of binding of a domain of a "non-target" protein to fluorescence. Determined by activated cell sorting (FACS) analysis or radioactive immunoprecipitation (RIA) Approximately 10%, 9%, 8%, and 7% of the domain binding to that specific target protein is determined. It is understood that it will be less than 6%, 5%, 4%, 3%, 2%, or 1%. Regarding the binding of the domain to the target protein, it is referred to as "specific binding" or "specific binding". "Specifically" to a specific polypeptide or epitope on a specific polypeptide target. The term "nonspecific interaction" refers to a bond that is measurably different from a nonspecific interaction. Heterogeneous binding, for example, involves the binding of a target protein to a similar structure that generally lacks binding activity. It can be measured by comparing it with the binding of a control protein, which is a protein. For example, specific binding occurs when there is an excess of unlabeled targets or other control proteins that are similar to the target. This can be determined by competition. In this case, excessive binding of the labeled target to the probe is... If competitive inhibition occurs by an unlabeled target, it suggests specific binding.

[0280] As used herein, "specific binding" or "specifically binding" or "specifically binding" refers to a particular type of polyp The term "specific" to a peptide or epitope on a particular polypeptide target For example, at least about 10 -4 M, or at least about 10 -5 M, or less Approximately 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 a small amount at least about 10 -11 M, or at least about 10 -12 Against targets M or larger This can be shown by a binding domain having Kd (which may be determined as described above). In one embodiment, the term "specific binding" means that the binding domain is not limited to any other polyp Without substantially binding to a peptide or polypeptide epitope, a specific polypeptide or This refers to a bond that binds to an epitope on a specific polypeptide.

[0281] Simultaneous application of heterobifunctional molecules to transmembrane E3 ubiquitin ligases and membrane-bound proteins Binding leads to ubiquitination and degradation of transmembrane proteins. Preferably, membrane-bound tubules Proteins are transmembrane proteins. Therefore, preferably, transmembrane E3 ubiquitin ligatures are used. The simultaneous binding of heterobifunctional molecules to enzymes and transmembrane proteins is a function of transmembrane proteins. This results in ubiquitination and degradation. Preferably, the degradation involves proteasome degradation and regeneration. It is at least one type of sososomal degradation. Preferably, the degradation is lysosomal degradation.

[0282] The heterobifunctional molecules defined herein are, therefore, transmembrane E3 ubiquitin ligators. By bringing the enzyme closer to its target, i.e., a membrane-bound protein, the membrane-bound protein on the cell membrane This may knock down or eliminate the presence of the protein. In other words, membrane binding This will result in a decrease in the steady-state level of the protein.

[0283] In this specification, the steady-state level is defined as the amount of protein present per cell. In some cases, the steady-state levels of membrane-bound proteins are at least approximately compared to reference cells. 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 Decreases by %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. In some cases, or in some cases, it may decrease by about 100%, that is, the heterobifunctional molecule The binding process results in the complete absence of membrane-bound proteins.

[0284] In a preferred embodiment, the heterobifunctional molecule is a bispecific antibody. The body is described, for example, in Wu X and Demarest SJ, Methods. (2019)154:3-9. Therefore, preferably the first binding domain is a transmembrane E3 ubiquitin ligase, k is defined in the section above, “Transmembrane E3 ubiquitin ligase to which the first binding domain binds.” This antibody can specifically bind to the transmembrane E3 ubiquitin ligase. Furthermore, the second binding domain is also an antibody, and this antibody is a membrane-bound protein, preferably This refers to a transmembrane protein, preferably the protein to which the second binding domain binds, as described above. It can specifically bind to transmembrane proteins defined by . Two antibodies (i.e., The first and second binding domains can bind directly to each other, or to two antibodies. A linker may be present between them, preferably a linker as defined herein. It can exist.

[0285] In preferred embodiments, the heterobifunctional molecule is a bispecific nanobody. Isomer nanobodies are, for example, described in International Publication No. 2015 / 044386 and Conrath et al. (Camel Single-domain Antibodies as Modular Building Units in Bispecific and Biva This is disclosed in Lent Antibody Constructs, JBC, 2001). Preferably, the first bonded The main component is a transmembrane E3 ubiquitin ligase, preferably the one described above with the "first binding domain". The transmembrane E3 ubiquitin ligase specified in the section "Combined transmembrane E3 ubiquitin ligase" It is a nanobody that can bind specifically. Preferably, the second binding domain is also a nanobody. Yes, this nanobody is a membrane-bound protein, preferably a transmembrane protein, preferably This is the transmembrane protein defined in the section "Protein to which the second binding domain binds" above. It can bind specifically. Two nanobodies (i.e., the first and second binding bodies) The main bodies can bond directly to each other, or a linker can be between the two nanobodies. A linker may be present, preferably one as defined herein. Cut.

[0286] In preferred embodiments, the heterobifunctional molecule is a bicyclic peptide. Preferably, The first binding domain is a transmembrane E3 ubiquitin ligase, preferably the "first binding domain" described above. The transmembrane E3 ubiquitin ligase to which the domain binds is defined in the section "Transmembrane E3 ubiquitin ligase". It is a cyclic peptide that can specifically bind to ligase. Preferably, a second binding The combined domain is also a cyclic peptide, and this cyclic peptide is a membrane-bound protein, preferably or a transmembrane protein, preferably the "protein to which the second binding domain binds" described above. It can specifically bind to the transmembrane proteins defined in the section. Two cyclic peptides ( That is, the first and second binding domains use, for example, the same cross-linking portion. Therefore, they can directly bind to each other, or a linker exists between the two cyclic peptides. A linker may be present, preferably one as defined herein. ru.

[0287] The heterobifunctional molecule of the present invention has a first binding domain and a second binding domain. A linker may be included. The linker is any suitable linker known in the art. -This may also be the case. Preferably, the linker is a Gly-Ser sequence. Those skilled in the art will know I know how to select a linker depending on the first and second linking domains. The linker is, for example, of the form (GGGGS)n, (GGS)n, and (G)n. From the highly flexible linker, (EAAAK)n, (SPKKKRKVEAS)n (array number) (code 81), or (SGSETPGTSESATPES)n (code 82), or ( KSGSETPGTSESATPES)n (Sequence ID 83), or any of those variants It may also be a more rigid linker in the shape of an ant, where n is preferably between 1 and 7. That is, 1, 2, 3, 4, 5, 6, or 7.

[0288] The linker preferably consists of 2 to 30 amino acids, or 3 to 23 amino acids, It has a length between 3 and 18 amino acids.

[0289] therapeutic use The heterobifunctional molecules defined herein are transmembrane ubiquitin E3 ligases and selected The simultaneous binding of selected membrane-bound proteins allows for the level of any selected membrane-bound protein. It can be used to reduce.

[0290] In one embodiment, the heterobifunctional molecule as defined herein is used for pharmaceutical purposes. The medical uses described herein involve the administration of an effective amount of the heterobifunctional molecule. For use as a medicine for the treatment of the specified(s) diseases, as specified herein It is formulated as a heterodifunctional molecule as defined herein, but is heterodifunctional as defined herein. A method of treating specified diseases (multiple diseases may be specified) using a sex molecule, wherein an effective amount of the substance is administered to the target. Telobifunctional molecules, used in the preparation of medicines to treat specified diseases (multiple diseases may be specified). The step of administering a heterobifunctional molecule as defined herein, where heterobifunctional The sex molecule is administered in an effective dose, and the administration of the effective dose may result in the specified disease(s) Similar to the method which includes the use of heterobifunctional molecules as defined herein for the treatment of ) It can be formulated into a pharmaceutical product. All such medical uses are envisioned by the present invention. It can be done.

[0291] A person skilled in the art could determine any membrane-bound protein involved in the onset, severity, or duration of a disease. The increased activity may indicate that it is a suitable target for heterobifunctional molecules as defined herein. Understand. Therefore, heterobifunctional molecules are not any particular membrane-bound protein or Not limited to any specific disease. Preferably, the disease is a membrane-bound protein, preferably The increase in receptor activity is characterized by an increase in the activity of membrane-bound receptors, and the increase in the activity of membrane-bound receptors is preferably It is something that affects or determines the onset, severity, or duration of a disease. .

[0292] As a non-limiting example, heterobifunctional molecules have been shown to reduce cancer, dementia, heart disease, and infections. It may be used in at least one procedure.

[0293] Increased membrane-bound receptor activity is significant, for example, in the onset, severity, or duration of cancer. It is well known in the art that it plays an important role. Therefore, in one embodiment, Heterobifunctional molecules are used to treat, prevent, alleviate, or suppress cancer-related symptoms. It is used.

[0294] Preferably, cancer is the protein to which the "second binding domain" in this specification binds. It is a type of cancer as defined in the section.

[0295] Preferably, the cancer is a solid tumor or a blood cancer. Alternatively, or even better, The body may be involved in cancer angiogenesis.

[0296] Preferably, the solid tumor is the protein to which the "second binding domain" described herein binds. It is a solid tumor as defined in the section "...".

[0297] Preferably, blood cancer is a protein to which the "second binding domain" described herein binds. It is a type of blood cancer as defined in the section "...".

[0298] In one embodiment, the present invention relates to a composition comprising a heterobifunctional molecule as defined herein. Related. The composition is cell culture, preferably animal cell culture, more preferably mammalian cell culture. It may be suitable for use in culture. Furthermore, or alternatively, the composition is preferable The above is a pharmaceutical composition or a cosmetic composition.

[0299] The composition may contain one type of heterobifunctional molecule, for example, two or more. To knock down or knock out the presence of the above different membrane-bound proteins, The composition may contain at least two different heterobifunctional molecules. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different types of heterobiform angiotensin It may contain potent molecules.

[0300] Compositions containing the heterobifunctional molecules described above are used in pharmaceutical or cosmetic compositions. or other foods for humans or animals, including medical foods and nutritional supplements. It can be prepared in various media.

[0301] "Medical food" refers to food that is formulated to meet specific nutritional requirements for diseases or conditions. These products are intended for dietary management. For example, medical foods are supplied through a feeding tube. This includes, but is not limited to, vitamin and mineral preparations administered enterally (known as enteral administration). It's not something that can be done.

[0302] A "dietary supplement" is a product intended to supplement a person's diet. This refers to products that are typically provided in the form of formulations such as pills, capsules, and tablets. For example, but not limited to, a nutritional supplement shall contain one or more of the following ingredients. May contain: vitamins, minerals, herbs, plant-based medicines; amino acids, total dietary intake Dietary substances intended to supplement meals by increasing their quantity, and any of the aforementioned concentrations Abridged products, metabolites, ingredients, extracts, or combinations thereof. Dietary supplements are available in the form of food bars, beverages, and powders. Foods including, but not limited to, cereals, prepared foods, food additives, and candies. It may be incorporated into it.

[0303] Therefore, the composition in question includes, but is not limited to, food, other ingestible physiological It may be compounded with materials permitted in this specification. Furthermore, or instead, as described herein The composition for use may be administered orally in combination with (individual) administration of food.

[0304] The composition may be administered alone or in combination with other pharmaceutical or cosmetic agents. It can be combined with physiologically acceptable carriers. In particular, hetero Bifunctional molecules are pharmaceutically or physiologically acceptable excipients, carriers, and vehicles. By using additives in the formulation process, it can be formulated as a pharmaceutical or cosmetic composition. It is possible.

[0305] Appropriate pharmaceutically or physiologically acceptable excipients, carriers, and vehicles include processing agents. and drug delivery modifiers and enhancers, such as calcium phosphate and magnesium stearate. Talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, ka Sodium hydroxymethylcellulose, dextrose, hydroxypropyl-P-methylcellulose Rodextrin, polyvinylpyrrolidinone, low-melting-point wax, ion exchange resin, etc. Any combination of two or more of these is also included. Other pharmaceutically acceptable excipients Regarding this, "Remington's Pharmaceutical Sciences," Mack Pub. Co., New Jersey (1 991), and "Remington: The Science and Practice of Pharmacy," by Lippincott Willi ams & Wilkins, Philadelphia, 20th edition (2003), 21 st edition (2005) and 22 nd e This is described in dition (2012), which is incorporated herein by reference.

[0306] Pharmaceutical or cosmetic composition comprising a heterobifunctional molecule for use as described in the present invention. This includes, for example, solutions, suspensions, or emulsions, suitable for the intended method of administration. It may also be in any form. In a preferred embodiment, the heterobifunctional molecule may be in solid form or It is administered in liquid form.

[0307] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. There are such solid dosage forms, and heterobifunctional molecules such as sucrose, lactose, and Alternatively, it may be miscible with at least one inert diluent such as starch. Such dosage form This also includes additional substances other than inert diluents, such as smoothing agents like magnesium stearate. It may also contain a buffer. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. Tablets and pills may be further prepared using enteric coating. ru.

[0308] Liquid dosage forms for oral administration are commonly used in the art, such as water or saline solution. pharmaceutically acceptable emulsions, solutions, suspensions, syrups containing an inert diluent, And may contain elixirs. Such compositions may contain wetting agents, emulsifiers and suspending agents. It contains adjuvants such as cyclodextrin, as well as sweeteners, flavorings, and fragrances. That's fine.

[0309] Liquid carriers are typically used in the preparation of solutions, suspensions, and emulsions. In embodiments, the liquid carrier / liquid dosage form intended for use in the implementation of the present invention is, for example, Water, saline solution, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oil or This includes fats, etc., and mixtures of two or more thereof. In preferred embodiments, as specified herein The heterobifunctional molecule of the present invention, as defined, is miscible with an aqueous solution before administration. It is preferable that the aqueous solution is suitable for water, and such aqueous solutions are well known in the art. It is further known in this art that the suitability of a solution for administration may depend on the route of administration. Yes, they are.

[0310] In a preferred embodiment, the aqueous solution is an isotonic aqueous solution. The isotonic aqueous solution is preferably , is nearly (or completely) isotonic with respect to plasma. In a more preferred embodiment, isotonic The aqueous solution is physiological saline.

[0311] Liquid carriers include solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickeners, and viscosity modifiers. It may also contain other suitable pharmaceutically acceptable additives such as fertilizers, stabilizers, and flavorings. 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, Examples include cottonseed oil.

[0312] For parenteral administration, the carrier is an oily substance such as ethyl oleate or isopropyl myristate. It may also be a stealth. The composition for use in the present invention is fine particles, microcapsules, and This may be in the form of psosome capsules, or any combination of two or more thereof.

[0313] Time-release, sustained-release, or controlled-release delivery systems include, for example, Lee's "Diffusion-Control lled Matrix Systems", pp. 155-198 and Ron and Langer, "Erodible Systems", pp. 1 99-224, in "Treatise on Controlled Drug Delivery", A. Kydonieus Ed., Marcel Dek As described in Ker, Inc., New York 1992, diffusion-controlled matrix systems Alternatively, an erosive system may be used. The matrix may be subjected to, for example, hydrolysis or enzymatic cleavage. Degradation, for example, by proteases, occurs spontaneously in situ and in vivo. It may be a biodegradable material that can be used. The delivery system may be, for example, a naturally occurring material. It may be a polymer or a synthetic copolymer, for example, in the form of a hydrogel. Exemplary polymers having cleavable bonds include polyesters and polyorthoesters. Poly(phosphoester), poly(anhydrous), poly(phosphoester), polyamide, polyurethane, poly((phosphoester) Examples include midocarbonates and poly(phosphazenes).

[0314] The heterobifunctional molecule of the present invention can also be administered in the form of liposomes. As is known in the field of science, liposomes are generally phospholipids or other lipid substances. Liposomes are obtained by a single-layer or multi-layer hydrated liquid crystal dispersed in an aqueous medium. It is formed by being non-toxic, physiologically acceptable, and capable of forming liposomes. Any lipid that can be metabolized can be used. The composition of the present invention in liposome form is as described above. In addition to the heterobifunctional molecules defined in the details, the following may be included: stabilizers, preservatives, excipients, etc. Yes, it is possible. Preferred lipids are natural and synthetic phospholipids and phosphatidylcholine (reci (Chin) is. Methods for forming liposomes are known in the art. For example, Press cott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NY, See p. 33 et seq (1976).

[0315] A pharmaceutical composition or cosmetic composition may include a unit dose formulation, and the unit dose is as described above. A dose sufficient to have a therapeutic or inhibitory effect on the disorder or condition as defined in the details, Effective in reducing or knocking out the expression of and / or membrane-bound proteins The unit dose is the therapeutic or inhibitory effect of the disorder or condition as defined herein. and / or a single dose in an amount effective to reduce the expression of the target membrane-bound protein It may be sufficient. Instead, the unit dose is sufficient for the impairment or condition as defined herein. The dose may be administered regularly during the course of treatment or suppression. The concentration of the target composition may be monitored to ensure that the desired level is maintained. stomach.

[0316] A heterobifunctional molecule or a composition comprising a heterobifunctional molecule as defined herein is: Enteral, oral, parenteral, sublingual, inhalation (e.g., as a mist or spray), rectal, and It may be administered topically, and if desired, conventional non-toxic pharmacochemical or physiologically acceptable drugs may be used. It may also be preferably administered in a dosage unit formulation comprising a carrier, an adjuvant, and a vehicle. For example, preferred modes of administration include oral, subcutaneous, transdermal, transmucosal, and iontophoresis. Intravenous, intraarterial, intramuscular, intraperitoneal, nasal (e.g., via nasal mucosa), subdural, rectal, stomach Direct administration to the intestines and other specific or affected organs or tissues, such as cancerous tissue. These include: administration to the central nervous system, spinal administration, epidural administration, and administration to the ventricles, etc. Local administration is possible, such as transdermal administration using transdermal patches or iontophoresis devices. This may also involve the use of [unspecified]. As used herein, the term parenteral means subcutaneous injection, intravenous injection. This includes intravenous injection, intramuscular injection, or intravenous infusion techniques.

[0317] Heterobifunctional molecules are pharmaceutically acceptable carriers and adjuvants suitable for the desired administration route. It can be mixed with a gastric tube and a vehicle. The heterobifunctional molecule of the present invention can be mixed with a gastric tube or It may be administered via transdermal tube replacement.

[0318] In preferred embodiments, the present invention provides an effective total daily dose for treating cancer-related symptoms. For use in the treatment, prevention, or suppression of a condition, the heterogeneous compounds defined above herein It relates to functional molecules.

[0319] The dosage form for oral administration may be a solid oral dosage form. The class of solid oral dosage forms is mainly tablets. The formulation consists of a compound and a capsule, but other forms are also known in the art and are equally suitable. It is possible. When used as a solid oral dosage form, the heterobifunctional molecule as defined herein is For example, immediate-release tablets (or capsules, etc.) or sustained-release tablets (or capsules, etc.) It may be administered in the form of ) as will be obvious to those skilled in the art, any appropriate immediate-release A single-release or sustained-release solid dosage form can be used in the context of the present invention.

[0320] The heterodifunctional molecules described for use as described herein are available in solid form, liquid form, etc. aerosol form, or tablets, pills, powder mixtures, capsules, granules, injections, creams M, solutions, suppositories, enemas, colon cleansers, emulsions, dispersions, food premixes, and others It can be administered in an appropriate form. The heterobifunctional molecule of c is also liposomal formulation. It can be administered as a prodrug. Then, the prodrug is converted in the treated subject into a therapeutically effective form. Additional administration methods are known in the art.

[0321] Injectable preparations, such as sterile aqueous or oily suspensions for injection, may be provided with a suitable dispersant or moisture. It can be formulated using humectants and suspensions according to known techniques. It is sterile injectable. The preparations are also non-toxic and parenterally acceptable, such as a solution in propylene glycol. It may be a sterile injectable solution or suspension in a diluent or solvent. Among the acceptable vehicles and solvents are water, Ringer's solution, and isotonic sodium chloride. It contains a um solution. Furthermore, sterile fixative oil has traditionally been used as a solvent or suspension medium. It is used. For this purpose, any brand of solid containing synthetic mono or diglycerides is used. A constant oil may be used. Furthermore, fatty acids such as oleic acid are used in the preparation of injectable drugs. ru.

[0322] Rectal suppositories for heterobifunctional molecules contain heterobifunctional molecules with cocoa butter or poly Ethylene glycol and similar substances are solid at room temperature but liquid at rectal temperature, and therefore dissolve in the rectum. Prepared by mixing with a suitable non-irritating excipient that releases heterobifunctional molecules. It is possible.

[0323] The heterobifunctional molecules for use described herein are single active pharmaceutical products (or cosmetics). They can be administered as a drug, but they are also used to treat or suppress diseases or disorders. It can be used in combination with one or more other medications used for the disease or disorder. The heterobifunctional molecules of the present invention are used in combination with the treatment, prevention, or suppression of symptoms related to the present invention. Some representative drugs that are useful for this purpose include coenzyme Q, vitamin E, idebenone, and MitoQ EPI-743, vitamin K and its analogues, naphthoquinone and its derivatives, other Examples include, but are not limited to, vitamins and antioxidants.

[0324] When additional activators are used in combination with the heterobifunctional molecule of the present invention, additional activity The agents are generally referred to in Physicians' Desk Reference (PDR) 5, which is incorporated herein by reference. The therapeutic dose shown in the 3rd Edition (1999), or a therapeutically useful dose known to those skilled in the art. The heterobifunctional molecule of the present invention and other therapeutically active agents or drugs ( The combination of the present invention may be administered at the recommended maximum clinical dose or at a lower dose. The dose level of the active compound in the product depends on the route of administration, the severity of the disease, and the patient's response. It may be modified to obtain the desired therapeutic response. It may be administered in combination with other therapeutic agents. In such cases, the therapeutic agent may be formulated as separate compositions to be administered simultaneously or at different times. The therapeutic agent can be administered as a single composition.

[0325] Further aspects Furthermore, or alternatively, the heterobifunctional molecules described in the present invention may be used for research purposes. There are cases where this occurs.

[0326] Therefore, in one embodiment, the present invention relates to a membrane-bound protein, preferably a transmembrane protein. The present invention relates to a method for identifying a target for treatment, preferably for cancer treatment. Preferably, Cancer is a type of cancer as defined above.

[0327] Preferably, the method involves adding one or more members of a library of heterobifunctional molecules to the cells. The process includes the step of exposing the heterobifunctional molecule to a first binding domain and a second binding domain. Includes the main dish.

[0328] The first binding domain is a transmembrane E3 ubiquitin ligase, preferably the "first binding domain Transmembrane E3 ubiquitin ligase to which the main binding agent is defined in the section "Transmembrane E3 ubiquitin ligase to which the main binding agent is located". It can bind to ligases. Preferably, the first binding domain is "first binding domain This is the binding domain defined in the "Main" section. Preferably, the first binding domain is the antibody Preferably, the heterobifunctional molecules in the library are It contains the same or nearly identical first binding domain.

[0329] Preferably, the second binding domain is the binding domain defined in the section "Second Binding Domain" Preferably, the second binding domain is an antibody, preferably a nanobody. The second binding domain of the heterobifunctional molecule preferably includes a scramble sequence, for example. For example, it contains at least an unknown amino acid residue (X). Preferably, the second binding domain is For example, (X) n Annotated as n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, with at least 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more unknown amino acids It may also contain a scrambled sequence having acid residues. The second binding domain contains one or more scrambled sequences. Crumbled arrays, for example, 2, 3, 4, 5, 6, 7, 8 or more scrambled arrays It may include columns.

[0330] The scramble sequence in the second binding domain is preferably the membrane binding of the second binding domain. It affects the binding affinity to the combined protein. Scrambling in the second binding domain. By altering the sequence of the sequence, the second binding domain is preferably a different membrane binding domain. It binds to protein. Preferably, the heterobifunctional molecules in the library are unique or It includes a unique second binding domain.

[0331] Preferably, the heterobifunctional molecules in the library are bispecific antibodies, and preferably This is a bispecific nanobody.

[0332] Preferably, cells are exposed to a library of heterobifunctional molecules under appropriate conditions, and then... It is possible to determine at least one of the cell viability, differentiation ability, stem cell properties, and proliferative ability. Those skilled in the art know how to evaluate the viability, differentiation ability, stem cell properties, and proliferative capacity of cells. It is.

[0333] One or more members of the library of heterobifunctional molecules as defined herein The viability, differentiation ability, stem cell properties, and proliferative capacity of cells exposed to the substance are preferably compared to control cells. The survival rate, differentiation ability, stem cell properties, and proliferative capacity are compared, respectively. Preferably, the control A cell, or reference cell, is one or more members of a library of heterobifunctional molecules. These are cells that have the same or substantially the same genetic background as the cells exposed to the substance. The cells are preferably cultured under the same or essentially the same conditions as the exposed cells. The method involves each well of the culture plate containing one of the heterobifunctional molecules from the library. This may be performed in a high-throughput assay involving cells exposed to more than one member. .

[0334] Preferably, this method allows for comparison of the viability and differentiation ability of exposed cells compared to control cells. A heterobifunctional molecule that reduces or increases at least one of stem cell properties and proliferative capacity. This further includes the step of identification.

[0335] The reduction may be compared to control cells, for example, the viability and differentiation ability of the exposed cells. A reduction in at least one of the stem cell properties and proliferative capacity is at least approximately compared to control cells. 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 Reduced by %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. It may be reduced by approximately 100%.

[0336] Furthermore, or alternatively, at least one of the following: viability, differentiation ability, stem cell properties, and proliferative capacity. One increase may be compared to control cells, for example, the viability of exposed cells, differentiation ability, etc. An increase in at least one of stem cell properties and proliferative capacity is at least approximately compared to control cells. 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 10 It may increase by 0%, 110%, 120%, 150%, 200%, or 500%.

[0337] The method preferably involves comparing the viability, differentiation ability, and stem cell count of exposed cells with those of control cells. Identify heterobifunctional molecules that reduce or increase at least one of cystiness and proliferative capacity. The step includes the following: A person skilled in the art can identify heterobifunctional molecules using any conventional method You may use steps. As a non-limiting example, heterobifunctional molecules can be used in DNA barcoding. Therefore, they may be conjugated, and functionally related heterozygotes can be determined by DNA sequencing. Functional molecules can be rapidly identified and validated (e.g., Franzini et al, Angewandte Chemi). e, 2015; Zimmerman and Neri, Drug Discovery Today, 2016). In another non-restrictive example... The heterobifunctional molecule may also contain a protein tag used for detection and isolation. The purified molecules may then be analyzed using mass spectrometry.

[0338] Following the identification of heterobifunctional molecules, target membrane-bound proteins are found in the art. They can be identified using any conventional means. As a non-limiting example, membrane-bound proteins It can be identified using biotinylation of the cell surface and identification based on mass spectrometry. By comparing the cell surface proteomes of inoculated and treated cells, we can determine which targets can be removed from the cell surface. It becomes clear whether they have been removed. Such methods are well established, for example, RNF4 Previously used in the art to identify the endogenous substrate of 3 (Koo et al, Natur e 2012). In another non-limiting example, heterobifunctional molecules interact with cell surface proteins. It is used to perform immunoprecipitation of the substance. The precipitated molecules are then analyzed using mass spectrometry. It may be analyzed in this way.

[0339] One or more members of the library of heterobifunctional molecules as defined herein The cells exposed may be cell cultures, cell lines, biopsies, and organoids, and That could be part of it.

[0340] Preferably, the cells are patient-derived tissue, preferably a portion of cultured patient-derived tissue. or derived therefrom. Preferably, the cells are biopsies or organoids, preferably tumor cells. It is part of or derived from a ulcer organoid.

[0341] The biopsy may be an excisional biopsy, an incisional biopsy, or a core biopsy. Organoids are preferably These are cancer organoids. The organoids are preferably patient-derived organoids. Preferably, it is a tumor organoid.

[0342] Manufacturing of heterobifunctional molecules In one embodiment, the present invention relates to a method for producing the heterobifunctional molecule of the present invention, - A step of selecting a transmembrane E3 ubiquitin ligase and a membrane-bound protein; - A first binding domain that can specifically bind to transmembrane E3 ubiquitin ligase. Steps to construct the n; - Construct a second binding domain that can specifically bind to membrane-bound proteins. Step and; and - A step of joining a first binding domain to a second binding domain, preferably The bond is either a direct bond or via a linker, preferably a linker as defined herein. step It relates to methods that include this.

[0343] In this specification, the step of constructing the first binding domain and the second binding domain is: It is understood that this can be carried out using any conventional means in the art. As a typical example, at least one of the first and second binding domains is in the art. In this context, conventionally known binding domains, such as transmembrane E3 ubiquitin ligases, Regarding specific binding to antibodies or membrane-bound proteins known in this art, This antibody is known in the field for its specific binding properties.

[0344] Instead, at least one of the first and second binding domains is a de novo binding domain. For example, an antibody or nanobody-binding domain discovered by immunology research These are some examples, but are not limited to them.

[0345] Before constructing heterobifunctional molecules, transmembrane E3 ubiquitin ligase and membrane-bound proteins You can choose a combination of qualities: transmembrane E3 ubiquitin ligase and membrane-bound protein. The step of selecting targets the transmembrane E3 ubiquitin ligase to the membrane-bound protein. To evaluate the ability to do so, first, any heterodimerization system known in the art, This is carried out using, for example, Takara Bio USA's A / C dimerization system. This may be the case, but is not limited to this. The ability depends on how well E3 ubiquitin ligase binds to membranes. This is evaluated by determining whether proteins can be ubiquitinated and internalized. It may also be used. Furthermore, or instead, a heterodimerization system can be used to create E3 ubiquitous After forcing the interaction between the ligase and the membrane-bound protein, the membrane-bound protein's cell By determining how much the surface level and / or total protein level decreases, This ability should be appreciated. For this purpose, transmembrane E3 ubiquitin ligase is FK It may be fused with a BP or FRB domain. Similarly, membrane-bound proteins are FRB It may be merged with the domain or the FKBP domain, respectively. In this system, The most common dimerizing compound is the A / C heterodimerizer. This is a heterodimer that binds to specific transmembrane E3 ubiquitin ligases and membrane-bound proteins. This provides a simple approach to evaluate the effects of this child.

[0346] Alternatively, or even further, select transmembrane E3 ubiquitin ligases and membrane-bound proteins. The next step involves incorporating a first non-natural epitope tag into the transmembrane E3 ubiquitin ligase. This can be achieved by incorporating a second non-natural epitope tag into the membrane-bound protein. Good. When expressed in cells, the first and second epitope tags are preferably expressed in each other. It is presented in the extracellular domain, i.e., outside the cell. It can bind to a first binding domain and a second epitope tag. Heterobifunctional molecules possessing a second binding domain can then bind to membrane-bound proteins. It may also be used to evaluate the ability to target transmembrane E3 ubiquitin ligases. Using heterobifunctional molecules, E3 ubiquitin ligase and membrane-bound proteins are forced to interact. After interaction, the ubiquitination and internalization of membrane-bound proteins are performed via transmembrane E3 ubiquitination. The efficacy may be evaluated by determining the extent to which gauze is produced. Alternatively, use this selection system to select E3 ubiquitin ligase and membrane-bound protein After forcing the interaction of the proteins, the cell surface level and / or total level of membrane-bound proteins The ability may be assessed by determining the extent to which protein levels decrease. Furthermore, the step of selecting the transmembrane E3 ubiquitin ligase and membrane-bound protein is as follows: As detailed herein, methods for reducing the surface level of membrane-bound proteins in cells and It's worth considering.

[0347] After selecting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein, ( Naturally, the first transmembrane E3 ubiquitin ligase can specifically bind to the extracellular portion. The binding domain can specifically bind to the extracellular portion of (natural) membrane-binding proteins. A heterobifunctional molecule containing a second binding domain may be constructed.

[0348] Ubiquitination / Screening Method The present inventors have identified an appropriate combination of transmembrane E3 ubiquitin ligase and membrane-bound protein. For example, combinations that can be effectively targeted by heterobifunctional molecules as defined herein We developed an effective screening method.

[0349] In one embodiment, the present invention thus reduces the surface level of cell membrane-bound proteins. This relates to the method. The method is preferably, a) Express transmembrane E3 ubiquitin ligase and membrane-bound proteins on the cell surface. The step of preparing the cells; and b) A step of exposing cells to a heterobifunctional molecule as defined herein, Terror-bifunctional molecules are, i) A transmembrane E3 ubiquitin ligase that can specifically bind to the extracellular portion. 1 binding domain; and ii) A second binding agent that can specifically bind to the extracellular portion of membrane-bound proteins. main Steps and Includes.

[0350] The method preferably involves step c) determining the surface level of the cell membrane-bound protein. Furthermore, it includes: The reduction is preferably before step b), on the surface of the cell membrane-bound protein. This is a decrease compared to the level. Preferably, the decrease in protein level indicates heterobifunctionality. Protein levels of membrane-bound proteins in identical or similar cells that have not been exposed to the molecule A reduction in comparison, for example, the cell membrane provided in step a) of the method of the present invention. This is a decrease compared to the protein level of the binding protein.

[0351] Step b) Exposing cells to heterobifunctional molecules is performed by the heterobifunctional molecules passing through the transmembrane E 3. It is preferable that conditions allow for simultaneous binding of ubiquitin ligase and transmembrane proteins. stomach.

[0352] The transmembrane E3 ubiquitin ligase is preferably the transmembrane E3 ubiquitin ligase described herein. It is gauze.

[0353] Membrane-bound proteins are preferably transmembrane proteins. Transmembrane proteins are... Transmembrane proteins as described in the specification may also be used.

[0354] At least one of the transmembrane E3 ubiquitin ligase and membrane-bound protein is wild-type The protein may be, for example, a protein naturally present in the provided cell. E3 ubiquitin ligase and membrane-bound proteins are preferably expressed within the same cell. If necessary, at least one wild-type protein is superimposed in the cells being supplied. It is expressed in excess. The heterobifunctional molecule for use in the method of the present invention is preferably It can bind to epitopes present in wild-type transmembrane E3 ubiquitin ligase. It contains a first binding domain and / or is naturally present in wild-type transmembrane proteins. It contains a second binding domain that can bind to a pitope.

[0355] 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 located on the cell surface of the provided cell. It is exposed above. Preferably, the first non-natural epitope tag is a transmembrane E3 ubiquitin ligator. It is located at the N-terminus of the -ase. Transmembrane E3 ubiquitin ligase is the first unnatural epitope tag. If present, the heterobifunctional molecule is preferably selective to the first non-natural epitope tag. It includes a first binding domain that binds to [the other domain].

[0356] 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. Preferably, the second non-natural epitope tag is located at the N-terminus of the membrane-bound protein. If the binding protein contains a second non-natural epitope tag, heterobifunctional molecules are preferred. Alternatively, it includes a second binding domain that selectively binds to a second non-natural epitope tag.

[0357] "Non-natural epitope tags" as used herein refer to wild-type proteins, naturally occurring proteins, and It is understood as an epitope that is not normally present in a protein. The term "tag" may be used interchangeably in this specification.

[0358] Unnatural epitopes for transmembrane E3 ubiquitin ligases and membrane-bound proteins, respectively. Tag embedding can be done using any conventional molecular biological method known in the art. This can be done. The first and second epitope tags can be any suitable tags. Preferably, the tag is a short amino acid sequence. Preferably, the tag is an antibody or antibody cleavage. One, preferably with respect to VHH, can be produced using any conventional means known to those skilled in the art. This is an amino acid sequence that can be used. Non-natural epitope tags are publicly available tags. It may be a tag or a newly discovered sequence. The first and second epitope tags are the same tag. It may be a single tag, or it may be a different tag. Preferably, the first and second epitomes Epitope tags are different tags. Non-natural epitope tags include Alpha tags, E6 tags, myc tag, FLAG tag, His tag, V5 tag, VSV tag, GFP protein The group consisting of RFP proteins may be selected.

[0359] The first epitope tag is preferably Goetzke et al (2019, Nature Communications). Alternatively, the Alpha tags described in 10(1), 1-12) may be used. The UBC described in Ling et al. (2019, Molecular Immunology, 114(July), 513-523) It may also be a 6e tag (E6 tag). The second epitope tag is preferably Goetzke Alternatively, the Alpha tag described by et al (cited above) may be used. The tag may also be the UBC6e tag (E6 tag) as described in Ling et al. (cited above). Alternatively, the first tag may be an Alpha tag, and the second tag may be an E6 tag. Good. Alternatively, the first tag could be an E6 tag, and the second tag could be an Alpha tag. It's okay to have it.

[0360] Epitope tag and any suitable corresponding antibody or antibody fragment that recognizes the epitope Any combination may be used in the manner defined herein. Preferred antibody fragments are: It is a nobody (VHH). Therefore, an epitope and a correspondence that recognizes the epitope. Any suitable combination of nanobodies (VHHs) is used in the manner defined herein. It's okay.

[0361] Those skilled in the art can select any suitable combination of epitope-antibody or antibody fragment. For example, a person skilled in the art could use a suitable epitope-antibody or antibody known in the art. A combination of fragments may be selected. Alternatively, or further, an epitope-antibody or antibody may be used. The fragments may be newly discovered combinations and may be used in the manner defined herein. It may be used.

[0362] The antibody or antibody fragment specifically binds to any of the first or second epitope tags. It may be a fine antibody or antibody fragment. A preferred antibody fragment is a nanobody. Alternatively, the heterobifunctional molecule used in the method of the present invention is a bispecific antibody, Preferably, it is a bispecific nanobody.

[0363] Preferably, the first epitope tag is an Alpha tag, and the second epitope tag is If it is an E6 tag, the first binding domain of the heterobifunctional molecule is anti-Alpha VHH It may also be the second binding domain, and the second binding domain may be anti-E6 VHH. Preferably, the If the first epitope tag is the E6 tag and the second epitope tag is the Alpha tag The first binding domain of the heterobifunctional molecule may also be anti-E6 VHH (Ling e (t al, cited above), the second binding domain may be anti-Alpha VHH (Goetzke et al.) al., cited above). Therefore, the preferred combination of epitope tag-binding domain is, i) Alpha tag - anti-Alpha VHH (Goetzke et al, (cited above)); and ii) E6 tag-anti-E6 VHH (Ling et al, supra) It is at least one of the following.

[0364] Preferred Alpha tags are sequence number 96 and at least approximately 80%, 85%, 90%, 9 It has 5%, 96%, 97%, 98%, 99%, or 100% sequence identity. The E6 tag has sequence number 97 and at least approximately 80%, 85%, 90%, 95%, 96%, It has 97%, 98%, 99%, or 100% sequence identity. Preferred anti-Alpha VHH is at least approximately 80%, 85%, 90%, 95%, 96%, and 9% of sequence number 98. It has 7%, 98%, 99%, or 100% sequence identity. Preferred anti-E6 VHH The CDR3 sequence of sequence number 99 is at least approximately 80%, 85%, 90%, 95%, and 96. It has sequence identity of %, 97%, 98%, 99%, or 100%.

[0365] Preferably, the cell surface level of membrane-bound proteins is exposed to heterobifunctional molecules. The same cells, for example, the cells provided in step a) of the method of the present invention, do not have membrane-bound tannins. It is reduced compared to the level of the protein on the cell surface.

[0366] The terms “(protein) level” and “(protein) quantity” are used interchangeably in this specification. It may be used alternatively. Preferably, the cell surface level is in step b) of the method of the present invention. Compared to the cell surface level of membrane-bound proteins before, at least about 5%, 10%, and 15%. %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 Reduced by % or approximately 100%. In this specification, a 100% reduction is defined as a transmembrane reduction. This is understood to indicate that the protein is no longer detectable on the cell surface. The decrease in levels is due to the protein remaining on the cell surface after exposure to heterobifunctional molecules. It may also be determined by directly determining the level or quantity.

[0367] Ubiquitination preferably involves the internalization and separation of ubiquitinated membrane-bound proteins. This leads to a solution. Therefore, instead, or even further, the surface level of membrane-bound proteins The decrease occurs after exposure to heterobifunctional molecules, for example after step b), in membrane-bound proteins. This may be determined by determining the total protein level, or amount, of the cells. The total protein level of the cell is preferably the membrane-bound protein level prior to step b) of the method of the present invention. Compared to the total protein level of the cell, at least about 5%, 10%, 15%, 2% 0%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 7 0%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% decrease It is reduced by approximately 100%. In this specification, a 100% reduction is defined as a transmembrane protein This is understood to indicate that the substance is no longer detectable within the cell.

[0368] Alternatively, or even further, a decrease in the cell surface level of membrane-bound proteins is a consequence of the membrane-bound protein This determines the increase in the intracellular localization of proteins, preferably the increase in the endosomal localization of membrane-bound proteins. It may be determined by the following: Ubiquitination of membrane-bound proteins is preferably performed by This leads to the internalization of membrane-bound proteins. The internalized proteins are then degraded. It is also often, and preferably, degraded by lysosomes. Increased intracellular protein localization of the protein. To determine this, the method involves the step of determining the increased intracellular localization of membrane-bound proteins. Previously, for example, by treating cells with bafilomycin, The procedure may include a step of inhibiting lysosome turnover. Preferably, the membrane The intracellular localization of the binding protein is determined prior to step b) of the method of the present invention. Compared to intracellular localization, it increases by at least approximately 1.5, 2, 3, 4, 5, 6 times or more. Add.

[0369] The membrane-bound protein may contain a third non-natural epitope tag. Epitope tags are used to determine the protein level of membrane-bound proteins, preferably at the membrane. The binding protein at the cell surface level, total protein level, and intracellular level. It may be used to determine at least one of them. Third non-natural epitope tag Preferably, the extracellular portion of the membrane-bound protein is located. Therefore, preferably, The third non-natural epitope tag is exposed on the cell surface of the provided cell. Preferably, The third unnatural epitope tag is the N-terminus of the membrane-bound protein and the second unnatural epitope tag It is located between the two.

[0370] The transmembrane E3 ubiquitin ligase may contain a fourth non-natural epitope tag. The fourth non-natural epitope tag determines the protein level of transmembrane E3 ubiquitin ligase. It may be used to determine. The fourth non-natural epitope tag is preferably a transmembrane E 3. Located in the extracellular portion of ubiquitin ligase. Therefore, preferably, the fourth non-natural enzyme The pitope tag is exposed on the cell surface of the provided cells. Preferably, a fourth non-natural element The pitope tag is the N-terminus of the transmembrane E3 ubiquitin ligase and the first unnatural epitope tag. It is located between the two.

[0371] Therefore, in one embodiment, the transmembrane E3 ubiquitin ligase is used for the first and fourth non- They may contain natural epitope tags, and membrane-bound proteins are second and third non-natural proteins. It may include an epitope tag. The third and / or fourth non-natural epitope tag is Any conventional tag known to those skilled in the art for protein detection, e.g., myc, h is, whether it is a FLAG tag, V5 tag, VSV tag, HA tag, GFP or RFP. Yes, but not limited to these.

[0372] The preferred combination of the first and fourth tags is the myc tag and the Alpha tag, or FL. These are the AG tag and the E6 tag. The preferred combination of the second and third tags is the Flag tag and the E6 tag. 6 tags, or the myc tag and Alpha tag.

[0373] The level or amount of membrane-bound protein can be determined using any conventional means known to those skilled in the art. The determination may be made using immunofluorescence, Western blotting, etc. , if necessary, quantitative immunofluorescence and / or quantitative Western blotting, cells This includes, but is not limited to, surface biotinylation, FACS analysis, and quantitative mass spectrometry. stomach.

[0374] The absolute amount or level of membrane-bound proteins is, for example, before exposure to heterobifunctional molecules. Alternatively, a direct comparison with subsequent membrane-bound protein levels, for example, on the cell surface before and after exposure. It may also be determined by determining the fluorescence intensity. Alternatively, or further, by determining the membrane-bound protein The relative level or amount of household tampers before and after exposure is, for example, This may be determined by comparison with glycemic or total cellular protein levels.

[0375] Transmembrane E3 ubiquitin ligase containing non-natural epitopes and membrane ligase containing non-natural epitopes The synthetic protein is preferably expressed in the same cell. Transmembrane E3 ubiquitin ligase and Any cells suitable for the expression of membrane-bound proteins may be used in the method of the present invention. Immortalized cells are, preferably, immortalized cells, preferably cell lines, preferably human cell lines, preferably human The cancer cell lineage is preferably HEK239T cells.

[0376] The cell line contains wild-type or "natural" transmembrane E3 ubiquitin ligase and wild-type membrane-bound ligase. It may express at least one of the following proteins: wild-type transmembrane E3 ubiquitin ligase. And at least one wild-type membrane-bound protein may be overexpressed in the cell. If necessary, reduce the amount of wild-type transmembrane E3 ubiquitin ligase and wild-type membrane-bound proteins. At the very least, one of them may be constitutively overexpressed within the cell.

[0377] Instead, the cells use engineered transmembrane E3 ubiquitin ligase and engineered membrane binding. At least one protein may be expressed. Transmembrane E3 ubiquitin ligase is the first It is manipulated to include, and optionally, a fourth non-natural epitope tag. Membrane-bound proteins contain a second, and optionally a third, non-natural epitope tag. The cells are manipulated with manipulated transmembrane E3 ubiquitin ligase and manipulated membrane. At least one of the binding proteins may be transiently overexpressed. If necessary, the cells may , less of the manipulated transmembrane E3 ubiquitin ligase and manipulated membrane-bound proteins Either one may be constitutively overexpressed. Constitutive expression is, for example, transmembrane E3 ubiquitin. An expression cassette expressing at least one ligase and membrane-bound protein is used in cell genotherapy. This can sometimes be achieved by incorporating it into the system.

[0378] A small amount of manipulated transmembrane E3 ubiquitin ligases and manipulated membrane-bound proteins Both single expression and, if necessary, constitutive expression are known in the art to those skilled in the art. This can be achieved using any conventional means.

[0379] Transmembrane E3 ubiquitin ligase (including non-natural epitopes as needed) and (as needed) Depending on the situation, at least one membrane-bound protein (including non-natural epitopes) The sequence can be introduced into cells for transient or constitutive expression, as needed. The coding sequence(s) is part of the expression cassette that is introduced into the cell. It may also be part of the expression vector. Preferred expression vectors are naked DNA, DNA compound. It is a compound or viral vector. Preferred naked DNA is linear or circular nucleic acid components. It is a offspring, for example, a plasmid. Plasmids are created using standard molecular cloning techniques. A circular double-stranded DNA loop into which additional DNA segments can be inserted. This refers to a DNA complex, which is a DNA complex bound to any carrier suitable for delivering DNA into a cell. It can be an NA molecule. Preferred carriers are lipoplex, liposome, polymerosome, and Replexes, viral vectors, dendrimers, inorganic nanoparticles, virosoms and cells Selected from the group consisting of transconductive peptides.

[0380] The cells contain transmembrane E3 ubiquitin ligases and non-natural epitope tags. To express membrane-bound proteins containing non-natural epitope tags (multiple tags possible) at an endogenous level. It may be modified as follows: As a non-limiting example, the first and, if necessary, the second non-natural The sequence encoding the pitope tag may be incorporated into the genome sequence of the provided cell. Furthermore, in the same cells, a second, and optionally a third, non-natural epitope tag is added. The sequence to be included may be incorporated into the genome sequence of the provided cell.

[0381] Therefore, the genome sequence of the provided cells encoding transmembrane E3 ubiquitin ligase It incorporates sequences encoding a first, and optionally a fourth, non-natural epitope tag. It can be modified in such a way. The modified genome sequence is preferably the one defined herein. Transmembrane E3 ubiquitin ligators containing one, and optionally a fourth, non-natural epitope tag. It encodes and expresses an enzyme. Preferably, in the same cell, it encodes a membrane-bound protein. The genome sequences of the provided cells are used to identify a second, and optionally a third, non-natural epitope. The genome sequence can be modified to incorporate a tag-coding sequence. or a second, and optionally third, non-natural epitope tag as defined herein It encodes and expresses membrane-bound proteins, including those included in the protein.

[0382] Code the first, second, and, if necessary, third and fourth non-natural epitope tags. Methods for target genome modification to incorporate sequences are well known to those skilled in the art, and the first, and And, if necessary, to incorporate a sequence encoding a fourth non-natural epitope tag, or To incorporate sequences encoding a second, and optionally a third, non-natural epitope tag. It contains site-specific endonucleases that generate double-strand breaks at genomic locations, but this Not limited. Preferred site-specific nucleases are those in the CRISPR-Cas system. ru.

[0383] Therefore, the first, and optionally the fourth, non-natural epitope tag is i) transmembrane E 3. Site-specific nuclei that generate double-strand breaks in sequences encoding ubiquitin ligases. ii) an oligonucleotide containing an array encoding the first and, optionally, the fourth tag. The step of introducing a creotide or donor plasmid into the cell leads to the cell's genome. It may be included. The double-strand break preferably occurs when the mature transmembrane protein has a first strand at its N-terminus. And, if necessary, it is positioned to include a fourth tag, preferably the double-strand break is the first And, if necessary, a fourth tag, the signal peptide of mature transmembrane ubiquitin E3 ligase. It is located between and the N-terminus. Preferably, within the same cell, the second and If necessary, a third non-natural epitope tag is added i) within the sequence encoding the membrane-bound protein. ii) a site-specific nuclease that generates double-strand breaks, and ii) second and as needed An oligonucleotide or donor plasmid containing a sequence encoding a third tag is injected into the cell. Depending on the introduction step, it may be introduced into the cell's genome. Double-strand breaks are preferred. This is a position in which the mature membrane-bound protein contains a second and optionally a third tag at its N-terminus. It is located in [location].

[0384] Oligonucleotides or donor plasmids are preferably homologous sequence-dependent repair (ho Contains sequences to promote mology-directed repair.

[0385] Alternatively, or furthermore, the first, second, and, if necessary, third and fourth non-natural substances Epitope tags may be introduced using CRISPR-Cas prime editing technology. .

[0386] The methods defined above in this specification also involve transmembrane E3 ubiquitin ligases and membrane-bound proteins. This may be considered a method for selecting a combination with a component. Preferably, as defined herein. The method involves selecting an effective combination of transmembrane E3 ubiquitin ligase and membrane-bound protein. This is a method for selection. Preferably, the combination is such that when the two are in close proximity, the transmembrane E3 A biquitin ligase is effective when it can ubiquitinize membrane-bound proteins. It is a combination. Ubiquitination of membrane-bound proteins is preferably internalized. This results in the following: Preferably, the transmembrane E3 ubiquitin ligase and membrane-bound protein are It is brought into close proximity by simultaneous binding to the heterobifunctional molecule as defined in the specification.

[0387] The methods of the present invention as defined above herein also involve the surface level of membrane-bound proteins in cells. Heterobifunctional molecules for reducing, preferably heterobifunctional as defined herein This can also be considered a method for determining the efficiency of molecules. Preferably, the method is as outlined above. The method includes the following steps. Preferably, the method is a) Express transmembrane E3 ubiquitin ligase and membrane-bound proteins on the cell surface. The step of preparing the cells; and b) Cells are heterobifunctional molecules, preferably heterobifunctional molecules as defined herein. Steps to be exposed to Includes.

[0388] The method preferably involves step c) determining the surface level of the cell membrane-bound protein. Furthermore, it includes: The reduction is preferably before step b), on the surface of the cell membrane-bound protein. This is a decrease compared to the level.

[0389] The methods defined herein also include, - A method for selecting effective combinations of transmembrane E3 ubiquitin ligase and transmembrane protein. And if the protein levels of transmembrane proteins decrease after step c) How to select that combination, - Screening effective combinations of transmembrane E3 ubiquitin ligase and transmembrane proteins How to do it; - A method for producing a defined heterobifunctional molecule, wherein the heterobifunctional component The offspring selectively bind to selected combinations of transmembrane E3 ubiquitin ligase and transmembrane proteins. How to combine; - Determining the ability of transmembrane E3 ubiquitin ligases to ubiquitinate membrane-bound proteins. A method in which the protein level of transmembrane proteins decreases after step c). If present, this is a way in which transmembrane E3 ligases can ubiquitinate membrane-bound proteins. ; - Methods for targeting membrane-bound proteins for degradation by heterobifunctional molecules and - A method for determining the ubiquitination of membrane-bound proteins, wherein the membrane-bound protein table A decrease at the surface level indicates ubiquitination of membrane-bound proteins, and this decrease is preferable. Method b) is a decrease compared to the surface level of membrane-bound proteins in the cell prior to the measurement. It may be considered to be at least one of the following.

[0390] As shown above in this specification, the method defined herein is effective heterobifunctional compound For example, targeting effective combinations of transmembrane E3 ubiquitin ligase and membrane-bound proteins. It may be used to construct heterobifunctional molecules. Therefore, the present invention Furthermore, relating to heterobifunctional molecules, preferably heterobifunctional molecules as defined herein. Furthermore, the transmembrane E3 ubiquitin ligase and membrane to which the heterobifunctional molecule selectively binds. The binding protein is selected using the selection method defined above herein. The selection method preferably involves the following steps: a) Express transmembrane E3 ubiquitin ligase and membrane-bound proteins on the cell surface. This is a step in which cells are prepared, - Transmembrane E3 ubiquitin ligase has a first non-natural epitope tag in its extracellular region. Implied; - Membrane-bound proteins contain a second non-natural epitope tag in their extracellular portion, step Pu and; b) A step of exposing cells to a heterobifunctional molecule, wherein the heterobifunctional molecule is - A first binding domain that can specifically bind to the first non-natural epitope tag n; 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) The surface level of membrane-bound proteins is at least approximately 10%, 20%, 30%, and 40%. Transmembrane E 3. A step of selecting ubiquitin ligases and transmembrane proteins, wherein the reduction is Step b) is a decrease compared to the surface level of the cell membrane-bound protein. Pu and Includes.

[0391] In one aspect, the present invention relates to the first and optionally fourth non-as defined herein. It is related to transmembrane E3 ubiquitin ligases containing natural epitope tags.

[0392] In another aspect, the present invention relates to a second, and optionally third, as defined herein. It relates to membrane-bound proteins that contain unnatural epitope tags.

[0393] In one aspect, the present invention is - First, and optionally fourth, non-natural epitope tags as defined herein. Transmembrane E3 ubiquitin ligase containing; and - Second, and optionally third, non-natural epitope tags as defined herein. Membrane-bound proteins including It relates to the combination of these. [Brief explanation of the drawing]

[0394] [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 an A / C dimerizer. HEK293T cells were transfected with RNF43-FKBP and TβRII-Flag-FRB and treated overnight with either 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 the 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 7-1]This figure illustrates the effect of bi-VHH on E3 ligase and target combinations at the endogenous level. (A) Strategy for generating endogenously tagged proteins. (B) Schematic diagram of a bi-VHH approach to target cell surface removal using endogenously tagged versions of E3 ligase and target combinations. [Figure 7-2] This figure illustrates the effect of bi-VHH on E3 ligase and target combinations at the endogenous level. (A) Strategy for generating endogenously tagged proteins. (B) Schematic diagram of a bi-VHH approach to target cell surface removal using endogenously tagged versions of E3 ligase and target combinations. [Examples]

[0395] [Example 1] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were converted to 10% fetal bovine serum (GE Health). hcare), 2 mM UltraGlutamine (Lonza), 100 units / m L-penicillin and 100 μg / mL streptomycin (Invitrogen) are included. Cells were cultured in RPMI (Invitrogen). The cells were kept at 37°C and 5% CO2. The cells were cultured. For transfection, follow the manufacturer's protocol for microscopic observation. Therefore, use FuGENE 6 (Promega), and for biochemistry, use PEI. And so, it was implemented. A / C Heterodimerizer (Takara Bio, # (635056) was 1 μM and left overnight at 37°C, with the control condition being an equivalent amount of 100% ethanol. The treatment was administered using a specific method. TGFβ was administered at a dose of 1.5 ng / mL for 45 minutes.

[0396] Constructs and antibodies TGF-βII type serine / threonine kinase receptor (TβRII)-Flag-FK BP and -Flag-FRB are Peter ten Dijke (LUMC, Leid Provided by (en). RNF43-FKBP and -FRB Q5 High-Fi Using delity 2× Master Mix (NEB), human RNF43 C FKBP at the end 36V Alternatively, it can be obtained by inserting the FRB code sequence. All constructs were sequenced. CD63-GFP was identified by J. Klumperman. (Presented by UMCU, Utrecht. Immunoblotting (IB), immunofluorescence ( For IF, the following primary antibody was used for immunoprecipitation (IP): Rabbit anti-FLAG (Sig ma-Aldrich), rat anti-HA (Roche), mouse anti-FLAG (M2;Si gma-Aldrich), mouse anti-Actin (MP Biomedicals) Rabbit anti-RNF43 (Sigma-Aldrich). Primary antibody is as described by the manufacturer. Dilution was performed according to the instructions. The secondary antibody used for IB or IF was 1:8000 or At 1:300, obtained from either Rockland or Invitrogen. I used that.

[0397] Immunofluorescence and confocal microscopy HEK293T cells were coated with laminin (Sigma) in a 24-well plate. The cells were grown on a raschelating cover slip. After overnight transfection, the cells were phosphate-reduced. The cells were fixed with 4% formaldehyde in saline (PBS). The sample was then blocked with a buffer containing 0.1% saponin at room temperature (RT) for 30 minutes. The cells were incubated with primary and secondary antibodies in a blocking buffer at room temperature for 1 hour. The cells were treated with Prolong Diamond (Life Technologies). The image was mounted on a microscope and acquired using an LSM700 confocal microscope. The image was then processed using ImageJ. Analyzed and processed.

[0398] Immunoprecipitation and Western blotting After transfection, the cells are left in a 10cm dish until they reach 80% confluence. The cells were grown using PBS. After washing the cells with PBS, the cells were scraped off and treated with 100 mM NaCl and 50 ml of water. M Tris pH7.5, 0.25% Triton X-100, 10% Glycerol 50 mM NaF, 10 mM Na3VO4, 10 μM leupeptin, 10 μM Apro The cells were lysed with a cell lysis buffer containing tinine and 1 mM PMSF. The lysate was 16.00 The solution was clarified by centrifugation at 0×g for 15 minutes at 4°C. The dissolved material was then used as an SDS sample buffer. The solution was taken and heated at 37°C for 1 hour. For immunoprecipitation, 25 μl of the lysate was pre-bound. Incubate with Flag-M2 beads (Sigma) and incubate overnight at 4°C. The beads were baited. After washing, the beads were eluted with sample buffer and heated at 37°C for 1 hour. SDS- After PAGE, the protein was identified by Western blotting using Immobilon-FL. Transferred to a PVDF membrane (Milipore). Odyssey blocking buffer (LI After blocking with -COR, the protein was processed using Amersham Typhoon Using a Biomolecular Imager (GE Health Care) Goat anti-mouse / rabbit Alexa 680 (Invitrogen), donkey anti-rat Al exa 680 (Invitrogen) or goat anti-mouse / rabbit IRDye 80 Labeled with a specified primary antibody detected at 0 (Rockland).

[0399] Results and Discussion RNF43 and TGF-βII type serine / threonine kinase receptor (TβRII) forced Dimerization induces lysosomal localization and degradation of TβRII. The membrane targets selected cell surface proteins for internalization and lysosomal degradation. To demonstrate the concept of reorienting penetrating E3 ligases, FKBP / FRB II A merization system was used. The FKBP domain was added to the C-terminus of both TβRII and RNF43. One of the FRB domains was fused to it. When co-expressed in HEK293T cells, this These proteins do not interact (Figure 2). However, when an A / C dimerizing agent is added, R Co-immunoprecipitation of NF43 and TβRII is induced (Figure 2). The dimerizing agent itself is TβRII It does not inhibit the stability of RNF43 (Figure 2, whole cell lysate). Next, RNF43 and TβR We investigated whether forced interaction with II alters the intracellular localization of TβRII. In the absence of a dimerizing agent, TβRII is present in the absence of RNF43 (Figure 3A) and RNF4 In all cases of co-expression of compound 3 (Figure 3B), it was mainly localized to the cell membrane. However, dimerization occurred. When the agent is added, both TβRII and RNF43, as well as the lysosomal marker CD63, This strongly induced relocalization to perinuclear vesicles of the positive cell (Figure 3C). These findings suggest that RN Forced dimerization of F43 and TβRII leads to increased levels of TβRII being delivered to lysosomes. This indicates that guidance is being provided.

[0400] Did the increased lysosomal localization of TβRII result in a decrease in the amount of functional TβRII? To determine this, we analyzed protein levels using Western blotting. RNF43 expression itself does not affect the stability of TβRII, but RNF43 and TβRI When dimerization of I was induced, the amount of TβRII protein clearly decreased (Figure 4). These results together suggest that transmembrane E3 ligase RNF43 is not normally associated with transmembrane receptor T We demonstrated that forced dimerization of βRII targets TβRII for lysosomal degradation. It is.

[0401] [Example 2] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were converted to 10% fetal bovine serum (GE Health). hcare), 2 mM UltraGlutamine (Lonza), 100 units / m L-penicillin and 100 μg / mL streptomycin (Invitrogen) are included. Cells were cultured in RPMI (Invitrogen). The cells were kept at 37°C and 5% CO2. The cells were cultured in FuGEN according to the manufacturer's protocol. This was done using E 6 (Promega).

[0402] Constructs and antibodies E6-Flag-TGF-βII type serine / threonine kinase receptor (TβRII) and -Epidermal Growth Factor Receptor (EGF) R) and Alpha-myc-RNF43 and RNF167 are Q5 High-F Subcloning using idelity 2× Master Mix (NEB) Obtained from the following. All constructs were sequenced. Immunofluorescence (IF) was used to obtain the following primary antibodies Body samples used: Rabbit anti-Flag (Sigma-Aldrich) and mouse anti-Myc (Hybridoma 9E10). The primary antibody was diluted according to the manufacturer's instructions. The secondary antibody used in IF was used at a 1:300 ratio (Life Technologies). ).

[0403] Immunofluorescence and confocal microscopy HEK293T cells were sterilized in laminin (Sigma-Aldrich) in a 24-well plate. They were propagated on glass coverslips coated with ). After overnight transfection, Cells are treated with 100 nM bi-VHH(VHH Alpha-(G4S)3-VHH E6 Before and during a 5-hour treatment, 20n Incubate M Bafilomycin A1 (Sigma-Aldrich) for 1 hour The cells were then washed twice with warmed culture medium and treated with 0.05M phosphate buffer at pH 7. Cells were fixed with 4% formaldehyde in 4. Cells were then mixed with 2% BSA and 0.1% saponin in PBS. The cells were blocked with a buffer containing , at room temperature (RT) for 30 minutes. Afterward, the cells were blocked. In a buffer, with a primary antibody against either Flag or Myc, leave at room temperature for 1 hour. The cells were incubated, followed by incubation with a secondary antibody at room temperature for 1 hour. Mounted on Rolong Diamond (Life Technologies), Images were acquired using an LSM700 confocal microscope. The images were analyzed and processed using ImageJ. .

[0404] Results and Discussion RNF43 and RNF167 can be dimerized by forced dimerization using bispecific VHH, T It induces the removal of βRII and EGFR from the cell surface. To further confirm the functionality of the heterobifunctional molecule of the present invention, the extracellular region VHH is By mediating dimerization, the selected receptor was targeted with E3 ligase. For this purpose This includes cells that target epitope tags (E6 tags) and E3 ligases (Alpha tags). It was merged into the external domain. These epitope tags were selected so that VHH would recognize them (Go etzke et al., 2019, Nature Communications, 10(1), 1-12; Ling et al., 2019, Molec (Urical Immunology, 114(July), 513-523), dual specificity for these two epitopes V We created HH(bi-VHH) and enabled dimerization mediated by VHH. To determine the changes in the localization of the receptor, the E3 ligase was tagged with a Myc epitope tag, and the receptor A Flag epitope tag was incorporated into it. When co-expressed in HEK293T cells, None of the receptors co-localized with any of the E3 ligases. E3 ligases are primarily intracellular co-localized. Within the compartment, the receptors were mainly localized to the cell membrane (data not shown). However, After treatment with bi-VHH for 5 hours, both RNF43 and RNF167 were TβRII This induced the removal of EGFR from the cell membrane. Furthermore, it induced lysosome turnover. In bafilomycin-treated cells, the internalized protein coclassifies in the perinuclear region. It is teratogenic, and E3 ligase and its targets accumulate in late endosome / lysosome structures. This was shown (Figures 5A-D). These findings suggest that heterogeneous groups such as bi-VHH are involved. Using functional molecules, transmembrane E3 ligases are intentionally dimerized with selected transmembrane receptors. This has shown that it is possible to induce the removal of receptors from the cell surface by causing this change. ru.

[0405] [Example 3] material and method Cell culture and transfection Human fetal kidney (HEK) 293T cells were converted to 10% fetal bovine serum (GE Health). hcare), 2 mM UltraGlutamine (Lonza), 100 units / m L-penicillin and 100 μg / mL streptomycin (Invitrogen) are included. Cells were cultured in RPMI (Invitrogen). The cells were kept at 37°C and 5% CO2. The cells were cultured in FuGEN according to the manufacturer's protocol. Perform the test using E 6 (Promega) or Effectene (Qiagen). Ta.

[0406] Constructs and antibodies E6-Flag-Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4), Receptor-type Tyrosine FLT3 (FLT-3), programmed cell death protein 1 (PD) -1) and programmed cell death ligand 1 (PD-L1) and Alpha-myc -RNF43, RNF128, RNF130 and RNF167 are Q5 High-Fi Subcloning using delity 2× Master Mix (NEB) All constructs were sequenced. Immunofluorescence (IF) was performed using the following primary antibodies. Using: Rabbit anti-Flag or mouse anti-Flag (Sigma-Aldrich) The primary antibody was diluted according to the manufacturer's instructions. The secondary antibody used for IF was diluted 1:30. Used in version 0 (Life technologies).

[0407] Immunofluorescence and confocal microscopy HEK293T cells were sterilized in laminin (Sigma-Aldrich) in a 24-well plate. They were propagated on glass coverslips coated with ) 6 hours after transfection. , cells were treated with 50 nM bi-VHH (VHH Alpha-(G4S)3-VHH E6) They were incubated overnight with [the substance]. After treatment, the cells were washed twice with warmed culture medium and 0.05 [of the substance]. Cells were fixed with 4% formaldehyde in M ​​phosphate buffer at pH 7.4. The cells were blocked with a buffer containing BSA at room temperature (RT) for 30 minutes. In a buffering solution, the primary antibody against Flag was incubated with the primary antibody at room temperature for 1 hour, and then with the secondary antibody. The cells were incubated at room temperature for 1 hour. Mounted on e technologies, the LSM700 uses a 5x objective lens. Images are taken using a confocal microscope or an EVOS-M5000 microscope with a 20x objective lens. The images were acquired. The images were analyzed and processed using ImageJ.

[0408] Results and Discussion The specific combination of E3 ligase and target enables forced dilution using bispecific VHH. Embodied material allows for surface removal of the target. To screen for further candidate combinations of E3 ligases and receptors, previously created In addition to the constructed structures, Alpha-Myc-RNF128, Alpha-Myc-RN F130, E6-Flag-CTLA-4, E6-Flag-FLT-3, E6-Fla g-PD-1 and E6-Flag-PD-L1 were generated. Co-development occurred in HEK293T cells. When revealed, CTLA-4, FLT-3, PD-1, and PD-L1 are all localized on the cell surface. It was present. When treated overnight with bi-VHH, the surface targets were removed in the following E3-target combinations. This will be done with: CTLA-4 and RNF167; FLT-3 and RNF43, RNF12 8 or RNF167, PD-1 and RNF128, RNF130 or RNF167, and PD-L1 and RNF43, RNF128 or RNF130 (Figures 6A-D). The findings suggest that various transmembrane E3 ligases can be intentionally dimerized with selected transmembrane receptors. Therefore, bi-VHH is used to induce the removal of these receptors from the cell surface. This expands the range of applications for heterobifunctional molecules. Furthermore, these findings are This emphasizes that not all combinations are necessarily effective.

[0409] [Example 4] 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. To create a cancer cell line, the inventors used guide RNA and Alpha-Myc or E6- It is used in combination with Flag-tagged donor DNA, and E3 ligase or endogenous target Between the signal peptide (SP) of the gene locus and the coding sequence of the first mature amino acid, these This facilitates the insertion of tags (Figure 7A). Using these cell lines, bi-VHH Removal of endogenous targets from the cell surface by forced dimerization is performed using microscopy or Western spectroscopy. Evaluate using one of the following methods: blotting (Figure 7B).

Claims

1. A heterobifunctional molecule comprising a first and a second binding domain, i) The first binding domain specifically binds to transmembrane E3 ubiquitin ligase. It is possible to do; ii) The second binding domain can specifically bind to transmembrane proteins, The heterobifunctional relationship between the transmembrane E3 ubiquitin ligase and the transmembrane protein. The simultaneous binding of molecules preferably results in the ubiquitination and internalization of the transmembrane protein. A heterobifunctional molecule.

2. The molecule is the extracellular component of the transmembrane E3 ubiquitin ligase and the transmembrane protein A heterobifunctional molecule according to claim 1, which binds to the extracellular portion of a substance.

3. Simultaneous binding of the molecule to the transmembrane E3 ubiquitin ligase and the transmembrane protein. Claim 1, the combination results in the degradation of the transmembrane protein, preferably lysosomal degradation. Or the heterobifunctional molecule described in 2.

4. The aforementioned transmembrane E3 ubiquitin ligase contains monoubiquitin, multiubiquitin, and Lys4 The transmembrane protein is ubiquitinated by an 8-linked or Lys63-linked polyubiquitin chain. A heterobifunctional molecule according to any one of claims 1 to 3, which is modified.

5. The claim states that the transmembrane protein is a receptor, preferably a receptor involved in cancer. A heterobifunctional molecule as described in any one of items 1 to 4.

6. The aforementioned transmembrane E3 ubiquitin ligase contains RNF43, RNF167, ZnRF3, and RN F13, AMFR, MARCH1, MARCH2, MARCH4, MARCH8, MAR CH9, RNF149, RNF145, RNFT1, RNF130 and RNF128 Selected from the group and / or the transmembrane protein is TGFβR1, TG FβR2, EGFR, ERBB2, ERBB3, IGF1R, MET, VEGFR2, K IT, FLT3, PDGFRA, PDGFRB, GHR, FZD1, FZD2, FZD3 , FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP 5. LRP6, PD-1, PD-L1, CTLA4, CMTM6, CMTM4 and WL A heterobifunctional molecule selected from the group consisting of S, according to any one of claims 1 to 5. 。

7. The transmembrane E3 ubiquitin ligase is RNF43, and the transmembrane protein is PD - Selected from the group consisting of L1, FZD7, FLT3, TGFβR2, and EGFR, The heterobifunctional molecule according to claim 6.

8. The transmembrane E3 ubiquitin ligase is RNF167, and the transmembrane protein is P Selected from the group consisting of D-1, CTLA4, FLT3, TGFβR2, and EGFR. The heterobifunctional molecule according to claim 6.

9. The transmembrane E3 ubiquitin ligase is RNF128, and the transmembrane protein is P The H-type according to claim 6, which is at least one of D-1, PD-L1, and FLT3. A terobifunctional molecule.

10. The transmembrane E3 ubiquitin ligase is RNF130, and the transmembrane protein is PD- The heterobifunctional component according to claim 6, which is at least one of 1 and PD-L1. child.

11. The molecule includes a linker between the first binding domain and the second binding domain. or the heterobifunctional molecule according to any one of claims 1 to 10.

12. At least one of the first domain and the second domain is an organic small molecule Alternatively, it is a protein molecule, preferably the heterobifunctional molecule is a bicyclic peptide. A heterobifunctional molecule according to any one of claims 1 to 11.

13. At least one of the first domain and the second domain is an antibody or A functional fragment, preferably the functional fragment is a nanobody, according to claims 1 to 12. A heterobifunctional molecule as described in any one of the following items.

14. The heterobifunctional molecule is a bispecific antibody, preferably a bispecific nanobody. The heterobifunctional molecule according to claim 13.

15. At least one of the first domain and the second domain is an aptamer A heterobifunctional molecule according to any one of claims 1 to 14.

16. A heterobifunctional component according to any one of claims 1 to 15 for use as a pharmaceutical. child.

17. A heterobifunctionality according to any one of claims 1 to 16 for use in the treatment of cancer. A molecule, wherein the cancer is preferably colorectal cancer, ovarian cancer, breast cancer, esophageal cancer, From the group consisting of stomach cancer, prostate cancer, lung cancer, melanoma, leukemia, pancreatic cancer, and bladder cancer Selected heterobifunctional molecules.

18. The aforementioned transmembrane E3 ubiquitin ligase and membrane-bound protein a) Express transmembrane E3 ubiquitin ligase and membrane-bound proteins on the cell surface. This is a step in which cells are prepared, - The transmembrane E3 ubiquitin ligase has a first non-natural epitope in its extracellular portion. Includes tags; - The membrane-bound protein contains a second non-natural epitope tag in its extracellular portion. Step and; b) A step of exposing the cells to a heterobifunctional molecule, wherein the heterobifunctional The molecule is, - A first binding domain that can specifically bind to the first non-natural epitope tag. In; and - A second binding domain capable of binding to the second non-natural epitope tag. Steps including; c) the step of determining the surface level of the membrane-bound protein in the cell; d) The surface level of the membrane-bound protein is at least about 10%, 20%, 30%, 4% When the amount decreases by 0%, 50%, 60%, 70%, 80%, 90%, or approximately 100%, the above A step of selecting a transmembrane E3 ubiquitin ligase and the membrane-bound protein, The reduction is compared to the surface level of the membrane-bound protein of the cell prior to step b). The step is a decrease compared to the step. A heterobiometric pair according to any one of claims 1 to 17, selected using a selection method including functional molecule.

19. A method for identifying transmembrane proteins as targets for cancer treatment, wherein the method 、 i) Expose cells to one or more members of a library of heterobifunctional molecules. The step is to have the heterobifunctional molecule have a first binding domain and a second binding domain The main component is included, and the first binding domain binds to a transmembrane E3 ubiquitin ligase. The second domain contains a scrambled sequence, and the steps; ii) The least of the viability, differentiation ability, stem cell properties and proliferative capacity of the exposed cells The first step is to decide on at least one; iii) Among the viability, differentiation ability, stem cell properties and proliferation ability of the exposed cells, the least At least one of the following: viability of control cells, differentiation ability, stem cell properties, and proliferative capacity. The step of comparing with one; iv) Compared to the control cells, the viability, differentiation ability, and stem cell properties of the exposed cells The heterobifunctional molecule reduces or increases at least one of the following: The steps to identify; v) The heterobifunctional molecule identified in step iv) can be bound The step of identifying the transmembrane protein Includes, Preferably, the heterobifunctional molecule is a bispecific antibody, and preferably, bispecific A method for creating sexual nanobodies.

20. The cells are patient-derived tissue, preferably a part of cultured patient-derived tissue. or derived therefrom, preferably the cells are biopsy or organoids, preferably, The method according to claim 19, wherein the method is part of or derived from a tumor organoid.

21. A method for reducing the surface level of cell membrane-bound proteins, wherein the method is a) Distributing transmembrane E3 ubiquitin ligase and the membrane-bound protein on the cell surface The steps include: preparing the cells to be expressed; b) A step of exposing the cells to a heterobifunctional molecule, wherein the heterobifunctional The molecule is, i) It can specifically bind to the extracellular portion of the transmembrane E3 ubiquitin ligase. The first binding domain; and ii) A second binding agent that can specifically bind to the extracellular portion of the membrane-bound protein. Combined domain Steps including; c) If necessary, the step of determining the surface level of the membrane-bound protein in the cell. and Includes, The aforementioned decrease is compared to the surface level of the membrane-bound protein in the cell prior to step b). A method that represents a comparative decrease.

22. - The transmembrane E3 ubiquitin ligase has a first non-natural epitope in the extracellular region. The tag is included, and the first binding domain of the heterobifunctional molecule is the first non-natural Binding to pitope tags; and - The membrane-bound protein contains a second non-natural epitope tag in its extracellular portion. The second binding domain of the heterobifunctional molecule is the second non-natural epitope tag Combine The method according to claim 21, wherein at least one of the following is the method according to claim 21.

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