Phase change adjustment element and use thereof

Abstract

The present application provides a novel phase transition regulating element, which comprises a part containing a multivalent phase transition domain and at least two ligands, wherein at least one ligand is covalently connected to the part containing the multivalent phase transition domain, and the rest of the ligands are covalently connected to the part containing the multivalent phase transition domain or to the other ligands, and each of the at least two ligands can specifically bind to its corresponding cell surface molecule. The phase transition regulating element of the present application can effectively enrich cell surface molecules by driving phase separation after binding to cell surface molecules, enhance the condensation of cell surface molecules (such as receptor oligomerization, etc.), and further regulate a series of cell physiological and biochemical activities, such as receptor downstream signal transmission, cell endocytosis, etc.

Description

Technical Field

[0001] This invention relates to a novel phase transition regulating element that, after binding to cell surface molecules, can effectively enrich cell surface molecules by driving phase separation, enhance the aggregation of cell surface molecules (e.g., receptor oligomerization), and thereby regulate a series of cellular physiological and biochemical activities, such as downstream receptor signal transduction and endocytosis. Background Technology

[0002] Cell surface molecules are common candidates for drug targets, but many cell surface molecules ultimately fail to be successfully developed into useful drugs. As an example, death receptor 5 (DR5), a member of the tumor necrosis factor (TNF) receptor superfamily, has yet to be developed into a drug.

[0003] Research has found that multivalent macromolecules can aggregate due to intermolecular or intramolecular interactions, thereby separating from the surrounding ordinary solution phase and forming an independent liquid phase enriched with the macromolecules. This process is called "liquid-liquid phase separation (LLPS, also referred to as "phase separation" or "phase transition")". Numerous small droplets enriched with these macromolecules exist within the liquid phase formed by phase separation, with diameters reaching several micrometers or even larger. These highly identifiable droplets are called "phase transition droplets".

[0004] Multivalent ligands, synthesized chemically, have been reported as tools for studying the molecular function of cell surfaces, used to induce or stabilize the dimerization or oligomerization of specific receptors. For example, multivalent ligands based on complex macrocyclic peptide backbones have been used (Science, 1993, 262(5136):1019-1024). However, the enhancing effect of such multivalent ligands on downstream receptor signaling is strictly limited by the ligand valence. Based on this, IgM Biosciences reported using natural multivalent antibodies to increase the oligomerization of receptor molecules, for example, by transplanting the VH domain binding to DR5 into the IgM backbone, which is a natural multivalent ligand, to obtain pentamer DR5-binding IgM (Mol Cancer Ther; 20(12):2483-2494). However, such polymeric IgMs not only have receptor-binding activity strictly limited by antibody valence, but also can only recognize a single target, thus posing a high risk of systemic toxicity.

[0005] To date, there have been no reports of using multivalent ligands containing multivalent phase transition domains with phase transition regulation functions, let alone using such multivalent ligands to manipulate corresponding cell surface molecules, thereby improving the drug-like properties of cell surface molecules such as receptors. Summary of the Invention

[0006] Through in-depth research, the inventors discovered that a multivalent phase transition modulatory element is obtained by covalently linking a portion containing a multivalent phase transition domain capable of driving liquid-liquid phase separation with a plurality of ligand molecules capable of specifically binding to different candidate cell surface molecular targets. Using this multivalent phase transition modulatory element can not only improve the specificity and selectivity for candidate targets, but also, compared with ligand molecules without phase separation modulatory function, the multivalent phase transition modulatory element of the present invention exhibits a significantly enhanced cell surface molecular aggregation effect and / or improved signaling pathway manipulation activity.

[0007] Therefore, in one aspect, the present invention provides a phase transition modulation element, characterized in that the phase transition modulation element includes a portion containing a multivalent phase transition domain, and the remaining portion includes at least two ligands, wherein at least one of the ligands is connected to the portion containing the multivalent phase transition domain, and the remaining ligands are connected to the portion containing the multivalent phase transition domain or to other ligands contained in the same phase transition modulation element, each of the at least two ligands being capable of specifically binding to its corresponding cell surface molecule, wherein the cell surface molecule may be the same or different.

[0008] In a preferred embodiment, the portion containing the multivalent phase transition domain is covalently connected to the at least one ligand or between ligands, optionally via a peptide linker or a non-peptide linker, preferably a peptide linker.

[0009] In yet another preferred embodiment, the phase transition modulation element of the present invention is a fusion protein comprising the portion containing the multivalent phase transition domain and at least two of the ligands.

[0010] In one embodiment, the phase change regulating element provided by the present invention comprises three or more ligands.

[0011] In another embodiment, the number of ligands in the phase change regulating element is 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0012] In one embodiment, the phase change modulation element of the present invention, after its contained ligand binds to one or more cell surface molecules recognized by the ligand, exhibits a significantly increased degree of aggregation of the one or more cell surface molecules compared to the case where the phase change modulation element of the present invention is not bound, or compared to the case where it is bound to other multivalent ligands of the same valence as the phase change modulation element of the present invention.

[0013] In a preferred embodiment, the multivalent phase transition structural domain in the phase transition regulating element of the present invention is composed of at least two, for example three, four, five, six, seven, eight, nine or ten motifs connected in series, and the motifs may be the same or different.

[0014] In a more preferred embodiment, the multivalent phase transition domain in the phase transition modulation element of the present invention is formed by at least one SUMO3 motif connected in series with at least one SIM motif, or by at least one PRMH motif connected in series with at least one SH3 motif.

[0015] In a further preferred embodiment, the multivalent phase transition domain in the phase transition modulating element is formed by one, two, three or four SUMO3 motifs connected in series with one, two, three or four SIM motifs, or by one, two, three or four PRMH motifs connected in series with one, two, three or four SH3 motifs.

[0016] In a further preferred embodiment, the multivalent phase transition domain comprises an amino acid sequence as shown in SEQ ID NO:18 or SEQ ID NO:19, or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO:18 or SEQ ID NO:19. In another preferred embodiment, the multivalent phase transition domain is composed of an amino acid sequence as shown in SEQ ID NO:18 or SEQ ID NO:19, or is composed of an amino acid sequence having at least 80%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO:18 or SEQ ID NO:19.

[0017] In another preferred embodiment, the phase change modulation element of the present invention includes a ligand capable of specifically binding to tumor-associated antigens.

[0018] In a more preferred embodiment, the tumor-associated antigen is selected from the group consisting of CXC motif chemokine receptor 4 (CXCR4), hepatocyte growth factor receptor (c-Met or HGFR), epidermal growth factor receptor (EGFR), epidermal growth factor receptor 2 (HER2), prostate-specific membrane antigen (PSMA), fibroblast activating protein (FAP), carcinoembryonic antigen (CEA), folate receptor α (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), p95HER2, EpCAM, HER3, CD30 or TPBG (5T4), CD19, CD79b, CD20, CD22, CD37, CD38, BCMA, and GPRC5D.

[0019] In another preferred embodiment, the phase transition modulation element of the present invention contains another ligand that can specifically bind to a tumor necrosis factor receptor, preferably death receptor 5 (DR5) or Fas, more preferably death receptor 5 (DR5).

[0020] In another preferred embodiment, the ligand in the phase transition regulating element of the present invention may be selected from peptide ligands or non-peptide ligands. The peptide ligand is preferably selected from one or more of the group consisting of: antibodies or antigen-binding fragments thereof that specifically bind to cell surface molecules, cytokines, growth factors, adhesion molecules, peptide hormones, or polypeptides randomly selected by phage display or yeast display. In a more preferred aspect, the antibody may be selected from monoclonal antibodies, polyclonal antibodies, human antibodies, or humanized antibodies; the antigen-binding fragment may be selected from F(ab')2, Fab, single-chain variable fragments (scFv), single-domain antibody fragments (VHH or nanobodies); and the non-peptide ligand is preferably selected from one or more of the group consisting of small molecule agonists or antagonists, antisense oligonucleotides, or small interfering RNA (siRNA).

[0021] In a further preferred embodiment, one or more ligands in the phase change regulating element of the present invention are single-chain variable segments.

[0022] In a more preferred embodiment, the phase transition regulating element of the present invention comprises a single-chain variable fragment comprising an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 80%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO:21, and / or another ligand comprising a single-chain variable fragment comprising an amino acid sequence selected from those shown in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:23 or an amino acid sequence having at least 80%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:23. In other preferred embodiments, the phase transition regulating element of the present invention comprises one ligand consisting of an amino acid sequence as shown in SEQ ID NO:21 or an amino acid sequence having at least 80%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO:21, and / or another ligand consisting of any of the amino acid sequences selected from those shown in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:23 or an amino acid sequence having at least 80%, 90%, 95%, or 99% identity with the sequence shown in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:23.

[0023] In a preferred embodiment, the phase change modulation element of the present invention may further include a tag and / or a polypeptide that cleaves the tag without affecting the function of the ligand and the portion containing the multivalent phase change domain.

[0024] In some implementations, the tag is used to separate, purify, and identify the portion containing the multivalent phase transition domain.

[0025] In some implementations, the tag may be selected from polyhistidine (His) x The tags include maltose-binding protein (MBP) tags, glutathione S-transferase (GST) tags, and small molecule ubiquitin-like modified protein (SUMO) tags. In the phase transition modulation element of the present invention, the tags may be attached to the N-terminus and / or C-terminus of the portion containing the multivalent phase transition domain.

[0026] The phase change regulating element according to the present invention comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:25, or has at least 80%, 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:25.

[0027] In another aspect, the present invention provides a method for screening phase change regulating elements, comprising the following steps:

[0028] Step A: Generate a library containing multiple candidate phase transition modulating elements, wherein each candidate phase transition modulating element comprises a portion containing a multivalent phase transition domain and at least two ligands, wherein at least one ligand is covalently linked to the portion containing the multivalent phase transition domain, and the remaining ligands are covalently linked to the portion containing the multivalent phase transition domain or to other ligands, each of the at least two ligands being specifically bound to its corresponding cell surface molecule, optionally, the portion containing the multivalent phase transition domain in the same phase transition modulating element is covalently linked to the at least one ligand or to the ligands via a linker, wherein the cell surface molecules specifically bound by the ligands to each candidate phase transition modulating element may be the same or different;

[0029] Step B: Using a reference material that is distinguished only by the absence of the multivalent phase transition domain as a negative control, under the same conditions suitable for phase transition, determine the activity level of the candidate phase transition regulating element and the negative control in generating phase transition droplets in Step A above.

[0030] Step C: Select the candidate phase change regulating element that produces more phase change droplets than the negative control in step B above as the target phase change regulating element.

[0031] In another aspect, the present invention provides a phase change regulating element obtained by the above-described method for screening phase change regulating elements.

[0032] In another aspect, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the aforementioned phase change regulating element, and optionally a pharmaceutically acceptable carrier.

[0033] In another aspect, the present invention provides a method for treating a disease using any of the aforementioned phase change regulating elements or pharmaceutical compositions.

[0034] In some embodiments, the method of treating a disease according to the present invention includes administering an effective amount of the phase transition regulating element of the present invention or the pharmaceutical composition of the present invention to a subject in need. In a preferred embodiment, the disease is selected from the group consisting of chronic autoimmune disorders, inflammatory conditions, diseases related to abnormal cell proliferation or apoptosis, sepsis, or viral infections. In a more preferred embodiment, the disease related to abnormal cell proliferation or apoptosis is cancer. In a further preferred embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), glioma (e.g., neuroblastoma), neuroblastoma, melanoma, breast cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, cervical cancer, esophageal cancer, sarcoma, esophageal cancer (e.g., squamous cell carcinoma of the esophagus), thyroid cancer (e.g., papillary thyroid carcinoma), or prostate cancer, B-cell lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute monocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), mixed lineage leukemia, NUT midline carcinoma, Burkitt lymphoma, or mycosis fungoides (MF), or metastatic forms thereof.

[0035] In another aspect, the present invention provides the use of the aforementioned phase transition regulating element or the aforementioned pharmaceutical composition in the preparation of an agent for treating a disease. In a preferred embodiment, the disease is selected from the group consisting of chronic autoimmune disorders, inflammatory conditions, diseases related to abnormal cell proliferation or apoptosis, sepsis, or viral infections. In a more preferred embodiment, the disease related to abnormal cell proliferation or apoptosis is cancer. In a further preferred embodiment, the cancer is selected from the group consisting of lung cancer, colorectal cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), glioma (e.g., neuroblastoma), neuroblastoma, melanoma, breast cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, cervical cancer, esophageal cancer, sarcoma, esophageal cancer (e.g., squamous cell carcinoma of the esophagus), thyroid cancer (e.g., papillary thyroid carcinoma), or prostate cancer, B-cell lymphoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute monocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), mixed lineage leukemia, NUT midline carcinoma, Burkitt lymphoma, or mycosis fungoides (MF), or metastatic forms thereof.

[0036] According to the present invention, a novel phase transition modulating element is provided, which, upon binding to cell surface molecules, can increase the aggregation of these molecules on the cell membrane by driving phase separation, thereby enhancing the regulation of physiological and biochemical activities associated with this aggregation. This enhancement is not limited by the valence of the ligands contained in the modulating element itself. Furthermore, because this novel modulating element contains two or more ligands, it can achieve richer and more refined targeting specificity, as well as more effective manipulation performance, through combinations of different ligands.

[0037] More specifically, the present invention provides a phase transition modulation element comprising a portion containing a multivalent phase transition domain and at least a first ligand and a second ligand connected thereto. Compared to multivalent ligands that do not contain a multivalent phase transition domain and cannot induce phase separation, the phase transition modulation element of the present invention significantly enhances the regulatory effect on cell surface molecules that interact with the first ligand and the second ligand. Attached Figure Description

[0038] Figure 1 The results of detecting various synthetic ligands and their truncated variants transiently expressed in Escherichia coli BL21(DE3) using polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions were obtained. Color development was performed by Coomassie brilliant blue staining. Figure 1 Figure A shows the electrophoresis results of the synthetic ligand CN-P2S2-DN and its various truncated variants. Figure 1B shows the SDS-PAGE results of synthetic ligands EN-P2S2-DN, MN-P2S2-DN, PN-P2S2-DN, HN-P2S2-DN, and CN-P2S2-DN targeting different receptors. Markers represent molecular weight markers (purchased from Beijing Polymer Biotechnology Co., Ltd.).

[0039] Figure 2 : Figure 2 A shows the laser confocal imaging results obtained from in vitro liquid-liquid phase separation experiments using CN-P2S2-DN protein labeled with the fluorescent dye Alexa 488 at concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. The scale bar for each inset is 5 μm. Figure 2 Image B shows the droplet formation and fusion process of 12.5 μM CN-P2S2-DN protein during in vitro phase separation experiments, captured using a NIKON A1R HD25 laser confocal microscope. 0 min indicates the start point of the observation period.

[0040] Figure 3 : Figure 3 The results of the fluorescence recovery after bleaching (FRAP) experiment, using a NIKON A1R HD25 laser confocal microscope, are shown. The top image is a snapshot of the droplet's change over time; the bottom image shows the corresponding fluorescence intensity statistical analysis results. Time 0 indicates the starting time point for observation and measurement immediately after photobleaching pulse stimulation. Data points in the bottom image represent the average values ​​of three independent experiments. The vertical axis shows the average fluorescence intensity of the photobleached region after standardization correction, based on the average fluorescence intensity of the unbleached region; the horizontal axis shows the elapsed time (s) after photobleaching pulse stimulation.

[0041] Figure 4 : Figure 4 This image shows a laser confocal microscopy live-cell imaging snapshot of HEK293T cells co-expressing CXCR4-mCherry and DR5-GFP treated with 200 nM CN-P2S2-DN protein (labeled with the fluorescent dye Alexa 647), and normal HEK293T cells as a control. In the images, CXCR4-mCherry, DR5-GFP, CN-P2S2-DN, and DAPI represent the fluorescence excitation images of exogenous CXCR4, exogenous DR5, CN-P2S2-DN protein, and the cell nucleus, respectively. The Merge image is the result of overlaying the four images in situ. The scale bar in each inset is 5 μm. Figure 4 A represents HEK293T cells with added control solution. Figure 4B represents HEK293T cells supplemented with CN-P2S2-DN protein. Figure 4 C is the general Figure 4 The image in B, magnified from the white dashed box area, shows the live-cell imaging results of co-localization analysis of fluorescence signals representing CN-P2S2-DN, CXCR4, and DR5 in the spots (puncta) marked by white arrows. The scale bar in each small image is 1 μm.

[0042] Figure 5 : Figure 4 The statistical results of the grayscale values ​​of the fluorescence signal at the location marked by the arrow in Figure C. The horizontal axis in the figure represents the relative distance along the direction indicated by the arrow, and the vertical axis represents the relative fluorescence intensity measured at the corresponding location.

[0043] Figure 6 Live-cell imaging colocalization analysis of the truncated variant P2S2-CN of CN-P2S2-DN with exogenous receptors CXCR4-mCherry and DR5-GFP expressed on the surface of HEK293T cells. Figure 6 Image A shows a fluorescence colocalization image of 200 nM P2S2-CN (Alexa 647 labeled) with CXCR4-mCherry and DR5-GFP, captured using a NIKON A1R HD25 laser confocal microscope. The scale bar in each inset is 1 μm. Figure 6 B represents the statistical results of the fluorescence signal grayscale values ​​measured at the locations marked by the white arrows. In the figure, the horizontal axis represents the relative distance along the direction indicated by the arrows, and the vertical axis represents the relative fluorescence intensity measured at the corresponding locations. Each data point represents the average of three independent experiments.

[0044] Figure 7 Results of live-cell imaging co-localization analysis of the truncated variant P2S2 with exogenous receptors CXCR4-mCherry and DR5-GFP on the surface of HEK293T cells. Figure 7 Image A shows a fluorescence colocalization image of 200 nMP2S2 (Alexa 647 labeled) with CXCR4-mCherry and DR5-GFP, obtained using a NIKON A1R HD25 laser confocal microscope. The scale bar in each inset is 1 μm. Figure 7 B represents the statistical results of the fluorescence signal grayscale values ​​measured at the locations marked by the white arrows. In the figure, the horizontal axis represents the relative distance along the direction indicated by the arrows, and the vertical axis represents the relative fluorescence intensity measured at the corresponding locations. Each data point represents the average of three independent experiments.

[0045] Figure 8Results of live-cell imaging co-localization analysis of the truncated variant P2S2-DN with exogenous receptors CXCR4-mCherry and DR5-GFP on the surface of HEK293T cells. Figure 8 Image A shows a fluorescence colocalization image of 200 nM P2S2-DN (Alexa 647 labeled) with CXCR4-mCherry and DR5-GFP, obtained using a NIKON A1R HD25 laser confocal microscope. The scale bars in each inset are 1 μm. Figure 8 B represents the statistical results of the fluorescence signal grayscale values ​​measured at the locations marked by the white arrows. In the figure, the horizontal axis represents the relative distance along the direction indicated by the arrows, and the vertical axis represents the relative fluorescence intensity measured at the corresponding locations. Each data point represents the average of three independent experiments.

[0046] Figure 9 This section presents time-series laser confocal microscopy imaging results of HEK293T cells co-expressing CXCR4-mCherry and DR5-GFP, after treatment with 200 nM CN-P2S2-DN protein (labeled with the fluorescent dye Alexa 647). CXCR4-mCherry, DR5-GFP, and CN-P2S2-DN are shown as fluorescence images of exogenous CXCR4, exogenous DR5, and CN-P2S2-DN proteins, respectively. The merged image shows the result of in-situ overlay of these three images. The upper images are fluorescence images of intact cells. The scale bar in each sub-image is 5 μm. The lower image is a magnified view of the image within the white dashed box in the upper images, showing the fusion and division of puncta formed by CN-P2S2-DN binding to cell surface receptors CXCR4 and DR5 over time. The scale bar in each sub-image is 1 μm.

[0047] Figure 10 This study presents the time-series statistical analysis of fluorescence recovery of CN-P2S2-DN, CXCR4-mCherry, and DR5-GFP on the cell surface of HEK293T cells treated with CN-P2S2-DN (labeled with the fluorescent dye Alexa 647) after FRAP treatment. Each data point represents the average of three independent experiments. The vertical axis of the figure represents the average fluorescence intensity of the photobleached region after normalization, based on the average fluorescence intensity of the unbleached region; the horizontal axis represents the elapsed time (s) after photobleaching pulse stimulation.

[0048] Figure 11 The interaction between CN-P2S2-DN and endogenous receptors on the surface of SJSA-1 cells. Figure 11A shows a snapshot of live-cell imaging of SJSA-1 cells treated with 200 nM CN-P2S2-DN protein (labeled with the fluorescent dye Alexa 561) using a NIKON A1R HD25 laser confocal microscope. The scale bar in the figure is 10 μm. Figure 11 B shows the time-series imaging results of SJSA-1 cells treated with 200 nM CN-P2S2-DN protein (labeled with the fluorescent dye Alexa 561) using a NIKON A1R HD25 laser confocal microscope. The scale bar in the figure is 1 μm.

[0049] Figure 12 Immunofluorescence co-localization analysis results of the synthetic ligand CN-P2S2-DN with endogenous receptors CXCR4 and DR5 expressed on the surface of SJSA-1 cells. Figure 12 Image A shows a fluorescence colocalization image of 200 nm CN-P2S2-DN (Alexa 561 labeled) with endogenous CXCR4 and DR5 receptors, obtained using a NIKON A1R HD25 confocal microscope. The scale bars in each inset are 1 μm. Figure 12 B represents the statistical analysis results of the fluorescence signal grayscale values ​​measured at the straight lines marked with white arrows. In the figure, the horizontal axis represents the relative distance along the direction indicated by the arrow, and the vertical axis represents the relative fluorescence intensity measured at the corresponding locations. Each data point represents the average of three independent experiments.

[0050] Figure 13 Immunofluorescence colocalization analysis results of the truncated variant P2S2 of CN-P2S2-DN with the endogenous receptors CXCR4 and DR5 on the surface of SJSA-1 cells. Figure 13 Image A shows fluorescence colocalization images of the 200 nM truncated variant P2S2 (Alexa 561 labeled) with endogenous CXCR4 and DR5 receptors, obtained using a NIKON A1R HD25 confocal microscope. Scale bars in each inset are 1 μm. Figure 13 B represents the statistical analysis results of the fluorescence signal grayscale values ​​measured at the straight lines marked with white arrows. In the figure, the horizontal axis represents the relative distance along the direction indicated by the arrow, and the vertical axis represents the relative fluorescence intensity measured at the corresponding locations. Each data point represents the average of three independent experiments.

[0051] Figure 14This section presents snapshots of the fluorescence recovery of CN-P2S2-DN on the cell surface, taken using a NIKON A1R HD25 laser confocal microscope, after FRAP treatment of SJSA-1 cells treated with 200 nM CN-P2S2-DN (labeled with the fluorescent dye Alexa 561). The corresponding time-series statistical analysis results are also included. Time 0 indicates the starting time point immediately after photobleaching pulse stimulation. The scale bars in the upper subplots are all 0.5 μm. In the lower subplots, the vertical axis represents the average fluorescence intensity of the photobleached area after standardization, based on the average fluorescence intensity of the unbleached area; the horizontal axis represents the elapsed time (s) after photobleaching pulse stimulation. Each data point represents the average of three independent experiments.

[0052] Figure 15 Imaging results obtained using an Olympus IX83 fluorescence inverted microscope after treating SJSA-1 cells with 100 nM CN-P2S2-DN protein, its various truncated variants, or combinations of truncated variants for 3 hours.

[0053] Figure 16 : Figure 15 Western blot analysis of caspase-3 expressed in treated SJSA-1 cells.

[0054] Figure 17 Western blotting analysis of caspase-3 expressed in HEK293T and SJSA-1 cells treated with different concentrations of CN-P2S2-DN protein. The cells were labeled with 0.1 μg / mL rabbit polyclonal antibody against caspase-3, and β-tubulin was used as an internal control.

[0055] Figure 18 Effect of 100 nM CN-P2S2-DN protein treatment on the time-dependent proliferation of SJSA-1 cells. SJSA-1 cells were treated with CellTracker... TM Green CMFDA Dye marking, and using Zoom System is used for monitoring.

[0056] Figure 19Evaluation of the apoptosis-inducing activity of CN-P2S2-DN protein, its various truncated variants, and combinations of truncated variants against SJSA-1 cells. SJSA-1 cells were treated with 100 nM CN-P2S2-DN protein, its truncated variants, or combinations thereof for 3 hours. The percentage of apoptotic SJSA-1 cells was determined by flow cytometry based on Annexin V-FITC / PI double staining. Ctrl in the figure represents the blank solution control.

[0057] Figure 20 Comparison of apoptosis-inducing activity of CN-P2S2-DN protein against SJSA-1 and HEK293T cells. Under the same conditions, SJSA-1 and HEK293T cells were treated with 100 nM CN-P2S2-DN protein for 3 hours, respectively. The percentage of SJSA-1 and HEK293T cells distributed in different quadrants was determined by flow cytometry based on Annexin V-FITC / PI double staining. NC in the figure represents the blank solution control.

[0058] Figure 21 The results show the expression levels of the dr5 gene in SJSA-1 and HEK293T cells.

[0059] Figure 22 Evaluation results of the apoptosis-inducing activity of CN-P2S2-DN protein against SJSA-1 cells. SJSA-1 cells were treated with different concentrations of CN-P2S2-DN protein solution, and apoptosis was measured by flow cytometry based on Annexin V-FITC / PI double staining. The horizontal axis represents the concentration of CN-P2S2-DN protein solution used, and the vertical axis represents the percentage of SJSA-1 cells undergoing apoptosis.

[0060] Figure 23 Evaluation of the apoptosis-inducing activity of CN-P2S2-DN protein and its truncated variants (P2S2-DN, P2S2) against four tumor cell lines and HEK293T cells. Depending on the cell line, cells were treated with 100 nM or 200 nM CN-P2S2-DN protein or its truncated variants for 3 or 5 hours, and the percentage of apoptotic cells was determined by flow cytometry based on Annexin V-FITC / PI double staining. In the figure, ctrl represents the blank solution control.

[0061] Figure 24This figure compares the apoptosis-inducing activity of CN-S2S2-DN protein in SJSA-1 cells and negative control cells. Under the same conditions, SJSA-1 cells and control cells were treated with 100 nM CN-S2S2-DN protein for 3 hours, respectively. The percentage of SJSA-1 cells and control cells distributed in different quadrants was then determined by flow cytometry based on Annexin V-FITC / PI double staining. Ctrl in the figure represents HEK293T cells as a negative control.

[0062] Figure 25 Comparative results of the apoptosis-inducing activity of EN-P2S2-DN protein against different cell lines. Flow cytometry was used to detect the apoptosis-inducing activity of EN-P2S2-DN on two tumor cell lines and HEK293T cells. Depending on the cell line, cells were treated with 100 nM or 200 nM EN-P2S2-DN for 3 or 5 hours, respectively, and stained using Annexin V-FITC / PI double staining.

[0063] Figure 26 Comparison of apoptosis-inducing activities of MN-P2S2-DN protein against different cell lines. Flow cytometry was used to detect the apoptosis-inducing activity of MN-P2S2-DN on three tumor cell lines and HEK293T cells. Depending on the cell line, cells were treated with 100 nM or 200 nM MN-P2S2-DN for 3 or 5 hours, respectively, and stained using Annexin V-FITC / PI double staining. Detailed Implementation

[0064] definition

[0065] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0066] As used herein, the terms “comprising” or “including” mean that a composition and method comprises the described components, but does not exclude other components. “Mainly composed of”, when used to define a composition and method, should exclude any other components that are obviously essential to the composition. Therefore, compositions defined herein as mainly composed of these components will not exclude trace contamination from separation and purification methods and pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Composed of” should exclude trace components of other ingredients used in the application of the compositions of the invention and of substantial process. Examples defined by these provisional terms are within the scope of this invention.

[0067] As used herein, the term "about" refers to a common range of error for a corresponding value that is readily known to those skilled in the art. Values ​​or parameters described herein in the manner of "about" include the value or parameter itself.

[0068] As used herein, the term “liquid-liquid phase separation” (LLPS, also referred to herein as “phase separation” or “phase transition”) refers to the following transformation process that occurs between a multivalent macromolecule and its multivalent ligand: under suitable solution conditions, the multivalent macromolecule and its multivalent ligand aggregate through interaction to form a larger complex, which reaches a certain solubility and separates from the ordinary solution phase to form an independent liquid phase enriched with the complex.

[0069] The term "phase change droplet" refers to a highly recognizable small droplet with a diameter of several micrometers or even larger that exists within the liquid phase formed by phase separation. In this article, "phase change droplet" is sometimes simply referred to as "droplet".

[0070] As used herein, the term "phase transition modulator" refers to a chemical entity capable of modulating cell surface molecules through phase separation, thereby modulating a range of cellular events associated with those molecules. For example, a phase transition modulator, through its multivalent phase transition domains, recruits bound cell surface molecules into phase transition condensates, significantly increasing the local concentration of cell surface molecules, thereby enabling manipulation of various cellular events associated with cell surface molecules, including but not limited to recognizing external stimuli and promoting downstream signal transduction of receptors, participating in enzymatic reactions, endocytosis, cell proliferation and differentiation, and intercellular communication.

[0071] As used herein, the term "multivalent phase transition domain" refers to a domain containing multiple structural modules or motifs associated with phase separation. As previously described, biomacromolecules can aggregate and undergo phase separation due to intermolecular or intramolecular interactions. Modules or motifs capable of causing the aforementioned intermolecular or intramolecular interactions include, but are not limited to: (1) linearly arranged and functionally similar structural modules or motifs in proteins or peptides; (2) structural modules or motifs that promote oligomerization of proteins or peptides; (3) multipolymer binding sites resulting from post-translational modifications; and (4) inherently disordered regions or low-complexity domains in proteins or peptides. See, for example, Wang et al., Cell 174(3):688-699, 2018; Nott, Timothy J et al., Molecular Cell 57(5):936-947, 2015. The number of structural modules or motifs contained in the multivalent phase transition domain of the present invention that are necessary to cause the said intermolecular or intramolecular interactions is the valence of the multivalent phase transition domain. For example, the multivalent phase transition domain P2S2, which is obtained by the tandem repetition of two PRMH motifs and two SH3 motifs, has a valence of four.

[0072] In a preferred aspect, the multivalent phase transition domain of the present invention is formed by tandem of one or more SUMO3 motifs and one or more SIM motifs, or by tandem of one or more PRMH motifs and one or more SH3 motifs. More preferably, the multivalent phase transition domain comprises an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:12, or comprises an amino acid sequence having 80% or more, for example, 80%, 90%, 95%, or 99% sequence identity with the sequence shown in SEQ ID NO:1 or SEQ ID NO:12, as long as the sequence can still cause phase separation of the regulatory element.

[0073] The term "ligand" is used in its broadest sense to refer to the other party in a pair that has a specific interaction. More specifically, the term "ligand" refers to a chemical entity capable of interacting with cell surface molecules. The interaction between the ligands involved in this invention and the cell surface molecules they target includes covalent bonds, ionic bonds, hydrogen bonds, or other bonding mechanisms known in the art. The ligands of this invention can be naturally occurring, such as natural molecules isolated from target cell populations or organisms from which the cell surface molecules originate, for example, certain small molecule metabolites, or macromolecules such as antibodies. The ligands of this invention can also be non-natural, such as chemical entities obtained through artificial synthesis or modification that are distinct from natural molecules. That is, the term "ligand" herein encompasses various ligand structures, including but not limited to small molecules, antibodies, antigen-binding fragments, peptides, peptide mimics, antisense oligonucleotides, or small interfering RNA (siRNA), as long as they exhibit the specific binding activity required for their pairing. In preferred embodiments, the ligand herein refers to a peptide or peptide mimic composed of natural or non-natural amino acids linked by peptide bonds, such as an antibody or its antigen-binding fragment, a cytokine, a growth factor, an adhesion molecule, a peptide hormone, etc.

[0074] In this paper, "multivalent ligand" refers to a single ligand capable of binding two or more molecules (e.g., receptors), where the molecules bound by the same multivalent ligand may be the same or different. The valence of a ligand refers to the number of interacting binding regions contained in a macromolecule or its ligands.

[0075] The term "cell surface molecule" herein has the meaning as commonly understood in the art. Non-limiting examples of cell surface molecules include protein molecules, sugar molecules, glycoproteins, etc., and receptors such as cytokine receptors, histocompatibility receptors, T-cell receptors, etc., ion channels, etc. In some cases, a cell surface molecule can be a molecule present in a target cell population but substantially absent (or present at a low concentration) in other populations, thereby indicating or defining the type of the target cell population. In other cases, a cell surface molecule can be a molecule present in a target cell population that is directly or indirectly related to the pathological state or condition to be corrected, and substantially absent or present at a low concentration in cells or tissues unaffected by that pathology. The ligands in this invention can bind to the cell surface through interaction with cell surface molecules, preferably to the surface of cells (e.g., tumor cells) having the pathological state or condition to be corrected.

[0076] In this document, "receptor" can include endogenous receptors or exogenous receptors. Endogenous receptors include receptors that are naturally present in cells. Exogenous receptors include receptors that are introduced into cells from outside. In some respects, an exogenous receptor can be a naturally present sequence contained in other cells of the same individual. In other respects, an exogenous receptor can be a receptor from a different organism or a different species. Exogenous receptors also include synthetic receptors that are not naturally present in any organism. Exogenous receptors include chimeric receptors, which are receptors constructed by connecting regions (e.g., extracellular, transmembrane, intracellular, etc.) of different molecules (e.g., different proteins, homologous proteins, orthologous proteins, etc.).

[0077] In this paper, "tandem connection" includes connecting units directly via covalent bonds or indirectly via spacer sequences (or connectors, connection sequences). In the structure obtained by tandem connection, the connecting units can be arranged in the same direction or in different directions.

[0078] The terms “increase” or “activation” as used herein refer to the ability to cause an overall increase, for example, an overall increase of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 75%, 85%, 90%, 95% or more. In some respects, increase or activation can refer to the downstream activity of ligand-cell surface molecule interactions.

[0079] The terms “reduction” or “inhibition” in this document refer to the ability to cause an overall reduction, for example, an overall reduction of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 75%, 85%, 90%, 95% or more. In some respects, reduction or inhibition can refer to downstream activity of ligand-cell surface molecule interactions.

[0080] As used in this article, the term “oligomerization” refers to the non-covalent aggregation of several biomacromolecules, such as several receptor molecules, into a complex, in which their functional state may be altered.

[0081] The term "antibody" in this document encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., dual-Fab).

[0082] The term "intact antibody" is used to refer to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein. In this document, "intact antibody" may be used interchangeably with "full-length antibody" and "whole antibody".

[0083] The terms "antigen-binding fragment" or "antibody fragment" refer to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antigen-binding fragments include, but are not limited to: dual Fab, Fv, Fab, Fab'-SH, F(ab')2, bispecific antibodies, linear antibodies, single-chain antibodies (e.g., scFv, ScFab), single-domain antibodies (e.g., VH domain, VHH domain, or nanobodies), and multispecific antibodies formed from antibody fragments.

[0084] The term "single-chain antibody," also known as "single-chain Fv," "single-chain variable fragment," "sFv," or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired antigen-binding structure. For a review of scFv, see Pluckthun's *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, Springer Verlag, New York, pp. 269–315 (1994); Malmborg et al., *J. Immunol. Methods* 183:7 13, 1995.

[0085] The term "single-domain antibody" refers to an antibody fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain. In some respects, single-domain antibodies are human single-domain antibodies (see, for example, U.S. Patent No. 6,248,516B1). Examples of single-domain antibodies include, but are not limited to, VHH.

[0086] The term "small molecule" refers to any molecule with a molecular weight of about 2000 Daltons or less, such as about 1000 Daltons or less. In some respects, small molecules can be organic molecules. In other respects, small molecules can be inorganic molecules.

[0087] As used herein, the term "molecular mimic" or "molecular mimic" refers to a polypeptide that is sufficiently similar in conformation and / or binding ability (e.g., secondary structure, tertiary structure) to a given polypeptide or a portion thereof to bind to a binding partner of the polypeptide. The mimic may bind to the binding partner with an affinity equal to, less than, or greater than that of the polypeptide it mimics. The molecular mimic may or may not have significant amino acid sequence similarity to the polypeptide it mimics. The mimic may be naturally occurring or engineered. In some respects, the mimic may perform all the functions of the mimicked polypeptide. In other respects, the mimic does not perform all the functions of the mimicked polypeptide.

[0088] As used herein, “tumor-associated antigen” or “TAA” refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor (e.g., a cancer cell, a cell in the tumor stroma). In some respects, target cell antigens are antigens on the surface of tumor cells. In one respect, TAAs are selected from the group consisting of: CXC motif chemokine receptor 4 (CXCR4), hepatocyte growth factor receptor (c-Met or HGFR), epidermal growth factor receptor (EGFR), epidermal growth factor receptor 2 (HER2), and prostate-specific membrane antigen (PSMA), fibroblast activating protein (FAP), carcinoembryonic antigen (CEA), folate receptor α (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), p95HER2, EpCAM, HER3, CD30 or TPBG (5T4), CD19, CD79b, CD20, CD22, CD37, CD38, BCMA, and GPRC5D.

[0089] Unless otherwise specified, the terms “CXC motif chemokine receptor 4” or “CXCR4” as used herein broadly refer to any naturally occurring CXCR4 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses the full-length CXCR4 and dissociated regions or domains of CXCR4, such as the extracellular domain of CXCR4. The term also encompasses naturally occurring variants of CXCR4, such as splice variants or allelic variants. An exemplary amino acid sequence of human CXCR4 is shown in Uniprot ID: P61073. The invention also contemplates minor sequence variations, particularly conserved amino acid substitutions of CXCR4 that do not affect CXCR4 function and / or activity.

[0090] Unless otherwise specified, the terms “death receptor 5” or “DR5” as used herein broadly refer to any naturally occurring DR5 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses the full-length DR5 and isolated regions or domains of DR5, such as the extracellular domain of DR5. The term also encompasses naturally occurring variants of DR5, such as splice variants or allelic variants. An exemplary amino acid sequence of human DR5 is shown in Uniprot ID: O14763. The invention also considers minor sequence variations, particularly conserved amino acid substitutions of DR5 that do not affect DR5 function and / or activity.

[0091] The term "epidermal growth factor receptor (EGFR)" is also known as the proto-oncogene c-ErbB-1 or receptor tyrosine protein kinase erbB-1. Unless otherwise stated, this term refers broadly to any naturally occurring EGFR from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses the full-length EGFR and dissociated regions or domains of EGFR, such as the extracellular domain of EGFR. The term also encompasses naturally occurring variants of EGFR, such as splice variants or allelic variants. The amino acid sequence of human EGFR is shown in UniProt accession number P00533 (version 211). This invention also considers minor sequence variations, particularly conserved amino acid substitutions of EGFR that do not affect EGFR function and / or activity.

[0092] Unless otherwise specified, the term "hepatocyte growth factor receptor (Met)" as used herein refers to any naturally occurring hepatocyte growth factor receptor from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses the full-length hepatocyte growth factor receptor and its isolated regions or domains, such as the extracellular domain of the hepatocyte growth factor receptor. The term also encompasses naturally occurring variants of the hepatocyte growth factor receptor, such as splice variants or allelic variants. The invention also considers minor sequence variations, particularly conserved amino acid substitutions of the hepatocyte growth factor receptor that do not affect its function and / or activity.

[0093] In this document, "effective amount" refers to the amount required to effectively achieve the desired result at the stated dose and the necessary elapsed time. The desired result may be, for example, symptom relief, prolonged survival, improved activity, etc. In preferred embodiments, the administration includes, but is not limited to, topical application, non-gastrointestinal application, mucosal application, intranasal application, intraocular application, intrathecal application, subdural application, subcutaneous application, and enteral application.

[0094] As used herein, "disease" means any condition from which treatment would be beneficial, including but not limited to chronic and acute illnesses or diseases, including those pathological conditions that predispose mammals to said diseases. In some aspects, said disease is a disease associated with modifications to the function or activity of cell surface molecules. In some preferred aspects, said disease is a chronic autoimmune disorder, an inflammatory condition, a disease associated with abnormal cell proliferation, a disease associated with abnormal cell apoptosis, sepsis, or a viral infection. In the most preferred aspect, said disease is cancer.

[0095] The term "cancer" in this document refers to or describes a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. Cancer includes both solid tumor cancers and non-solid tumor cancers. Solid tumors include, but are not limited to, lung cancer, colorectal cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), glioma (e.g., neuroblastoma), neuroblastoma, melanoma, breast cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, cervical cancer, esophageal cancer, sarcoma, esophageal cancer (e.g., squamous cell carcinoma of the esophagus), thyroid cancer (e.g., papillary thyroid carcinoma), or prostate cancer, or their metastatic forms. In some respects, cancer is colorectal cancer (CRC). In some respects, cancer is lung cancer. Further aspects of lung cancer include epidermal growth factor receptor-positive (EGFR) cancer. + Lung cancer. Other aspects of lung cancer include non-small cell lung cancer (e.g., squamous or non-squamous lung cancer) and small cell lung cancer. In some respects, the cancer is prostate cancer. A further aspect of prostate cancer includes castration-resistant prostate cancer (CRPC). In some respects, the cancer is breast cancer. A further aspect of breast cancer includes HER2-positive (HER2... +Breast cancer. Other aspects of breast cancer include ductal carcinoma of the breast. In some cases, breast cancer is early-stage breast cancer. In some cases, the cancer is a metastatic form of a solid tumor. In some cases, metastatic forms of solid tumors include lung cancer, colorectal cancer, head and neck cancer, glioma, neuroblastoma, melanoma, breast cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, cervical cancer, esophageal cancer, sarcoma, thyroid cancer, and prostate cancer. In some cases, the cancer is a non-solid tumor cancer. Non-solid tumor cancers include, but are not limited to, B-cell lymphoma. Further aspects of B-cell lymphoma include, for example, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), acute monocytic leukemia, multiple myeloma, acute myeloid leukemia (AML), mixed lineage leukemia, NUT midline carcinoma, Burkitt lymphoma, or mycosis fungoides (MF).

[0096] The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that can be administered to a patient together with the phase change modulating element of the present invention without impairing the functional activity of the modulating element. In some embodiments, a "pharmaceutically acceptable" substance is suitable for contact with cells, tissues, or organs of animals or humans without excessive toxicity, irritation, allergic reactions, immunogenicity, or other adverse reactions, and is used in dosage forms in amounts according to an administration schedule and in proportion to a reasonable benefit / risk ratio. In some embodiments, a "pharmaceutically acceptable" substance as a component of a pharmaceutical composition is also compatible with other components of the composition. In some embodiments, the term "pharmaceutically acceptable carrier" includes, but is not limited to, pharmaceutically acceptable inactive ingredients, materials, compositions, and carriers, such as liquid fillers, solid fillers, diluents, excipients, carriers, solvents, and encapsulating materials. Carriers also include all pharmaceutically acceptable dispersion media, coatings, buffers, isotonic agents, stabilizers, absorption delay agents, antimicrobial agents, antibacterial agents, antifungal agents, adjuvants, etc. Unless any conventional carrier is incompatible with the phase change modulating element, this disclosure covers the use of conventional carriers in pharmaceutical compositions. See, for example, Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania, 2005); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd ed., Ash et al., Gower Publishing Co. (2007); and Pharmaceutical Preformulation and Formulation, Gibson, ed., CRC Press LLC (Boca Raton, Florida, 2004).

[0097] The preferred embodiments for carrying out the present invention will now be described. It should be noted that the embodiments described below are examples illustrating representative embodiments of the present invention, but the present invention is not limited to these embodiments.

[0098] Two or more of the methods described below can be combined, and such combinations are also included in this invention.

[0099] Example

[0100] Material:

[0101] Fetal bovine serum: purchased from GIBCO, ThermoFisher Scientific

[0102] Pen-Strep: Purchased from GIBCO, ThermoFisher Scientific

[0103] DMEM: Purchased from HyClone and Cytiva

[0104] RMPI-1640: Purchased from GIBCO, ThermoFisher Scientific

[0105] Cell lines:

[0106] All cell lines used in this application were purchased from the China National Biomedical Laboratory Cell Resource Bank (http: / / www.crcpumc.com / ).

[0107] A.HEK293T cell line

[0108] The human kidney epithelial cell line HEK293T was maintained at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum and 100 units / ml Pen-Strep.

[0109] B.SJSA-1 cell line

[0110] Human osteosarcoma cell line SJSA-1 (formerly known as OsA-CL) was maintained at 37°C and 5% CO2 in RMPI-1640 containing 10% fetal bovine serum and 100 Units / ml Pen-Strep.

[0111] C.COLO-205 cell line

[0112] Human colon adenocarcinoma cell line COLO-205 was maintained at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum and 100 Units / ml Pen-Strep.

[0113] D.PC-3 cell line

[0114] Human prostate adenocarcinoma cell line PC-3 was maintained at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum and 100 Units / ml Pen-Strep.

[0115] E.NCI-H226 cell line

[0116] Human lung squamous cell carcinoma line NCI-H226 was maintained at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum and 100 Units / ml Pen-Strep.

[0117] FT-47D cell line

[0118] Human breast cancer ductal cell line T-47D was maintained at 37°C and 5% CO2 in DMEM containing 10% fetal bovine serum, 0.2 Units / ml insulin (purchased from Life Technologies), and 100 Units / ml Pen-Strep.

[0119] G.THP-1 cell line

[0120] The human leukemia monocytic cell line THP-1 was maintained at 37°C and 5% CO2 in RMPI-1640 containing 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol (purchased from Sigma-Aldrich), and 100 Units / ml Pen-Strep.

[0121] H.OCI-AML3 cell line

[0122] Human acute myeloid leukemia cell line OCI-AML3 was maintained at 37°C and 5% CO2 in RMPI-1640 containing 20% ​​fetal bovine serum and 100 Units / ml Pen-Strep.

[0123] Antibody:

[0124] Table 1: Antibodies used in this invention

[0125]

[0126] Example 1: Construction of expression vector

[0127] DNA molecules encoding the amino acid sequences shown in sequence numbers 1–13 and 16–17 in Table 2 were artificially synthesized. The obtained DNA molecules were inserted into the pRSFDuet-1 vector (a Novagen product of Merck), replacing the DNA fragment (containing the restriction enzyme recognition sequence) between the NcoI and XhoI restriction site recognition sequences, to obtain recombinant prokaryotic expression vectors.

[0128] DNA molecules encoding CXCR4-mCherry or DR5-GFP were synthesized artificially (sequence numbers 14-15 in Table 2 below). The obtained DNA molecules were inserted into the pcDNA3.1 vector (Invitrogen), replacing the DNA fragment (containing the restriction enzyme recognition sequence) between the HindIII and XhoI restriction site recognition sequences, to obtain the recombinant eukaryotic expression vectors pcDNA3.1-CXCR4-mCherry and pcDNA3.1-DR5-GFP, respectively. Plasmids were extracted using an endotoxin-free plasmid extraction kit (purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.) according to the manufacturer's instructions and stored at -20℃.

[0129] Table 2: Constructs used in this invention

[0130]

[0131]

[0132] Example 2: Expression and purification of recombinant proteins

[0133] The prokaryotic expression vector prepared in Example 1 was purified using methods generally known in the art, and the purified vector was transformed into Escherichia coli BL21(DE3). See, for example, Maryam B, Hossein KS et al., Expression and Purification of Biologically Active Recombinant Rabbit Monocyte Chemoattractant Protein1 in Escherichia coli. FEMS Microbiology Letters, 2018(9):9.

[0134] Select identified single colonies and transfer them to LB liquid medium. Incubate at 37°C with shaking until dissolved. 600Approximately 0.8. After cooling to 18°C, 0.1 mM isopropyl β-D-thiogalactoside (IPTG, purchased from INALCO, USA) was added to induce expression for 16 hours. The mixture was centrifuged at 4000 x g for 30 minutes, and the precipitate was collected. It was resuspended in a basal buffer containing 40 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 10 mM imidazole, and then sonicated. The mixture was then centrifuged at 20,000 × g at 4°C for 1 hour, and the supernatant was collected. The supernatant was sequentially processed using a Ni-NTA column (purchased from Nanjing Genscript Biotech Co., Ltd.) and an MBP Taprap HP column (purchased from Cytiva Biotech Co., Ltd.). Then, the protease was added at a rate of 2 μL TEV protease (1 U / μL) per 8 μg of recombinant protein, and the mixture was digested overnight at 4°C to remove the MBP tag. Finally, the recombinant protein was purified using a Superdex 200 Increase 10 / 300GL column (purchased from Cytiva Biotechnology Co., Ltd.). The resulting target recombinant protein had only one additional Gly / Ser residue attached to its N-terminus, minimizing the impact on the structure and function of the recombinant protein.

[0135] The obtained recombinant protein was stored in a buffer solution containing 40 mM HEPES, pH 7.5, 500 mM NaCl, and 5% glycerol, and refrigerated at -80°C until use.

[0136] Using methods known in the art, endotoxins were further removed from the purified protein solution, see, for example, M. Teodorowicz et al., Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. PloS one 12, e0173778 (2017); S. Liu et al., Removal of endotoxin from recombinant protein preparations. Clinical biochemistry 30, 455-463 (1997).

[0137] Additionally, in this paper, the concentration of the protein solution can be adjusted to no more than 1 mM as needed, as described below. Triton X-114 (purchased from Sigma-Aldrich) was added to the protein solution to a final concentration of 1% v / v. The solution containing Triton X was incubated at 4°C with continuous stirring for 60 min. Subsequently, the solution was transferred to a 30°C water bath and incubated for 30 min. Then, at room temperature, the solution was centrifuged at 20,000 × g for 20 min to separate the protein-containing supernatant layer from the Triton X-114 layer. The supernatant layer was transferred by pipetting to endotoxin-free tubes (purchased from Shanghai Sangon Biotech Co., Ltd.).

[0138] The obtained recombinant protein was analyzed by SDS-PAGE as described below:

[0139] Electrophoresis buffer powder (Tris-MOPS-SDS Running Buffer Powder, purchased from Nanjing GenScript Biotech Co., Ltd.) was dissolved in 1L of deionized water to prepare 1× electrophoresis buffer. SurePAGE precast gel (purchased from Nanjing GenScript Biotech Co., Ltd.) was prepared according to the manufacturer's instructions using Bio-Rad... Tetra System 5 (purchased from Bio-Rad) was used for gel electrophoresis in 1×MOPS electrophoresis buffer. After electrophoresis, the gel was cleaned according to the manufacturer's instructions. A rapid protein staining system (purchased from Nanjing Genscript Biotech Co., Ltd.) was used to stain the gel with Coomassie Brilliant Blue. The staining results are shown below. Figure 1 middle.

[0140] The results show that CN-P2S2-DN and its various truncated variants ( Figure 1 A), and synthetic ligands targeting different tumor-associated antigens ( Figure 1 B) All showed uniform electrophoretic bands and the correct molecular weight.

[0141] Example 3: In vitro liquid-liquid phase separation experiments of synthetic ligands and their truncated variants

[0142] The purified recombinant protein CN-P2S2-DN (SEQ ID NO:24) prepared in Example 2 was used to perform in vitro liquid-liquid phase separation (LLPS) as described below.

[0143] (1) Fluorescently labeled recombinant protein

[0144] According to the manufacturer's instructions, the purified recombinant protein CN-P2S2-DN or its various truncated variants are mixed with the fluorescent dye Alexa Fluor. TM 488C5 maleimide, or Alexa FluorTM 488C5 maleimide, or Alexa Fluor TM 546C5 maleimide (both purchased from Thermo Fisher Scientific) were mixed in a 1:1 molar ratio, rotated to mix at room temperature, and incubated for 1 hour. Then, the mixture was analyzed using Zeba... TM Spin Desalting Columns (purchased from Thermo Scientific TM (Catalog number 89882) was centrifuged to remove free dye, yielding fluorescently labeled recombinant protein. The obtained recombinant protein was stored at -80°C until use.

[0145] (2) In vitro liquid-liquid phase separation

[0146] At room temperature, the Alexa 488-labeled CN-P2S2-DN protein solution prepared as described above was seeded onto a 0.17 mm 384 low-binding multi-well microplate (In Vitro Scientific) and recorded, then sealed with optical clear film (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The recombinant protein was diluted with reaction buffer (20 mM HEPS, pH 7.4, 150 mM NaCl, and 10% congestant PEG8000) to final concentrations of 50 μM, 25 μM, 12.5 μM, 6.35 μM, and 3.125 μM, respectively, in a total volume of 10 μl, for observation of phase separation.

[0147] Example 4: Fluorescence confocal imaging

[0148] The microplate prepared in Example 3 for inducing in vitro phase separation was placed under a NIKON A1R HD25 laser confocal microscope (purchased from Nikon, Inc.). Using the microscope's built-in 100× oil lens, fluorescence confocal imaging was performed on the above-mentioned CN-P2S2-DN protein droplets according to fluorescence confocal imaging methods known to those skilled in the art, to observe the induced in vitro liquid-liquid phase separation.

[0149] Specifically, for droplets containing 12.5 μM CN-P2S2-DN protein, fluorescence images were captured at specified time points (0, 1, 2, 3, and 25 min) with the observation time start set at 0 min, to observe the droplets and their fusion. The results are shown in... Figure 2 In A and 2B.

[0150] The results show that, in in vitro liquid-liquid phase separation experiments, the CN-P2S2-DN recombinant protein can form multiple spherical droplets, which adhere to the surface of the coverslip by gravity (see [link to study]). Figure 2A). During time-lapse observation, the droplet-like condensates formed by CN-P2S2-DN can fuse together into larger condensates through cross-linking (see [link]). Figure 2 B).

[0151] Example 5: Fluorescent Recovery After Bleaching (FRAP) Experiment

[0152] Fluorescent recovery after bleaching (FRAP) is a common assay used to verify liquid-liquid phase separation; see, for example, Alberti, Simon et al., Journal of Molecular Biology 430(23):4806-4820, 2018; McSwiggen, David T et al., Genes & Development 33(23-24):1619-1634, 2019. Therefore, FRAP was performed on droplets containing 12.5 μM CN-P2S2-DN protein, identical to that in Example 4. After bleaching with a 488 nm laser at 50% intensity for 1 second, fluorescence images were captured using a NIKON A1R HD25 laser confocal microscope (Nikon, Inc.) with the microscope's 100x oil immersion objective. The captured images were analyzed and exported using NIS-Elements ARAnalysis (Nikon, Inc.) and ImageJ (National Institutes of Health) software.

[0153] Throughout the observation period, the time when the photobleaching pulse was applied to the droplet was designated as time 0, and fluorescence intensity was recorded at each indicated time point. The average fluorescence intensity of the photobleached region was normalized based on the average fluorescence intensity of the unbleached region, and a fluorescence intensity curve was plotted using Graphpad Prism8 (GraphPad, Inc.). The results are shown below. Figure 3 The data is the average of three independent measurements.

[0154] The results showed that the average fluorescence intensity in the photobleached region gradually recovered over time after the laser pulse ended. This indicates that CN-P2S2-DN protein molecules can still diffuse freely in the condensed phase, allowing fluorescence to be restored in the bleached region.

[0155] Based on the results of droplet fusion experiments and photobleaching recovery experiments, the droplets induced by the CN-P2S2-DN recombinant protein of the present invention prepared in Example 3 have liquid-like properties and are liquid-liquid phase separated.

[0156] Example 6: Intracellular liquid-liquid phase separation experiment

[0157] (1) Phase separation based on exogenous receptor expression

[0158] HEK293T cells were used at a rate of 5 × 10⁻⁶ 5 Cells were seeded at a density of approximately 60-70% confluence in 4-well 35mm culture dishes (In Vitro Scientific) and cultured at 37°C and 5% CO2. Then, 0.5 μg each of plasmids pCDNA3.1-CXCR4-mCherry and pCDNA3.1-DR5-GFP were added to each well in a sterile centrifuge tube. The plasmids were gently mixed with antibiotic- and serum-free Opti-MEM Medium (Invitrogen, Inc.) using a pipette, and then Lipo8000 was added. TM The transfection reagent (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was gently mixed again to obtain the plasmid solution for transfection (i.e., each 100 μl of Opti-MEM Medium contained 0.5 μg each of plasmids pCDNA3.1-CXCR4-mCherry and pCDNA3.1-DR5-GFP, and 1.6 μl of Lipo8000). The mixed plasmid solution was evenly added dropwise to each well, gently mixed, and then cultured at 37℃ and 5% CO2 for 24–36 hours to obtain HEK293T cells co-expressing CXCR4-mCherry and DR5-GFP.

[0159] 200 nM CN-P2S2-DN protein or its different truncated variants P2S2-CN, P2S2, and P2S2-DN (all labeled with the fluorescent dye Alexa 647) were added to the cell culture medium described above. HEK293T cells treated in the same way with blank solution under the same conditions were used as a negative control.

[0160] According to the manufacturer's instructions, HEK293T cells co-expressing CXCR4-mCherry and DR5-GFP were stained with Hoechst 33258 dye (purchased from Invitrogen, Inc.). Then, a microdisc was placed in the live-cell culture apparatus of a NIKON A1R HD25 laser confocal microscope. After adding the Alexa 647-labeled recombinant protein, time-series imaging was immediately initiated, obtaining fluorescence images corresponding to CXCR4-mCherry, DR5-GFP, the Alexa 647-labeled recombinant protein, and DAPI, as well as co-localization images obtained by superimposing these fluorescence images. The results are shown below. Figures 4-8 middle.

[0161] Then, for HEK293T cells treated with 200 nM CN-P2S2-DN (Alexa 647), the recovery process of fluorescence corresponding to CN-P2S2-DN (Alexa 647), CXCR4-mCherry, and DR5-GFP was measured using the same method as in Example 5. The results are shown in... Figures 9-10 .

[0162] Figures 4-8 The results showed that in HEK293T cells co-expressing CXCR4-mCherry and DR5-GFP, the addition of CN-P2S2-DN (Alexa 647) resulted in the enrichment and localization of GFP and mCherry signals within aggregates formed by CN-P2S2-DN protein. Conversely, no such colocalization and enrichment were observed in the addition of either the control solution or the truncated variant.

[0163] Figure 9 and Figure 10 The spatiotemporal analysis results of CN-P2S2-DN protein, CXCR4-mCherry, and DR5-GFP in photobleaching experiments are shown. For CN-P2S2-DN and CXCR4-mCherry, both can rapidly redistribute from unbleached areas to bleached areas. For DR5-GFP, once recruited into agglomerates containing the above three components, it exhibits relatively solid-like characteristics.

[0164] The above results indicate that the CN-P2S2-DN protein can not only bind to the corresponding CXCR4 and DR5 receptors simultaneously through its contained ligands CN and DN, but also induce phase separation through its contained phase separation motif P2S2, thereby driving the aggregation of unbound receptors in the surrounding region in a manner conducive to the oligomerization of these receptors.

[0165] (2) Phase separation based on endogenous receptors

[0166] Known CXCR4 + / DR5 + Human osteosarcoma cell line SJSA-1 cells were used at 5x10 5Cells were seeded at a density of approximately 60-70% confluence in 4-well 35mm culture dishes (In Vitro Scientific). Cells were cultured at 37°C and 5% CO2 until the cell density reached approximately 60-70% confluence. Then, 200 nM of CN-P2S2-DN protein labeled with Alexa 561 fluorescence was added to the culture medium, and the cells were incubated at 37°C and 5% CO2 for 4-6 hours. A snapshot of the treated SJSA-1 cells was taken using a NIKON A1R HD25 laser confocal microscope (Nikon, Inc.) through the microscope's 100× oil immersion objective lens. The results are shown below. Figure 11 A. The results show that CN-P2S2-DN is also compatible with DR5. + / CXCR4 + The corresponding endogenous receptors in double-positive SJSA-1 cells bind to the puncta, which then accumulate on the cell surface and form micron-sized puncta structures characteristic of liquid-liquid phase separation.

[0167] Microscopic observation of specific spot structures was performed. The instant of cell treatment with CN-P2S2-DN protein added to the culture medium was designated as time 0. Time-series imaging was conducted on the same specific region on the surface of SJSA-1 cells at 0 min, 0.5 min, 1 min, and 1.5 min. The observed results are shown in... Figure 11 B. The results showed that the spots exhibited the same contact fusion phenomenon observed in in vitro phase change droplets.

[0168] Example 7: Colocalization of recombinant protein with endogenous receptor

[0169] Known CXCR4 + / DR5 + SJSA-1 cells at 5 × 10 5 Cells were seeded at a density of 35 mm in 4-well culture dishes (purchased from In Vitro Scientific) and cultured at 37°C until the cell density reached approximately 70-80% confluence.

[0170] 200 nM CN-P2S2-DN recombinant protein and its truncated variant P2S2 (both labeled with Alexa 647) were added to the cell culture medium. Cells were incubated at 37°C and 5% CO2 for 4–6 hours. Cells were then fixed with 4% paraformaldehyde (Beyotime Biotechnology Co., Ltd.), permeabilized with 0.1% Triton X-100 (Beyotime Biotechnology Co., Ltd.), and blocked with 0.5% BSA / PBS (Sangon Biotech Co., Ltd.). Cells were washed 5 times with 1×PBS at room temperature for 5 min each time. Rabbit anti-DR5-specific polyclonal antibody (Proteintech, catalog number 15497-1-AP) and mouse anti-CXCR4 monoclonal antibody (Proteintech, catalog number 60042-1-Ig) were added as primary antibodies, and the cells were incubated overnight at 4°C. Cells were washed 5 times with 1×PBS at room temperature for 5 min each time. Finally, a donkey anti-rabbit IgG (H+L) highly cross-linked secondary antibody (Alexa Fluor) was added. TM The secondary antibody, Plus488 (purchased from Thermo Fisher Scientific, catalog number A32790) and donkey anti-mouse IgG (H+L), is highly cross-adsorbed. Alexa Fluor TM Plus 568 (Thermo Fisher Scientific, catalog number A10037) was used as the secondary antibody, and the cells were incubated at room temperature for 1 hour. The cells were washed five times with 1×PBS for 5 minutes each time. Then, the cells were sealed in 4',6'-diamidinyl-2-phenylindole (DAPI) (Beyotime) and observed and immunofluorescence was captured using a NIKON A1RHD25 laser confocal microscope. Results are shown in... Figure 12 A, 13A. Using the same software and parameters as in Example 5, measurements were taken from the captured fluorescence images. Figure 12 A. Fluorescence intensity at the straight line marked with a white arrow in 13A, the results are shown in... Figure 12 B, 13B.

[0171] The results show that, compared with the truncated variant, only the recombinant protein CN-P2S2-DN can colocalize with the cell's endogenous receptor, and this colocalization pattern is almost identical to the pattern observed in the extracellular experiments in Example 3.

[0172] Example 8: Photobleaching recovery of recombinant proteins in tumor cells

[0173] SJSA-1 cells were treated with 200 nM CN-P2S2-DN (Alexa 561 label) for 4 hours using the same method as in Example 5, followed by photobleaching analysis. Results are shown in... Figure 14 .

[0174] The FRAP assay results showed that CN-P2S2-DN specifically binds to endogenous CXCR4 and DR5 receptors on tumor cells, inducing the formation of phase-separation-driven co-aggregating spots with liquid-like dynamic properties. This means that after the CN-P2S2-DN protein binds to endogenous receptors on the cell membrane surface via its two ligands CN and DN, it can further induce phase separation based on the phase-separation motif P2S2, thereby driving the aggregation of unbound endogenous receptors in the surrounding cell membrane region in a manner conducive to the oligomerization of these receptors.

[0175] Example 9: Modification of Recombinant Proteins for Receptor-Receptor Communication

[0176] The manipulation activity of various recombinant proteins prepared in Example 2 on downstream signaling proteins of the receptor was observed by cell morphology and analyzed by Western blot.

[0177] (1) Changes in cell morphology

[0178] Known CXCR4 + / DR5 + SJSA-1 cells at 5 × 10 5 Cells were seeded at a density of approximately 70–80% confluence in 4-well 35 mm culture dishes (purchased from In Vitro Scientific) and cultured at 37°C until the cell density reached approximately 70–80%. 100 nM CN-P2S2-DN, its truncated variants (P2S2-CN, P2S2-DN, P2S2, CN, DN, CN-DN), or combinations of variants (combinations of P2S2-CN and P2S2-DN, combinations of CN and DN) were added to the cell culture medium, and the cells were incubated at 37°C with 5% CO2 for 3 hours. Cells were treated with the appropriate blank buffer as a solution control. Live cells were photographed using an Olympus IX83 fluorescence inverted microscope; the results are shown below. Figure 15 The results showed that, compared with various truncated variants, variant combinations and solution controls, a series of significant morphological changes, such as shrinkage and rapid phagocytosis by neighboring cells, were observed only in SJSA-1 cells incubated with CN-P2S2-DN for 3 hours.

[0179] (2) Changes in signal transmission pathways

[0180] Activation mediated by cleavage of full-length cysteine ​​protease 3 is a key event in apoptosis (see, for example, Nicholson, Donald W. et al., "Identification and inhibition of the ICE / CED-3 protease necessary for mammalian apoptosis." Nature 376.6535(1995):37-43; Hsia, Jiun-Yi et al., "Prognostic significance of caspase-3 expression in primary-resected esophageal squamous cell carcinoma." European Journal of Surgical Oncology (EJSO) 29.1(2003):44-48). Using Western blotting, a method well-known in the art, changes in pro-caspase-3 expression levels in SJSA-1 cells after the addition of the recombinant protein of this invention were analyzed. See, for example, Zhang, T. et al., "Discovery of a novel third-generation EGFRinhibitor and identification of a potential combination strategy to overcomeresistance." Molecular Cancer 19.1 (2020). .

[0181] Specifically, endotoxin-free CN-P2S2-DN recombinant protein, its truncated variants (P2S2-CN, P2S2-DN, P2S2, CN, DN, CN-DN), or combinations of variants (combinations of P2S2-CN and P2S2-DN, combinations of CN and DN) were added to SJSA-1 cells (1×10⁻⁶ cells per cell line) at a final concentration of 100 nM. 6 Incubate in a culture medium containing (number of cells / well) at 37°C for 3 hours. Then, follow the manufacturer's instructions using a minute filter. TMSJSA-1 cell lysates were prepared using the Total Protein Extraction Kit for Animal Cultured Cells / Tissues (purchased from Invent Biotech). The lysates were resuspended in 2×Laemmli loading buffer (purchased from Sigma-Aldrich, Inc.). Protein samples were transferred to a PVDF membrane via SDS-PAGE using the same method as in Example 2. After transfer, the PVDF membrane was washed several times with deionized water, and then blocking buffer (TBST containing 5% skim milk) was added, and the membrane was blocked at room temperature for 2 hours. The blocking buffer was discarded, and rabbit anti-Caspase-3 polyclonal antibody (purchased from CST, catalog number 9662) diluted with the blocking buffer was added as the primary antibody, and the membrane was incubated overnight at 4°C. Then, the primary antibody solution was discarded, and the membrane was washed five times with TBST for 6 minutes each time. F(ab')2-goat anti-rabbit IgG (H+L), HRP was used as the secondary antibody, and the membrane was incubated at room temperature for 1 hour. Finally, the protein bands were visualized using enhanced chemiluminescence reagents. The results are shown below. Figure 16 .

[0182] The results showed that, compared with any of the cases involving the addition of various truncated variants, variant combinations, and blank controls, only the addition of CN-P2S2-DN recombinant protein significantly reduced the level of full-length cysteine ​​protease 3 in SJSA-1 cells.

[0183] In addition, endotoxin-free CN-P2S2-DN recombinant protein was added to the culture medium of SJSA-1 cells and HEK293T cells at final concentrations of 0, 50, 100, 200, and 500 nM, respectively. Rabbit anti-Caspase-3 polyclonal antibody (purchased from CST, catalog number 9662) was used as the primary antibody, and the expression level of procysteine ​​protease 3 in the cells was determined by Western blotting. The results are shown in... Figure 17 The results show that, in the known CXCR4... + / DR5 + In SJSA-1 cells, the level of procysteine ​​protease 3 was significantly reduced in a concentration-dependent manner according to the CN-P2S2-DN recombinant protein, indicating that procysteine ​​protease 3 was significantly activated intracellularly. In contrast, procysteine ​​protease 3 activation was not detected in the non-tumor cell line HEK293T.

[0184] Based on the above results, it can be confirmed that the CN-P2S2-DN of the present invention strongly forms aggregates based on ligand-receptor binding and can regulate communication between endogenous receptors as a multivalent binary system.

[0185] Example 10: Induction of synthetic apoptosis by recombinant protein CN-P2S2-DN

[0186] To confirm that the phase transition regulating element of the present invention promotes phase separation and thereby manipulates tumor cell apoptosis by binding to the DR5 receptor and tumor-associated antigen on the cell surface, this embodiment mainly uses flow cytometry to analyze the apoptosis-inducing activity of the recombinant protein CN-P2S2-DN prepared in Example 2 and its various truncated variants (P2S2-CN, P2S2-DN, P2S2, CN, DN, CN-DN, P2-CN, S2-DN) and combinations of truncated variants (combination of P2S2-CN and P2S2-DN, combination of CN and DN, combination of P2-CN and S2-DN) on the DR5-expressing tumor cell line SJSA-1. As a negative control, a blank solution and non-tumor HEK293T cells were used.

[0187] (1) Apoptosis-inducing activity against SJSA-1 cells

[0188] With 1×10 5 SJSA-1 cells were seeded per well in 96-well plates (Falcon). Endotoxin-free CN-P2S2-DN protein was added to the cell culture medium at a final concentration of 100 nM, and the cells were incubated at 37°C for 4–6 hours. Then, following the manufacturer's instructions, CellTracker was used… TM Green CMFDA Dye (purchased from Invitrogen, Inc.) was used to label SJSA-1 cells, and... The Zoom System (purchased from Essen, USA) monitors cell proliferation through... The analysis software records and analyzes the cell growth status. The results are shown below. Figure 18 The results showed that, at a dose of 100 nM, cell death was observed as early as 3 hours after the addition of the recombinant protein and reached a plateau phase 8 hours after addition.

[0189] SJSA-1 cells were loaded at 1 x 10⁻⁶. 6 Cells were seeded in 24-well plates (Falcon) and recombinant protein CN-P2S2-DN and its truncated variants and combinations thereof were added at a final concentration of 100 nM. The plates were incubated at 37°C and 5% CO2 for 3 hours. After washing the cells with PBS buffer, Annexin V-FITC / PI double staining was performed using the Annexin V-FITC apoptosis detection kit (Beyotime) according to the manufacturer's instructions. Fluorescence intensity was measured using an LSRFortessa SORP flow cytometer (BD) to determine the apoptotic proportion of SJSA-1 cells. Results are shown in […]. Figure 19 middle.

[0190] Flow cytometry results showed that in SJSA-1 cells, the proportion of Annexin V-positive apoptotic cells induced by CN-P2S2-DN was significantly higher than that of the blank solution used as a negative control, and also exhibited significantly higher apoptosis-inducing activity than the truncated variant CN-DN obtained by linking with known adapter techniques, the combination of truncated variants CN+DN without linkers, and all other truncated variants and combinations thereof. Notably, no significant apoptosis was observed in SJSA-1 cells containing the combination of the truncated variants P2-CN and S2-DN. This suggests that the regulatory element of the present invention, obtained by linking motifs required to induce phase separation in a tandem manner, can significantly improve the phase separation effect compared to the kit form where motif separation exists, thereby enhancing the element's function in regulating cell surface molecular activity.

[0191] (2) Comparison of apoptosis-inducing activities between SJSA-1 cells and HEK293T cells

[0192] The apoptosis-inducing activity of 100 nM recombinant protein CN-P2S2-DN against tumor cells SJSA-1 and non-tumor cells HEK293T was determined under the same conditions using the same method as in (1) of this embodiment. Simultaneously, the expression level of DR5 in SJSA-1 cells and HEK293T cells was determined using Q-PCR, a method commonly known in the art. See, for example, Van Guilder, Heather D et al., Biotechniques 44(5):619-626, 2008. The results of apoptosis induction and Q-PCR are shown in [references to be inserted here]. Figure 20 and 21 middle.

[0193] Figure 20 The results showed that CN-P2S2-DN induced apoptosis in 78.8% of SJSA-1 tumor cells, significantly higher than that in HEK293T cells (4.9%) and the blank control (3.6%). On the other hand, there was no statistically significant difference between HEK293T cells and the blank control group.

[0194] CN-P2S2-DN exhibited significantly different apoptosis-inducing effects in the SJSA-1 and HEK293T cell lines, which is speculated to be due to the significant differences in the expression levels of cell surface receptor molecules between the two cell lines. Compared with the known CXCR4... + / DR5 + The double-positive tumor cell line SJSA-1 differs from the non-tumor cell line HEK293T in that it lacks DR5 expression (see [link]). Figure 21This suggests that interacting with cell surface molecules (such as specific ligands) by recognizing and binding to them is crucial for the recombinant fusion protein of the present invention to manipulate cell surface molecule-related cellular events.

[0195] (3) Activity concentration curve of recombinant protein CN-P2S2-DN

[0196] Similarly, flow cytometry was used to evaluate the apoptosis-inducing activity of different concentrations of CN-P2S2-DN recombinant protein. A series of endotoxin-free CN-P2S2-DN recombinant protein solutions of different concentrations were prepared and added to SJSA-1 cells (1×10⁻⁶ cells per cell line). 6 Cells / well were incubated at 37°C for 4-6 hours. The apoptosis rate of SJSA-1 cells under different concentration conditions was determined as described above, and dose-response curves were plotted. Results are shown in... Figure 22 The results show that CN-P2S2-DN in CXCR4 + / DR5 + SJSA-1 cells exhibit strong apoptosis-inducing activity, IC50 50 It is 10.14 nM.

[0197] (4) Apoptosis-inducing activity against other tumor cells

[0198] Other known double-positive tumor cell lines expressing both cxcr4 and dr5 genes were selected, and the apoptosis-inducing activity of the CN-P2S2-DN recombinant protein in these cell lines was determined.

[0199] The apoptosis-inducing activity of 100 or 200 nM CN-P2S2-DN in the above cell lines was determined by flow cytometry and compared with the results of the same concentration of truncated variants (P2S2-DN, P2S2) or a blank solution as a negative control. Results are shown in... Figure 23 .

[0200] As a result, no difference in apoptosis rate was observed between the non-tumor cell line HEK293T and the control group. On the other hand, CXCR4... + / DR5 + Double-positive tumor cells (including colorectal, sarcoma, lung, and prostate cell lines) all showed a significant response to CN-P2S2-DN, with an apoptosis ratio Ratio min >40%. Among them, the maximum response was observed in the human colon adenocarcinoma cell line COLO-205, with an apoptosis rate of approximately 80%. Therefore, it is confirmed that the phase change modulation element of the present invention does not depend on a specific cell type for the manipulation of cell surface molecules and their related functions, but is effective in a variety of cancer cell lines.

[0201] Example 11: Activity of recombinant protein CN-S2S2-DN

[0202] Using another recombinant protein, CN-S2S2-DN (as shown in SEQ ID NO:25), the same method as in Examples 3-5 above was repeated to further confirm the phase transition regulation fusion protein's manipulative activity against apoptosis through phase separation. The results confirmed that although different multivalent phase transition domains were used, the recombinant protein CN-S2S2-DN was also able to form multiple spherical droplets characteristic of liquid-liquid phase separation in vitro and exhibited typical photobleaching recovery in FRAP experiments. This indicates that the recombinant protein can also effectively induce phase separation.

[0203] Using the same method as in Example 10, flow cytometry was further employed to analyze and compare the apoptosis-inducing activity of the recombinant protein CN-S2S2-DN against SJSA-1 cells and HEK293T cells. The results are shown in... Figure 24 The results showed that the apoptosis-inducing activity of CN-S2S2-DN differed significantly between SJSA-1 cells (21.6%), which are tumor cells, and HEK293T cells (2.3%), which are non-tumor cells. This indicates that altering the multivalent phase transition domain did not substantially affect the activity of the phase transition regulating element of the present invention in manipulating cell surface molecules (such as inducing tumor cell apoptosis).

[0204] Example 12: Induction of synthetic apoptosis by other recombinant proteins

[0205] The study reported that various tumor-associated antigens (TAAs) were expressed significantly higher in cancer cells than in normal tissues, including but not limited to hepatocyte growth factor receptor (also referred to as c-Met, HGFR in this paper) and epidermal growth factor receptor (EGFR).

[0206] Following the method for constructing synthetic ligands as described in Example 1, the apoptosis-inducing activity of multivalent binary recombinant ligands targeting different tumor-associated antigens and cell surface receptors was evaluated in different tumor cell lines. The recombinant ligands used and their truncated variants were prepared using the methods described in Examples 1 and 2.

[0207] (1) Recombinant protein EN-P2S2-DN

[0208] The recombinant protein EN-P2S2-DN, prepared and purified using the same method as in Example 2, is a synthetic ligand obtained by covalently linking an anti-EGFR nanobody (also referred to herein as EN, SEQ ID NO:4) and an anti-DR5 nanobody to both ends of P2S2, respectively.

[0209] Lung squamous cell carcinoma line NCI-H226 and colon adenocarcinoma line COLO-205, along with the renal epithelial cell line HEK293T as a negative control, were used at 1 x 10⁻⁶. 6 Cells were seeded in 24-well plates (Falcon) and endotoxin-free EN-P2S2-DN recombinant protein was added to each well. Figure 25 The concentrations shown were reacted at 37°C and 5% CO2 for 3 or 5 hours. Then, flow cytometry analysis based on Annexin V-FITC / PI double staining (Beyotime) was performed using the same method as in Example 10 to determine the apoptosis-inducing activity of EN-P2S2-DN in different cell lines. The results are shown in... Figure 25 .

[0210] The results showed that EGFR + / DR5 + Both positive tumor cells showed a significant response to EN-P2S2-DN, with the strongest response observed in the human colon adenocarcinoma cell line COLO-205, where the apoptosis rate exceeded 80%. In contrast, only 1.2% of non-tumor cells (HEK293T cells) exhibited apoptosis, a significantly low proportion. Therefore, it is confirmed that EN-P2S2-DN can induce tumor cell apoptosis by manipulating cell surface receptor-mediated signaling pathways.

[0211] (2) Recombinant protein MN-P2S2-DN

[0212] The recombinant protein MN-P2S2-DN, prepared and purified using the same method as in Example 2, is a synthetic ligand obtained by covalently linking an anti-c-Met nanobody (also referred to herein as MN, SEQ ID NO:5) and an anti-DR5 nanobody to both ends of P2S2, respectively.

[0213] Using the same method described above, flow cytometry was employed to determine the apoptosis-inducing activity of MN-P2S2-DN in three different tumor cell lines (osteosarcoma cell line SJSA-1, prostate adenocarcinoma cell line PC-3, and acute myeloid leukemia cell line OCI-AML3) and the HEK293T cell line as a negative control. Results were shown in... Figure 26 .

[0214] The results show that c-Met + / DR5 +Double-positive tumor cells all showed a significant response to MN-P2S2-DN, with an apoptosis rate of approximately 30% to 50%. In contrast, in the non-tumor cell line HEK293T cells, this apoptosis rate was only 1.3%, significantly lower than all tumor cell lines. Therefore, it was confirmed that MN-P2S2-DN can also induce tumor cell apoptosis by manipulating cell surface receptor-mediated signaling pathways.

[0215] The two different recombinant fusion proteins described above further confirm that the phase transition modulation element of the present invention manipulates the activity of cell surface molecules (such as inducing tumor cell apoptosis) without depending on a specific ligand. Those skilled in the art will anticipate that phase transition modulation elements obtained using ligand combinations of other cell surface molecules can also achieve similar manipulation effects on cellular events.

[0216] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <120> A phase change regulating element and its application <130> Pending <140> Pending <141> 2022-04-09 <160> 25 <170> SIPOSequenceListing 1.0 <210> 1 <211> 607 <212> PRT <213> Artificial sequence <220> <223> The PRM and SH3 domains are cascaded repeats, with MBP and His6 tags fused to the N-terminus and C-terminus, respectively. <400> 1 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Ser Ala Ser 370 375 380 Met Lys Gly Gly Ser Trp Gly Gly Ser Lys Lys Thr Pro Pro Pro Val 385 390 395 400 Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser 405 410 415 Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser 420 425 430 Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Leu Asn 435 440 445 Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu 450 455 460 Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser 465 470 475 480 Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro 485 490 495 Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Ala Ser Gly Ala 500 505 510 Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Ser Gly Ala Thr 515 520 525 Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg 530 535 540 Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu 545 550 555 560 Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly 565 570 575 Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Gly 580 585 590 Ser Met Gly Glu Phe Pro Ala Gly Asn His His His His His 595 600 605 <210> 2 <211> 520 <212> PRT <213> Artificial sequence <220> <223> The anti-CXCR4 nanobody has an MBP tag fused to its N-terminus and a His6 tag to its C-terminus. <400> 2 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp 405 410 415 Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val Ala Ala Ile Thr 420 425 430 Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp Ser Val 435 440 445 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 450 455 460 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys 465 470 475 480 Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp 485 490 495 Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Phe Pro Ala 500 505 510 Gly Asn His His His His His 515 520 <210> 3 <211> 516 <212> PRT <213> Artificial sequence <220> <223> The anti-DR5 nanobody has an MBP tag fused to its N-terminus and a His6 tag fused to its C-terminus. <400> 3 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Met Gly Glu Val 370 375 380 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 385 390 395 400 Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys Ile Asn Asn Met 405 410 415 Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Gln 420 425 430 Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg 435 440 445 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 450 455 460 Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Glu 465 470 475 480 Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn Tyr Trp Gly Gln 485 490 495 Gly Thr Leu Val Thr Val Ser Ser Glu Phe Pro Ala Gly Asn His His 500 505 510 His His His His 515 <210> 4 <211> 519 <212> PRT <213> Artificial sequence <220> <223> The anti-EGFR nanobody has an MBP tag fused to its N-terminus and a His6 tag fused to its C-terminus. <400> 4 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly Trp 405 410 415 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Val Ala Ile Asn 420 425 430 Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 435 440 445 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met Asn 450 455 460 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Tyr 465 470 475 480 Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr Glu Tyr Asp Tyr 485 490 495 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Phe Pro Ala Gly 500 505 510 Asn His His His His His His 515 <210> 5 <211> 508 <212> PRT <213> Artificial sequence <220> <223> The anti-C-Met nanobody has an MBP tag fused to its N-terminus and a His6 tag fused to its C-terminus. <400> 5 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 370 375 380 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu 385 390 395 400 Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 405 410 415 Glu Gly Val Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala 420 425 430 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 435 440 445 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val 450 455 460 Tyr Tyr Cys Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu 465 470 475 480 Tyr Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 485 490 495 Glu Phe Pro Ala Gly Asn His His His His His His 500 505 <210> 6 <211> 663 <212> PRT <213> Artificial Sequence <400> 6 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp 405 410 415 Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val Ala Ala Ile Thr 420 425 430 Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp Ser Val 435 440 445 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 450 455 460 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys 465 470 475 480 Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp 485 490 495 Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Gly 500 505 510 Gly Ser Gly Gly Ser Gly Gly Ser Ala Ser Ser Arg Ser Met Gly Met 515 520 525 Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 530 535 540 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys Ile 545 550 555 560 Asn Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu 565 570 575 Val Ala Gln Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser Val 580 585 590 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 595 600 605 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 610 615 620 Asn Ala Glu Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn Tyr 625 630 635 640 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Phe Pro Ala Gly 645 650 655 Asn His His His His His His 660 <210> 7 <211> 869 <212> PRT <213> Artificial Sequence <400> 7 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp 405 410 415 Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val Ala Ala Ile Thr 420 425 430 Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp Ser Val 435 440 445 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 450 455 460 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys 465 470 475 480 Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp 485 490 495 Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Gly 500 505 510 Gly Ser Gly Gly Ser Gly Gly Ser Ala Ser Met Lys Gly Gly Ser Trp 515 520 525 Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser 530 535 540 Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Lys Thr 545 550 555 560 Pro Pro Pro Val Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly 565 570 575 Ser Gly Gly Ser Gly Gly Ser Asp Leu Asn Met Pro Ala Tyr Val Lys 580 585 590 Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu Leu Ser Leu Ile Lys Gly 595 600 605 Thr Lys Val Ile Val Met Glu Lys Ser Ser Asp Gly Trp Trp Arg Gly 610 615 620 Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro Ser Asn Tyr Val Thr Glu 625 630 635 640 Glu Gly Asp Ser Pro Leu Ala Ser Gly Ala Gly Gly Ser Glu Gly Gly 645 650 655 Gly Ser Glu Gly Gly Thr Ser Gly Ala Thr Asp Leu Asn Met Pro Ala 660 665 670 Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu Leu Ser Leu 675 680 685 Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser Asp Gly Trp 690 695 700 Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro Ser Asn Tyr 705 710 715 720 Val Thr Glu Glu Gly Asp Ser Pro Leu Gly Ser Met Gly Met Gly Glu 725 730 735 Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 740 745 750 Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys Ile Asn Asn 755 760 765 Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val Ala 770 775 780 Gln Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser Val Lys Gly 785 790 795 800 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 805 810 815 Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala 820 825 830 Glu Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn Tyr Trp Gly 835 840 845 Gln Gly Thr Leu Val Thr Val Ser Ser Glu Phe Pro Ala Gly Asn His 850 855 860 His His His His His 865 <210> 8 <211> 868 <212> PRT <213> Synthetic Sequence <400> 8 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr Ala Met Gly Trp 405 410 415 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Val Ala Ile Asn 420 425 430 Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 435 440 445 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu Gln Met Asn 450 455 460 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Tyr 465 470 475 480 Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr Glu Tyr Asp Tyr 485 490 495 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Gly Gly 500 505 510 Ser Gly Gly Ser Gly Gly Ser Ala Ser Met Lys Gly Gly Ser Trp Gly 515 520 525 Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser Lys 530 535 540 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Lys Thr Pro 545 550 555 560 Pro Pro Val Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser 565 570 575 Gly Gly Ser Gly Gly Ser Asp Leu Asn Met Pro Ala Tyr Val Lys Phe 580 585 590 Asn Tyr Met Ala Glu Arg Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr 595 600 605 Lys Val Ile Val Met Glu Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser 610 615 620 Tyr Asn Gly Gln Val Gly Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu 625 630 635 640 Gly Asp Ser Pro Leu Ala Ser Gly Ala Gly Gly Ser Glu Gly Gly Gly 645 650 655 Ser Glu Gly Gly Thr Ser Gly Ala Thr Asp Leu Asn Met Pro Ala Tyr 660 665 670 Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu Leu Ser Leu Ile 675 680 685 Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser Asp Gly Trp Trp 690 695 700 Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro Ser Asn Tyr Val 705 710 715 720 Thr Glu Glu Gly Asp Ser Pro Leu Gly Ser Met Gly Met Gly Glu Val 725 730 735 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 740 745 750 Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys Ile Asn Asn Met 755 760 765 Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Gln 770 775 780 Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg 785 790 795 800 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 805 810 815 Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Glu 820 825 830 Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn Tyr Trp Gly Gln 835 840 845 Gly Thr Leu Val Thr Val Ser Ser Glu Phe Pro Ala Gly Asn His His 850 855 860 His His His His 865 <210> 9 <211> 866 <212> PRT <213> Artificial Sequence <400> 9 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr Ala Ile Gly Trp 405 410 415 Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Leu Cys Ile Asp 420 425 430 Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 435 440 445 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn 450 455 460 Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Thr Pro Ile 465 470 475 480 Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr Asp Tyr Trp Gly 485 490 495 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly 500 505 510 Gly Ser Gly Gly Ser Ala Ser Met Lys Gly Gly Ser Trp Gly Gly Ser 515 520 525 Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser Lys Gly Gly 530 535 540 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Lys Thr Pro Pro Pro 545 550 555 560 Val Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly 565 570 575 Ser Gly Gly Ser Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr 580 585 590 Met Ala Glu Arg Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val 595 600 605 Ile Val Met Glu Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn 610 615 620 Gly Gln Val Gly Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp 625 630 635 640 Ser Pro Leu Ala Ser Gly Ala Gly Gly Ser Glu Gly Gly Gly Ser Glu 645 650 655 Gly Gly Thr Ser Gly Ala Thr Asp Leu Asn Met Pro Ala Tyr Val Lys 660 665 670 Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu Leu Ser Leu Ile Lys Gly 675 680 685 Thr Lys Val Ile Val Met Glu Lys Ser Ser Asp Gly Trp Trp Arg Gly 690 695 700 Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro Ser Asn Tyr Val Thr Glu 705 710 715 720 Glu Gly Asp Ser Pro Leu Gly Ser Met Gly Met Gly Glu Val Gln Leu 725 730 735 Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 740 745 750 Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys Ile Asn Asn Met Gly Trp 755 760 765 Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Gln Ile Thr 770 775 780 Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 785 790 795 800 Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 805 810 815 Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Glu Ile Leu 820 825 830 Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn Tyr Trp Gly Gln Gly Thr 835 840 845 Leu Val Thr Val Ser Ser Glu Phe Pro Ala Gly Asn His His His His 850 855 860 His His 865 <210> 10 <211> 735 <212> PRT <213> Artificial Sequence <400> 10 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Ser Ala Ser 370 375 380 Met Lys Gly Gly Ser Trp Gly Gly Ser Lys Lys Thr Pro Pro Pro Val 385 390 395 400 Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser 405 410 415 Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser 420 425 430 Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Leu Asn 435 440 445 Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu 450 455 460 Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser 465 470 475 480 Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro 485 490 495 Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Ala Ser Gly Ala 500 505 510 Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Ser Gly Ala Thr 515 520 525 Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg 530 535 540 Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu 545 550 555 560 Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly 565 570 575 Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Gly 580 585 590 Ser Met Gly Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln 595 600 605 Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe 610 615 620 Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala Pro Lys Gly Glu Arg 625 630 635 640 Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val Arg Ser Gly Val Ser 645 650 655 Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp 660 665 670 Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu 675 680 685 Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly Ser Gly Ala 690 695 700 Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr Gln Val Thr 705 710 715 720 Val Ser Ser Glu Phe Pro Ala Gly Asn His His His His His His 725 730 735 <210> 11 <211> 731 <212> PRT <213> Artificial Sequence <400> 11 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Gly Ser Ala Ser 370 375 380 Met Lys Gly Gly Ser Trp Gly Gly Ser Lys Lys Thr Pro Pro Pro Val 385 390 395 400 Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser 405 410 415 Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser 420 425 430 Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Leu Asn 435 440 445 Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu 450 455 460 Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser 465 470 475 480 Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro 485 490 495 Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Ala Ser Gly Ala 500 505 510 Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Ser Gly Ala Thr 515 520 525 Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg 530 535 540 Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu 545 550 555 560 Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly 565 570 575 Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Gly 580 585 590 Ser Met Gly Met Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 595 600 605 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr 610 615 620 Phe Asp Lys Ile Asn Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 625 630 635 640 Gln Arg Asp Leu Val Ala Gln Ile Thr Pro Gly Gly Ile Thr Asp Tyr 645 650 655 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 660 665 670 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala 675 680 685 Val Tyr Tyr Cys Asn Ala Glu Ile Leu Lys Arg Ala Tyr Ile Asp Val 690 695 700 Tyr Val Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu 705 710 715 720 Phe Pro Ala Gly Asn His His His His His His 725 730 <210> 12 <211> 680 <212> PRT <213> Artificial Sequence <400> 12 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Ala Ser Met Ser 370 375 380 Glu Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asp His Ile Asn 385 390 395 400 Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe Lys Ile Lys 405 410 415 Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys Glu Arg Gln 420 425 430 Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly Gln Pro Ile 435 440 445 Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp Glu Asp Thr 450 455 460 Ile Asp Val Phe Gln Gln Gln Thr Val Val Gly Gly Ser Gly Gly Ser 465 470 475 480 Gly Gly Ser Gly Gly Ser Met Ser Glu Glu Lys Pro Lys Glu Gly Val 485 490 495 Lys Thr Glu Asn Asp His Ile Asn Leu Lys Val Ala Gly Gln Asp Gly 500 505 510 Ser Val Val Gln Phe Lys Ile Lys Arg His Thr Pro Leu Ser Lys Leu 515 520 525 Met Lys Ala Tyr Cys Glu Arg Gln Gly Leu Ser Met Arg Gln Ile Arg 530 535 540 Phe Arg Phe Asp Gly Gln Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln 545 550 555 560 Leu Glu Met Glu Asp Glu Asp Thr Ile Asp Val Phe Gln Gln Gln Thr 565 570 575 Val Val Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Val 580 585 590 Asp Val Ile Asp Leu Thr Ile Glu Ser Ser Ser Asp Glu Glu Glu Asp 595 600 605 Pro Pro Ala Lys Arg Gly Gly Ser Gly Gly Ser Gly Gly Arg Gly Gly 610 615 620 Ser Lys Val Asp Val Ile Asp Leu Thr Ile Glu Ser Ser Ser Asp Glu 625 630 635 640 Glu Glu Asp Pro Pro Ala Lys Arg Gly Ala Met Arg Cys Trp Gly Gly 645 650 655 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Gly Glu Phe Pro Ala 660 665 670 Gly Asn His His His His His His 675 680 <210> 13 <211> 944 <212> PRT <213> Synthetic Sequence <400> 13 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Glu Val Gln Leu 370 375 380 Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu 385 390 395 400 Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp 405 410 415 Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val Ala Ala Ile Thr 420 425 430 Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp Ser Val 435 440 445 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 450 455 460 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys 465 470 475 480 Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp 485 490 495 Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Gly 500 505 510 Gly Ser Gly Gly Ser Gly Gly Ser Ala Ser Met Ser Glu Glu Lys Pro 515 520 525 Lys Glu Gly Val Lys Thr Glu Asn Asp His Ile Asn Leu Lys Val Ala 530 535 540 Gly Gln Asp Gly Ser Val Val Gln Phe Lys Ile Lys Arg His Thr Pro 545 550 555 560 Leu Ser Lys Leu Met Lys Ala Tyr Cys Glu Arg Gln Gly Leu Ser Met 565 570 575 Arg Gln Ile Arg Phe Arg Phe Asp Gly Gln Pro Ile Asn Glu Thr Asp 580 585 590 Thr Pro Ala Gln Leu Glu Met Glu Asp Glu Asp Thr Ile Asp Val Phe 595 600 605 Gln Gln Gln Thr Val Val Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 610 615 620 Gly Ser Met Ser Glu Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn 625 630 635 640 Asp His Ile Asn Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln 645 650 655 Phe Lys Ile Lys Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr 660 665 670 Cys Glu Arg Gln Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp 675 680 685 Gly Gln Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu 690 695 700 Asp Glu Asp Thr Ile Asp Val Phe Gln Gln Gln Thr Val Val Gly Gly 705 710 715 720 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Val Asp Val Ile Asp 725 730 735 Leu Thr Ile Glu Ser Ser Ser Asp Glu Glu Glu Asp Pro Pro Ala Lys 740 745 750 Arg Gly Gly Ser Gly Gly Ser Gly Gly Arg Gly Gly Ser Lys Val Asp 755 760 765 Val Ile Asp Leu Thr Ile Glu Ser Ser Ser Asp Glu Glu Glu Asp Pro 770 775 780 Pro Ala Lys Arg Gly Ala Met Arg Cys Trp Gly Gly Ser Gly Gly Ser 785 790 795 800 Gly Gly Ser Gly Gly Ser Met Gly Met Gly Glu Val Gln Leu Leu Glu 805 810 815 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 820 825 830 Ala Ala Ser Gly Thr Phe Asp Lys Ile Asn Asn Met Gly Trp Tyr Arg 835 840 845 Gln Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Gln Ile Thr Pro Gly 850 855 860 Gly Ile Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 865 870 875 880 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 885 890 895 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Glu Ile Leu Lys Arg 900 905 910 Ala Tyr Ile Asp Val Tyr Val Asn Tyr Trp Gly Gln Gly Thr Leu Val 915 920 925 Thr Val Ser Ser Glu Phe Pro Ala Gly Asn His His His His His His 930 935 940 <210> 14 <211> 601 <212> PRT <213> Artificial sequence <400> 14 Met Glu Gly Ile Ser Ile Tyr Thr Ser Asp Asn Tyr Thr Glu Glu Met 1 5 10 15 Gly Ser Gly Asp Tyr Asp Ser Met Lys Glu Pro Cys Phe Arg Glu Glu 20 25 30 Asn Ala Asn Phe Asn Lys Ile Phe Leu Pro Thr Ile Tyr Ser Ile Ile 35 40 45 Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val Ile Leu Val Met Gly 50 55 60 Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Lys Tyr Arg Leu His Leu 65 70 75 80 Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe Trp Ala Val 85 90 95 Asp Ala Val Ala Asn Trp Tyr Phe Gly Asn Phe Leu Cys Lys Ala Val 100 105 110 His Val Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile Leu Ala 115 120 125 Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His Ala Thr Asn Ser 130 135 140 Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Val Val Tyr Val Gly Val 145 150 155 160 Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe Ile Phe Ala Asn 165 170 175 Val Ser Glu Ala Asp Asp Arg Tyr Ile Cys Asp Arg Phe Tyr Pro Asn 180 185 190 Asp Leu Trp Val Val Val Phe Gln Phe Gln His Ile Met Val Gly Leu 195 200 205 Ile Leu Pro Gly Ile Val Ile Leu Ser Cys Tyr Cys Ile Ile Ile Ser 210 215 220 Lys Leu Ser His Ser Lys Gly His Gln Lys Arg Lys Ala Leu Lys Thr 225 230 235 240 Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cys Trp Leu Pro Tyr Tyr 245 250 255 Ile Gly Ile Ser Ile Asp Ser Phe Ile Leu Leu Glu Ile Ile Lys Gln 260 265 270 Gly Cys Glu Phe Glu Asn Thr Val His Lys Trp Ile Ser Ile Thr Glu 275 280 285 Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pro Ile Leu Tyr Ala Phe 290 295 300 Leu Gly Ala Lys Phe Lys Thr Ser Ala Gln His Ala Leu Thr Ser Val 305 310 315 320 Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Lys Gly Lys Arg Gly Gly 325 330 335 His Ser Ser Val Ser Thr Glu Ser Glu Ser Ser Ser Phe His Ser Ser 340 345 350 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Lys Val Ser 355 360 365 Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe 370 375 380 Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu 385 390 395 400 Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu 405 410 415 Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser 420 425 430 Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro Ala Asp 435 440 445 Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu 450 455 460 Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp 465 470 475 480 Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu Arg Gly 485 490 495 Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly 500 505 510 Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys 515 520 525 Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His Tyr Asp 530 535 540 Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro 545 550 555 560 Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His Asn Glu 565 570 575 Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His Ser 580 585 590 Thr Gly Gly Met Asp Glu Leu Tyr Lys 595 600 <210> 15 <211> 691 <212> PRT <213> Artificial Sequence <400> 15 Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Leu 20 25 30 Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45 Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60 Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu 65 70 75 80 Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175 Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 185 190 Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205 Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215 220 Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270 Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285 Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300 Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser 305 310 315 320 Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu 325 330 335 Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp 340 345 350 Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu Ile 355 360 365 Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr 370 375 380 Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His 385 390 395 400 Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410 415 Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430 Gly Asn Ala Asp Ser Ala Met Ser Gly Gly Ser Gly Gly Ser Gly Gly 435 440 445 Ser Gly Gly Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val 450 455 460 Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe 465 470 475 480 Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr 485 490 495 Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr 500 505 510 Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro 515 520 525 Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly 530 535 540 Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys 545 550 555 560 Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile 565 570 575 Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His 580 585 590 Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp 595 600 605 Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile 610 615 620 Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro 625 630 635 640 Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr 645 650 655 Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val 660 665 670 Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu 675 680 685 Leu Tyr Lys 690 <210> 16 <211> 184 <212> PRT <213> Synthetic Sequence <400> 16 Met Lys Gly Gly Ser Trp Gly Gly Ser Lys Lys Thr Pro Pro Pro Val 1 5 10 15 Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser 20 25 30 Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser 35 40 45 Lys Ala Ser Gly Ala Gly Gly Ser Glu Val Gln Leu Met Glu Ser Gly 50 55 60 Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 65 70 75 80 Ser Gly Arg Thr Phe Asn Asn Tyr Ala Met Gly Trp Phe Arg Arg Ala 85 90 95 Pro Lys Gly Glu Arg Glu Phe Val Ala Ala Ile Thr Arg Ser Gly Val 100 105 110 Arg Ser Gly Val Ser Ala Ile Tyr Gly Asp Ser Val Lys Asp Arg Phe 115 120 125 Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn 130 135 140 Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala Ser Ala 145 150 155 160 Ile Gly Ser Gly Ala Leu Arg Arg Phe Glu Tyr Asp Tyr Ser Gly Gln 165 170 175 Gly Thr Gln Val Thr Val Ser Ser 180 <210> 17 <211> 274 <212> PRT <213> artificial sequence <400> 17 Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg 1 5 10 15 Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu 20 25 30 Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly 35 40 45 Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Ala 50 55 60 Ser Gly Ala Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Ser 65 70 75 80 Gly Ala Thr Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met 85 90 95 Ala Glu Arg Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile 100 105 110 Val Met Glu Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly 115 120 125 Gln Val Gly Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser 130 135 140 Pro Leu Gly Ser Met Gly Met Gly Glu Val Gln Leu Leu Glu Ser Gly 145 150 155 160 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 165 170 175 Ser Gly Thr Phe Asp Lys Ile Asn Asn Met Gly Trp Tyr Arg Gln Ala 180 185 190 Pro Gly Lys Gln Arg Asp Leu Val Ala Gln Ile Thr Pro Gly Gly Ile 195 200 205 Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 210 215 220 Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 225 230 235 240 Asp Thr Ala Val Tyr Tyr Cys Asn Ala Glu Ile Leu Lys Arg Ala Tyr 245 250 255 Ile Asp Val Tyr Val Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 260 265 270 Ser Ser <210> 18 <211> 208 <212> PRT <213> Artificial Sequence <400> 18 Met Lys Gly Gly Ser Trp Gly Gly Ser Lys Lys Thr Pro Pro Pro Val 1 5 10 15 Pro Pro Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser 20 25 30 Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser 35 40 45 Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Leu Asn 50 55 60 Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu 65 70 75 80 Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser 85 90 95 Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro 100 105 110 Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Ala Ser Gly Ala 115 120 125 Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Ser Gly Ala Thr 130 135 140 Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg 145 150 155 160 Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu 165 170 175 Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly 180 185 190 Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Gly 195 200 205 <210> 19 <211> 196 <212> PRT <213> Artificial sequence <400> 19 Met Ser Glu Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asp His 1 5 10 15 Ile Asn Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe Lys 20 25 30 Ile Lys Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys Glu 35 40 45 Arg Gln Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly Gln 50 55 60 Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp Glu 65 70 75 80 Asp Thr Ile Asp Val Phe Gln Gln Gln Thr Val Val Gly Gly Ser Gly 85 90 95 Gly Ser Gly Gly Ser Gly Gly Ser Met Ser Glu Glu Lys Pro Lys Glu 100 105 110 Gly Val Lys Thr Glu Asn Asp His Ile Asn Leu Lys Val Ala Gly Gln 115 120 125 Asp Gly Ser Val Val Gln Phe Lys Ile Lys Arg His Thr Pro Leu Ser 130 135 140 Lys Leu Met Lys Ala Tyr Cys Glu Arg Gln Gly Leu Ser Met Arg Gln 145 150 155 160 Ile Arg Phe Arg Phe Asp Gly Gln Pro Ile Asn Glu Thr Asp Thr Pro 165 170 175 Ala Gln Leu Glu Met Glu Asp Glu Asp Thr Ile Asp Val Phe Gln Gln 180 185 190 Gln Thr Val Val 195 <210> 20 <211> 128 <212> PRT <213> Artificial Sequence <400> 20 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr 50 55 60 Gly Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 65 70 75 80 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 85 90 95 Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg 100 105 110 Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 21 <211> 124 <212> PRT <213> Artificial Sequence <400> 21 Met Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys 20 25 30 Ile Asn Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp 35 40 45 Leu Val Ala Gln Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Asn Ala Glu Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 127 <212> PRT <213> artificial sequence <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Val Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys Asp Tyr 100 105 110 Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 23 <211> 125 <212> PRT <213> artificial sequence <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Leu Cys Ile Asp Ala Ser Asp Asp Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Ala Thr Pro Ile Gly Leu Ser Ser Ser Cys Leu Leu Glu Tyr Asp Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 24 <211> 477 <212> PRT <213> Artificial sequence <400> 24 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr 50 55 60 Gly Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 65 70 75 80 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 85 90 95 Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg 100 105 110 Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ala Ser Met Lys 130 135 140 Gly Gly Ser Trp Gly Gly Ser Lys Lys Thr Pro Pro Pro Val Pro Pro 145 150 155 160 Arg Thr Thr Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 165 170 175 Ser Lys Lys Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser Lys Gly 180 185 190 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asp Leu Asn Met Pro 195 200 205 Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp Glu Leu Ser 210 215 220 Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser Ser Asp Gly 225 230 235 240 Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe Pro Ser Asn 245 250 255 Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Ala Ser Gly Ala Gly Gly 260 265 270 Ser Glu Gly Gly Gly Ser Glu Gly Gly Thr Ser Gly Ala Thr Asp Leu 275 280 285 Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg Glu Asp 290 295 300 Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu Lys Ser 305 310 315 320 Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly Trp Phe 325 330 335 Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Gly Ser Met 340 345 350 Gly Met Gly Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 355 360 365 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp 370 375 380 Lys Ile Asn Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg 385 390 395 400 Asp Leu Val Ala Gln Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp 405 410 415 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 420 425 430 Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 435 440 445 Tyr Cys Asn Ala Glu Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val 450 455 460 Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 465 470 475 <210> 25 <211> 552 <212> PRT <213> artificial sequence <400> 25 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Arg Ala Pro Lys Gly Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Arg Ser Gly Val Arg Ser Gly Val Ser Ala Ile Tyr 50 55 60 Gly Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 65 70 75 80 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 85 90 95 Val Tyr Thr Cys Ala Ala Ser Ala Ile Gly Ser Gly Ala Leu Arg Arg 100 105 110 Phe Glu Tyr Asp Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ala Ser Met Ser 130 135 140 Glu Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asp His Ile Asn 145 150 155 160 Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe Lys Ile Lys 165 170 175 Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys Glu Arg Gln 180 185 190 Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly Gln Pro Ile 195 200 205 Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp Glu Asp Thr 210 215 220 Ile Asp Val Phe Gln Gln Gln Thr Val Val Gly Gly Ser Gly Gly Ser 225 230 235 240 Gly Gly Ser Gly Gly Ser Met Ser Glu Glu Lys Pro Lys Glu Gly Val 245 250 255 Lys Thr Glu Asn Asp His Ile Asn Leu Lys Val Ala Gly Gln Asp Gly 260 265 270 Ser Val Val Gln Phe Lys Ile Lys Arg His Thr Pro Leu Ser Lys Leu 275 280 285 Met Lys Ala Tyr Cys Glu Arg Gln Gly Leu Ser Met Arg Gln Ile Arg 290 295 300 Phe Arg Phe Asp Gly Gln Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln 305 310 315 320 Leu Glu Met Glu Asp Glu Asp Thr Ile Asp Val Phe Gln Gln Gln Thr 325 330 335 Val Val Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys Val 340 345 350 Asp Val Ile Asp Leu Thr Ile Glu Ser Ser Ser Asp Glu Glu Glu Asp 355 360 365 Pro Pro Ala Lys Arg Gly Gly Ser Gly Gly Ser Gly Gly Arg Gly Gly 370 375 380 Ser Lys Val Asp Val Ile Asp Leu Thr Ile Glu Ser Ser Ser Asp Glu 385 390 395 400 Glu Glu Asp Pro Pro Ala Lys Arg Gly Ala Met Arg Cys Trp Gly Gly 405 410 415 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Gly Met Gly Glu Val 420 425 430 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 435 440 445 Arg Leu Ser Cys Ala Ala Ser Gly Thr Phe Asp Lys Ile Asn Asn Met 450 455 460 Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Asp Leu Val Ala Gln 465 470 475 480 Ile Thr Pro Gly Gly Ile Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg 485 490 495 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 500 505 510 Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Glu 515 520 525 Ile Leu Lys Arg Ala Tyr Ile Asp Val Tyr Val Asn Tyr Trp Gly Gln 530 535 540 Gly Thr Leu Val Thr Val Ser Ser 545 550

Claims

1. A phase change regulating element, characterized in that, The phase transition modulating element comprises a multivalent phase transition domain and at least two ligands. One ligand is covalently linked to the multivalent phase transition domain via a linker, and the remaining ligands are covalently linked to the multivalent phase transition domain or to other ligands contained in the same phase transition modulating element via linkers. Each ligand is capable of specifically binding to its corresponding cell surface molecule. The multivalent phase transition structural domain is composed of at least two motifs connected in series and can cause phase separation of the phase transition regulating element; The multivalent phase transition domain is formed by at least one SUMO3 motif connected in series with at least one SIM motif, or by at least one PRMH motif connected in series with at least one SH3 motif; The amino acid sequence of the SUMO3 motif is MSEEKPKEGVKTENDHINLKVAGQDGSVV QFKIKRHTPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDV FQQQTVV; The amino acid sequence of the SIM motif is KVDVIDLTIESSSDEEEDPPAKR; The amino acid sequence of the PRMH motif is KKTPPPVPPRTTSK; The amino acid sequence of the SH3 motif is DLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPL; The ligand is an antibody or its antigen-binding fragment that specifically binds to cell surface molecules; The antibody is selected from monoclonal antibodies, human antibodies, or humanized antibodies, and the antigen-binding fragment is selected from F(ab')2, Fab, single-chain variable fragments, and single-domain antibody fragments.

2. The phase change regulating element according to claim 1, wherein the multivalent phase change structural domain is composed of two, three, four, five, six, seven, eight, nine, or ten motifs connected in series.

3. The phase transition modulation element of claim 1, wherein the multivalent phase transition structural domain is formed by one, two, three or four SUMO3 motifs connected in series with one, two, three or four SIM motifs, or by one, two, three or four PRMH motifs connected in series with one, two, three or four SH3 motifs.

4. The phase change regulating element according to claim 1, wherein, The multivalent phase transition domain is composed of an amino acid sequence as shown in SEQ ID NO:18 or SEQ ID NO:

19.

5. The phase change regulating element according to claim 1, wherein, The single-domain antibody fragment is a VHH or a nanobody.

6. The phase change regulating element according to any one of claims 1 to 5, comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 ligands.

7. The phase transition modulation element according to any one of claims 1 to 5, wherein of the at least two cell surface molecules specifically bound by the at least two ligands, one cell surface molecule is a tumor-associated antigen and the other cell surface molecule is a tumor necrosis factor receptor; in, The tumor-associated antigens are selected from the group consisting of CXC motif chemokine receptor 4 (CXCR4), hepatocyte growth factor receptor (c-Met or HGFR), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate-specific membrane antigen (PSMA), fibroblast activating protein (FAP), carcinoembryonic antigen (CEA), folate receptor α (FolR1), melanoma-associated chondroitin sulfate proteoglycan (MCSP), p95HER2, EpCAM, HER3, CD30 or TPBG (5T4), CD19, CD79b, CD20, CD22, CD37, CD38, BCMA, and GPRC5D.

8. The phase change regulating element according to claim 7, wherein, The tumor necrosis factor receptor is either death receptor 5 (DR5) or Fas receptor.

9. The phase change regulating element according to claim 8, wherein, The tumor necrosis factor receptor is death receptor 5 (DR5).

10. The phase change modulation element of claim 1, wherein the at least two ligands included in the phase change modulation element are single-chain variable segments.

11. The phase change regulating element according to claim 10, wherein, One ligand is a single-stranded variable fragment consisting of an amino acid sequence as shown in SEQ ID NO:21, and / or another ligand is a single-stranded variable fragment consisting of an amino acid sequence selected from SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:

23.

12. The phase transition modulation element of claim 1, further comprising a tag and / or a polypeptide cleaved from the tag that do not affect the function of the ligand and the portion containing the multivalent phase transition domain.

13. The phase transition regulating element of claim 1, comprising the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:

25.

14. The phase transition regulating element of claim 1, wherein it is a fusion protein.

15. A method for screening phase change regulating elements, the method comprising the following steps: Step A: Generate a library containing multiple candidate phase transition modulators, wherein each candidate phase transition modulator includes a multivalent phase transition domain and at least two ligands, wherein at least one ligand is covalently linked to the multivalent phase transition domain, and the remaining ligands are covalently linked to the multivalent phase transition domain in the same candidate phase transition modulator or to other ligands contained in the same phase transition modulator. Each of the at least two ligands is capable of specifically binding to its corresponding cell surface molecule, wherein the cell surface molecule specifically bound by the ligands in each candidate phase transition modulator is the same or different cell surface molecules. Step B: Using a reference that is distinguished only by the absence of the multivalent phase transition domain as a negative control, under the same conditions suitable for phase transition, determine the activity level of the candidate phase transition regulating element and the negative control in generating phase transition droplets in Step A; Step C: Select the candidate phase change regulating element that produces more phase change droplets than the negative control in step B as the target phase change regulating element; The multivalent phase transition domain is formed by at least one SUMO3 motif and at least one SIM motif in series, or by at least one PRMH motif and at least one SH3 motif in series. The amino acid sequence of the SUMO3 motif is MSEEKPKEGVKTENDHINLKVAGQDGSVV QFKIKRHTPLSKLMKAYCERQGLSMRQIRFRFDGQPINETDTPAQLEMEDEDTIDV FQQQTVV; The amino acid sequence of the SIM motif is KVDVIDLTIESSSDEEEDPPAKR; The amino acid sequence of the PRMH motif is KKTPPPVPPRTTSK; The amino acid sequence of the SH3 motif is DLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPL.

16. The screening method of claim 15, wherein, The portion of the multivalent phase transition structural domain contained in the same phase transition regulating element is covalently connected to the at least one ligand or between ligands via a connector.

17. The screening method according to claim 15, wherein, The connector is a peptide connector.

18. A phase change regulating element obtained by the method of any one of claims 15 to 17.

19. A pharmaceutical composition comprising the phase change regulating element of any one of claims 1 to 14 and 18, and a pharmaceutically acceptable carrier.

20. Use of the phase change regulating element according to any one of claims 1 to 14 and 18 in the preparation of an agent for treating a disease selected from the group consisting of lung cancer, colorectal cancer, sarcoma or prostate cancer, or metastatic forms thereof.

Images