Protein artificially causing phagocytosis in vivo

Abstract

The present disclosure addresses the problem of providing technology for phagocytizing unwanted cells or the like in vivo.　This protein includes, from the N-terminus to the C-terminus, (A) a target binding region that binds to a target, and (C) an SHBG-like domain of ProS.

Description

Proteins that artificially induce phagocytosis in living organisms

[0001] This disclosure relates to a protein that artificially induces phagocytosis of unwanted cells and other substances in living organisms.

[0002] Unwanted cells are constantly generated within the body. When unwanted cells are generated, a series of removal systems are activated that selectively induce cell death in these unwanted cells, followed by the elimination of the dead cells. It is known that if there is a defect in this unwanted cell removal system, unwanted cells accumulate in the body with age, causing various diseases such as cancer and autoimmune diseases (Non-patent documents 1-3). Therefore, removing unwanted cells in the body is extremely important from the perspective of suppressing the accumulation of unwanted cells and, as a result, extending healthy life expectancy.

[0003] Traditionally, many advanced approaches have been developed to eliminate unwanted cells by utilizing the body's immune system, such as cellular immunity and humoral immunity, to kill cells. For example, CAR-T cells, which utilize the chimeric antigen receptor (CAR) system, have been successful in lymphoma treatment as a system for killing unwanted cells using cellular immunity (Non-Patent Literature 4-6). CAR-T cells identify surface proteins that are specifically expressed on unwanted cells such as cancer cells using CARs, and selectively kill these unwanted cells. Similarly, technologies using antibodies are widely known as systems for killing unwanted cells using the humoral immune system. For example, antibodies targeting PD-L1 or PD-1 inhibit the function of PD-L1 or PD-1, thereby forcibly releasing the brake on the cell-killing activity of T cells brought about by the action of PD-1, and as a result, induce the killing of target cells, including cancer cells, by T cells (Non-Patent Literature 7-9). Similarly, bispecific antibodies against CD3 in T cells and CD19 in B-cell lymphomas also promote cell death mediated by T cells (Non-Patent Documents 10 and 11).

[0004] Furthermore, as a system for killing unwanted cells without utilizing the immune system, we can cite technologies that utilize the signaling pathways inherent in cells to kill unwanted cells. For example, small molecules that target tyrosine kinases in the cell membrane or cytoplasm can directly kill unwanted cells by inhibiting proliferation signaling pathways in cancer cells (Non-Patent Documents 12 and 13).

[0005] As described above, once unwanted cells die, in the body, the dead cells (apoptotic cells) are subsequently removed by phagocytic cells and disappear from the body (Non-Patent Literature 14). Phagocytic cells rapidly recognize and take up apoptotic cells and break them down into components such as nucleotides and amino acids (Non-Patent Literature 15). This phagocytosis of apoptotic cells by phagocytic cells is initiated by an "Eat Me" signal from phosphatidylserine (PtdSer) exposed on the cell surface (Non-Patent Literature 16). Protein S, a humoral factor (Non-Patent Literature 17), binds to PtdSer exposed on the cell surface. Furthermore, TAM family receptors present on the surface of phagocytic cells selectively bind to protein S attached to the surface of apoptotic cells via the SHBG domain of this molecule. In this way, apoptotic cells are recognized by phagocytic cells via protein S and TAM family receptors (e.g., Tyro3, ​​Axl, and MerTK). Subsequently, the phagocytic signaling pathway of TAM family receptors is activated, and apoptotic cells are phagocytosed and eliminated by phagocytic cells (Non-Patent Literature 18-20). It should be noted that TAM family receptors are expressed not only in professional phagocytic cells such as macrophages and microglia, but also in non-professional phagocytic cells such as epithelial cells, endothelial cells, and fibroblasts (Non-Patent Literature 14).

[0006] Soussi, T. & Wiman, K. G. Shaping Genetic Alterations in Human Cancer: The p53 Mutation Paradigm. Cancer Cell 12, 303-312 (2007).Rahman, A. & Isenberg, D. A. Systemic lupus erythematosus. NEngl J Med 358, 929-939 (2008).Smolen, J. S. et al. Rheumatoid arthritis. Nat Rev Dis Primers 4, 1-23 (2018).Huston, J. S. et al. Protein engineering of antibody bindingsites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences 85, 5879-5883 (1988).Brudno, J. N. & Kochenderfer, J. N. Current understanding and management of CAR T cell-associated toxicities. Nat Rev Clin Oncol 21, 501-521(2024).Baker, D. J., Arany, Z., Baur, J. A., Epstein, J. A. & June,C. H. CAR T therapy beyond cancer: the evolution of a living drug. Nature 619, 707-715 (2023).Y, I., Y, A., K, S. & T, H. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death.The EMBO journal 11, (1992).Iwai, Y. et al. Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proceedings of the National Academy of Sciences 99, 12293-12297 (2002).Bader, J. E. et al. Obesity induces PD-1 on macrophages to suppress anti-tumour immunity. Nature 630, 968-975 (2024).Loffler, A. et al. A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 95, 2098-2103 (2000).Dreier, T. et al. Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. International Journal of Cancer 100, 690-697 (2002).Kris, M. G. et al. Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs. JAMA 311, 1998-2006 (2014).Thress, K. S. et al.Acquired EGFR C797S mutation mediatesresistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 21, 560-562 (2015).Arandjelovic, S. & Ravichandran, K. S. Phagocytosis of apoptotic cells in homeostasis. Nat Immunol 16, 907-917 (2015).Nagata, S. & Tanaka, M. Programmed cell death and the immune system. Nat Rev Immunol 17, 333-340 (2017).Suzuki, J., Denning, D. P., Imanishi, E., Horvitz, H. R. &Nagata, S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science 341, 403-406 (2013).Nyberg, P., He, X., Hardig, Y., Dahlback, B. & Garcia De Frutos, P. Stimulation of Sky Tyrosine Phosphorylation by Bovine Protein S. European Journal of Biochemistry 246, 147-154 (1997).Lai, C. & Lemke, G. An extended family of protein-tyrosinekinase genes differentially expressed in the vertebrate nervous system. Neuron 6, 691-704 (1991).Graham, D. K., Dawson, T. L., Mullaney, D. L., Snodgrass, H. R. & Earp, H. S.Cloning and mRNA expression analysis of a novel human protooncogene, c-mer. Cell Growth Differ 5, 647-657 (1994).Prasad, D. et al. TAM receptor function in the retinal pigment epithelium. Mol Cell Neurosci 33, 96-108 (2006).

[0007] As described above, the removal of unwanted cells in living organisms involves two steps: cell death and subsequent phagocytosis and elimination of the dead cells. Systems utilizing the former mechanism of cell death, such as those using CAR-T cells and antibodies, have already been developed. On the other hand, systems utilizing the latter mechanism of phagocytosis and elimination of dead cells have yet to be developed. Furthermore, phagocytosis by phagocytic cells has the potential to eliminate unwanted substances other than unwanted cells, such as viruses and LDL cholesterol, which cannot utilize the cell death system, so there are high expectations for systems that utilize this mechanism. In light of these circumstances, the objective of this disclosure is to provide a technology for eliminating unwanted substances such as unwanted cells in living organisms.

[0008] Based on the idea that modifying the PtdSer binding ability of protein S (ProS) to the binding ability to targets contained in unwanted cells would enable unwanted cells to be recognized and phagocytosed by phagocytic cells, the inventors conducted various studies on modified protein S for phagocytosis of unwanted cells in the body.

[0009] As a result, the inventors have discovered that a protein having (A) a target-binding region that binds to unwanted cells, and (C) an SHBG-like domain of ProS, can bridge the gap between unwanted cells and phagocytic cells in vivo, enabling the phagocytosis of unwanted cells. This disclosure was completed by further investigation based on these findings.

[0010] This disclosure provides inventions in the following embodiments: 1-1. A protein comprising (A) a target-binding region that binds to a target (excluding the γ-carboxyglutamic acid domain of protein S), and (C) a sex hormone-binding globulin-like domain of protein S. 1-2. The fusion protein according to 1-1, further comprising 2 to 4 epidermal growth factor-like domains of (B) protein S between the target-binding region that binds to the target (A) and the sex hormone-binding globulin-like domain of protein S (C). 1-3. The fusion protein according to 1-1 or 1-2, wherein the recognized object containing the target is an unwanted cell, virus, or bacterium. 1-4. The fusion protein according to any one of 1-1 to 1-3, wherein the target-binding region (A) is bound to the N-terminus of an amino acid sequence shown in any of (i) to (xii) below. (i) The amino acid sequence shown in SEQ ID NO: 28 (ii) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28 (iii) The amino acid sequence shown in SEQ ID NO: 29 (iv) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 29 (v) The amino acid sequence shown in SEQ ID NO: 30 (vi) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30 (vii) The amino acid sequence shown in SEQ ID NO: 31 (viii) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31 (ix) The amino acid sequence shown in SEQ ID NO: 32 (x) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32 (xi) The amino acid sequence shown in SEQ ID NO: 33 (xii) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33 Items 1-5. The protein described in any of items 1-1 to 1-4, wherein the measured value of the Crunch binding assay below is 0.8 times or more than the measured value of the control protein below. <Control Protein> A control protein is a protein in which a target binding region with the same sequence as the target binding region of the protein being measured is bound to the N-terminus of any of the amino acid sequences shown in (i), (iii), (v), (vii), (ix), and (xi) above.<Crunch Binding Assay> (1) Prepare cells that express the target on the cell membrane. (2) If the tag peptide is not attached to the C-terminus of the protein, prepare the protein to be measured with the tag peptide attached to the C-terminus. (3) Place 5 × 10 cells in 100 μl of DPBS (Dulbecco's Phosphate Buffered Saline; containing 1 mg / ml BSA) containing 10 μg / ml of tagged protein. 4 Cells are added and incubated on ice for 30 minutes, after which the cells are harvested. (4) An anti-tagged antibody is used to measure the amount of tagged protein bound to the harvested cells, and this amount of tagged protein is taken as the measured value. Item 1-6. A pharmaceutical composition comprising the protein described in any of Items 1-1 to 1-5. Item 1-7. A nucleic acid comprising a polynucleotide encoding the protein described in any of Items 1-1 to 1-5. Item 1-8. The nucleic acid described in Item 1-7 further comprising a promoter operably linked to the polynucleotide encoding the protein. Item 1-9. Cells comprising the nucleic acid described in Item 1-8. Item 1-10. A viral vector comprising the nucleic acid described in Item 1-8.

[0011] The protein disclosed herein can phagocytose recognized substances (such as unwanted cells) containing a target within the body. Furthermore, since the target-binding region of the protein disclosed herein can be designed according to the type of target contained in the recognized substance to be removed, it can exert phagocytic activity against various cell types and viruses.

[0012] This is the result of analysis of an unwanted cell model that expresses GFP on the cell membrane. (a) shows GFPm obtained by transfecting BDKO cells (in which Xkr8 and TMEM 16F were knocked out) with GFP (GFPm) fused with the CD8a signal sequence and transmembrane region. + This image shows BDKO cells observed using a confocal microscope. (b) shows BDKO cells and GFPm + This figure shows the detection of GFP in BDKO cells by flow cytometry using an anti-GFP antibody. c represents GFPNbm + K562 cells and GFPm +It is a schematic diagram showing that cell-cell association is observed when co-cultured with BDKO cells. d is an image observed by a confocal microscope after co-culturing the two types of cells overnight. Above d, GFP Nb (GFPNbm) in which the signal sequence and transmembrane region of CD8a are fused to K562 cells transfected with BDKO cells + Results of co-culture with K562 cells; in the middle of d, GFPm + Results of co-culture of BDKO cells and GFPNbm + Results of co-culture with K562 cells; at the bottom of d, GFPNbm +This shows the results of co-culture of BDKO cells and K562 cells. In d, associated cells are indicated by arrows. This figure shows the structure and properties of the ProS variant (Crunch) of this disclosure. a is a schematic diagram of the structure of ProS (top panel) and Crunch that binds to GFP (GFPNb-Crunch). In a, SS refers to the signal sequence and E refers to the EGF-like domain. b is the result of screening of the signal sequence (SS) of Crunch by a Crunch binding assay. In b, Mock refers to the mock control, Alb refers to the SS of albumin, IL-2 refers to the SS of IL-2, EGF refers to the SS of EGF, FGF refers to the SS of fibroblast growth factor-9, IgG refers to the SS of IgG, and CD8 refers to the SS of CD8a. b shows the mean value (n=3) and error bars (standard deviation) of the mean fluorescence intensity (MFI) measured by the Crunch binding assay. c shows the results of purifying GFPNb-Crunch with various SSs using nickel beads from a condition medium containing GFPNb-Crunch secreted from HEK293T cells, followed by Western blotting (top panel) and CBB staining (bottom panel). d is a diagram showing the procedure for establishing GFPNb-Crunch-highly expressing Chinese hamster ovary (CHO) cells (CHO / GFPNb-Crunch-S3). e shows the results of purifying GFPNb-Crunch from the condition medium of CHO / GFPNb-Crunch-S3 cells, followed by SDS-PAGE and CBB staining. f shows the results of size exclusion chromatography (SEC) analysis of BSA (top panel) and purified GFPNb-Crunch (bottom panel). g shows the results of a Crunch binding assay for GFPNb-Crunch + This shows the results of measuring the binding affinity of purified GFPNb-Crunch to BDKO cells and BDKO cells. It also shows the measurement results during the establishment of GFPNb-Crunch high-expression CHO cells and the results of measuring the properties of GFPNb-Crunch. a shows the results of Western blotting using anti-FLAG antibody after SDS-PAGE of the culture supernatant of each cell. b shows the results of the Crunch binding assay. +This shows the results of measuring the binding affinity of GFPNb-Crunch in the culture supernatant to BDKO cells. In a and b, CHO refers to CHO cells; CHO S2 refers to cells obtained by twice infecting CHO cells with a lentivirus encoding GFPNb-Crunch-IRES (Internal ribosome entry site)-RFP (red fluorescent protein) and selecting RFP-highly expressing cells by flow cytometry; CHO I3 refers to cells obtained by infecting CHO S2 cells with the aforementioned lentivirus; and CHO S3 refers to cells obtained by selecting RFP-highly expressing cells from CHO I3 cells by flow cytometry. The culture supernatant used for measurement was obtained by culturing each cell in serum medium for 16 hours, then switching to serum-free medium and culturing for a further 32 hours. c shows the results of measuring the GFPNb-Crunch titer in the supernatant by ELISA after seeding GFPNb-Crunch-highly expressing CHO cells in serum medium, culturing for 16 hours, and then changing to serum-free medium, with the supernatant collected daily. d shows the results of performing Western blotting with anti-FLAG antibody after subjecting purified GFPNb-Crunch from the culture supernatant collected on day 6 after the medium change to SDS-PAGE. e shows the results of SDS-PAGE followed by CBB staining of 0.25–1.00 μl of BSA (standard) and purified GFPNb-Crunch. f shows the results of evaluating the glycosylation of the purified GFPNb-Crunch, which was treated with PNGaseF, subjected to SDS-PAGE, and then Western blotting with anti-FLAG antibody. g shows the results of ELISA analysis of the binding of GFP nanobodies and GFPNb-Crunch to a GFP-coated plate. g also shows the dissociation constant (KD) estimated by performing nonlinear regression analysis using R. h is the result of measuring the thermal stability of the purified GFPNb-Crunch and 1D3 antibody by differential scanning fluorescence (DSF) scan (n=3, mean value). i is the result of measuring the aggregation onset temperature (Tm) of the purified GFPNb-Crunch and 1D3 antibody by DSF scan.Figure j shows the results of treating GFPNb-Crunch produced in the presence or absence of vitamin K with PNGaseF, subjecting it to SDS-PAGE, and then performing Western blotting with an anti-FLAG antibody. This figure shows that Crunch induces dimerization and phosphorylation of MerTK. Figure a shows NIH3T3 cells and NIH3T3 cells into which codon-optimized MerTK has been introduced (MerT3). + The results of detecting mMerTK in NIH3T3 cells using flow cytometry with an anti-mMerTK antibody are shown. b shows NIH3T3 cells and MerT + The image shows the results of Western blotting using anti-mMerTK antibody and anti-α-tubulin antibody after subjecting lysates of NIH3T3 cells to SDS-PAGE. c is a schematic diagram of a cell capable of detecting MerTK dimerization. The cells shown in c are NIH3T3 cells expressing MerTK-sfGFP, in which a split superfolder GFP, consisting of sfGFP1-10 at the N-terminus and sfGFP11 at the C-terminus, is fused to the C-terminus of mMerTK (MerTK-sfGFP). + (NIH3T3 cells). d is MerTK-sfGFP + NIH3T3 cells were co-cultured with living or apoptotic thymocytes in the presence of fetal bovine serum (FBS) containing ProS, and MerTK-sfGFP was produced. + The following shows the results of flow cytometry analysis of the fluorescence of NIH3T3 GFP. The bar graph in section d represents MerTK-sfGFP. + The percentage of GFP-positive cells in NIH3T3 cells (n=4) is shown. e represents MerTK-sfGFP in the presence of mock HEK293T cells or HEK293T cells expressing GFPNb-Crunch in a controlled medium. + NIH3T3 cells and GFPm + BDKO cells were co-cultured, and MerTK-sfGFP was introduced. + The following shows the results of flow cytometry analysis of GFP fluorescence in NIH3T3 cells. The bar graph labeled 'e' represents MerTK-sfGFP. + The percentage of GFP-positive cells in NIH3T3 cells (n=4) is shown. f and g are MerTK cells in the presence or absence of GFPNb-Crunch. + NIH3T3 cells and GFPm + After co-culturing BDKO cells, MerTK + The following shows the results of Western blotting performed using anti-phospho-MerTK antibody and anti-MerTK antibody after subjecting NIH3T3 cell lysates to SDS-PAGE. f is the Western blotting image, and g is a bar graph showing pMerTK expression levels analyzed in ImageJ. h shows MerTK expression levels in the presence or absence of GFPNb-Crunch and in the presence or absence of anti-GFP antibody. + NIH3T3 cells and GFPm + After co-culturing BDKO cells, MerTK + The results of Western blotting using anti-phospho-MerTK antibody and anti-MerTK antibody after subjecting NIH3T3 cell lysates to SDS-PAGE are shown. Figures i-l show the results of cell viability assays performed on IL-3-dependent Ba / F3 cells expressing both GFPm and MerTK under IL-3 deficiency and in or without GFPNb-Crunch. i is the mean number of viable cells, j is the mean survival rate, k is a bar graph showing the number of viable cells at 48 and 78 hours, and l is a bar graph showing the survival rate at 48 and 78 hours (n=3). In Figure 4, all data are shown as mean ± standard deviation. Statistical analysis in d, e, and i-l was performed using Student's unpaired t-test. Statistical analysis in g was performed using a one-way ANOVA with Tukey-Kramer t-test. Significant differences are indicated by *P<0.001. This figure shows that MerTK dimerization is induced by Crunch. a and b show NIH3T3 cells into which MerTK-sfGFP and tagRFP have been introduced in the presence of mock HEK293T cells or HEK293T cells expressing GFPNb-Crunch (RFP + MerTK-sfGFP + NIH3T3 cells) and BDKO cells or GFPm+ Co-culture BDKO cells and RFP + MerTK-sfGFP + The following shows the results of flow cytometry analysis of GFP fluorescence in NIH3T3 cells. a) shows the dotted results obtained by flow cytometry, and b) shows the GFP fluorescence obtained using BDKO cells. + MerTK-sfGFP + This is a bar graph (n=4) showing the percentage (%) of GFP-positive cells in NIH3T3 cells. It shows that Crunch induces phosphorylation of MerTK. a) shows the phosphorylation of NIH3T3 cells or MerTK by binding ProS to viable thymocytes or apoptotic thymocytes. + After co-culturing NIH3T3 cells, NIH3T3 cells or MerTK + The results of Western blotting using anti-phospho-MerTK antibody (top panel) and anti-MerTK antibody (bottom panel) after subjecting NIH3T3 cell lysates to SDS-PAGE are shown. b shows Ba / F3 cells or Ba / F3 cells with PtdSer exposed (Xkr4 + By attaching ProS to Ba / F3 cells, MerTK + After co-culturing NIH3T3 cells, MerTK + The results of Western blotting using anti-phospho-MerTK antibody (top panel) and anti-MerTK antibody (bottom panel) after subjecting NIH3T3 cell lysates to SDS-PAGE are shown. c represents MerTK in the presence or absence of GFPNb-Crunch. + NIH3T3 cells and GFPm + BDKO cells were co-cultured for 0-30 minutes, and MerTK was measured over time. + The results of Western blotting using anti-phospho-MerTK antibody and anti-MerTK antibody after preparing a lysate of NIH3T3 cells and subjecting it to SDS-PAGE are shown. The figure shows that MerTK signaling is activated by Crunch. Figure a shows an overview of the cell survival assay. Ba / F3 cells, Ba / F3 cells expressing MerTK (MerTK +Ba / F3 cells), Ba / F3 cells expressing aXkr4 (Ba / F3 cells with PtdSer exposed, Xkr4 + Ba / F3 cells), Ba / F3 cells expressing both MerTK and aXkr4 (Ba / F3 cells with PtdSer exposed, MerTK + Xkr4 + When Ba / F3 cells are cultured in 500 μl of IL-3(-) RPMI1640 containing FBS (Pros), MerTK is activated. + Xkr4 + Only Ba / F3 cells can survive. Figures b to e show the results of cell survival assays performed on each cell type cultured under IL-3 deficiency. b is the average number of viable cells, c is the average survival rate, d is a bar graph showing the number of viable cells after 48 and 78 hours, and e is a bar graph showing the survival rate after 48 and 78 hours (n=3). Figures f to i show the results of cell survival assays performed on Ba / F3 cells introduced with MerTK, cultured in a medium containing GFPNb-Crunch or FBS (containing Pros) supplemented with PBS. f is the average number of viable cells, g is the average survival rate, h is a bar graph showing the number of viable cells after 48 and 78 hours, and i is a bar graph showing the survival rate after 48 and 78 hours (n=3). In Figure 7, all data are shown as mean ± standard deviation. Statistical analysis for d, e, and g was performed using a one-way ANOVA with the Tukey-Kramer t-test. Statistical analysis in f-i was performed using Student's unpaired t-test. Significant differences are indicated by NS P>0.05, * P<0.05, ** P<0.01, and ** P<0.1. This figure shows that GFPNb-Crunch can recognize GFP present on the surface of primary cells derived from mice. a is ROSA26 in mice. GFPm-OVA This is a schematic diagram. ROSA26 is a gene in C57BL / 6 mice in which chicken ovalbumin (OVA) and GFPm, linked by a CMV enhancer, chicken β-actin promoter, and GGGGS linker, and a rabbit β-globin polyadenylation signal (pA) are inserted into the Rosa26 locus. GFPm-OVA Mice were created. b is a wild-type mouse and ROSA26 GFPm-OVAThe results of flow cytometry analysis of GFP on the cell membrane of mouse-derived thymocytes, spleen cells, bone marrow (BM) cells, and blood cells are shown. c represents the results of Crunch binding assays for wild-type mice and ROSA26 GFPm-OVA The results of measuring the binding affinity of GFPNb-Crunch to mouse-derived thymocytes, spleen cells, bone marrow (BM) cells, and blood cells are shown. The figure shows that Crunch induces phagocytosis in a MerTK-dependent manner. a-c are ROSA26 GFPm-OVA Mouse-derived thymocytes were stained with pHrodo-Red, and then MerTK was applied in the presence or absence of GFPNb-Crunch. + Co-culture NIH3T3 cells with MerTK in a medium containing 0-10% FBS. + This shows the results of measuring pHrodo-Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells. a is MerTK after co-culture. + Image of NIH3T3 cells observed under a microscope; b shows MerTK cells after co-culture. + NIH3T3 cells were subjected to flow cytometry to detect pHrodo-Red-positive cells (phagocytosis-positive cells). The result shows that c represents MerTK cells after co-culture. + This is a bar graph (n=3) showing the percentage of pHrodo-Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells. d-f represent ROSA26. GFPm-OVA Mouse-derived thymocytes were stained with pHrodo-Red, and then NIH3T3 cells or MerTK cells were selected in the presence or absence of GFPNb-Crunch. + Co-culture NIH3T3 cells with MerTK in a medium containing 0-10% FBS. + This shows the results of measuring pHrodo-Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells. d represents NIH3T3 cells and MerTK after co-culture. + NIH3T3 cells were subjected to flow cytometry to detect pHrodo-Red-positive cells (phagocytosis-positive cells). The result shows that e represents NIH3T3 cells and MerTK after co-culture. +This is a bar graph (n=3) showing the percentage of pHo-Red positive cells (phagocytosis positive cells) in NIH3T3 cells. f is ROSA26 GFPm-OVA After staining mouse-derived thymocytes with pHrodo-Red, NIH3T3 cells expressing human MerTK in the presence of humanized GFPNb-Crunch (hMerTK) were selected. + After co-culturing with NIH3T3 cells in a medium containing 5% FBS, the post-co-culturing hMerTK + This bar graph shows the percentage of pHo-Red positive cells (phagocytosis-positive cells) in NIH3T3 cells (n=3). g represents ROSA26. GFPm-OVA The results shown are from co-culturing mouse-derived thymocytes with peritoneal macrophages in a medium containing 0-10% FBS, either in the presence or absence of GFPNb-Crunch, followed by staining with anti-CD11b-APC antibody and analysis of the macrophages by flow cytometry. The bar graph shown in g represents CD11b + Engulfment in macrophages + This represents the proportion of the cell population (n=3). All data are shown as mean ± standard deviation. Statistical analysis for b-g was performed using Student's unpaired t-test. Significant differences are indicated by * P<0.01. This figure shows the pharmacokinetics and immunogenicity of Crunch. a is ROSA26 in the presence of 10 μg / ml GFPNb-Crunch. GFPm-OVA MerTK targeting mouse-derived thymocytes +These are the results of a phagocytosis assay of NIH3T3 cells. 'a' shows the results using GFPNb-Crunch produced in the presence or absence of vitamin K. 'b' to 'd' show the results of measuring plasma GFPNb-Crunch concentration after intravenous (iv) or intraperitoneal (ip) injection of 150 μg of GFPNb-Crunch into mice. 'b' shows the experimental protocol, 'c' shows the results of determining plasma GFPNb-Crunch levels by ELISA, and 'd' shows the remaining percentage and half-life of plasma GFPNb-Crunch (n=4 mice). 'e' shows the results of predicting the epitopes of anti-mouse protein S (mProS) antibody and anti-GFPNb-Crunch antibody using BepiPred-2.0. In 'e', ​​the epitope score is shown with a threshold of 0.5. f and g show the results of measuring antibodies against GFP, GFPNb-Crunch, GFPNb (anti-GFP nanobody), and Gla domain-deficient mProS (Gla del mProS) in plasma after intravenous administration of 150 μg of GFPNb-Crunch or GFP to mice, with plasma samples collected 24 days later (n=4 mice for GFPNb-Crunch, n=3 mice for GFP). In the measurement shown in g, 100 ng / ml of mouse anti-GFP antibody or mouse anti-FLAG antibody was used as a control. h shows the results of measuring the expression levels of GAPDH, IFNγ, IL-6, CCL5, IL-10, and Arg1 mRNA in macrophages stimulated in D-MEM with 10 μg / ml GFPNb-Crunch, mouse IgG2a, 10% FBS, or 10 ng / ml LPS at 37°C for 24 hours (n=3 independent biological samples). h, Macrophage stimulation. Macrophages were stimulated in D-MEM at 37°C for 24 hours with 10 μg / ml GFPNb-Crunch, mouse IgG2a, 10% FBS, or 10 ng / ml LPS. RNA was extracted from macrophages, converted to cDNA, and then RT-PCR was performed to examine the expression levels of GAPDH, IFNγ, IL-6, CCL5, IL-10, and Arg1. Data are shown as -2ΔCt, normalized by the Ct value of GAPDH (n=3 independent biological samples). All data are shown as mean ± SD.The statistics for g were calculated using Student's unpaired t-test. The statistics for h were calculated using one-way ANOVA with Tukey-Kramer t-test. Significance is indicated by *P < 0.05. This figure shows that Crunch removes target cells in vivo. a is the experimental protocol used to verify the spleen cell removal effect of GFPNb-Crunch using mice transplanted with spleen cells. b-f are wild-type spleen cells (WT) or GFPm. + The following shows the results of flow cytometry analysis of GFP-positive cells in the blood and spleen of mice that were intravenously injected with spleen cells and then administered GFPNb-Crunch or PBS, four days after spleen cell injection. b is a dot plot from the flow cytometry analysis, c is a bar graph showing the percentage of GFP-positive cells in the spleen (n=4 mice), d is a bar graph showing the percentage of GFP-positive cells in the blood (n=4 mice), e is a bar graph showing the number of GFP-positive cells in the spleen (n=4 mice), and f is a bar graph showing the number of spleen cells (n=4 mice). g is the protocol of an experiment that investigated the phagocytic induction effect of GFPNb-Crunch using mice transplanted with spleen cells. h and i are GFPm stained with pHrodo Red. + The following shows the results of flow cytometry analysis of phagocytic cells in the spleen of mice that were intravenously injected with spleen cells and then administered GFPNb-Crunch or PBS, 24 hours after spleen cell injection. h is a bar graph showing the percentage of GFP-positive cells in the spleen (n=4 mice), and i is MerTK. + Cells and macrophage cells (F4 / 80 + This is a bar graph (n=4 mice) showing the percentage of pHo-Red positive cells (phagocytosis positive cells) in a cell. All data are shown as mean ± standard deviation. Statistical analysis for c-f was performed using a one-way ANOVA with the Tukey-Kramer t-test. Statistical analysis for h-i was performed using Student's unpaired t-test. Significant differences are indicated by NS P>0.05. This figure shows the tumor suppressor effect of Crunch and its effect on effector cells in melanoma cells. a is GFPm +This is an experimental protocol that investigated the inhibitory effect of GFPNb-Crunch on tumor engraftment and proliferation using an allogeneic transplantation model with B16.F10 melanoma cells. b and c are GFPm + The following shows the results of measuring tumor volume over time in mice that were subcutaneously injected with B16.F10 melanoma cells and then intraperitoneally administered with GFPNb-Crunch or physiological saline. b is the average tumor volume, and c is the tumor volume for each individual mouse (physiological saline n=6, Crunch n=4 mice). d and e show t-SNE (t-stochastic neighbor embedding) of non-malignant cells in the tumor microenvironment (TME) of human melanoma cells. d shows single-cell data (4857 cells, GSE115978, non-malignant cells) including B cells, T cells, cancer-associated fibroblasts (CAFs), endothelial cells, macrophages, NK cells, and CD4 cells. + T cells, CD8 + This is a diagram showing clusters of T cells. 'e' shows MerTK expression in each cell. 'f' to 'h' are 5 × 10⁻⁶ cells. 5 The following are the results of a study in which individual B16.F10 melanoma cells were transplanted into 6-week-old C57BL / 6 female mice. On day 9 after transplantation, the primary tumor was excised and isolated into single cells. These cells were stained with anti-MerTK antibody, CD45 antibody, CD11b antibody, Ly6C antibody, Ly6G antibody, or F4 / 80 antibody and analyzed by flow cytometry. Column f plots the single-cell population with and without anti-MerTK antibody, Column g shows the MerTK expression level in each cell population, and Column h shows the intensity of Ly6C and F4 / 80, or the intensity of Ly6C and Ly6G, as indicators of CD45 expression. + CD11b + Cells and CD45 + CD11b + MerTK + This shows the distribution of cells. This figure demonstrates that Crunch is effective as a therapeutic agent in an allogeneic transplantation model. a is GFPm + This is an experimental protocol that investigated the inhibitory effect of GFPNb-Crunch on tumor engraftment and proliferation using an allogeneic transplantation model with B16.F10 melanoma cells. b and c are GFPm+ Results showing the intraperitoneal administration of GFPNb-Crunch or saline to mice subcutaneously injected with B16.F10 melanoma cells and the measurement of tumor volume over time are presented. b is the average value of tumor volume, and c is the tumor volume for each individual mouse (saline n = 6, Crunch n = 4 mice). d - g are GFPm + In mice subcutaneously injected with B16.F10 melanoma cells, when the tumor size reached 4 mm 3 (7 days after injection of melanoma cells), intraperitoneal administration of GFPNb-Crunch or saline was performed 3 times every 2 days, and the results of measuring the volume and weight of the tumors are shown. d is a macroscopic photograph of the tumor excised on day 16 after injection of melanoma cells, e is the average value of tumor volume, f is the tumor volume for each individual mouse, and g is a bar graph showing the weight of the tumor excised on day 16 after injection of melanoma cells normalized by body weight (saline n = 5, Crunch n = 6 mice). P = 0.00034 on day 10, P = 0.0015 on day 13, and P = 0.0027 on day 16. g is GFPm + Kaplan-Meier curves obtained by intraperitoneal administration of GFPNb-Crunch or saline to mice subcutaneously injected with B16.F10 melanoma cells are shown (saline n = 9, Crunch n = 8 mice). In g, the comparison between groups was performed using the Log-rank test. h and i are GFP + B16.F10 melanoma cells, or GFPm + In mice subcutaneously injected with B16.F10 melanoma cells, when the tumor size reached 4 mm 3 (7 days after injection of melanoma cells), intraperitoneal administration of GFPNb-Crunch or saline was performed 3 times every 2 days, and the results of measuring the volume and weight of the tumors are shown. h is the average value of tumor volume, and i is the tumor volume for each individual mouse. On day 16, GFPm + Compared with the group using B16.F10 melanoma cells and GFPNb-Crunch, GFP + The group using B16.F10 melanoma cells and saline had P = 0.032, GFP +The group using B16.F10 melanoma cells and GFPNb-Crunch had P = 0.023, GFPm + The group using B16.F10 melanoma cells and saline had P = 0.015. j is GFPm + This is the protocol of an experiment to verify the inhibitory effect of GFPNb-Crunch on tumor metastasis using an orthotopic transplantation model with B16.F10 melanoma cells. k and l are GFPm + B16.F10 melanoma cells were intravenously injected into mice, and starting from the next day, 100 μg of GFPNb-Crunch or saline was administered 3 times every 2 days. The results of analyzing the lungs of the mice 14 days after the injection of melanoma cells are shown. k is a macroscopic photograph of the excised lungs, and l is a bar graph showing the number of metastatic nodules on the surface of the excised lungs. All data are shown as mean ± standard deviation. The statistics for b, e - i, and l were performed using Student's unpaired t-test. The statistics for h were performed using one-way ANOVA by Tukey-Kramer t-test. The significant differences are indicated as * P < 0.05, ** P < 0.01, *** P < 0.001. This is a figure showing that Crunch containing a single-chain variable fragment (scFv) as a target-binding sequence has an effect of removing target cells. a is a schematic diagram of the structure of scCD19-Crunch containing an scFv against mouse CD19 (mCD19) as a target-binding sequence. In a, SS is the signal sequence, 1D3 VL is the variable light chain region of the scFv, 1D3 VH is the variable heavy chain region of the scFv, and E refers to the EGF-like domain. b is the structure of scCD19-Crunch predicted by AlphaFold2. The variable light chain region (1D3 VL) is shown in red, the variable heavy chain region (1D3 VH) is shown in blue, and the other regions are shown in green. c is MerTK under the condition of the presence or absence of scCD19-Crunch + NIH3T3 cells and BDKO cells or GFPm + After co-culturing BDKO cells, MerTK +The results of Western blotting performed using anti-phospho-MerTK antibody (top panel), anti-MerTK antibody (middle panel), and anti-α-tubulin antibody (bottom panel) after subjecting lysates of NIH3T3 cells to SDS-PAGE are shown. d shows the results of Western blotting using anti-phospho-MerTK antibody (top panel), anti-MerTK antibody (middle panel), and anti-α-tubulin antibody (bottom panel) after staining wild-type mouse thymocytes with pHrodo-Red. + Co-cultured with NIH3T3 cells in serum-free medium, MerTK + This shows the results of measuring pHrodo-Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells. The bar graph shows the results for NIH3T3 cells and MerTK after co-culture. + This shows the percentage of pHrodo-Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells (n=3). e-h show the results of flow cytometry analysis of spleen cells and blood cells 3 days after intraperitoneal administration of PBS or 100 μg of scCD19-Crunch to mice. e is CD45 + CD19 contained in cells + B220 + Dot plot showing B cells; f represents CD45 cells found in the spleen and blood. + CD19 in cells + B220 + The bar graph shows the percentage of B cells, g is the total number of spleen cells, and h is the CD19 cells contained in the spleen. + B220 + This is a bar graph showing the number of B cells (n=4 mice). 'i' represents mice injected intraperitoneally (ip) or intravenously (iv) with 100 μg of scGFP-Crunch, scCD19-Crunch, or PBS, and the blood CD45 count was measured 3 days after injection. + CD19 contained in cells + B220 + The results of flow cytometry analysis of B cells are shown (PBS; n=4 mice, Crunch; n=3 mice). 'j' represents the results of intraperitoneal injection (ip) of 100 μg of scGFP-Crunch, scCD19-Crunch, anti-CD19 antibody (1D3), or PBS into mice, and blood CD45 levels from day 1 to day 7 after injection. +B220 contained in cells + The results of flow cytometry analysis of B cells (n=3 mice) are shown. k and l are the results of (1) a single intraperitoneal injection of 25 μg, 50 μg, 100 μg, 200 μg, or 300 μg of scCD19-Crunch into the peritoneal cavity of mice, (2) three intraperitoneal injections of 100 μg of scCD19-Crunch at 12-hour intervals (the third injection was after sample collection on day 1), (3) a single intraperitoneal injection of 100 μg of anti-CD19 antibody (1D3) into the peritoneal cavity of mice, followed by a single intraperitoneal injection of 100 μg of mouse anti-rat κ light chain antibody (100Abs) the following day, or (4) a single intraperitoneal injection of 300 μg of anti-CD19 antibody (1D3) into the peritoneal cavity of mice, followed by blood CD45 up to day 7 from the injection. + CD19 contained in cells + B220 + This is the result of flow cytometry analysis of B cells (n=3 mice). k represents the results for all groups on day 3, and l represents the results for mice administered 0–300 μg of scCD19-Crunch on days 1–3. m represents 1 × 10⁻⁶ 11 The vg AAV9-EF1-tagRFP (RFP) or AAV-EF1-scCD19-Crunch (Crunch) was injected intravenously (iv) into mice, and blood CD45 levels were measured at 1 week (P=0.39), 4 weeks (P=0.0030), 8 weeks (P=0.0017), 12 weeks (P=0.0012), 16 weeks (P=0.00067), 20 weeks (P=0.0000047), and 24 weeks (P=0.0000044). + CD19 contained in cells + B220 +This figure shows the results of flow cytometry analysis of B cells (week 1: n=3 mice, weeks 4-24: RFP n=3 mice, Crunch n=4 mice). All data are shown as mean ± SD. Statistics for d, f-h, and m were performed using Student's unpaired t test. Statistics for i were performed using one-way ANOVA with Tukey-Kramer t test. Significance is indicated by NS P>0.05 and *P<0.00001. This figure shows the effect of Crunch, which includes scFv as the target binding region. Figure a shows the results of flow cytometry analysis of scCD19-Crunch binding to spleen cells and CD19 expression in spleen cells using anti-FLAG antibody and anti-CD19-APC antibody, respectively, after incubation of C57BL / 6 mouse spleen cells with 10 μg / ml scCD19-Crunch. b shows the results of co-culturing spleen cells with macrophages in D-MEM with 10 μg / ml scCD19-Crunch, followed by staining with anti-CD11b-APC antibody and analysis of the macrophages by flow cytometry. c shows the results of measuring the thermal stability of scGFP-Crunch by differential scanning fluorescence (DSF) scan. d shows the results of analyzing the binding of scGFP-Crunch to GFP on a GFP-coated plate by ELISA. e shows GFPm + BDKO cells were incubated with 10 μg / ml GFPNb-Crunch, scGFP-Crunch, or Gla del mProS, and the GFPm of these proteins was obtained. + This is the result of detecting binding to BDKO cells using flow cytometry with an anti-FLAG antibody. f is ROSA26. GFPm-OVA Mouse-derived thymocytes were stained with pHrodo-Red, and then MerTK was applied in the presence of 10 μg / ml of GFPNb-Crunch (GFPNb), scCD19-Crunch (scCD19), scGFP-Crunch (scGFP), or PBS. + Co-culture with NIH3T3 cells and MerTK +This shows the results of measuring pHrodo Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells (n=3 independent biological samples: thymocytes from different mice). g is the result of predicting the epitopes of mProS and scGFP-Crunch using BepiPred-2.0. h is the result of intravenous administration of 150 μg of GFP, GFPNb-Crunch, GFPNb (anti-GFP nanobody), scGFP-Crunch, or Gla domain-deficient mProS (Gla del mProS) to mice, followed by plasma collection 24 days later, and measurement of antibodies against GFP, GFPNb-Crunch, GFPNb, and Gla del mProS in the plasma by ELISA (GFPNb-Crunch n=4 mice, scGFP-Crunch n=3 mice, GFP n=3 mice). All data are shown as mean ± SD. Statistics for f and h were calculated using one-way ANOVA with the Tukey-Kramer t-test. Significance is indicated by NS P>0.05, *P<0.00001. This figure compares scCD19-Crunch and anti-CD19 antibodies. a is representative data from the putt on day 3 in Figure 14j. b is a histogram obtained by measuring the anti-rat IgG-488 (1D3-bound) signal in DAPI-lymphocytes after incubation of mouse blood cells treated with anti-CD19 antibody (1D3) with anti-rat IgG-488 antibody. c is a histogram obtained by incubation of mouse blood cells treated with anti-CD19 antibody (1D3) with anti-rat IgG-488 antibody, anti-CD45-PE antibody, anti-CD19 APC antibody, or anti-B220-PE-Cy7 antibody, and then measuring the CD45 +This is a dot plot showing the measurement of 1D3-bound cells contained in cells. d is a schematic diagram of an adeno-associated virus 9 (AAV9) vector into which scCD19-Crunch or tagRFP is inserted. In this AAV9, scCD19-Crunch or tagRFP is incorporated so that it can be expressed under the control of the EF1 core promoter. e and f show the results of incubating AAV9-EF1-tagRF at different titers in the human neuroblastoma cell line SH-SY5Y and measuring tagRFP by flow cytometry. + This is the result of analyzing a population of cells. e is a histogram, and f contains tagRFP. + This is a plot of cells and viral titers. g and h represent AAV9-EF1-scCD19-Crunch or AAV9-EF1-tagRF at 1 × 10⁻¹⁰ 11 This is the result of analyzing liver cells and blood cells two weeks after intravenous injection of vg into mice. g stands for tagRFP. + This is a histogram of liver cells, where h represents the CD19 cells contained in CD45+ cells. + B220 + This is a dot plot of B cells. i and j show the results of analyzing the scCD19-Crunch in plasma collected 24 weeks after injection from mice injected with AAV9-EF1-tagRFP or AAV9-EF1-scCD19-Crunch. i is CD19 +The results of analyzing scCD19-Crunch in plasma using a Crunch binding assay with BDKO are shown, with j representing the calculated plasma scCD19-Crunch concentration (RFP n=3 mice, scCD19-Crunch n=4 mice). Data are shown as mean ± SD. Statistical analysis was performed using Student's unpaired t-test. Figure 14k shows the dose-dependency of B cell removal by scCD19-Crunch. Specifically, (1) 25 μg, 50 μg, 100 μg, 200 μg, or 300 μg of scCD19-Crunch is injected once into the peritoneal cavity of a mouse; (2) 100 μg of scCD19-Crunch is injected three times into the peritoneal cavity of a mouse at 12-hour intervals (the third injection is after sample collection on day 1); (3) 100 μg of anti-CD19 antibody (1D3) is injected once into the peritoneal cavity of a mouse, followed by a single injection of 100 μg of mouse anti-rat κ light chain antibody into the peritoneal cavity the following day (100 μg + κ-rat kappa); or (4) 300 μg of anti-CD19 antibody (1D3) is injected once into the peritoneal cavity of a mouse, and blood CD45 levels are monitored until day 7 after injection. + CD19 contained in cells + B220 + This is the result of flow cytometry analysis of B cells (n=3 mice). 'a' shows the blood CD45 levels from day 1 to day 7 after injection. + CD19 contained in cells + B220 + This is the result of flow cytometry analysis of B cells. b represents the amount of B220 in the blood. + The MFI of CD19 in B cells was compared to the B220 in the blood of mice that were not injected. + This is a figure normalized by MFI for CD19 in B cells. c is representative data from day 2 after injection. d is the data for the day shown and the previous day (day 0 is CD45). +This is a scatter plot of the difference in the ratio of B cells (assuming a 55% B cell ratio) and the normalized MFI of CD19. In d, the X-axis indicates that a larger value indicates a decrease in scCD19-Crunch binding. This figure shows the results of the B cell removal assay and AAV assay for scCD19-Crunch in plasma. a and b show the results of analyzing B cells and scCD19-Crunch in the blood 24 hours after 100 μg of scCD19-Crunch was injected intraperitoneally into mice (n=3 mice). a is CD45 + b is the percentage of B cells contained in the cells, and c is the plasma scCD19-Crunch concentration. c is the plasma scCD19-Crunch concentration and CD45 after injection of AAV9-EF1-RFP or AAV9-EF1-scCD19-Crunch. + CD19 contained in cells + B220 + This is a scatter plot showing the proportion of B cells (RFP; n=3, Crunch n=4). This figure shows the results for Crunch, which targets endogenous proteins for disease treatment. a is a figure of AlphaFold2 structural prediction of the scFv of anti-TYRP antibody (20D7S, purple) and anti-CD19 antibody (1D3, gray). b is the result of staining B16.F10 melanoma cells with scTYRP1-Crunch (right is scTYRP1-Crunch, left is PBS) or anti-TYRP1 antibody (TA99) (right is TA99, left is PBS). c-g are the results of subcutaneous injection of B16.F10 melanoma cells into mice with a tumor size of 4 mm. 3 From the point at which the tumor reached a certain stage, scTYRP1-Crunch, anti-TYRP1 antibody (TA99), or physiological saline were administered intraperitoneally every two days for a total of three times, and the tumor volume, tumor weight, and body weight were measured (n=4 mice; one mouse treated with scGFP-Crunch died 15 days after injection). c is the average tumor volume for all mice, d is the tumor volume for each individual mouse, and e is the body weight of the mouse excluding tumor weight. h-j are 8-week-old MRL mice, a spontaneous SLE model mouse. lpr / lprThe following shows the results of flow cytometry analysis of spleen cells and blood cells after intraperitoneal administration of 150 μg of scCD19-Crunch, scGFP-Crunch, or physiological saline to mice twice a week until 13 weeks of age. The experimental protocols for h-j are shown in Figure 20a. h is CD45 + CD19 contained in cells + B220 + Dot plot showing B cells; i represents CD45 cells found in the spleen and blood. + CD19 in cells + B220 + A bar graph showing the percentage of B cells; j represents CD19 contained in the spleen. + B220 + This is a bar graph showing the number of B cells (n=3 mice). k is an image of a glomerular region section prepared from a kidney excised after the above test, stained with hematoxylin and eosin (H&E) or DAPI. The original magnification is 400x. The bars represent 50 μm. l is an image of a glomerular region section prepared from a kidney excised after the above test and from a kidney excised from a wild-type MRL mouse, stained with DAPI and anti-mouse IgG-Alexa647. The original magnification is 400x. The bars represent 50 μm. m is the result of measuring the UPC (urinary protein-creatinine ratio) after urine samples were collected after the above test was completed (physiological saline; n=7 mice, scGFP-Crunch; n=3 mice, scCD19-Crunch; n=9 mice). n and o are the results of measuring ANA (antinuclear antibody) and anti-dsDNA antibody in serum collected at the end of the above test by ELISA (physiological saline; n=7 mice, scGFP-Crunch; n=3 mice, scCD19-Crunch; n=9 mice). All data are shown as mean ± standard deviation. Statistics for c, f, g, i, j, m-o were calculated using one-way ANOVA with Tukey-Kramer t-test. Significant differences are indicated by NS P>0.05, *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001, *****p<0.00001, ******p<0.000001. This figure shows a model diagram and biological data for the treatment of SLE using scCD19-Crunch. a is an 8-week-old MRL, a spontaneously developing SLE model mouse. lpr / lprThis is the protocol for experiments using mice. Figure b is a bar graph showing the number of spleen cells in mice after treatment (MRL n=1, saline n=3, Crunch n=3). Figure c is a bar graph showing the body weight of mice after treatment. Figure d is a bar graph showing the spleen weight (normalized by body weight) of mice after treatment. Figures e and f are histopathological scores of glomerular lesions. Figure e is a score calculated from the average of 5-6 glomeruli in each mouse (saline n=7 mice, scGFP-Crunch n=3 mice, scCD19-Crunch n=9 mice). Figure f is the amount of IgG deposition in glomerular lesions measured by Alexa647 MFI in Figure 19l (saline n=4 mice, scGFP-Crunch n=3 mice, scCD19-Crunch n=6 mice). In figures b-e, MRL refers to wild-type MRL mice used as controls, and Saline refers to MRL mice administered with saline. lpr / lpr Mice, scGFP is MRL treated with scGFP-Crunch. lpr / lpr Mice, scCD19 was treated with scCD19-Crunch in MRLs. lpr / lprThis refers to mice. Figures b-d all represent MRL (n=1 mice), saline (n=3 mice), scGFP-Crunch (n=3 mice), and scCD19-Crunch (n=9 mice). Compared to scCD19-Crunch, saline (e: P=0.00000081, f: P=0.0019) and scGFP-Crunch (e: P=0.00025, f: P=0.0041) are shown. All data are shown as mean ± standard deviation. Statistics for b-f were performed using one-way ANOVA with Tukey-Kramer t-test. Significant differences are indicated by NS P > 0.05 and *P < 0.01. This figure shows the results of modifying Crunch to suppress phagocytosis inhibition by C4BP. Figure a shows the results of predicting the three-dimensional structure of the human C4BPB Sushi1 domain bound to the mouse Pros LG-like domain, and the three-dimensional structure of the mouse Pros LG-like domain bound to MerTK Ig-like domain 1, using Alphafold, and predicting the site in the mouse Pros LG-like domain that is involved in binding to the human C4BPB Sushi1 domain but not in binding to MerTK Ig-like domain 1. The amino acid numbers shown in the lower panel of a (e.g., Glu21) correspond to the amino acid numbers of mouse ProS (SEQ ID NO: 3). Figure b shows the results of detecting Sushi-Fc bound to GFPNb-Crunch after adding Sushi-Fc to each well of a 96-well plate coated with each GFPNb-Crunch and incubating. Figure c shows ROSA26 GFPm-OVA After staining mouse-derived thymocytes with pHrodo-Red, NIH3T3 cells expressing mouse MerTK (mMerTK) were selected in the presence or absence of GFPNb-Crunch and Sushi-Fc. + After co-culturing with NIH3T3 cells in a medium containing 5% FBS, mMerTK was obtained after co-culturing. +This bar graph shows the percentage of pHrodo Red-positive cells (phagocytosis-positive cells) in NIH3T3 cells (n=2). In b and c, WT refers to GFPNb-Crunch (SEQ ID NO: 20) without amino acid substitution, while A432A, 465A, 3A, 3A432A, and 3A465A refer to GFPNb-Crunch with specified amino acid substitutions. In c, "Ctrl" refers to the condition in which PBS was added instead of GFPNb-Crunch, "-" refers to the condition without Sushi-Fc, and "+" refers to the condition with Sushi-Fc added. This figure shows the relationship between the structure and function of Crunch. a is a schematic diagram of the structure of six Crunch cells (GP1-6) with different numbers of EGF-like domains. b is the result of evaluating the secretion amount of GP1-6 by performing a Crunch binding assay using the culture supernatant of HEK293T cells transfected with nucleic acids encoding GP1-6. The bar graph in b shows the mean fluorescence intensity (MFI) measured by the Crunch binding assay. c shows MerTK-sfGFP in the presence of the culture supernatant of HEK293T cells transfected with nucleic acids encoding GP1-6. + NIH3T3 cells and BDKO cells or GFPm + Co-culture with BDKO cells and MerTK-sfGFP + The following shows the results of flow cytometry analysis of GFP fluorescence in NIH3T3 cells. The bar graph labeled 'e' represents MerTK-sfGFP. + This shows the percentage of GFP-positive cells in NIH3T3 cells. The diagram shows that four GFPNb-Crunch proteins (GP1-GP4) with different numbers of EGF-like domains phosphorylate MerTK. a) shows GFPm with GP1-GP4 attached. + BDKO cells and MerTK + After co-culturing NIH3T3 cells, MerTK + The results of Western blotting using phospho-MerTK antibody and anti-MerTK antibody after subjecting the lysate of NIH3T3 cells to SDS-PAGE are shown. Figure b shows the result of calculating the ratio of phospho-MerTK to MerTK (phospho-MerTK / MerTK) from the image shown in figure a, using ImageJ to obtain fluorescence intensity data for phospho-MerTK and MerTK. In figures a and b, "nt" refers to GFPNb without GFPNb-Crunch binding. + This shows a negative control using BDKO cells. The data in b are shown as mean ± standard deviation. This figure shows the results of producing scFAP-Crunch and scPDGFRβ-Crunch. a shows the results of purifying scFAP-Crunch or scPDGFRβ-Crunch from the culture supernatant of HEK293T cells transfected with scFAP-Crunch or scPDGFRβ-Crunch using an anti-His antibody, followed by Western blotting with an anti-FLAG antibody. b shows the results of a Crunch binding assay using the culture supernatant of CHO cells transfected with scFAP-Crunch or scPDGFRβ-Crunch, targeting FAP-expressing HEK293T cells, PDGFRβ-expressing HEK293T cells, or mock HEK293T cells. This is a schematic diagram of how the fusion protein of this disclosure bridges the gap between the target (cell) and the phagocytic cell. Figure 25 is merely a schematic diagram, and the sizes of cells, molecules, domains, regions, etc., are not accurately represented.

[0013] 1. Terminology Unless otherwise specified, terms used in this disclosure have the meanings generally understood by those skilled in the art in the fields of medicine, pharmacy, molecular biology, microbiology, organic chemistry, etc. If a term defined herein does not have the same meaning as it is generally understood, the definition in this disclosure shall prevail.

[0014] In this disclosure, amino acid residues in amino acid sequences are denoted by a single letter abbreviation. Specifically, glycine is G, alanine is A, valine is V, leucine is L, isoleucine is I, phenylalanine is F, tyrosine is Y, tryptophan is W, serine is S, threonine is T, cysteine ​​is C, methionine is M, aspartic acid is D, glutamic acid is E, asparagine is N, glutamine is Q, lysine is K, arginine is R, histidine is H, and proline is P.

[0015] In this disclosure, unless otherwise specified, amino acid sequences are described from left to right, starting from the N-terminus and ending at the C-terminus.

[0016] In this disclosure, "sequence identity" with respect to amino acid sequences refers to the percentage of amino acid residues that match between a reference sequence and a candidate sequence that are optimally aligned (to the state of maximum match). The sequence identity of a candidate sequence to a reference sequence is calculated by the following formula: Sequence identity (%) = [(Number of matching amino acid residues between the reference sequence and the candidate sequence) / (Number of amino acid residues in the reference sequence)] × 100. Additions or deletions (e.g., gaps) may exist in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W, which are available in public databases (e.g., DDBJ (http: / / www.ddbj.nig.ac.jp)).

[0017] In this disclosure, "recognized substance" refers to a substance (such as unwanted cells) that is phagocytosed by a phagocytic cell, and "target" refers to a site contained in the recognized substance that binds to the target binding region of the protein of this disclosure.

[0018] 2. Structure and Sequence of Protein S (ProS) Protein S (ProS) has the function of removing apoptotic cells and consists of a γ-carboxyglutamic acid (Gla) domain, a thrombin-sensitive region (TSR), an epidermal growth factor (EGF)-like domain, and a sex hormone-binding globulin (SHBG)-like domain. The Gla domain recognizes and binds to PtdSer exposed on the cell surface of apoptotic cells. The SHBG-like domain binds to TAM receptors, particularly MerTK and Tyro3 in phagocytic cells. The EGF-like domain contains four domains, EGF1, EGF2, EGF3, and EGF4, from the N-terminus to the C-terminus. The SHBG-like domain contains two laminin G-like domains, laminin G-like domain 1 (LG-like domain 1) and laminin G-like domain 2 (LG-like domain 2), from the N-terminus to the C-terminus.

[0019] The amino acid sequence of human ProS is shown in Sequence ID No. 1. In Sequence ID No. 1, positions 1-24 are the signal sequence, positions 25-41 are the propeptide sequence, positions 42-87 are the Gla domain, positions 88-116 are the thrombin-sensitive region (TSR), positions 117-283 are four EGF-like domains, positions 284-298 are spacers, positions 299-666 are SHBG-like domains, and positions 667-676 are sequences added to the C-terminus. In addition, in the four EGF-like domains in Sequence ID No. 1, positions 117-155 Position 1 corresponds to EGF1, positions 157-200 to EGF2, positions 201-242 to EGF3, and positions 243-283 to EGF4. In addition, in the SHBG-like domain of Sequence ID No. 1, positions 299-475 correspond to LG-like domain 1, positions 476-483 are spacers linking LG-like domain 1 and LG-like domain 2, and positions 484-666 correspond to LG-like domain 2. The base sequence of the cDNA encoding human ProS is shown in Sequence ID No. 2.

[0020] The amino acid sequence of mouse ProS is shown in Sequence ID No. 3. In Sequence ID No. 3, positions 1-24 are the signal sequence, positions 25-41 are the propeptide sequence, positions 42-87 are the Gla domain, positions 88-116 are the TSR, positions 117-283 are the four EGF-like domains, positions 284-298 are the spacer, positions 299-665 are the SHBG-like domain, and positions 666-675 are the sequence added to the C-terminus. Furthermore, in the four EGF-like domains in Sequence ID No. 3, positions 117-155 correspond to EGF1, positions 157-200 to EGF2, positions 201-242 to EGF3, and positions 243-283 to EGF4. Furthermore, in the SHBG-like domain in Sequence ID No. 3, positions 299-475 correspond to LG-like domain 1, positions 476-483 are spacers linking LG-like domain 1 and LG-like domain 2, and positions 484-665 correspond to LG-like domain 2. The nucleotide sequence of the cDNA encoding mouse ProS is shown in Sequence ID No. 4.

[0021] 3. Protein Targets In this disclosure, the target substance can be anything that needs to be removed from the body. The type of target substance is not particularly limited, but examples include unwanted cells, viruses, bacteria, and oxidized low-density lipoprotein (oxidized LDL).

[0022] Unwanted cells that are recognized are cells that need to be removed from the body. The types of unwanted cells are not particularly limited, but examples include cancer cells, autoreactive B cells, autoreactive T cells, IgE-producing B cells, activated cardiac fibroblasts and other disease-causing cells; and senescent cells.

[0023] The types of viruses to be recognized are not particularly limited, as long as they are those that need to be removed from the body, but examples include human immunodeficiency virus, poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus, rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, hepatitis B virus, parvovirus, herpesvirus, varicella-zoster virus, cytomegalovirus, herpesvirus, smallpox virus, vaccinia virus, poxvirus), hepatitis C virus, SARS-CoV-2, influenza virus, etc.

[0024] The types of bacteria to be recognized are not particularly limited, as long as they are those that need to be removed from the body, but examples of infectious bacteria include Helicobacter pylori, Salmonella, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium avianum, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocystis, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria tetanus, and Bordetella pertussis.

[0025] Among these recognized substances, unwanted cells are a preferred example of a target for removal by the protein of this disclosure.

[0026] 4. Protein Structure and Sequence The protein of this disclosure has (A) a target-binding region that binds to a target, and (C) an SHBG-like domain of ProS. Preferably, the protein of this disclosure has (A) a target-binding region that binds to a target and (C) an SHBG-like domain of ProS arranged from the N-terminus to the C-terminus. Preferably, the protein of this disclosure further contains (B) 2 to 4 EGF-like domains of ProS, and more preferably, the 2 to 4 EGF-like domains of (B) ProS are arranged between (A) the target-binding region that binds to a target and (C) the SHBG-like domain of ProS.

[0027] The target-binding domain of the protein disclosed binds to a target contained in the substance to be recognized (e.g., unwanted cells). On the other hand, the SHBG-like domain of the protein disclosed binds to TAM family receptors located on the cell membrane of phagocytic cells. Through these two bindings, the protein disclosed functions as a connector bridging the gap between the substance to be recognized and the phagocytic cell, resulting in the substance being recognized by the phagocytic cell. The binding of the SHBG-like domain to the TAM family receptor then triggers phagocytosis of the substance by the phagocytic cell. As an example of this mechanism, as shown in Figure 25, two molecules of the protein disclosed bind to targets contained in the substance to be recognized and associate with each other. In this state, they bind to the TAM family receptor of the phagocytic cell, inducing dimerization. This activates or suppresses the signaling pathway from the TAM family receptor, thereby inducing phagocytosis of the substance by the phagocytic cell. The protein disclosed will be described in detail below.

[0028] [(A) Target-binding region] The proteins of this disclosure include a target-binding region that binds to a target contained in the substance to be recognized. For example, if the substance to be recognized is an unwanted cell, the target-binding region may be composed of a molecule that can specifically bind to a protein present on the surface of the unwanted cell. However, the target-binding region is other than the Gla domain of ProS.

[0029] If the target substance is a non-essential cell, the target proteins present on the surface of the non-essential cell may be appropriately selected from known proteins. For example, if the non-essential cell is a cancer cell, potential target proteins present on the surface of the cancer cell include 5T4, α5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin, Bcr-abl, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27, CD30, CD33, CD52, CD56, CD80, CDK4, C EA, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / neu, HPV-E7, HSP70, HST-2, hTERT, hTRT, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski, MC1R, Myosin, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, Proteinase-3, minor Examples of target proteins include BCR-ABL, PD-L1, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1, RU2, SAGE, SART-1, SART-3, Survivin, TEL / AML1, TGFβ, TPI, TRP-1, TRP-2, VEGF, WT1, NY-Eso-1, and NY-Eso-B. If the unwanted cells are autoreactive B cells, potential target proteins present on the surface of autoreactive B cells include CD19 and CD20. If the unwanted cells are autoreactive T cells, potential target proteins present on the surface of autoreactive T cells include CD3 and CD7. If the unwanted cells are IgE-producing B cells, potential target proteins present on the surface of IgE-producing B cells include IgE. If the unwanted cells are senescent cells, potential target proteins present on the surface of senescent cells include urokinase-type plasminogen activator receptors (uPARs).If the unwanted cells are activated or fibrous fibroblasts, potential target proteins present on the surface of these fibroblasts include FAP, PDGFRα, and PDGFRβ.

[0030] The type of constituent molecule of the target-binding domain is not particularly limited, as long as it can bind to the target. Examples include proteins (with 50 or more amino acid residues) and peptides (with fewer than 50 amino acid residues). Furthermore, the target-binding domain may be modified by adding DNA, RNA, small molecule compounds, etc. (More details on modification will be provided later).

[0031] If the target-binding region is composed of a protein, an example of such a target-binding region is a domain having the amino acid sequence of the antigen-binding region of an antibody that binds to a target. Specifically, examples include domains having the amino acid sequences of antibodies such as single-chain antibodies (scFv, single-chain Fv), Fab fragment antibodies, VHH antibodies (nanobody, Variable domain of heavy chain of heavy chain antibody), and VNAR (variable region of cartilaginous fish-derived heavy chain antibodies). Techniques for producing these antibodies are well known, and those skilled in the art can design the amino acid sequences of these antibodies according to the type of target protein (antigen). Furthermore, it is preferable that the scFv and Fab fragment antibodies used as target-binding regions correspond to the species of organism to which the protein disclosed herein is applied. For example, if the protein disclosed herein is applied to humans, it is preferable that the scFv and Fab fragment antibodies are human antibodies or fragments of humanized antibodies.

[0032] If the target-binding domain is composed of a protein, other examples of such target-binding domains include a receptor that binds to a ligand present as a target on the surface of unwanted cells that are to be recognized, or a domain having the amino acid sequence of the ligand-binding domain of such receptor; or a domain having the amino acid sequence of a ligand that binds to a receptor present as a target on the surface of unwanted cells that are to be recognized. Specific examples of receptors and ligands used as constituent molecules of the target-binding domain include CD4 ectodomain, CD8α, CD8β, CD11A, CD11B, CD11C, CD18, CD29, CD49A, CD49B, CD49D, CD49E, CD49F, CD61, CD41, CD51, TCR (TCRα, TCRβ, TCRγ, TCRδ), FcεR, etc. It is preferable that the receptor or its ligand-binding domain and ligand used as the target-binding domain correspond to the species of organism to which the protein of this disclosure is applied. For example, if the protein of this disclosure is applied to humans, it is preferable that the receptor or its ligand-binding domain and ligand that constitute the target-binding domain are of human origin.

[0033] If the target binding region is composed of a peptide, then the target binding region specifically refers to a domain having an amino acid sequence such as an HLH peptide (a molecular target peptide with a helix-loop-helix structure). The technology for producing HLH peptides is well known, and those skilled in the art can design the amino acid sequence of the HLH peptide according to the type of target protein (antigen).

[0034] Furthermore, if the target binding region is modified by DNA or RNA, the DNA or RNA is specifically in the form of an aptamer. Techniques for producing aptamers are well known, and those skilled in the art can design the nucleotide sequence of the aptamer according to the target.

[0035] [(C) SHBG-like domain of ProS] The protein disclosed herein contains the SHBG-like domain of ProS. If the protein disclosed herein does not contain the (B) EGF-like domain of ProS described later, the target binding region may be linked to either the N-terminal or C-terminal side of the SHBG-like domain of ProS. If the protein disclosed herein contains the (B) EGF-like domain of ProS described later, it is preferable that the target binding region, the EGF-like domain of ProS, and the SHBG-like domain of ProS are arranged from the N-terminus to the C-terminus. The SHBG-like domain of ProS plays a role in binding to TAM receptors present on the surface of phagocytic cells.

[0036] The amino acid sequence of the SHBG-like domain is preferably corresponding to the species of organism to which the protein disclosed herein is applied. For example, if the protein disclosed herein is applied to humans, the SHBG-like domain is preferably a human-derived amino acid sequence. Specifically, examples of the SHBG-like domain amino acid sequences include the amino acid sequence shown in SEQ ID NO: 23 for human ProS and the amino acid sequence shown in SEQ ID NO: 24 for mouse ProS.

[0037] The amino acid sequence of the SHBG-like domain may be the wild-type amino acid sequence, or it may be modified. Examples of modified sequences of the SHBG-like domain of ProS include the amino acid sequences shown in (1) below: (1) Amino acid sequences having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 24

[0038] In the amino acid sequence described in (1) above, the sequence identity with respect to the corresponding pre-modification amino acid sequence should be 80% or higher, but more specifically, it should be 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0039] The amino acid sequence in (1) above only needs to have a function equivalent to or better than the corresponding pre-modification amino acid sequence (SEQ ID NO: 23 or 24). Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that in a protein with the same sequence except for the amino acid sequence of the SHBG-like domain, the case in which the SHBG-like domain contains the amino acid sequence in (1) above has a target binding ability and / or binding ability to phagocytic cells that is equivalent to or better than the case in which the SHBG-like domain contains the pre-modification amino acid sequence (SEQ ID NO: 23 or 24). For example, when the "Crunch binding assay (condition 1)" described below is performed on a control protein having the amino acid sequence shown in SEQ ID NO: 23 or 24 as an SHBG-like domain, and a control protein in which the SHBG-like domain of the control protein has been replaced with a control sequence, if the average fluorescence intensity (MFI) of the control protein is set to 100% and the average fluorescence intensity of the control protein is 80% or higher (preferably 90% or higher), and / or when the "Split GFP assay" shown in the Examples section is performed, if the percentage of GFP-positive cells for the control protein is set to 100% and the percentage of GFP-positive cells for the control protein is 80% or higher (preferably 90% or higher), then the control sequence is determined to have a function equivalent to or better than the original amino acid sequence (SEQ ID NO: 23 or 24). <Crunch binding assay (condition 1)> (1) Prepare cells that express the target on the cell membrane. (2) If a tag peptide is not attached to the C-terminus of the protein, prepare a protein to be measured with a tag peptide attached to the C-terminus. (3) Add 5 × 10 cells to 100 μl of DPBS (Dulbecco's Phosphate Buffered Saline; containing 1 mg / ml BSA) containing 10 μg / ml of tagged protein. 4 (4) Add the cells and incubate on ice for 30 minutes, then collect the cells. (4) Use an anti-tagged antibody to measure the amount of tagged protein bound to the collected cells, and this amount of tagged protein is taken as the measurement value.

[0040] When evaluating a modified amino acid sequence of the sequence shown in SEQ ID NO: 23, the protein with the amino acid sequence shown in SEQ ID NO: 19 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 19 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - four EGF-like domains of human ProS - spacer B - SHBG-like domain of human ProS (positions 349-716 of SEQ ID NO: 23 and SEQ ID NO: 19) - C-terminal sequence of human ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0041] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 24, the protein with the amino acid sequence shown in Sequence ID No. 20 is used as a control protein. The amino acid sequence shown in Sequence ID No. 20 (control protein) is an amino acid sequence consisting of, from the N-terminus to the C-terminus, the signal sequence of mouse CD8a - EF - anti-GFP VHH antibody - four EGF-like domains derived from mouse - spacer B (positions 349-715 of Sequence ID No. 14 and Sequence ID No. 20) - SHBG-like domain derived from mouse ProS - C-terminal sequence of mouse ProS - EF - His tag - EF - Flag tag.

[0042] Since C4BPB (C4b-binding protein) is known to exhibit ProS-dependent phagocytic inhibitory activity, the amino acid sequence of the SHBG-like domain in the protein disclosed herein may be modified to avoid C4BP binding in order to exert a more effective phagocytic effect.

[0043] The modifications introduced to avoid C4BP binding in the amino acid sequence of the SHBG-like domain are not particularly limited, but for example, in the amino acid sequence shown in SEQ ID NO: 23 or 24, at least one of the lysine at position 16, arginine at position 18, lysine at position 126, arginine at position 134, and glutamic acid at position 167 may be replaced with another amino acid. Specifically, the types of amino acids to be substituted for each of the amino acids at positions 16, 18, 126, 134, and 167 include neutral amino acids, preferably alanine, glycine, valine, leucine, or isoleucine, and more preferably alanine. The amino acids at positions 16, 18, 126, 134, and 167 in the amino acid sequence shown in SEQ ID NO: 23 or 24 are located in LG-like domain 1 (SEQ ID NO: 36 or 37), which constitutes the SHBG-like domain, and correspond to positions 16, 18, 126, 134, and 167, respectively, in the amino acid sequence (LG-like domain 1) shown in SEQ ID NO: 36 or 37.

[0044] More specifically, the following amino acid sequences (a1), (a2), (b1), (b2), (c1), (c2), (d1), and (d2) are examples of SHBG-like domain amino acid sequences that can avoid C4BP binding. (a1) An amino acid sequence in which the glutamic acid at position 167 of the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (a2) An amino acid sequence in which the glutamic acid at position 167 of the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 24. (b1) An amino acid sequence in which the lysine at position 16, arginine at position 18, and lysine at position 126 are each replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (b2) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 24. (c1) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (c2) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 24.(d1) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, in which case the lysine at position 16, arginine at position 18, lysine at position 126, and glutamic acid at position 167 are each replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, in which case the amino acid sequence shown in SEQ ID NO: 23 or 24 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23 or 24.

[0045] In the amino acid sequences of (a2), (b2), (c2), and (d2) above, sequence identity with respect to the corresponding pre-modification amino acid sequence should be 80% or higher, but more specifically, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher are acceptable. Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that in a protein with the same sequence except for the amino acid sequence of the SHBG-like domain, the case in which the SHBG-like domain includes (a2), (b2), (c2), or (d2) above has a target binding ability and a binding ability to phagocytic cells that is equivalent to or better than the case in which the SHBG-like domain includes the pre-modification amino acid sequence (the amino acid sequence of (a1), (b1), (c1), or (d1) above). Whether the "target binding ability and binding ability to phagocytic cells are equivalent to or better" can be determined by the method described above.

[0046] [EGF-like domain of (B)ProS] In the protein of this disclosure, it is preferable to include the EGF-like domain of (B)ProS, more preferably to include 1 to 4 or 2 to 4 EGF-like domains of (B)ProS, even more preferably to include 3 or 4, and most preferably to include 4.

[0047] In the protein disclosed herein, if it contains one EGF-like domain, it may be any of EGF1, EGF2, EGF3, and EGF4, but EGF4 is preferred.

[0048] In the protein disclosed herein, if two EGF-like domains are present, it is sufficient for two of them to be selected from EGF1, EGF2, EGF3, and EGF4. The combination is not particularly limited, however, it is preferable that EGF3 and EGF4 are arranged from the N-terminus to the C-terminus.

[0049] Furthermore, in the protein disclosed herein, if three EGF-like domains are present, it is sufficient for three of EGF1, EGF2, EGF3, and EGF4 to be present, and it is preferable that EGF2, EGF3, and EGF4 are arranged from the N-terminus to the C-terminus.

[0050] Furthermore, in the protein of this disclosure, if four EGF-like domains are included, it is preferable that EGF1, EGF2, EGF3, and EGF4 are arranged from the N-terminus to the C-terminus.

[0051] Furthermore, the amino acid sequence of the EGF-like domain is preferably corresponding to the species of organism to which the protein disclosed herein is applied. For example, if the protein disclosed herein is applied to humans, the EGF-like domain is preferably a human-derived amino acid sequence.

[0052] Specifically, examples of amino acid sequences in which two EGF-like domains (EGF3 and EGF4) are linked include the amino acid sequence shown in SEQ ID NO: 9 for human ProS and the amino acid sequence shown in SEQ ID NO: 10 for mouse ProS.

[0053] Specifically, examples of amino acid sequences in which three EGF-like domains (EGF2, EGF3, and EGF4) are linked include the amino acid sequence shown in SEQ ID NO: 11 for human ProS and the amino acid sequence shown in SEQ ID NO: 12 for mouse ProS.

[0054] Specifically, examples of amino acid sequences in which four EGF-like domains (EGF1, EGF2, EGF3, and EGF4) are linked include the amino acid sequence shown in SEQ ID NO: 13 for human ProS and the amino acid sequence shown in SEQ ID NO: 14 for mouse ProS.

[0055] The amino acid sequence of the EGF-like domain may be a wild-type amino acid sequence, or it may be modified. Examples of modified EGF-like domain sequences are the amino acid sequences shown in (2) below. (2) Amino acid sequences that have 80% or more sequence identity with any of the amino acid sequences shown in Sequence ID No. 9 to 14

[0056] In the amino acid sequence described in (2) above, the sequence identity with respect to the corresponding pre-modification amino acid sequence should be 80% or higher, but more specifically, it should be 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0057] The amino acid sequence in (2) above only needs to have a function equivalent to or better than the corresponding pre-modification amino acid sequence (any of 9 to 14). Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that in a protein with the same sequence except for the amino acid sequence of the EGF-like domain, the case in which the EGF-like domain contains the amino acid sequence in (2) above has a target binding ability and / or binding ability to phagocytic cells that is equivalent to or better than the case in which the EGF-like domain contains the pre-modification amino acid sequence (any of SEQ ID NOs. 9 to 14). For example, when the "Crunch binding assay (condition 1)" is performed on a control protein having the amino acid sequence shown in any of sequence numbers 9 to 14 as an EGF-like domain, and a control protein in which the EGF-like domain of the control protein is replaced with a control sequence, if the average fluorescence intensity (MFI) of the control protein is set to 100% and the average fluorescence intensity of the control protein is 80% or higher (preferably 90% or higher), and / or when the "Split GFP assay" shown in the Examples section is performed, if the percentage of GFP-positive cells of the control protein is set to 100% and the percentage of GFP-positive cells of the control protein is 80% or higher (preferably 90% or higher), then the control sequence is determined to have a function equivalent to or better than the original amino acid sequence (any of sequence numbers 9 to 14).

[0058] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 9, the protein with the amino acid sequence shown in Sequence ID No. 15 is used as a control protein. The amino acid sequence shown in Sequence ID No. 15 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - two EGF-like domains of human ProS (positions 167-249 in Sequence ID No. 9 and Sequence ID No. 15) - spacer - SHBG-like domain derived from human ProS - C-terminal sequence of human ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0059] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 10, the protein with the amino acid sequence shown in Sequence ID No. 16 (GP4) is used as a control protein. The amino acid sequence shown in Sequence ID No. 16 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - two mouse-derived EGF-like domains (positions 167-249 in Sequence ID No. 10 and Sequence ID No. 16) - spacer - SHBG-like domain from mouse ProS - C-terminal sequence of mouse ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0060] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 11, the protein with the amino acid sequence shown in Sequence ID No. 17 is used as a control protein. The amino acid sequence shown in Sequence ID No. 17 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - three EGF-like domains of human ProS (positions 167-293 in Sequence ID No. 11 and Sequence ID No. 17) - spacer - SHBG-like domain derived from human ProS - C-terminal sequence of human ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0061] When evaluating a modified amino acid sequence of the sequence shown in SEQ ID NO: 12, the protein with the amino acid sequence shown in SEQ ID NO: 18 (GP3) is used as a control protein. The amino acid sequence shown in SEQ ID NO: 18 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - three mouse-derived EGF-like domains (positions 167-293 in SEQ ID NO: 12 and SEQ ID NO: 18) - spacer - SHBG-like domain from mouse ProS - C-terminal sequence of mouse ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0062] When evaluating a modified amino acid sequence of the sequence shown in SEQ ID NO: 13, the protein with the amino acid sequence shown in SEQ ID NO: 19 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 19 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - four EGF-like domains of human ProS (positions 167-333 of SEQ ID NO: 13 and 19) - spacer B - SHBG-like domain derived from human ProS - C-terminal sequence of human ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0063] When evaluating a modified amino acid sequence of the sequence shown in SEQ ID NO: 14, the protein with the amino acid sequence shown in SEQ ID NO: 20 (GP2) is used as a control protein. The amino acid sequence shown in SEQ ID NO: 20 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - four mouse-derived EGF-like domains (positions 167-333 of SEQ ID NO: 14 and SEQ ID NO: 20) - spacer - SHBG-like domain from mouse ProS - C-terminal sequence of mouse ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0064] [Spacer A between (A) target binding domain and (B) EGF-like domain of ProS] In the protein of this disclosure, if (B) ProS has an EGF-like domain, the C-terminus of the (A) target binding domain and the N-terminus of the (B) ProS EGF-like domain may be directly linked, or they may be linked via one or more amino acids as a spacer (hereinafter referred to as Spacer A).

[0065] When spacer A is provided, the number of constituent amino acids of spacer A is not particularly limited, but examples include 1 to 60, 1 to 50, or 1 to 40.

[0066] The amino acid sequence of spacer A is not particularly limited, but an example of spacer A is the TSR of ProS or a modified sequence thereof. The TSR of human ProS consists of the amino acid sequence shown in SEQ ID NO: 5, and the TSR of mouse ProS consists of the amino acid sequence shown in SEQ ID NO: 6.

[0067] Examples of modified sequences of ProS TSR include the amino acid sequences shown in (3) below. (3) Amino acid sequences that have 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 5 or 6

[0068] In the amino acid sequence described in (3) above, the sequence identity with respect to the corresponding pre-modification amino acid sequence should be 80% or higher, but more specifically, it should be 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0069] The amino acid sequence in (3) above only needs to have a function equivalent to or better than the corresponding pre-modification amino acid sequence (SEQ ID NO: 5 or 6). Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that, in a protein with the same sequence except for the amino acid sequence of spacer A, the case in which spacer A contains the amino acid sequence in (3) above has a binding ability to the target and / or to phagocytic cells that is equivalent to or better than the case in which spacer A contains the pre-modification amino acid sequence (SEQ ID NO: 5 or 6). For example, when the "Crunch binding assay (condition 1)" is performed on a control protein having the amino acid sequence shown in SEQ ID NO: 5 or 6 as spacer A, and a control protein in which spacer A of the control protein is replaced with a control sequence, if the average fluorescence intensity (MFI) of the control protein is set to 100% and the average fluorescence intensity of the control protein is 80% or higher (preferably 90% or higher), and / or when the "Split GFP assay" shown in the Examples section is performed, if the percentage of GFP-positive cells of the control protein is set to 100% and the percentage of GFP-positive cells of the control protein is 80% or higher (preferably 90% or higher), then the control sequence is determined to have a function equivalent to or better than the original amino acid sequence (SEQ ID NO: 5 or 6).

[0070] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 5, the protein with the amino acid sequence shown in Sequence ID No. 7 is used as a control protein. The amino acid sequence shown in Sequence ID No. 7 consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - human ProS-derived TSR (positions 167-195 in Sequence ID No. 5 and Sequence ID No. 7) - four human ProS-derived EGF-like domains - spacer - human ProS-derived SHBG-like domain - human ProS-C-terminal sequence - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0071] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 6, the protein with the amino acid sequence shown in Sequence ID No. 8 is used as a control protein. The amino acid sequence shown in Sequence ID No. 8 consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - TSR from mouse ProS (positions 167-195 in Sequence ID No. 6 and Sequence ID No. 8) - four mouse-derived EGF-like domains - spacer - SHBG-like domain from mouse ProS - C-terminal sequence of mouse ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0072] A preferred example of the protein of this disclosure is that it does not contain spacer A, i.e., (A) the C-terminus of the target binding region and (B) the N-terminus of the EGF-like domain are directly linked.

[0073] [Spacer B between the EGF-like domain of (B)ProS and the SHBG-like domain of (C)ProS] In the protein of this disclosure, if the EGF-like domain of (B)ProS is present, the C-terminus of the EGF-like domain of (B)ProS and the N-terminus of the SHBG-like domain of (C)ProS may be directly linked, but it is preferable that they are linked via one or more amino acids as a spacer (hereinafter referred to as Spacer B).

[0074] When spacer B is provided, the number of constituent amino acids in spacer B is not particularly limited, but examples include 1 to 50, 5 to 50, 10 to 50, 1 to 40, 5 to 40, 10 to 40, 1 to 30, 5 to 30, 10 to 30, 1 to 20, 5 to 20, 10 to 20, or 14 to 16.

[0075] The amino acid sequence of spacer B is not particularly limited, but an example of spacer B is the amino acid sequence of the spacer located between the EGF-like domain and the SHBG-like domain of ProS, or a modified version thereof. In human ProS, the spacer located between the EGF-like domain and the SHBG-like domain consists of the amino acid sequence shown in Sequence ID No. 21. In mouse ProS, the spacer located between the EGF-like domain and the SHBG-like domain consists of the amino acid sequence shown in Sequence ID No. 22.

[0076] Examples of the modified sequences include the amino acid sequences shown in (4) below. (4) Amino acid sequences having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 21 or 22

[0077] In the amino acid sequence described in (4) above, the sequence identity with respect to the corresponding pre-modification amino acid sequence should be 80% or higher, but more specifically, it should be 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0078] The amino acid sequence in (4) above only needs to have a function equivalent to or better than the corresponding pre-modification amino acid sequence (SEQ ID NO: 21 or 22). Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that, in a protein with the same sequence except for the amino acid sequence of spacer B, the case in which spacer B contains the amino acid sequence in (4) above has a target binding ability and / or binding ability to phagocytic cells that is equivalent to or better than the case in which spacer B contains the pre-modification amino acid sequence (SEQ ID NO: 21 or 22). For example, when the "Crunch binding assay (condition 1)" is performed on a control protein having the amino acid sequence shown in SEQ ID NO: 21 or 22 as spacer B, and a control protein in which spacer B of the control protein is replaced with a control sequence, if the average fluorescence intensity (MFI) of the control protein is set to 100% and the average fluorescence intensity of the control protein is 80% or higher (preferably 90% or higher), and / or when the "Split GFP assay" shown in the Examples section is performed, if the percentage of GFP-positive cells of the control protein is set to 100% and the percentage of GFP-positive cells of the control protein is 80% or higher (preferably 90% or higher), then the control sequence is determined to have a function equivalent to or better than the original amino acid sequence (SEQ ID NO: 21 or 22).

[0079] When evaluating a modified amino acid sequence of the sequence shown in SEQ ID NO: 21, the protein with the amino acid sequence shown in SEQ ID NO: 19 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 19 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - four EGF-like domains of human ProS - spacer B (positions 334-348 of SEQ ID NO: 21 and SEQ ID NO: 19) - SHBG-like domain derived from human ProS - C-terminal sequence of human ProS - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0080] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 22, the protein with the amino acid sequence shown in Sequence ID No. 20 is used as a control protein. The amino acid sequence shown in Sequence ID No. 20 (control protein) is an amino acid sequence consisting of, from the N-terminus to the C-terminus, the signal sequence of mouse CD8a - EF - anti-GFP VHH antibody - four EGF-like domains derived from mouse - spacer B (positions 334-348 of Sequence ID No. 14 and Sequence ID No. 20) - SHBG-like domain derived from mouse ProS - C-terminal sequence of mouse ProS - EF - His tag - EF - Flag tag.

[0081] [Additional amino acid sequence at the C-terminus of the SHBG-like domain of (C)ProS] An amino acid sequence (hereinafter referred to as the C-terminal additional sequence) may be attached to the C-terminus of the SHBG-like domain of (C)ProS.

[0082] When a C-terminal addition sequence is provided, the number of constituent amino acids in the C-terminal addition sequence is not particularly limited, but examples include 1 to 50, 5 to 50, 1 to 40, 5 to 40, 1 to 30, 5 to 30, 1 to 20, 5 to 20, 5 to 15, or 8 to 12.

[0083] The amino acid sequence of the C-terminal addition sequence is not particularly limited, but an example of a C-terminal addition sequence is the amino acid sequence or a modified version thereof that is linked to the C-terminal side of the SHBG-like domain of ProS. In human ProS, the amino acid sequence linked to the C-terminal side of the SHBG-like domain is the amino acid sequence shown in SEQ ID NO: 25. In mouse ProS, the amino acid sequence linked to the C-terminal side of the SHBG-like domain is the amino acid sequence shown in SEQ ID NO: 26.

[0084] Examples of the modified sequences include the amino acid sequences shown in (5) below. (5) Amino acid sequences having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 25 or 26

[0085] In the amino acid sequence of (5) above, the sequence identity with respect to the corresponding pre-modification amino acid sequence should be 80% or higher, but more specifically, it should be 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0086] The amino acid sequence in (5) above only needs to have a function equivalent to or better than the corresponding pre-modification amino acid sequence (SEQ ID NO: 25 or 26). Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that, in a protein with the same sequence except for the amino acid sequence of the C-terminal addition sequence, the case in which the amino acid sequence in (5) above is included as the C-terminal addition sequence has a target binding ability and / or binding ability to phagocytic cells that is equivalent to or better than the case in which the pre-modification amino acid sequence (SEQ ID NO: 25 or 26) is included as the C-terminal addition sequence. For example, when the "Crunch binding assay (condition 1)" is performed on a control protein having the amino acid sequence shown in SEQ ID NO: 25 or 26 as the C-terminal addition sequence, and a control protein in which the C-terminal addition sequence of the control protein is replaced with the control sequence, if the average fluorescence intensity (MFI) of the control protein is set to 100% and the average fluorescence intensity of the control protein is 80% or higher (preferably 90% or higher), and / or when the "Split GFP assay" shown in the Examples section is performed, if the percentage of GFP-positive cells of the control protein is set to 100% and the percentage of GFP-positive cells of the control protein is 80% or higher (preferably 90% or higher), then the control sequence is determined to have a function equivalent to or better than the original amino acid sequence (SEQ ID NO: 25 or 26).

[0087] When evaluating a modified amino acid sequence of the sequence shown in SEQ ID NO: 25, the protein with the amino acid sequence shown in SEQ ID NO: 19 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 19 (control protein) consists of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - four EGF-like domains of human ProS - spacer B - SHBG-like domain derived from human ProS - C-terminal addition sequence of human ProS (positions 717-726 in SEQ ID NO: 25 and SEQ ID NO: 19) - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0088] When evaluating a modified amino acid sequence of the sequence shown in Sequence ID No. 26, the protein with the amino acid sequence shown in Sequence ID No. 20 is used as a control protein. The amino acid sequence shown in Sequence ID No. 20 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - four EGF-like domains derived from mouse - spacer B - SHBG-like domain derived from mouse ProS - C-terminal sequence of mouse ProS (positions 716-725 of Sequence ID No. 26 and Sequence ID No. 20) - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0089] [Sequence to be added to the N-terminus of the target binding region] In the protein of this disclosure, (A) one or more amino acids may be added to the N-terminus of the target binding region, as needed.

[0090] The number of amino acids added to the N-terminal side of the target binding region is not particularly limited, but examples include 1 to 60, 1 to 50, or 1 to 40.

[0091] In one embodiment of the fusion protein of this disclosure, a signal sequence is added to the N-terminus of the target binding region. In the protein of this disclosure, when the signal sequence of mouse CD8a is added to the N-terminus of the target binding region, the production amount of the protein of this disclosure can be increased when animal cells (particularly CHO cells) are used as the host. The signal sequence of mouse CD8a is the amino acid sequence shown in SEQ ID NO: 27.

[0092] [Sequences to be added to the C-terminus of the protein] In the protein of this disclosure, if a C-terminal addition sequence is linked to the C-terminus of the (C)SHBG-like domain, a tag peptide such as a His tag or a FLAG® tag may be added to the C-terminus of the C-terminal addition sequence as needed.

[0093] [Preferred Examples of Proteins of the Disclosure] A preferred example of a protein of the Disclosure is a protein in which a target-binding region is attached to the N-terminus of any of the following amino acid sequences. (i) The amino acid sequence shown in SEQ ID NO: 28 (i-1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28 (i-2) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 has glutamic acid at position 349 replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine (i-3) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 has glutamic acid at position 349 replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28 (i-4) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 has lysine at position 198, arginine at position 200, and lysine at position 308 replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine (i-5) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 28, the lysine at position 198, the arginine at position 200, and the lysine at position 308 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28. (i-6) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 28, the lysine at position 198, the arginine at position 200, and the arginine at position 316 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (i-7) An amino acid sequence in which, in the amino acid sequence shown in Sequence ID No. 28, lysine at position 198, arginine at position 200, and arginine at position 316 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 28.(i-8) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (i-9) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (ii) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28. (ii-1) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 29 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (ii-2) An amino acid sequence in which the 309th position of glutamic acid in the amino acid sequence shown in SEQ ID NO: 29 is replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (ii-3) An amino acid sequence in which the 309th position of glutamic acid in the amino acid sequence shown in SEQ ID NO: 29 is replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 29. (ii-4) An amino acid sequence in which the 158th position of lysine, the 160th position of arginine, and the 268th position of lysine are each replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (ii-5) An amino acid sequence in which the amino acid sequence shown in Sequence ID No. 29 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 29.(ii-6) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 29, the lysine at position 158, the arginine at position 160, and the arginine at position 276 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (ii-7) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 29, the lysine at position 158, the arginine at position 160, and the arginine at position 276 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 29. (ii-8) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 29, the lysine at position 158, the arginine at position 160, the lysine at position 268, and the glutamic acid at position 309 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (ii-9) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 29, lysine at position 158, arginine at position 160, lysine at position 268, and glutamic acid at position 309 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 29. (iii) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 30. (iii-1) An amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30. (iii-2) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 30, glutamic acid at position 265 is substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iii-3) An amino acid sequence in which the 265th position of glutamic acid in the amino acid sequence shown in SEQ ID NO: 30 is replaced with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30.(iii-4) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 30 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iii-5) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 30 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30. (iii-6) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 30 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iii-7) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 30, the lysine at position 114, the arginine at position 116, and the arginine at position 232 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30. (iii-8) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 30, the lysine at position 114, the arginine at position 116, the lysine at position 224, and the glutamic acid at position 265 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iii-9) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 30 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30. (iv) An amino acid sequence shown in SEQ ID NO: 31. (iv-1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31.(iv-2) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 31 has glutamic acid at position 349 replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iv-3) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 28 has glutamic acid at position 349 replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31. (iv-4) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 31 has lysine at position 198, arginine at position 200, and lysine at position 308 replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iv-5) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 31, the lysine at position 198, the arginine at position 200, and the lysine at position 308 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31. (iv-6) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 31, the lysine at position 198, the arginine at position 200, and the arginine at position 316 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (iv-7) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 31, the lysine at position 198, the arginine at position 200, and the arginine at position 316 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31. (iv-8) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 31, the lysine at position 198, the arginine at position 200, the lysine at position 308, and the glutamic acid at position 349 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine.(iv-9) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 31, lysine at position 198, arginine at position 200, lysine at position 308, and glutamic acid at position 349 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31. (v) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 32. (v-1) An amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32. (v-2) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 32, glutamic acid at position 309 is substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (v-3) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 32 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32. (v-4) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 32 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, at position 158, arginine at position 160, and lysine at position 268, at position 158, arginine at position 160, and lysine at position 268, at position 158, arginine at position 160, and lysine at position 268, at position 158, arginine, or isoleucine, more preferably alanine, at position 158, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32. (v-6) An amino acid sequence in which the amino acid sequence shown in Sequence ID No. 32 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, in the amino acid sequence shown in Sequence ID No. 32.(v-7) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 32, lysine at position 158, arginine at position 160, and arginine at position 276 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32. (v-8) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 32, lysine at position 158, arginine at position 160, lysine at position 268, and glutamic acid at position 309 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (v-9) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 32, lysine at position 158, arginine at position 160, lysine at position 268, and glutamic acid at position 309 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32. (vi) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33. (vi-1) An amino acid sequence that has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33. (vi-2) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33, glutamic acid at position 265 is substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (vi-3) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 33 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33. (vi-4) An amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 33 is replaced by a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine.(vi-5) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33, the lysine at position 114, the arginine at position 116, and the lysine at position 224 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33. (vi-6) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33, the lysine at position 114, the arginine at position 116, and the arginine at position 232 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (vi-7) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33, the lysine at position 114, the arginine at position 116, and the arginine at position 232 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33. (vi-8) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33, the lysine at position 114, the arginine at position 116, the lysine at position 224, and the glutamic acid at position 265 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine. (vi-9) An amino acid sequence in which, in the amino acid sequence shown in SEQ ID NO: 33, lysine at position 114, arginine at position 116, lysine at position 224, and glutamic acid at position 265 are each substituted with a neutral amino acid, preferably alanine, glycine, valine, leucine, or isoleucine, more preferably alanine, and which has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33.

[0094] The amino acid sequence shown in Sequence ID No. 28 consists of four EGF-like domains of human ProS, a human-derived spacer B, a human-derived SHBG-like domain, and the C-terminal sequence of human ProS, from the N-terminus to the C-terminus. This is the amino acid sequence of human Pros (Sequence ID No. 1) from position 117 onwards.

[0095] The amino acid sequence shown in Sequence ID No. 29 is an amino acid sequence consisting of three EGF-like domains of human ProS, a human-derived spacer B, a human ProS-derived SHBG-like domain, and the C-terminal sequence of human ProS, from the N-terminus to the C-terminus. This is the amino acid sequence of human Pros (Sequence ID No. 1) from position 157 onwards.

[0096] The amino acid sequence shown in Sequence ID No. 30 is an amino acid sequence consisting of two EGF-like domains of human ProS, a human-derived spacer B, a human-derived SHBG-like domain, and the C-terminal sequence of human ProS, from the N-terminus to the C-terminus. This is the amino acid sequence of human Pros (Sequence ID No. 1) from position 201 onwards.

[0097] The amino acid sequence shown in Sequence ID No. 31 is an amino acid sequence consisting of four EGF-like domains of mouse ProS, a mouse-derived spacer B, a human-derived SHBG-like domain, and the C-terminal sequence of mouse ProS, from the N-terminus to the C-terminus. This is the amino acid sequence of mouse Pros (Sequence ID No. 3) from position 117 onwards.

[0098] The amino acid sequence shown in Sequence ID No. 32 is an amino acid sequence consisting of three EGF-like domains of mouse ProS, a mouse-derived spacer B, a human-derived SHBG-like domain, and the C-terminal sequence of mouse ProS, from the N-terminus to the C-terminus. This is the amino acid sequence of mouse Pros (Sequence ID No. 3) from position 157 onwards.

[0099] The amino acid sequence shown in Sequence ID No. 33 is an amino acid sequence consisting of two EGF-like domains of mouse ProS, a mouse-derived spacer B, an SHBG-like domain of mouse ProS, and the C-terminal sequence of mouse ProS, from the N-terminus to the C-terminus. This is the amino acid sequence of mouse Pros (Sequence ID No. 3) from position 201 onwards.

[0100] The sequence identity values ​​specified in (i-1), (i-3), (i-5), (i-7), (i-9), (ii-1), (ii-3), (ii-5), (ii-7), (ii-9), (iii-1), (iii-3), (iii-5), (iii-7), (iii-9), (iv-1), (iv-3), (iv-5), (iv-7), (iv-9), (v-1), (v-3), (v-5), (v-7), (v-9), (vi-1), (vi-3), (vi-5), (vi-7), and (vi-9) above should be 80% or higher, but more specifically, values ​​of 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher are acceptable.

[0101] The amino acid sequences of (i-1), (i-3), (i-5), (i-7), (i-9), (ii-1), (ii-3), (ii-5), (ii-7), (ii-9), (iii-1), (iii-3), (iii-5), (iii-7), (iii-9), (iv-1), (iv-3), (iv-5), (iv-7), (iv-9), (v-1), (v-3), (v-5), (v-7), (v-9), (vi-1), (vi-3), (vi-5), (vi-7), and (vi-9) only need to have a function equivalent to or better than the corresponding pre-modification amino acid sequence (any of sequence numbers 28 to 33). Here, "having a function equivalent to or better than the corresponding pre-modification amino acid sequence" means that in proteins with the same sequence of target binding region, the above (i-1), (i-3), (i-5), (i-7), (i-9), (ii-1), (ii-3), (ii-5), (ii-7), (ii-9), (iii-1), (iii-3), (iii-5), (iii-7), (iii-9), (iv-1), (iv-3), (iv-5), (iv-7), This refers to cases where the target-binding region is linked to the N-terminus of the amino acid sequences (iv-9), (v-1), (v-3), (v-5), (v-7), (v-9), (vi-1), (vi-3), (vi-5), (vi-7), and (vi-9), and the ability to bind to targets and / or phagocytic cells is equal to or greater than that of the original amino acid sequence (any of sequence numbers 28-33) when the target-binding region is linked to the N-terminus.For example, when the "Crunch binding assay (1)" is performed on a control protein in which a target binding region is bound to the N-terminus of the amino acid sequence shown in any of SEQ ID NOs. 28 to 33, and on a control fusion protein in which the amino acid sequence of the control protein is replaced with a control sequence, if the average fluorescence intensity (MFI) of the control protein is set to 100% and the average fluorescence intensity of the control protein is 80% or higher (preferably 90% or higher), and / or when the "Split GFP assay" shown in the Examples section is performed, if the GFP-positive cell percentage of the control protein is set to 100% and the GFP-positive cell percentage of the control protein is 80% or higher (preferably 90% or higher), then the control sequence is determined to have a function equivalent to or better than the original amino acid sequence (any of SEQ ID NOs. 28 to 33).

[0102] When evaluating the amino acid sequences of (i-1), (i-3), (i-5), (i-7), and (i-9), the protein with the amino acid sequence shown in SEQ ID NO: 19 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 19 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - SEQ ID NO: 28 - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0103] When evaluating the amino acid sequences of (ii-1), (ii-3), (ii-5), (ii-7), and (ii-9), the protein with the amino acid sequence shown in SEQ ID NO: 17 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 17 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - SEQ ID NO: 29 - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0104] When evaluating the amino acid sequences of (iii-1), (iii-3), (iii-5), (iii-7), and (iii-9), the protein with the amino acid sequence shown in SEQ ID NO: 15 is used as a control protein. The amino acid sequence shown in SEQ ID NO: 15 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - SEQ ID NO: 30 - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0105] When evaluating the amino acid sequences of (iv-1), (iv-3), (iv-5), (iv-7), and (iv-9), the protein with the amino acid sequence shown in SEQ ID NO: 20 (GP2) is used as a control protein. The amino acid sequence shown in SEQ ID NO: 20 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - SEQ ID NO: 31 - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0106] When evaluating the amino acid sequences of (v-1), (v-3), (v-5), (v-7), and (v-9), the protein with the amino acid sequence shown in SEQ ID NO: 18 (GP3) is used as a control protein. The amino acid sequence shown in SEQ ID NO: 18 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - SEQ ID NO: 32 - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0107] When evaluating the amino acid sequences of (vi-1), (vi-3), (vi-5), (vi-7), and (vi-9), the protein (GP4) with the amino acid sequence shown in SEQ ID NO: 16 is used. The amino acid sequence shown in SEQ ID NO: 16 (control protein) is an amino acid sequence consisting of the mouse CD8a signal sequence - EF - anti-GFP VHH antibody - SEQ ID NO: 33 - EF - His tag - EF - Flag tag, from the N-terminus to the C-terminus.

[0108] Proteins in which a target-binding region (A) is bound to the N-terminus of each of the aforementioned amino acid sequences may have a signal sequence or the like added to the N-terminus of the target-binding region, as described above, and may also have a tag peptide or the like added to the C-terminus of each of the aforementioned amino acid sequences.

[0109] Furthermore, another preferred example of a protein in this disclosure is one in which the measured value of the "Crunch binding assay (condition 1)" is 0.8 times or more, preferably 0.9 times or more, compared to the measured value of the control protein described below. The upper limit of the ratio of the measured values ​​is not particularly limited, but for example, it may be 1.0 times, 1.2 times, or 1.5 times. <Control protein> A control protein is a protein in which a target binding region with the same sequence as the target binding region of the protein to be measured is bound to the N-terminus of the amino acid sequence shown in any of (i) to (vi) above.

[0110] 5. Modification of Proteins The proteins of this disclosure may be bound with other proteins, small molecules, etc., as needed. For example, by binding a drug to the proteins of this disclosure, the reaction after phagocytosis can be controlled.

[0111] 6. Method for producing the protein The protein of this disclosure can be produced by known methods such as genetic engineering methods and organic chemical methods.

[0112] For example, if the constituent molecules of the (A) target binding domain in the protein of the disclosure are proteins or peptides, the nucleic acid encoding the protein of the disclosure can be transfected into a host cell and the host cell can be cultured. The nucleic acid encoding the fusion protein of the disclosure can be easily prepared by those skilled in the art based on the amino acid sequence of the protein. Alternatively, to transfect a host cell with the nucleic acid encoding the protein of the disclosure, a recombinant vector containing the nucleic acid encoding the protein of the disclosure can be used. The recombinant vector should contain a promoter operably linked to the nucleic acid encoding the protein of the disclosure. Operable linkage means that the promoter and the nucleic acid of the disclosure are linked in a manner that allows them to function in the host cell. The host cell may be any of the following: animal cells, yeast, bacteria, insect cells, plant cells, etc., but animal cells are preferred. In particular, if the protein of the disclosure has the signal sequence of mouse CD8a, the protein of the disclosure can be efficiently produced by using CHO cells as the host.

[0113] Furthermore, if the constituent molecule of the target-binding domain (A) in the protein of this disclosure is not a protein or peptide, the protein other than the target-binding domain may be produced by genetic engineering, and then the constituent molecule of the target-binding domain may be attached to the N-terminus of the protein by organic chemical methods or the like.

[0114] 7. Formulation of Proteins The proteins of this disclosure are formulated into desired dosage forms using at least one pharmaceutically acceptable carrier and provided as pharmaceutical compositions. The type of carrier used for formulation is not particularly limited, as long as it is non-toxic to the recipient at the dose and concentration used, and known carriers (RemIngton's Pharmaceutical Sciences, 18) thThe appropriate choice of carrier may be selected from those listed in Ed. Mack Printing Company, 1990, depending on the dosage form. Pharmaceutically acceptable carriers are not limited to these, but include, for example, aqueous media such as sterile water and physiological saline; buffering agents such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); antioxidants such as ascorbic acid and methionine; octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, and benzyl alcohol; preservatives such as methylparaben, propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; and polysorbate. Examples include surfactants such as 80, polysorbate 20, and poloxamer 188; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; sugars such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran; chelating agents such as EDTA and EGTA; low molecular weight (less than approximately 10 residues) oligopeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes; and nonionic surfactants such as polyethylene glycol.

[0115] The dosage form of the pharmaceutical composition containing the protein disclosed herein may be set as appropriate depending on the method of administration, etc., and may be solid, semi-solid, liquid, etc. Examples include parenteral preparations such as injections, infusions, suppositories, aerosol inhalants, eye drops, lotions, gels, sprays, and ointments; and oral preparations such as capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, lozenges, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, and emulsions. Among these, parenteral preparations are a preferred example.

[0116] 8. Uses of the Proteins The proteins of this disclosure can be used to remove targets in vivo by phagocytic cells. The proteins of this disclosure may be used alone if they have one target-binding sequence, or they may be used in combination if they have two or more different target-binding sequences.

[0117] The animals to which the proteins disclosed herein can be applied are not limited to mammals such as humans, mice, rats, guinea pigs, rabbits, hamsters, dogs, cats, weasels, cattle, and pigs. Among these, humans are a preferred example.

[0118] The types of diseases to which the proteins disclosed herein are applicable are not particularly limited, and any disease in which the progression of symptoms can be suppressed, improved, or cured by removing the target is expected.

[0119] For example, if the target of the protein disclosed herein is cancer cells, the protein disclosed herein can be used in the treatment of cancer. The types of cancer are not particularly limited, but include solid cancers such as pancreatic cancer, lung cancer, osteosarcoma, colorectal cancer, colon cancer, gastric cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, melanoma, and brain tumors; and hematological cancers such as leukemia and malignant lymphoma.

[0120] Furthermore, for example, if the target of the protein disclosed herein is autoreactive B cells or autoreactive T cells, the protein disclosed herein can be used to treat autoimmune diseases. The types of autoimmune diseases are not particularly limited, but include, for example, collagen diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, dermatomyositis, polymyositis, systemic sclerosis (scleroderma), and polyarteritis; chronic thyroiditis (Hashimoto's disease), myasthenia gravis, autoimmune hepatitis, and pancreatitis.

[0121] Furthermore, for example, if the target of the protein disclosed herein is IgE-producing B cells, the protein disclosed herein can be used to treat allergic diseases. The types of allergic diseases are not particularly limited, but examples include bronchial asthma, atopic dermatitis, allergic rhinitis, hyper-IgE syndrome, and hay fever.

[0122] Furthermore, for example, if the target of the protein disclosed herein is bacteria, the protein disclosed herein can be used to treat bacterial infections. The types of bacterial infections are not particularly limited, but examples include infections caused by Helicobacter pylori, Salmonella, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium avianum, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocystis, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria tetanus, Bordetella pertussis, and others.

[0123] Furthermore, for example, if the target of the protein disclosed herein is a virus, the protein disclosed herein can be used to treat viral infections. The types of viral infections are not particularly limited, but include, for example, infections caused by human immunodeficiency virus, poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus, rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, hepatitis B virus, parvovirus, herpesvirus, varicella-zoster virus, cytomegalovirus, herpesvirus, smallpox virus, vaccinia virus, poxvirus), hepatitis C virus, SARS-CoV-2, influenza virus, and others.

[0124] The method of administering the protein described herein is not particularly limited and may be set appropriately depending on the type of disease to which it is applied, etc. Examples include oral administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, interarticular administration, intrafocal administration, pulmonary administration, nasal administration, and ophthalmic administration. Among these, intravascular administration, intramuscular administration, subcutaneous administration, and intraperitoneal administration are preferred.

[0125] The dosage of the protein disclosed herein should be appropriately determined according to the type of disease being treated, the patient's age and weight, etc., to determine an effective therapeutic dose. For example, as an example of the dosage of the prophylactic or therapeutic agent disclosed herein, for adults, the protein disclosed herein may be administered once or several times a day at a dose of approximately 0.001 to 100 mg / kg-body weight or approximately 0.01 to 10 mg / kg-body weight per day. The administration interval of the protein disclosed herein should be appropriately set within the range that produces a therapeutic effect, but examples include daily, every other day, weekly, every other week, every two to three weeks, monthly, every other month, or every two to three months.

[0126] 9. A drug comprising a nucleic acid encoding a protein. One embodiment of the nucleic acid drug of the present disclosure comprises the nucleic acid encoding the protein of the present disclosure. The nucleic acid drug of the present disclosure, when administered to a living organism in which the removal of a target is required, expresses a protein in the living organism, and the target can be removed in the living organism by the action of the protein. The nucleic acid encoding the protein can be readily prepared by a person skilled in the art based on the amino acid sequence of the protein.

[0127] Each nucleic acid contained in the nucleic acid drug of this disclosure may be either cDNA or mRNA, but cDNA is a preferred example. If the nucleic acid contained in the nucleic acid drug of this disclosure is cDNA, the cDNA may be operably ligated to a promoter. Operable ligation means that the promoter and the nucleic acid of this disclosure are ligated in a manner that allows them to function in a host cell.

[0128] In one embodiment of the nucleic acid drug of this disclosure, each of the nucleic acids is formulated together with a nucleic acid delivery aid. Specific examples of nucleic acid delivery aids include lipofectamine, oligofectamine, liposomes, polyamines, DEAE dextran, calcium phosphate, and dendrimers.

[0129] In another embodiment of the nucleic acid drug of this disclosure, each of the nucleic acids is introduced into a vector. Examples of vector types include viral vectors such as adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, Sendai virus vectors, adenovirus vectors, Simian virus vectors, vaccinia virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors; plasmid vectors, etc.

[0130] In the nucleic acid drugs disclosed herein, the animals to which they are applied, the diseases they are affected, the methods of administration, etc., are the same as those described for the proteins. Therefore, the information regarding the animals to which they are affected, the diseases they are affected, and the methods of administration, etc., as described for the proteins disclosed herein, applies to the nucleic acid drugs disclosed herein.

[0131] The dosage of the nucleic acid drugs disclosed herein should be appropriately determined according to the type of disease being treated, the patient's age and weight, etc. For example, as an example of the dosage of the nucleic acid drugs disclosed herein, the amount of nucleic acid administered per day to an adult should be approximately 10 to 5000 μg / body, or approximately 20 to 4000 μg / body, administered once or several times a day. Another example of the dosage of the nucleic acid drugs disclosed herein is, if each nucleic acid is introduced into a vector, the amount of viral vector administered per day to an adult should be 1 × 10⁶ 10 ~1 x 10 16 vg / body size, or 1x10 12 ~1 x 10 14 It is sufficient to administer the drug once or several times a day at a dose of approximately vg / body. The administration interval for the nucleic acid drug disclosed herein may be set as appropriate within the range that produces a therapeutic effect, but examples include daily, every other day, weekly, every other week, every two to three weeks, monthly, every other month, or every two to three months.

[0132] This disclosure is not limited in any way to the embodiments of the invention described above and the examples shown below. Various modifications are also included in this invention, provided that they do not depart from the scope of the claims and are easily conceivable by those skilled in the art. All references to documents and other materials cited herein are incorporated herein by reference.

[0133] 1. Experiment Overview: To develop a protein modality that selectively eliminates unwanted target cells, we created a fusion protein (ProS modified) containing an amino acid sequence in which a target-binding region, a ProS-derived EGF-like domain, and a ProS-derived SHBG-like domain are linked in that order from the N-terminus to the C-terminus, and evaluated its functionality.

[0134] Hereinafter, the aforementioned fusion protein will be referred to as Crunch (Connector for Removal of Unwanted Cell Habitat). Furthermore, a Crunch having an amino acid sequence of a nanobody (Nb) that binds to GFP (Green Fluorescent Protein) as its target binding domain will be referred to as "GFPNb-Crunch". A Crunch having an amino acid sequence of scFv that binds to GFP as its target binding domain will be referred to as "scGFP-Crunch". A Crunch having an amino acid sequence of scFv that binds to mouse CD19 (mCD19) as its target binding domain will be referred to as "scCD19-Crunch". A Crunch having an amino acid sequence of scFv that binds to TYRP1 as its target binding domain will be referred to as "scTYRP1-Crunch". A Crunch having an amino acid sequence of scFv that binds to FAP as its target binding domain will be referred to as "scFAP-Crunch". A Crunch having an amino acid sequence of scFv that binds to PDGFRβ as its target binding domain will be referred to as "scPDGFRβ-Crunch".

[0135] 2. Experimental Materials and Methods 2-1. Mice C57BL / 6, MRL / Mpj, and MRL-lpr mice were purchased from Nippon SLC Co., Ltd. (Shizuoka, Japan) and raised under specific pathogen-free conditions. All experimental protocols were carried out in accordance with institutional guidelines on animal welfare and were approved by the Kyoto University Animal Experiment Committee.

[0136] 2-2. Cell Culture Ba / F3 cells were cultured in PRMI1640 (Wako) containing 10% fetal bovine serum (FBS, Gibco), antibiotic (Nacalai), 55 μM β2-mercaptoethanol, and 45 units / ml IL-3 under the conditions described in Reference 1. HEK293T cells and IH3T3 cells were cultured in DMEM (Wako) containing 10% FBS and antibiotic. K562 cells and B16.F10 cells were cultured in PRMI1640 containing 10% FBS, antibiotic, and 55 μM β2-mercaptoethanol. CHO cells and SH-SY5Y cells were cultured in DMEM / Ham's F-12 (Wako) containing 10% FBS and antibiotic. Each cell type was cultured at 37°C and 5% CO2.

[0137] 2-3. Plasmid Construction To express green fluorescent protein (GFPm) on the cell membrane, the mouse (m)CD8a signal sequence (SS) and the mCD8a transmembrane region (TM) were fused to the N-terminus and C-terminus of monomeric EGFP, respectively. mCD8a cDNA was isolated from mouse spleen cDNA. GFPm cDNA was introduced into pNEF and plenti vectors. ROSA26 GFPm-OVATo generate mice, a sequence encoding GFPm, conjugated with chicken volubumin (OVA) (Reference 2) using a GGGGS linker, was introduced into a pbluescript vector. A sequence encoding anti-GFP VHH antibody (GFP nanobody, GFPNb) (produced by Integrated DNA Technologies (IDT)) was inserted between mCD8a SS and mCD8a TM, and introduced into a plenti-IRES-tagRFP vector to express GFPNb on the cell membrane. For Crunch signal sequence screening, signal sequences of albumin (Reference 3), EGF (Reference 4), fibroblast growth factor-9 (FGF-9) (Reference 5), immunoglobulin G (IgG), and interleukin-2 (IL-2) (Reference 6), produced by Eurofins Japan, were cloned into a pNEF vector. To establish high-Crunch-expressing CHO cells, Crunch was cloned into a plenti-IRES-tagRFP vector. Mouse MerTK (mMerTK) was synthesized by IDT after codon optimization, tagged with FLAG at the C-terminus, and introduced into plenti, plenti-IRES-tagRFP, and plenti-IRES-puro vectors. Human MerTK (hMerTK) was also synthesized by IDT after codon optimization, tagged with FLAG at the C-terminus, and introduced into plenti-IRES-puro vectors. To produce recombinant protein in E. coli, GFP nanobodies fused with GFP and a FLAG tag were introduced into the pCold I (Takara, 3360) vector. This pCold vector has a translation-promoting element (TEE), 6 histidine, and a Factor Xa site at the N-terminus. For mouse CD19 expression, mouse CD19 (NM_009844.3) was isolated from mouse spleen cDNA, tagged with FLAG at the C-terminus, and introduced into the plenti-IRES-tagRFP vector. Split superfolder GFP (sfGFP) is composed of sfGFP1-10 at the N-terminus and sfGFP11 at the C-terminus.These two sfGFPs were fused to the C-terminus of mMerTK via the (GGGGS)3 linker and introduced into plenti-IRES-puro or plenti-IRES-tagRFP vectors.

[0138] 2-4. Establishment of Cell Lines Ba / F3 cells lacking Xkr8 (Reference 7) and TMEM 16F (Reference 8) (BDKO), and Ba / F3 cells expressing activated scramblase human Xkr4 (aXkr4) were previously established (Reference 1). Lentiviruses were prepared under the conditions described in Reference 1. Lentivirus components plenti, plenti-IRES-tagRFP, or plenti-IRES-puro, along with pCMV-VSVG-RSV-Rev (RIKEN) and pCAG-HIVgp (RIKEN), were transfected into HEK293T cells using polyethyleneimine MAX (PEI) (Polysciences). The supernatant was collected 48 hours after transfection, filtered, and centrifuged (20,000 × g, 4 °C, 2 hours). After centrifugation, the supernatant was removed, and the lentivirus pellet was resuspended in fresh medium. Using this concentrated lentivirus supernatant, 1 × 10 5 Ba / F3, BDKO and K562 cells, and 1 × 10 4 NIH3T3, CHO, and B16.F10 cells were infected. In some cases, virus-infected cells were selected with puromycin (1–10 μg / ml).

[0139] 2-5. ROSA26 GFPm-OVA Mouse Establishment ROSA26 GFPm-OVAThe mice were generated targeting the ROSA26 locus of C57BL / 6 mice. Briefly, the target construct was injected into fertilized eggs along with a Cas9 / sgRNA complex and transplanted into female mice. The knock-in mice were generated at the Animal Experiment Facility of the Institute for Biomedical Sciences, Kyoto University. The mouse genotypes were determined using the following primers: Fw, 5'-GCTGAGCCAGACCTCCATCGCGCAC-3' Rv WT, 5'-CGGTCCTCAGAAGCCAGGAG-3' Rv KI, 5'-ATGTACTGCCAAGTGGGCAGTTTAC-3'

[0140] 2-6. Crunch Production Crunch consists of a signal sequence (SS), a target binding region, an EGF-like domain (E), and an SHBG domain arranged from the N-terminus, followed by eight histidines and a FLAG tag (Figure 2a). The target binding region is the sequence of an anti-GFP VHH antibody (GFP nanobody), an anti-mouse CD19 single-strand variable fragment (scFv) (Addgene, 107227), an anti-GFP scFv (Reference 21) (Addgene, 182095), an anti-FAP scFv (Reference 31), or an anti-PDGFRβ scFv (Reference 32). In addition, to target tyrp1, the V of an anti-TYRP antibody (Reference 24) (20D7S, KEGG D10124) is used. L and V H scFv(V L and V HThe sequence used was a linked (GGGGS) × 3 linker (synthesized by IDT). The mouse ProS (mProS) sequence (NM_011173.3) was isolated from mouse eye cDNA. The human ProS (hProS) sequence (NM_000313.4) was synthesized by IDT. High-crunch-expressing CHO cells were established by lentivirus infection and selection of high-crunch-expressing cells (Figure 2d). To purify crunch from the condition medium, high-crunch-expressing CHO cells were cultured in serum-free DMEM / Ham's F-12 containing antibiotics. After 5-7 days, the condition medium was collected and filtered through a 0.2 μm filter (Thermo Fisher Scientific). The final concentration of 25 mM HEPES-NaOH (pH 7.5) and Ni-NTA agarose beads (Fujifilm) were added to the condition medium and incubated overnight at 4°C. The following day, Crunch was eluted from the Ni-NTA beads, replaced with DPBS (Dulbecco's Phosphate Buffered Saline) (Nacalai), and concentrated using Amicon Ultra (Merck). Protein concentration was measured by SDS-PAGE and CBB (Coomassie Brilliant Blue) staining (Figure 3f), and protein purity was confirmed by size exclusion chromatography (SEC).

[0141] 2-7. Expression and Purification of Recombinant Proteins from E. coli GFP and GFP nanobodies (GFPNb) were produced in BL21 (DE3, NEB, C2527). BL21 was transformed with a pCold plasmid and cultured in LB medium at 37°C until the OD600 reached 0.4–0.8. Protein expression was then induced by incubation in 0.125 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 15°C for 16 hours. After incubation, BL21 was centrifuged (6,000 xg, 4°C, 5 min), washed with DPBS (Dulbecco's Phosphate Buffered Saline), and then sonicated in TNE buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA). The soluble fraction was recovered after centrifugation (20,000 xg, 4°C, 10 min), and the His-tagged binding protein was purified using Ni-NTA beads as described in the "Crunch Preparation" section.

[0142] 2-8. Production of Sushi-Fc A fusion protein (Sushi-Fc) was produced using genetic engineering techniques, in which the mouse CD8 signal sequence, EcoRI, the Sushi1 domain of human C4BPB, BamHI, the Hinge-Fc domain of mouse IgG2a, and the His tag are linked from the N-terminus to the C-terminus. The complete amino acid sequence of Sushi-Fc is shown in SEQ ID NO: 62. In SEQ ID NO: 62, positions 1-31 correspond to the mouse CD8 signal sequence, positions 34-91 correspond to the Sushi1 domain of human C4BPB, and positions 94-325 correspond to the Hinge-Fc domain of mouse IgG2a.

[0143] 2-9. HPLC: Fusion proteins were loaded into SEC (Superdex Increase 200 5 / 150 GL, GE Healthcare) at 0.15 ml / min using D-PBS(-)(Nacalai) and analyzed.

[0144] 2-10. Cells were washed with DPBS before flow cytometry antibody staining. NIH3T3 cells or MerTK +NIH3T3 cells were stained with anti-MerTK-biotin (R&D, BAF591, 1 μg / ml) in 1 mg / ml BSA-enriched DPBS for 30 minutes on ice. BDKO cells or GFPm + BDKO cells were stained with rabbit anti-GFP antibody (MBL, 568, 1:1000) in DPBS containing 1 mg / ml BSA for 30 minutes on ice. After staining, the cells were washed and incubated with donkey anti-rabbit IgG (H+L) antibody containing Streptavidin-Alexa647 (Jackson, 016 600-084, 1:1000) or Alexa-647 (Invitrogen, A-31573, 1:1000) for 30 minutes on ice. After staining, these cells were washed and analyzed using FACS Lyric (BD Biosciences) or FACS Aria (BD Biosciences) and FlowJo (BD Biosciences). ROSA26 GFPm-OVA GFPm expression in the mouse thymus, spleen, bone marrow (BM), and blood was confirmed using Fc block (Biosciences, 553142, 1:1000) along with the primary antibody.

[0145] 2-11. Crunch Binding Assay: 5 × 10 units in 100 μl of DPBS (containing 1 mg / ml BSA (Nacalai)) with Crunch-containing medium, 10 μg / ml purified Crunch, or 10-fold diluted mouse plasma. 4 GFPm + BDKO cells were added and incubated on ice for 30 minutes. GFPm after incubation. +BDKO cells were harvested and Crunch binding was detected. Specifically, Crunch binding was detected using the following procedure: First, rabbit anti-FLAGtag immunoglobulin (Ig) (MBL, PM020, 1:1000) was incubated with cells on ice for 30 minutes in 1 mg / ml BSA-containing DPBS. Next, donkey anti-rabbit IgG (H+L) antibodies labeled with Alexa-647 (Invitrogen, A-31573, 1:1000), Alexa-555 (Invitrogen, A-21428, 1:1000), or Alexa-488 (Invitrogen, A-32731, 1:1000) were incubated with cells on ice for 30 minutes in 1 mg / ml BSA-containing DPBS. Subsequently, the fluorescence intensity of the cells was analyzed using FACS Lyric (BD Biosciences) and FlowJo (BD Biosciences), and the mean fluorescence intensity (MFI) was determined.

[0146] Crunch binding assays using thymocytes, spleen cells, bone marrow cells, and blood cells showed that GFPm + The procedure was the same as described above, except that these cells were used instead of BDKO cells. In addition, in the Crunch binding assay to evaluate scFAP-Crunch or scPDGFRβ-Crunch, GFPm + The procedure was the same as described above, except that HEK293T cells transfected with FAP or PDGFRβ were used instead of BDKO cells.

[0147] 2-12. SDS-PAGE and Western blotting cell lysates were prepared using RIPA buffer containing a protease inhibitor cocktail (Nacalai) (50 mM HEPES-NaOH [pH 8.0], 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). The cell lysates were incubated on ice for 15 minutes and then centrifuged (20,000 xg, 4°C, 15 minutes). Protein concentration in the supernatant was measured using the BCA Protein Assay kit (ThermoFisher Scientific). Sample protein levels were adjusted using RIPA buffer and 5× SDS sample buffer (200 mM Tris-HCl [pH 6.8], 10% SDS, 25% glycerol, 5% 2-ME, 0.05% bromophenol blue). These samples were loaded onto Extra PAGE One Precast Gel 5-10% or 10-20% (Nacalai) and electrophoresed at 35 mA for 40 minutes. After electrophoresis, the samples were transferred to a PVDF membrane at 0.1 A for 60 minutes. After transfer, the samples were incubated with blocking buffer containing 5% skim milk or 5% BSA (Nacalai) in TBS-T (Tris Buffered Saline with Tween 20) or PVDF Blocking Reagent for Can Get Signal (Toyobo). After blocking, the primary antibody was incubated overnight at 4°C in blocking buffer or Can Get Signal (TOYOBO). The following day, the membrane was washed with TBS-T and incubated with the secondary antibody in blocking buffer or Can Get Signal at room temperature for 1 hour. Then, it was incubated with Immobilon Western chemiluminescent HRP (Millipore) and the signal was detected using the FUSION chemiluminescence imaging system (Vilber). The data were analyzed and quantified using ImageJ.The primary antibodies used were: anti-FLAG-HRP (Sigma, A8592, 1:6000), anti-MerTK-biotin (0.1 μg / ml), mouse anti-α-tubulin antibody (Sigma, T6199, 0.5 μg / ml), and rabbit anti-phospho MerTK antibody (CST, 44463S, 1:1000). The secondary antibodies used were: goat anti-rabbit Ig-HRP antibody (DAKO, P0448, 0.025 μg / ml), streptavidin HRP (Proteintech, SA00001-0, 1:20000), and goat anti-mouse Ig-HRP (DAKO, P0447, 0.1 μg / ml).

[0148] 2-13. ELISA 10 μg of protein was dissolved in 100 μl of bicarbonate buffer (an aqueous solution containing 0.15 w / v% Na2CO2 and 0.293 w / v% NaHCO3, pH 9.6), spread onto a 96-well plate, and incubated overnight at 4°C. The following day, the wells were washed with TBS-T (Tris Buffered Saline with Tween 20) and incubated for 1 hour in ELISA blocking buffer (2 w / v% BSA in TBS-T). After incubation, the wells were washed with TBS-T and subjected to an ELISA assay to detect bound crunch or nanobodies using anti-FLAG-HRP antibody (1:5000 in ELISA blocking buffer) or goat anti-mouse Ig-HRP antibody (0.5 μg / ml in ELISA blocking buffer). The HRP signal was visualized by adding a TMB substrate (Nacalai, 05298-80), followed by the addition of 1 M sulfuric acid to terminate the HRP reaction. The absorption value at 450 nm was measured as the signal using Synergy H1 (BioTek). Kd was calculated using R (OD / ODmax = X / [Kd + X], where OD is OD450 at each protein concentration, and X is the protein concentration).

[0149] 2-14. Glycosylation of Crunch: 1 μg of protein was incubated in denaturation buffer (NEB, P0704S) at 100°C for 10 minutes. After incubation, PNGaseF (NEB, P0704S) was added and incubated at 37°C for 1 hour. The PNGaseF-treated samples were subjected to SDS-PAGE and Western blotting analysis.

[0150] 2-15. Differential Scanning Fluorescence (DSF) DSF was performed using a dye-based method (Reference 25). 2.5 μg of protein and SYPRO Orange (Invitrogen, S6650, 1:1000) were incubated in a total of 25 μl of DPBS. Data were collected as fluorescence signals using a ROX filter on a Thermal Cycler Dice TP700 (Takara). The temperature was stabilized at 25°C for 10 minutes, then increased from 25°C to 95°C (1°C / min). The apparent melting temperature (Tm) was determined from the maximum value of the first derivative of the fluorescence intensity curve.

[0151] 2-16. A control experiment using thymocytes as the target cells for the Split GFP assay was performed using the following procedure: NIH3T3 cells expressing the split superfolder GFP conjugate MerTK (MerTK-sfGFP) (MerTK-sfGFP + NIH3T3 cells) 1 x 10 5 Cells were seeded in 12-well plates and cultured overnight. The following day, MerTK-sfGFP was cultured in DMEM containing 10% fetal bovine serum (FBS). + In NIH3T3 cells, 1 × 10 6 Viable or apoptotic thymocytes from cells were added and co-cultured at 37°C for 2 hours. Thymocytes were removed by washing three times with DPBS, and MerTK-sfGFP was added. + GFP fluorescence in NIH3T3 cells was analyzed using FACS Aria and FlowJo. The thymocytes used were mouse-derived. Viable thymocytes were treated with PBS at 37°C for 3 hours. Apoptotic thymocytes were treated with Fas ligand at 37°C for 3 hours (Reference 7).

[0152] GFPNb-Crunch and GFPm + MerTK-sfGFP in the presence of BDKO cells + The following procedure was used to confirm the dimerization of MerTK in NIH3T3 cells: MerTK-sfGFP + NIH3T3 cells 1 x 10 5 Cells were seeded in 12-well plates and cultured overnight. The following day, MerTK-sfGFP was cultured in DMEM containing 10% fetal bovine serum (FBS). + In NIH3T3 cells, 1 × 10 6 Target cells (GFPm + BDKO cells (or BDKO cells) and mock-transfected HEK293T cells or Crunch-transfected HEK293T cells were added to the appropriate culture medium and co-cultured at 37°C for 2 hours. Target cells were removed by washing three times with DPBS, and MerTK-sfGFP was produced. + GFP fluorescence in NIH3T3 cells was analyzed using FACS Aria and FlowJo.

[0153] 2-17. A control experiment using thymocytes as the target cells for the MerTK phosphorylation assay was performed using the following procedure: 1 × 10⁻⁶ 6 Viable or apoptotic thymocytes from cells were mixed with D-MEM containing 10% FBS (containing Pros) to bind Pros to PtdSer (phosphatidylserine) on the thymocytes. The cells were then washed with D-MEM to remove free ProS from the FBS, and the viable or apoptotic thymocytes were resuspended in serum-free D-MEM. Subsequently, NIH3T3 cells or MerTK cells were used. + NIH3T3 cells were added and cultured overnight. Then, the cells were centrifuged (300×g, 37°C, 2 minutes) to form a pellet, and co-cultured for a further 15 minutes. After co-culture, viable or apoptotic thymocytes were removed by washing three times with cold HEPES buffer (20 mM HEPES-NaOH [pH 7.4], 137 mM NaCl). The recovered NIH3T3 cells or MerTK cells were then processed. +NIH3T3 cells were lysed in RIPA buffer containing a protease inhibitor cocktail and Phos-STOP (Roche). The resulting cell lysate was incubated on ice for 15 minutes, then transferred to a 1.5 ml tube and incubated on ice for 60 minutes. After incubation, the samples were centrifuged (20,000 × g, 4°C, 15 mins), and the supernatant was subjected to SDS-PAGE. Western blotting was performed using anti-phospho-MerTK antibody and anti-MerTK antibody. The thymocytes used were mouse-derived. Viable thymocytes were those treated with PBS at 37°C for 3 hours. Apoptotic thymocytes were those treated with Fas ligand at 37°C for 3 hours (Reference 7).

[0154] Furthermore, a control experiment using live cultured cells with exposed PtdSer was performed as the target cells according to the following procedure: Ba / F3 cells 1 × 10⁶ 6 Ba / F3 cells 1×10⁶ with PtdSer exposed by expression of cells or aXkr4 6 Cells were mixed with D-MEM containing 10% FBS (containing Pros) to bind Pros to PtdSer on Ba / F3 particles. The cells were then washed with D-MEM to remove free ProS from the FBS, and the cells were resuspended in serum-free D-MEM before being subjected to MerTK. + NIH3T3 cells were added and cultured overnight. Then, the cells were centrifuged (300×g, 37°C, 2 minutes) to form a pellet and co-cultured for another 15 minutes. MerTK after co-culture was collected using the same procedure as above. + NIH3T3 cells were harvested and Western blotting was performed using anti-phospho-MerTK antibodies and anti-MerTK antibodies.

[0155] GFPNb-Crunch and GFPm + MerTK in the presence of BDKO cells + The following procedure was used to confirm the phosphorylation of MerTK in NIH3T3 cells: 1 × 10⁻⁶ 5 MerTK for cells +NIH3T3 cells were seeded in 12-well plates, and 6 hours after seeding, the culture medium was replaced with D-MEM containing 5 mg / ml BSA. The following day, BDKO cells or GFPm cells were used. + BDKO cells 1 x 10 6 Cells and GFPNb-Crunch 0 μg / ml or 5 μg / ml were added and cultured overnight. Then, the cells were centrifuged (300 × g, 37°C, 2 minutes) to form a pellet and co-cultured for another 15 minutes. MerTK after co-culture was collected using the same procedure as above. + NIH3T3 cells were harvested and Western blotting was performed using anti-phospho-MerTK antibody and anti-MerTK antibody. In experiments to confirm the time dependence, the amount of GFPNb-Crunch added was changed to 0 μg / ml or 1 μg / ml, and the co-culture time after centrifugation was changed to 0 to 30 minutes, and the same procedure as above was performed. In experiments with the addition of anti-GFP antibody, 5 μg / ml of anti-GFP antibody was added along with 0 μg / ml or 1 μg / ml of GFPNb-Crunch, and the other procedures were the same as above.

[0156] GFPm + MerTK in the presence of BDKO cells + The following procedure was used to confirm the phosphorylation of MerTK in NIH3T3 cells: 1 × 10⁻⁶ 5 MerTK for cells + NIH3T3 cells were seeded in a 12-well plate, incubated at 37°C for 24 hours, then the medium was removed and washed with PBS. Serum-free medium was then added to each well and incubated at 37°C for 8–16 hours to induce MerTK + NIH3T3 cells were starved. Separately, 1 × 10⁶ 6 GFPm + BDKO cells and purified GFPNb-Crunch (GP1-4) at 0 μg / ml or 5 μg / ml are mixed in 1 ml of PBS containing 0.1% BSA and incubated at 4°C for 30 minutes to obtain GFPm +GFPNb-Crunch was conjugated to BDKO cells. MerTK cells were then subjected to starvation. + Each well containing NIH3T3 cells was treated with GFPNb-Crunch-conjugated GFPm + BDKO cells 1 x 10 6 Cells were added and co-cultured at 37°C for 15 minutes. MerTK after co-culture was collected using the same procedure as described above. + NIH3T3 cells were harvested and Western blotting was performed using anti-phospho-MerTK antibody and anti-MerTK antibody. The experiment was performed six times. Fluorescence intensity data for phospho-MerTK and MerTK were obtained using ImageJ, and the ratio of phospho-MerTK to MerTK (phospho-MerTK / MerTK) was calculated. A negative control (GFPm without GFPNb-Crunch binding) was used. + The relative ratio was calculated by setting the aforementioned ratio (when using BDKO cells) to 1.

[0157] Experiments using scCD19-Crunch, which has a single-strand variable fragment (scFv) as the target binding domain, were performed using the following procedure: 1 × 10⁻¹⁶ 5 MerTK for cells + NIH3T3 cells were cultured overnight in serum-free D-MEM. The following day, the D-MEM was replaced with one containing 5 mg / ml BSA, and BDKO cells or BDKO cells (mCD19) introduced with scCD19 were cultured. + BDKO cells) 1 x 10 6 Cells and scCD19-Crunch 0 μg / ml or 5 μg / ml were added and cultured overnight. Then, the cells were centrifuged (300 × g, 37°C, 2 minutes) to form a pellet and co-cultured for another 15 minutes. MerTK after co-culture was collected using the same procedure as above. + NIH3T3 cells were harvested and subjected to Western blotting using anti-phospho-MerTK antibody, anti-MerTK antibody, and anti-α-tubulin antibody.

[0158] 2-18. On the day before the cell viability assay experiment, the cells were placed in RPMI1640 (containing FBS) containing IL-3 for 1 × 10⁶ days. 6The cells were seeded to a concentration of cells / ml. The following day, the cells were washed with D-PBS and placed in RPMI1640 (containing FBS, IL-3-free) containing 0 μg / ml or 10 μg / ml Crunch, with a total dose of 2.5 × 10⁶. 5 Cells were seeded in 12-well plates at a concentration of cells / 500μl and cultured for 72 hours. The number of viable cells was measured daily using trypan blue.

[0159] 2-19. Phagocytosis assay 1×10 5 MerTK for cells + NIH3T3 cells or parental NIH3T3 cells were seeded in a 12-well plate and cultured overnight. The following day, 1 × 10⁶ cells were cultured. 6 Target cells were stained with 0.1 μg / ml pHrodo Red (Thermo Fisher Scientific) for 30 minutes, then suspended in D-MEM containing 0-10% FBS, and further treated with Crunch 0 μg / ml or 10 μg / ml. After co-culturing for 2 hours, the target cells were removed by washing three times with D-PBS, and then MerTK was used. + NIH3T3 cells or parental NIH3T3 cells were resuspended in CHES buffer (20 mM CHES [pH 9.0], 150 mM NaCl, 2% FBS), and phagocytosis-positive cells (pHrodo Red-positive cells) were measured using FACS Aria and FlowJo.

[0160] Furthermore, when evaluating phagocytic activity in the presence of Sushi-Fc, 10 μg / ml of Sushi-Fc was added simultaneously with 10 μg / ml of Crunch under the aforementioned experimental conditions.

[0161] Experiments using scCD19-Crunch, which has a single-strand variable fragment (scFv) as a target-binding domain, were performed using the following procedure: Splenocytes isolated from wild-type C57BL / 6 male mice were stained with 0.1 μg / ml pHrodo Red for 30 minutes. The pHrodo Red-stained spleen cells and MerTK + NIH3T3 cells were co-cultured for 3 hours in serum-free D-MEM containing 10 μg / ml scCD19-Crunch. After co-culture, the same procedure as described above was performed to obtain MerTK +Phagocytosis-positive cells (pHrodo Red-positive cells) in NIH3T3 cells were measured.

[0162] Macrophages used in the phagocytosis assay were prepared as described in the "Macrophage Stimulation Assay" section below. Briefly, 1 × 10⁻⁶ macrophages were used. 5 Peritoneal cells from cells were seeded in 12-well plates and incubated in D-MEM containing 10% FBS and antibiotics for 3 hours. After culturing, the cells were washed three times with DPBS, and the adherent cells were used as phagocytic cells for co-culture as described above. After co-culture, target cells were removed by washing three times with DPBS. Macrophages were detached using acetylacetase (ICT, AT104) and stained with anti-CD11b-APC (1:200) in CHES buffer for 30 minutes on ice. After staining, macrophages were washed, resuspended in CHES buffer containing DAPI (1:2000), and analyzed using FACS Aria.

[0163] 2-20. Spleen cell transplantation: 6-10 week old wild-type C57BL / 6 male mice and ROSA26 GFPm-OVA Splenocytes were collected from male mice. 1 × 10 7 wild-type spleen cells or GFPm cells + Splenocytes were intravenously injected into 6-10 week old male C57BL / 6 mice. One day after injection, 100 μg GFPNb-Crunch or PBS was injected into the peritoneal cavity of the mice. Four days after splenocyte injection, the mice's splenocytes and blood cells were collected. The blood cells were washed three times with RBC Lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). GFP-positive cells in these cells were analyzed using FACS 421 Lyric and FlowJo.

[0164] 2-21. In vivo phagocytosis assay of ROSA26 6-10 weeks old. GFPm-OVA GFPm from male mice + Spleen cells were collected. 1 × 10 7 GFPm cells +Splenocytes were stained with 0.1 μg / ml pHrodo Red for 30 minutes and intravenously injected into 6-10 week old male C57BL / 6 mice. Two hours after injection, 100 μg GFPNb-Crunch or PBS was injected into the peritoneal cavity of the mice. Splenocytes were collected from the mice 24 hours after injection. These cells were stained with anti-MerTK-biotin (R&D, BAF591, 1 μg / ml) on ice for 30 minutes. After staining, the cells were washed and stained with streptavidin-Alexa647 (Jackson, 016-600-084, 1:1000) on ice for 30 minutes. For F / 80 staining, these cells were treated with Fc block (Biosciences, 553142, 1:1000) for 15 minutes and then stained with anti-F4 / 80-APC / Cy7 (Biolegend, 157315, 1:200) on ice for 30 minutes. After staining, these cells were washed, resuspended in CHES buffer, and analyzed using FACS Aria and FlowJo with DAPI.

[0165] 2-22. In vivo half-life and anti-drug antibody (ADA) assays. Plasma half-life measurements and ADA assays were performed using 8-10 week old C57BL / 6 female mice. After introducing 150 μg of protein intravenously or intraperitoneally into the mice, blood samples were collected from the tail using heparin-coated hematocrit tubes to prevent coagulation. For plasma half-life measurements, blood was collected from the tail at 10 minutes, 1, 4, 12, 24, 48, 72, and 120 hours after injection. For the ADA assay, blood was collected from the tail 24 days after injection. These blood samples were centrifuged (17,000 xg, 4°C, 5 min) to obtain plasma, which was diluted 1:10 (for plasma half-life) or 1:200 (for ADA measurement) with 50% glycerol in PBS and stored at -80°C until analysis. Crunch or antibodies in the plasma were measured using ELISA. Crunch concentrations were determined by analyzing standard crunch samples used for plasma half-life experiments. Using data points from 12 to 72 hours, the half-life was calculated as the β phase (t1 / 2 = tlog[0.5] / log[Ac / A0], t1 / 2; half-life, t; elapsed time, Ac; remaining crunch, A0; amount of crunch at 12 hours) (Reference 26). 100 ng / ml mouse anti-GFP IgG2a (Invitrogen, A-11120) or mouse anti-FLAG IgG1 (Sigma, F1804) antibody was used as a control in the ELISA for the ADA assay.

[0166] 2-23. Estimation of Crunch's Immunogenicity To predict B cell epitopes, the Crunch sequence was analyzed using BepiPred-2.0 (Reference 27) with a threshold of 0.5. T cell epitopes were predicted using NetMHC4.1 (Reference 28) with an 8-mer peptide for H-2-kb. The thresholds were set to the default ranks: strong (0.5) and weak (2.0).

[0167] 2-24. Macrophage Stimulation Assay Macrophages were prepared from 8-10 week old C57BL / 6 female mice. Specifically, 2 ml of 3% thioglycolate was injected into the peritoneal cavity of the mice. Three days after injection, peritoneal cells were collected from the mice using 10 ml of DPBS and 1 × 10⁶ cells were extracted.6 Cells were seeded in 12-well plates and cultured for 3 hours in D-MEM containing 10% FBS and antibiotics. After culturing, the cells were washed three times with DPBS. Subsequently, the cells were treated with Fc block (1:1000), and then rat anti-CD45-PE (Biolegend, 147711, 1:200), rat anti-CD11b-APC antibody (Biolegend, 101211, 1:200), rat anti-Ly6G-APC / Cy7 antibody (Biolegend, 127623, 1:200), and rat anti-Ly6C-PerCP / Cy5.5 antibody (Biolegend, 128011, 1:200) were added and incubated on the plates to confirm that the purity of macrophages was 95% or higher. In the stimulation assay, macrophages were stimulated in D-MEM at 30°C with 10 μg / ml Crunch, mouse IgG2a (BioXCell, BE0122), 10% FBS, or 10 ng / ml LPS (Sigma, L4516). After 24 hours, the cells were washed with PBS, macrophage RNA was extracted using the RNeasy Mini Kit (QIAGEN), and 500 ng of total RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen). The resulting cDNA was adjusted to 10 ng / μl, and the expression of each gene was measured using a Thermal Cycler Dice TP700 (Takara) with a target primer set and TB Green Premix Ex Taq (Takara).

[0168] 2-25. To confirm the inhibitory effect of Crunch on the engraftment and proliferation of ectopic transplanted tumors, the following experiment was conducted: 1 × 10 5 GFPm + B16.F10 melanoma cells were subcutaneously injected into the hind limbs of 6-8 week old female C57BL / 6 mice. 100 μg of GFPNb-Crunch or saline was administered intraperitoneally to the mice 1, 8, and 15 days after tumor cell injection to confirm tumor engraftment and proliferation. Tumor volume was measured every 3 days using calipers (V = πLW² / 6, V: volume, L: tumor long diameter, W: tumor short diameter).

[0169] Furthermore, the following experiment was conducted to confirm the therapeutic effect of Crunch after tumor formation: 1 × 10 5 Melanoma cells (B16.F10 melanoma cells, GFP) + B16.F10 melanoma cells, or GFPm + B16.F10 melanoma cells were subcutaneously injected into the hind limbs of 6-8 week old female C57BL / 6 mice. Tumor size was 4 mm. 3 From the point when the tumor reached a certain stage (7 days after melanoma cell injection), 100 μg of GFPNb-Crunch, scGFP-Crunch, scTYPR-Crunch, or physiological saline was administered intraperitoneally every two days for a total of three times. Tumor volume was measured every three days using calipers (V = πLW² / 6, V: volume, L: tumor long diameter, W: tumor short diameter). The weight of the tumor excised on the 16th day after melanoma cell injection was measured.

[0170] 2-26. Tumor metastasis 1×10 5 GFPm + B16.F10 melanoma cells were intravenously injected into 6-8 week old C57BL / 6 female mice. 100 μg of GFPNb-Crunch or physiological saline was administered intraperitoneally 1, 3, and 5 days after melanoma cell injection. Mice were euthanized 14 days after melanoma cell injection, and their lungs were collected. Tumor metastasis was quantified by measuring metastatic nodules.

[0171] 2-27. Analysis of immune cells in the tumor microenvironment of human acral lentiginous melanoma. To search for Crunch effector cells in melanoma cells, a human acral lentiginous melanoma dataset from eight patients was obtained from the GEO database under accession number GSE11597841. These data were annotated by cell type using the Single Cell Portal (Reference 29). MerTK expression in each cell was visualized.

[0172] 2-28. Flow cytometry of MerTK-expressing cells in a B16F10 ectopic transplantation model. B16.F10 melanoma cells were injected into mice, and tumors were excised from the mice 9 days later. The tumors were treated with PBS containing 1 mg / ml Collagenase I (Sigma, C0130) and 10 Kunitz / ml DNase I (Sigma, D4263) at 37°C for 1 hour. The treated tumors were resuspended and passed through a 70 μm filter (PLS, 43-57070-51) for flow cytometry. Cells for flow cytometry were treated with Fc block (1:1000) for 15 minutes, and then stained with anti-MerTK-biotin antibody (1 μg / ml) in DPBS containing 1 mg / ml BSA on ice for 30 minutes. After staining, the cells were washed and incubated with Streptavidin-Alexa488 (Jackson, 016-540-084, 1:1000), anti-CD45-PE antibody (1:200), anti-CD11b-APC antibody (1:200), and anti-Ly6C-PerCP / Cy5.5 antibody (1:200) with either anti-Ly6G-APC / Cy7 antibody (1:200) or anti-F4 / 80-APC / Cy7 antibody (1:200). After staining, the cells were washed, resuspended in PBS containing DAPI (1:2000), and analyzed by FACS Lyric.

[0173] 2-29. Prediction of Protein Structure: The FASTA sequence files of Crunch and scFv were analyzed using AlphaFold (v 2.3.2) to predict their structures. The obtained structures were further analyzed using PyMOL software.

[0174] 2-30. B-cell removal assay PBS, scGFP-Crunch, scCD19-Crunch, and antibodies were injected intraperitoneally or intravenously into 6-11 week old C57BL / 6 male mice. Rat anti-mouse CD19 IgG2a (1D3, BioXCell, BE0150) and mouse anti-rat κ light chain IgG2a (MAR18.5, BioXCell, BE0122) were used for antibody injection. The 1D3 antibody was injected 24 hours before the injection of the MAR18.5 antibody. When scCD19-Crunch was injected three times, injections were given every 12 hours, with the third injection given after sample collection on day 1. For cell analysis, spleen cells were treated once with RBC Lysis buffer, and hematopoietic cells were treated three times with the same buffer. These cells were treated with Fc block (Biosciences, 553142, 1:1000) for 15 minutes and stained on ice with anti-CD45-PE (Biolegend, 147711, 1:200), anti-CD19-APC (Biolegend, 115511, 1:200), and anti-B220-PE / Cy7 (Biolegend, 103221, 1:200) for 30 minutes. After staining, these cells were washed and analyzed using FACS Aria and FlowJo with DAPI.

[0175] 2-31. Adeno-associated virus 9 (AAV9) The pAAV-EF1 vector shown in Figure 16d was constructed. Specifically, the pAAV-U6-sgRNA-CBh mCherry (Addgene #91947) vector was excised between two ITRs using NotI, and the EF1a core promoter region of plenti-Cas9 (Addgene, #52962) was inserted. By substituting the promoter, the sequence between the two ITRs was reduced to 1444 bp, allowing the pAAV-EF1 vector to contain sequences up to 2500-3000 bp.

[0176] AAVs were prepared by transfecting HEK293T cells with pAAV-EF1-tagRFP or pAAV-EF1-scCD19-Crunch, pAdDeltaF6 (Addgene, 112867), and the pAAV2 / 9n (Addgene, 112865) vector using polyethyleneimine (PEI). 24 hours after transfection, the supernatant was changed to serum-free D-MEM containing 2 mM L-glutamine and 1 mM sodium pyruvate. 72 hours after the medium change, the supernatant containing the cells was collected. A final concentration of 10% chloroform was added to the supernatant, and it was incubated at 4°C for 5 minutes with rotation. After incubation, a final concentration of 1.2 M sodium chloride was added, and the mixture was vortexed and centrifuged (2,000 × g, 4°C, 10 minutes). The aqueous phase after centrifugation was transferred to a new tube and mixed with 20% PEG 8000 (Sigma, P2139), and incubated overnight at 4°C. The following day, the virus pellet was recovered by centrifugation (2,000 × g, 4°C, 30 minutes) and suspended in HEPES buffer (20 mM HEPES NaOH [pH 7.4], 137 mM NaCl). The AAV in HEPES buffer was treated with 400 units / ml DNase I (NEB, M3030) and 5 μg / ml RNase A (Nacalai, 30142-04) at 37°C for 30 minutes. Subsequently, the AAV was purified three times using chloroform. Chloroform purification was performed by adding an equal volume of chloroform to the AAV, centrifugation (12,000 × g, 4°C, 5 minutes), and recovery of the aqueous phase. Finally, AAV was enriched and replaced with DPBS using Amicon Ultra 100 kDa (Merck). AAV titer was measured by RT-qPCR on a Thermal Cycler Dice TP 700 using specific primers for WPRE (Fw, 5'-GTCCTTTCCATGGCTGCTC-3', Rv, 5'CCGAAGGGACGTAGCAGA-3') and TB Green Premix Ex Taq. pAAV plasmids were used as standards.

[0177] In vitro infections: 1 × 10 8 ~1 x 10 10vg AAV9-EF1-tagRFP was sown the previous day in a 1x10 5 Individual SH-SY5Y cells were added, and tagRFP was performed using FACS Aria and FlowJo 5 days after infection. + The proportion was analyzed. In vivo infection was 1 × 10⁻⁶. 11 vg AAV9-EF1-tagRFP or AAV9-EF1-scCD19-Crunch was intravenously injected into 8-10 week old C57BL / 6 male mice. The excised livers were treated with Trypsin / EDTA at 37°C for 30 minutes, and hepatocytes were collected from the livers perfused with DPBS using a 70 μm filter. B cells in the blood were analyzed according to the procedure described in section 2-30, "B cell removal assay."

[0178] 2-32. Systemic lupus erythematosus (SLE) model: 150 μg of scCD19-Crunch, scGFP-Crunch, or saline solution is administered to the MRL. lpr / lpr Mice were administered intraperitoneally twice a week from 8 to 13 weeks of age. After the study, body weight and spleen weight were measured, and spleen cells and blood cells were collected from the mice. Spleen cells and blood cells were treated with RBC Lysis buffer and stained with Fc block, anti-CD45-PE, anti-CD19-APC, and anti-B220-PE / Cy7. After staining, these cells were washed and analyzed using FACS Aria and FlowJo with DAPI.

[0179] 2-33. Renal Histology and Immune Complex Deposition: Kidneys were removed from MRL mice. MRLs were also administered with physiological saline, scCD19-Crunch, or scGFP-Crunch. lpr / lpr Mouse kidneys were removed. The removed kidneys were fixed with 10% buffered formalin and embedded in paraffin. Kidney sections (4 μm) were stained with hematoxylin and eosin (H&E) or DAPI (1:1000) and anti-mouse IgG-Alexa647 (Invitrogen, A31571, 1:1000). The histopathological score of glomerular lesions was semi-quantitatively evaluated on a scale of 0–3 (Reference 30). The score was calculated from the average of 5–6 glomeruli from each mouse. Stained sections were analyzed using a confocal microscope and ImageJ.

[0180] 2-34. Uric protein / creatinine (UPC) ratio: The UPC ratio was measured in the urine of mice before sacrificing them, using the QuantiChrom Protein Creatinine Ratio Assay Kit (BioAssay Systems, DPCR-100).

[0181] 2-35. Serum was collected from the hearts of mice at the end of the ANA and anti-dsDNA antibody tests. Mouse antinuclear antibody (ANA) in the serum was measured using the Mouse ANA ELISA kit (FineTest, EM1607-CM). For the detection of anti-dsDNA antibodies in the serum, a 96-well plate coated with 1 μg of plasmid using DNA Coating Solution and DNA High-Binding Plate (Cell biolabs, AKR-5182) was used. The serum was diluted 1:100 and the anti-dsDNA antibody was measured by ELISA.

[0182] 2-36. Binding assay of Crunch and Sushi-Fc A 100 μl solution of bicarbonate buffer (aqueous solution containing 0.15 w / v% Na2CO2 and 0.293 w / v% NaHCO3, pH 9.6) containing Crunch protein (5 μg / 100 μl) was added to each well of a 96-well plate and incubated overnight at 4°C. The following day, the wells were washed with TBS-T (Tris Buffered Saline with Tween 20) and incubated for 1 hour with ELISA blocking buffer (2 w / v% BSA in TBS-T). After incubation, the wells were washed with TBS-T, and then 100 μl of ELISA blocking buffer containing Sushi-Fc (10 μg / ml) was added and incubated for 1 hour. After incubation, the wells were washed with TBS-T, and Sushi-Fc bound to Crunch was detected using goat anti-mouse Ig-HRP antibody (0.5 μg / ml in ELISA blocking buffer). The HRP signal was visualized by adding TMB substrate (Nacalai, 05298-80), and the HRP reaction was terminated by adding 1 M sulfuric acid. The absorption value at 450 nm was measured as the signal using Synergy H1 (BioTek).

[0183] 2-37. Statistical analysis data were expressed as mean ± SD. Student's unpaired t-test was used for comparisons between two groups. One-way ANOVA using the Tukey-Kramer t-test was used for comparisons between three or more groups. Kaplan-Meier and log-rank tests were used for comparisons of survival rates. All p-values ​​were calculated using two-tailed tests. A p-value of less than 0.05 was considered statistically significant.

[0184] 3. Experimental Results 3-1. Creation of Crunch and Verification of Crunch's Binding Ability to Target Cells To demonstrate the function of Crunch, an experimental model was used in which cells expressing GFP as a cell surface protein were used as target cells, and GFPNb-Crunch (Figure 2a), which has the amino acid sequence of the anti-GFP VHH antibody (GFP nanobody, GFPNb) (References 9, 10), was used as the target binding region. The GFPNb-Crunch used consisted of an amino acid sequence in which the following were linked from the N-terminus in this order: signal sequence-EF-GFPNb (SEQ ID NO: 40)-four EGF-like domains derived from mouse ProS (SEQ ID NO: 14)-spacer B (SEQ ID NO: 22)-SHBG-like domain derived from mouse ProS (SEQ ID NO: 24)-C-terminal addition sequence of mouse ProS (SEQ ID NO: 26)-EF-His tag (HHHHHHHH)-EF-Flag tag (DYKDDDDK).

[0185] First, cells expressing GFP as a cell membrane protein were established as a model cell for unwanted cells. Additionally, cells expressing GFPNb as a cell membrane protein were established as a model cell for phagocytic cells. To prevent the influence of PtdSer-ProS interaction on phagocytosis, two ubiquitous scramblases, Xkr8 (Reference 7) and TMEM 16F (Reference 8), were knocked out in Ba / F3 cells to create BDKO cells (Reference 1). These BDKO cells were transfected with GFP (GFPm), which is a fused GFP with the CD8a signal sequence (SS) and transmembrane region (TM), to create BDKO cells expressing GFPm (GFPm + BDKO cells were created. GFPm +In BDKO cells, confocal microscopy and flow cytometry analysis confirmed that GFP was localized to the cell membrane (Figure 1a,b). Furthermore, transfection of K562 cells with GFPNb (GFPNbm), which fuses the CD8a signal sequence (SS) and transmembrane domain, resulted in K562 cells expressing GFPNbm (GFPNbm). + K562 cells were generated. GFPNbm + K562 cells and GFPm + When BDKO cells were co-cultured with GFPNbm cells, association between the two cell types was observed (Figure 1c,d), confirming that GFPm is recognized by GFPNbm.

[0186] Next, we investigated whether GFPm could be recognized by GFPNb in Crunch. Since extracellular secretion of ProS was difficult in HEK293T cells, we screened for signal sequences (SS) that could efficiently secrete Crunch. As candidate SSs, we selected albumin (Reference 3), EGF (Reference 4), fibroblast growth factor-9 (FGF-9) (Reference 5), immunoglobulin G (IgG), interleukin-2 (IL-2) (Reference 6), and CD8a. All of these SSs were mouse-derived. GFPNb-Crunch plasmids containing each SS were transfected into HEK293T cells, and the culture medium was collected after 48 hours of incubation. Crunch binding assays were performed using each culture medium to confirm the amount of GFPNb-Crunch secreted. As a result, GFPNb-Crunch containing the CD8 SS was recognized by GFPm. + It showed the highest binding strength to BDKO cells (Figure 2b). Furthermore, when the amount of GFPNb-Crunch secreted in the purified medium using the His tag was examined, similarly, GFPNb-Crunch with CD8a SS showed the highest binding strength (Figure 2c).

[0187] Therefore, in the following experiment, we used a GFPNb-Crunch containing mouse CD8 SS. Specifically, the GFPNb-Crunch used had an amino acid sequence consisting of, from the N-terminus, the signal sequence of mouse CD8a (SEQ ID NO: 27) - EF-GFPNb (SEQ ID NO: 40) - four EGF-like domains derived from mouse ProS (SEQ ID NO: 14) - spacer B (SEQ ID NO: 22) - SHBG-like domain derived from mouse ProS (SEQ ID NO: 24) - C-terminal addition sequence of mouse ProS (SEQ ID NO: 26) - EF-His tag (HHHHHHHH) - EF-Flag tag (DYKDDDDK). The complete amino acid sequence is shown in SEQ ID NO: 20. The nucleotide sequence of the cDNA encoding the GFPNb-Crunch is shown in SEQ ID NO: 41.

[0188] Next, we attempted to establish GFPNb-Crunch-highly expressing Chinese hamster ovary (CHO) cells for mass production of GFPNb-Crunch. A lentivirus encoding GFPNb-Crunch-IRES (Internal ribosome entry site)-RFP (red fluorescent protein) was introduced into CHO cells, and RFP-highly expressing cells were selected by flow cytometry. After repeating this virus infection and selection cycle three times (Figure 2d), the amount of Crunch secreted, as confirmed by Western blotting, increased (Figure 3a,b), and GFPNb-Crunch-highly expressing CHO cells were obtained. In GFPNb-Crunch-highly expressing CHO cells, the amount of GFPNb-Crunch secreted increased to a concentration of 9.54 ± 0.38 mg / L on day 6 of culture (Figure 3c). Using the obtained GFPNP-Crunch high-expression CHO cells, GFPNb-Crunch was purified from the conditional medium using His tagging. The purification efficiency from the supernatant was close to 100% as determined by Western blotting, and the production amount was estimated to be approximately 9.61 mg / L from CBB staining of the purified protein (Figure 3d,e). Furthermore, the purified GFPNb-Crunch showed a single band on SDS-PAGE and a single peak on size exclusion chromatography, confirming that high-purity GFPNb-Crunch was obtained (Figure 2e,f), and it was also uniformly glycosylated (Figure 3f). In addition, when the purified GFPNb-Crunch was subjected to a Crunch binding assay, it showed a concentration-dependent GFPm + The increased binding strength to BDKO cells (Figure 2g) and the recognition of GFP (Figure 3d) confirmed that GFPNb-Crunch can efficiently recognize target cells. Regarding biochemical properties, GFPNb-Crunch showed thermal stability comparable to that of antibodies (Figure 3h,i). Furthermore, because the Gla domain of GFPNb-Crunch is substituted with GFPNb, it was unaffected by vitamin K in the culture medium (Figure 3j).

[0189] 3-2. Verification of Crunch's effect on phagocytic cells Next, we verified whether Crunch could recognize phagocytic cells. MerTK, one of the TAM receptors, recognizes PtdSer exposed on the cell surface of apoptotic cells via ProS, causing MerTK dimerization and self-decomposition of tyrosine residues. To investigate whether Crunch, like ProS, recognizes specific markers on apoptotic cells and induces MerTK signaling, we introduced MerTK with optimized codons into immortalized fibroblast cell line NIH3T3 cells (MerTK + We established NIH3T3 cells (Figure 4a,b). Furthermore, to detect dimerized MerTK, we introduced Split superfolder GFP conjugate MerTK (MerTK-sfGFP) into NIH3T3 cells (MerTK-sfGFP). + NIH3T3 cells were established (Figure 4c) (Reference 11). MerTK-sfGFP + In a Split GFP assay using NIH3T3 cells, MerTK-sfGFP + We investigated whether the dimerization of MerTK that occurs when NIH3T3 cells bind to target cells can be detected by GFP luminescence. Specifically, we investigated MerTK-sfGFP. + NIH3T3 cells were incubated with apoptotic thymocytes in the presence of fetal bovine serum (FBS) containing ProS to confirm whether the MerTK-sfGFP system functions upon binding to target cells. The results showed that MerTK-sfGFP exhibited GFP positivity after incubation with apoptotic cells. + An increase in NIH3T3 cells was observed (Figure 4d). Based on these results, MerTK-sfGFP + In a Split GFP assay using NIH3T3 cells, MerTK-sfGFP was observed. + We confirmed that MerTK dimerization in NIH3T3 cells can be detected as a GFP signal.

[0190] Next, the Split GFP assay was performed to determine GFPNb-Crunch and GFPm + MerTK-sfGFP in the presence of BDKO cells + We confirmed the dimerization of MerTK in NIH3T3 cells. As a result, in the presence of GFPNb-Crunch, MerTK-sfGFP was observed. + NIH3T3 cells and GFPm + MerTK-sfGFP, when co-cultured with BDKO cells, exhibits GFP positivity. + An increase in NIH3T3 cells was observed (Figure 4e and Figure 5a,b), confirming that GFPNb-Crunch induces MerTK dimerization in phagocytic cells in the presence of target cells.

[0191] Furthermore, we investigated whether Crunch could induce phosphorylation of MerTK (Reference 12). First, we conjugated ProS to viable thymocytes or apoptotic thymocytes and then induced phosphorylation of parental NIH3T3 cells or MerTK. + NIH3T3 cells were co-cultured with MerTK cells for 15 minutes. After co-culture, NIH3T3 cells or MerTK cells were used. + Lysates of NIH3T3 cells were prepared, and the degree of MerTK phosphorylation was analyzed by Western blotting using anti-phospho-MerTK antibody and MerTK antibody. As a result, phosphorylated MerTK was twice as high in apoptotic thymocytes compared to viable thymocytes (Figure 6a). Furthermore, MerTK was also increased not only in apoptotic cells but also in viable Ba / F3 cells expressing activated scramblase Xkr4 (aXkr4) (Reference 1) to expose PtdSer. + We were able to confirm that it induces phosphorylation of MerTK in NIH3T3 cells (Figure 6b). These results confirm that the MerTK phosphorylation assay used in this experiment can measure phosphorylated MerTK in phagocytic cells.

[0192] Next, MerTK in the presence of GFPNb-Crunch + NIH3T3 cells and GFPm + A phosphorylation assay was performed using co-cultured BDKO cells. The results showed that MerTK was produced in the presence of GFPNb-Crunch. + NIH3T3 cells and GFPm + When BDKO cells are co-cultured, MerTK +It was confirmed that the phosphorylation level of MerTK in NIH3T3 cells increased over time (Figure 4f,g and Figure 6c). Furthermore, MerTK was elevated in the presence of GFPNb-Crunch and anti-GFP antibody. + NIH3T3 cells and GFPm + Co-culturing BDKO cells suppressed MerTK phosphorylation (Figure 4h). This result indicates that GFPNb-Crunch specifically binds to GFP on target cells.

[0193] Furthermore, we investigated whether MerTK signaling is activated by Crunch. It is known that IL-3-dependent Ba / F3 cells can survive when MerTK signaling is activated under IL-3 depletion conditions (Reference 13). Therefore, we created Ba / F3 cells into which MerTK was introduced and examined their survival rate under IL-3 depletion conditions (Figure 7a). When cultured for 48 hours under IL-3 deficiency, parental Ba / F3 cells showed decreased cell number and survival rate even in the presence of FBS containing ProS (Figure 7b,c), and similar results were obtained for Ba / F3 cells expressing either aXkr4 or MerTK. However, Ba / F3 cells expressing both aXkr4 and MerTK showed improved survival rate under IL-3 deficiency (Figure 7d,e). This result indicates that PtdSer-mediated MerTK signaling promoted cell survival. Furthermore, even after culturing under IL-3 deficiency for 72 hours, a similar trend was observed, except for a slight increase in the cell viability of Ba / F3 cells expressing MerTK (Figure 7d,e).

[0194] Next, we investigated whether GFPNb-Crunch increased the survival rate of Ba / F3 cells expressing both GFPm and MerTK. As a result, these Ba / F3 cells were confirmed to survive even in the presence of GFPNb-Crunch under IL-3 deficiency (Figure 4i-l). In contrast, Ba / F3 cells expressing MerTK but not GFPm did not show an increase in cell number or survival rate even in the presence of GFPNb-Crunch under IL-3 deficiency (Figure 7f-i).

[0195] These results clearly demonstrate that Crunch induces MerTK dimerization, MerTK phosphorylation, and MerTK downstream signaling.

[0196] 3-3. Crunch removes target cells ex vivo. Next, we investigated whether GFPNb-Crunch could induce phagocytosis of target cells. Knock-in mice were created in which GFPm conjugated with chicken ovalbumin (OVA) (Reference 2) was inserted into the ROSA26 locus (ROSA26 GFPm-OVA A mouse was created (Figure 8a). ROSA26 GFPm-OVA Flow cytometry analysis of mouse-derived thymocytes, spleen cells, bone marrow cells, and blood cells confirmed that GFP was expressed on the cell surface of all cell types (Figure 8b). Furthermore, a Crunch binding assay using these cells confirmed that GFPNb-Crunch bound to these cells (Figure 8c). These results indicate that GFPNb-Crunch can recognize GFP present on the surface of primary mouse cells.

[0197] Also, ROSA26 GFPm-OVA Thymocytes were collected from the mouse thymus, labeled with pHrodo-Red, and then subjected to MerTK in the presence of GFPNb-Crunch. + Co-culture NIH3T3 cells with MerTK in a medium containing 0-10% FBS. + The percentage of pHrodo Red-positive cells in NIH3T3 cells was determined. The results showed that GFPNb-Crunch was associated with MerTK + It was confirmed that phagocytosis-positive cells in NIH3T3 cells were increased in a serum concentration-dependent manner (Figure 9a-c). Furthermore, MerTK + When NIH3T3 cells expressing BFP (blue fluorescent protein) were used as a control instead of NIH3T3 cells, no increase in phagocytic-positive cells was observed in the presence of GFPNb-Crunch (Figure 9d,e), indicating that the effect of GFPNb-Crunch is not limited to MerTK. +It was found to be specific to NIH3T3 cells. Furthermore, while vitamin K affects the γ-carboxylation of the Gla domain of ProS (Reference 20), the function of Crunch lacking the Gla domain was not affected even in the absence of vitamin K during the Crunch production process (Figure 10a).

[0198] Furthermore, a humanized GFPNb-Crunch was created by replacing the four EGF-like domains and SHBG-like domains derived from mouse ProS in the GFPNb-Crunch used above with four EGF-like domains and SHBG-like domains derived from human ProS. Specifically, the amino acid sequence of this humanized GFPNb-Crunch consists of, from the N-terminus, the mouse CD8a signal sequence (SEQ ID NO: 27) - EF-GFPNb (SEQ ID NO: 40) - four EGF-like domains derived from human ProS (SEQ ID NO: 13) - spacer B (SEQ ID NO: 21) - SHBG-like domain derived from human ProS (SEQ ID NO: 23) - C-terminal addition sequence of human ProS (SEQ ID NO: 25) - EF-His tag (HHHHHHHH) - EF-Flag tag (DYKDDDDK), and its complete amino acid sequence is shown in SEQ ID NO: 19. The nucleotide sequence of the cDNA encoding this humanized GFPNb-Crunch is shown in SEQ ID NO: 42.

[0199] A phagocytosis assay was performed using the aforementioned humanized GFPNb-Crunch. Specifically, ROSA26 GFPm-OVA Mouse-derived thymocytes were labeled with pHrodo-Red, and then NIH3T3 cells expressing human MerTK in the presence of the humanized GFPNb-Crunch (hMerTK) were created. + Co-cultured with NIH3T3 cells in a medium containing 5% FBS, and hMerTK + The percentage of pHrodo Red-positive cells in NIH3T3 cells was determined. The results showed an increase in phagocytic-positive cells even when using humanized GFPNb-Crunch, indicating induction of phagocytosis of target cells (Figure 9f).

[0200] Furthermore, GFPNb-Crunch is CD11b +It was also confirmed that it induces phagocytosis by macrophages (Figure 9g).

[0201] These results revealed that Crunch induces phagocytosis of target cells in a MerTK-dependent manner ex vivo, and that it can be applied to human cells by humanizing Crunch.

[0202] 3-4. Pharmacokinetics and Immunogenicity of Crunch Next, to evaluate the in vivo applicability of Crunch, its pharmacokinetic (PK) profile was assessed. GFPNb-Crunch was administered intraperitoneally (ip) and intravenously (iv) to mice by injection, and the plasma half-life was measured from 10 minutes to 120 hours (Figure 10b). As a result, the plasma half-lives of Crunch were 11.4 hours (iv) and 15.0 hours (ip) (Figure 10c,d).

[0203] Although Crunch's immunogenicity is expected to be low because the anti-inflammatory response is induced by the ProS-MerTK pathway (Reference 21), we then investigated the potential immunogenicity of Crunch. Analysis using NetMHC4.1 (Reference 22) and BepiPred-2.0 (Reference 23) showed that replacing the Gla domain of ProS with a nanobody did not increase the number of T cell epitopes and B cell epitopes in the ProS module (Figure 10e). However, the replacement of the Gla domain with a nanobody slightly increased the number of epitopes in the target-binding module. To evaluate this, mice were injected with GFPNb-Crunch and GFP, and antibody production was examined 24 days after injection. As a result, antibodies against GFPNb were generated after injection of GFPNb-Crunch, but antibodies against the ProS module were not generated (Figure 10f,g). Taken together, these results indicate that the immunogenicity of Crunch is mainly dependent on the target-binding module. Furthermore, when we examined the immune response to Crunch in macrophages, we found that it did not induce an increase in cytokine expression (Figure 10h).

[0204] 3-5. Crunch was tested to see if it could remove target cells in mice that were already eliminating target cells in vivo. ROSA26GFPm-OVA Mouse spleen cells (ROSA26) GFPm-OVA Splenocytes from wild-type (WT) mice or spleen cells from wild-type mice were intravenously injected into wild-type mice, and one day later, 100 μg of GFPNb-Crunch or PBS was intraperitoneally injected into the mice (Figure 11a). ROSA26 GFPm-OVA When mice injected with spleen cells were administered PBS, GFP-positive cells (GFP) were detected 4 days after injection of spleen cells. + The number of ROSA26 cells increased in the spleen and blood (Figure 11b). However, ROSA26 GFPm-OVA When GFPNb-Crunch was administered to mice injected with spleen cells, the GFP levels in the spleen and blood were reduced. + The proportion of cells decreased (Figure 11c-e), and GFPNb-Crunch contained ROSA26 GFPm-OVA It was found to have the effect of removing spleen cells. On the other hand, since GFPNb-Crunch did not affect the number of spleen cells in the host cells (Figure 11f), it was suggested that GFPNb-Crunch is not toxic.

[0205] Next, to verify whether the aforementioned effect of GFPNb-Crunch is dependent on phagocytosis, an in vivo phagocytosis assay was performed. ROSA26 GFPm-OVA Splenocytes were labeled with pHrodo Red and intravenously injected into WT mice. Two hours after splenocyte injection, 100 μg of GFPNb-Crunch or PBS was injected intraperitoneally (Figure 11g). Phagocytic cells in the host spleen were analyzed 22 hours after GFPNb-Crunch injection. Although GFP-positive cells did not decrease sharply 22 hours after GFPNb-Crunch administration (Figure 11h), MerTK + Cells and macrophage cells (F4 / 80 + The number of cells was increased (Figure 11i). These results confirm that GFPNb-Crunch induces phagocytosis in vivo via phagocytic cells expressing MerTK and / or F4 / 80.

[0206] 3-6. Crunch demonstrates therapeutic effects in xenograft models. We investigated whether Crunch exerts therapeutic effects on disease by removing target cells. Using the B16.F10 mouse melanoma cell line (References 14, 15), we confirmed the effects of Crunch on tumor engraftment and proliferation in a xenograft model. GFPm + B16.F10 melanoma cells were subcutaneously injected into the hind limbs of mice, and 100 μg of GFPNb-Crunch or physiological saline was administered intraperitoneally three times, 1 day, 8 days, and 15 days after tumor cell injection (Figure 12a). As a result, it was confirmed that GFPNb-Crunch administration suppressed tumor engraftment and proliferation (Figures 12b,c and Table 1).

[0207] Next, we investigated the therapeutic effect of Crunch administration after tumor formation. Specifically, GFPm + B16.F10 melanoma cells were subcutaneously injected into the hind limbs of mice, and tumor size was 4 mm. 3 From the point when this was reached (7 days after melanoma cell injection), 100 μg of GFPNb-Crunch or physiological saline was administered intraperitoneally every two days for a total of three times (Figure 13a). As a result, GFPNb-Crunch administration was effective against GFPm + The proliferation of B16.F10 melanoma was dramatically suppressed (Figure 13b,c). When tumors were excised from mice after the end of the experiment and observed (Figure 13d), the size (Figure 13e) and weight (Figure 13f) of the tumors were reduced by GFPNb-Crunch administration. Consistent with this, it was also confirmed that GFPNb-Crunch administration extended the survival period of the mice (Figure 13g).

[0208] Next, we investigated whether GFP expression on the cell surface is important for targeting by GFPNb-Crunch. Specifically, we investigated whether GFP expression in cells expressing GFP. + B16.F10 melanoma cells, or GFPm cells expressing GFP on their cell surface. + B16.F10 melanoma cells were subcutaneously injected into the hind limbs of mice, and tumor size was 4 mm. 3From the point when the threshold was reached, 100 μg of GFPNb-Crunch or physiological saline was administered intraperitoneally every two days for a total of three times. As a result, GFPm + B16.F10 melanoma cells are GFPm + Unlike B16.F10 melanoma cells, GFPNb-Crunch did not disappear upon administration (Figure 13h,i). This result indicates that GFP expression on the cell surface is important for GFPNb-Crunch targeting.

[0209] Next, to gain insight into effector cells within melanoma cells, we analyzed scRNA data from human melanoma cells (Reference 24). The results confirmed that macrophages in the human tumor environment express MerTK (Figure 12d,e). Consistent with this, CD11b was found in the tumor environment of B16.F10 melanoma cells. + Ly6C int F4 / 80 high Macrophages expressed MerTK (Figure 12f-h), suggesting that tumor-associated macrophages are involved in phagocytosis.

[0210] Next, we investigated the effect of Crunch administration on tumor metastasis. Specifically, GFPm + B16.F10 melanoma cells were intravenously injected into mice, and starting the following day, 100 μg of GFPNb-Crunch or physiological saline was administered every two days for a total of three times (Figure 13j). Analysis of the mouse lungs 14 days after melanoma cell injection revealed a reduction in the number of metastatic nodules in mice administered GFPNb-Crunch (Figure 13k,l).

[0211] 3-7. Crunch containing scFv demonstrates target cell removal effect. The effect of Crunch containing a single-strand variable fragment (scFv) (Reference 4) as a target binding region was investigated. Specifically, scCD19-Crunch was constructed using scFv (scFv-mCD19) from a 1D3 rat monoclonal antibody clone against mouse CD19 (mCD19) (Reference 17) (Figure 14a). The scCD19-Crunch has an amino acid sequence consisting of, from the N-terminus, the scFv-mCD19 signal sequence (SEQ ID NO: 43) - scFv-mCD19 (SEQ ID NO: 44) - four EGF-like domains derived from mouse ProS (SEQ ID NO: 14) - spacer B (SEQ ID NO: 22) - SHBG-like domain derived from mouse ProS (SEQ ID NO: 24) - C-terminal addition sequence of mouse ProS (SEQ ID NO: 26) - EF-His tag (HHHHHHHH) - EF-Flag tag (DYKDDDDK). The complete amino acid sequence is shown in SEQ ID NO: 45. The nucleotide sequence of the cDNA encoding the scCD19-Crunch is shown in SEQ ID NO: 46.

[0212] AlphaFold2 confirmed that scFv in scCD19-Crunch does not interfere with the Crunch backbone (Figure 14b).

[0213] Furthermore, after incubating C57BL / 6 mouse spleen cells with 10 μg / ml scCD19-Crunch, the binding of scCD19-Crunch to spleen cells was analyzed by flow cytometry using an anti-FLAG antibody, and CD19 expression in spleen cells was analyzed using an anti-CD19-APC antibody. As a result, scCD19-Crunch was found to bind to CD19 + It was confirmed that it binds to spleen cells (Figure 15a).

[0214] Also, MerTK in the presence of scCD19-Crunch + NIH3T3 cells and BDKO cells or mCD19 + A phosphorylation assay was performed using co-cultured BDKO cells. The results showed that in the presence of scCD19-Crunch, MerTK +Increased phosphorylation levels of MerTK were observed in NIH3T3 cells (Figure 14c). Furthermore, in the presence of scCD19-Crunch, mouse thymocytes labeled with pHrodo-Red and MerTK + NIH3T3 cells were co-cultured in serum-free medium, and MerTK + The percentage of pHrodo Red-positive cells in NIH3T3 cells was determined. The results showed that scCD19-Crunch was associated with MerTK + Increase the proportion of phagocytic-positive cells in NIH3T3 cells, MerTK + It was confirmed that scCD19-Crunch induces phagocytosis of mouse thymocytes by both NIH3T3 cells and macrophages (Figure 14d, Figure 15b). These results confirm that scCD19-Crunch induces phosphorylation of MerTK and phagocytosis of mouse spleen cells.

[0215] Furthermore, in mice administered scCD19-Crunch, the amount of CD19 in the blood and spleen was reduced. + B220 + The proportion of B cells was significantly reduced (Figure 14e,f). Consistent with this, the number of B cells was reduced in the spleen of mice administered scCD19-Crunch (Figure 14g,h). These results confirm that Crunch functions in vivo even when scFv is used as the target binding domain.

[0216] To compare the properties of scFv and Nb (nanobody) in the target binding region of Crunch, scGFP-Crunch was constructed using scFv (scFv-GFP) (Reference 21) from an N86 / 38 mouse monoclonal antibody clone against GFP. The amino acid sequence of this scGFP-Crunch consists of, from the N-terminus, the scFv-GFP signal sequence (SEQ ID NO: 43) - scFv-GFP (SEQ ID NO: 55) - four EGF-like domains derived from mouse ProS (SEQ ID NO: 14) - spacer B (SEQ ID NO: 22) - SHBG-like domain derived from mouse ProS (SEQ ID NO: 24) - C-terminal addition sequence of mouse ProS (SEQ ID NO: 26) - EF-His tag (HHHHHHHH) - EF-Flag tag (DYKDDDDK). The complete amino acid sequence is shown in SEQ ID NO: 56. The nucleotide sequence of the cDNA encoding this scGFP-Crunch is shown in SEQ ID NO: 57.

[0217] scGFP-Crunch and GFPNb-Crunch were confirmed to have equivalent stability (Figure 3h, Figure 15c). On the other hand, scGFP-Crunch had a higher affinity for GFP in ELISA than GFPNb-Crunch (K D (1.24 ± 0.24 nM, Figure 15d), high binding affinity to target cells (Figure 15e), and efficient clearance of target cells was observed (Figure 15f). Furthermore, similar to GFPNb-Crunch, scGFP-Crunch showed a slight increase in immunogenicity in the target-binding region consisting of scFv, but no increase in immunogenicity in the ProS portion (Figure 15g). However, when antibody production was examined 24 days after injection of scGFP-Crunch or GFPNb-Crunch into mice, the amount of antibody against scGFP-Crunch was much lower than that against GFPNb-Crunch, indicating that crunch with scFv fused as the target-binding region has lower immunogenicity (Figure 15h).

[0218] Next, to confirm the specificity of scCD19-Crunch to target B cells, scCD19-Crunch and scGFP-Crunch were injected intraperitoneally (ip) and intravenously (iv) into mice, and three days later, CD45 + CD19 in cells + B220 + The percentage of B cells was measured by flow cytometry. As a result, a decrease in B cell count was observed only with scCD19-Crunch, and there was little difference due to the different administration routes (Figure 14i).

[0219] Next, to compare the effects of Crunch and the antibody, we conducted an investigation using an anti-CD19 antibody (1D3) that has the same epitope as scCD19-Crunch. Anti-CD19 antibody (1D3) is known to eliminate B cells in a mouse model (References 22, 23). First, anti-CD19 antibody (1D3) was injected into the peritoneal cavity of mice, and three days later, the CD19 + B220 + B cells were measured using flow cytometry. The result was CD19 + B220 + B cells were not detected even after injection of anti-CD19 antibody (1D3) (Figure 16a). However, this was due to the effect of the 1D3 antibody masking the anti-CD19-APC epitope used for CD19 detection (Figure 16b,c). Most B220 + Since cells show CD19 expression in lymphocytes in the blood, B220 + Cells were considered target B cells. When 100 μg of scCD19-Crunch was injected into mice, scCD19-Crunch reduced the number of target B cells until day 3 after injection, but after day 3, the number of B cells gradually increased (Figure 14j). On the other hand, in mice injected with anti-CD19 antibody (1D3), the number of target B cells did not decrease until day 3 after injection, but there was a slight decrease on day 6 (Figure 16j).

[0220] Since the anti-CD19 antibody (1D3) is a rat antibody (IgG2a) against mouse CD19, we investigated whether injecting a mouse anti-rat κ light chain IgG2a antibody after injecting the 1D3 anti-CD19 antibody (1D3) would improve the elimination of target B cells. As a result, injection of the anti-rat κ light chain antibody slightly promoted the elimination of target B cells (Figure 14k).

[0221] Furthermore, the effect of scCD19-Crunch in mice increased in a dose-dependent manner, saturating at a dose of 200 μg on day 3 after injection (Figure 14I). It is noteworthy that three injections of 100 μg of scCD19-Crunch showed a better elimination effect than a single injection of 300 μg (Figure 14k).

[0222] To continuously produce scCD19-Crunch in mice, the scCD19-Crunch construct (SEQ ID NO: 45) or tagRFP (SEQ ID NO: 58) was inserted into an adeno-associated virus 9 (AAV9) vector (Figure 16d). Infection of SH-SY5Y cells confirmed the successful production of AAV9 (Figures 16e,f). Next, AAV9 encoding tagRFP (AAV9-EF1-tagRFP) or AAV9 encoding scCD19-Crunch (AAV9-EF1-scCD19-Crunch) was intravenously injected into mice, and hepatocytes and blood B cells were analyzed two weeks after injection. The results showed that tagRFP expression was observed in hepatocytes of mice injected with AAV9-EF1-tagRFP (Figure 16g), and 14 days after injection, CD19 expression was observed in the blood of mice injected with AAV9-EF1-scCD19-Crunch. + B220 + A decrease in the number of cells was observed (Figure 16h). In mice injected with AAV9-EF1-scCD19-Crunch, scCD19-Crunch was detected in the plasma 24 weeks after injection (Figure 16i,j), and the B-cell depletion effect persisted for 24 weeks (Figure 15m).

[0223] These results confirm that Crunch is effective in eliminating target cells whether administered as a protein or nucleic acid.

[0224] 3-8. Pharmacokinetics of Crunch Next, we investigated the relationship between the amount of Crunch and the efficiency of target cell removal. As shown in Figure 15a, when mouse spleen cells were incubated with scCD19-Crunch and then stained with anti-CD19-APC antibody, a decrease in mean fluorescence intensity (MFI) was observed. This suggests that scCD19-Crunch bound to CD19 on B cells reduces the accessibility of anti-CD19-AP antibody. Similarly, in vivo, administration of scCD19-Crunch reduced B220 + A decrease in CD19 MFI was observed in B cells (Figure 17b,c), which is attributed to B220 + This correlated significantly with a decrease in B cells (Figure 17d). These findings suggest that if scCD19-Crunch binding does not reach a critical threshold, B cell removal becomes ineffective.

[0225] To further evaluate crunch function, plasma scCD19-crunch concentrations were measured. Intraperitoneal injection of 100 μg of scCD19-Crunch into mice resulted in a plasma scCD19-Crunch concentration of 71.5 ± 7.3 ng / ml one day later (Figure 18b). Furthermore, intravenous injection of AAV9 encoding scCD19-Crunch (AAV9-EF1-scCD19-Crunch) into mice resulted in plasma scCD19-Crunch concentrations of 91.2–572.3 ng / mL 24 weeks later (Figure 16j). B cell depletion correlated with plasma crunch concentration, and B cell recovery in mice was observed when plasma scCD19-Crunch concentrations were 91.2 ng / ml or less (Figure 18c). These results suggest that the threshold for scCD19-Crunch concentration in plasma is approximately 50–100 ng / ml (0.5–1 nM) for B cell removal via scCD19-Crunch.

[0226] 3-9. Crunch aims to demonstrate its therapeutic effect by targeting endogenous antigens in mouse disease models that exhibit therapeutic effects against diseases. First, tests were conducted targeting the endogenous antigen TYRP1 on melanoma cells. scTYRP1-Crunch was created using scFv (scFv-TYRP1) of the anti-TYRP1 antibody (20D7S, Flanvotumab46). The scTYRP1-Crunch has an amino acid sequence consisting of, from the N-terminus, the scFv-GFP signal sequence (SEQ ID NO: 43) - scFv-TYRP1 (SEQ ID NO: 59) - four EGF-like domains derived from mouse ProS (SEQ ID NO: 14) - spacer B (SEQ ID NO: 22) - SHBG-like domain derived from mouse ProS (SEQ ID NO: 24) - C-terminal addition sequence of mouse ProS (SEQ ID NO: 26) - EF-His tag (HHHHHHHH) - EF-Flag tag (DYKDDDDK). The complete amino acid sequence is shown in SEQ ID NO: 60. The nucleotide sequence of the cDNA encoding the scTYRP1-Crunch is shown in SEQ ID NO: 61. The constructed scTYRP1-Crunch was confirmed to bind to B16.F10 cells expressing TYRP1 (Figure 19b). B16.F10 melanoma cells were subcutaneously injected into the hind limbs of mice, and the tumor size was 4 mm. 3 After growth, scTYRP1-Crunch was administered intraperitoneally every two days for a total of three times. As a result, administration of scTYRP1-Crunch significantly suppressed the proliferation of B16.F10 melanoma (Figure 19c,d). After the end of the experiment, tumors were excised from the mice and observed, and the tumor weight had decreased (Figure 19e,f). However, the body weight of the mice at the end of the experiment had not decreased (Figure 19g).

[0227] Next, the therapeutic effect of Crunch on systemic lupus erythematosus (SLE) (References 18, 19) was investigated. Specifically, 150 μg of scCD19-Crunch or saline was administered intraperitoneally to 8-week-old MRLlpr / lpr mice, a spontaneous SLE model mouse. Administration of 150 μg of scCD19-Crunch or saline was continued twice a week from 8 to 13 weeks of age (5 weeks). As a result, CD19 levels in the spleen and blood of mice administered scCD19-Crunch were reduced. + B220 + A decrease in the proportion of B cells was observed (Figures 19h-j and 20b). Furthermore, while administration of scCD19-Crunch did not affect body weight or spleen weight (Figures 20c,d), glomerulonephritis and lymphocyte infiltration were suppressed in the kidneys of mice administered scCD19-Crunch (Figures 19k, 20e). On the other hand, while immune complex deposition increased in the glomeruli of mice administered physiological saline, IgG deposition decreased in mice administered scCD19-Crunch (Figures 19l, 20f). The urinary protein-creatinine ratio also returned to normal after scCD19-Crunch administration (Figure 19m). Similarly, serum antinuclear antibodies (ANA) and anti-dsDNA antibodies, autoantibodies detected in SLE, decreased with scCD19-Crunch administration (Figures 19n,o). These results confirm that Crunch, which binds to CD19, is effective in treating SLE.

[0228] 3-10. Modification of Crunch to suppress phagocytic inhibition by C4BP The Sushi1 domain of human C4BPB has been shown to bind to human, mouse, and bovine ProS. Furthermore, human C4BP is known to exhibit ProS-dependent phagocytic inhibitory effects. Therefore, we attempted to develop a Crunch that is less susceptible to phagocytic inhibition by human C4BPB. First, we predicted the binding sites of the Sushi1 domain of human C4BPB and the LG-like domain of mouse Pros, and the binding sites of the LG-like domain of mouse Pros and the Ig-like domain 1 of MerTK, using Alphafold's predictive structural model (Figure 21a). Based on these results, we predicted the site in the mouse Pros LG-like domain that is involved in binding to the human C4BPB Sushi1 domain but not in binding to the MerTK Ig-like domain 1. We then created five variants (432A, 465A, 3A, 3A432A, and 3A465A) by making the following amino acid substitutions to mouse GFPNb-Crunch (SEQ ID NO: 20).432A: A modified version in which the R at position 482 is replaced with A in the amino acid sequence shown in SEQ ID NO: 20 (GFPNb-Crunch containing modified LG-like domain 1 in which the R at position 134 in the amino acid sequence of SEQ ID NO: 37 is replaced with A) 465A: A modified version in which the E at position 515 is replaced with A in the amino acid sequence shown in SEQ ID NO: 20 (GFPNb-Crunch containing modified LG-like domain 1 in which the E at position 167 in the amino acid sequence of SEQ ID NO: 37 is replaced with A) 3A: A modified version in which the K at position 364, the R at position 366, and the K at position 474 are each replaced with A in the amino acid sequence shown in SEQ ID NO: 20 (modified LG in which the K at position 16, the R at position 18, and the K at position 126 are each replaced with A) GFPNb-Crunch containing LG-like domain 1)3A432A: A modified version in which the K at position 364, the R at position 366, and the R at position 482 in the amino acid sequence shown in SEQ ID NO: 20 are replaced with A (GFPNb-Crunch containing modified LG-like domain 1 in which the K at position 16, the R at position 18, and the R at position 134 in the amino acid sequence of SEQ ID NO: 37 are replaced with A). 3A465A: A modified version in which the K at position 364, the R at position 366, the K at position 474, and the E at position 515 in the amino acid sequence shown in SEQ ID NO: 20 are replaced with A (GFPNb-Crunch containing modified LG-like domain 1 in which the K at position 16, the R at position 18, the K at position 126, and the E at position 167 in the amino acid sequence of SEQ ID NO: 37 are replaced with A).

[0229] Using each of the GFPNb-Crunch obtained above, we performed binding assays between Crunch and Sushi-Fc. Sushi-Fc is a protein possessing the Sushi1 domain of human C4BPB and the Hinge-Fc domain of mouse IgG2a. As a result, the unmodified GFPNb-Crunch (WT, SEQ ID NO: 20) bound to Sushi-Fc, and binding to Sushi-Fc was maintained with GFPNb-Crunch 432A and 465A. However, in GFPNb-Crunch 3A, 3A432A, and 3A465A, Sushi-Fc binding was significantly suppressed compared to WT (Figure 21b).

[0230] Furthermore, each GFPNb-Crunch obtained above was subjected to a phagocytosis assay in the presence of Sushi-Fc. Specifically, ROSA26 GFPm-OVA After labeling mouse-derived thymocytes with pHrodo-Red, NIH3T3 cells expressing human MerTK (hMerTK) were created in the presence of each of the aforementioned GFPNb-Crunch and Sushi-Fc. + Co-cultured with NIH3T3 cells in a medium containing 5% FBS, and hMerTK + The percentage of pHrodo Red-positive cells in NIH3T3 cells was determined. The results showed that in the control group (Ctrl), which used PBS instead of GFPNb-Crunch, the addition of Sushi-Fc reduced phagocytosis, confirming that Sushi-Fc inhibits phagocytosis mediated by Protein S present in PBS. Similarly, in the unmodified GFPNb-Crunch (WT, SEQ ID NO: 20), the addition of Sushi-Fc reduced phagocytosis (Figure 21c). In contrast, with GFPNb-Crunch 465A, 3A, 3A432A, and 3A465A, phagocytosis was maintained at over 60% even after the addition of Sushi-Fc (Figure 21c). Furthermore, under conditions without the addition of Sushi-Fc, no difference in phagocytic activity was observed in any of the GFPNb-Crunch variants (465A, 3A, 3A432A, and 3A465A) compared to the wild type (WT), indicating that these amino acid substitutions do not affect the phagocytic function of Crunch (Figure 21c).

[0231] These results suggest that the human C4BPB Sushi1 domain binds to Crunch containing the unmodified LG-like domain 1, inhibiting phagocytosis and interfering in human blood. However, it was revealed that by introducing amino acid substitutions in GFPNb-Crunch at 465A, 3A, 3A432A, and 3A465A, binding to human C4BPB can be suppressed, thus avoiding phagocytosis inhibition and interference in human blood.

[0232] 3-11. Influence of EGF-like Domains in Crunch The effect of the number of EGF-like domains in Crunch on Crunch production was investigated. HEK293T cells were transfected with nucleic acids encoding GFPNb-Crunch, as shown in Table 2 and Figure 22a, and Crunch binding assays and Split GFP assays were performed using the culture supernatant of these cells. The GFPNb-Crunch used in the above experiments corresponds to GP2.

[0233] As a result, binding to GFPm and dimerization of MerTK in phagocytic cells were observed with all GP1-6, but GP1-4 showed the highest levels of GFPm binding and MerTK dimerization promotion in phagocytic cells, with GP2 showing particularly high levels of GFPm binding and MerTK dimerization promotion (Figure 22b,c). This suggests that Crunch is easily secreted from transformants during the manufacturing process.

[0234] Next, GFPm with GP1-4 attached. + BDKO cells and MerTK + NIH3T3 cells were co-cultured and phosphorylation assays were performed. The results showed that regardless of whether GP1-4 was used, MerTK + It was confirmed that MerTK in NIH3T3 cells is phosphorylated (Figure 23a,b). In particular, it was found that MerTK phosphorylation was more pronounced when GP1 or GP2 was used (Figure 23a,b).

[0235] These results suggest that in Crunch, the presence of 2 to 4 (particularly 3 or 4) EDF-like domains between the target-binding region and the ProS-derived SHBG-like domain can enhance the phagocytic activity of phagocytic cells.

[0236] 3-12. Creation of scFAP-Crunch and scPDGFRβ-Crunch FAP (Fibroblast Activation Protein) and PDGFRβ (Platelet-derived growth factor receptor β) are known to be expressed on the surface of activated fibroblasts and fibrous fibroblasts. Therefore, we created crunches targeting FAP or PDGFRβ.

[0237] The scFAP-Crunch was constructed using the scFv (scFv-FAP) of the anti-FAP antibody (Reference 31). The amino acid sequence of this scFAP-Crunch consists of, from the N-terminus, a signal sequence (SEQ ID NO: 43) - scFv-FAP (SEQ ID NO: 63) - GS - four EGF-like domains derived from mouse ProS (SEQ ID NO: 14) - spacer B (SEQ ID NO: 22) - SHBG-like domain derived from mouse ProS (SEQ ID NO: 24) - C-terminal addition sequence of mouse ProS (SEQ ID NO: 26) - His tag (HHHHHHHH) - Flag tag (DYKDDDDK). The complete amino acid sequence is shown in SEQ ID NO: 64. The nucleotide sequence of the cDNA encoding this scFAP-Crunch is shown in SEQ ID NO: 65.

[0238] Furthermore, sc PDGFRβ-Crunch was constructed using scFv (scFv-PDGFRβ) of the anti-PDGFRβ antibody (Reference 32). The amino acid sequence of this sc PDGFRβ-Crunch consists of, from the N-terminus, a signal sequence (SEQ ID NO: 43) - scFv - PDGFRβ (SEQ ID NO: 66) - GS - four EGF-like domains derived from mouse ProS (SEQ ID NO: 14) - spacer B (SEQ ID NO: 22) - SHBG-like domain derived from mouse ProS (SEQ ID NO: 24) - C-terminal addition sequence of mouse ProS (SEQ ID NO: 26) - His tag (HHHHHHHH) - Flag tag (DYKDDDDK). The complete amino acid sequence is shown in SEQ ID NO: 67. The nucleotide sequence of the cDNA encoding this scPDGFRβ-Crunch is shown in SEQ ID NO: 68.

[0239] HEK293T cells transfected with a plasmid containing cDNA encoding scFAP-Crunch or scPDGFRβ-Crunch were cultured for 48 hours, and the supernatant was collected. Next, scFAP-Crunch or scPDGFRβ-Crunch was purified using His tagging, and the purified product was subjected to Western blotting, confirming the presence of scFAP-Crunch or scPDGFRβ-Crunch (Figure 24a).

[0240] Furthermore, CHO cells transfected with scFAP-Crunch or scPDGFRβ-Crunch were prepared, and the supernatant was collected after culturing these CHO cells. Separately, a plasmid containing cDNA encoding FAP-HA (a protein in which an HA tag is linked to the C-terminus of mouse FAP, amino acid sequence number 69, nucleotide sequence number 70) or PDGFRβ-HA (a protein in which an HA tag is linked to the C-terminus of mouse PDGFβR, amino acid sequence number 71, nucleotide sequence number 72) was transfected into HEK293T cells, and cells cultured for 48 hours were used as target cells, and the supernatant was subjected to a Crunch binding assay. As a result, binding to FAP or PDGFRβ was observed in the supernatant (Figure 24b).

[0241] These results confirm that we were able to create scFAP-Crunch, which binds to FAP, and scPDGFRβ-Crunch, which binds to PDGFRβ.

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Claims

1. A protein comprising (A) a target-binding region that binds to a target (excluding the γ-carboxyglutamic acid domain of protein S), and (C) a sex hormone-binding globulin-like domain of protein S.

2. The protein according to claim 1, further comprising 2 to 4 epidermal growth factor-like domains of protein (B) between the target-binding region that binds to the target (A) and the sex hormone-binding globulin-like domain of protein (C) of protein S.

3. The protein according to claim 1 or 2, wherein the recognized substance containing the target is an unwanted cell, virus, or bacterium.

4. The protein according to claim 1 or 2, wherein the (A) target binding region is bound to the N-terminus of any of the amino acid sequences shown in (i) to (xii) below: (i) the amino acid sequence shown in SEQ ID NO: 28 (ii) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 28 (iii) the amino acid sequence shown in SEQ ID NO: 29 (iv) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 29 (v) the amino acid sequence shown in SEQ ID NO: 30 (vi) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 30 (vii) the amino acid sequence shown in SEQ ID NO: 31 (viii) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 31 (ix) the amino acid sequence shown in SEQ ID NO: 32 (x) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 32 (xi) the amino acid sequence shown in SEQ ID NO: 33 (xii) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 33 5. The protein according to claim 1 or 2, wherein the measured value of the Crunch binding assay described below is 0.8 times or more than the measured value of the control protein described below. <Control Protein> The control protein is a protein in which a target binding region having the same sequence as the target binding region of the protein to be measured is bound to the N-terminus of any of the amino acid sequences shown in (i), (iii), (v), (vii), (ix), and (xi) above. <Crunch Binding Assay> (1) Prepare cells that express the target on the cell membrane. (2) If a tag peptide is not attached to the C-terminus of the protein, prepare a protein to be measured with a tag peptide attached to the C-terminus. (3) Add 5 × 10 cells to 100 μl of DPBS (Dulbecco's Phosphate Buffered Saline; containing 1 mg / ml BSA) containing 10 μg / ml of tagged protein. 4 (4) Add the cells and incubate on ice for 30 minutes, then collect the cells. (4) Use an anti-tagged antibody to measure the amount of tagged protein bound to the collected cells, and this amount of tagged protein is taken as the measurement value.

6. A pharmaceutical composition comprising the protein described in claim 1 or 2.

7. A nucleic acid comprising a polynucleotide encoding the protein described in claim 1 or 2.

8. The nucleic acid according to claim 7, further comprising a promoter operably linked to the polynucleotide encoding the protein.

9. A cell containing the nucleic acid described in claim 8.

10. A viral vector comprising the nucleic acid described in claim 8.

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