FGFR agonist VHH and composition containing FGFR agonist vhh

Abstract

PROBLEM TO BE SOLVED: The present invention, the present invention, provides a humanized FGFR agonist VHH and a composition containing a humanized FGFR agonist VHH. SOLUTION: The humanized FGFR agonist VHH comprises CDR1, CDR2, and CDR3 selected from the group consisting of (1) CDR1, CDR2, and CDR3 comprising amino acid sequences of SEQ ID NOS: 32, 33, and 1, respectively, (2) CDR1, CDR2, and CDR3 comprising amino acid sequences of SEQ ID NOS: 34, 35, and 2, respectively, (3) CDR1, CDR2, and CDR3 comprising amino acid sequences of SEQ ID NOS: 36, 37, and 3, respectively, and (4) CDR1, CDR2, and CDR3 comprising amino acid sequences of SEQ ID NOS: 38, 39, and 4, respectively. This VHH is an FGFR agonist VHH. This composition containing VHH can be a composition for a cell culture medium, a cosmetic composition, or a pharmaceutical composition, particularly a pharmaceutical composition for the treatment and prevention of fibrosis of various organs and for the inhibition of cancer metastasis.

Description

FGFR agonist VHH and compositions comprising FGFR agonist VHH

[0001] The present invention relates to humanized FGFR agonist VHHs and compositions comprising humanized FGFR agonist VHHs.

[0002] Animals have the ability to heal damaged tissues. Tissue healing is achieved by fibroblasts and mesenchymal cells differentiating into myofibroblasts, which then close the wound and synthesize and deposit extracellular matrix such as type I collagen. After the injury heals, myofibroblasts undergo apoptosis and disappear. However, due to persistent damage such as inflammation, myofibroblasts may persist without undergoing apoptosis. In this case, extracellular matrix synthesis continues indefinitely, resulting in an abnormal increase in extracellular matrix and tissue fibrosis. Diseases in which tissue loses elasticity due to the abnormal accumulation of extracellular matrix are generally referred to as fibrosis. This type of tissue fibrosis can occur in all organs except nervous tissue (e.g., heart, lungs, pancreas, liver, kidneys, etc.) (Non-Patent Document 1).

[0003] Treatment of fibrosis is important because it stiffens organs and ultimately leads to organ dysfunction. For example, idiopathic pulmonary fibrosis, a type of pulmonary fibrosis, causes lung stiffness, leading to respiratory distress and ultimately death. Patients with this disease have an average life expectancy of 3-5 years. Nonalcoholic fatty liver disease (NAFLD), a liver disease, is estimated to affect approximately 15 million people in Japan. Nonalcoholic fatty liver (NAFL) is a type of NAFLD, and its advanced stage, nonalcoholic steatohepatitis (NASH), is called NAFL. Ten percent of NAFLD patients progress from NASH to liver fibrosis, cirrhosis, and liver cancer. The number of patients with this condition is even higher in the United States, where 25-35% of the population (approximately 100 million people) are estimated to have NAFLD, and approximately 20% of these patients develop NASH.

[0004] COVID-19, caused by SARS-CoV-2, is known to cause various respiratory diseases. While many COVID-19 patients achieve remission, some suffer from sequelae such as palpitations, shortness of breath, and dyspnea. A follow-up study of patients with sequelae using CT scans found ground-glass opacities in the lungs in 44.1% of cases and fibrous stripes in 33.9% (Non-Patent Document 2). Pathological analysis of lung tissue from patients who recovered from COVID-19 also revealed fibrosis in areas of pneumonia (Non-Patent Document 3). In severe acute respiratory syndrome (SARS), which is caused by the same coronavirus as SARS-CoV-2, CT scans have confirmed fibrosis in lung tissue even seven years after onset. Therefore, it is expected that COVID-19 will also result in patients suffering from long-term sequelae due to fibrosis.

[0005] Given the large number of fibrosis patients and the severity of its symptoms, there has been a strong desire for a treatment for fibrosis. However, until now, fibrosis has been considered an irreversible phenomenon and treatment was considered impossible. However, recent advances in fibrosis research have led to a partial elucidation of its pathogenesis. It has been known that endothelial cells and immune cells present at the site of injury secrete various cytokines (Non-Patent Document 4). It is now believed that fibrosis occurs through a dynamic process in which these cytokines act on immune cells, myofibroblasts, and other cells to promote or suppress fibrosis. However, the detailed mechanism remains unknown.

[0006] Fibroblast growth factors (hereinafter sometimes abbreviated as "FGF") are cell growth factors discovered in 1973 and form a family of cell growth factors. 22 or 23 members have been identified in humans. This difference is due to whether human FGF15 and FGF19 are considered separate species, since human FGF19 is the mouse ortholog. All FGFs identified to date are signaling molecules with structural similarities and are known to be multifunctional proteins with a wide range of effects.

[0007] FGFs generally act as cell mitogens, but are known to have other effects and are sometimes referred to as "promiscuous growth factors." In biochemistry, "promiscuity" refers to the degree to which a single receptor or enzyme can bind to and react with a wide variety of molecules. In the case of FGFs, four receptor subtypes are activated by more than 20 different FGF ligands. FGFs are known to have many functions during development, including mesoderm induction, anterior-posterior axis patterning, limb formation, nervous system induction, and neurogenesis, as well as in mature tissues, including angiogenesis, keratinocyte organization, and wound healing. Various compositions, including cosmetics, have been developed based on the cell proliferation effects of FGFs.

[0008] FGFs 1 to 10 are known to bind to all fibroblast growth factor receptors (FGFRs). Of these, FGF1 is known as acidic FGF (aFGF) and FGF2 is known as basic FGF (bFGF). FGFs 11 to 14 are also known as FGF homologous factors 1 to 4 (FHF1 to 4). Despite their significant sequence similarity to other FGFs, they do not bind to FGFRs. Furthermore, because they are involved in intracellular processes not mediated by other FGFs, they are also known as intracellular FGFs and are said to function differently from other FGFs.

[0009] Furthermore, FGFs are classified into a subfamily containing FGF19, FGF21, and FGF23 as members. While other FGF family members exhibit local effects, the members of these subfamilies are known to act as metabolic regulators, i.e., to exert systemic effects. Among these, FGF21 is known to specifically bind to FGFR1, FGFR2, and FGFR3 and, together with its membrane-bound co-receptor β-klotho, improve insulin resistance and type 2 diabetes through these receptors. It has also been reported as a potential disease-modifying agent that reverses obesity and obesity-induced fatty liver and hyperglycemia. Anti-FGFR1 agonist antibodies have also been proposed as candidate drugs for the treatment of diabetes (see Patent Document 1, hereinafter referred to as "Prior Art 1").

[0010] There are four types of FGFRs (FGFR1-4), each of which has three extracellular immunoglobulin-like loop domains (domains 1-3) and an intracellular tyrosine kinase domain; these four types form the receptor tyrosine kinase family. When a ligand binds to the extracellular domain, the intracellular tyrosine kinase domain positively induces cell proliferation, and its activity is normally strictly controlled. However, in cancer cells, fusion with EML4-ALK or other proteins, or mutation of the tyrosine kinase gene sequence, can lead to a constantly activated state, like an activated EGF receptor, and induce "oncogenic transformation."

[0011] Patent Document 2 describes a single domain antibody (VHH antibody) that is a tyrosine kinase agonist for human FGFR1 to 4. This VHH antibody is obtained in a cell-free system from peptides obtained from a library, and has cell proliferation activity. Patent Document 1 also discloses that this agonist VHH antibody can be used in cell culture media for the proliferation and maintenance of undifferentiated states of fibroblasts, mesenchymal stem cells, iPS cells, and the like.

[0012] Tumor tissue contains not only cancer cells but also various types of cells, such as tumor blood vessels and cancer-associated fibroblasts (CAFs), which constitute the cancer microenvironment. CAFs are fibroblasts that make up the cancer stroma and are known to produce various growth factors that promote cancer cell proliferation. CAFs are further classified into cancer-promoting CAFs and non-myofibroblasts, and it is known that non-myofibroblasts contain cancer-suppressing CAFs that suppress tumor formation. In particular, intractable cancers such as pancreatic cancer are characterized by severe fibrosis in the stroma. CAFs play a central role in this fibrosis. Extracellular matrix such as collagen produced by CAFs and collagen fibers formed by lysyl oxidase (LOX) induce hardening of cancer tissue, increased internal pressure, and vascular collapse. In particular, intractable cancers, fibrosis is thought to inhibit the penetration of anticancer drugs, preventing them from reaching cancer cells and resulting in their ineffectiveness.

[0013] Patent Document 3 describes a method for treating gastric cancer, which comprises administering an anti-fibroblast growth factor receptor 2 IIIb (anti-FGFR2-IIIb) antibody and modified FOLFOX6 (mFOLFOX6) chemotherapy. This anti-FGFR2-IIIb antibody inhibits the binding of FGF2 or FGF7 to human FGFR2, thereby blocking the binding of FGF ligands to FGFR2.

[0014] Metastasis occurs when cancer cells migrate from the site of origin (primary tumor) and form tumors again in distant sites. The cure rate for cancer treatment has improved when the cancer is confined to the primary tumor and has not yet metastasized. However, the prognosis for advanced cases after metastasis remains poor, so early detection of cancer and prevention of metastasis are essential for cancer treatment. However, there are not enough known drugs that can effectively suppress cancer metastasis.

[0015] Furthermore, from the perspective of addressing environmental impact and animal ethics, the production of cultured meat, which is obtained by culturing animal cells in vitro, has recently attracted attention. Compared to the supply of meat through traditional livestock farming and the farming of crustaceans and fish, cultured meat is expected to reduce land use, water pollution, methane emissions, and carbon dioxide emissions, thereby reducing the environmental impact. Furthermore, from the perspective of animal ethics, it is expected that cultured meat will enable the supply of meat without factory farming and slaughterhouses.

[0016] However, compared with traditional livestock farming and aquaculture, cultured meat is expensive, hindering its widespread adoption. Most of the production cost is due to the high cost of growth factors and other components contained in the culture medium. For example, fetal bovine serum (FBS) is sometimes used in cell culture media, but serum-free media is preferred due to concerns about animal-derived products and animal ethics. Essential 8™, a serum-free medium containing eight components developed for human pluripotent stem cells (PSCs) and fibroblasts, is currently capable of culturing bovine myoblasts and is expected to be applied to cultured meat. However, since FGF2 and TGFβ account for 96% of the cost of Essential 8's eight components, the production cost of growth factors presents a major challenge for cost reduction.

[0017] JP 2020-125299 A International Publication No. 2022 / 270518 JP 2020-520903 A

[0018] Zhao, M., Wang, L., Wang, M. et al. Targeting fibrosis: mechanisms and clinical trials. Sig Transduct Target Ther 7, 206 (2022). https: / / doi.org / 10.1038 / s41392-022-01070-3So, M., Kabata, H., Fukunaga, K. et al. Radiological and functional lung sequelae of COVID-19: a systematic review and meta-analysis. BMC Pulm Med 21, 97 (2021). https: / / doi.org / 10.1186 / s12890-021-01463-0Sakai, T., Azuma, Y., Aoki, K. et al. Elective lung resection after treatment for COVID-19 pneumonia. Gen Thorac Cardiovasc Surg 69, 1159-1162 (2021). https: / / doi.org / 10.1007 / s11748-021-01630-4Gao C-C, Bai J, Han H and Qin H-Y (2022) The versatility of macrophage heterogeneity in liver fibrosis. Front. Immunol. 13:968879. doi: 10.3389 / fimmu.2022.968879Xiuqin Zhang, Omar A. Ibrahimi, Shaun K. Olsen, Hisashi Umemori, Moosa Mohammadi, David M.Ornitz, Receptor Specificity of the Fibroblast Growth Factor Family: THE COMPLETE MAMMALIAN FGF FAMILY*, Journal of Biological Chemistry, Volume 281, Issue 23, 2006, Pages 15694-15700, ISSN 0021-9258, https: / / doi.org / 10.1074 / jbc.M601252200.

[0019] In recent years, research and development into the treatment of fibrosis has been active, and in Japan, drugs for the treatment of pulmonary fibrosis, such as pirfenidone in 2008 and nintedanib in 2015, were approved. Pirfenidone inhibits the production of TGF-β, which is involved in the differentiation of lung epithelial cells into fibroblasts; it inhibits the production of inflammatory cytokines (TNF-α, IL-1, IL-6, etc.) and enhances the production of anti-inflammatory cytokines (IL-10); it suppresses the decline of IFN-γ, which leads to a correction of the Th1 / 2 balance; and it inhibits the production of growth factors (FGF2, PDGF) involved in fibrosis formation. Nintedanib is a tyrosine kinase inhibitor that inhibits PDGFRs, EGFRs, and FGFRs, thereby suppressing the recruitment of lymphocytes and fibrocytes and inhibiting their differentiation into fibroblasts.

[0020] While these drugs are effective in inhibiting the progression of fibrosis, they cannot cure fibrosis, nor can they completely halt the progression of the disease. Furthermore, pirfenidone causes liver dysfunction in some patients and photosensitivity in more than half of those taking it, making it unsuitable for patients with impaired liver function. Patients taking it must always wear sunscreen, long sleeves, and sunscreen. Nintedanib causes diarrhea, including severe diarrhea, in more than half of patients. These side effects are thought to be due to the multiple targets of pirfenidone and nintedanib.

[0021] Despite these side effects that significantly reduce the quality of life of those who take them, these drugs only suppress the progression of fibrosis, and do not provide a fundamental cure. Therefore, there is a need for therapeutic drugs with fewer side effects that can fundamentally treat fibrosis or more effectively suppress the progression of the disease. Furthermore, no effective drugs have yet been developed for NASH, a type of liver fibrosis.

[0022] As mentioned above, immune cell-derived cytokines at the site of injury are thought to promote fibrosis by acting on myofibroblasts. Therefore, it is thought that controlling these factors could potentially cure fibrosis. However, the detailed relationship between cytokines and fibrosis has not been elucidated, so it is unclear which cytokines are effective targets. For example, FGF21, which is involved in hepatic fat metabolism, is expected to be useful in the treatment of NASH, and research is ongoing. However, as of September 9, 2022, the PEGylated NASH treatment BMS-986036 failed to produce effective results in Phase II clinical trials (https: / / clinicaltrials.gov / ct2 / history / NCT03486912?V_35&embedded=true). Therefore, there is a need for a treatment that targets cytokines that have not previously been targeted and that can fundamentally cure fibrosis rather than simply inhibit its progression. Alternatively, there is a need for a treatment that more effectively inhibits the progression of fibrosis. Furthermore, there is also a need for the development of effective treatments that inhibit cancer metastasis.

[0023] Furthermore, cytokines, which are endogenous substances in the body, are known to have a short half-life in the blood even when administered, making them unsuitable as drugs. Therefore, there is a need for drugs that can serve as alternatives to cytokines, which have a long half-life in the blood, are stable enough for long-term storage, and can be easily prepared.

[0024] Under these circumstances, the inventors of the present invention conducted extensive research and completed the present invention. Specifically, one aspect of the present invention is a VHH-Fc conjugate (hereinafter sometimes simply referred to as "VHH-Fc") comprising a humanized VHH that binds to the extracellular domain of fibroblast growth factor receptor (FGFR) 1 and an Fc sequence of a human antibody. Preferably, the VHH comprises a CDR3 selected from the group consisting of the amino acid sequences represented by SEQ ID NOS: 1 to 4 in the Sequence Listing. Furthermore, it is more preferable that the binding activity of the VHH is in the order of FGFR2, FGFR4, FGFR3, FGFR1, FGFR1, FGFR4, and FGFR3, but not FGFR2; or in the order of FGFR2, FGFR4, and FGFR1, but not FGFR3; or in the order of FGFR4, FGFR2, and FGFR1, but not FGFR3.

[0025] The VHH-Fc conjugate may also be a dimer. In this case, each VHH contained in the conjugate may have the same CDR3 sequence or may have a different CDR3 sequence. Another aspect of the present invention is a pharmaceutical composition for treating fibrosis, which contains the humanized VHH-Fc conjugate as an active ingredient. The dissociation constant of the VHH with the extracellular domain of FGFR1 is 10 －８ It is preferable that the CDR3 sequence is less than M. Furthermore, the humanized VHH preferably has a CDR3 sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 4 in the Sequence Listing, and more preferably has a full-length amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 5 to 8 in the Sequence Listing.

[0026] Here, VHH (variable domain of heavy chain of heavy chain antibody) refers to an antibody composed only of the variable domain of a single-chain antibody consisting only of the heavy chain of llama, alpaca, etc. VHH has a simpler structure than antibodies possessed by humans and other animals, and therefore is highly thermostable and can be easily produced in large quantities in cells other than human cells such as Escherichia coli, making it preferable for use as a drug. Furthermore, the VHH of the present invention has a specific CDR3, making it excellent in that it has high target specificity and few side effects as a drug.

[0027] The VHH of the present invention is preferably linked to an Fc sequence derived from a human antibody, which allows for efficient dimerization of the target receptor and efficient signal transduction of the receptor. Furthermore, the pharmaceutical composition for treating fibrosis of the present invention is characterized in that the organ affected by fibrosis is an organ other than the brain. Furthermore, the organ affected by fibrosis is preferably the liver. Another aspect of the present invention is a pharmaceutical preparation containing the pharmaceutical composition for treating fibrosis.

[0028] Another aspect of the present invention is a VHH-Fc conjugate comprising a VHH having an amino acid sequence selected from the group consisting of the CDR3 amino acid sequences represented by SEQ ID NOS: 1 to 4 in the Sequence Listing, and a human Fc amino acid sequence. The VHH is preferably a VHH selected from the group consisting of VHHs having the amino acid sequences represented by SEQ ID NOS: 5 to 8 in the Sequence Listing, and more preferably the amino acid sequence of the conjugate is at least 90%, 95%, or 98% homologous to any one selected from the group consisting of the amino acid sequences represented by SEQ ID NOS: 11 to 14 in the Sequence Listing, or to any one of the amino acid sequences represented by SEQ ID NOS: 11 to 14.

[0029] The present invention provides a pharmaceutical composition that can fundamentally treat fibrosis in various organs with minimal side effects. Fibrotic organs include the heart, lungs, pancreas, liver, and kidneys. In particular, the heart affected by myocardial fibrosis, the lungs affected by idiopathic pulmonary fibrosis (IPF), the pancreas affected by cystic fibrosis, the liver affected by NAFLD, including NASH, and the kidneys affected by nephrosclerosis.

[0030] Figure 1 shows a scheme for hit compound screening using the cDNA display method. Figure 2 shows the structure of the linker used in the cDNA display method. Figure 3 is a graph showing the results of a single-point binding assay using Octet for screening products obtained using the extracellular domain of human FGFR1 as the target protein. The vertical axis represents response (nm), and the horizontal axis represents each clone VHH. A thick solid line indicates a response of 0.1.

[0031] Figure 4 is a graph showing the results of a single-point binding assay using Octet for screening products obtained using the extracellular domain II of human FGFR1 as the target protein. The vertical axis represents response (nm), and the horizontal axis represents each clone VHH. A thick solid line indicates a response of 0.1. Figure 5 is a graph showing the results of affinity measurements of VHH clones for the target molecule FGFR1. Figure 5(A) shows the results of affinity measurements for VM46, and Figure 5(B) shows the results for VM1637. The vertical axis represents response, and the horizontal axis represents time (seconds). The numbers in the graphs indicate the concentrations of the analytes used.

[0032] Figure 6 is a graph showing the results of affinity measurements of each VHH clone for target molecules. The vertical axis is the same as in Figure 5. Figure 6(A) shows the results of affinity measurements for the target molecule FGFR1(IIIb). Figure 6(B) shows the results of affinity measurements for the target molecule FGFR1(IIIc). Figure 6(C) shows the results of affinity measurements for the target molecule FGFR2(IIIb). Figure 6(D) shows the results of affinity measurements for the target molecule FGFR2(IIIc). Figure 7 is a graph showing the results of affinity measurements of each VHH clone for target molecules. The vertical axis is the same as in Figure 5. Figure 7(A) shows the results of affinity measurements for the target molecule FGFR3(IIIb). Figure 7(B) shows the results of affinity measurements for the target molecule FGFR3(IIIc). Figure 7(C) shows the results of affinity measurements for the target molecule FGFR4. Figure 8 is a graph showing the results of affinity measurements of each VHH clone for target molecules. The vertical axis is the same as in Figure 5. Figure 8(A) shows the results of affinity measurements for the target molecule FGFR1(IIIb). Figure 8(B) shows the results of affinity measurements for the target molecule FGFR1(IIIc). Figure 8(C) shows the results of affinity measurements for the target molecule FGFR2(IIIb). Figure 8(D) shows the results of affinity measurements for the target molecule FGFR2(IIIc).

[0033] Figure 9 is a graph showing the results of affinity measurements of each VHH clone for the target molecule. The vertical axes are the same as those in Figure 5. Figure 9(A) shows the results of affinity measurements for the target molecule FGFR3(IIIb). Figure 9(B) shows the results of affinity measurements for the target molecule FGFR3(IIIc). Figure 9(C) shows the results of affinity measurements for the target molecule FGFR4. Figure 10 is an electrophoretic image showing the results of SDS-PAGE performed after purification of VHH-Fc. Outside the figure, the numbers on the left indicate molecular weight. VF151, VF152, VF155, and VF156 indicate the numbers of the VHH-Fc binders used, respectively.

[0034] Figure 11 is a graph showing the results of affinity measurements of VHH-Fc clones for the target molecule FGFR1. Figure 11(A) shows the results of affinity measurements for VF151, Figure 11(B) for VF152, Figure 11(C) for VF155, and Figure 11(D) for VF156. The vertical axis shows response, and the horizontal axis shows time (seconds). The numbers in the graphs indicate the concentration of the analyte used. Figure 12 is a graph showing the quantification of cell proliferation when cultured cells were stimulated with VHH-Fc, along with the EC calculated based on the graph. 50 The vertical axis of the graph shows relative fluorescence intensity (RLU), and the horizontal axis shows the concentrations (mg / mL) of the samples and subjects used. Figure 12(A) shows the growth curve and EC50 values ​​for FGF, and Figures 12(B) to (F) show the growth curves and EC50 values ​​for VF151 to VF156, respectively. Figure 13 is a graph showing the results of quantitative PCR analysis of gene expression levels. Figure 13(A) shows the relative expression levels of ACTA2, and Figure 13(B) shows the relative expression levels of COL1A1. Figure 14 is a graph showing the thermal stability of each VHH-Fc. Figure 14(A) shows the raw data obtained when thermal stability was analyzed using UNcle. The vertical axis of the graph shows scattered light intensity (SLS) at 266 nm. Figure 14(B) shows the thermal stability of each VHH-Fc (VF151 to VF154) and the subject FGF2, calculated from the measured data.

[0035] Photographs showing observation of cancer metastasis to the liver on the 7th and 14th days after organoid transplantation. Photographs showing fluorescence microscopic images of the liver and its sections obtained on the 14th day after organoid transplantation. Photographs showing fluorescence microscopic images of liver sections obtained on the 14th day after organoid transplantation and a graph showing the area ratio of metastatic tumors to the entire liver in the liver sections. Photographs of liver sections obtained on the 14th day after organoid transplantation, showing hematoxylin and eosin (H&E) staining (top row), anti-αSMA antibody staining (middle row), and immunohistochemical staining with anti-Transgelin antibody (bottom row).

[0036] The present invention is described in further detail below using embodiments. The present invention relates to a humanized agonist VHH for treating fibrosis. The VHH may further comprise the Fc region of a human antibody at its C-terminus. The VHH has the agonistic property of binding to FGFR. The Fc region may be linked to the VHH via a linker.

[0037] An "agonist" is a drug that acts on receptor molecules in the body and exhibits functions similar to those of neurotransmitters and hormones. Substances that actually work in the body are called ligands to distinguish them. If an agonist acts on only one specific receptor out of multiple receptors and not on others, it is called a selective agonist. An example of such a selective agonist is NMDA, which binds to only one of the four receptors for glutamate, the main excitatory neurotransmitter in the central nervous system.

[0038] Agonists that are less active and less effective than biomolecules are called partial agonists. Examples of partial agonists include dopamine D2 receptor antagonists such as beta-blockers, opioids, benzodiazepine hypnotics, aripiprazole (Abilify), and phencyclidine (PCP anesthetic), which are used in medicine.

[0039] Furthermore, "VHH" refers to an antibody fragment consisting of a single domain of approximately 15 kDa contained in IgG, which is composed only of a heavy chain possessed by animals of the Camelidae family. In this specification, the term "single domain antibody" refers to a molecule having an effect equivalent to the smallest antibody whose diagnostic or therapeutic usefulness has been proven, and which is a heavy chain antibody (VHH) of camelids. H H) or cartilaginous fish IgNAR (V NAR VHHs are antibody fragments derived from a single monomeric variable domain of a VHH. VHHs are approximately one-tenth the molecular weight of a normal antibody, and have excellent stability, affinity, and tissue permeability, as well as faster blood clearance than antibody molecules.

[0040] Furthermore, the term "antibody" refers to a substance that is produced in vivo in response to an antigen and specifically binds to the antigen, as well as artificially modified versions thereof. Examples of antibodies include immunoglobulin G (IgG), as well as artificially modified IgGs, such as those in which the framework sequence (hereinafter sometimes referred to as "FR") of a non-human IgG has been partially or completely replaced with that of a human IgG, or those in which an artificial framework has been partially bonded to the FR. An "antibody fragment" refers to a portion of an antibody, such as one that includes one of the multiple domains that make up the heavy chain.

[0041] "Fibroblast growth factor receptor" (FGFR) refers to a receptor to which a fibroblast growth factor (FGF) binds. Here, the FGFR is preferably any receptor selected from the group consisting of fibroblast growth factor receptor 1 (hereinafter sometimes referred to as "FGFR1"), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), and fibroblast growth factor receptor 4 (FGFR4).

[0042] FGFRs 1-4 share a common structure: they contain three immunoglobulin loops (I, II, III) in the extracellular domain and a tyrosine kinase domain within the cell. It is said that even if immunoglobulin loop I is missing, it has almost no effect on signal transduction. Loop II is highly conserved across FGFRs 1-4 and is believed to be essential for ligand binding. Ligand binding promotes dimerization of FGFRs. This brings the tyrosine kinase domains into close physical proximity, resulting in mutual phosphorylation of tyrosine residues within the kinase domains. Effector molecules then bind to the activated kinase domain, and the effector molecules are activated by phosphorylation of their tyrosine residues. In this way, various proteins within the cell are activated one after another.

[0043] The Fc region refers to the portion of an antibody obtained by digesting it with papain that does not contain the variable region. Fc regions bind to each other and contribute to protein dimerization. The Fc region of an antibody bound to an antigen is recognized by Fc receptors on phagocytes such as leukocytes and macrophages, promoting phagocytosis by these cells (opsonization). It also has functions such as complement activation and antibody-dependent cellular cytotoxicity (ADCC). The Fc region-added agonist VHH of the present invention is preferred because it can efficiently dimerize at least FGFR and activate intracellular signaling.

[0044] Furthermore, although peptide linkers and chemical linkers can generally be used as linkers connecting VHH and the Fc portion, the use of peptide linkers is preferred when using production systems that employ cells, etc. In particular, when an Fc sequence is added, it is preferable to use the hinge of the antibody from which the Fc is derived as the linker, and this may be modified as appropriate, for example by introducing a mutation to stabilize the VHH-Fc dimer. For example, the peptide linker set forth in SEQ ID NO: 10 in the Sequence Listing is preferred in terms of the flexibility of the linker that ensures the movement of the VHH.

[0045] VHH sequences of the present invention can be obtained by immunizing animals with heavy chain antibodies, such as camels and llamas, or by screening artificial VHH libraries using techniques such as phage display, mRNA display, and cDNA display. From the viewpoints of ease of acquisition and diversity, it is preferable to use the cDNA display method.

[0046] 1. Preparation of cDNA Display Molecules In the cDNA display method, a linker with the structure shown in Figure 2 is used to form a display molecule having mRNA, a peptide encoded by the mRNA, and a cDNA encoding the peptide. As shown in Figure 2, the cDNA display linker consists of a main chain and side chains. The main chain has an mRNA binding site to which mRNA binds, a photocrosslinking site for linking the mRNA to the main chain, a solid-phase binding site for binding to a solid phase after mRNA binding, a solid-phase cleavage site for subsequently releasing the cDNA molecule from the solid phase, a side-chain binding site for linking a side chain, and a reverse transcription site for synthesizing cDNA corresponding to the peptide corresponding to the mRNA.

[0047] Here, the photo-crosslinking moiety is preferably composed of a photo-crosslinking base that forms a crosslink when exposed to long-wavelength light, as this causes less damage to the mRNA to which it is linked. Examples of such photo-crosslinking bases include 3-cyanovinylcarbazole (hereinafter sometimes abbreviated as "cnvK") and analogs of cnvK. Examples of solid-phase binding moieties include biotin and streptavidin.

[0048] The solid-phase cleavage site is preferably composed of a nucleotide that can be specifically cleaved by an enzyme or the like. For example, it is preferably composed of a ribonucleotide containing a guanine base (hereinafter sometimes abbreviated as "rG") or a nucleotide containing a modified base, because these can be site-specifically cleaved by a specific enzyme. The photo-crosslinking site is located between the solid-phase binding site and the side chain binding site, and the reverse transcription site is located near the 5' end of the main chain. The solid-phase binding site is located near the 3' end of the main chain, and the solid-phase cleavage site is located between the solid-phase binding site and the mRNA binding site.

[0049] The side chain is bound at one end to the side chain binding site of the main chain, and a peptide presentation site for presenting a peptide translated from the mRNA is provided at the free end. Fluorescein, FITC, or other fluorescent molecules can be linked between the peptide presentation site and the side chain binding site to facilitate detection.

[0050] In the cDNA display method, the sequence of the mRNA-binding site of the main chain is first designed based on the nucleotide sequence of the target peptide or other target molecule, and the cDNA display linker is then prepared. mRNA from the desired library is bound to the mRNA-binding site, and then light is irradiated to link the mRNA to the main chain at the photocrosslinking site. The mRNA-cDNA display linker conjugate is added to a cell-free translation system solution, whereby the mRNA linked to the main chain is translated into a peptide and presented at the peptide presentation site of the cDNA display linker, producing an mRNA display molecule in which the mRNA and a peptide with a corresponding sequence are linked to the cDNA display linker. The peptide displayed here can have the desired sequence. In the present invention, this can be used as the target VHH to obtain a peptide with FGFR agonist activity.

[0051] Next, the mRNA display molecule is bound to a solid phase at the solid-phase binding site, and cDNA is synthesized on the cDNA display linker using reverse transcriptase to produce a cDNA display molecule. Here, in the cDNA display molecule, mRNA and cDNA form a double strand (see Figure 1). The solid phase is not particularly limited as long as it can bind to the solid-phase binding site. For example, when the solid-phase binding site is composed of biotin, the solid-phase surface is preferably coated with streptavidin. The solid phase may be in the form of a plate or beads; however, a bead-shaped solid phase is preferred for ease of handling during selection, etc., as described below. The synthesized cDNA display molecule is then cleaved from the solid phase using an enzyme to produce a free molecule. For example, an endonuclease can be used as the enzyme. When the solid-phase cleavage site is composed of rG, endonuclease V (hereinafter sometimes referred to as "EndoV") is preferred because it can specifically cleave at the solid-phase cleavage site.

[0052] A selection step is performed to select display molecules that present peptides that bind to the target molecule from the various cDNA display molecules formed as described above. The process of removing display molecules other than the cDNA display molecules that present peptides that bind to the target molecule and increasing the content of display molecules that bind to the target molecule is called "enrichment."

[0053] In the selection step, the target molecule bound to the solid phase is contacted with the cDNA display molecule obtained as described above, and only the cDNA display molecules bound to the target molecule are collected to obtain molecules that bind to the target molecule from the cDNA display molecules. When streptavidin-coated beads (hereinafter sometimes referred to as "SA beads") are used as the solid phase, the target molecule can be immobilized on the SA beads by binding biotin to the target molecule, and used for selection.

[0054] The library composed of cDNAs in the cDNA display molecules obtained in the selection step is called the 1st library. By repeating the same procedure as above, peptides that bind to the target molecule contained in the library are enriched. The library obtained after the Nth iteration is called the Nth library. The library enrichment procedure is preferably repeated a desired number of times, for example, 3 to 5 times. Enrichment is insufficient if repeated up to two times, while repeating six or more times does not improve the enrichment rate.

[0055] 2. Analysis of Screening Products by NGS The enriched library, which is the screening product obtained above, is analyzed using a next-generation sequencer (hereinafter sometimes abbreviated as "NGS"). First, the enriched library is subjected to PCR and amplified under the following conditions to obtain a PCR product. The reaction mixture was prepared by placing approximately 2.5 to 10 μL of PrimeSTAR MAX, approximately 0.5 to 2 μL of the enriched screening product, and approximately 1 to 4 pmol each of the primers PL_prRd-N4_NL_FW (SEQ ID NO: 15) and PL_prRd-N4_Ytag_RV (SEQ ID NO: 16) in a tube and adjusting the volume to approximately 5 to 20 μL with ultrapure water. The sequences of these two primers are shown below.

[0056] 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNATGGAAGTACAATTAGTTGAATCTGGTGGTGGGCTTG-3' (SEQ ID NO: 15) 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNTGAAGAGACTGTCACCAACGTGCCTTG-3' (SEQ ID NO: 16)

[0057] The PCR program involves approximately 10 to 14 cycles with an annealing temperature of approximately 60 to 64°C and an extension time of approximately 10 to 20 seconds to obtain a PCR product. The resulting PCR product is purified, and index PCR is performed using the purified product as a template. The reaction mixture for index PCR consists of approximately 10 to 15 μL of PrimeSTAR MAX, approximately 0.5 to 2 μL of the purified product described above, approximately 0.25 to 1 μL of approximately 2.5 to 10 μM forward / reverse primers (Nextera XT Index 1 Primers (N7XX) and Nextera XT Index 2 Primers (S5XX)), and approximately 5 to 20 μL of ultrapure water.

[0058] The PCR program was the same as above, except for the annealing temperature of approximately 50 to 54°C and the extension time of approximately 10 to 20 seconds. Approximately 7 to 9 cycles were performed to obtain PCR products. The Index PCR products were purified to obtain purified products, and the DNA concentrations of these purified products were measured using a NanoPad DS-11. The Index PCR purified products were diluted with RNase-free water to approximately 8 to 12 nM, and approximately 4 to 6 μL of each product was collected and mixed in one tube. Next, an NGS sample library was created according to the Miseq (Illumina) instruction manual and analyzed using the MiSeq Reagent Nano Kit v2 (500 Cycles) (Illumina). The DNA sequences obtained from NGS were translated into amino acid sequences to obtain VHH sequences that bind to each target molecule.

[0059] 3. Production of VHH (monomer) The VHH clone obtained as described above is expressed in an expression system. For such an expression system, it is preferable that the culture supernatant contains very little HCP (host cell protein), a protein naturally secreted by the producing bacteria. This ensures that the target protein secreted into the culture supernatant is present in very high purity. Furthermore, it is preferable to use bacteria that secrete the target protein with the correct higher-order structure.

[0060] The use of such bacteria eliminates the need for bacterial disruption and results in almost no detectable protease activity in the culture supernatant, which has the advantage of virtually eliminating the degradation of the secreted target protein, a major problem in many protein secretion production systems.

[0061] Examples of such producers include Corynebacterium glutamicum (hereinafter sometimes referred to as "C. glutamicum"). C. glutamicum has been used for industrial amino acid production for over 60 years, and has a wealth of experience and proven track record. A method for culturing high cell counts using inexpensive and simple media has been established. In addition, the C. glutamicum strain itself is highly safe. This is because C. glutamicum does not produce toxic substances and is not pathogenic. The following explanation will be given using C. glutamicum as an example.

[0062] 3-1. Construction of a plasmid for VHH (monomer) expression. Using the enriched library as a template, sequences for restriction enzyme digestion are added by PCR in the usual manner to obtain a PCR product. Primers should be able to add restriction enzyme recognition sequences that are not contained in the plasmid vector or enriched library.

[0063] This PCR product and the C. glutamicum expression plasmid vector are each treated with a restriction enzyme that recognizes one of the insertion sites at the enzyme's optimal temperature for approximately 1 hour, and then with a restriction enzyme that recognizes the other insertion site at the enzyme's optimal temperature for approximately 1 hour. The reaction mixture derived from the PCR product, which will become the insert DNA, is purified. The reaction product derived from the plasmid vector is subjected to 1% agarose gel electrophoresis at approximately 80 to 120 V for approximately 25 to 35 minutes, and then excised from the gel after electrophoresis and purified to obtain the purified product.

[0064] The purified plasmid vector DNA is dephosphorylated using a dephosphorylating enzyme at about 35 to about 39°C for about 1 hour. The insert DNA and plasmid vector DNA are then mixed at a molar ratio of about 1:10, and a ligation reaction is carried out overnight at about 16°C using a ligation enzyme such as Ligation High (Toyobo Co., Ltd.) to obtain a plasmid library into which the selected VHH library has been introduced.

[0065] 3-2. Expression of VHH (monomer) using a producer bacterium. The following describes the process using the plasmids obtained in 3-1 above, for example, by electroporation, using C. glutamicum as the producer bacterium. First, transformants are prepared by introducing the transformants into a desired medium, such as CM2G medium, and pre-cultured overnight at approximately 28 to 32°C. The pre-culture solution is then subcultured in PM1S medium placed in each well of a 96-deep-well plate and cultured at approximately 20 to 30°C for approximately 60 to 84 hours. Since VHH (monomer) is secreted into the culture supernatant, after the culture is complete, the plate is centrifuged at approximately 3,000 to 5,000 x g for approximately 15 to 45 minutes at approximately 20°C, and the supernatant is recovered. The centrifuged supernatant is passed through a 0.22 μm filter to remove the bacterial cells in the supernatant, and the supernatant is stored frozen at −80° C. as a stock.

[0066] 4. Octet Single-Point Binding Assay: The binding activity of the VHH clones obtained as described above to each target molecule is measured using biolayer interferometry. For example, OctetRED384 (Fortebio) can be used for this assay, as shown below. The concentration of a ligand, e.g., a portion of FGFR1, is adjusted to approximately 100 to approximately 400 nM, and approximately 60 to approximately 80 μL of assay solution is added to a 384-well plate to measure the binding of each target molecule to the VHH clones immobilized on the well plate. Prior to measurement, the tip of a Dip and Read® His1K Biosensor (Fortebio) is immersed in approximately 200 μL of PBS-T (PBS containing 0.05% Tween 20, sometimes referred to as "PBS-T" hereinafter, pH approximately 7.4) for approximately 5 to 15 minutes to hydrate the sensor chip.

[0067] The measurement order for each run is as follows: 1) Baseline step: Measurement in PBS-T for approximately 30 seconds. 2) Loading step: Measurement for approximately 60 seconds using VHH diluted approximately 50-fold in PBS-T. 3) Baseline step: Measurement for approximately 30 seconds in PBS-T. 4) Association step: Measurement for approximately 100 seconds using each target molecule diluted in PBS-T. 5) Dissociation step: Measurement for approximately 100 seconds in PBS-T. 6) Regeneration step: Measurement for 5 seconds in glycine-HCl (pH approximately 2.2) and approximately 30 seconds in PBS-T. These steps are repeated at least three times. The data obtained are processed using Octet software (Molecular Devices) to obtain the results of the single-point binding assay. For example, clones showing a binding response of approximately 0.1 nm or greater can be considered hit clones.

[0068] 5. Sequence analysis of hit clones Each VHH gene in the hit clones is identified by sequence analysis. The hit clone transformant is cultured overnight at approximately 37°C, and colony PCR is performed on the culture medium. The resulting PCR product is purified using AMPure XP, and then each DNA sequence of the purified DNA is analyzed using a sequencer. Any duplicated sequences among the hit clones obtained are counted as one, and the resulting clone sequence is designated as a unique VHH clone.

[0069] 6. Multipoint binding assay using Octet Biolayer Interferometry. The interaction between purified VHH clones and target proteins was analyzed using OctetRED384 (Fortebio). Purified VHHs were immobilized on a His1K sensor chip (Fortebio), and measurements were performed by binding to target proteins prepared at approximately 100-200 nM and diluted two-fold. Prior to measurements, the tip of the Dip and Read SA Biosensor was immersed in approximately 200 μL of PBS-T (PBS containing 0.05% Tween 20, pH approximately 7.4) for approximately 10 minutes to hydrate the sensor chip. Approximately 40-80 μL of each measurement solution was then added to a 384-well plate, and measurements were performed following steps 1) to 6) below.

[0070] 1) Baseline step: Measurement for approximately 60 seconds in PBS-T. 2) Loading step: Measurement for approximately 120 seconds using purified VHH diluted approximately 100-fold in PBS-T. 3) Baseline step: Measurement for approximately 60 seconds in PBS-T. 4) Association step: Measurement for approximately 120 seconds using FGFR1 extracellular domain diluted in PBS-T. 5) Dissociation step: Measurement for approximately 120 seconds in PBS-T. 6) Regeneration step: Measurement for approximately 5 seconds in glycine-HCl (pH approximately 2.2) and approximately 5 seconds in PBS-T, repeated at least three times. After measurement, the reference value (PBS-T only) was subtracted from the actual measurement value, and global fitting was performed using a 1:1 binding model using Octet software to calculate affinity.

[0071] 8. Preparation of VHH-Fc The Fc sequence of a human antibody is added to the unique VHH clone obtained above using standard methods. 8-1. Construction of a VHH-Fc Expression Plasmid A VHH-Fc expression plasmid can be prepared by, for example, requesting synthesis of a human Fc sequence from a gene synthesis service and inserting it into a plasmid containing a VHH clone sequence. Alternatively, if an Fc expression plasmid is available, it can be prepared by inserting the VHH nucleotide sequence into that plasmid. The insertion can be performed, for example, using the restriction enzyme method described above, or using a system utilizing homologous recombination such as Gibson Assembly (New England Biolabs). In these cases, insertion can be performed after adding a restriction enzyme recognition sequence or a homologous recombination sequence by PCR using conventional methods.

[0072] The resulting VHH-Fc expression plasmid is used to transform competent cells, such as Escherichia coli JM109. The transformed E. coli is plated on an agar medium plate and cultured overnight at approximately 36 to 38°C. The resulting colonies are picked and cultured overnight under the same conditions. The VHH-Fc expression plasmid is then extracted and further purified. This procedure may be performed using, for example, the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.). The DNA of the extracted plasmid is then sequenced.

[0073] 8-2. Expression of VHH-Fc VHH-Fc can be obtained by introducing the prepared VHH-Fc expression plasmid into appropriate cultured cells, such as Expi293F cells (Thermo Fisher Scientific), and secreting it into the culture medium. Plasmid introduction can be performed using a standard chemical transfection method, such as Expifectamine® 293 (Thermo Fisher Scientific) and Opti-MEM (Gibco) according to the accompanying instructions. The cells are seeded into a flask and cultured at approximately 35-39°C in an environment of approximately 6-10% CO2. The transfected cells are cultured for approximately 84-108 hours, after which the culture supernatant is recovered. Cells can be removed from the culture supernatant by passing the recovered culture supernatant through a 0.22 μm filter or similar. Hereinafter, the culture supernatant obtained after the treatment described above will sometimes be simply referred to as the "supernatant."

[0074] 8-2. Purification of VHH-Fc VHH-Fc is purified from the supernatant via Protein A. For this purification procedure, a column packed with Amsphere A3 (JSR Corporation, hereafter referred to as "carrier") can be used. The supernatant is applied to a column packed with approximately 400 to approximately 600 μL of carrier. The carrier is washed by adding approximately 4 to approximately 6 mL of PBS, approximately 4 to approximately 6 mL of high-salt PBS (PBS containing approximately 1 M NaCl), and approximately 4 to approximately 6 mL of PBS, in that order. The carrier is then eluted with approximately 4 to approximately 6 mL of approximately 100 mM Glycine-HCl, pH approximately 2.2 (Fujifilm Wako Pure Chemical Industries, Ltd.), and neutralized with approximately 400 to approximately 600 μL of approximately 1 M Tris-HCl buffer (pH approximately 8.5). The eluted VHH-Fc is transferred to, for example, an Amicon Ultra 10 kDa (Millipore) and centrifuged at approximately 3,000 to 4,000 xg for approximately 25 to 35 minutes at approximately 4°C to concentrate the VHH-Fc. Approximately 3 to 5 mL of PBS is then added, and the eluate is centrifuged under the same conditions as above to replace the solvent with PBS. This PBS replacement procedure is repeated at least three times. The purity of the purified VHH-Fc can be confirmed by SDS-PAGE. The concentration of the purified VHH-Fc can be quantified by the BCA method using, for example, the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) with bovine serum albumin as the standard protein. VHH-Fc can be obtained in this manner.

[0075] The VHH-Fc obtained by the above method is thought to be secreted into the culture supernatant as a homodimer, but heterodimeric VHH-Fc can also be obtained by simultaneously transfecting two different VHH-Fc expression plasmids prepared above.

[0076] Compositions Comprising FGFR1 Agonist VHHs The present invention also relates to pharmaceutical and cosmetic compositions comprising FGFR1 agonist VHHs. The term "cosmetic composition" as used herein encompasses pharmaceuticals and medicinal drugs. Furthermore, the term "cosmetic composition" as used herein also encompasses quasi-drugs.

[0077] The excipients disclosed herein may include, but are not limited to, water, glycerol, saline, vegetable oils, fruit oils, flower extracts, mineral oils, synthetic oils, sugar compounds, silicates, calcium salts, magnesium salts, sodium chloride, potassium chloride, lactic acid, starch, sugar alcohols, cellulose, activated carbon, glycerin, butter, amino acids, paraffin, honey, wax, beeswax, agar, calcium carbonate, citric acid, tartaric acid, stearic acid, xanthan gum, benzoic acid, polyethylene glycol, silicones, derivatives thereof, salts thereof, or any combination thereof. The cosmetic composition may further include fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, lubricants, glidants, antioxidants, surfactants, flavoring agents, solvents, buffers, chelating agents, viscosity modifiers, surface active agents, humectants, or any combination thereof.

[0078] The compositions of the present invention can be administered by various routes, including, but not limited to, direct application to the skin, oral administration, intra-arterial administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, intracardiac administration, intraventricular administration, intratracheal administration, oral administration, rectal administration, intraperitoneal administration, intradermal administration, topical administration, transdermal administration, and intrathecal administration, or by implantation or inhalation. The FGFR1 agonist VHH can be in the form of a solid, semi-solid, gel, liquid, or gas. Specific dosage forms include, but are not limited to, capsules, powders, granules, ointments, liquids, suppositories, enemas, injections, inhalants, liposomes, and aerosols. In another embodiment, a nucleic acid molecule encoding an FGFR1 agonist VHH can be carried in a liposome or on a microparticle and delivered to the skin, organs, tissues, or the like.

[0079] Pharmaceutical Compositions Comprising FGFR Agonist VHHs Pharmaceutical compositions comprising the FGFR agonist VHHs of the present invention are effective against diseases that respond to FGF, particularly FGF2. For example, pharmaceutical compositions comprising the FGFR agonist VHHs of the present invention can be used for the treatment and prevention of fibrosis, particularly pulmonary fibrosis. "Treatment" refers to therapeutic treatment, and refers to reducing the severity, slowing the progression, or stopping the progression of the targeted disease or disorder. In particular, in the present invention, "treatment" includes shrinking or stopping the growth of lesions, slowing the growth, or slowing the progression of symptoms, inhibiting progression, or stopping the progression of a disease, or slowing the progression of a disease, inhibiting symptoms, or completely or partially alleviating symptoms, or reducing the severity. "Treatment" also includes promoting the effect of other drugs used in combination.

[0080] "Subject" includes, but is not limited to, humans and other mammals, laboratory mammals, domestic mammals, sport mammals, and pet mammals. An example of a subject is a patient suffering from fibrosis.

[0081] A "therapeutically effective amount" refers to an amount of an active substance that is effective for treating a disease or disorder in a subject. In the present invention, for example, it refers to an amount of an FGFR agonist VHH that is effective for slowing the progression of fibrosis in a subject. The therapeutically effective amount of the FGFR agonist VHH of the present invention may vary depending on the condition, age, sex, and weight of the subject, the amount and type of other drugs used in combination, and the activity of the FGFR agonist VHH that produces the desired response in the subject. A therapeutically effective amount also includes a case where the beneficial effects of treatment outweigh any undesirable effects that may be caused by the FGFR agonist VHH.

[0082] The dosage and frequency of administration of the FGFR agonist VHH can be changed as appropriate depending on the condition of the patient, etc. An example of the dose of the FGFR agonist VHH can be about 0.3 to about 1.7 mg / kg, preferably about 0.5 to about 1.5 mg / kg, and more preferably about 0.7 to about 1.3 mg / kg. An example of the frequency of administration of the FGFR agonist VHH can be once about every 6 to about 7 days, once about every 8 to about 9 days, once about every 10 to about 12 days, or once about every 12 to about 14 days.

[0083] Cosmetics containing FGFR agonist VHH The FGFR agonist VHH of the present invention can be incorporated into cosmetics. For example, by incorporating 0.1 ppm or more of an FGFR agonist VHH into cosmetics, skin firmness can be improved and wrinkles caused by aging or ultraviolet rays can be reduced.

[0084] Medium Compositions Comprising FGFR Agonist VHHs. FGFR agonist VHHs can be used as a substitute for FGF2 or in addition to FGF2 to form medium compositions. In particular, FGFR agonist VHHs can be used as medium compositions for cultivated meat. The medium of the present invention is particularly useful as a medium for cultivated meat. While FGFs added to culture media as growth factors are very expensive, the FGFR1 agonist VHHs of the present application possess biological activity equivalent to that of natural FGF2 ligands, promoting the proliferation and differentiation of pluripotent stem cells (PSCs) for regenerative medicine and human mesenchymal stem cells while maintaining the properties of PSCs. Furthermore, their aggregation onset temperature (Tag) is approximately 20°C higher than that of FGF2, demonstrating excellent thermostability. Therefore, the FGFR1 agonist VHH antibodies of the present invention have the potential to be used as a cost-effective, thermostable alternative to FGF2 in the cell preparation of stem cells in regenerative medicine and cultivated meat production.

[0085] Inhibition of Cancer Metastasis "Cancer" refers to a cell mass that proliferates independently of the body's autonomous control, and is a malignant tumor that infiltrates surrounding tissue and metastasizes, including epithelial carcinomas and non-epithelial sarcomas. Primary tumors and primary lesions are lesions that arise in the primary site or tissue and are not the result of progression, recurrence, or metastasis from elsewhere. Metastatic tumors and metastatic lesions are lesions that have metastasized from the primary site.

[0086] "Treatment" refers to therapeutic treatment, and refers to reducing the severity, slowing the progression, or stopping the progression of a targeted disease or disorder. In particular, in the case of the present invention, "treatment" includes shrinking or stopping the growth of cancer or tumor lesions, or slowing the growth, or slowing the progression, inhibiting the progression, or stopping the progression of cancer or tumors, or slowing, inhibiting, or stopping metastasis, or completely or partially alleviating symptoms or reducing the severity. "Treatment" also includes promoting the effect of other anticancer drugs used in combination.

[0087] "Metastasis" of cancer refers to the migration of cancer cells from a primary focus to other organs or tissues via the blood or lymph, where they settle and proliferate again to form a tumor. Without being bound by any particular theory, in the present invention, it is believed that FGFR1 agonist VHH inhibits fibrosis in metastatic focus, thereby inhibiting the colonization of cancer cells in other organs and thereby preventing metastasis.

[0088] "Subject" includes, but is not limited to, humans and other mammals, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, laboratory mammals, livestock mammals, sport mammals, and pet mammals. Examples of subjects include patients with cancer who are at risk of metastasis, and patients who already have cancer with metastatic lesions.

[0089] "Suppressing cancer metastasis" means delaying or preventing the re-formation of tumors at distant sites by delaying or preventing the migration, invasion, or adhesion of tumor cells from a primary focus or an already existing metastatic focus, compared to when an FGFR1 agonist VHH is not administered. Without being bound by any particular theory, it is thought that this is because the FGFR1 agonist VHH suppresses fibrosis of cancer lesions, thereby inhibiting the adhesion of cancer cells at distant sites, and / or, when an anticancer drug is used in combination, the anticancer drug more effectively prevents the adhesion of cancer cells at distant sites. In one embodiment, cancer metastasis is inhibited by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to when the FGFR1 agonist VHH is not administered.

[0090] Administration of an FGFR1 agonist VHH in combination with one or more other anticancer agents includes simultaneous administration of the FGFR1 agonist VHH and the anticancer agent, as well as sequential administration. Sequential administration can be performed in any order, and refers to administration of the FGFR1 agonist VHH followed by administration of the anticancer agent, or administration of the anticancer agent before administration of the FGFR1 agonist VHH. Simultaneous or sequential administration of the anticancer agent and the FGFR1 agonist VHH can also be performed multiple times consecutively.

[0091] The term "pharmaceutically acceptable carrier" refers to a molecule or composition that is physiologically acceptable and does not normally cause adverse reactions when administered, and includes non-toxic solid, semi-solid, gel-like, or liquid excipients, diluents, formulation aids, encapsulating materials, and the like.

[0092] In some embodiments, the pharmaceutical composition of the present invention is used to treat esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, pancreatic cancer, colorectal cancer, breast cancer, lung cancer, various bone and soft tissue tumors, bone metastasis, prostate cancer, bladder cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, penile cancer, retroperitoneal tumor, adrenal cancer, head and neck cancer, thyroid cancer, cervical cancer, uterine cancer, ovarian cancer, or skin cancer, particularly to suppress metastasis from pancreatic cancer. For example, the pharmaceutical composition of the present invention suppresses liver metastasis from pancreatic cancer. The cancer may be primary, locally advanced, unresectable, or metastatic.

[0093] In the present invention, any type of anticancer drug can be used in combination. Examples of anticancer drugs include paclitaxel, actinomycin, cetuximab, bevacizumab, irinotecan, epirubicin, etoposide, oxaliplatin, asparaginase, alectinib, pemetrexed, ifosfamide, gefitinib, oxaliplatin, cyclophosphamide, nivolumab, carboplatin, cabazitaxel, imatinib, and cyclophosphamide. Pecitabine, amrubicin, trastuzumab, gemcitabine, crizotinib, cisplatin, afatinib, regorafenib, capecitabine, ramucirumab, cytarabine, gemcitabine, sunitinib, lapatinib, erlotinib, docetaxel, degafur-uracil, temozolomide, liposomal doxorubicin, dacarbazine, dasatinib, paclitaxel Examples of such anti-cancer drugs include cefotaxime, tegafur / gimeracil / oteracil potassium, doxorubicin, vinorelbine, nimustine, nedaplatin, sorafinib, nogitecan, paclitaxel, eribulin, veltuzumab, vincristine, bleomycin, trastuzumab, vinorelbine, vinblastine, fluorouracil, panitumumab, mitomycin, melphalan, methotrexate, ramucirumab, levofolinate, trifluridine / tipiracil, rituximab, lenvatinib, anastrozole, exemestane, enzalutamide, goserelin, degarelix, bicalutamide, leuprorelin, abiraterone, ethinyl, chlormazine, tamoxifen, toremifene, flutamide, and torozole.

[0094] A "therapeutically effective amount" refers to an amount of an active substance that is effective for treating a disease or disorder in a subject. In the present invention, it refers to an amount of an FGFR1 agonist VHH that is effective for suppressing cancer metastasis in a subject. The therapeutically effective amount of the FGFR1 agonist VHH of the present invention may vary depending on the condition, age, sex, and weight of the subject, the amount and type of anticancer drug used in combination, and the activity of the FGFR1 agonist VHH that brings about the desired response in the subject, etc. A therapeutically effective amount also includes a case where the beneficial effects of treatment outweigh any undesirable effects that may be caused by the FGFR1 agonist VHH.

[0095] The dosage and frequency of administration of the FGFR1 agonist VHH can be changed as appropriate depending on the condition of the patient, the state of the cancer lesion, etc. An example of the dose of the FGFR1 agonist VHH can be about 0.3 to about 1.7 mg / kg, preferably about 0.5 to about 1.5 mg / kg, and more preferably about 0.7 to about 1.3 mg / kg. An example of the frequency of administration of the FGFR1 agonist VHH can be once about every 6 to about 7 days, once about every 8 to about 9 days, once about every 10 to about 12 days, or once about every 12 to about 14 days.

[0096] The FGFR1 agonist VHH can be administered by various routes, including but not limited to oral, intraarterial, parenteral, intranasal, intravenous, intramuscular, intracardiac, intracerebroventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal administration, or by implantation or inhalation. The route can be freely selected depending on the location of the lesion, and is not particularly limited. The FGFR1 agonist VHH can be formulated in solid, semisolid, liquid, or gas form. Specific dosage forms include, but are not limited to, capsules, powders, granules, ointments, liquids, suppositories, enemas, injections, inhalants, liposomes, and aerosols. In another embodiment, a nucleic acid molecule encoding the FGFR1 agonist VHH can be carried in a liposome or on a microparticle and delivered to the lesion.

[0097] Examples of embodiments of the present invention are listed below. [1] A humanized fibroblast growth factor receptor (FGFR) agonist VHH, comprising CDR1, CDR2, and CDR3 selected from the group consisting of: (1) CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 33, and 1, respectively; (2) CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 34, 35, and 2, respectively; (3) CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 37, and 3, respectively; and (4) CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 38, 39, and 4, respectively. [2] The FGFR agonist VHH according to item [1], comprising an amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8. [3] The FGFR agonist VHH according to item [1] or [2], which is a VHH-Fc to which an Fc sequence of a human antibody has been added. [4] The FGFR agonist VHH according to any one of items [1] to [3], which is a dimer. [5] A composition comprising the FGFR agonist VHH according to any one of items [1] to [4]. [6] A pharmaceutical composition comprising the FGFR agonist VHH according to any one of items [1] to [5]. [7] The pharmaceutical composition according to item [6], which is for treating fibrosis. [8] The pharmaceutical composition according to item [7], wherein the fibrosis is hepatic fibrosis. [9] The pharmaceutical composition according to any one of items [1] to [5], which is used to inhibit cancer metastasis.

[10] The pharmaceutical composition according to item [9], wherein the metastasis is hepatic metastasis.

[0098]

[11] The FGFR agonist VHH according to any one of items [1] to [5] for use as a pharmaceutical.

[12] The FGFR agonist VHH according to item

[11] for use in treating fibrosis.

[13] The FGFR agonist VHH according to item

[12] , wherein the fibrosis is hepatic fibrosis.

[14] The FGFR agonist VHH according to any one of items [1] to [5], for use in inhibiting cancer metastasis.

[15] The FGFR agonist VHH according to item

[14] , wherein the metastasis is hepatic metastasis.

[0099]

[16] Use of the FGFR agonist VHH according to any one of items [1] to [5] for the manufacture of a medicament.

[17] Use of the FGFR agonist VHH according to item

[16] for the manufacture of a medicament for the treatment of fibrosis.

[18] The use according to item

[17] , wherein the fibrosis is liver fibrosis.

[19] Use of the FGFR agonist VHH according to any one of items [1] to [5] for the manufacture of a medicament for inhibiting cancer metastasis.

[20] The use according to item

[19] , wherein the metastasis is liver metastasis.

[0100]

[21] A method for treating fibrosis in a patient, comprising administering to the patient the FGFR agonist VHH of any of items [1] to [5].

[22] The method according to item

[21] , wherein the fibrosis is liver fibrosis.

[23] A method for inhibiting cancer metastasis in a patient, comprising administering to the patient the FGFR agonist VHH of any of items [1] to [5].

[24] The method according to item

[23] , wherein the metastasis is liver metastasis.

[0101] The present invention will be further described below using examples, but the scope of the present invention is not limited to the following examples.

[0102] (Example 1) Synthesis of cDNA display using PharmaLogical (PL) library Synthesis of cDNA display using PharmaLogical (PL) library was carried out in the following steps, similar to the scheme in Figure 1. (1) cnvPreparation of KrG Linker (hereinafter sometimes simply referred to as "cnvK linker") The cnvK linker has a main chain and a side chain. The base sequence of the biotin fragment that forms the main chain is 3'-AAgAATTTCCAKGCCGCCCCCCGVCCT-3' (SEQ ID NO: 17). Here, BioTEG is bound to the 5' end of the main chain. In the base sequence, g represents guanosine, V represents Amino C6-dT, and K represents 3-cyanovinylcarbazole. The puromycin segment that forms the side chain of the cnvK linker has the structure 5'-(5S)TCTFZZCCP-3'. The free end of the side chain sequence, P, represents puromycin as a protein binding site. (5S) represents 5' Thiol C6, F represents FITC-dT, and Z represents SpaceR18. The chemical synthesis of the main chain and side chains was outsourced to Tsukuba Oligo Service Co., Ltd. (Ushiku City, Ibaraki Prefecture).

[0103] First, 15 nmol of biotin fragment (final concentration 150 μM) and EMCS (Dojindo Laboratories, final concentration 16.7 mM) were added to 0.2 M sodium phosphate buffer (pH 7.2) and incubated at 37°C for 30 minutes. The mixture was then ethanol precipitated using Quick-Precip Plus Solution (Edge BioSystems). Next, 37.5 nmol of puromycin segment was dissolved in 1 M aqueous disodium hydrogen phosphate solution containing 50 mM DTT to a final concentration of 417 μM, and the mixture was stirred at room temperature for 1 hour. The buffer was then exchanged into 0.02 M sodium phosphate buffer (pH 7.0) containing 0.03 M NaCl using a NAP5 column (GE Healthcare Biosciences).

[0104] The buffer-exchanged reduced puromycin segment solution was mixed with the ethanol precipitate of the EMCS-modified biotin fragment and left overnight at 4°C. DTT was then added to the reaction mixture to a final concentration of 50 mM, and the mixture was stirred at room temperature for 30 minutes. Ethanol precipitation was then performed using Quick-Precip Plus Solution (Edge BioSystems). The ethanol precipitate was dissolved in 100 μL of nuclease-free water (Nacalai Tesque).

[0105] The lysate was separated by 12% polyacrylamide gel electrophoresis, and the cnvK linker fraction was excised. The excised gel was crushed using a BioMasher II set (Nippi), and 500 μL of nuclease-free water was added. The mixture was stirred overnight at 4°C to extract the cnvK linker. The stirred solution was transferred to a Costar® Spin-X® centrifuge tube filter, 0.22 μm cellulose acetate (corning), and then centrifuged at 16,000 x g for 15 minutes to separate the gel from the extract. Ethanol precipitation was then performed using Quick-Precip Plus Solution to obtain the desired cnvK linker (Figure 2). The resulting cnvK linker was dissolved in nuclease-free water and stored at -20°C.

[0106] (2) Preparation of cDNA for VHH-presenting cDNA display (2-1) Transcription The applicant's PharmaLogical DNA library (full-length VHH-encoding DNA library, manufactured by Epsilon Molecular Engineering) was transcribed into mRNA using the T7 RiboMAX Express Large Scale RNA Production System (Promega) according to the attached manual. The amount of DNA used was 6.6 μg. The amount of DNA used for each subsequent selection was 0.1 to 1 μg from the second round onwards. The resulting transcription product was purified using RNAClean XP (Beckman Coulter) according to the attached manual to obtain a purified product. The concentration of the purified product was quantified using a NanoPad DS-11 FX (DeNovix).

[0107] (2-2) Ligation To 20 pmol of purified mRNA and 20 pmol of cnvK linker, NaCl (final concentration 0.2 M) and Tris-HCl buffer (pH 7.5, final concentration 50 mM) were added and incubated at 90°C for 1 minute. The mixture was then cooled to 70°C at a rate of 0.1°C / sec and incubated at 70°C for 1 minute. The mixture was then cooled to 25°C at a rate of 0.1°C / sec and subsequently cooled to 10°C at a rate of 2°C / sec, allowing the cnvK linker to hybridize to the 3' end of the mRNA. Subsequently, a UVP CRossLinker (catalog number CL-3000, 365 nm, 100-115 V (Analytik Jena) was used to illuminate the mixture with 4,060 μJ / cm of 365 nm UV light. 2 The cnvK linker was photocrosslinked to the mRNA by irradiation, resulting in an mRNA-linker complex.

[0108] (2-3) Preparation of mRNA Display Using a 50 μL-scale cell-free translation system (PUREfrex® 1.0, Gene Frontier), 20 pmol of mRNA-linker complex was incubated in a tube at 37°C for 30 minutes. Next, MgCl2 and KCl were added to the tube to final concentrations of 75 mM and 900 mM, respectively, and the tube was incubated at 37°C for 1 hour to display the peptide corresponding to the mRNA on puromycin in the mRNA-linker conjugate. Next, EDTA (pH 8.0) was added to the tube to a final concentration of 70 mM, and the tube was incubated at 4°C for 5 minutes to prepare mRNA display.

[0109] (2-4) Preparation of cDNA display: 60 μL of Dynabeads Myone streptavidin C1 (Thermo Fisher Scientific) was placed in a new tube, and 200 μL of binding buffer was added and washed. The mRNA display prepared by methods (2-1)-(2-3) above was then added and stirred at 25°C for 30 minutes. 200 μL of binding buffer was added and washed, and then the mixture was incubated at 42°C for 30 minutes in a reaction solution with the composition shown in Table 1 below for reverse transcription to prepare mRNA / cDNA-VHH conjugates.

[0110]

[0111] After the reverse transcription reaction was completed, 200 μL of binding buffer was added for washing, followed by the addition of 39 μL of His tag binding / washing buffer and 1 μL of 1,000 U / μL RNase T1, and the mixture was stirred at 37°C for 15 minutes to elute the mRNA / cDNA-VHH conjugate (hereinafter sometimes referred to as "cDNA display molecule") from the Dynabeads Myone streptavidin C1.

[0112] Example 2: Selection of VHHs against the FGFR1 extracellular domain and analysis of their sequences (1) Biotinylation of the FGFR1 extracellular domain. 1.5 nmol of biotinylation reagent (EZ-Link® Sulfo-NHS-LC-Biotin, Thermo Fisher Scientific) was added to 30 μL of PBS containing 300 pmol of recombinant human FGFR1 protein (hereinafter sometimes abbreviated as "FGFR1"; Abcam) and incubated at room temperature for 30 minutes. Zeba® Spin Desalting Columns, 7K MWCO, (Thermo Fisher Scientific) were inserted into the column according to the manufacturer's instructions, and 0.5 mL of PBS-T was added to the column. The reaction mixture obtained above was added to the column and centrifuged at 1,500 × g for 2 minutes. The buffer in the reaction mixture was then replaced with PBS-T to remove unreacted biotinylation reagent, yielding biotinylated FGFR1.

[0113] (2) Selection against FGFR Selection was performed using the cDNA display prepared in (1) above. Selection was performed in rounds 1 to 5. The synthesis scale of the VHH-presenting cDNA display library used in each selection round is shown in Table 2. Round 1 shown in Table 2 below is sometimes referred to as "R1."

[0114]

[0115] (3) Screening procedure for selection cycle 1 (Round 1) 50 pmol of biotinylated FGFR1 prepared in (1) above was added to a tube containing 100 μL of Dynabeads Myone streptavidin C1, and the mixture was mixed by inversion at 4°C for 30 minutes to prepare biotinylated FGFR1-immobilized beads.

[0116] The cDNA display molecules prepared at the synthesis scale shown in Table 2 above were diluted to 100 μL with PBS-T. This diluted solution was added to the tube containing the biotinylated FGFR1-immobilized beads and mixed by inversion at 25°C for 30 minutes. The tube was left to stand, and the supernatant was collected. The beads were washed four times with PBS-T.

[0117] The supernatant collected above and 50 pmol of biotinylated FGFR1 were mixed in a new tube and mixed by inversion at 25°C for 30 minutes to obtain a mixed solution. The mixed solution was then added to a tube containing 100 μL of Dynabeads Myone streptavidin C1 and mixed by inversion at 25°C for 30 minutes to immobilize the biotinylated FGFR1 containing the cDNA display-biotinylated FGFR1 complex to the beads. The tube containing the beads was washed with PBS-T. This washing procedure was repeated four times. The beads were then added to this tube, and all the beads were collected in one tube.

[0118] The cDNA-displayed molecules bound to the beads were then collected and purified using AMpure XP (Beckman Coulter) according to the accompanying manual. The purified solution was amplified by PCR using cnvK NewYtag for poly A (SEQ ID NO: 18: 5'-TTTCCACGCCGCCCCCCGTCCT-3') and PL_T7pro (SEQ ID NO: 19: 5'-GATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTC-3') as primers with PrimeSTAR Max (Takara Bio Inc.) under the conditions shown in Table 3 to obtain PCR product 1.

[0119]

[0120] The PCR products were then analyzed by 4% denaturing PAGE. The PCR products were purified using AMpure XP according to the attached manual, and the purified product was used as the R1 library for the second round of selection.

[0121] (4) Screening Procedure for Selection Cycle 2 (R2) 10 pmol of biotinylated FGFR1 was added to a tube containing 10 μL of Dynabeads Myone streptavidin C1, and the mixture was mixed by inversion at 4° C. for 30 minutes to prepare biotinylated FGFR1-immobilized beads.

[0122] A cDNA display library was prepared from the R1 library obtained in (3) above using the same method as above, using the synthesis scale shown in Table 2 above. The library was diluted to 100 μL with PBS-T containing 2.5 μL of 200 mg / mL heparin solution, and 50 μL was added to a tube containing biotinylated FGFR1-immobilized beads and mixed by inversion at 25°C for 30 minutes.

[0123] The remaining 50 μL of supernatant was added to a tube containing 10 pmol of biotinylated FGFR1, mixed, and then mixed by inversion at 25°C for 30 minutes to obtain a mixed solution. This mixture was added to a new tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by inversion at 25°C for 30 minutes to immobilize the biotinylated FGFR1 containing the cDNA display-biotinylated FGFR1 complex to the beads. All of the beads were combined into one tube and washed with 200 μL of PBS-T containing 0.2% BSA. This washing procedure was repeated three times.

[0124] The beads were then washed with 20 μL of 0.2% BSA-containing PBS-T by inversion at room temperature for 15 minutes. The cDNA display molecules bound to the beads were collected and purified using AMpure XP according to the attached manual to obtain a purified solution. This purified solution was amplified by PCR under the same conditions as R1, except for 20 cycles, to obtain PCR product 2. PCR product 2 was purified using AMpure XP according to the attached manual to create the R2 library.

[0125] (5) Screening Procedure for Selection Cycle 3 (R3): Using the same method as above, a cDNA display library was prepared from the R2 library in a tube at the synthesis scale shown in Table 2 above. The tube was diluted to 100 μL with PBS-T containing 0.05% BSA to prepare a diluted solution. This diluted solution was then added to 20 μL of Dynabeads Myone Streptavidin T1 and mixed by end-over-end at 4°C for 30 minutes to prepare a mixed solution. After mixing, the mixture was allowed to stand, and the supernatant was recovered. This recovered supernatant was mixed with 10 pmol of biotinylated FGFR1 and mixed by end-over-end at 4°C for 30 minutes to obtain a mixed solution. This mixed solution was added to a tube containing 20 μL of Dynabeads Myone Streptavidin T1 and mixed by end-over-end at 4°C for 30 minutes to immobilize the biotinylated FGFR1 containing the cDNA display-biotinylated FGFR1 complex to the beads.

[0126] The tube containing the beads was washed with 200 μL of PBS-T containing 0.05% BSA. This washing procedure was repeated three times. Then, 20 μL of PBS-T containing 0.05% BSA was added to the tube containing the beads, and the beads were further washed by mixing by inversion at room temperature for 15 minutes. The cDNA display molecules bound to the beads were collected and purified using AMpure XP according to the attached manual to obtain a purified solution. This purified solution was subjected to PCR amplification under the same conditions as R1, except for the number of cycles, which was 22, to obtain PCR product 3. PCR product 3 was purified using AMpure XP according to the attached manual to create the R3 library.

[0127] (6) Screening procedure for selection cycle 4 (R4): Using the same method as above, the R3 library was transferred to a cDNA display library tube and prepared at the synthesis scale shown in Table 2. The procedure was then repeated as in R3, except that Dynabeads Myone streptavidin C1 was used instead of Dynabeads Myone streptavidin T1, and the PCR cycle number was 12, to obtain an enriched library (R4 library).

[0128] (7) Analysis of Screening Products by NGS The enriched library, which is the screening product obtained in step (6) above, was analyzed using a next-generation sequencer (hereinafter sometimes abbreviated as "NGS") by the following treatment. First, the enriched library was subjected to PCR and amplified under the following conditions to obtain a PCR product. The reaction solution was prepared by placing 5 μL of PrimeSTAR MAX, 1 μL of the enriched screening product, and 2 pmol each of primers PL_prRd-N4_NL_FW (SEQ ID NO: 15 in the Sequence Listing) and PL_prRd-N4_Ytag_RV (SEQ ID NO: 16 in the Sequence Listing) in a tube and adjusting the volume to 10 μL with ultrapure water.

[0129] PCR was performed under the same conditions as in Table 3, except that the annealing temperature was 62°C and the number of cycles including denaturation was 12. The PCR product was obtained under the same conditions as in Table 3. The resulting PCR product was purified using AMPure XP, and Index PCR was performed using purified product 4 as a template. The reaction solution for Index PCR consisted of 12.5 μL of PrimeSTAR MAX, 1 μL of purified product 4, 0.5 μL of 5 μM forward and reverse primers (Nextera XT Index 1 Primers (N7XX) and Nextera XT Index 2 Primers (S5XX)), and 10.5 μL of ultrapure water.

[0130] PCR products were obtained using the same PCR program conditions as in Table 3, except for an annealing temperature of 52°C and eight cycles including denaturation. The Index PCR product was similarly purified using AMPure XP to obtain purified product 5, and the DNA concentration of this purified product 5 was measured using a NanoPad DS-11. The Index PCR purified product was diluted to 10 nM with RNase-free water, and 5 μL of each product was collected and mixed in a single tube. Next, an NGS sample library was created according to the MiSeq (Illumina) instruction manual and analyzed using the MiSeq Reagent Nano Kit v2 (500 cycles) (Illumina). The DNA sequences obtained from NGS were translated into amino acid sequences to obtain the VHH sequences that bind to each target molecule.

[0131] (8) Evaluation of Primary Binding of VHHs (8-1) Preparation of a VHH Expression Plasmid Library To evaluate the primary binding of VHHs, a VHH expression plasmid library was prepared as follows. The DNA library encoding the VHHs selected by the above screening was cloned into a plasmid vector for VHH expression. First, sequences for restriction enzyme digestion were added to the DNA library obtained by screening by PCR according to standard methods to obtain PCR products. To prepare the reaction solution, 25 μL of PrimeSTAR MAX, 1 μL of the screening product, and 10 pmol each of the primers PL_VHH_SfiI-NcoI_FW (SEQ ID NO: 20) and PL_VHH_BamHI-NotI_RV (SEQ ID NO: 21) were added to a tube, and the volume was adjusted to 50 μL with ultrapure water. The sequences of the two primers are shown below.

[0132] 5'-ccggcCatggccACTGCggccGAAGTACAATTAGTTGAATCTGGTGGTGGGCTTG-3' (SEQ ID NO: 20) 5'-AAAAgcggccgcggatccTGAAGAGACTGTCACCAACGTGCC-3' (SEQ ID NO: 21)

[0133] The PCR program was the same as that shown in Table 3, except that the annealing temperature was 55°C and the number of cycles including denaturation was 25. This PCR product and the C. glutamicum expression plasmid vector were treated with the restriction enzyme BamHI at 37°C for 1 hour, and then with the restriction enzyme SfiI at 50°C for 1 hour. The reaction solution derived from the PCR product that would become the insert DNA was purified using AMPureXP, and the reaction product derived from the plasmid vector was subjected to 1% agarose gel electrophoresis at 100 V for 30 minutes. After electrophoresis, the product was excised from the gel and purified to obtain a purified product.

[0134] The purified plasmid vector DNA was dephosphorylated using the dephosphorylating enzyme Fast AP Thermosensitive Alkaline Phosphatase (Thermo Scientific) at 37°C for 1 hour. Then, the insert DNA and the plasmid vector DNA were mixed at a molar ratio of 1:10, and a ligation reaction was carried out overnight at 16°C using Ligation High (Toyobo Co., Ltd.). A plasmid library incorporating the selected VHH library was obtained.

[0135] (8-2) Production of VHH (culture supernatant) Each of the plasmids obtained in (8-1) above was introduced into C. glutamicum by electroporation to produce transformants. The resulting transformants were inoculated into CM2G medium and pre-cultured overnight at 30°C. The transformants contained in the CM2G culture were then subcultured in PM1S medium for VHH expression and cultured at 25°C for 72 hours to allow secretion and expression of VHH in the culture supernatant. The culture supernatant was centrifuged at 4,000 x g for 30 minutes to collect the culture supernatant, which was then passed through a 0.22 μm filter to remove bacterial cells, yielding a VHH clone.

[0136] (8-3) Octet Single-Point Binding Assay Using OctetRED384 (Fortebio), the VHH clones obtained as described above were immobilized on a His1K sensor chip, and their binding activity to FGFR1(IIIc)-Fc (R&D) was measured. The analyte, FGFR1(IIIc)-Fc (R&D), was adjusted to a concentration of 200 nM, and 70 μL of the assay solution was added to a 384-well plate to measure binding to the VHH clones. Prior to measurement, the tip of a Dip and Read® His1K Biosensor (Fortebio) was immersed in 200 μL of PBS-T (PBS containing 0.05% Tween 20, pH 7.4) for 10 minutes to hydrate the sensor chip.

[0137] The measurement order for each run was as follows: 1) Baseline step: 30 seconds in PBS-T. 2) Loading step: 60 seconds using VHH diluted 50-fold in PBS-T. 3) Baseline step: 30 seconds in PBS-T. 4) Association step: 100 seconds using FGFR1(IIIc)-Fc diluted in PBS-T. 5) Dissociation step: 100 seconds in PBS-T. 6) Regeneration step: 5 seconds in glycine-HCl (pH 2.2) and 30 seconds in PBS-T. These steps were repeated three times. The data were processed using Octet software version 1.2.1.5 (Molecular Devices). The binding responses of each clone VHH to FGFR1(IIIc)-Fc obtained from the single-point binding assay using Octet are shown in Figure 3. Thirty-five clones showing a binding response of 0.1 nm or more were obtained and designated as hit clones.

[0138] (8-4) Sequence analysis of hit clones. Each VHH gene in the hit clones was identified by sequence analysis. Hit clone transformants were cultured overnight at 37°C, and colony PCR was performed on the culture medium. The resulting PCR products were purified using AMPure XP, and then each DNA was analyzed by Eurofins genomics. Among the hit clones obtained, only one duplicated sequence was counted, and these clones were designated as unique VHH clones.

[0139] Example 3: Selection of VHHs for FGFR1 domain II and sequence analysis thereof (1) Biotinylation of FGFR1 domain II To 400 pmol of human FGFR1 domain II-FC protein (hereinafter sometimes abbreviated as "FGFR1 domain II-Fc") in 40 μL of PBS, 20 nmol of biotinylation reagent (EZ-Link® Sulfo-NHS-LC-Biotin, Thermo Fisher Scientific) was added and allowed to react at room temperature for 30 minutes. Zeba® Spin Desalting Columns, 7K MWCO, (Thermo Fisher Scientific) were used according to the manufacturer's instructions, and 0.5 mL of PBS-T was substituted. The reaction solution obtained above was added to the columns and centrifuged at 1,500 × g for 2 minutes. The buffer in the reaction solution was then substituted with PBS-T to remove unreacted biotinylation reagent, yielding biotinylated FGFR1 domain II-Fc.

[0140] (2) Selection against FGFR1 domain II Selection was performed using the cDNA display prepared in (1) above. Selection was performed in rounds 1 to 4. The synthesis scale of the VHH-presenting cDNA display library used in each selection round is shown in Table 2 above.

[0141] (3) Screening procedure for selection cycle 1 (Round 1) 50 pmol of biotinylated FGFR1 domain II-Fc prepared in (1) above was added to a tube containing 50 μL of Dynabeads Myone streptavidin C1, and the mixture was mixed by inversion at 25°C for 30 minutes to prepare biotinylated FGFR1 domain II-Fc-immobilized beads.

[0142] The cDNA display molecule prepared at the synthesis scale shown in Table 2 above was diluted to 100 μL with PBS-T, added to a tube containing biotinylated FGFR1 domain II-Fc-immobilized beads, and mixed by inversion for 30 minutes at 25°C. The tube was left to stand, the supernatant was collected, and the beads were washed four times with PBS-T.

[0143] The supernatant collected above and 100 pmol of biotinylated FGFR1 domain II-Fc were mixed in a new tube and mixed by inversion at 25°C for 30 minutes to obtain a mixed solution. The mixed solution was then added to a tube containing 200 μL of Dynabeads Myone streptavidin C1 and mixed by inversion at 4°C for 30 minutes to immobilize the biotinylated FGFR1 domain II-Fc containing the cDNA display-biotinylated FGFR1 domain II-Fc complex to the beads. The tube containing the beads was washed with PBS-T. This washing procedure was repeated four times. The beads were then added to this tube, and all the beads were collected in one tube.

[0144] The cDNA-displayed molecules bound to the beads were then collected and purified using AMpure XP (Beckman Coulter) according to the attached manual. The purified solution was amplified by PCR using cnvK NewYtag for poly A (SEQ ID NO: 18) and PL_T7pro (SEQ ID NO: 19) as primers with PrimeSTAR Max (Takara Bio Inc.) under the conditions listed in Table 3 to obtain PCR product 1.

[0145] The PCR products were then analyzed by 4% denaturing PAGE. The PCR products were purified using AMpure XP according to the attached manual, and the purified product was used as the R1 library for the second round of selection.

[0146] (4) Screening procedure for selection cycle 2 (R2) A cDNA display library was prepared from the R1 library obtained in (3) above using the same method as above, using the synthesis scale shown in Table 2 above, and diluted to 100 μL with PBS-T. The resulting solution was then added to a tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by inversion at 25°C for 30 minutes.

[0147] After mixing, the tube was left to stand, and the supernatant was collected and diluted to 1000 μL with PBS-T containing 10 pmol of biotinylated FGFR1 domain II-Fc, 0.4% Block Ace (KAC Corporation), and 100 pmol of IgG1 protein (human, recombinant). The mixture was mixed by end-over-end at 25°C for 30 minutes to obtain a mixed solution. This mixture was added to a new tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by end-over-end at 25°C for 30 minutes to immobilize the biotinylated FGFR1 domain II-Fc containing the cDNA display-biotinylated FGFR1 domain II-Fc complex to the beads. All of the beads were combined into one tube and then washed with 200 μL of PBS-T. This washing procedure was performed three times.

[0148] The beads were then washed with 40 μL of PBS-T by inverting and mixing for 15 minutes at room temperature. The cDNA display molecules bound to the beads were collected and purified using AMpure XP according to the attached manual to obtain a purified solution. This purified solution was subjected to PCR amplification under the same conditions as R1, except for the number of cycles, which was 20, to obtain PCR product 2. The obtained PCR product 2 was purified using AMpure XP according to the attached manual to create the R2 library.

[0149] (5) Screening procedure for selection cycle 3 (R3) Using the same method as above, a cDNA display library was prepared from the R2 library in a tube using the synthesis scale shown in Table 2 above, and diluted to 100 μL with PBS-T to prepare a diluted solution. This diluted solution was then added to a tube containing 20 μL of Dynabeads Myone streptavidin C1, and mixed by inversion at 25°C for 30 minutes.

[0150] After mixing, the tube was left to stand, and the supernatant was collected and diluted to 1000 μL with PBS-T containing 10 pmol of biotinylated FGFR1 domain II-Fc, 0.4% Block Ace (KAC Corporation), and 100 pmol of IgG1 protein (human, recombinant). The mixture was mixed by end-over-end at 25°C for 30 minutes to obtain a mixed solution. This mixture was added to a new tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by end-over-end at 25°C for 30 minutes to immobilize the biotinylated FGFR1 domain II-Fc containing the cDNA display-biotinylated FGFR1 domain II-Fc complex to the beads. All of the beads were combined into one tube and then washed with 200 μL of PBS-T. This washing procedure was performed three times.

[0151] The beads were then washed with 20 μL of PBS-T by inverting and mixing at room temperature for 15 minutes. The cDNA display molecules bound to the beads were collected and purified using AMpure XP according to the attached manual to obtain a purified solution. This purified solution was subjected to PCR amplification under the same conditions as R1, except for the number of cycles, which was 20, to obtain PCR product 3. PCR product 3 was purified using AMpureXP according to the attached manual to create the R3 library.

[0152] (6) Screening Procedure for Selection Cycle 4 (R4): Using the same method as above, a cDNA display library was prepared from the R3 library in a tube using the synthesis scale shown in Table 2 above, diluted to 100 μL with PBS-T, and then added to a tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by end-over-end mixing at 25°C for 30 minutes. This diluted solution was then added to a tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by end-over-end mixing at 4°C for 30 minutes to obtain a mixed solution. The procedure was then repeated as for R3, except that Dynabeads Myone streptavidin C1 was used instead of Dynabeads Myone streptavidin C1 and the PCR cycle number was changed to 15, to obtain an enriched library (R4 library).

[0153] (7) Screening Procedure for Selection Cycle 5 (R5): Using the same method as above, a cDNA display library was prepared from the R4 library in a tube using the synthesis scale shown in Table 2 above, diluted to 100 μL with PBS-T, and then added to a tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by end-over-end mixing at 25°C for 30 minutes. This diluted solution was then added to a tube containing 20 μL of Dynabeads Myone streptavidin C1 and mixed by end-over-end mixing at 4°C for 30 minutes to obtain a mixed solution. The procedure was then repeated as for R4, except that Dynabeads Myone streptavidin C1 was used instead of Dynabeads Myone streptavidin C1 and the PCR cycle number was changed to 12, to obtain an enriched library (R5 library).

[0154] (8) Analysis of Screening Products by NGS The enriched library, which is the screening product obtained in step (7) above, was subjected to the following treatment in order to analyze it by NGS. First, the enriched library was subjected to PCR and amplified under the following conditions to obtain a PCR product. The reaction solution was prepared by placing 5 μL of PrimeSTAR MAX, 1 μL of the enriched screening product, and 2 pmol each of the primers PL_prRd-N4_NL_FW (SEQ ID NO: 15) and PL_prRd-N4_Ytag_RV (SEQ ID NO: 16) in a tube and adjusting the volume to 10 μL with ultrapure water.

[0155] The PCR program was the same as that shown in Table 3, except that the annealing temperature was 62°C and the number of cycles including denaturation was 12. The PCR product was obtained by purifying the PCR product using AMPure XP, and index PCR was performed using purified product 4 as a template. The reaction solution for index PCR consisted of 12.5 μL of PrimeSTAR MAX, 1 μL of purified product 4, 0.5 μL of 5 μM forward and reverse primers (Nextera XT Index 1 Primers (N7XX) and Nextera XT Index 2 Primers (S5XX)), and 10.5 μL of ultrapure water.

[0156] The PCR program was the same as in Table 3 above, except that the annealing temperature was 52°C and the number of cycles including denaturation was 8. The Index PCR product was similarly purified using AMPure XP to obtain purified product 5, and the DNA concentration of this purified product 5 was measured using a NanoPad DS-11. The Index PCR purified product was diluted to 10 nM with RNase-free water, and then 5 μL of each product was collected and mixed in one tube. Next, an NGS sample library was created according to the Miseq (Illumina) instruction manual and analyzed using the MiSeq Reagent Nano Kit v2 (500 Cycles) (Illumina). The DNA sequences obtained from NGS were translated into amino acid sequences to obtain the VHH sequences that bind to each target molecule.

[0157] (9) Evaluation of Primary Binding of VHHs (9-1) Preparation of a VHH Expression Plasmid Library To evaluate the primary binding of VHHs, a VHH expression plasmid library was prepared as follows. The DNA library encoding the VHHs selected by the above screening was cloned into a plasmid vector for VHH expression. First, a sequence for restriction enzyme digestion was added to the DNA library obtained by screening by PCR as per standard methods, and a PCR product was obtained. To prepare the reaction solution, 25 μL of PrimeSTAR MAX, 1 μL of the screening product, and 10 pmol each of the primers PL_VHH_SfiI-NcoI_FW (SEQ ID NO: 20) and PL_VHH_BamHI-NotI_RV (SEQ ID NO: 21) were added to a tube, and the volume was adjusted to 50 μL with ultrapure water.

[0158] The PCR program was the same as that shown in Table 3, except that the annealing temperature was 55°C and the number of cycles including denaturation was 25. This PCR product and the C. glutamicum expression plasmid vector were treated with the restriction enzyme BamHI at 37°C for 1 hour, and then with the restriction enzyme SfiI at 50°C for 1 hour. The reaction solution derived from the PCR product that would become the insert DNA was purified using AMPureXP, and the reaction product derived from the plasmid vector was subjected to 1% agarose gel electrophoresis at 100 V for 30 minutes. After electrophoresis, the product was excised from the gel and purified to obtain a purified product.

[0159] The purified plasmid vector DNA was dephosphorylated using the dephosphorylating enzyme Fast AP Thermosensitive Alkaline Phosphatase (Thermo Scientific) at 37°C for 1 hour. Then, the insert DNA and the plasmid vector DNA were mixed at a molar ratio of 1:10, and a ligation reaction was carried out overnight at 16°C using Ligation High (Toyobo Co., Ltd.). A plasmid library incorporating the selected VHH library was obtained.

[0160] (9-2) Production of VHH (culture supernatant) Each of the plasmids obtained in (9-1) above was introduced into C. glutamicum by electroporation to produce transformants. The resulting transformants were inoculated into CM2G medium and pre-cultured overnight at 30°C. The transformants contained in the CM2G culture were then subcultured in PM1S medium for VHH expression and cultured at 25°C for 72 hours to allow secretion and expression of VHH in the culture supernatant. The culture supernatant was centrifuged at 4,000 x g for 30 minutes to collect the supernatant, which was then passed through a 0.22 μm filter to remove bacterial cells, yielding a VHH clone.

[0161] (9-3) Octet Single-Point Binding Assay Using OctetRED384 (Fortebio), the VHH clones obtained as described above were immobilized on a His1K sensor chip, and their binding activity to FGFR1 domain II-Fc was measured. The analyte, FGFR1 domain II-Fc, was adjusted to a concentration of 100 nM, and 70 μL of the assay solution was added to a 384-well plate to measure binding to the VHH clones. Prior to measurement, the tip of a Dip and Read® His1K Biosensor (Fortebio) was immersed in 200 μL of PBS-T (0.05% Tween 20, pH 7.4) for 10 minutes to hydrate the sensor chip.

[0162] The measurement order for each run was as follows: 1) Baseline step: 30 seconds of measurement in PBS-T. 2) Loading step: 60 seconds of measurement using VHH diluted 50-fold in PBS-T. 3) Baseline step: 30 seconds of measurement in PBS-T. 4) Association step: 100 seconds of measurement using FGFR1 domain II-Fc diluted in PBS-T. 5) Dissociation step: 100 seconds of measurement in PBS-T. 6) Regeneration step: 5 seconds of measurement in glycine-HCl (pH 2.2) and 30 seconds of measurement in PBS-T. These steps were repeated three times. The data obtained were processed using Octet software version 1.2.1.5 (Molecular Devices). Figure 4 shows the binding response of each VHH clone to FGFR1 domain II protein obtained from the single-point binding assay using Octet. Thirty-nine clones with binding responses of 0.1 nm or greater were identified.

[0163] (9-4) Sequence Analysis of Hit Clones The VHH genes of the hit clones obtained as described above were identified by gene sequence analysis. Hit clone transformants were cultured overnight at 37°C in the same medium as above, and colony PCR was performed on the resulting culture medium to obtain PCR products. 10 units of Exonuclease I (E. coli) and 0.5 units of Shrimp Alkaline Phosphatase (rSAP) (both from New England Biolabs) were added to the resulting PCR products, and the mixture was incubated at 37°C for 45 minutes. The enzymes were then inactivated by heating at 80°C for 15 minutes. DNA sequence analysis using each reaction mixture after this enzyme inactivation treatment was outsourced to Eurofins genomics. Among the hit clones obtained, only one duplicated sequence was counted, and these clones were designated as unique VHH clones.

[0164] (Example 4) Obtaining Each Unique VHH Clones (1) Obtaining Purified VHHs For each unique VHH clone identified by sequence analysis in Examples 2 and 3 above, transformed C. glutamicum was pre-cultured overnight at 30°C in CM2G medium. This pre-culture was then subcultured in PM1S medium for VHH expression and cultured at 25°C for 72 hours to allow secretion and expression of VHHs in the culture supernatant. The culture supernatant was centrifuged at 4,000 x g, and the supernatant was collected. Next, samples were purified using His Multi Trap HP (Cytiva) according to the manufacturer's instructions. 100 μL of elution buffer (50 mM Tris-HCl buffer (pH 7.5) containing 300 mM NaCl and 500 mM imidazole) was added to the well containing the sample, and the mixture was centrifuged at 500 x g for 2 minutes at 4°C. The eluate was collected and used as a purified VHH clone sample.

[0165] (2) Octet Multipoint Binding Assay: The interaction between purified VHH clones and the FGFR1 extracellular domain was analyzed using the Octet RED384. Purified VHHs were immobilized on a His1K sensor chip (Fortebio) and bound to FGFR1 extracellular domain (prepared at 2-fold dilutions from 200 nM) or FGFR1 extracellular domain II (prepared at 2-fold dilutions from 100 nM). Prior to measurement, the tip of a Dip and Read SA Biosensor was immersed in 200 μL of PBS-T (PBS containing 0.05% Tween 20, pH 7.4) for 10 minutes to hydrate the sensor chip. Then, 70 μL of each test solution was added to a 384-well plate, and measurements were performed as follows:

[0166] 1) Baseline step: Measurement for 60 seconds in PBS-T. 2) Loading step: Measurement for 120 seconds using purified VHH diluted 100-fold in PBS-T. 3) Baseline step: Measurement for 60 seconds in PBS-T. 4) Association step: Measurement for 120 seconds using FGFR1 extracellular domain diluted in PBS-T. 5) Dissociation step: Measurement for 120 seconds in PBS-T. 6) Regeneration step: Measurement for 5 seconds in glycine-HCl (pH 2.2) and 5 seconds in PBS-T, repeated three times.

[0167] After the above measurements were completed, the reference value (PBS-T only) was subtracted from the actual measured value, and the affinity was measured using Octet software, performing global fitting using a 1:1 binding model. The measurement results for representative clones from each VHH are shown in Figure 5, and the affinities are listed in Tables 4 and 5.

[0168]

[0169]

[0170] Example 5: Single-point binding assay for each FGFR extracellular domain using Octet. Using Octet RED384, interaction analysis was performed between purified VHH clones and the extracellular domains of FGFR1(IIIb), FGFR1(IIIc), FGFR2(IIIb), FGFR2(IIIc), FGFR3(IIIb), FGFR3(IIIc), or FGFR4. IIIb and IIIc for each receptor represent splicing variants of the receptor extracellular domain III (all manufactured by R&D). Purified VHHs were immobilized on a His1K sensor chip (manufactured by Fortebio), and each FGFR extracellular domain was adjusted to 100 nM. 70 μL of the assay solution was added to a 384-well plate to measure binding to the VHH clones. Before measurement, the tip of the Dip and Read SA Biosensor was immersed in 200 μL of PBS-T (PBS containing 0.05% Tween 20, pH 7.4) for 10 minutes to hydrate the sensor chip.

[0171] The measurement order for each run was as follows: 1) Baseline step: 30-second measurement in PBS-T. 2) Loading step: 120-second measurement using purified VHH diluted 100-fold in PBS-T. 3) Baseline step: 30-second measurement in PBS-T. 4) Association step: 120-second measurement using each FGFR extracellular domain diluted in PBS-T. 5) Dissociation step: 120-second measurement in PBS-T. 6) Regeneration step: 5-second measurement in glycine-HCl (pH 2.2) and 5-second measurement in PBS-T, repeated three times.

[0172] After the above measurements were completed, the responses of the VHH clones to each FGFR extracellular domain were graphed to analyze receptor specificity. The results are shown in Figures 6 to 9. Because the extracellular domains of each receptor have similar Ig-like structures, it was found that even products screened using FGFR1 also bind to other receptors. It was also found that the receptors to which they strongly bind differed depending on the VHH clone.

[0173] (Example 6) Preparation of VHH-Fc bodies VHH-Fc bodies were prepared as follows. (1) Construction of expression plasmids VHHs VM44, VM46, VM1637, and VM1640 selected based on a multipoint binding assay using Octet were cloned into a plasmid vector for expressing Fc bodies. The dissociation constants (units: M) for each FGFR of each VHH clone obtained in Example 5 above are shown in Table 6.

[0174]

[0175] First, a sequence for homologous recombination was added to a glycerol stock of a unique VHH clone from C. glutamicum by PCR as described in the standard method, and a PCR product was obtained. The reaction mixture consisted of 1 U of KOD FX Neo, 25 μL of 2x PCR Buffer for KOD FX Neo, dNTPs at a final concentration of 0.4 mM, a small amount of glycerol stock, and primers PL_to_pcDNA_FW (SEQ ID NO: 22) and PL_to_pcDNA_RV (SEQ ID NO: 23) at a final concentration of 0.3 μM each, and the volume was adjusted to 50 μL with ultrapure water. The sequences of the two primers are shown below.

[0176] 5'- AAGGGCGTGCAGTGCGAAGTACAATTAGTTGAATCTGGTGGTG-3' (SEQ ID NO: 22) 5'- gctgctcttgggctcTGAAGAGACTGTCACCAACGTG-3' (SEQ ID NO: 23)

[0177] A two-step PCR program was performed with denaturation at 98°C for 10 seconds and denaturation and extension at 68°C for 30 seconds, for 30 cycles to obtain the PCR product. The resulting PCR product was purified as insert DNA using a Gel / PCR Extraction Kit (Nippon Genetics) according to the attached instructions. The Fc expression plasmid vector was treated with restriction enzymes EcoRI and BamHI at 37°C for 1 hour and subjected to 1% agarose gel electrophoresis at 100 V for 30 minutes. After electrophoresis, the gel was excised and purified using a Gel / PCR Extraction Kit (Nippon Genetics) according to the attached instructions to obtain the purified product.

[0178] Insert DNA and plasmid vector DNA were mixed at a molar ratio of 1:3, and a plasmid was obtained by SLiCE reaction at 37°C for 15 minutes. The resulting plasmid was transformed into Escherichia coli JM109, plated on an agar medium plate, and cultured overnight at 37°C. Colonies that appeared on the agar medium were picked and cultured overnight at 37°C. Plasmids were extracted using the FastGene Plasmid Mini Kit (Nihon Genetics) according to the attached instructions. DNA sequencing of the extracted plasmid was outsourced to Eurofins genomics, and the DNA sequence was analyzed.

[0179] (2) Expression of VHH-Fc using Expi293F cells. Expi293F cells (Thermo Fisher Scientific) were passaged in Expi293 Expression Medium (Thermo Fisher Scientific). Expi293F cells were seeded into T25 flasks (Sarstedt) and cultured at 37°C under 8% CO2. The Fc expression plasmid, ExpiFectamine® 293 (Thermo Fisher Scientific), was suspended in Opti-MEME (Gibco) and allowed to stand at room temperature for 15 minutes. This was then added to overnight-cultured Expi293F cells. After 96 hours of culture, the culture supernatant was collected. The collected culture supernatant was filtered through a 0.22 μm filter to remove cells from the supernatant.

[0180] (3) Purification of VHH-Fc The culture supernatant was applied to a column packed with 500 μL of Amsphere® A3 (JSR Corporation, hereafter referred to as "carrier"). The carrier was washed by sequentially adding 5 mL of PBS, 5 mL of high-salt PBS (PBS containing 1 M NaCl), and 5 mL of PBS. The column was then eluted with 5 mL of 100 mM Glycine-HCl pH 2.2 (Fujifilm Wako Pure Chemical Industries, Ltd.) and neutralized with 500 μL of 1 M Tris-HCl pH 8.5. The eluted VHH-Fc was transferred to an Amicon Ultra 10 kDa column (Millipore) and centrifuged at 3,500 × g for 30 minutes at 4°C to concentrate the VHH-Fc. Next, 4 mL of PBS was added, and the column was centrifuged under the same conditions. This PBS replacement procedure was repeated three times. The purity of the purified VHH-Fc was confirmed by SDS-PAGE (Figure 10). SDS-PAGE was performed using a 4% concentrated, 10% separating gel. Five μL of sample was applied to each well and electrophoresed at 150 V for 1 hour. Precision Plus Protein Standard (BioRad) was used as a molecular weight marker. The concentration of VHH-Fc was quantified by the BCA method using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) with bovine serum albumin as the standard protein. The Fc forms of VM44, VM46, VM1637, and VM1640 thus obtained were designated VF151, VF152, VF155, and VF156 (SEQ ID NOs: 11 to 14, respectively). VF151, VF152, VF155, and VF156, as well as the human Fc sequence and the sequence of the Hinge between VHH and Fc, are as follows:

[0181] Full-length amino acid sequence of VF151: EVQLVESGGG LVQPGGSLRL SCAASGSISS INIMGWFRQA PGKGREFVAA ISRIGSSTAY ADSVKGRFTI SRDNAKNTVY LQMNSLRAED TAVYYCAASI HFLGQSYADY WGQGTLVTVS SEPKSSDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK (SEQ ID NO: 11)

[0182] Amino acid sequence of VF151 VHH (VM44) EVQLVESGGG LVQPGGSLRL SCAASGSISS INIMGWFRQA PGKGREFVAA ISRIGSSTAY ADSVKGRFTI SRDNAKNTVY LQMNSLRAED TAVYYCAASI HFLGQSYADY WGQGTLVTVS S (SEQ ID NO: 5)

[0183] Amino acid sequence of CDR1 of VF151 (VM44) GSISSINIMG (SEQ ID NO: 32) Amino acid sequence of CDR2 of VF151 (VM44) AISRIGSSTA YADSVKG (SEQ ID NO: 33) Amino acid sequence of CDR3 of VF151 (VM44) SIHFLGQSYA DY (SEQ ID NO: 1)

[0184] Full-length amino acid sequence of VF152: EVQLVESGGG LVQPGGSLRL SCAASGQTFS SYNMGWFRQA PGKGREFVAS ISRSGGLTYY ADSVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCAADY VLDLKRYRTQ HNYWGQGTLV TVSSEPKSSD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 12)

[0185] Amino acid sequence of VF152 VHH (VM46) EVQLVESGGG LVQPGGSLRL SCAASGQTFS SYNMGWFRQA PGKGREFVAS ISRSGGLTYY ADSVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCAADY VLDLKRYRTQ HNYWGQGTLV TVSS (SEQ ID NO: 6)

[0186] Amino acid sequence of CDR1 of VF152 (VM46) GQTFSSYNMG (SEQ ID NO: 34) Amino acid sequence of CDR2 of VF152 (VM46) SISRSGGLTY YADSVKG (SEQ ID NO: 35) Amino acid sequence of CDR3 of VF152 (VM46) DYVLDLKRYR TQHNY (SEQ ID NO: 2)

[0187] Full-length amino acid sequence of VF155: EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYDMSWYRQA PGKGLEWVAA ITIGGSTNYA ASVKGRFTIS RDNAKNTLYL QMNSLRAEDT AVYYCNAWQH SWHGKDKDYW GQGTLVTVSS EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ ID NO: 13)

[0188] Amino acid sequence of VF155 VHH (VM1637) EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYDMSWYRQA PGKGLEWVAA ITIGGSTNYA ASVKGRFTIS RDNAKNTLYL QMNSLRAEDT AVYYCNAWQH SWHGKDKDYW GQGTLVTVSS (SEQ ID NO: 7)

[0189] Amino acid sequence of CDR1 of VF155 (VM1637) GFTFSRYDMS (SEQ ID NO: 36) Amino acid sequence of CDR2 of VF155 AITIGGSTNY AASVKG (SEQ ID NO: 37) Amino acid sequence of CDR3 of VF155 (VM1637) WQHSWHGKDK DY (SEQ ID NO: 3)

[0190] Full-length amino acid sequence of VF156: EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYDMSWYRQA PGKGLEWVAT ITSGGSTNYA DSVKGRFTIS RDNAKNTLYL QMNSLRAEDT AVYYCNAYHH SWHDVDADYW GQGTLVTVSS EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK (SEQ ID NO: 14)

[0191] Amino acid sequence of VF156 VHH (VM1640): EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYDMSWYRQA PGKGLEWVAT ITSGGSTNYA DSVKGRFTIS RDNAKNTLYL QMNSLRAEDT AVYYCNAYHH SWHDVDADYW GQGTLVTVSS (SEQ ID NO: 8)

[0192] Amino acid sequence of CDR1 of VF156 (VM1640): GFTFSRYDMS (SEQ ID NO: 38) Amino acid sequence of CDR2 of VF156 (VM1640): TITSGGSTNY ADSVKG (SEQ ID NO: 39) Amino acid sequence of CDR3 of VF156 (VM1640): YHHSWHDVDA DY (SEQ ID NO: 4)

[0193] Human Fc sequence SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK (SEQ ID NO: 9) Hinge sequence between VHH and Fc EPKSSDKTHT CPPCPAPELL GGP (SEQ ID NO: 10)

[0194] (4) The affinity of the VHH-Fc obtained as described above for FGFR1(IIIc)-Fc was measured using the Octet RED384 multipoint binding assay (Fortebio). FGFR1(IIIc)-Fc-Avi (R&D) was immobilized on an SA sensor chip, and VHH-Fc prepared at a 2-fold dilution from 25 nM was allowed to bind to the VHH-Fc. Prior to measurement, the tip of a Dip and Read® SA Biosensor (Fortebio) was immersed in 200 μL of PBS containing 0.05% Tween 20 (hereinafter sometimes referred to as "PBS-T") for 10 minutes to hydrate the sensor chip. Then, 70 μL of each test solution was added to a 384-well plate, and measurements were performed according to steps 1) to 6) below.

[0195] 1) Baseline step: 30-second measurement in PBS-T. 2) Loading step: 300-second measurement with FGFR1(IIIc)-Fc-Avi in ​​PBS-T. 3) Baseline step: 30-second measurement in PBS-T. 4) Association step: 120-second measurement with VHH-Fc diluted in PBS-T. 5) Dissociation step: 120-second measurement in PBS-T. 6) Regeneration step: 5-second measurement in glycine-HCl (pH 2.2) and 5-second measurement in PBS-T, repeated three times. After the above measurements, the reference value (PBS-T only) was subtracted from the actual measurement value, and global fitting was performed using a 1:1 binding model using Octet software to calculate the affinity (Figure 11). The affinity of VHH-Fc is shown in Table 7. It was found that the addition of the Fc sequence tended to improve binding affinity.

[0196]

[0197] (Example 7) Evaluation of agonistic activity of VHH-Fc using NIH3T3 cells The agonistic activity of VHH-Fc was evaluated using mouse embryonic fibroblast cells NIH3T3 (ATCC) as follows.

[0198] (1) Cell culture Mouse embryonic fibroblasts NIH3T3 were subcultured in DMEM (High glucose) (Sigma, hereafter simply referred to as “DMEM”) containing 10% Fatal Calf Serum (Biowest, hereafter referred to as “CS”) and 1% Penicillin-Streptomycin Mixed Solution (containing 100 units / mL penicillin G and 100 μg / mL streptomycin sulfate) (Nacalai Tesque, hereafter referred to as “PS(+)”).

[0199] (2) Cell proliferation assay: 100 μL of the cells were added to a 96-well white cell culture plate (Thermo Fisher Scientific) to a final concentration of 2.5 × 10 3Cells were seeded at 0.01 cells / well and cultured overnight at 37°C in a 5% CO2 environment. The medium was removed from each well and replaced with DMEM containing various test substances, 1% CS, and 1% PS(+), followed by further culture for 72 hours. The 96-well plate was left at room temperature for 5 minutes, after which viable cell counts were measured using the Cell Titer Glo® 2.0 Cell Viability Assay (Promega) according to the attached instructions. Test substances were measured at four points for each concentration, and a 3-fold dilution series starting from 500 nM was prepared for nine concentrations. The measured data were subjected to four-parameter logistic curve analysis using GraphPad Prism 9, and the EC values ​​were calculated from the resulting sigmoid curve. 50 The mean and standard error were calculated (Fig. 12). All VHH-Fc had cell proliferation activity, demonstrating that VHH-Fc with agonistic activity had been prepared.

[0200] (Example 8) Evaluation of the anti-fibrotic activity of VHH-Fc using HHSC cells Human hepatic stellate cells (HHSCs) are known to be activated by TGFβ and promote fibrosis by secreting collagen fibers, etc. Therefore, the anti-fibrotic activity of VHH-Fc was evaluated using human hepatic stellate cells as follows.

[0201] (1) Cell Culture Human hepatic stellate cells (HHSC, Cell Applications) were cultured in human hepatic stellate cell growth medium (basal medium + additives) containing fetal bovine serum (FBS) (Cell Applications, hereafter referred to as "HSCM") at 37°C in a 5% CO environment, and subcultured using a subculture reagent set.

[0202] (2) Evaluation of anti-fibrotic activity 2 mL of the solution was added to a 6-well cell culture plate (Falcon) at a final concentration of 5 × 10 5Cells were seeded at 1000 cells / well and cultured at 37°C in a 5% CO2 environment. After confirming cell adhesion, the medium was removed from each well and replaced with HSCM basal medium without FBS, followed by overnight culture. TGFβ (Acrobyosystems) alone or TGFβ and VHH-Fc were then added to the medium, and the cells were cultured for an additional 24 hours. TGFβ was diluted to a final concentration of 50 ng / mL, and VHH-Fc was diluted to 1 μM.

[0203] (3) Gene Expression Analysis Cells were collected in a 1.5 mL tube, and RNA was purified using the RNeasy mini Kit (QIAGEN) according to the manufacturer's instructions. 30 μL of RNase-free water was added to the column to elute the RNA.

[0204] RNA concentration was measured using a NanoDrop 2000 (Thermo Scientific). After denaturing the RNA at 65°C for 5 minutes, reverse transcription was performed using 1 μg of RNA and a ReverTra Ace® qPCR RT Kit (Toyobo) according to the accompanying instructions. Quantitative PCR was then performed using KOD SYBR qPCR Mix (Toyobo) and an Applied Biosystems™ StepOnePlus™ Real-Time PCR System (Applied Biosystems) according to the accompanying instructions, and relative quantification was performed using the ΔΔCt method using the expression level of r28S. Quantitative PCR was performed using the primers listed in Table 8 below for ACTA2 (protein name: Actin, aortic smooth muscle, also known as αSMA) and COL1A1 (protein name: Collagen alpha-1(I) chain).

[0205]

[0206] The results of quantitative PCR are shown in Figure 13. The expression levels of ACTA2 and COL1A1 were elevated in the group in which hepatic stellate cells were stimulated with TGFβ alone. These genes indicate the degree of hepatic stellate cell fibrosis and the level of fibroprotein production, respectively, demonstrating that TGFβ promotes hepatic stellate cell fibrosis. On the other hand, the expression levels of these genes were reduced by the simultaneous addition of VF156. This indicates that VF156 inhibited TGFβ-dependent hepatic stellate cell fibrosis. These results suggest that VF156 can be used to treat fibrosis or slow the progression of the disease. Furthermore, VF151, VF152, and VF155, which have similar agonistic activity, are also expected to have similar effects.

[0207] Example 9: Measurement of Thermal Stability The thermal stability of VHH-Fc and FGF2 was measured using UNcle (Unchained Labs). VHH-Fc was prepared at 0.5 mg / mL in PBS, and FGF2 (R&D) was prepared at 1 mg / mL. For dynamic light scattering (DLS) and selective light scattering (SLS), a temperature ramp of 1°C / min was performed from 25°C to 95°C while monitoring. SLS was measured at 266 nm and 473 nm. DLS was measured at the start and end of the temperature ramp. Tm, Tag, and DLS measurements were calculated and analyzed using UNcle analysis software. SLS 266 nm and Tag are shown in Figure 14 against the temperature ramp. As shown in Figure 14, VHH-Fc exhibited higher thermal stability than FGF2. As described above, a VHH-Fc complex can be obtained in which the Fc sequence of a human antibody is added to a humanized VHH of the present invention that binds to the extracellular domain of fibroblast growth factor receptor (FGFR) 1.

[0208] Example 10: Example of a culture medium composition for cultivated meat containing an FGFR agonist VHH. A culture medium containing an FGFR agonist VHH, particularly a culture medium composition for cultivated meat, is prepared. Instead of the FGF2 typically added to culture media, an FGFR agonist VHH is added to the culture medium to create a culture medium composition. Alternatively, the amount of FGF2 added is reduced, and an FGFR agonist VHH is added to compensate. Because VHHs have an aggregation onset temperature (Tagg) approximately 20°C higher than FGF2, they exhibit excellent thermal stability, resulting in the culture medium composition of the present invention having excellent thermal stability. The cell culture medium of the present invention, particularly in the preparation of stem cells for regenerative medicine and cultivated meat production, has the potential to be used as a cost-effective alternative to FGF2 with excellent thermal stability.

[0209] Example 11: Example 1 of a cosmetic composition containing an FGFR agonist VHH The cosmetic composition contains an FGFR1 agonist VHH. The concentration of the FGFR1 agonist VHH is, for example, 0.1 ppm or more. The cosmetic composition further contains cosmetically acceptable excipients, such as water, glycerol, lipids, antioxidants, preservatives, etc. The cosmetic composition may be a low-viscosity liquid, or may be made into a liquid or cream with increased viscosity by adding other excipients. The cosmetic composition is applied to the face, neck, hands, and feet to prevent wrinkles caused by aging and ultraviolet rays and to prevent the progression of wrinkles.

[0210] Example 12 Example 2 of a cosmetic composition containing an FGFR1 agonist VHH As a variation of Example 4, a liposome carrier encapsulating an FGFR1 agonist VHH can be used. By encapsulating the VHH in such a liposome carrier, its absorption into the skin is enhanced. As a further alternative, an injectable solution can be prepared together with an excipient such as physiological saline, and the composition can be injected into the skin using a delivery device such as a syringe.

[0211] Example 13: Inhibition of metastasis by FGFR1 agonist VHH using a mouse model of cancer metastasis The effect of inhibiting cancer metastasis was investigated using the Fc forms of VF151 and VF156. Evaluation of VHH-Fc using a mouse model of cancer liver metastasis transplanted with mouse-derived colon cancer organoids. Activation of fibroblasts at the metastatic site is important for cancer metastasis. To verify whether the Fc form of FGFR1 agonist VHH has the effect of inhibiting cancer metastasis, a non-clinical trial was conducted using a mouse model of cancer liver metastasis transplanted with mouse-derived colon cancer organoids. The liver, along with lymph nodes and the peritoneal cavity, is known to be a common site of metastasis from various primary tumors, and pancreatic cancer is particularly susceptible to liver metastasis.

[0212] (1) Organoid Culture. 1C9 cells were established by introducing mutations into four driver genes in mouse colon cancer cells. These cells (hereafter referred to as "AKTP cells") were used for organoid formation. The four driver gene mutations, ApcD716 (A), Kras+ / LSL-G12D (K), Tgfbr2flox / flox (T), and Trp53+ / LSL-R270H (P), were introduced. AKTP cells were subcultured in Advanced DMEM / F12 (Gibco) supplemented with 10% Fetal Bovine Serum (FBS), 10 μM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.), 500 nM A-8301 (Sigma), and 5 μM CHIR99021 (Sigma).

[0213] (2) Transplantation of organoids into mice and administration of VHH-Fc NSG mice were purchased from the Central Institute for Experimental Animals. 5AKTP cells in a mixture of organoid cells and 50 μL of PBS were transplanted into the spleens of anesthetized NSG mice. The day before organoid injection, mice were randomly assigned to three groups by weight stratification to ensure equal mean body weights. VHH-Fc was administered intraperitoneally using PBS as the vehicle. Administration was performed at a dose of 1 mg / kg on day 3 after organoid transplantation. A negative control, vehicle alone without VHH-Fc, was also administered. A second administration of VF151 and vehicle was performed on day 9 after organoid transplantation.

[0214] (3) Observation of Liver Metastasis of Colorectal Cancer Using IVIS Imaging Liver metastasis was observed using the IVIS Lumina LT on days 7 and 14 after organoid transplantation. Anesthetized mice were intraperitoneally administered 150 mg / kg of D-Luciferin Potassium Salt (Fujifilm Wako Pure Chemical Industries, Ltd.). After administration, luminescence from luciferase introduced into the cancer cells was observed using IVIS imaging. The results are shown in Figure 15. Luciferase luminescence intensity was significantly reduced in the VF151- and VF156-treated groups compared to the vehicle group. These results suggest that the FGFR1 agonist VHH-Fc may have the effect of suppressing cancer metastasis to the liver.

[0215] (4) Observation of the Liver by Fluorescence Microscopy On day 14 after organoid transplantation, mice were dissected and livers were isolated. The obtained livers were observed under a fluorescence microscope. The results are shown in Figure 16. Venus fluorescence was significantly reduced in the VF151- and VF156-administered groups compared to the vehicle group. These results suggest that the FGFR1 agonist VHH-Fc may have the effect of suppressing cancer metastasis to the liver.

[0216] (5) Preparation of pathological tissue specimens. Livers were immersed in fixative, fixed at room temperature for 24 hours, and embedded in paraffin. Paraffin blocks were sliced ​​to a thickness of 4 μm using a rotary microtome. The paraffin sections were stretched, attached to glass slides, and dried using a hot plate. The paraffin sections were then subjected to hematoxylin-eosin staining and immunostaining.

[0217] (6) Hematoxylin-eosin staining and calculation of tumor percentage. Paraffin sections were deparaffinized and hydrophilized using xylene, a 100% to 70% alcohol series, and RO water, then immersed in Lilly-Meyer's hematoxylin solution for 10 minutes. After rinsing with RO water and allowing the sections to develop in running water for 10 minutes, they were immersed in 1% eosin Y ethanol solution for 5 minutes and then immersed in RO water. The stained sections were dehydrated and cleared using a 70% to 100% alcohol series and xylene, then mounted in Entelaneu and subjected to observation. The specimens were observed under a bright-field microscope. The percentage of metastatic tumor relative to the total liver area was calculated (Figure 17). The percentage of metastatic tumor was significantly reduced in the VF151 and VF156 groups compared to the vehicle group, with VF151 showing particularly significant resistance. These results suggest that FGFR1 agonist VHHs may have the effect of suppressing cancer metastasis to the liver.

[0218] (7) Immunostaining with Anti-αSMA and Anti-Transgelin Antibodies. Paraffin sections were deparaffinized and hydrophilized using xylene, a 100% to 70% alcohol series, and RO water, and then infiltrated in a solution containing anti-αSMA or anti-Transgelin antibodies. αSMA is a marker for CAFs, which are involved in cancer malignancy, and Transgelin is thought to be a protein involved in metastasis in many different cancers. After washing, the paraffin sections were infiltrated in a solution containing Alexa 594- or Alexa 488-conjugated secondary antibodies for immunohistochemistry. Images of the staining results are shown in Figure 18. αSMA and Transgelin expression was observed in the vehicle-treated group. In contrast, αSMA and Transgelin expression was relatively suppressed in the FGFR1 agonist VHH-treated group, and αSMA expression was not detected in the VF151-treated group. These results suggest that FGFR1 agonist VHHs may suppress fibroblast activation and thus inhibit cancer metastasis to the liver.

[0219] SEQ ID NO: 1: Amino acid sequence of VF151 CDR3 SEQ ID NO: 2: Amino acid sequence of VF152 CDR3 SEQ ID NO: 3: Amino acid sequence of VF155 CDR3 SEQ ID NO: 4: Amino acid sequence of VF156 CDR3 SEQ ID NO: 5: Amino acid sequence of VM44

[0220] SEQ ID NO: 6: Amino acid sequence of VM46 SEQ ID NO: 7: Amino acid sequence of VM1637 SEQ ID NO: 8: Amino acid sequence of VM1640 SEQ ID NO: 9: Human Fc sequence SEQ ID NO: 10: Amino acid sequence of Hinge between VHH and Fc

[0221] SEQ ID NO: 11: Amino acid sequence of VF151 SEQ ID NO: 12: Amino acid sequence of VF152 SEQ ID NO: 13: Amino acid sequence of VF155 SEQ ID NO: 14: Amino acid sequence of VF156 SEQ ID NO: 15: Nucleotide sequence of primer

[0222] SEQ ID NO: 16: Nucleotide sequence of primer SEQ ID NO: 17: Backbone of linker for cDNA display SEQ ID NO: 18: Nucleotide sequence of primer SEQ ID NO: 19: Nucleotide sequence of primer SEQ ID NO: 20: Nucleotide sequence of primer

[0223] SEQ ID NO: 21: Nucleotide sequence of primer SEQ ID NO: 22: Nucleotide sequence of primer SEQ ID NO: 23: Nucleotide sequence of primer SEQ ID NO: 24: Nucleotide sequence of primer for quantitative PCR SEQ ID NO: 25: Nucleotide sequence of primer for quantitative PCR

[0224] SEQ ID NO: 26: Nucleotide sequence of a primer for quantitative PCR SEQ ID NO: 27: Nucleotide sequence of a primer for quantitative PCR SEQ ID NO: 28: Nucleotide sequence of a primer for quantitative PCR SEQ ID NO: 29: Nucleotide sequence of a primer for quantitative PCR

[0225] SEQ ID NO: 32: Amino acid sequence of CDR1 of VF151 SEQ ID NO: 33: Amino acid sequence of CDR2 of VF151 SEQ ID NO: 34: Amino acid sequence of CDR1 of VF152 SEQ ID NO: 35: Amino acid sequence of CDR2 of VF152

[0226] SEQ ID NO: 36: Amino acid sequence of CDR1 of VF155 SEQ ID NO: 37: Amino acid sequence of CDR2 of VF155 SEQ ID NO: 38: Amino acid sequence of CDR1 of VF156 SEQ ID NO: 39: Amino acid sequence of CDR2 of VF156

Claims

1. A humanized fibroblast growth factor receptor (FGFR) agonist VHH, said VHH comprising CDR1, CDR2 and CDR3 selected from the group consisting of: (1) CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 33 and 1, respectively; (2) CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 34, 35 and 2, respectively; (3) CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 36, 37 and 3, respectively; and (4) CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 38, 39 and 4, respectively.

2. The FGFR agonist VHH according to claim 1, comprising an amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8, or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8.

3. The FGFR agonist VHH of claim 1, which is a VHH-Fc to which the Fc sequence of a human antibody has been added.

4. The FGFR agonist VHH of claim 1, which is a dimer.

5. A composition comprising the FGFR agonist VHH of claim 1.

6. A pharmaceutical composition comprising the FGFR agonist VHH of claim 1.

7. The pharmaceutical composition according to claim 6 for the treatment of fibrosis.

8. The pharmaceutical composition according to claim 7, wherein the fibrosis is liver fibrosis.

9. The pharmaceutical composition according to claim 6, which is used to inhibit cancer metastasis.

10. The pharmaceutical composition according to claim 9, wherein the metastasis is liver metastasis.

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