Anti-FGF19 Antibody

Abstract

Anti-FGF19 antibodies and antigen-binding fragments thereof that selectively target the N-terminus of fibroblast growth factor 19 (FGF19) are provided. Also provided are compositions and uses of the antibodies, as well as methods for treating diseases or disorders caused by or associated with aberrant FGF19-FGFR4 signaling, such as cancer, by administering the anti-FGF19 antibodies.

Description

[Technical field]

[0001] Disclosed herein are antibodies and antigen-binding fragments thereof that bind to fibroblast growth factor 19 (FGF19), specifically to key residues in the N-terminus of FGF19 that contain the epitope of the antibody. Also provided herein are compositions and uses of the antibodies, as well as methods of treating diseases or disorders caused by or associated with aberrant FGF19-FGFR4 signaling, such as cancer, by administering anti-FGF19 antibodies. [Background technology]

[0002] Fibroblast growth factors (FGFs) are a family of proteins with diverse functions in the development of organisms from nematodes to humans. In many normal developmental and physiological processes, FGF-FGFR (FGF receptor) signaling is crucial, and it has been found that its dysregulation may play an important role in tumor development and progression (Non-Patent Document 1).

[0003] In humans, fibroblast growth factor 19 (FGF19) is a bile acid-induced and ileum-derived peptide growth factor that functions to regulate bile acid metabolism by binding hepatocyte-expressed FGF receptor 4 (FGFR4) and β-klotho (KLB) to repress hepatic transcription of the gene encoding cholesterol-7-a-hydroxylase 1 (CYP7A1), an essential enzyme for bile acid biosynthesis (Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). In addition to its bile acid regulatory function, clinical observations have demonstrated that both FGF19 and its cognate receptor FGF4R are highly expressed in tumors compared to adjacent non-tumor tissues (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9, Non-Patent Document 2, Non-Patent Document 10, Non-Patent Document 11).

[0004] FGF19 has been proposed to be a driver gene in the development of hepatocellular carcinoma (HCC), the most common type of liver cancer, accounting for approximately 90% of all liver cancer cases (Non-Patent Document 12). Liver cancer is the sixth most commonly diagnosed cancer worldwide, but ranks as the third most common cause of cancer-related deaths worldwide (Non-Patent Document 13). Several clinical studies have shown that HCC is characterized by highly localized amplification of fibroblast growth factor (FGF) 19 (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9). High expression of FGF19 and / or FGFR4 was found to promote tumor progression and could also predict poor prognosis of HCC patients (Non-Patent Document 14, Non-Patent Document 15). In transgenic mice, overexpression of FGF19 causes hepatocellular metaplasia, neoplasia and ultimately HCC (Non-Patent Document 16), which was abolished in FGFR4 knockout mice (Non-Patent Document 17); therefore, mechanistic confirmation of the tumorigenic activity of abnormal FGF19 / FGFR4 signaling provides a theoretical basis for the development of antagonists targeting the FGF19 / FGFR4 pathway as potential cancer treatments. However, several selective FGFR4 inhibitors under development for the treatment of HCC have led to increased bile acid synthesis and hepatotoxicity in either preclinical animal models or early human clinical trials (Non-Patent Document 18, Non-Patent Document 19, Non-Patent Document 20, Non-Patent Document 21, Non-Patent Document 22). Efforts have been made to develop drugs that directly target FGF19 as a therapeutic target for treating HCC, resulting in a neutralizing anti-FGF19 antibody (Patent Document 1 Genentech, Inc.). Treatment with this anti-FGF19 antibody (1A6) prevented mice overexpressing FGF19 from developing HCC, and such treatment also suppressed the growth of HCC xenografts in mice (Non-Patent Document 6, Non-Patent Document 10). Unfortunately, however, in toxicity studies, administration of humanized 1A6 antibody to cynomolgus monkeys increased the hepatic transcription of CYP7A1, elevated bile acid synthesis, and thus dramatically altered bile acid metabolism, causing severe dose-dependent side effects such as weight loss, severe diarrhea, and reduced food intake (Non-Patent Document 23).Given the role of FGF19 in regulating bile acid metabolism, antibodies targeting FGF19 may impair its normal physiological function in this respect, resulting in unacceptable side effects. Given these facts, clinical therapies targeting FGF19 with antibodies will need to address such side effects. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2007136893 [Non-patent literature]

[0006] [Non-Patent Document 1] Turner N, Grose R. Nature Reviews Cancer, 2010, 10(2): 116, https: / / www.nature.com / articles / nrc2780 [Non-Patent Document 2] MH Xie et al., Cytokine 11, 729-735 (1999) [Non-Patent Document 3] JA Holt et al., Genes & development 17, 1581-1591 (2003) [Non-Patent Document 4] H. Kurosu et al., The Journal of biological chemistry 282, 26687-26695 (2007) [Non-Patent Document 5] BC Lin et al., The Journal of biological chemistry 282, 27277-27284 (2007) [Non-Patent Document 6] ET Sawey et al., Cancer Cell 19, 347-358 (2011) [Non-Patent Document 7] K. Wang et al., Hepatology 58, 706-717 (2013)

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

[0007] The present invention fulfills that need by providing an anti-FGF19 antibody that specifically targets the N-terminus of FGF19, which is highly effective as an anti-HCC therapy without adversely affecting the normal bile acid regulatory function of FGF19.

[0008] All references, including scientific publications, patent application publications, and patent publications, are incorporated herein by reference in their entirety for all purposes.

[0009] The present invention is based in part on the discovery of a new binding region on FGF19, specifically on the N-terminus of FGF19, and the identification of an anti-FGF19 antibody that exhibits strong anti-tumor activity against HCC and has a desirable safety profile that does not affect FGF19 regulation of bile acid synthesis by specifically binding to the binding region on the N-terminus of FGF19. Thereby, the present invention also provides a new therapeutically potential target on the N-terminus of FGF19, and an antibody that specifically binds to said target for the diagnosis, prevention, treatment and / or prognosis of pathological conditions associated with or caused by abnormal FGF19 / FGFR4 signaling, specifically cancers associated with or caused by abnormal FGF19 / FGFR4 signaling, including but not limited to HCC. Also provided are nucleic acid molecules, expression vectors and host cells comprising nucleic acid sequences encoding the antibody or an antigen-binding fragment thereof. The present invention also provides pharmaceutical compositions, methods, kits, products and pharmaceutical uses related to modulation of the FGF19 / FGFR4 signaling pathway.

[0010] In a first aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to human fibroblast growth factor 19 (FGF19), the binding being dependent on the N-terminus of FGF19. In one embodiment, the antibody or antigen-binding fragment of the present application binds to an epitope located at amino acid residues 38-45 of SEQ ID NO: 1. More specifically, the epitope includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven of the following residues of human FGF19 (SEQ ID NO: 1): W38, D40, P41, I42, R43, L44, and R45. In another embodiment, the antibody or antigen-binding fragment of the present application binds to an epitope consisting of amino acid residues 38-45 of SEQ ID NO: 1 and at least one of the following residues of human FGF19 (SEQ ID NO: 1): E81, P161, and L169. In further embodiments, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or similar techniques; -9 M ~ approx. 1×10-12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0011] In another embodiment of the first aspect, the anti-FGF19 antibody or antigen-binding fragment of the present application comprises at least one, two, or three heavy chain variable (VH) domain complementarity determining regions (CDRs) comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, 13, or 19, or a variant thereof comprising one or more conservative substitutions. In another embodiment, the anti-FGF19 antibody or antigen-binding fragment of the present application comprises at least one, two, or three light chain variable (VL) domain CDRs comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 14, or a variant thereof comprising one or more conservative substitutions. In another embodiment, the anti-FGF19 antibody or antigen-binding fragment of the present application comprises at least one, two, or three heavy chain variable (VH) domain CDRs comprising the amino acid sequence of SEQ ID NO: 3, 4, 5, 13, or 19, or a variant thereof comprising one or more conservative substitutions, and at least one, two, or three light chain variable (VL) domain CDRs comprising the amino acid sequence of SEQ ID NO: 6, 7, 8, or 14, or a variant thereof comprising one or more conservative substitutions. In a further embodiment, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a nucleotide sequence of about 1×10 to human FGF19 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D(ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0012] In one embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable (VH) domain comprising an HCDR1 (heavy chain complementarity determining region 1) having the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising one or more conservative substitutions, an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising one or more conservative substitutions, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 19, or a variant thereof comprising one or more conservative substitutions; and / or (b) a light chain variable (VL) domain comprising an LCDR1 (light chain complementarity determining region 1) having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising one or more conservative substitutions, an LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising one or more conservative substitutions, and an LCDR3 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 14, or a variant thereof comprising one or more conservative substitutions.

[0013] In one embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 13, or SEQ ID NO: 19; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO: 8, or SEQ ID NO: 14.

[0014] In one embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising one or more conservative substitutions, an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising one or more conservative substitutions, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof comprising one or more conservative substitutions; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising one or more conservative substitutions, an LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising one or more conservative substitutions, and an LCDR3 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof comprising one or more conservative substitutions. In a more specific embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO: 8.

[0015] In one embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising one or more conservative substitutions, an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising one or more conservative substitutions, and an HCDR3 having the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising one or more conservative substitutions; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising one or more conservative substitutions, an LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising one or more conservative substitutions, and an LCDR3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising one or more conservative substitutions. In a more specific embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 13; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0016] In one embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof comprising one or more conservative substitutions, an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising one or more conservative substitutions, and an HCDR3 having the amino acid sequence of SEQ ID NO: 19 or a variant thereof comprising one or more conservative substitutions; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof comprising one or more conservative substitutions, an LCDR2 having the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising one or more conservative substitutions, and an LCDR3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof comprising one or more conservative substitutions. In a more specific embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises (a) a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4, and an HCDR3 having the amino acid sequence of SEQ ID NO: 19; and / or (b) a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 6, an LCDR2 having the amino acid sequence of SEQ ID NO: 7, and an LCDR3 having the amino acid sequence of SEQ ID NO: 14.

[0017] In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 9, 15 or 20; and / or a light chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 10, 16 or 21. In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable domain having the amino acid sequence of SEQ ID NO: 9, 15 or 20; and / or a light chain variable domain having the amino acid sequence of SEQ ID NO: 10, 16 or 21.

[0018] In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:9 and / or a light chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:10; (b) a light chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:15. or (c) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 20 and / or a light chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 21. In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises (a) a heavy chain variable domain having the amino acid sequence of SEQ ID NO:9 and / or a light chain variable domain having the amino acid sequence of SEQ ID NO:10; (b) a heavy chain variable domain having the amino acid sequence of SEQ ID NO:15 and / or a light chain variable domain having the amino acid sequence of SEQ ID NO:16; or (c) a heavy chain variable domain having the amino acid sequence of SEQ ID NO:20 and / or a light chain variable domain having the amino acid sequence of SEQ ID NO:21.

[0019] In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 11, 17 or 22. chain; and / or a light chain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 12, 18 or 23. Chains In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises a polypeptide having the amino acid sequence of SEQ ID NO: 11, 17 or 22. chain;and / or a light molecule having the amino acid sequence of SEQ ID NO: 12, 18 or 23. Chains include.

[0020] In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a polypeptide having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:11; chain, and / or a light chain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 12. chain; (b) a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 17; chain, and / or a light chain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 18. chain; or (c) a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:22. chain, and / or a light chain having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 23. Chains In one preferred embodiment, the anti-FGF19 antibody or antigen-binding fragment thereof of the present application comprises: (a) a polypeptide having the amino acid sequence of SEQ ID NO:11; chain, and / or a light molecule having the amino acid sequence of SEQ ID NO:12 chain; (b) a polymer having the amino acid sequence of SEQ ID NO: 17; chain, and / or a light molecule having the amino acid sequence of SEQ ID NO:18 chain; or (c) a clone having the amino acid sequence of SEQ ID NO:22. chain, and / or a light molecule having the amino acid sequence of SEQ ID NO:23 Chains include.

[0021] In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment thereof that competes for binding to human FGF19 with an antibody comprising a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO:3, an HCDR2 having the amino acid sequence of SEQ ID NO:4, and an HCDR3 having the amino acid sequence of SEQ ID NO:5; and a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:8. In a more specific embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment thereof that competes for binding to human FGF19 with an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO:9 and a light chain variable domain having the amino acid sequence of SEQ ID NO:10. More preferably, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0022] In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that competes for binding to human FGF19 with an antibody comprising a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO:3, an HCDR2 having the amino acid sequence of SEQ ID NO:4, and an HCDR3 having the amino acid sequence of SEQ ID NO:13; and a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:14. In a more specific embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that competes for binding to human FGF19 with an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO:15 and a light chain variable domain having the amino acid sequence of SEQ ID NO:16. More preferably, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0023] In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that competes for binding to human FGF19 with an antibody comprising a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO:3, an HCDR2 having the amino acid sequence of SEQ ID NO:4, and an HCDR3 having the amino acid sequence of SEQ ID NO:19; and a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:14. In a more specific embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that competes for binding to human FGF19 with an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO:20 and a light chain variable domain having the amino acid sequence of SEQ ID NO:21. More preferably, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0024] In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that binds to the same epitope of human FGF19 as an antibody comprising a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO:3, an HCDR2 having the amino acid sequence of SEQ ID NO:4, and an HCDR3 having the amino acid sequence of SEQ ID NO:5; and a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:8. In a more specific embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that binds to the same epitope of human FGF19 as an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO:9 and a light chain variable domain having the amino acid sequence of SEQ ID NO:10. More preferably, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0025] In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that binds to the same epitope of human FGF19 as an antibody comprising a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO:3, an HCDR2 having the amino acid sequence of SEQ ID NO:4, and an HCDR3 having the amino acid sequence of SEQ ID NO:13; and a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:14, for binding to human FGF19. In a more specific embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that binds to the same epitope of human FGF19 as an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO:15 and a light chain variable domain having the amino acid sequence of SEQ ID NO:16, for binding to human FGF19. More preferably, the antibody or antigen-binding fragment has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0026] In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment thereof that binds to the same epitope of human FGF19 as an antibody comprising a heavy chain variable (VH) domain comprising an HCDR1 having the amino acid sequence of SEQ ID NO:3, an HCDR2 having the amino acid sequence of SEQ ID NO:4, and an HCDR3 having the amino acid sequence of SEQ ID NO:19; and a light chain variable (VL) domain comprising an LCDR1 having the amino acid sequence of SEQ ID NO:6, an LCDR2 having the amino acid sequence of SEQ ID NO:7, and an LCDR3 having the amino acid sequence of SEQ ID NO:14, for binding to human FGF19. In a more specific embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment thereof that binds to the same epitope of human FGF19 as an antibody comprising a heavy chain variable domain having the amino acid sequence of SEQ ID NO:20 and a light chain variable domain having the amino acid sequence of SEQ ID NO:21, for binding to human FGF19. More preferably, the antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of about 1×10 as measured by surface plasmon resonance (e.g., BIACORE) or a similar technique; -9 M ~ approx. 1×10 -12 K of M D (ii) cross-react with cynomolgus monkey FGF19; (iii) block binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibit FGF19-induced cell proliferation; (v) have no or only a small effect on FGF-suppressed CYP7A1 expression; and (vi) have no or only a small effect on FGF19-maintained bile acid homeostasis.

[0027] In one embodiment, the anti-FGF19 antibody is a human antibody. In one embodiment, the anti-FGF19 antibody is a human monoclonal antibody (mAb).

[0028] In one embodiment, the anti-FGF19 antibody is a Fab, F(ab')2, Fv, or single chain Fv (ScFv).

[0029] In one embodiment, the anti-FGF19 antibody comprises a heavy chain constant region of the subclass IgG1, IgG2, IgG3, IgG4 or a variant thereof, and a light chain constant region of the type kappa or lambda or a variant thereof.

[0030] In one embodiment, the anti-FGF19 antibody is an isolated antibody. In one embodiment, the anti-FGF19 antibody is a recombinant antibody.

[0031] In a preferred embodiment, the antibody or antigen-binding fragment of the first aspect of the present application is for treating disease or disorder caused by or associated with abnormal FGF19-FGFR4 signal transduction, and comprises administering to a subject in need thereof.In a specific embodiment, the disease or disorder is cancer caused by or associated with abnormal FGF19-FGFR4 signal transduction.In a more specific embodiment, the cancer is hepatocellular carcinoma (HCC).

[0032] In a second aspect, the present disclosure provides a composition, e.g., a pharmaceutical composition, comprising an anti-FGF19 antibody or antigen-binding fragment of the first aspect of the invention and a pharma- ceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the anti-FGF19 antibody or antigen-binding fragment.

[0033] In a third aspect, the present disclosure provides a kit comprising an anti-FGF19 antibody or antigen-binding fragment of the first aspect of the present application, or a composition of the second aspect of the present application. In a preferred embodiment, the kit comprises a therapeutically effective amount of the anti-FGF19 antibody or antigen-binding fragment.

[0034] In a fourth aspect, the present disclosure provides a method for preventing or treating disease or disorder caused by or associated with abnormal FGF19-FGFR4 signal transduction, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of the first aspect of the present application or the pharmaceutical composition of the second aspect of the present application.In a specific embodiment, the disease or disorder is cancer caused by or associated with abnormal FGF19-FGFR4 signal transduction.In a more specific embodiment, the cancer is hepatocellular carcinoma (HCC).

[0035] In a fifth aspect, the present disclosure provides a method for preventing recurrence of disease or disorder caused by or associated with abnormal FGF19-FGFR4 signal transduction, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of the first aspect of the present application, or the pharmaceutical composition of the second aspect of the present application.In a specific embodiment, the disease or disorder is cancer caused by or associated with abnormal FGF19-FGFR4 signal transduction.In a more specific embodiment, the cancer is hepatocellular carcinoma (HCC).In one embodiment, the recurrence of disease or disorder occurs after surgery.

[0036] In a sixth aspect, the present disclosure provides the use of the antibody or antigen-binding fragment of the first aspect of the present application or the pharmaceutical composition of the second aspect of the present application for treating various disorders or diseases described herein or for preventing the recurrence of various disorders or diseases described herein.In a specific embodiment, the disease or disorder is a disease or disorder caused by or associated with abnormal FGF19-FGFR4 signal transduction, preferably a cancer caused by or associated with abnormal FGF19-FGFR4 signal transduction.In a more specific embodiment, the cancer is hepatocellular carcinoma (HCC).In one embodiment, the recurrence of the disease or disorder occurs after surgery.

[0037] In a seventh aspect, the present disclosure provides the use of the antibody or antigen-binding fragment of the first aspect of the present application, or the pharmaceutical composition of the second aspect of the present application, in the manufacture of a medicament for treating various disorders or diseases described herein, or for preventing the recurrence of various disorders or diseases described herein.In a specific embodiment, the disease or disorder is a disease or disorder caused by or associated with abnormal FGF19-FGFR4 signaling, preferably a cancer caused by or associated with abnormal FGF19-FGFR4 signaling.In a more specific embodiment, the cancer is hepatocellular carcinoma (HCC).In one embodiment, the recurrence of the disease or disorder occurs after surgery.In one embodiment, the recurrence of the disease or disorder occurs after surgery. [Brief description of the drawings]

[0038] [Figure 1A] Figure 1 shows the deletable role of the N-terminus of FGF19 in mediating FGF19 activity. The binding affinity of FGF19 or FGF19ΔNT (N-terminal deletion mutant of FGF19) to FGFR4 or FGFR4-KLB complex is shown. The N-terminal deletion mutant FGF19ΔNT showed reduced binding affinity to FGFR4. FGFR4-hFc was captured as a ligand on a sensor chip in a Biacore. The interaction between the ligand FGFR4 and the analytes FGF19 or FGF19ΔNT was measured in the presence of 20 μg / ml heparin. The analytes FGF19 and FGF19ΔNT were serially diluted 2-fold from 1000 nM. [Figure 1B] Figure 1 shows the role of the deletion possible N-terminus of FGF19 in mediating FGF19 activity. N-terminal deletion mutant FGF19ΔNT showed reduced activity in inducing cell proliferation. Hep3B was cultured with different concentrations of FGF19 or FGF19ΔNT in DMEM containing 1% FBS. [Figure 1C]Figure 1. Role of the truncable N-terminus of FGF19 in mediating FGF19 activity. The N-terminal deletion mutant FGF19ΔNT retained the ability to suppress CPY7A1 gene expression in vivo. C57BL / 6 mice were fasted before intraperitoneal injection with the indicated treatments. Hepatic gene expression of CPY7A1 was analyzed by qPCR. Each treatment group consisted of 3 male and 3 female mice. [Diagram 2] Schematic diagram illustrating the generation of antibody G1A8 using antibody phage display technology. N-terminal peptides of FGF19 or full-length FGF19 protein were used as antigens to select against our large-scale non-immune antibody phage display library (D. Li et al., A potent human neutralizing antibody Fc-dependently reduces established HBV infections. Elife 6, 2017). Through subsequent ELISA screening with both full-length FGF19 and the N-terminal deletion mutant FGF19ΔNT, antibodies were identified that bound to FGF19 with higher affinity than FGF19ΔNT. Wells shaded in dark grey indicate higher affinity compared to wells shaded in light grey or white wells. The protein sequences of the CDR3 regions of both the heavy and light chains of one identified antibody 31A3 and its affinity-enhanced antibody G1A8 are shown in parallel, with different amino acids underlined. [Figure 3A] Figure 1 shows the improved binding affinity and higher N-terminus dependency of G1A8. Kinetic analysis of the binding of anti-FGG19 antibody 31A3 or G1A8 to FGF19 using SPR. FGF19 was serially diluted 2-fold from 100 nM. [Figure 3B]Figure 1 shows the improved binding affinity and higher N-terminus dependency of G1A8. The binding of both 31A3 and G1A8 to FGF19 is dependent on the N-terminus of FGF19, as shown by the different binding profiles when FGF19 or FGF19ΔNT is used as the target. The binding activity of the antibodies to FGF19 and FGF19ΔNT was analyzed by ELISA. [Figure 4A] Characterization of G1A8. G1A8 and 31A3 showed inhibition of FGF19-induced cell proliferation. Hep3B were cultured with 20 ng / ml FGF19 and 100 nM anti-FGF19 antibody or isotype control antibody. [Figure 4B] Figure 1 shows the characterization of G1A8. G1A8 inhibited the interaction of FGF19 with FGFR5. Antibody G1A8 and isotype control antibody were mixed with 100 nM FGFR4-Fc and 20 μg / ml heparin. The inhibition of FGFR4 binding to FGF19 was analyzed by ELISA. [Figure 4C] Characterization of G1A8. G1A8 did not impair FGF19-downregulated hepatic CYP7A1 gene expression in mice. C57BL / 6 mice were fasted before intraperitoneal injection with the indicated treatments. Hepatic CYP7A1 gene expression levels were analyzed by qPCR. Each group consisted of 3 male and 3 female mice. FGF19, 0.1 mg / kg; G1A8, 5-fold molar excess of FGF19. [Figure 5A] Figure 1 shows the anti-tumor activity of G1A8 in a human hepatocellular carcinoma xenograft mouse model. G1A8 inhibited tumor growth at the early stage of tumor development. Hep3B-luciferase stably expressing luciferase was subcutaneously (sc) injected into NOD SCID mice. Mice were divided into groups (n=6 / group) based on equivalent mean tumor bioluminescence intensity and administered 200 μg of G1A8 or isotype control antibody treatment. Tumor growth was measured by calipers. [Figure 5B]Figure 1 shows the anti-tumor activity of G1A8 in a human hepatocellular carcinoma xenograft mouse model. G1A8 inhibited tumor growth at the early stage of tumor development. Hep3B-luciferase stably expressing luciferase was subcutaneously (sc) injected into NOD SCID mice. Mice were divided into groups (n=6 / group) based on equivalent mean tumor bioluminescence intensity and administered 200 μg of G1A8 or isotype control antibody treatment. Tumor growth was measured by bioluminescence. [Figure 5C] Figure 1 shows the anti-tumor activity of G1A8 in a human hepatocellular carcinoma xenograft mouse model. G1A8 inhibited tumor growth at the early stage of tumor development. Hep3B-luciferase stably expressing luciferase was subcutaneously (sc) injected into NOD SCID mice. Mice were divided into groups (n=6 / group) based on equivalent mean tumor bioluminescence intensity and administered 200 μg of G1A8 or isotype control antibody treatment. Tumor growth was measured by bioluminescence. [Figure 5D] Figure 1 shows the anti-tumor activity of G1A8 in a human hepatocellular carcinoma xenograft mouse model. G1A8 inhibited tumor growth at the end stage of tumor development. NSG mice bearing Hep3B (sc) tumors were divided into groups (n=6 / group) based on equivalent mean tumor volume and administered 200 μg of G1A8 or isotype control antibody treatment. Tumor growth was measured by calipers. The days when antibody treatment was administered are marked with arrows. [Figure 5E] Figure 1 shows anti-tumor activity of G1A8 in a human hepatocellular carcinoma xenograft mouse model. G1A8 inhibited tumor growth at the end stage of tumor development. NSG mice bearing Hep3B(sc) tumors were divided into groups (n=6 / group) based on equivalent mean tumor volume and administered 200 μg of G1A8 or isotype control antibody treatment. Survival was analyzed by Kaplan-Meier survival analysis. Days when antibody treatment was administered are marked with arrows. [Figure 6A]Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Schematic diagram illustrating the schedule of G1A8 administration and sample collection from animals. Four cynomolgus monkeys were divided into two groups receiving intravenous (iv) administration of control saline (grey) or G1A8-hIgG1 (black). Each group consisted of one male (triangle) and one female (circle) monkey. [Figure 6B] 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. [Figure 6C] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Blood biochemistry of cynomolgus monkeys. Serum TBA of cynomolgus monkeys was measured at various time points. The days of intravenous administration of G1A8 or control saline are marked with arrows. The dashed and dotted lines indicate the normal reference intervals of biochemical parameters in male and female cynomolgus monkeys, respectively. [Figure 6D] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Blood biochemistry of cynomolgus monkeys. Serum TBIL of cynomolgus monkeys was measured at various time points. The days of intravenous administration of G1A8 or control saline are marked with arrows. The dashed and dotted lines indicate the normal reference intervals of biochemical parameters in male and female cynomolgus monkeys, respectively. [Figure 6E] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Blood biochemistry of cynomolgus monkeys. Serum ALT of cynomolgus monkeys was measured at various time points. The days of intravenous administration of G1A8 or control saline are marked with arrows. The dashed and dotted lines indicate the normal reference intervals of biochemical parameters in male and female cynomolgus monkeys, respectively. [Figure 6F] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Blood biochemistry of cynomolgus monkeys. Serum AST of cynomolgus monkeys was measured at various time points. The days of intravenous administration of G1A8 or control saline are marked with arrows. The dashed and dotted lines indicate the normal reference intervals of biochemical parameters in male and female cynomolgus monkeys, respectively. [Figure 6G]Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Gene expression related to bile acid metabolism is shown. Cynomolgus monkey tissue samples were taken at the end of the study. Expression of selected genes in liver was analyzed by qPCR. [Figure 6H] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Gene expression related to bile acid metabolism is shown. Cynomolgus monkey tissue samples were taken at the end of the study. Expression of selected genes in the ileum was analyzed by qPCR. [Figure 6I] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Gene expression related to bile acid metabolism is shown. Tissue samples from cynomolgus monkeys were taken at the end of the study. Expression of selected genes in kidneys was analyzed by qPCR. [Figure 6J] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Pharmacokinetic profile of G1A8. G1A8 was measured after administration of 10 mg / kg G1A8 on day 1. [Figure 6K] Figure 1 shows the safety evaluation of G1A8 in cynomolgus monkeys. Pharmacokinetic profile of G1A8. G1A8 was measured after administration of 30 mg / kg G1A8 on day 16. [Figure 6L] Figure 1 shows safety evaluation of G1A8 in cynomolgus monkeys. Kinetic analysis of binding of G1A8 to cynomolgus monkey FGF19. Cynomolgus monkey FGF19 was serially diluted 2-fold from 100 nM. [Figure 7A] FIG. 1 shows a structural analysis of the FGF19-G1A8 complex. A ribbon representation of FGF-G1A8 in orthogonal view is shown. FGF19 is shown in black with the variable heavy (VH) and variable light (VL) chains of G1A8 in grey. The N- and C-termini of FGF19 are labelled. The constant region of the G1A8 Fab has been removed for clarity. [Figure 7B] 1 shows a structural analysis of the FGF19-G1A8 complex. A surface view of the FGF19-G1A8 complex is shown. The structure reveals perfect shape complementarity at first glance. [Figure 7C]Figure 1 shows a structural analysis of the FGF19-G1A8 complex, a detailed view of the G1A8-FGF19 interface, dashed lines represent hydrogen bonds. [Figure 7D] 1 shows the structural analysis of the FGF19-G1A8 complex. The binding activity of G1A8 at 50 nM to wild-type FGF19 and FGF19 alanine mutants was analyzed using SPR. [Figure 8A] Figure 1 shows the activity of HS29. Binding analysis between anti-FGF19 antibody G1A8 and HS29 to FGF19 alanine mutants was performed using SPR. FGF19 alanine mutants were tested at 50 nM. [Figure 8B] Figure 1 shows the activity of HS29. Kinetic analysis of the binding of HS29 to FGF19. FGF19 was serially diluted 2-fold from 100 nM. [Figure 8C] Figure 1 shows the activity of HS29. Inhibition of FGF19-induced cell proliferation by HS29. Hep3B were cultured with 20 mg / ml FGF19 and 100 nM of anti-FGF19 antibody or isotype control antibody. [Figure 9A] Figure 1 shows the evaluation of anti-tumor activity of HS29 in a patient-derived xenograft (PDX) model. BALB / c nude mice bearing FGF19-expressing PDX LI6677 were divided into groups (n=5 / group) with equivalent mean tumor volume and administered 10 mg / kg HS29 intraperitoneally or no treatment as control. [Figure 9B] Figure 1 shows the evaluation of anti-tumor activity of HS29 in a patient-derived xenograft (PDX) model. BALB / c nude mice bearing non-FGF19-expressing PDX LI6646 were divided into groups (n=5 / group) with equivalent mean tumor volume and administered 10 mg / kg HS29 intraperitoneally or no treatment as control. [Figure 9C]Figure 1 shows the evaluation of anti-tumor activity of HS29 in patient-derived xenograft (PDX) models. FGF19 mRNA expression in PDX models is shown. Tumor samples were taken at the end of the study and FGF19 expression was analyzed by qPCR. Each data point represents one mouse tumor sample, and the level of FGF19 mRNA expression in Hep3B cell line was used as reference. The dashed line indicates the reliable detection limit of the assay. [Figure 9D] Figure 1 shows the evaluation of anti-tumor activity of HS29 in patient-derived xenograft (PDX) models. The body weight of PDX-bearing mice was measured at the time of study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] definition Unless specifically defined elsewhere in this specification, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] As used in this specification, including the appended claims, the singular forms of words such as "a," "an," and "the" include the corresponding plural references unless the context clearly dictates otherwise.

[0041] The term "or" is used to mean, or is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0042] In the context of this disclosure, unless otherwise indicated, the expression "comprise" and its variations, such as "comprises" and "comprising", imply the inclusion of the specified elements, e.g., amino acid sequences, nucleotide sequences, properties, steps, or groups thereof, and do not imply the exclusion of any other elements, e.g., amino acid sequences, nucleotide sequences, properties, and steps. As used herein, the term "comprise" or any variation thereof can be replaced with the terms "contain", "include" or sometimes "have" or equivalent variations thereof. In certain embodiments, the expression "comprise" also includes the scenario of "consisting of".

[0043] As used herein, the term "FGF19" refers to the ileum-derived hormone fibroblast growth factor 19, which is a ligand with high affinity to FGFR4 (fibroblast growth factor receptor 4). As mentioned above, FGF19 plays an important role in the regulation of bile acid synthesis, among others, by inhibiting the transcription of CYP7A1 in the liver through the FGFR4 / Klotho-β receptor complex. In the context of this application, unless otherwise indicated, FGF19 refers to human FGF19. The orthologue of human FGF19 in mice is FGF15. The amino acid sequence of FGF19, such as human FGF19, and the nucleotide sequence encoding it are known in the art. FGF has a homologous core region formed by 12 antiparallel β-strands flanked by different N- and C-termini (A. Beenken and M. Mohammadi, Nature reviews. Drug discovery 8, 235-253 (2009)). Primary sequence variations within the N- and C-termini of various FGFs account for their distinct biological activities (N. Itoh and DM Ornitz, J Biochem 149, 121-130 (2011); R. Goetz and M. Mohammadi, Nat Rev Mol Cell Biol 14, 166-180 (2013)).

[0044] Antibodies and antigen-binding fragments thereof Unless otherwise specified, the term "antibody" as used herein includes antibodies as well as antibody fragments in the broadest sense, so long as it recognizes and binds to human FGF19. In particular, the antibody of the present application binds to the N-terminus of human FGF19, specifically, amino acid residues 38-45 of FGF19 having the amino acid sequence of SEQ ID NO: 1. The antibody of the present application generally refers to a monospecific antibody. However, the present application also contemplates heterospecific (heterospecific) or multispecific antibodies. An antibody binds to a specific antigenic determinant or epitope by a specific binding site. An "antibody fragment" refers to a portion of a full-length antibody, and typically includes a binding or variable region for an antigen. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0045] The most common basic structure of antibodies is a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one small chain (about 25 kDa) designated "light" and one large chain (about 50-70 kDa) designated "heavy". The amino terminus of each chain contains a variable domain of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The heavy chains Carboxy The terminal portion may be defined as a constant region involved in effector functions. Typically, the light chains of human antibodies are classified as kappa and lambda light chains. Furthermore, the heavy chains of human antibodies are typically classified as α, δ, ε, γ, or μ, which define the antibody isotype as IgA, IgD, IgE, IgG, and IgM, respectively, and its subclasses, such as IgG1, IgG2, IgG3, and IgG4. Each light / heavy chain variable region / domain (VL / VH) pair forms an antibody binding site. Thus, a complete antibody usually has two binding sites.

[0046] The term "hypervariable domain" refers to the amino acid residues of an antibody that are involved in antigen binding. The hypervariable domain comprises amino acid residues from the "complementarity determining regions (CDRs)" (i.e., LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2 and HCDR3 in the heavy chain variable domain). Typically, both heavy and light chain variable domains comprise three hypervariable domains, i.e., CDRs, located between relatively conserved "framework regions" or "FRs". The CDRs are usually aligned by the framework regions, allowing binding to a specific epitope. Usually, from N-terminus to C-terminus, both light and heavy chain variable domains comprise FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (FR3), CDR-3 (CDR3), and FR-4 (FR4). The Kabat numbering system is used for amino acid residues of antibodies unless otherwise specified (Kabat et al., Sequence of proteins of immunological interest, 5th ed., Public Health Service, National Institute of Health, Bethesda, MD (1991)).

[0047] Unless otherwise specified, the terms "antibody fragment", "target-binding fragment" and "antigen-binding fragment" are interchangeable in the context of this application and refer to antibody fragments that retain the ability to specifically bind to the antigen bound by the full-length antibody (FGF19, or in particular the N-terminus of FGF19), e.g., antibody fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., single-chain Fv (ScFv); nanobodies; and multispecific antibodies formed from antibody fragments.

[0048] By "specific binding" or "specifically binds" is meant that an antibody exhibits preferential binding to a particular target compared to other proteins, although this specificity does not require absolute binding specificity. An antibody is considered "specific" for its intended target if its binding determines the presence of the target protein in a sample without producing undesired results, e.g., false positives. The antibodies or antigen-binding fragments thereof of the present invention will bind to the target protein with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold stronger than its affinity to non-target proteins. Alternatively or additionally, the antibodies or antigen-binding fragments thereof of the present invention will bind to the target protein with an affinity of at least 1×10 -8 Lower than M, 1×10 -9 M(1nM) less than 1×10 -10 Lower than M, 1×10 -11 Lower than M or 1×10 -12 The antibody of the present application will have a binding affinity represented by a KD value lower than M (1 pM). An antibody of the present application is said to specifically bind to a given amino acid sequence, e.g., the amino acid sequence of mature human FGF19, or a polypeptide containing the N-terminal residues 38-45 of human FGF19, if it binds to a given amino acid sequence, e.g., the amino acid sequence of mature human FGF19, or a polypeptide containing the N-terminal residues 38-45 of human FGF19, but does not bind to a protein lacking that sequence.

[0049] As used herein, the term "human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse carbohydrate chains if produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0050] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, meaning that the antibody molecules within the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in small amounts. "Monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by a particular method. Monoclonal antibodies (mAbs) can be obtained by conventional methods known in the art. See, e.g., Kohler G et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel FM et al., CURRENT PROTPCOLS IN MOLECULAR BIOLOGY 1992; Harlow E et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan JE et al., CURRENT PROTPCOLS IN IMMUNOLOGY 1993.

[0051] In one embodiment, the antibody of the present invention that specifically binds to human FGF19 also shows cross-reactivity with the cynomolgus monkey orthologue of human FGF19.As used herein, the term "cross-reactivity" refers to the ability of an antibody to react with homologues or orthologues from other species.The cross-reactivity of an antibody can be determined by any method known in the art.For example, it can be determined by measuring the binding affinity by surface plasmon resonance (e.g., BIACORE) or similar technology (e.g., KinExa or OCTET).

[0052] In some embodiments, the antibody or antigen-binding fragment of the present application does not affect or only slightly affects FGF19-suppressed CYP7A1 expression. In other words, CYP7A1 expression is significantly inhibited by FGF19 upon administration of the antibody or antigen-binding fragment. More specifically, the change, or specifically increase, in CYP7A1 expression level is less than 5%, 10%, 15% or 20% after administration of the antibody or antigen-binding fragment in the presence of FGF19.

[0053] In some embodiments, the antibody or antigen-binding fragment of the present application does not impair or only slightly impairs FGF19-maintained bile acid biosynthesis. Stable bile acid homeostasis can be determined by monitoring the transcription or expression level of one or more genes involved in bile acid metabolism in relevant tissues. For example, the gene can be a gene encoding a bile acid transporter protein. The specific genes measured can be ASBT, IBABP, CYP7A1, NTCP, OATP2, BSEP, OSTα, OSTβ, MRP2, MRP3, and / or MRP4 in the liver, kidney, and / or ileum. The change in the expression level of one or more of ASBT, IBABP, CYP7A1, NTCP, OATP2, BSEP, OSTα, OSTβ, MRP2, MRP3, and / or MRP4 in the liver, kidney, and / or ileum is limited to, for example, 5%, 10%, 15%, or 20% or less after administration of the antibody or antigen-binding fragment in the presence of FGF19.

[0054] The antibody of the present application may be subjected to a purification process to remove undesired materials and obtain a purified antibody. Conventional methods for purifying antibodies include, but are not limited to, column chromatography methods well known in the art.

[0055] The antibody or antigen-binding fragment of the present invention may be an isolated antibody. The term "isolated" means that the antibody or antigen-binding fragment is at least partially free of other biological or non-biological materials from the cells, cell cultures, growth media, expression systems that produce them. Such materials include nucleic acids, proteins, lipids, carbohydrates, buffers, salts, or other materials such as cell debris and growth media.

[0056] The present application also contemplates an antibody or antigen-binding fragment thereof that contains one or more conservative substitutions, so long as the antibody or antigen-binding fragment binds to FGF19 and has at least one property of the antibody described herein. The term "conservative substitution" of amino acids is well known in the art and refers to the replacement of an amino acid residue with another amino acid residue that has a similar side chain in structure or function. For example, an exemplary list of conservative substitutions is provided in the following table. TIFF0007643727000001.tif180126

[0057] The present application also provides isolated nucleic acid sequences comprising nucleotide sequences encoding the antibodies or fragments thereof of the present application. "Isolated nucleic acid" or "isolated polynucleotide" refers to DNA or RNA in which the isolated polynucleotide is removed from all or a portion of a polynucleotide found in nature, or in which the isolated polynucleotide is linked to a polynucleotide with which it is not naturally linked. An isolated nucleic acid molecule "comprising" a particular nucleotide sequence may include, in addition to the specified sequence, operably linked regulatory sequences that control the coding region of the recited nucleic acid sequence. Due to codon degeneracy, one of skill in the art can understand that a particular amino acid sequence may be encoded by different nucleotide sequences.

[0058] therapeutic use The present disclosure provides a method for preventing, treating or preventing recurrence of disease or disorder caused by or associated with abnormal FGF19-FGFR4 signal transduction, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment of the first aspect of the present application or the pharmaceutical composition of the second aspect of the present application.In a specific embodiment, the disease or disorder is cancer caused by or associated with abnormal FGF19-FGFR4 signal transduction.In a more specific embodiment, the cancer is hepatocellular carcinoma (HCC).

[0059] As used herein, the terms "administration," "administering," "treating," and "treatment," when applied to a subject, e.g., an animal, including a human, or to a cell, tissue, organ, or biological fluid, refer to the contact of an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the subject, cell, tissue, organ, or biological fluid. Treatment of a cell includes contact of a reagent with a cell, as well as contact of a reagent with a fluid, where the fluid is in contact with the cell. The terms "administration" and "treatment" also include in vitro and ex vivo treatments, e.g., treatment of a cell with a reagent, diagnostic, binding compound, or another cell.

[0060] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject to treat a disease or at least one clinical symptom of a disease or disorder, is sufficient to effect such treatment for the disease, disorder or condition. A "therapeutically effective amount" may vary depending on the antibody, the disease, disorder, and / or symptoms of the disease or disorder, the severity of the disease, disorder, and / or symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case may be evident from scratch or may be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combined subjects for effective treatment of a disease, disorder, or condition.

[0061] In the context of the present application, a "subject" refers to an animal, preferably a mammal, such as a primate, preferably a higher primate, such as a human.

[0062] The term "cancer" or "tumor" as used herein refers to or describes a physiological condition in mammals that is typically characterized by upregulated cell proliferation. In a preferred embodiment, the cancer is associated with abnormal FGF19-FGFR4 signaling. The antibody or antigen-binding fragment of the present application can be considered that the anti-FGF19 antibody of the present application can be used to treat, for example, breast cancer, prostate cancer, colon cancer, lung cancer, and gastric cancer. EXAMPLES

[0063] material and method The following materials and methods are used in the examples.

[0064] cell line Human hepatocellular carcinoma cell line Hep3B was a gift from Dr. Fengming Lu's laboratory at Peking University Health Science Center. Hep3 cells were cultured in DMEM medium (Gibco, DMEM, C11965500BT) supplemented with 10% fetal bovine serum. Cells were cultured in a humidified incubator at 37°C with a 5% CO2 atmosphere.

[0065] Hep3-Luc23 stable cell lines expressing a firefly luciferase reporter gene were generated by lentiviral transduction and selected with puromycin.

[0066] The FreeStyle 293-F cell line was cultured in FreeStyle™ 293 Expression Medium (both cell line and medium from Thermo Fisher Scientific) at 37° C. in an 8% CO2 atmosphere on a humidified orbital shaker platform.

[0067] Protein expression and purification Human FGF19 (SEQ ID NO:1), and N-terminal deletion mutant FGF19 ΔNT For FGF19 (having an amino acid sequence corresponding to Arg43 to Lys216 of SEQ ID NO:1) and its variants, including FGF19 alanine substitution mutants, the coding sequences of the proteins were cloned into mammalian cell expression vectors with C-terminal His-Avi tags and these were transiently transfected into FreeStyle293-F cells (Fisher Scientific) either alone or co-transfected at a 1:1 ratio with an expression vector encoding the protein sequence of E. coli BirA biotin-protein ligase. Three to five days after transfection, cell supernatants were harvested and the proteins or biotinylated proteins were purified using Ni-NTA affinity chromatography (Qiagen).

[0068] For full-length human IgG1 antibody production, the coding sequences of VH and VL were cloned into human IgG1 heavy chain (HC) mammalian cell expression vector and light chain (LC) mammalian cell expression vector, respectively. FreeStyle293-F cells were transiently co-transfected with the two expression vector plasmids (HC and LC) at a 1:1 ratio. Three to five days after transfection, cell culture supernatants were harvested for purification of human IgG antibodies using affinity chromatography with protein A beads (GE Healthcare Life Sciences).

[0069] Screening of antibody libraries for FGF19 FGF19 N-terminal peptide (SEQ ID NO: 24), containing FGF19 N-terminal residues Arg23 to Ile42 followed by five amino acids with biotin modification at the C-terminus (SGSGK), was synthesized with a purity of over 95% by Scilight-peptide (Beijing, China). The N-terminal FGF19 peptide or biotinylated full-length FGF19 protein was captured on streptavidin-conjugated magnetic M-280 Dynabeads® (Thermo Fisher Scientific) as target proteins, and then analyzed using a human non-immune antibody phage display library (size: 1.0 × 10) constructed from peripheral blood mononuclear cells of 93 healthy donors. 10 ) prepared from 5 × 10 12 The phage-scFv (single-chain fragment of variable domain) particles were incubated. Two rounds of selection were performed. For the second round of selection, less target protein was used and extensive washing steps were applied. Phages showing specific binding to the target protein were eluted using a conventional basic triethanolamine solution.

[0070] Single clones were then picked and rescued to generate phage-scFvs in bacterial culture supernatants; these were shown to bind to full-length FGF19 in an enzyme-linked immunosorbent assay (ELISA) and to an N-terminally deleted version of FGF19 (FGF19 ΔNT ) to screen for specific binding to the N-terminus of FGF19. ΔNT Clones that bound to FGF19 with higher affinity than were selected as candidates with specific binding to the N-terminus of FGF19, and the heavy chain variable region (VH) and light chain variable region (VL) genes of these clones were sequenced. The corresponding amino acid sequences of the selected clones were aligned to eliminate overlapping clones, thereby identifying unique antibodies for further characterization.

[0071] 31A3 antibody sub-library construction and selection for affinity improvement To improve the affinity of antibody 31A3 using CDR3-targeted randomization, a sub-library with random mutagenesis for HCDR3 and LCDR3 of 31A3 was constructed using NNK degenerate codons. The size of the constructed antibody sub-library was 1.23×10 8 Antibody sub-library selection and screening were done similarly to that described above for screening of the antibody library for FGF19. Competitive elution with 31A3 was used to obtain high affinity hits from magnetic beads. Single clones were then picked, recovered to generate phage-scFvs in bacterial culture supernatants and screened for binding to FGF19. Only hits with higher binding affinity than 31A3 were retained.

[0072] ELISA assay NeutrAvidin (Sigma-Aldrich) at 2 μg / mL in phosphate buffered saline (PBS) was coated onto 96-well U-bottom plates (Nunc, MaxiSorp™) at 100 μL per well and incubated overnight at 4°C. Then, biotinylated FGF19 or FGF variants at 2 μg / mL at 100 μL per well were captured onto the plates by incubation at 30°C for 0.5-1 h. FGF19 or FGF19 variants were also coated directly onto 96-well U-bottom plates at 100 μL per well and incubated overnight at 4°C. For ELISA based on hIgG1 antibodies, 100 μL per well of 3-fold serially diluted antibodies in PBS containing 2% nonfat milk were added. For competitive ELISA, test antibodies were diluted 3-fold and mixed with competitor FGFR4-hFc. Bound antibodies were then detected using HRP-conjugated goat anti-hFc antibody (Thermo Fisher Scientific).

[0073] Binding kinetics analysis by surface plasmon resonance (SPR) assay Kinetic analysis of the present anti-FGF19 human IgG1 antibody was performed at 25°C using Biacore T200 (GE Healthcare Life Sciences). Anti-hFc antibody (GE Healthcare Life Sciences) was immobilized on a CM5 sensor chip using an amine-coupling kit (GE Healthcare Life Sciences). The present anti-FGF19 human IgG1 antibody was captured on the sensor chip as a ligand, and then the analyte FGF19 (in two-fold serial dilutions from 100 nM) or FGF19 alanine mutant (50 nM) was injected into each flow cell. An injection of buffer (HBS with 0.5% Tween 20 and 0.3 mM EDTA) served as a negative control. 3 M MgCl2 was used as a regeneration buffer between each binding and dissociation cycle. The binding rate (ka), dissociation rate (kd), and affinity constant (KD) were calculated using the Biacore T200 evaluation software.

[0074] Cell proliferation assay Human HCC cell line Hep3 was treated with various concentrations of FGF19 or FGF19 mutants in DMEM supplemented with 1% fetal bovine serum to induce excessive cell proliferation. To evaluate the inhibitory activity of anti-FGF19 antibodies against FGF19-induced cell proliferation, 15 μg / ml (100 nM) hIgG1 antibody was added to the cell culture medium. After 72 hours, cell proliferation was measured using Cell Counting Kit-8 (Dojindo Molecular Technologies) according to the manufacturer's product instructions.

[0075] Expression of CYP7A1 in the liver Five- to six-week-old C57BL / 6 mice were fasted overnight before intraperitoneal (ip) injection of 2 μg of (1) FGF19, (2) FGF19 mutants, or (3) FGF19 plus 60 μg anti-FGF19 antibody. Mice were euthanized 3 h after ip injection, and livers were harvested. For subsequent analysis of CYP7A1 mRNA expression levels, total liver RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), and complementary DNA (cDNA) was generated by reverse transcription using the Prime Script RT-PCR Kit (Takara). CYP7A1 mRNA expression levels (relative to GAPDH) were assessed by qPCR using an ABI Fast 7500 real-time instrument (Applied Biosystems).

[0076] Animal testing NOD SCID and NSG mice were used to evaluate the anti-tumor activity of anti-FGF19 antibodies. 6- to 8-week-old mice were inoculated with 5 × 10 6 Hep3 cells (100 μl per mouse) were injected subcutaneously. Mice were divided into three groups (n=6 per group) based on equivalent mean tumor bioluminescence intensity or tumor volume and were injected intraperitoneally (ip) with 200 μg of G1A8 or control IgG twice a week. In vivo, tumor bioluminescence intensity of tumor-bearing mice was measured using an IVIS Lumina III In Vivo Imaging System (PerkinElmer) after ip injection of 15 mg / kg D-luciferin (PerkinElmer). Tumor volumes were measured using electronic calipers and calculated using the formula 3.14×L×W2 / 6, where L and W are the maximum and minimum measured diameters, respectively. All animal protocols were carried out according to the Chinese National Guidelines for Housing and Care of Laboratory Animals and were approved by the IACUC at NIBS and were performed in accordance with institutional regulations. Studies to evaluate the anti-tumor efficacy of antibodies in patient-derived xenografts in BALB / c nude mice were performed under an approved IACUC protocol at Crown Bioscience Inc.

[0077] Safety evaluation of the present antibodies, specifically G1A8, was performed at JOINN Research Institute (Beijing). Healthy, experimentally naive, 3-4 year old cynomolgus monkeys weighing ~3 kg were intravenously injected with G1A8 antibody at 30 mg / kg body weight and saline at 30 mg / kg body weight on day 16 as a control. Clinical observations, body weight, temperature, blood chemistry, and anatomic pathology were evaluated. Blood samples were collected at various time points (days 1, 8, 15, 23, and 30) for pharmacokinetic analysis of G1A8. Liver, ileum, and kidney samples were collected at the end of the study for RNA analysis of genes related to bile acid metabolism.

[0078] Crystallization and structure determination of the FGF19-G1A8 complex FGF19 and G1A8-Fab were expressed separately in FreeStyle 293-F cells and purified independently by Ni-NTA flow cytometry (Qiagen). To obtain the FGF19-G1A8 complex, FGF19 and G1A8-Fab were mixed in a 1:1 molar ratio, incubated overnight at 4°C, and further purified on a Superdex S200 10 / 300 column (GE Healthcare) in a buffer containing 10 mM Tris-HCl, pH 8.0, and 500 mM NaCl. The resulting FGF19-G1A8 complex was then concentrated to 18 mg / mL and crystallized at 20°C using the hanging drop diffusion method described above by mixing 1 μL of protein with 1 μL of a reservoir solution containing 0.2 M lithium sulfate monohydrate, 0.1 M Bis-Tris pH 6.9, and 26% w / v polyethylene glycol 3350. Quadrilateral shaped crystals appeared in 7 days and were flash frozen in liquid nitrogen.

[0079] X-ray diffraction data were collected at the Shanghai Synchrotron Radiatin Facility (SSRF) beamline BL17U. Data were processed with HKL2000 and XDS. The crystals belong to the P212121 space group and contain two copies of the FGF19-G1A8 complex per asymmetric unit. The structure was determined by molecular replacement using Phaser in Phenix with the following structures as search models: FGF19 (PDB ID: 2P23) and the anti-steroid Fab5F2 structure (PDB ID: 3KDM). The model was iteratively built in Coot and refined in PHENIX.

[0080] statistical analysis All analyses were performed using GraphPad Prism version 6.00. Ordinary one-way ANOVA or unpaired Student's t-test was used for comparison between groups. Two-way ANOVA and Tukey's multiple comparison test were used for evaluation of continuous variables. Kaplan-Meier survival analysis and log-rank test were used for survival analysis.

[0081] Example 1. Identification of binding targets at the N-terminus of FGF19 We performed a variety of assays to investigate whether functional differences exist between full FGF19 and its N-terminally deleted versions.

[0082] Compared to full-length FGF19, FGF19 ΔNT Mutant (FGF19 ΔNT (having an amino acid sequence corresponding to residues Arg43 to Lys216 of SEQ ID NO:1) had significantly weaker binding affinity for FGFR4 and exerted a significantly reduced activity for inducing tumor cell proliferation in in vitro assays (Figures 1A and 1B).

[0083] Furthermore, full-length FGF19 and the above-mentioned N-terminal deletion mutant FGF19 ΔNTWe examined the bile acid regulatory function of FGF19. A mouse model was used in this study because exogenous FGF19 exerts its bile acid regulatory function by binding to the mouse receptor FGFR4, which shares 90% amino acid identity with human FGFR4, and suppresses mouse hepatic transcription of CYP7A1 (M. Zhou et al., 2014, supra; R. Goetz et al., Molecular and cellular biology 27, 3417-3428 (2007)). ΔNT Hepatic CYP7A1 gene expression levels were evaluated in these mice after intraperitoneal injection of FGF19. Statistically significant differences were observed in the bile acid regulatory function by suppressing hepatic CYP7A1 gene expression levels, indicating that full-length and N-terminal deletion mutant FGF19 ΔNT In light of this finding, it is reasonable to expect that therapeutic agents targeting the N-terminus of FGF19 could potentially inhibit its oncogenic activity without adversely affecting its bile acid-regulated physiological functions.

[0084] Example 2. Generation of antibodies that specifically target the N-terminus of human FGF19 The following two different selection strategies using SEQ ID NO:1 or SEQ ID NO:24 as binding targets were designed to identify antibodies that specifically target the N-terminus of FGF19 (Figure 2). Antibody selection was performed by using either the FGF19 N-terminal peptide (SEQ ID NO:24) or full-length FGF19 (SEQ ID NO:1) as the binding target, and a large non-immune human antibody phage display library (D. Li et al., Elife 6, 2017, supra) was selected. Antibodies that showed binding affinity to both the N-terminal fragment of FGF19 and full-length FGF19 were selected for the next screening step.

[0085] In the next screening step using an ELISA assay, full-length FGF19 and N-terminal deletion mutant FGF19 were ΔNT Both were used as binding targets, and they were able to bind to full-length FGF19 but not to FGF19. ΔNTAmong the many identified antibodies, an antibody designated 31A3 that showed relatively high binding affinity and specifically recognized the N-terminus of FGF19 was selected for further analysis (Figures 2 and 3).

[0086] Example 3. In vitro evaluation of the inhibitory effect of 31A3 on the tumorigenic activity of FGF19 Anti-proliferation assay was performed to evaluate the ability of 31A3 to inhibit the tumorigenic activity of FGF19 in vitro. To evaluate whether 31A3 can inhibit HCC cell proliferation specifically induced by FGF19, HCC cell line Hep3 was treated with 31A3 in the presence of exogenous FGF19. As shown in Figure 4A, 31A3 inhibited the FGF19-induced proliferation of Hep3 cells.

[0087] Example 4. Creation of 31A3 derived mutants with improved binding affinity To improve the affinity of 31A3 for the N-terminus of FGF19, site-directed alanine scanning mutagenesis was performed within the HCDR3 and LCDR3 regions of 31A3. This indicates that four amino acid residues, namely V96 and W97 in HCDR3 and Y91 and T95 in LCDR3, are potential candidates for improving binding affinity by substitution with other amino acids. We then constructed an antibody phage display library containing 31A3-derived antibodies with random mutations at the above four positions in the HCDR3 and LCDR3 regions. During this 31A3-derived sublibrary selection, 31A3-hIgG1 protein was used as a competitor to eliminate binders with higher binding affinity for FGF19. After a stringent biopanning selection step, a small panel of antibodies with improved affinity was obtained. One of the resulting antibodies, G1A8, which differs from 31A3 by three amino acids, showed a 27-fold increase in affinity, a 60-fold slower off-rate, and stronger anti-proliferative activity than the parent antibody 31A3 (Figures 2, 3A, 3B, and 4A).

[0088] Another mutant antibody, HS29, was generated based on sequence analysis of a small panel of antibodies obtained from the above panning. HS29 has a similar binding epitope, anti-proliferative activity, and binding affinity as G1A8 (Figure 8).

[0089] Example 5. In vitro and in vivo efficacy In in vitro testing of activity, G1A8 had an IC 50 G1A8 also exerted a strong anti-proliferative effect in exogenous FGF19-treated Hep3 cells (Fig. 4A).

[0090] Its anti-tumor activity in vivo was then evaluated using a xenograft mouse model (Figure 5). We first established a Hep3B-Luc23 cell line stably expressing luciferase, which allowed quantification of tumor growth in vivo using bioluminescence imaging. Mice were subcutaneously injected with these Hep3B-Luc23 cells in the right flank, then divided into three groups with equivalent mean tumor bioluminescence intensity, and then administered intraperitoneal injections of antibody treatment (200 μg of G1A8 or isotype control) twice a week for 4 weeks. Monitoring tumor growth over time by measuring tumor volume and relative tumor cell bioluminescence intensity showed that G1A8 significantly suppressed tumor growth compared to the isotype control antibody (Figures 5A-C).

[0091] Anti-tumor efficacy was also evaluated against xenograft tumors formed from transplanted wild-type Hep3B cells. Antibody treatment was administered to tumors with a tumor volume of approximately 100 mm 3The treatment started when the tumor reached a size of 100 μg / ml. Mice bearing Hep3B were divided into three groups with equivalent mean tumor volume and received intraperitoneal injections of antibody (200 μg of G1A8 or isotype control antibody) twice a week for 3 weeks. Similar to the results above for Hep3B-Luc23 xenografts, G1A8 significantly inhibited Hep3B tumor progression (Figure 5D). Furthermore, treatment with G1A8 significantly extended the survival of mice compared to the isotype control group (Figure 5E).

[0092] HS29 showed strong anti-proliferative activity similar to G1A8 (Figure 8C). The anti-tumor activity of HS29 was tested in a patient-derived xenograft (PDX) tumor model (Figure 9A). Treatment with HS29 reduced tumor size by 10% with a mean tumor volume of approximately 150 mm. 3 The tumor growth was started when the tumor tissue size reached 10 mg / kg. Mice administered 10 mg / kg HS29 by intraperitoneal injection showed arrested tumor growth compared to control mice. Notably, the control mice showed significant weight loss while the tumors continued to grow, and two of them died due to poor physiological conditions due to tumor progression; on the other hand, the HS29-treated mice maintained a constant weight and remained healthy (Figure 9D). These data confirmed the anti-tumor activity of HS29 both in vitro and in vivo.

[0093] Example 6. Safety evaluation in mice and cynomolgus monkeys We found that the antibodies of the present application, especially G1A8, do not show cross-reactivity with mouse FGF15 (the orthologue of human FGF19), which shares only 49% amino acid sequence identity with FGF19 (TJ Wright et al., Dev Biol 269, 264-275 (2004)). Given this view, the above-mentioned mouse tumor xenograft model may not be suitable for evaluating the safety profile of G1A8 treatment. Nevertheless, recalling that human FGF19 exerts its bile acid regulatory function in mice via mouse FGFR4 to suppress hepatic CYP7A1 transcription (M. Zhou et al., 2014, supra; R. Goetz et al., 2007, supra) (Figure 1C), this mouse model was used to evaluate whether G1A8 affects hepatic CYP7A1 transcription. Surprisingly, G1A8 did not affect the FGF19-induced repression of hepatic CYP7A1 transcription (Fig. 4C ), indicating that G1A8 probably does not interfere with the bile acid regulatory function of FGF19.

[0094] Previous studies (Non-Patent Document 23) showed that administration of humanized anti-FGF19 antibody (1A6) in cynomolgus monkeys caused disruption of bile acid metabolism by disturbing FGF19 function and increasing CYP7A1 gene expression, along with clinical signs such as weight loss, decreased food intake, severe diarrhea, and ultimately resulted in unscheduled euthanasia of all animals in the 10 and 30 mg / kg treatment groups. The amino acid sequences of cynomolgus monkey FGF19 and human FGF19 were nearly identical (sharing 98% amino acid identity). The inventors also tested the cross-reactivity of the present antibody and demonstrated that G1A8 binds to both cynomolgus monkey FGF19 and human FGF19 with similar binding affinity (Figure 3A and Figure 6L). Therefore, cynomolgus monkeys were determined to be a good model for evaluating the safety profile of G1A8, especially whether it impairs the bile acid regulatory function of FGF19.

[0095] Four treatment-naive cynomolgus monkeys were randomly divided into two groups and intravenously administered control saline or 10 mg / kg G1A8 on day 1 and 30 mg / kg G1A8 on day 16. Each group contained one male and one female. Blood samples were collected throughout the study period (Figure 6A).

[0096] All monkeys completed treatment with two doses of G1A8, and none of them showed any clinical side effects reported for 1A6 (binding to the C-terminus of FGF19), such as weight loss, reduced food intake, liquid feces, or diarrhea (Figure 6B). A previous study of 1A6 treatment (single dose) in monkeys reported a significant increase in serum total bile acid (TBA) levels, alanine transaminase (ALT), and aspartate transaminase (AST) (Non-Patent Document 23). Monkeys in the G1A8 treatment group initially showed a slight increase in serum TBA concentrations on day 15 (first examination time point) after treatment with G1A8 (10 mg / kg), but no further increase was observed on day 16 after the second dose of a higher dose of 30 mg / kg, but rather a decrease was observed (Figure 6C). This finding suggests that the slight increase in serum TBA levels observed on day 15 is likely explained by normal physiological changes in the animals. Serum levels of total bilirubin (TBIL), alanine transaminase (ALT), and aspartate transaminase (AST) showed no significant differences in mice treated with either control saline or G1A8, suggesting that administration of G1A8 did not result in liver injury (Figures 6G-I).

[0097] We also collected monkey tissue samples from organs involved in bile acid recycling (liver, ileum, and kidney) to assess the expression levels of multiple genes known to affect bile acid metabolism (Figure 6D-6F). In the liver of animals treated with G1A8, no increase was observed in CYP7A1 gene expression, and the expression of other genes encoding bile acid transport proteins did not show a significant increase compared to control animals (Figure 6D). In the kidney and ileum, the expression of genes for bile acid transporters did not differ between the G1A8-treated and control groups (Figure 6E, Figure 6F), indicating that bile acid recycling and metabolism were maintained at normal physiological levels. Serum concentration-time profiles for the two different G1A8 doses revealed similar terminal half-lives for G1A8 of 174.29 and 188.38 hours in cynomolgus monkeys administered at 10 mg / kg and 30 mg / kg, respectively (Figure 6J and Figure 6K).

[0098] Taken together, these safety evaluation studies in cynomolgus monkeys demonstrated that administration of G1A8 was not associated with bile acid-related toxicity, thus suggesting that treatment with G1A8 in a therapeutic setting is unlikely to cause significant malabsorption of bile acids or other bile acid-related side effects.

[0099] Example 7. Structural analysis of the FGF19-G1A8 complex The structure of the Fab version of G1A8 in complex with FGF19 was determined at 2.6 A resolution by X-ray crystallography to understand the molecular basis of the interaction between the present antibody and FGF19. Antibody G1A8 was selected for this analysis.

[0100] The structure was solved by molecular replacement with an R of 0.216 / 0.278 with good alignment. work / R free The FGF19 and G1A8 structures were refined to the same extent (Table 1, see below). Residues 37–172 of FGF19, which show well-defined electron density in the structures, were used as a model. The FGF19 and G1A8 structures show seemingly perfect shape complementarity, with a total of 983 Å. 2The surface area covered by the variable heavy (VH) and variable light (VL) chains of G1A8 is nearly identical (495 Å 2 488Å 2 ). The key residues that comprise the epitope of G1A8 are located within the N-terminus of FGF19 and a fragment (SEQ ID NO:2) consisting of amino acid residues 38-45 of SEQ ID NO:1, above the cleft between VH and VL (Figures 7A and 7B). This finding is consistent with the specified characteristics of G1A8, which specifically targets the N-terminus of FGF19, as described above.

[0101] [Table 1]

[0102] Specifically, the eight residues from positions 38 to 45 of FGF19 engage extensively with all six complementarity determining region (CDR) loops from both VH and VL of G1A8 through a mixture of hydrophobic and polar contacts, as shown in Figure 7C and Table 2. The side chain of the N-terminal residue Trp38 of FGF19 is anchored in a hydrophobic pocket formed by Ala33, Ser52, Ser57, Tyr59, Gln102, and Leu104 of HCDR1-3, while Arg45 of FGF19 engages in salt-bridge interactions with Glu103 and Asp52 of HCDR3 and LCDR2, respectively (Figure 7C). The FGF19 / Trp38Ala mutant showed reduced binding affinity to G1A8, and mutation of Arg45 to Ala resulted in complete loss of binding (Figure 7D), confirming that these interactions are essential. Arg45 of FGF19 also makes an additional hydrogen-bonding interaction with Asn100 of HCDR3 (Figure 7C). G1A8's parent antibody 31A3 has a Val at this same position, and this difference is likely responsible for the lower affinity of 31A3 than G1A8 (Figures 2 and 3A). The G1A8-FGF19 complex structure also revealed that residues 168-172 of the C-terminus of FGF19 are in close proximity to the N-terminus at the interaction interface of FGF19 and G1A8. Residue Leu169 at the C-terminus along with the N-terminus of FGF19 forms hydrophobic interactions with Tyr51 and Pro57 of LCDR2. Hydrogen bonds between Tyr51 and the backbone carbonyl group of FGF19 / Pro167 further stabilize the C-terminal interaction of FGF19. Mutation of Leu169 to Ala results in a faster dissociation rate and therefore a decreased binding affinity for the antibody (Figure 7D).

[0103] [Table 2]

Claims

(a) HCDR1 having the amino acid sequence of SEQ ID NO:3; HCDR2 having the amino acid sequence of SEQ ID NO:4, HCDR3 having the amino acid sequence of SEQ ID NO: 19 A heavy chain variable domain (VH) comprising: LCDR1 having the amino acid sequence of SEQ ID NO:6, LCDR2 having the amino acid sequence of SEQ ID NO:7, LCDR3 having the amino acid sequence of SEQ ID NO: 14 A light chain variable domain (VL) comprising: (b) HCDR1 having the amino acid sequence of SEQ ID NO:3; HCDR2 having the amino acid sequence of SEQ ID NO:4, HCDR3 having the amino acid sequence of SEQ ID NO: 13 A heavy chain variable domain (VH) comprising: LCDR1 having the amino acid sequence of SEQ ID NO:6, LCDR2 having the amino acid sequence of SEQ ID NO:7, LCDR3 having the amino acid sequence of SEQ ID NO: 14 or a light chain variable domain (VL) comprising: (c) HCDR1 having the amino acid sequence of SEQ ID NO:3; HCDR2 having the amino acid sequence of SEQ ID NO:4, HCDR3 having the amino acid sequence of SEQ ID NO:5 A heavy chain variable domain (VH) comprising: LCDR1 having the amino acid sequence of SEQ ID NO:6, LCDR2 having the amino acid sequence of SEQ ID NO:7, LCDR3 having the amino acid sequence of SEQ ID NO:8 A light chain variable domain (VL) comprising: An antibody or an antigen-binding fragment thereof that binds to human fibroblast growth factor 19 (FGF19), comprising:

2. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, 15 or 20.

3. The antibody or antigen-binding fragment of claim 1, comprising a light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, 16 or 21.

4. (a) a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:9, and a light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10; (b) a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; or (c) a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20, and a light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

21. The antibody or antigen-binding fragment of claim 1 .

5. (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:9, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:10; (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 16; or (c) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:

21. The antibody or antigen-binding fragment of claim 1 .

6. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, 17 or 22.

7. The antibody or antigen-binding fragment of claim 1, comprising a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 12, 18 or 23.

8. (a) a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11, and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:12; (b) a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 18; or (c) a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:22, and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

23. The antibody or antigen-binding fragment of claim 1 .

9. (a) a heavy chain comprising the amino acid sequence of SEQ ID NO:11, and a light chain comprising the amino acid sequence of SEQ ID NO:12; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO: 18; or (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 22, and a light chain comprising the amino acid sequence of SEQ ID NO:

23. The antibody or antigen-binding fragment of claim 1 .

10. The antibody or antigen-binding fragment of any one of claims 1 to 9, wherein the antigen-binding fragment is a Fab, a Fab', an F(ab')2, an Fv, a single-chain Fv (ScFv) or a disulfide-stabilized Fv (dsFv).

11. The antibody or antigen-binding fragment thereof has the following characteristics: (i) a binding affinity to human FGF19 of 1×10 as measured by surface plasmon resonance -9 M~1×10 -12 K of M D (ii) cross-reacts with cynomolgus monkey FGF19; (iii) blocks binding of human FGF19 to human FGFR4 and / or human FGFR4-KLB complex; (iv) inhibits FGF19-induced cell proliferation; (v) in the presence of FGF19, there is an increase in CYP7A1 expression after administration of not more than 20%; and (vi) in the presence of FGF19, there is an increase in the expression level of one or more of ASBT, IBABP, CYP7A1, NTCP, OATP2, BSEP, OSTα, OSTβ, MRP2, MRP3 and / or MRP4 in the liver, kidney and / or ileum of not more than 20% after administration.

12. The antibody or antigen-binding fragment described in claim 1, which binds to an epitope including the following region of human FGF19 (sequence number 1): amino acids 38 to 45.

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment of any one of claims 1 to 12.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 12 and a pharma- ceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, for preventing or treating a disease or disorder caused by abnormal FGF19-FGFR4 signaling.

16. The pharmaceutical composition according to claim 14, for preventing the recurrence of a disease or disorder caused by abnormal FGF19-FGFR4 signaling.

17. The pharmaceutical composition according to claim 15 or 16, wherein the disease or disorder caused by abnormal FGF19-FGFR4 signaling is cancer.

18. The pharmaceutical composition according to claim 17, wherein the cancer is hepatocellular carcinoma (HCC).

Images