EA046377B1 - ANTIBODIES TO FGF19 - Google Patents

Description

Field of technology

Disclosed herein are antibodies and antigen-binding fragments thereof that bind to fibroblast growth factor 19 (FGF19), namely the majority of the residues constituting the antibody epitope at the N-terminus of FGF19. Also provided are compositions and uses of the antibody, as well as methods for treating a disease or disorder caused by or associated with abnormal FGF19-FGFR signaling, such as cancer, by administering anti-FGF19 antibodies.

State of the art

Fibroblast growth factors (FGFs) are a family of proteins that perform diverse functions during the development of organisms from nematodes to humans. The FGF/FGFR (fibroblast growth factor receptor) signaling pathway has been found to be particularly important in a number of normal developmental and physiological processes, and dysregulation of this pathway may play an important role in tumor development and progression (Turner N., Grose R., Nature Reviews Cancer, 2010, 10(2):116, https://www.nature.com/articles/nrc2780).

In humans, fibroblast growth factor 19 (FGF19) is a bile acid-inducible peptide growth factor that regulates bile acid metabolism by binding hepatocyte-expressed fibroblast growth factor receptor 4 (FGFR4) and β-klotho (KLD), resulting in repression of hepatic gene transcription , encoding cholesterol 7-αhydroxylase 1 (CYP7A1), an important enzyme in bile acid biosynthesis (M.H. Xie et al., Cytokine, 11, 729-735 (1999); J.A. Holt et al., Genes & development, 17, 1581-1591 (2003); H. Kurosu et al., The Journal of biological chemistry, 282, 26687-26695 (2007); and V. S. Lin et al., The Journal of biological chemistry, 282, 27277-27284 (2007)). In addition to its function in regulating fatty acid metabolism, clinical observations have demonstrated that both FGF19 and its cognate receptor FGFR4 are expressed at high levels in tumor tissue compared with adjacent non-tumor tissue (E.T. Sawey et al., Cancer Cell, 19, 347-358 (2011); K. Wang et al., Hepatology, 58, 706-717 (2013); S. M. Ahn et al., Hepatology, 60, 1972-1982 (2014); K. Schulze et al., Nat Genet., 47, 505-511 (2015); M. H. Xie et al. (1999); L. R. Desnoyers et al., Oncogene, 27, 85-97 (2008); and Y. Li et al., Oncotarget (2016 )).

FGF19 has been suggested to be a driver gene in the development of hepatocellular carcinoma (HCC), which is the most common type of liver cancer, accounting for approximately 90% of all liver cancers (J.M. Llovet et al., Nat. Rev. Dis. Primers , 2, 16018 (2016)). Liver cancer is the sixth most commonly diagnosed malignancy worldwide and the third most common cancer-related death worldwide (F. Bray et al., CA Cancer J. Clin., 68, 394-424 (2018)). Several clinical studies have demonstrated that HCC is characterized by highly focal amplification of fibroblast growth factor (FGF) 19 (ET Sawey et al., Cancer Cell, 19, 347-358 (2011); K. Wang et al., Hepatology, 58, 706-717 (2013)); S.M. Ahn et al., Hepatology, 60, 1972-1982 (2014); and K. Schulze et al., Nat. Genet., 47, 505-511 (2015)). High levels of FGF19 and/or FGFR4 expression have been found to stimulate tumor progression and may predict poor prognosis in patients with HCC (S. Miura et al., BMC Cancer, 12, 56 (2012); and J. Hyeon, S Ahn, J. J. Lee, D. H. Song, C. K. Park, Digestive diseases and sciences, 58, 1916-1922 (2013). In transgenic mice, overexpression of FGF19 led to the development of hepatocellular dysplasia, neoplasia and ultimately HCC (K. Nicholes et al., Am. J. Pathol., 160, 2295-2307 (2002)), which was eliminated in mice with FGFR4 knockout (D.M. French et al., PLoS One, 7, e36713 (2012)), which mechanistically confirmed the carcinogenic activity of the aberrant FGF19/FGFR4 signaling pathway, providing a theoretical basis for the development of antagonist drugs that target the FGF19/FGFR4 signaling pathway, in as a potential treatment for malignant neoplasms. However, several selective FGFR4 inhibitors being developed for the treatment of HCC resulted in increased bile acid synthesis and liver toxicity in either preclinical animal studies or early phase human clinical trials (M. Hagel et al., Cancer Discov., 5 , 424-437 (2015); R. Kim et al., European Journal of Cancer, 69, S41 (2016); S. L. Chan et al., Cancer research, 77, CT106-CT106 (2017); J. J. Joshi et al. , Cancer research, 77, 6999-7013 (2017); and B. Ruggeri et al., Cancer research, 77, 1234-1234 (2017)). Attempts have been made to develop drugs that specifically target FGF19 for the treatment of HCC, and a neutralizing antibody to FGF19 has been produced (WO 2007136893 A2, Genentech, Inc). Treatment with this anti-FGF19 antibody (1A6) prevented the development of HCC in transgenic mice overexpressing FGF19, and this therapy also resulted in decreased growth of HCC xenografts in mice (E.T. Sawey et al., 2011, supra; and L.R. Desnoyers et al. , 2008, supra). However, unfortunately, in a toxicological study, administration of a humanized 1A6 antibody to cynomolgus monkeys resulted in increased hepatic transcription of CYP7A1 and increased fatty acid synthesis, thereby significantly altering bile acid metabolism and causing severe dose-dependent side effects such as weight loss, severe diarrhea, and decreased appetite (R. Pai et al., Toxicological sciences: an official journal of the Society of Toxicology, 126, 446-456 (2012)). Considering the role of FGF19 in re

- 1 046377 regulation of bile acid metabolism, the action of antibodies that target FGF19 may adversely affect normal physiological functioning in this aspect and lead to unacceptable side effects. In view of these facts, clinical therapies targeting FGF19 with an antibody will need to address such side effects.

The present invention addresses these needs by providing anti-FGF19 antibodies that selectively target the N-terminus of FGF19, which exhibit high efficacy when used as a therapy for HCC without adversely affecting the normal function of FGF19 bile acid regulation.

All references, including scientific publications, patent application publications and patent publications, are incorporated herein in their entirety for all purposes.

Brief description of the invention

The present invention is based in part on the discovery of a new binding region on FGF19, namely the N-terminus of FGF19, and the isolation of anti-FGF19 antibodies that exhibit potent antitumor activity against HCC and also have the desired safety of not interfering with FGF19-mediated synthesis regulation bile acids by specifically binding to the specified binding region at the N-terminus of FGF19. Accordingly, the present invention provides a novel target at the N-terminus of FGF19 with therapeutic potential, as well as antibodies that specifically bind to said target for the diagnosis, prevention, treatment and/or prognosis of pathological conditions associated with or resulting from abnormal functioning of the FGF19/FGFR4 signaling pathway. in particular malignancies associated with or resulting from abnormal functioning of the FGF19/FGFR4 signaling pathway, including but not limited to HCC. It also provides nucleic acid molecules, expression vectors and host cells containing a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof. The present invention also provides pharmaceutical compositions, methods, kits, preparations associated with modulation of the FGF19/FGFR4 signaling pathway.

In a first aspect, the present application provides an isolated antibody or antigen binding fragment thereof that binds to human fibroblast growth factor 19 (FGF19) and the binding process is 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 at positions 38-45 of SEQ ID NO: 1. Specifically, the epitope contains at least one, at least two, at least three , at least four, at least five, at least six, at least seven of the following residues of FGF19 (SEQ ID NO: 1): W38, D40, P41, I42, R43, L44, R45. In a further embodiment, the antibody or antigen binding fragment of the present application binds to an epitope consisting of amino acid residues at positions 38-45 of SEQ ID NO: 1 and at least one of the following residues of human FGF19 (SEQ ID NO: 1): E81, P167 , L169. In a further embodiment of the study, the antibody or antigen-binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1 x 10 ^-9 M to about 1 x 10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

In another embodiment of the first aspect, an anti-FGF-19 antibody or antigen binding fragment according to the present application comprises at least one, two or three complementarity determining regions (CDRs) of a heavy chain variable domain (VH) containing the amino acid sequences of SEQ ID NO: 3, 4 , 5, 13 or 19 or variants thereof containing conservative substitutions. In another embodiment, an anti-FGF19 antibody or antigen binding fragment of the present application comprises at least one, two or three complementarity determining regions of a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 6, 7, 8 or 14 or variants thereof containing one or more conservative substitutions. In another embodiment, an anti-FGF19 antibody or antigen binding fragment according to the present application contains at least one, two or three complementarity determining regions of heavy chain variable domains (VH) containing the amino acid sequence of SEQ ID NO: 6, 7, 8 or 14 or variants thereof containing one or more conservative substitutions. In another embodiment, the antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a K _d value of about 1x10 ^-9 M to about 1x10 ^-12 M determined by surface plasmon resonance (e.g., BIACORE)

- 2 046377 or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

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

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

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

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

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

- 3 046377

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

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

In a more particular embodiment, an anti-FGF19 antibody or antigen binding fragment of the present application comprises (a) a heavy chain variable domain (VH) containing 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; and/or (b) a light chain variable domain (VL) comprising LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 14.

In one preferred embodiment, an anti-FGF19 antibody or antigen binding fragment 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 sequences 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 sequences of SEQ ID NO: 10, 16, or 21. In one preferred embodiment, an anti-FGF9 antibody or antigen binding fragment according to the present the application contains a heavy chain variable domain having at least 95, 96, 97, 98, 99 or 100% identity to the amino acid sequences 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.

In one preferred embodiment, the anti-FGF19 antibody and antigen binding fragment of the present application comprises (a) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence 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% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10;

(b) a heavy chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence SED ID NO: 16; or (c) a heavy chain variable domain having at least 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20, and a light chain variable domain having at least 95, 96 , 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21.

In one preferred embodiment, an anti-FGF19 antibody or antigen binding fragment of the present application comprises (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 and/or a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 10;

(b) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 15, and/or a light chain variable domain containing the amino acid sequence of SEQ ID NO: 16; or (c) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 20, and/or a light chain variable domain containing the amino acid sequence

- 4 046377 SEQ ID NO: 21.

In one preferred embodiment, the anti-FGF19 antibody or antigen binding fragment of the present application comprises a heavy chain having at least 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11, 17 or 22; and/or a light chain having at least 95, 96, 97, 98, 99, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, 18, or 23. In one preferred embodiment, an anti-FGF19 antibody or antigen binding fragment according to this application contains a heavy chain having the amino acid sequence SEQ ID NO: 11,17 or 22; and/or a light chain having the amino acid sequence of SEQ ID NO: 12, 18 or 23.

In one preferred embodiment, an anti-FGF19 antibody or antigen binding fragment of the present application comprises (a) a heavy chain having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11, and /or a light chain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 12;

(b) a heavy chain having at least 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17, and/or a light chain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to amino acid sequence SEQ ID NO: 18; or (c) a heavy chain having at least 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22, and/or a light chain having at least 95, 96, 97 , 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 23.

In one preferred embodiment, an anti-FGF9 antibody or antigen binding fragment of the present application comprises (a) a heavy chain having the amino acid sequence of SEQ ID NO: 11 and/or a light chain having the amino acid sequence of SEQ ID NO: 12;

(b) a heavy chain having the amino acid sequence of SEQ ID NO: 17, and/or a light chain having the amino acid sequence of SEQ ID NO: 18; and (c) a heavy chain having the amino acid sequence of SEQ ID NO: 22, and/or a light chain having the amino acid sequence of SEQ ID NO: 23.

In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that competes for binding to human FGF19 with a heavy chain variable domain (VH)-containing antibody comprising HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence SEQ ID NO: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 5; and a light chain variable domain (VL) containing LCDR1 having the amino acid sequence of SEQ ID NO: 6; LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 8. In a more particular 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 variable a heavy chain (VH) 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, said antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1x10 ^-9 M to about 1x10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

In one embodiment, the present application provides an anti-FGF19 antibody or antigen-binding fragment that competes for binding to human FGF19 with a heavy chain variable domain (VH)-containing antibody comprising HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence SEQ ID NO: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) comprising LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence

- 5 046377 sequence SEQ ID NO: 14. In a more particular embodiment, the present application provides an anti-FGF19 antibody or antigen binding fragment that competes for binding to human FGF19 with an antibody containing 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, said antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1x 10 ^-9 to about 1x 10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

In one embodiment, the present application provides an antibody or antigen binding fragment that competes for binding to human FGF19 with a heavy chain variable domain (VH) antibody containing HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 19, and a light chain variable domain (VL) containing LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 14. In a more particular embodiment, 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 the variable domain light chain having the amino acid sequence of SEQ ID NO: 21. More preferably, said antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1x10 ^-9 M to about 1x10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

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 a heavy chain variable domain (VH)-containing antibody containing HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2, having the amino acid sequence of SEQ ID NO: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 5; and a light chain variable domain (VL) comprising LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 8. In a more particular embodiment, the present The application provides an anti-FGF19 antibody or antigen-binding fragment that binds to the same epitope of human FGF19 as the antibody, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 9 10. More preferably, said antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1x10 ^-9 M to about 1x10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

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 the antibody containing

- 6 046377 heavy chain variable domain (VH) containing HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) comprising LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 14. In a more particular embodiment, the present The application provides an anti-FGF19 antibody or antigen-binding fragment that binds to the same epitope of human FGF19 as the antibody, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 15. 16. More preferably, said antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1x 10 ^-9 M to about 1x 10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

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 a heavy chain variable domain (VH)-containing antibody containing HCDR1 having the amino acid sequence of SEQ ID NO: 3, HCDR2, having the amino acid sequence of SEQ ID NO: 4, and HCDR3 having the amino acid sequence of SEQ ID NO: 13; and a light chain variable domain (VL) comprising LCDR1 having the amino acid sequence of SEQ ID NO: 6, LCDR2 having the amino acid sequence of SEQ ID NO: 7, and LCDR3 having the amino acid sequence of SEQ ID NO: 14. In a more particular embodiment, the present The application provides an anti-FGF19 antibody or antigen binding fragment that binds to the same epitope of human FGF19 as the antibody, comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 20, and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 20 21. More preferably, said antibody or antigen binding fragment thereof has at least one of the following properties:

(I) binds to human FGF19 with a Kd value of from about 1x10 ^-9 M to about 1x10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method;

(ii) cross-reacts with cynomolgus FGF19;

(III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex;

(IV) suppresses FGF19-induced cell proliferation;

(V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19.

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

In one embodiment, an anti-FGF19 antibody comprises a heavy chain constant region of the subclasses IgG1, IgG2, IgG3, IgG4, or a variation thereof, and a kappa or lambda light chain constant region, or a variation thereof.

In one embodiment, the anti-FGF19 antibody is an isolated antibody. In one embodiment, the anti-FGF19 antibody is a recombinant antibody. In a preferred embodiment, the present antibody or antigen binding fragment of the first aspect is for the treatment of diseases caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway, including administration to a subject in need thereof. In a particular embodiment, the disease is a malignancy caused by or associated with abnormal activation of the FGF19-FGFG4 signaling pathway. In a more particular embodiment, the malignancy is hepatocellular carcinoma (HCC). In a second aspect, the present invention provides a composition, that is, a pharmaceutical composition, comprising an anti-FGF19 antibody or antigen binding fragment according to the first aspect of the present application, as well as a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition contains a therapeutically effective amount of an anti-FGF19 antibody or antigen binding fragment.

- 7 046377

In a third aspect, the present invention provides a kit comprising an anti-FGF19 antibody or antigen binding fragment according to the first aspect of the present application, or a composition according to the second aspect of the present application. In a preferred embodiment, the kit contains a therapeutically effective amount of an anti-FGF19 antibody or antigen binding fragment.

In a fourth aspect, this discovery provides a method of preventing or treating a disease caused by or associated with abnormal activation of the FGF19-FGFG4 signaling pathway, including administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment according to the first aspect of the present application, or a pharmaceutical composition according to the second aspect aspect of this application. In a particular embodiment, the disease is a malignancy caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway. In a more particular embodiment, the malignancy is hepatocellular carcinoma (HCC).

In a fifth aspect, the present discovery provides a method for preventing the recurrence of a disease caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway, including administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment according to the first aspect of the present application, or a pharmaceutical composition according to the second aspect aspect of this application. In a particular embodiment, the disease is a malignancy caused by or associated with abnormal activation of the FGF19-FGFG4 signaling pathway. In a more particular embodiment, the malignancy is hepatocellular carcinoma (HCC). In one embodiment, disease recurrence was observed after surgery. In a sixth aspect, the present invention provides the use of an antibody or antigen binding fragment according to the first aspect of the present application or a pharmaceutical composition according to the second aspect of the present application for the treatment of various diseases described herein. In a particular embodiment, the disease is a disease caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway, preferably a malignancy caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway. In a more particular embodiment, the malignancy is hepatocellular carcinoma (HCC). In one embodiment, disease recurrence was observed after surgery.

In a seventh aspect, the present invention provides the use of an antibody or antigen binding fragment according to the first aspect of the present application or a pharmaceutical composition according to the second aspect of the present application in the manufacture of medicaments for the treatment of various diseases described herein or for the prevention of recurrence of various diseases described herein. In a particular embodiment, the disease is a disease caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway, preferably a malignancy caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway. In a more particular embodiment, the malignancy is hepatocellular carcinoma (HCC). In one embodiment, disease recurrence was observed after surgery. In one embodiment, disease recurrence was observed after surgery.

Brief description of graphic materials

In fig. 1A-1C show a minor role for the N-terminus of FGF19 in mediating FGF19 activity. (A) The binding affinity of FGF19 or FGF19'' ^NT (an N-terminal deletion variant of FGF19) to FGFR4 or the FGFR4-KLB complex is shown. In the case of FGF19 ^ANT (N-terminal deletion variant), reduced binding affinity to FGFR4 was observed. FGFR4-hFc was captured as a ligand on a sensor chip in Biacore. Interactions between the FGFR4 ligand and the FGF19 analyte or FGF19'' ^NT were analyzed in the presence of heparin (20 μg/ml). The analyzed components of FGF19 or FGF19 ^ant were taken in two-fold serial dilutions from 1000 nmol. (B) In the case of FGF19 ^ant (N-terminal deletion variant), decreased cell proliferation inducing activity was observed. Hep3B was cultured with various concentrations of FGF19 or FGF19'' ^NT in 1% PBS containing Dulbecco's modified Eagle's minimal essential medium. (C) FGF19'' ^NT (an N-terminal deletion variant of FGF19) retained the ability to suppress CYP7F1 gene expression in vivo. C57BL/6 mice were fasted before intraperitoneal injection of the indicated drugs. The expression of the hepatic CYP7A1 gene was analyzed using the qPCR method. Each treatment group consisted of 3 male and 3 female mice.

Fig. 2 is a schematic diagram that illustrates the formation of antibody G1A8 using antibody phase display technology. The N-terminal peptide of FGF19 or the full-length protein of FGF19 was used as an antigen for selection against a large library of non-immune phage display antibodies of the authors of the invention (D. Li et al., A potent human neutralizing antibody Fc-dependently reduces established HBV infections. Elife, 6 (2017 )). Through subsequent ELISA screening using both full-length FGF19 and the N-terminal deletion variant ( ^FGF19''NT )

- 8 046377 Have antibodies been identified that bind to FGF19 with higher affinity than ^FGF19'VI '. Dark gray shading indicates higher affinity compared to cells shaded in light gray or white. The CDR3 protein sequences of both the heavy chain and light chain of one identified antibody 31A3 and its affinity-enhanced antibody G1A8 are shown in parallel, with different amino acids underlined.

In fig. 3A, 3B show improved binding affinity and higher dependence of G1A8 on the N-terminus. (A) Kinetic analysis of binding of anti-FGF19 antibodies 31A3 or G1A8 to FGF19 using SPR. FGF was taken in two-fold serial dilutions from 100 nmol. (B) Binding of both 31A3 and G1A8 to FGF19 is dependent on the N terminus of FGF19, as demonstrated by different binding profiles when using FGF19 or ^FGF19'NI ' as the target. The binding activity of antibodies to FGF19 and FGF19 ' ^NI ' was analyzed using ELISA.

In fig. 4A-4C give a description of G1A8. (A) G1A8 and 31A3 showed the ability to inhibit FGF19-induced cell proliferation. NerZVs were cultured with FGF19 (20 ng/ml) and anti-FGF19 antibodies (100 nmol) or with an isotopic control antibody. (B) G1A8 inhibits the interaction between FGF19 and FGFR3. G1A8 antibody or isotopic control antibody was mixed with FGFR4-Fc (100 nmol) and heparin (20 μg/ml). Inhibition of FGFR4 binding to FGF19 was analyzed by ELISA. (C) G1A8 did not disrupt mouse liver CYP7A1 gene expression suppressed by FGF19. C57BL/6 mice were not fed before intraperitoneal injection of the indicated drugs. The expression of the hepatic CYP7A1 gene was analyzed using the qPCR method. Each treatment group consisted of 3 male and 3 female mice. FGF19, 0.1 mg/kg; G1A8, 5-fold molar excess of FGF19.

In fig. 5A-5E show the antitumor activity of G1A8 tested in mice bearing a human hepatocellular carcinoma xenograft. (A and B) G1A8 suppresses tumor growth early in tumor development. Stably expressing luciferase Hep3B-luciferase was administered subcutaneously (s.c.) to NOD SCID mice. Mice were divided into two groups (n=6/group) with equivalent mean tumor bioluminescence intensity and treated with 200 μg G1A8 or isotopic control antibody. Tumor growth was assessed using calipers (A) and bioluminescence (B and C). (D and E) G1A8 inhibited tumor growth at the late stage of tumor development. NSG mice bearing NerZV tumor cells were divided into two groups (n=6/group) with equivalent mean tumor volume and treated with 200 μg G1A8 or isotopic control antibody. Tumor growth was assessed using calipers (D), and survival was assessed using Kaplan-Meier survival analysis. (E) Days on which antibody treatment was administered are indicated by arrows.

In fig. 6A-6L provides a safety assessment of G1A8 in cynomolgus monkeys. (A) Schematic diagram illustrating the schedule of G1A8 administration and animal sampling. Four cynomolgus monkeys were divided into two groups, one of which received intravenous injections of saline control (in grey) or G1A8-hIgG1 (in black). Each group consisted of one male (indicated by a triangle) and one female (indicated by a circle) monkey. (B) Body mass of cynomolgus macaques. (C-F) Biochemical analysis of cynomolgus macaque blood. Total bile acids (C), total bilirubin (D), ALT (E) and AST (F) in cynomolgus serum were measured at different time points. The dotted line and dotted line indicate normal reference intervals for biochemical parameters in male and female cynomolgus monkeys, respectively. (G and H) Expression of genes associated with bile acid metabolism. At the end of the study, tissue samples were taken from cynomolgus macaques. The expression of selected genes in liver (G), ileum (H) and kidney (I) was analyzed using qPCR method. (J and K) Pharmacokinetic profile of G1A8. G1A8 concentrations were measured following administration of 10 mg/kg G1A8 on day 1 (J) or 30 mg/kg G1A8 on day 16 (K). (L) Kinetic analysis of G1A8 binding to cynomolgus FGF19. Cynomolgus FGF19 was taken in two-fold serial dilutions from 100 nmol.

In fig. 7A-7D show structural analysis of the FGF19-G1A8 complex. (A) Ribbon representation of FGF19-G1A8 in orthogonal view. FGF19 is shown in black, and the heavy chain (VH) and light chain (VL) variable domains of G1A8 are shown in gray. The N-terminus and C-terminus of FGF19 are marked. The G1A8 Fab constant domain has been removed for clarity. (B) Surface view of the FGF19-G1A8 complex. The structure reveals an outwardly perfect complementarity of forms. (C) Detailed view of the G1A8-FGF19 interface. The dotted lines indicate hydrogen bonds. (D) The binding activity of G1A8 to mutant FGF19 and alanine-substituted FGF19 was analyzed using SPR.

In fig. 8A-8C show the activity of HS29. (A) Binding analysis of anti-FGF19 antibodies G1A8 and HS29 to the alanine substitution mutant FGF19 was performed using SPR. Alanine-substituted FGF19 mutants were tested at a concentration of 50 nmol. (B) Kinetic analysis of HS29 binding to FGF19. FGF19 was taken in a two-fold serial dilution from 100 nmol. (C) Inhibition of FGF19-induced proliferation of HS29 cells. Hep3B was cultured with FGF19 (20 ng/ml) and anti-FGF19 antibodies or isotopic control antibodies (100 nmol).

- 9 046377

In fig. 9A-9D evaluate the antitumor activity of HS29 in patient-derived xenograft models. BALB/c athymic mice carrying the FGF19-expressing PDX LI667 gene (A) or the FGF19-non-expressing PDX LI667 gene (B) were divided into groups (n=5/group) with an equivalent average tumor volume; in one group, mice received intraperitoneal injection of HS29 (10 mg/kg), while the control group received no treatment. (C) FGF19 mRNA expression in mouse models. At the end of the study, tumor tissue samples were collected and FGF19 expression was analyzed by qPCR. Each point on the graph represents one mouse tumor sample, and the FGF19 mRNA expression level of the cultured Hep3B cell line is used as a reference. The dotted line indicates the reliable detection limit of the assay. (D) Body weight of mice carrying PDX was measured during the study.

Detailed Description of the Invention

Definitions.

Unless specifically defined herein, all other technical and scientific terms used herein have a meaning typically understood by one of ordinary skill in the art to which the invention pertains.

The term or is used to refer to and is used interchangeably with the term and/or unless the context clearly dictates otherwise.

In the context of the present discovery, unless otherwise indicated, the wording contains and variations thereof, such as containing, will be understood to imply the inclusion of the specified element, for example, an amino acid sequence, a nucleotide sequence, a property, a step or a group thereof, but not the exclusion of any other elements, for example amino acid sequences, nucleotide sequences, properties and steps. When used herein, the term contain or any variant thereof may be replaced by the term include or sometimes have or an equivalent variant thereof. In some embodiments, the statement to contain also includes a script consisting of.

As used herein, the term FGF19 refers to the ileal hormone fibroblast growth factor 19, which is a high affinity ligand for FGFR4 (fibroblast growth factor receptor 4). As stated above, FGF19 plays an important role in the regulation of bile acid synthesis, among other things, by inhibiting the transcription of CYP7A1 in the liver through the FGFR4/Klotho-e receptor complexes. As used herein, unless specifically stated otherwise, FGF19 refers to human FGF19. The ortholog of human FGF19 in mice is FGF15. The amino acid sequence of FGF19, eg human FGF19, and the nucleotide sequence encoding it are known in the art.

The fibroblast growth factor family has a homologous core region formed by 12 antiparallel (3-chains flanked by divergent N-terminus and C-terminus (A. Beenken, M. Mohammadi, Nature reviews. Drug Discovery, 8, 235-253 (2009)) Variations in the primary sequence between the N- and C-termini of different FGFs explain their different biological activities (N. Itoh, D.M. Ornitz, J. Biochem., 149, 121-130 (2011); R. Goetz, M. Mohammadi, Nat. Rev. Mol. Cell Biol., 14, 166-180 (2013)).

Antibody and its antigen-binding fragment.

Unless otherwise specified, the term antibody as used herein includes antibodies, as well as antibody fragments in the broadest sense when they recognize and bind to human FGF19. In particular, the antibody of the present application binds to the N-terminus of human FGF19, namely amino acid residues 38-45 of FGF19 having the amino acid sequence SEQ ID NO: 1. The antibody of the present application is generally a monospecific antibody. But this application also contemplates an antibody with heterologous specificity (heterospecific) or multispecific antibody. The antibody binds to specific antigenic determinants or epitotopes through specific binding sites. The term antibody fragment refers to a portion of a full-length antibody, typically containing a binding or variable region for the antibody. 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. The most commonly encountered basic antibody structure is a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair having one smaller chain, designated a light chain (about 25 kDa), and one larger chain, designated a heavy chain (about 50-70 kDa). At the amino end of each chain it has a variable domain consisting of 100-110 or more amino acids, primarily responsible for antibody recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, the light chains of human antibodies are classified into kappa and lambda light chains. In addition, human antibody heavy chains are usually classified into α, δ, ε, γ, or μ and define antibody isotypes (IgA, IgD, IgE, IgG, and IgM, respectively) and their subclasses (e.g., IgG1, IgG2, IgG3, IgG4) . Variable regions

- 10 046377 ti/domains of each light/heavy chain (VL/VH) pair form the antibody binding site. Accordingly, an intact antibody typically has two binding sites. The term hypervariable domain refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable domain contains amino acid residues of the complementarity determining region (CDR) (ie, LCDR1, LCDR2 and LCDR3 in the light chain variable domain and HCDR1, HCDR2, HCDR3 in the heavy chain variable domain). Typically, both the heavy and light chain variable domains contain three hypervariable domains, called CDRs, which are located between relatively conserved framework regions, or KUs. CDRs are typically aligned to framework regions to allow binding to a specific epitope. Typically, from the N-terminus to the C-terminus, the variable domains of both the light and heavy chains contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2) , FR-3 (FR3), CDR3 (CDR3) and FR-4 (or FR4). Kabat nomenclature is used for antibody amino acid residues unless otherwise noted (Kabat et al., Sequence of proteins of immunological interest, 5th ed., Public Health Service, National Institute of Health, Bethesda, MD (1991)).

Unless otherwise noted, the terms antibody fragment, target binding fragment, and antigen binding fragment are used interchangeably as used herein and refer to antibody fragments that retain the ability to specifically bind an antigen (FGF19 or, in particular, the N-terminus of FGF19) bound by the full-length antibody , i.e. fragments that have retained one or more CDRs. 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, eg single chain Fv fragment (ScFv); nanobodies and multispecific antibodies formed by antibody fragments.

By specific binding or binding-specificity is meant that an antibody exhibits preferential binding to a particular target over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered target specific if its binding detects the presence of the target protein in the sample, i.e. without producing unwanted results such as false positives. An antibody or antigen binding fragment thereof according to the present application will bind to a target protein with an affinity of at least 2 times higher, preferably at least 10 times higher, more preferably at least 20 times higher, and most preferably at least 100 times higher than the affinity of non-target proteins. Alternatively or additionally, an antibody or antigen binding fragment thereof according to the present application will have a binding affinity for its target protein, expressed in CD, lower than 1 x 10 ^-8 mol, lower than 1 x 10 ^-9 mol (1 nmol), lower than 1 x 10 ^-10 mol, lower than 1 x 10 ^-11 mol, or even lower than 1 x 10 ^-12 mol (1 pmol). The antibody of the present application is said to bind specifically to a polypeptide containing a given amino acid sequence, i.e. the amino acid sequence of mature human FGF19 or the N-terminal residues (38-45) of human FGF19, if it binds to polypeptides containing this sequence, but does not bind to proteins that lack this sequence. As used herein, the term human antibody refers to an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain murine carbohydrate chains if it is produced by a mouse, a mouse cell, or a hybridoma derived from a mouse cell. Likewise, mouse antibody or rat antibody refers to an antibody containing only the protein sequence of mouse or rat immunoglobulin alone, respectively. The terms monoclonal antibody, or mAb, or Mab as used herein refer to a population of essentially homogeneous antibodies, meaning that the antibody molecules comprising the population are identical in amino acid sequence, except for naturally occurring mutations that may be present in small quantities. The term monoclonal refers to the nature of the antibody obtained from an essentially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. Monoclonal antibodies (mAbs) can be produced by traditional methods known in the art. See, for example, Kohler G et al., Nature, 1975, 256:495-497; US Patent No. 4376110; Ausubel FM et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 1992; Harlow E. et al., ANTIBODIES: A LABORATORY MANUAL, Cold spring Harbor Laboratory, 1988; Colligan JE et al., CURRENT PROTOCOLS IN IMMUNOLOGY, 1993.

In one embodiment, an antibody of the present application that specifically binds to human FGF19 also shows cross-reactivity with the cynomolgus monkey ortholog of human FGF19. As used herein, the term cross-reactivity refers to the ability of an antibody to react with a homologous or orthologous protein from another species. Antibody cross-reactivity can be determined using any method known in the art. For example, it can be determined by measuring binding affinity using a surface plasmon resonance method (eg, BIACORE) or a similar method (eg, KinExa or OCTET).

- 11 046377

In some embodiments, the antibody or antigen-binding fragment of the present application does not bind or has minimal effect on FGF19-suppressive expression of CYP7A1. In other words, the expression of CYP7A1 is comparatively inhibited by FGF19 upon administration of an antibody or antigen binding fragment. More specifically, the change or specific increase in the expression level of CYP7A1 was no more than 5, 10, 15 or 20% after administration of the antibody or antigen-binding fragment in the presence of FGF19. In some embodiments, the antibody or antigen-binding fragment of the present application does not or only slightly disrupts FGF19-maintained bile acid homeostasis. Stable bile acid homeostasis can be determined by monitoring the level of transcription or expression of one or more genes involved in bile acid metabolism in relevant tissues. For example, these genes may be genes encoding bile acid transport proteins. Specific genes that may be analyzed include ASBT, IBABP, CYP7A1, NTCP, OATP2, BSEP, OSTa, OSTe, MRP2, MRP3, and/or MRP4 in the liver, kidney, and/or ileum. The change in the expression level of one or more genes ASBT, IBABP, CYP7A1, NTCP, OATP2, BSEP, OSTa, OSTe, MRP2, MRP3, and/or MRP4 in the liver, kidney and/or ileum is limited, for example, no more than 5, 10, 15 or 20% after administration of an antibody or antigen-binding fragment in the presence of FGF19.

The antibody of the present application can be subjected to a purification process to remove undesirable materials, resulting in a purified antibody. Conventional methods for purifying antibodies include, but are not limited to, column chromatography techniques, which are well known in the art.

The antibody or antigen binding fragment of the present application may be an isolated antibody. By isolated it is meant that the antibodies or antigen-binding fragments are at least partially free from other biological materials or non-biological materials from the cells, cell cultures, culture medium, expression system in which they are produced. These materials may include nucleic acids, proteins, lipids, carbohydrates, buffer, salt and other materials such as cellular debris and culture media.

The present application also contemplates an antibody or antigen binding fragment thereof containing one or more conservative substitutions, provided that the antibody or antigen binding fragment binds to FGF19 and has at least one of the antibody properties described herein. Conservative amino acid substitutions are well known in the art and generally refer to the replacement of one amino acid residue with another amino acid residue having a similar side chain in structure or function. For example, a sample list of conserved structures is presented in the table below.

Original amino acid residue Conservative substitution(s) Ala Gli; Ser April Liz; Gies Asn Gln; Gies Asp Gln; Asn Cis Ser; Ala Gln Asn Gln Asp; Gln Gli Ala Gies Asn; Gln Ile Lei; Shaft Lei Ile; Shaft

- 12 046377

Liz Apr; Gies Meth Lei; Ile; Shooting Range Hairdryer Shooting gallery; Meth; Lei About Ala Ser Tre Tre Ser Trp Shooting gallery; Hairdryer Shooting gallery Trp; Hairdryer Shaft Ile; Lei

The present application also provides an isolated nucleic acid sequence containing a nucleotide sequence encoding an antibody or fragment thereof. By isolated nucleic acid or isolated polynucleotide, we mean DNA or RNA that is removed from all or part of a polynucleotide to which the isolated polynucleotide occurs naturally, or is associated with a polynucleotide to which it is not naturally associated. An isolated nucleic acid molecule containing a particular nucleotide sequence may include, in addition to said sequence, operably linked regulatory sequences that control expression of the coding region of said nucleic acid sequences. One skilled in the art will appreciate that a particular amino acid sequence may be encoded by different nucleotide sequences due to codon degeneracy.

Use for therapeutic purposes.

The present discovery provides a method for preventing the recurrence of a disease caused by or associated with abnormal activation of the FGF19-FGFR4 signaling pathway, including administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen binding fragment according to the first aspect of the present application, or a pharmaceutical composition according to the second aspect of this application . In a particular embodiment, the disease is a malignancy caused by or associated with abnormal activation of the FGF19-FGFG4 signaling pathway. In a more particular embodiment, the malignancy is hepatocellular carcinoma (HCC). As used herein, administration and treatment, when applied to a subject, such as an animal, including a human, or a cell, tissue, organ or biological fluid, means contact of an exogenous pharmaceutical, therapeutic, diagnostic agent or composition with the subject, cell, tissue, organ or biological fluid . Treatment of a cell involves contact of the reagent with the cell, as well as contact of the reagent with the liquid, with the liquid in contact with the cell. The terms administration and treatment also include in vitro and ex vivo treatment, eg, with a cell, reagent, diagnostic, binding compound, or other cell.

The term therapeutically effective amount as used herein refers to an amount of antibody that, when administered to a subject to treat a disease or at least one clinical symptom of a disease, is sufficient to produce an effect of the disease or symptom. The therapeutically effective amount may vary depending on the antibody, the disease and/or symptom of the disease, the severity of the disease and/or symptoms, the age of the subject receiving treatment, and the weight of the subject receiving treatment. The appropriate amount in any particular case may be obvious to those skilled in the art or can be determined through routine experimentation. In the case of combination therapy, a therapeutically effective amount refers to the total number of entities combined to effectively treat the disease or condition.

In the context of this application, the subject is understood to be an animal, preferably a mammal, i.e. a primate, preferably a great ape, such as a human. The terms malignancy or tumor in this context describe a physiological condition of mammals that is typically characterized by unregulated cell growth. In a preferred embodiment, the malignancy is associated with aberrant signaling by the FGF19-FGFR4 signaling pathway. It can be envisaged that the antibody or antigen binding fragment of the present application can be used to treat, for example, breast cancer, prostate cancer, colon cancer, lung cancer and gastric cancer.

- 13 046377

Examples

Materials and methods.

The following materials and methods are used in the examples.

Cell lines.

The human hepatocellular carcinoma cell line Hep3B was a kind gift from Dr. Fengming Lu at the Peking University Health Science Center. Hep3B cells were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented with 10% fetal bovine serum (Gibco, DMEM, C11965500BT). Cells were cultured in a humidified incubator at 37°C in an atmosphere of 5% CO2. A stable Hep3B-Luc32 cell line expressing the firefly luciferase reporter gene was generated by lentivirus transduction and selected with puromycin.

The FreeStyle 293-F cell line was cultured in FreeStyle™ 293 expression medium (both cell line and medium are from Thermal Fisher Scientific) in a humidified orbital shaker at 37°C under 8% CO ₂ atmosphere.

Expression and purification of proteins.

For human FGF19 (SEQ ID NO: 1) and its variants, including the N-terminal deletion variant FGF19''NT (having the amino acid sequence corresponding to residues Arg43-Lys216 SEQ ID NO: 1) and mutated FGF19 with alanine substitution, encoding sequences for the proteins were cloned into a mammalian cell expression vector with a C-terminal His-Avi tag and transiently transfected alone or cotransfected with an expression vector encoding the E. coli biotin-protein ligase BirA protein sequence into a FreeStyle 293-F cell (Thermal Fisher Scientific) in a 1:1 ratio. To generate a full-length human IgG1 antibody, the VH and VL coding sequences were cloned, respectively, into a mammalian cell IgG1 heavy chain expression vector and a mammalian cell IgG1 light chain expression vector. FreeStyle cells were transiently cotransfected with two expression plasmids (heavy chain and light chain plasmids) in a 1:1 ratio. 3–5 days after transfection, cell culture supernatant was collected for purification of human IgG1 using protein A bead affinity chromatography (GE Healthcare Life Sciences).

Anti-FGF19 antibody library screening.

The FGF19 N-terminal peptide (SEQ ID NO: 24), consisting of residues Arg23 to Ile42 of the FGF19 N-terminal peptide followed by 5 amino acids (SGSGK) with biotin modification at the C-terminus, was synthesized by Scilight peptide (Beijing, China) with a purity of more than 95%. The N-terminal FGF19 peptide or biotinylated full-length FGF19 protein was captured as the target protein on streptavidin-conjugated magnetic M-280 Dynabeads® (Thermal Fisher Scientific) and then incubated with phage-scFv particles at 5x10 ¹² (single chain variable domain fragment ), prepared from a phage display library of non-immune human antibodies created from peripheral blood mononuclear cells of 93 healthy donors (size 1.0x10 ¹⁰ ). Two rounds of selection were conducted. For the second round of selection, a reduced amount of target protein was used and extensive washing steps were performed. A common triethanolamine stock solution was used to elute phages that specifically bound to the target protein. Subsequently, individual clones were selected and maintained to generate phage-scFv complexes in bacterial culture supernatant; A screen was performed analyzing the specific binding ability of the N-terminus of FGF19 in an enzyme-linked immunosorbent assay (ELISA) by comparing binding to full-length FGF19 and an N-terminal deletion variant of FGF19 (FGF19 ^ANT ). Clones that bind to FGF19 with higher affinity than FGF19 ''NT. were selected as candidates that specifically bind to the N-terminus of FGF19, and the heavy (VH) and light (VL) chain variable region genes of these clones were sequenced.

The corresponding amino acid sequences of the selected clones were aligned to eliminate duplicate clones and thereby identify unique antibodies for further characterization.

31A3 antibody sublibrary generation and affinity selection.

To improve the affinity of the 31A3 antibody through CDR3-targeted randomization, a random mutagenesis sublibrary was generated for HCDR3 and LCDR3 31A3 using degenerate codons of the NNK series. The size of the constructed antibody sublibrary was 1.23x108. Selection and screening of the sublibrary antibodies were carried out similarly to the method described above for screening the FGF19 antibody library. Competitive elution of human IgG1 31A3 was used to obtain high-affinity results from magnetic beads. Subsequently, individual clones were selected and maintained to produce phage-SCFV in bacterial culture supernatant to screen for FGF19 binding ability. Only results with higher affinity than 31A3 were retained.

- 14 046377

ELISA.

NeutrAvidin (Sigma Aldrich, 2 μg/ml) in phosphate-buffered saline was applied to a 96-well U-bottom plate (Nunc, MaxiSorp™) at 100 μl per well and incubated at 4°C overnight. Biotinylated FGF19 or FGF19 variants were then plated (100 μl per well) by incubation at 30°C for 0.5-1 hour. FGF19 or FGF19 variants were also applied directly to a 96-well U-bottom plate at the rate 100 μl per well and incubated at 4°C overnight. For ELISA, which uses human IgG1, threefold serially diluted antibodies in PBS containing 2% skim milk were added to 100 ml per well. In the case of competitive ELISA, the antibodies tested were taken in a three-fold serial dilution and mixed with the competing FGFR4-hFc. Bound antibodies were then detected using horseradish peroxidase-conjugated goat anti-human Fc antibody (Thermo Fisher Scientific). Surface Plasma Resonance (SPR) Binding Kinetic Analysis Kinetic analysis of human IgG1 antibodies to FGF19 according to the present application was performed using a Biacore T200 system (GE Healthcare Life Sciences) at 25°C. Anti-human Fc antibodies (GE Healthcare Life Sciences) were immobilized onto the CM5 sensor chip using an amino immobilization kit (GE Healthcare Life Sciences). Human IgG1 antibodies to FGF19 according to this application were captured as a ligand on the sensor chip, followed by injection of FGF19 analyte (2-fold serial dilution from 100 nmol) or alanine-switched mutant FGF19 (50 nmol) into each flow cell. The introduction of buffer (HBS with Tween20 and 0.3 mmol EDTA) served as a negative control. 3 mol MgCl ₂ was used as a renewal buffer between each association and dissociation cycle. Association rates (ka), dissociation rates (kd) and affinity constants ( _KD ) were calculated using BiacoreT200 software.

Cell proliferation assay.

The human HCC cell line NerZV was treated with various concentrations of FGF19 or FGF19 variants in Dulbecco's modified Eagle's minimal essential medium supplemented with 1% fetal bovine serum to induce excessive cell proliferation. To evaluate the inhibitory activity against FGF19-induced cell proliferation by anti-FGF19 antibodies, 15 μg/ml (100 nmol) of human IgG1 antibody was added to the cell culture medium. After 72 h, cell proliferation was analyzed using Cell Counting Kit-8 (Dojindo Molecular Technologies) according to the product manufacturer's instructions.

Expression of CYP7A1 in the liver.

C57BL/6 mice, 5 to 6 weeks old, were fasted overnight before intraperitoneal (i.p.) injection of 2 μg of (1) FGF19, (2) FGF19 variants, or (3) FGF19 combined with 60 μg of anti-FGF19 antibody. Mice were sacrificed 3 h after IP injection and liver collection was performed. For subsequent analysis of CYP7A1 mRNA expression levels, total liver RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), and complementary DNA (cDNA) was obtained by reverse transcription using the Prime Script RT-PCR Kit (Takara). The level of CYP7A1 mRNA expression (relative to GAPDH) was assessed by cNCR using an ABI Fast 7500 instrument (Applied Biosystems) in real time.

Experiments on animals.

NOD SCID and NSG mice were used to evaluate the antitumor activity of antibodies to FGF19. Mice at the age of 6-8 weeks were injected subcutaneously with 5x 10 ⁶ NerZV cells (100 μl per mouse) into the right flank. Mice were divided into three groups (n=6 per group) based on equivalent mean tumor bioluminescence intensity or tumor volume and received intraperitoneal (i.p.) injection of G1A8 (200 μg) or control IgG twice weekly. In vivo tumor bioluminescence intensity in tumor-bearing mice was measured using the IVIS Lumina III In Vivo Imaging System (PerkinElmer) after intraperitoneal injection of 15 mg/kg D-luciferin (PerkinElmer). Tumor volume was measured using an electronic caliper and calculated using the formula 3.14xLxW2/6, where L and W are the largest and smallest diameters, respectively. All animal experimental protocols were carried out in accordance with the national guidelines for the care and maintenance of laboratory animals in China and were performed in accordance with established regulations after approval by the Laboratory Animal Care and Use Committee of the National Institute of Biological Sciences. The study to evaluate the antitumor activity of antibodies in patient-derived xenograft BALD/c nude mice was conducted in accordance with approved protocols of the Laboratory Animal Care and Use Committee at Crown Bioscience Inc.

The safety evaluation of the antibody of this application, namely G1A8, was conducted at JOINN Laboratories (Nequin), Inc. 3- to 4-year-old healthy, treatment-naïve cynomolgus monkeys weighing ~3 kg were intravenously injected with G1A8 antibody at 30 mg/kg body weight or aqueous sodium chloride control on day 16 at 30 mg/kg body weight. Clinical observations, body weight, body temperature, blood chemistry and anatomical pathology were assessed.

- 15 046377 gi. Blood samples were collected at various time points (day 1, day 8, day 15, day 23 and day 30) for pharmacokinetic analysis of G1A8. Liver, ileal, and kidney tissue samples were collected at the end of the study for RNA analysis of genes associated with bile acid metabolism.

Crystallization and structural determination of the FGF19-G1A8 complex.

FGF19 and G1A8-Fab were separately expressed in FreeStyle 293-F cell line cells and individually purified by Ni-NTA flow chromatography (QIAGEN). To prepare the FGF-G1AB complex, FGF19 and G1A8-Fab were mixed in a 1:1 molar ratio, incubated at 4°C overnight, and then purified using a Superdex S200 10/300 column (GE Healthcare) with a buffer containing 10 mmol Tris-HCl (pH 8.0) and 500 mmol NaCl. The purified FGF19-G1A8 complex was then concentrated to 18 mg/ml and crystallized at 20°C using the hanging drop method via vapor diffusion by mixing 1 μl of protein and 1 μl of a reservoir solution containing 0.2 mol lithium sulfate monohydrate, 0.1 mol bis-tris (pH 6.9) and polyethylene glycol (w/v=26%). Quadrangular crystals appeared after 7 days. The crystals were quickly frozen in liquid nitrogen.

X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) BL17U. Data were processed in HKL2000 and XDS. The crystals belong to space group P212121 and contain two copies of the FGF19-G1A8 complex per asymmetric unit. The structure was determined by the molecular replacement method using Phaser and Phenix with the following structures as search models: FGF19 (PDB ID: 2P23) and Fab structure to steroid 5F2 (PDB ID: 3KDM). The model was iteratively developed in Coot and refined in PHENIX.

Statistical analysis.

All analyzes were performed using GraphPad Prism version 6.00. For comparisons between groups, ordinary one-way ANOVA or unpaired Student's T test was used. Two-way ANOVA and Tukey's multiple comparison test were used to evaluate continuous variables. Kaplan-Meier survival analysis and log-rank test methods were used for survival analysis.

Example 1 Detection of a binding target at the N-terminus of FGF19.

The present inventors performed multiple assays to determine whether there is a functional difference between full-length FGF19 and its N-terminal deletion version.

It was found that, compared to the full-length FGF19, the FGF19 ^ANT variant (FGF19''N ^T. having the amino acid sequence corresponding to the Arg43-Lys216 residues of SEQ ID NO: 1) had a significantly weaker binding affinity to FGFR4 and exhibited significantly less activity in inducing tumor proliferation cells in an in vitro assay (Figures 1A and 1C).

Subsequently, the function of regulation of bile acid metabolism carried out by full-length FGF19 and the indicated FGF19''N ^T variant with N-terminal deletion was assessed. Mice were used in this study because endogenous FGF19 was found to be capable of regulating bile acid metabolism by binding to the mouse FGFR4 receptor, which has 90% amino acid identity with human FGFR4, resulting in suppression of hepatic transcription of CYP7A1 in mice (M. Zhou et al., 2014, above; R. Goetz et al., Molecular and cellular biology, 27, 3417-3428 (2007)). Following intraperitoneal injection of FGF19 or FGF19'N ^T, the levels of CYP7A1 gene expression in the liver of these mice were assessed. There was no statistically significant difference between the full-length variant and the N-terminal variant of FGF19 ^ANT in the regulation of bile acid metabolism due to repression of the CYP7A1 gene expression level in the liver. In light of these results, it can be reasonably expected that a therapeutic agent targeting the N-terminus of FGF19 could potentially inhibit its tumorigenic activity without adversely affecting its physiological function in regulating bile acid metabolism.

Example 2: Generation of antibodies specifically targeting the N-terminus of human FGF19.

The following two strategies, using either SEQ ID NO: 1 or SEQ ID NO: 24 as the binding target, were developed to identify antibodies that specifically target the N-terminus of FGF19 (Fig. 2). Antibody selection was performed using either the N-terminal FGF19 peptide (SEQ ID NO: 24) or full-length FGF19 (SEQ ID NO: 1) as the binding target, and a large phage display library of non-immune human antibodies was selected (D. Li et al., Elife, 6, 2017, above). Antibodies that demonstrated the ability to bind both the N-terminal fragment of FGF19 and full-length FGF19 were selected for the next stage of screening. In the next step of ELISA screening, both full-length FGF19 and its N-terminal deletion variant (FGF19 ^ANT ) were used as binding targets to identify antibodies that were able to bind to full-length FGF19 but could not (or only weakly bind). ) with FGF19 ^ANT . Among a number of antibodies identified, an antibody designated 31A3, which showed relatively high binding affinity and specifically recognized the N-terminus of FGF19, was selected for further analysis (Figs. 2 and 3).

- 16 046377

Example 3. In vitro assessment of the inhibitory effect of 31A3 on the tumor activity of FGF19.

Antiproliferation assays were performed to evaluate the ability of 31A3 to inhibit the tumorigenic activity of FGF19 in vitro. To evaluate whether 31A3 could inhibit HCC cell proliferation specifically induced by FGF19, the HCC cell line Hep3B was treated with 31A3 in the presence of exogenous FGF19. As shown in Figure 4A, 31A3 inhibited FGF19-induced proliferation of Hep3B cells.

Example 4: Generation of 31A3-based mutant antibodies with improved binding affinity.

To enhance the affinity of 31A3 for the N-terminus of FGF19, site-directed alanine scanning mutagenesis was performed in the HCDR3 and LCDR3 regions of 31A3, showing that four amino acid residues, namely V96 and W97 in HCDR3 and Y91 and T95 in LCDR3, may be potential candidates. to improve binding affinity by substituting other amino acids. We then created a phage display library containing 31A3-derived antibodies with randomized mutations at the above four positions in the HCDR3 and LCDR3 regions. During the selection of a sublibrary of antibodies derived from 31A3, the 31A3-hIgG1 protein was used as a competitor to screen for binders with higher binding affinity to FGF19. After a stringent biopanning selection step, a small panel of antibodies with improved affinity was obtained. One of the resulting antibodies, G1A8, differing from 31A3 in three amino acids, demonstrated a 27-fold increase in affinity and a 60-fold decrease in dissociation rate, as well as more potent antiproliferative activity than the parent antibody 31A3 (Figs. 2, 3A, 3B and 4A).

Another antibody variant, HS29, was created based on sequence analysis of a small panel of antibodies generated by panning. HS29 has the same epitope, antiproliferative activity, and binding affinity as G1A8 (Fig. 8).

Example 5. Efficacy in vitro and in vivo.

In in vitro activity assays, G1A8 blocked FGF19-FGFR4 interaction with IC50 values of 1.39 nmol (Figure 4B). In addition, G1A8 showed a strong antiproliferative effect on Hep3B cells treated with exogenous FGF19 (Fig. 4A). Its in vivo antitumor activity was then assessed using xenograft mouse models (Figure 5). We are the first to create a Hep3B-Luc23 cell line that stably expresses luciferase and thus allows tumor growth to be quantified using in vivo bioluminescence imaging. Mice were injected subcutaneously with these Hep3B-Luc23 cells into the right flank and then divided into three groups with equivalent mean tumor bioluminescence intensity before intraperitoneal antibody injections (200 μg G1A8 or isotype control antibody) administered twice weekly for four weeks. Monitoring tumor growth over time by measuring tumor volumes and relative bioluminescence intensities of tumor cells showed that G1A8 significantly suppressed tumor growth compared to the isotopic control antibody (Figures 5A-5C).

Antitumor efficacy against xenograft tumors formed from transplanted wild-type Hep3B cells was also assessed. Antibody therapy was started when tumor volumes reached approximately 100 mm ³ in size. Hep3B-carrying mice were divided into three groups with equivalent mean tumor volumes and received an intraperitoneal injection of antibody (200 μg G1A8 or isotype control antibody) twice weekly for three weeks. Consistent with the above results for Hep3B-Luc23 xenografts, G1A8 significantly suppressed Hep3B tumor progression ( Fig. 5D ). Moreover, G1A8 treatment significantly prolonged the survival of mice compared with the isotype control antibody group (Figure 5E). HS29 exhibited potent antiproliferative activity similar to that of G1A8 (Fig. 8C). The antitumor activity of HS29 was examined in a patient-derived xenograft tumor model (Fig. 9A). HS29 therapy was initiated when the mean tumor volume was approximately 150 mm ³ . Mice that received an intraperitoneal injection of HS29 (10 mg/kg) showed suppressed tumor growth compared to control mice. Notably, the control mice experienced a significant decrease in body weight as the tumors continued to grow, and two of them died due to poor physiological condition due to tumor progression; whereas mice treated with HS29 maintained constant body weight and remained healthy (Figure 9D). These data support the antitumor activity of HS29 both in vitro and in vivo.

Example 6: Safety assessment in mice and cynomolgus monkeys.

The antibody of the present application, namely G1A8, was found to have no cross-reactivity with mouse FGF15, an ortholog of human FGF19, which shares only 49% amino acid identity with FGF19 (T. J. Wright et al., Dev. Biol., 269, 264-275 (2004)). In view of this observation, the aforementioned tumor xenograft mouse models may not be suitable for assessing the safety profiles of G1A8 therapy. However, keeping in mind that human FGF19 may exert its function in regulating bile acid metabolism in mice through

- 17 046377 murine FGFR4 suppresses CYP7A1 transcription in the liver (M. Zhou et al., 2014, supra; R. Goetz et al., 2007, supra) (Fig. 1C), this mouse model was used to evaluate whether G1A8 on CYP7A1 transcription in the liver. Surprisingly, G1A8 was found to have no effect on FGF19-induced transcriptional repression of CYP7A1 in the liver ( Fig. 4C ), indicating that G1A8 does not appear to affect the bile acid metabolism regulatory function of FGF19. A previous study (Pai et al., Toxicological sciences: an official journal of the Society of Toxicology, 126, 446-456 (2012)) showed that administration of a humanized antibody to FGF19 (1A6) to cynomolgus monkeys caused disruption of bile acid metabolism due to dysfunction FGF19 and increased CYP7A1 gene expression with the onset of clinical symptoms (decreased body weight, low food intake, severe diarrhea) and ultimately led to unscheduled euthanasia of all animals in the cynomolgus macaque treatment groups receiving 10 and 30 mg/kg, respectively. The amino acid sequences of cynomolgus FGF19 and human FGF19 are almost identical (98% amino acids identical). We also tested the cross-reactivity of the antibodies of the present application and confirmed that G1A8 binds to both cynomolgus FGF19 and human FGF19 with similar binding affinities (FIGS. 3A and 6L). Thus, cynomolgus monkeys were considered a good model to evaluate the safety profile of G1A8, particularly if it disrupts the regulatory function of FGF19 bile acid metabolism. Four treatment-naïve cynomolgus monkeys were randomized into two groups, one receiving saline control intravenously, the other receiving 10 mg/kg G1A8 on day 1 and 30 mg/kg G1A8 on day 16. Each group included one male and one female. Blood samples were collected throughout the study (Fig. 6A). All monkeys completed treatment with two doses of G1A8, and none showed any of the clinical side effects that have been reported for 1A6 (which binds to the C terminus of FGF19), including weight loss, low food intake, and loose stools. or diarrhea (Fig. 6B). A previous single-dose study of 1A6 therapy in monkeys reported marked increases in serum levels of total bile acids, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Pai et al., Toxicological sciences: an official journal of the Society of Toxicology, 126, 446-456 (2012)). Although monkeys in the G1A8 treatment group initially showed a slight increase in total serum bile acid levels on day 15 (1st time point tested) after treatment with G1A8 (10 mg/kg), there was a further increase after the second administration when higher dose (30 mg/kg), no observed on day 16; on the contrary, a decrease in this indicator was observed (Fig. 6C). This result suggests that the slight increase in serum bile acid levels observed at day 15 can likely be explained by normal physiological changes in this parameter in animals. There was no significant difference in total bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels between monkeys that received saline control and monkeys that received G1A8, indicating that there was no liver damage caused by G1A8 administration (Fig. 6 , 3rd).

We also collected monkey tissue samples from organs (liver, ileum, and kidney) responsible for bile acid recycling to assess the expression levels of several genes known to influence bile acid metabolism (Figures 6D-6F) . The livers of G1A8-treated animals did not show an increase in CYP7A1 gene expression, and the expression of other genes known to encode bile acid transport proteins did not show a significant increase compared with control animals (Fig. 6D). In the kidney and ileum, the expression of bile acid transporter genes was not different between the G1A8-treated group and the control group (Figures 6F, 6G), indicating that bile acid recycling and metabolism were maintained at normal physiological levels. Average concentration time courses for the two different doses of G1A8 showed similar terminal half-lives of G1A8 of 174.29 and 188.38 hours in cynomolgus monkeys dosed at 10 and 30 mg/kg, respectively (Figures 6J and 6K).

All of these safety studies in cynomolgus monkeys did not reveal toxicity due to effects on bile acid metabolism associated with G1A8 administration, and thus suggested that treatment with G1A8 in a therapeutic setting would not likely result in the development of significant malabsorption bile acids or other side effects associated with bile acid metabolism.

Example 7. Structural analysis of the FGF19-G1A8 complex.

The structure of the Fab version of G1A8 in complex with FGF19 was determined using X-ray crystallography at 2.6 resolution to understand the molecular basis of the interaction between the antibody of the present application and FGF19. Antibody G1A8 was selected for this analysis.

The structure was determined using the molecular replacement method and refined (R _work /R _fre e=0.216/0.278), its geometric properties were determined to be good. In the structure of FGF19 and G1A8, residues 37–172 of FGF19 were modeled with a well-defined electron density. The structure of FGF19 and G1A8 demonstrates outwardly ideal complementarity of form, occupying a common space

- 18 046377 area 983 A2. The hidden surface area for the heavy chain (VH) and light chain (VL) variable domains of G1A8 is almost identical (495 and 488 A ² ). Most of the residues constituting the G1A8 epitope are the N termini of FGF19 and are located within the fragment of SEQ ID NO: 2, consisting of amino acid residues 38-45 of SEQ ID NO: 1, which is located upstream of the cleft between VH and VL (Fig. 7A and 7C) . This finding is consistent with the designed property of G1A8 to specifically target the N terminus of FGF19, as described above.

Table 1

Crystallography data

FGF19-G1A8 Fab Data set Wavelength (A) 0.9789 Space group P212121 Unit cell parameters a, b, s (A) 85.0, 104.1, 165.4 α, β,γ( ^ο ) 90.0, 90.0, 90.0 Resolution (A) 48.73-2.6 (2.69 -2.60) Completeness(%) 99.7 (99.0) <Ι/σ(Ι)> 24.3 (3.3) CCi/ ₂ (%) 99.7 (95.2) Rmerge (%) 1.7(14.5) Plurality 2.0 (2.0) Optimization Resolution (A) 48.7-2.6 (2.66 -2.60) Copies 45834 (3207) Rwork/Rfree 20.0/25.2 (35.1/40.1) Atoms 8740 Debye-Waller factor (A ² ) 63.9 Standard deviation Link length 0.005 Bond angle 0.833 Ramachandran Map Percentage of residues in preferred area 96.37% Percentage of residues within acceptable region 3.63% Complete marginal balances 0

*Values in parentheses are for the highest electronic level.

More specifically, eight residues from positions 38-45 of FGF19 are involved in extensive interactions with all six loops of the complementarity determining regions from both VH and VL of G1A8 through a combination of hydrophobic and polar contacts, as shown in FIG. 2 and table. 2. Boko

-

Claims (12)

The chain of the Trp38 residue of FGF19 at the N-terminus is anchored in a hydrophobic pocket formed by Ala33, Ser52, Ser57, Tyr59, Gly102 and Leu104 from HCDR1-3, while Arg46 of FGF19 on the other side takes part in salt bridge interactions with Gly103 and Asp52 from HCDR3 and LCDR2, respectively (Fig. 7C). The FGF19/Trp38Ala mutant was shown to have reduced binding affinity for G1A8, and mutation of Arg45 to Ala resulted in complete loss of binding (Fig. 7D), confirming the importance of these interactions. FGF19 Arg45 also creates an additional hydrogen bonding interaction with Acn100 from HCDR3 (Figure 7C). The G1A8-related antibody 31A3 has Val at the same position, a difference that likely accounts for the lower affinity of 31A3 than G1A8 (Figs. 2 and 3A). The structure of the G1A8-FGF19 complex also showed that residues 168–172 of FGF19 at the C terminus are in close proximity to the N terminus at the interface between FGF19 and G1A8. The structure of the G1A8 FGF19 complex also revealed that residues 168–172 of FGF19 at the C terminus are in close proximity to the N terminus at the interface between FGF19 and G1A8. The Le169 residue of FGF19 at the C-terminus is in line with the N-terminus and forms a hydrophobic interaction with Tyr51 and Pro57 from LCDR2. The hydrogen bond between Tyr51 and the carbonyl group of the ECE19/Pro167 framework further stabilizes the C-terminal interaction of FGF19. Replacement of Leu169 with Ala results in a decrease in binding affinity for the antibody due to an increase in the dissociation rate (Fig. 7D). table 2 Contact residues between G1A8 and FGF19 G1A8 HCDR1 HCDR2 HCDR3 LCDR 3 LCDR2 LCDR3 AZZ S52 S57 N100 W101 Q102 E103 L104 Y34 Y51 D52 P57 W93 R95 FGF19 W38 W38 W38 R45 142, L44, E81 W38, 142 R45 W38 P41, R43 R45, P167 L169 R45 L169 P41 D40 * Contact residues with interatomic distances less than 4 A are summed. The underlined residues are involved in the formation of hydrogen bonds or salt bridges. CLAIM 1. An antibody or antigen-binding fragment thereof that binds to human fibroblast growth factor 19 (FGF19), containing any of (a), (b) and (c): (a) a heavy chain variable domain (VH) comprising 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, and a light chain variable domain (VL) containing 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; (b) a heavy chain variable domain (VH) comprising 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, and a light chain variable domain (VL) containing 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 (c) a heavy chain variable domain (VH) comprising 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, and a light variable domain chain (VL) containing 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. 2. The antibody or antigen binding fragment of claim 1, comprising a heavy chain variable domain having at least 95, 96, 97, 98, 99 or 100% 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%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, 16 or 21. 4. The antibody or antigen binding fragment of claim 1, comprising (a) a heavy chain variable domain having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, and - 20046377 light chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10; (b) a heavy chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain having at least 95, 96 , 97, 98, 99 or 100% sequence identity to the amino acid sequence SEQ ID NO: 16; or (c) a heavy chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 20, and a light chain variable domain having at least 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence SEQ ID NO: 21. 5. The antibody or antigen-binding fragment according to claim 1, comprising (a) a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 9, and a light chain variable domain containing 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. 6. The antibody or antigen binding fragment of claim 1, comprising a heavy chain having at least 95, 96, 97, 98, 99 or 100% 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, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, 18 or 23. 8. An antibody or antigen binding fragment according to claim 1, comprising (a) a heavy chain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 11, and a light chain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 12; (b) a heavy chain having at least 95, 96, 97, 98, 99 or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17, and a light chain having at least 95, 96, 97, 98 , 99 or 100% sequence identity to the amino acid sequence SEQ ID NO: 18; or (c) a heavy chain having at least 95, 96, 97, 98, 99 or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 22, and a light chain having at least 95, 96, 97, 98, 99 or 100% sequence identity to amino acid sequence SEQ ID NO: 23. 9. The antibody or antigen-binding fragment according to claim 1, comprising (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 11, and a light chain containing the amino acid sequence of SEQ ID NO: 12; (b) a heavy chain containing the amino acid sequence of SEQ ID NO: 17, and a light chain containing the amino acid sequence of SEQ ID NO: 18; (c) a heavy chain containing the amino acid sequence of SEQ ID NO: 22, and a light chain containing the amino acid sequence of SEQ ID NO: 23. 10. The antibody or antigen binding fragment of any one of the preceding claims, wherein the antigen binding fragment is a Fab, Fab', F(ab')2, Fv, single chain Fv (scFv) or disulfide stabilized Fv (dsFv). 11. An antibody or antigen-binding fragment as claimed in any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof has at least one of the following properties: (I) binds to human FGF19 with a K _D value of from about 1x10 -9 M to about 1x10 ^-12 M, determined by surface plasmon resonance (eg, BIACORE) or similar method; (ii) cross-reacts with cynomolgus FGF19; (III) blocks the binding of human FGF19 to human FGFR4 and/or to the human FGFR4-KLB complex; (IV) suppresses FGF19-induced cell proliferation; (V) has no or minimal effect on FGF19-suppressed CYP7A1 expression; and (VI) does not or slightly disrupt bile acid homeostasis maintained by FGF19. 12. An isolated nucleic acid molecule containing a nucleotide that encodes an antibody -