treatment method, isolated and anti-fgfr1 antibodies, isolated nucleic acid, host cell, method of pr

专利摘要:
METHOD OF TREATING A METABOLIC DISEASE OR CONDITION, ANTI-BODY, ANTI-FGFR1 ANTIBODIES, NUCLEIC ACID, HOST CELL, METHOD OF PRODUCTION OF AN ANTIBODY, PHARMACEUTICAL FORMULATION, USE OF THE ANTIBODY AND METHOD OF USING AGONIS TA DO FGFR1The invention provides FGFR1 agonists, including anti-FGFR1 agonist antibodies and methods of using them.
公开号:BR112013026266A2
申请号:R112013026266-4
申请日:2012-05-15
公开日:2020-11-10
发明作者:Junichiro SONODA；Yan Wu
申请人:Genentech, Inc；
IPC主号:

专利说明:

[001] [001] The current application contains a List of Sequences that was presented in ASCII format through EFS-Web and is fully incorporated into this application as a reference. The said copy of ASCII, created on April 27, 2012 is called P4653RUS.txt and is 26,083 bytes in size.
[002] [002] This application claims priority benefit to United States patent applications 61 / 486,731, filed on May 16, 2011, and 61 / 536,936, filed on September 20, 2011, the entire content of which is incorporated this application as a reference in its entirety. FIELD OF THE INVENTION
[003] [003] The present invention relates to FGFR1 agonists and methods of using them. BACKGROUND OF THE INVENTION
[004] [004] The inability to control blood glucose levels forms the basis of a variety of metabolic conditions. Diabetes is a hyperglycemic syndrome that results from a defect in insulin secretion in response to glucose (type 1 and type 2 diabetes) and effectiveness in decreasing insulin in the absorption of glucose in stimulating skeletal muscle and in the production of restrictive liver glucose ( type diabetes). Diabetes is a highly prevalent disease and, although therapeutic options are available for some diabetics, there is an urgent need for additional therapies.
[005] [005] Fibroblast growth factor 21 (FGF21) is a member of the endocrine FGF subfamily, which includes FGF19 and FGF23, and has been identified as a potential disease modifying agent for reversing obesity and obesity-induced hyperglycemia and hepatosteatosis (see , for example, Kharitonenkov and Larsen, Trends Endocrinol.
[006] [006] The invention is based, in part, on the discovery that FGFR1 activation improves diabetes. The invention provides FGFR1 agonists, including anti-FGFR1 agonist antibodies and methods of using them.
[007] [007] In one aspect, the invention provides a method of treating a metabolic disease or condition in an individual, which comprises administering to the individual an effective amount of an antifibroblast growth factor receptor (FGFR1) agonist, wherein the metabolic disease is selected from the group consisting of: polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, type diabetes 2, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), and maturity diabetes with onset in youth (MODY). In some embodiments, the FGFR1 agonist does not activate FGFR2 or FGFR3. In some embodiments, the FGFR1 agonist is an anti-FGFR1 antibody. In some embodiments, the anti-FGFR1 antibody has two FGFR1 binding sites, for example, an entire antibody or an F (ab ') 2 fragment. In some embodiments, the antibody binds to the peptide at 26 KLHAVPAAKTVKFKCP (SEQ ID NO: 28) or to the peptide at 28 FKPDHRIGGYKVRY (SEQ ID NO: 29). In some embodiments, the antibody binds to both peptide 26 and peptide 28. In some embodiments, the anti-FGFR1 antibody binds to both FGFR1b and FGFR1c. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody also binds to beta-Klotho.
[008] [008] In another aspect, the invention provides an isolated antibody that binds to FGFR1, wherein the antibody is an agonist of FGFR1 activity. In some embodiments, the antibody is neither an FGFR2 nor FGFR3 agonist. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody comprises (a) HVR-H3 which comprises an amino acid sequence selected from the group consisting of SSGYGGSDYAMDY (SEQ ID NO: 16), SGYGGSDYAMDY (SEQ ID NO: 17), EHFDAWVHYYVMDY (SEQ ID NO: : 18), TGTDVMDY (SEQ ID NO: 19) and GTDVMDY (SEQ ID NO: 20), (b) HVR-L3 comprising an amino acid sequence QQSYTTPPT (SEQ ID NO: 23), and (c) HVR-H2 comprising an amino acid sequence selected from the group consisting of X1X2IX3PX4DGX5TX6YADSVKG, where X1 is A or G, X2 is D or E, X3 is D or Y, X4 is N or Y, X5 is A or D, and X6 is D or Y (SEQ ID NO: 24) and X1IX2PX3DGX4TX5YADSVKG, where X1 is D or E, X2 is D or Y, X3 is N or Y, X4 is A or D, and X5 is D or Y (SEQ ID NO : 25). In some embodiments, the antibody comprises (a) HVR-H1 which comprises the amino acid sequence GFTFX1X2X3X4IX5, where X1 is S or T, X2 is N or
[009] [009] In some embodiments, the anti-FGFR1 antibody has two FGFR1 binding sites, for example, an entire antibody or an F (ab ') 2 fragment. In some embodiments, the antibody of the invention is a multispecific antibody. In some embodiments, the antibody also binds to beta-Klotho. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the invention provides an isolated nucleic acid that encodes an antibody of the invention. In some embodiments, the invention provides a host cell that comprises the nucleic acid according to claim 21. In some embodiments, the invention provides a method of producing an antibody that comprises culturing the host cell according to claim 22 , so that the antibody is produced. In some embodiments, the method further comprises recovering the antibody from the host cell.
[010] [010] In some embodiments, the invention provides a pharmaceutical formulation comprising an antibody of the invention and a pharmaceutically acceptable carrier.
[011] [011] In some embodiments, the invention provides an antibody of the invention for use as a medicament. In some embodiments, the antibody of the invention is for use in the treatment of a metabolic disease or condition selected from the group consisting of: polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH) , non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), and diabetes maturity beginning in youth (MODY). In some embodiments, the antibody of the invention is for use in sensitizing an individual to insulin.
[012] [012] In some embodiments, the invention provides the use of an antibody of the invention in the manufacture of a medicament. In some embodiments, the drug is for the treatment of a metabolic disease or condition selected from the group consisting of: polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), liver disease non-alcoholic fatty (NAFLD), hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), and diabetes maturity beginning in youth (MODY). In some embodiments, the drug is to sensitize an individual to insulin.
[013] [013] In some embodiments, the invention provides a method of treating diabetes in an individual, which comprises administering to the individual an effective amount of an antibody of the invention. In some embodiments, the method further comprises administering to the individual another agent to treat diabetes, as long as the other agent is not insulin. In some embodiments, the method further comprises administering to the individual an agent to treat cardiovascular disease. BRIEF DESCRIPTION OF THE FIGURES
[014] [014] Figure 1A shows an ELISA that measures the binding of anti-FGFR1 antibodies to purified FGFR ECD fragments.
[015] [015] Figure 1B shows surface plasmon resonance binding constants for R1MAb1 and R1MAb2.
[016] [016] Figure 1C shows a GAL-Elk1 luciferase assay in rat L6 cells. The cells were cotransfected with expression vectors for the FGFR isoform indicated together with GAL-Elk1, Renilla Luciferase SV40 and Gal responsive firefly luciferase reporter. The transfected cells were incubated with media containing increasing concentrations of R1Mab or acidic FGF (aFGF: positive control) for 6 hours before luciferase assays. Transcriptional activation was assessed by the relative firefly luciferase activity normalized by renilla luciferase activity and expressed as a relative luciferase unit (RLU).
[017] [017] Figure 1D shows an experiment similar to 1C, except that L6 cells expressed both FGFR1c and KLB.
[018] [018] Figure 1E shows an experiment similar to 1C, except that HEK293 cells were used.
[019] [019] Figure 1F shows western blot analysis of 3T3-L1 adipocytes treated with protein indicated at 0.5 µg / ml for the indicated time.
[020] [020] Figure 1G shows WAT taken from lean C57BL / 6 mice at the time indicated after i.p. at 1mpk of R1Mab (+) or control IgG (-), and submitted to western blot analysis.
[021] [021] Figure 2A shows blood glucose (left) and body weight (right) of db / db mice after a single i.p. of R1Mab1 (arrow) or control IgG at the indicated doses. Significance was observed in glucose (against control IgG) between days 1 to 30 for all groups and in weight (against control IgG) on day 8 for all groups, and between days 12 and 18 for group 50 mpk. N = 6 to approximately 14, * p <0.05, ** p <0.01.
[022] [022] Figure 2B shows blood glucose in randomly fed and fasted overnight mice (upper) and serum insulin levels after an overnight fast, and 30 minutes after i.p. of 1g / kg of glucose (lower). N = 6 to approximately 14, * p <0.05, ** p <0.01.
[023] [023] Figure 2C shows blood glucose (left) and body weight (right) of Ins2Akita mice after a single i.p. of R1Mab1 or control IgG at 3 mpk. N = 10. * p <0.01.
[024] [024] Figure 2D shows food intake (left), blood glucose (center) and body weight (right) of db / db mice after a single i.p. of R1Mab1 or control IgG at 1 mpk. PF: pair fed to group treated with R1MAb. N = 7. #p <0.001 (against IgG), * p <0.001 (against PF-IgG).
[025] [025] Figure 2E shows the quantification of the positive area for insulin in fixed pancreas sections. Tissues were collected on day 7 after a single i.p. 3 mpk (left) or 1 mpk (right) of R1MAb1 and stained for insulin and glucagon. N = 4 to approximately 7. ** P <0.002.
[026] [026] Figure 3A shows activation of MAPK signaling in mouse tissues. The indicated tissues were collected in 15 minutes (25 µg / mouse FGF21: superior) or 1 hour (R1Mab1 at 1 mpk: inferior) after i.p. of lean C57BL / 6 mice and submitted to Western blot analysis. PBS (upper) and IgG control (lower) were used as a negative control.
[027] [027] Figures B to D show representative H&E staining of liver (B), liver lipids (C) and serum lipids (D). Samples were collected on day 7 after a single i.p. from R1MAb1 to 1mpk.
[028] [028] Figure 3E shows metabolic parameters of ob / ob or transgenic mice ap2-SREBP1c (srebp) injected with Ab at 1mpk.
[029] [029] Figure 3F shows metabolic parameters of ap2-SREBP mice subcutaneously implanted with an osmotic pump for infusion of FGF21 (12 ng / day). ITT with i.p. at 1 U / kg was conducted on day 4. Serum was collected on day 6. N = 6 to approximately 8.
[030] [030] Figure 4A shows mRNA expression (red: highest expression and blue: lowest expression) in BAT for genes belonging to the indicated KEGG pathway. Samples were collected on day 4 after a single i.p. from R1MAb1 to 1mpk or mice fed in pairs injected with control IgG.
[031] [031] Figure 4B shows mRNA expression in BAT by qPCR. N = 6, * p <0.05, ** p <0.001.
[032] [032] Figure 4C shows a luciferase assay in HEK293 cells. The graph shows that both FGF21 and R1MAb1 induce transcription of a UAS-directed luciferase reporter gene in HEK293 cells via CREB fused to the GAL4 DNA binding domain (GAL-CREB) (two panels on the left), or a reporter gene of luciferase directed at CRE (two panels on the right), in a dose-dependent manner.
[033] [033] Figure 4D shows WAT collected 15 minutes after i.v. injection with 25 µg of FGF21 (+) or PBS (-), and submitted to western blot analysis.
[034] [034] Figure 4E shows western blot analysis of differentiated primary human adipocytes treated with FGF21 at 1 µg / ml for 30 minutes.
[035] [035] Figure 4F shows a model for the signaling pathway through which FGF21 and R1MAb activates the PGC-1alfa program in adipose tissues.
[036] [036] Figure 5 shows heparin-independent and FGFR1-dependent agonist activity of R1MAb1. The GAL-Elk1 luciferase assay in HEK293 cells. The cells were cotransfected with or without FGFR1c expression vectors as indicated, along with GAL-Elk1, renilla Luciferase SV40 and firefly luciferase reporter responsive to Gal. The transfected cells were incubated in medium containing increasing concentrations of R1Mab1 with or without 25 mg / L of pig heparin as indicated for 6 hours before the luciferase assays. Transcriptional activation was assessed by the relative firefly luciferase activity normalized by renilla luciferase activity and expressed as a relative luciferase unit (RLU).
[037] [037] Figure 6A shows food intake (left), body weight (center) w blood glucose (right) of db / db mice after a single i.p. of R1Mab2 or control IgG in doses of 3 mpk on day 0. Control mice were fed in pairs (PF) to adjust food intake until day 11. On day 11, food intake of mice treated with R1MAb2 returned to normal, and so all mice were fed at will after the 11th (AL). N = 7 to approximately 12. p <0.001.
[038] [038] Figure 6B shows the blood glucose level with the random feeding of mice used in Figure S2A on day 26.
[039] [039] Figure 6C shows GTT conducted using the same mice on day 28.
[040] [040] Figure 6D shows ITT conducted using the same mice on day 37.
[041] [041] Figure 7A shows blood glucose (left) and body weight (right) of ob / ob mice after a single i.p. of R1Mab1 or control IgG at doses of 1 mpk on day 0. The control mice were fed in pairs (PF) to the group treated with R1MAb. N = 7. * p <0.05 (against PF-IgG).
[042] [042] Figure 7B shows blood glucose (left) and body weight (right) of C57BL / 6 mice fed with HFD after a single i.p. of R1Mab1 or control IgG at doses of 1 mpk on day 0.
[043] [043] Figure 7C shows GTT conducted with HFD-fed mice used in S3B on day 8 after Ab injection. The mice were injected i.p. with 1g / kg of glucose after an overnight fast.
[044] [044] Figure 7D shows blood glucose (left) and body weight (right) of Ins2Akita mice on day 5 after a single i.p. of R1Mab1 or control IgG at 1 mpk. Control mice were fed in pairs (PF-IgG) to normalize body weight. * p <0.05.
[045] [045] Figure 8A shows the representative coloration of pancreatic islet in db / db mice analyzed in Figure 4E. Red: Insulin, Green: Glucagon, Blue: Cores. Note that R1MAb did not affect the general islet morphology.
[046] [046] Figure 8B shows the positive area distribution of insulin (%) in each islet, in each animal.
[047] [047] Figure 9A shows a schematic representation of IgG and IgG of an arm (OA). Blue: Heavy chain, Green: Light chain.
[048] [048] Figure 9B shows GAL-Elk-based luciferase assay on HEK293 cells expressing FGFR1c to compare R1MAb2 and the R1MAb2 DNA mutant (R1MAb2 DANA).
[049] [049] Figure 9C shows blood glucose in db / db mice before (pre) and on day 7 after i.p. of Ab indicated at 1 mpk. Control mice were fed in pairs to normalize body weight.
[050] [050] Figure 9D shows an ELISA measuring antibody binding to purified FGFR ECD fragments. OA-R1MAb1: OA-version of R1MAb1.
[051] [051] Figure 9E shows a GAL-Elk-based luciferase assay similar to S5B.
[052] [052] Figure 9F shows western blot analysis of 3T3-L1 adipocytes treated with protein indicated at 0.5 µg / ml for the indicated time.
[053] [053] Figure 9G shows blood glucose (left) and body weight (right) of db / db mice after a single i.p. Ab indicated on day 0 (arrow). N = 7. * p <0.05 (control IgG against R1MAb1 at 3 mpk), ** p <0.0005 (control IgG against R1MAb1 at 3 mpk and control IgG against R1MAb1 at 1mpk).
[054] [054] Figure 9H shows hepatic and serum lipids from samples that were collected on day 7 after Ab injection. N = 7. * p <0.05, ** p <0.0005 (against control IgG).
[055] [055] Figure 10 shows mRNA expression of KLB and FGFR isoforms in the liver and WAT. C57BL / 6 mice fed food and
[056] [056] Figure 11 shows the need for normal adipose function for FGF21 and R1MAb activity. Food intake (left), blood glucose (center) and body weight (right) of ob / ob or ap2-srebp1c transgenic mice after a single i.p. of R1Mab1 or control IgG at doses of 1 mpk on day 0. The same mice described Figure 3E. N = 7. #p <0.001 (against IgG), * p <0.001 (against PF-IgG). The post-treatment measurement was made 1 day after injection.
[057] [057] Figure 12A shows a HEK293 cell-based luciferase assay. The cells were cotransfected with expression vectors for indicated GAL fusion proteins, renilla luciferase SV40 and responsive luciferase reporter for GAL. Some cells were also cotransfected with KLB expression vector as indicated. The transfected cells were incubated with medium containing conditioned medium from HEK293 cells transfected with expression vector for FGF21 or empty control vector as indicated. After 6 hours of incubation, the cells were subjected to luciferase assays. Transcriptional activation was assessed by the relative activity of firefly luciferase normalized by renilla luciferase activity and expressed as number of inductions.
[058] [058] Figure 12B shows a similar luciferase assay where some cells were co-treated with the following nuclear receptor ligand: 1 M Wy14643 (PPAR), 5nM GW101516 (PPAR), 50nM rosiglitazone (PPAR), 50nM T0901317 (LXR ), 5nM T3 (TR), 30M CDCA (FXR). Note that FGF21 did not affect the activity of any of the nuclear receptors tested here with or without cognate ligand treatment.
[059] [059] Figure 12C shows western blot analysis of HEK293 cells treated with FGF21 at 0.5 µg / ml for 10 minutes. Some cells were pretreated with an inhibitor for FGFR (100nM PD173074), mTOR (100nM rapamycin) MEK1 / 2 (10 M U0126), PI3K (1 M wortmannin) as indicated.
[060] [060] Figure 12D shows downstream genes involved in oxidative metabolism in adipose tissues.
[061] [061] Figure 13 shows blood glucose (upper) and body weight (lower) of db / db mice after a single i.p. R1Mab2 (marked 182.2), R1Mab3 (marked 182.5) or control IgG (anti-Her2) at the indicated doses. N = 6, * p <0.05, ** p <0.005, *** p <0.0005. R1MAb3 comprises VH comprising SEQ ID NO: 6 and VH comprising SEQ ID NO: 4.
[062] [062] Figure 14A shows cumulative food intake, blood glucose and body weight change in lean C57BL / 6 mice after a single intraperitoneal injection of R1MAb1 or control IgG at 0.5 mpk. The mice were also implanted subcutaneously with a mini osmotic pump on day 0 for continuous infusion (c.i.) of recombinant FGF21 at 1.2 mpk / day or control vehicle (PBS). On day 3, the mice fasted overnight to conduct a glucose tolerance test (GTT) on day 4 (arrow).
[063] [063] Figure 14B shows glucose tolerance tests conducted with 2 g / kg of glucose injected intraperitoneally on day 4 after an overnight fast.
[064] [064] Figure 14C shows analysis of the serum of mice shown in Figures 2C, S10A and S10B. Serum samples were collected on day 5 after 4 hours of fasting. The data represent the mean ± SEM with n = 6 mice per group; * p <0.05, ** p <0.01, *** P <0.001 against PBS (c.i.) + control IgG, by two-tailed Student t test (n.s. = not significant).
[065] [065] Figure-15 shows the analysis of gene expression with the use of mRNA isolated from liver tissue of the mice used in Figures 2C and S10. Tissue samples were isolated on day 5 after 4 hours of fasting. The data represent the mean ± SEM with n = 6 mice per group; * p <0.05, ** p <0.001, *** P <0.001 against PBS (c.i.) + control IgG, by two-tailed Student t test (n.s. = not significant).
[066] [066] Figure-16 shows the analysis of gene expression with the use of mRNA isolated from the brown adipose tissue of the mice used in Figures 2C, S10 and S11. Tissue samples were isolated on day 5 after 4 hours of fasting. The data represent the mean ± SEM with n = 6 mice per group; * p <0.05, ** p <0.001, *** P <0.001 against PBS (c.i.) + control IgG, by two-tailed Student t test (n.s. = not significant).
[067] [067] Figure 17A shows ELISA results measuring antibody binding to biotinylated peptide fragments.
[068] [068] Figure 17B shows the amino acid sequences of FGFR1 (amino acids 161 to 212; SEQ ID NO: 27), the amino acid sequences of peptide no 26 (SEQ ID NO: 28) and peptide no 26 (SEQ ID NO: 29) together with the amino acids corresponding to peptide no. 26 of FGFR2 (SEQ ID NO: 30), FGFR3 (SEQ ID NO: 31) and FGFR4 (SEQ ID NO: 32) and the amino acids corresponding to peptide no. 28 of FGFR2 (SEQ ID NO: 33), FGFR3 (SEQ ID NO: 34) and FGFR4 (SEQ ID NO: 35). The differences between the sequences of peptides # 26 and peptides # 28 in FGFR1 and the corresponding region of FGFR2-4 are boxed.
[069] [069] Figure 17C shows ELISA results that measure the binding of His-tagged FGFR1 to the FGF2 protein in the presence of various concentrations of control R1MAb1 or IgG. Data are expressed as% His binding to FGFR1 and represent means ± SEM (n = 3).
[070] [070] Figure 17D shows the binding of iodinated FGF21 to HEK293 cells that stably express both KLB and FGFR1c in the presence of various concentrations of unlabeled R1MAb1 or FGF21 (the reaction also contained BSA (10 mg / ml) and control IgG ( 350 mM) to block non-specific binding Data are expressed as% bound FGF21 of the total radiolabelled FGF21 in the reaction. DETAILED DESCRIPTION OF THE ACCOMPLISHMENTS OF THE INVENTION I. DEFINITIONS
[071] [071] An "accepting human structure" for the purposes of the present application is a structure comprising the amino acid sequence of a light chain variable domain (VL) structure or a heavy chain variable domain (VH) structure derived from from a human immunoglobulin structure or a human consensus structure, as defined below. An accepting human structure "derived from" a human immunoglobulin structure or a human consensus structure may comprise the same amino acid sequence as this one or may contain exchanges in the amino acid sequence. In some embodiments, the number of amino acid exchanges is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less. In some embodiments, the human VL acceptor structure is identical in sequence to the human immunoglobulin VL structure sequence or human consensus structure sequence.
[072] [072] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise indicated, as used in the present application, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, antibody and antigen).
[073] [073] The term "anti-FGFR1 agonist antibody" refers to an antibody that is capable of binding to FGFR1 with sufficient affinity, so that the antibody is useful as a therapeutic agent in activating FGFR1.
[074] [074] The term "antibody" in the present application is used in the broadest sense and encompasses several antibody structures, which include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) and fragments of antibodies, as long as they exhibit the desired antigen binding activity.
[075] [075] An "antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds.
[076] [076] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or the light chain is derived from a specific source or species, while the remainder of the heavy chain and / or the light chain is derived from a different source or species.
[077] [077] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these can also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called,,,, and, respectively.
[078] [078] "Effector functions" refer to the biological activities attributable to the Fc region of an antibody, which vary with the isotype of the antibody.
[079] [079] An "effective amount" of an agent, for example, a pharmaceutical formulation, refers to an effective amount in dosages and for periods of time necessary to achieve the desired therapeutic or prophylactic result.
[080] [080] The term "Fc region" in the present application is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, an Fc region of the human immunoglobulin heavy chain extends from Cys226 or Pro230 to the carboxyl termination of the heavy chain. However, C- lysine
[081] [081] "Structure" or "FR" refers to residues of variable domain, except residues of the hypervariable region (HVR). The RF of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4.
[082] [082] The terms "whole antibody," "intact antibody" and "whole antibody" are used interchangeably in the present application to refer to an antibody that has a structure substantially similar to a native antibody structure or that has heavy chains containing an Fc region as defined in this application.
[083] [083] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to the cells into which the exogenous nucleic acid was introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells", which include the transformed primary cell and the progeny derived from them without regard to the number of passages. The progeny may not be completely identical to a parental cell in the nucleic acid content, but it may contain mutations. Mutant progenies that have the same biological function or activity as those screened or selected for the originally transformed cell are included in the present application.
[084] [084] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human cell or a human cell derived from a non-human source that uses repertoires of human antibodies or other sequences that encode human antibodies . This definition of a human antibody specifically excludes a humanized antibody that comprises non-human antigen binding residues.
[085] [085] A "human consensus structure" is a structure that represents amino acid residues most commonly occurring in a selection of human immunoglobulin VL or VH structure sequences.
[086] [086] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all HVRs correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody can optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has been subjected to humanization.
[087] [087] The term "hypervariable region" or "HVR", as used in the present application, refers to each of the regions of an antibody variable domain that are hypervariable in the sequence and / or forms structurally defined as loops ("loops" hypervariable ”). Generally, antibodies of four native chains comprise six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or "complementarity determining regions" (CDRs), the latter of which is of higher sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2) and 96 to 101 (H3). (Chothia and Lesk, J. Mol. Biol.
[088] [088] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule (s).
[089] [089] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (for example, cows, sheep, cats, dogs and horses), primates (for example, human and non-human primates such as monkeys), rabbits and rodents (for example, mice and rats) ). In certain embodiments, the individual or subject is a human.
[090] [090] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, an antibody is purified to more than 95% or 99% purity, as determined, for example, by electrophoresis (for example, SDS-PAGE, isoelectric focusing (IEF), capillarity electrophoresis) or chromatography (for example , ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).
[091] [091] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
[092] [092] "Isolated nucleic acid encoding an anti-FGFR1 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody (or fragments thereof), including nucleic acid molecule (s) in a single vector or separate vectors, and that nucleic acid molecule (s) presents itself in one or more locations in a host cell.
[093] [093] The term "monoclonal antibody", as used in the present application, refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that comprise the population are identical and / or bind to the same epitope, except for possible variant antibodies, for example, which contain mutations that occur naturally or that arise during the production of a monoclonal antibody preparation, these variants are generally present in smaller quantities. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant in an antigen. Thus, the “monoclonal” modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be interpreted as a requirement for production of the antibody by any specific method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods using animals transgenics containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for producing monoclonal antibodies are described in the present application.
[094] [094] "Native antibodies" refer to naturally occurring immunoglobulin molecules with variable structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked by disulfide. From the end N to C, each heavy chain has a variable region (VH), also called a heavy variable domain or a variable domain of the heavy chain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from the termination N to C, each light chain has a variable region (VL), also called a light variable domain or a light chain variable domain, followed by a light constant domain (CL). An antibody light chain can be assigned to one of two types, called kappa () and lambda (), based on the amino acid sequence of its constant domain.
[095] [095] The term “package insert” is used to refer to the instructions included as usual in commercial packaging of therapeutic products, which contain information on the indications, use, dosage, administration, combination therapy, against indications and / or warnings regarding the use of these therapeutic products.
[096] [096] “Percentage (%) of amino acid sequence identity” in relation to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence , after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitution as part of the sequence identity. Alignment, for the purposes of determining the percentage of amino acid sequence identity, can be achieved in several ways, which are within the technique, for example, with the use of publicly available computer programs, such as BLAST computer programs, BLAST-2, ALIGN or Megalign (DNASTAR).
[097] [097] In situations where ALIGN-2 is used for comparisons of amino acid sequences, the% amino acid sequence identity of a given A amino acid sequence in relation to a given B amino acid sequence (which can alternatively be formulated as a given sequence of amino acids A which has or comprises a certain% amino acid sequence identity with respect to a given sequence of amino acids B) is calculated as follows: 100 times the X / Y fraction where X is the number of residues of amino acids scored as identical comparisons by the sequence alignment program ALIGN-2 in that program alignment of A and B, and where Y is the total number of amino acid residues in B. It is considered that when the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the% identity of amino acid sequence A with respect to B is not equal to the% identity of amino acid sequence
[098] [098] The term “pharmaceutical formulation” refers to a preparation that is in that form to allow the biological activity of an active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to a subject to whom the formulation would be administered.
[099] [099] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, except an active ingredient, which is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[0100] [0100] The term “Fibroblast Growth Factor Receptor 1 or FGFR1”, as used in the present application, refers to any FGFR1 native from any source of vertebrates, including mammals, such as primates (eg humans) and rodents (for example, mice and rats), unless otherwise indicated. The term encompasses unprocessed “whole” FGFR1, as well as any form of FGFR1 that results from processing in the cell. The term also encompasses naturally occurring FGFR1 variants, for example, junction variants or allelic variants.
[0101] [0101] As used in the present application, "treatment" (and grammatical variations of the same as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed both for prophylaxis and during the course of clinical pathology. The desired treatment effects include, but are not limited to, preventing the occurrence or recurrence of the disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, improving or palliating the state of disease and prognosis for improvement. In some embodiments, the antibodies of the invention are used to slow the development of a disease or to slow the progression of a disease.
[0102] [0102] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody, generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain of an antibody that binds to the antigen to select a library of complementary VL or VH domains, respectively.
[0103] [0103] The term "vector", as used in the present application, refers to a nucleic acid molecule capable of reproducing another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure, as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are functionally linked. These vectors are referred to in this application as “expression vectors”. II. COMPOSITIONS AND METHODS
[0104] [0104] In one aspect, the invention is based, in part, on the discovery of agonistic anti-FGFR1 antibodies and the therapeutic activity of those antibodies. The antibodies of the invention are useful, for example, for the treatment of metabolic diseases, including diabetes.
[0105] [0105] In one aspect, the invention provides isolated antibodies that bind FGFR1. In certain embodiments, an anti-FGFR1 antibody binds to FGFR1b and / or FGFR1c and agonizes FGFR1 activity.
[0106] [0106] In one aspect, the invention provides an anti-FGFR1 antibody comprising at least one, two, three, four, five or six HVRs selected from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 25; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 20; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 22; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 23.
[0107] [0107] In some embodiments, an anti-FGFR1 antibody can be completely human, humanized or non-human. In one embodiment, an anti-FGFR1 antibody comprises HVRs as in any of the above embodiments, and further comprises an accepting human structure, for example, a human immunoglobulin structure or a human consensus structure.
[0108] [0108] In another aspect, an anti-FGFR1 antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2, 3 or 4 as follows: EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTWISWVPGKGLEWVGEIDPY
[0109] [0109] In another aspect, an anti-FGFR1 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) that is at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 6 as follows: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASF
[0110] [0110] In another aspect, an anti-FGFR1 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 2, 3 or 4 and SEQ ID NO: 6, respectively, including post-translational modifications of those sequences.
[0111] [0111] In a further aspect of the invention, an anti-FGFR1 antibody according to any of the above embodiments is a monoclonal antibody, which includes a chimeric, humanized or human antibody. In one embodiment, an anti-FGFR1 antibody is an antibody fragment, for example, an Fv, Fab, Fab ', scFv, diabody or F (ab') 2 fragment. In another embodiment, the antibody is an entire antibody, for example, an intact IgG1 antibody or another class of antibody or isotype as defined in the present application.
[0112] [0112] In an additional aspect, an anti-FGFR1 antibody according to any of the above embodiments can incorporate any of the characteristics individually or in combination, as described in Sections 1 to 7 below:
[0113] [0113] In certain embodiments, an antibody provided in the present application has a dissociation constant (Kd) 1 M, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM (for example, 10 -8 M or less, for example, from 10-8 M to 10-13 M, for example, from 10-9 M to 10-13.
[0114] [0114] In one embodiment, Kd is measured by a radiolabeled antigen (RIA) binding assay performed with the Fab version of an antibody of interest and its antigen, as described by the assay below. The binding affinity of the Fab solution to the antigen is measured by balancing Fab with a minimum concentration of (125I) -type antigen in the presence of a series of unlabeled antigen titrations, then capturing the bound antigen with a coated plate with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)).
[0115] [0115] According to another realization, Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25ºC immobilized antigen CM5 chips at approximately 10 response units (UK). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), according to the instructions of the Supplier. The antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 µg / mL (approximately 0.2 µM) before injection at a flow rate of 5 µL / minute to achieve approximately 10 response units (RU ) of coupled protein. After the antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold Fab serial dilutions (0.78 nM to 500 nM) are injected into PBS with 20 0.05% polysorbate surfactant (TWEEN 20TM) (PBST) at 25ºC at a flow rate of approximately 25 µL / min. The association rates (kon) and dissociation rates (koff) are calculated using a simple Langmuir one-to-one connection model (BIACORE® Evaluation Program version 3.2) by simultaneously adjusting the association and dissociation sensorgram. The dissociation equilibrium constant (Kd) was calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the association rate exceeds 106 M-1 S-1 by the surface plasmon resonance assay above, then the association rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in the intensity of emission of fluorescence (excitation = 295 nm; emission = 340 nm, 16 nm passage range) at 25ºC of 20nM of an antigen antigen in PBS (Fab form), pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a flow stop spectrometer (Aviv Instruments) or an SLM-AMINCOTM 8000 series spectrometer (ThermoSpectronic) with a mixing crucible.
[0116] [0116] In certain embodiments, an antibody provided in the present application is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab ', Fab'-SH, F (ab ') 2, Fv and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pages 269-315 (1994); see also document WO 93/16185; and US patents 5,571,894 and 5,587,458. For discussion of Fab and F (ab ') 2 fragments that comprise epitope residues that bind to rescue receptors and have an increased half-life in vivo, see US patent 5,869,046.
[0117] [0117] Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, for example, patent EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Tribodies and tetribodies are also described in Hudson et al. Nat. Med. 9: 129-134 (2003).
[0118] [0118] Single domain antibodies are fragments that comprise all or a portion of the variable domain of the heavy chain or all or a portion of the variable domain of the light chain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, for example, US patent 6,248,516 B1).
[0119] [0119] Antibody fragments can be produced by various techniques including, but not limited to, proteolytic digestion of an intact antibody, as well as production by recombinant host cells (e.g., E. coli or phage), as described in this application .
[0120] [0120] In certain embodiments, an antibody provided in the present application is a chimeric antibody. Certain chimeric antibodies are described, for example, in US patent 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.
[0121] [0121] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while maintaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody comprises one or more variable domains, where, for example, HVRs, for example, CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will comprise at least a portion of a human constant region. In some embodiments, some RF residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (for example, the antibody from which the HVR residues are derived), for example, to restore or improve specificity or antibody affinity.
[0122] [0122] Humanized antibodies and methods for their manufacture are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332: 323- 329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86: 10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (which describe SDR graft (a-CDR)); Padlan, Mol. Immunol.
[0123] [0123] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the “best fit” method (see, for example, Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from the human antibody consensus sequence of a specific subgroup of variable regions of the heavy chain (see, for example, Carter et al. Proc.
[0124] [0124] In certain embodiments, an antibody provided in the present application is a human antibody. Human antibodies can be produced using several known techniques. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).
[0125] [0125] Human antibodies can be prepared by administering an immunogen to a transgenic animal, which has been modified to produce intact human antibodies or intact antibodies with variable human regions in response to antigenic stimulation. Such animals typically contain all or a portion of the human immunoglobulin loci, which replaces the endogenous immunoglobulin loci, or which are present outside the chromosome or randomly integrated into the animal's chromosomes. In these transgenic mice, the endogenous immunoglobulin loci was generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see, Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example,
[0126] [0126] Human antibodies can also be manufactured by methods based on hybridoma. Human myeloma and mouse human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, for example, Kozbor, J.
[0127] [0127] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human derived phage display libraries. These variable domain sequences can then be combined with a desired human constant domain. Techniques for deleting human antibodies from antibody libraries are described below.
[0128] [0128] The antibodies of the invention can be isolated by selecting combinatorial libraries for antibodies with the desired activity or activities. For example, several methods are known in the art for generating phage-displayed antibody libraries and selecting those libraries for antibodies that have the desired binding characteristics. These methods are reviewed, for example, in Hoogenboom et al.
[0129] [0129] In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be selected for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phage typically exhibits antibody fragments, either as single chain Fv fragments (scFv) or as Fab fragments. Libraries from immunized sources provide antibodies with high affinity for the immunogen without the need for hybridoma construction. Alternatively, the virgin repertoire can be cloned (for example, from human) to provide a single source of antibodies to a wide variety of non-self and self-antigens without any immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, virgin libraries can also be produced synthetically by cloning the rearranged V gene segments from stem cells and using PCR primers that contain a random sequence to code highly variable CDR3 regions and to perform in vitro rearrangement , as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications that describe human antibody phage libraries include, for example: US patent 5,750,373, and US patent publications 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764 , 2007/0292936 and 2009/0002360.
[0130] [0130] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments described in the present application.
[0131] [0131] In certain embodiments, an antibody provided in the present application is a multispecific antibody, for example, a bispecific antibody.
[0132] [0132] Techniques for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs that have different specificities (see, Milstein and Cuello, Nature 305: 537 (1983)) document
[0133] [0133] Antibodies genetically engineered with three or more functional antigen binding sites, including "octopus antibodies", are also included in the present application (see, for example, US patent 2006 / 0025576A1).
[0134] [0134] The antibody or fragment in the present application also includes a "double-acting FAb" or "DAF" comprising an antigen binding site that binds FGFR1 as well as a different antigen, for example, beta-Klotho (see, US patent 2008/0069820, for example).
[0135] [0135] In certain embodiments, sequences of variant amino acids of the antibodies provided in the present application are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Variant amino acid sequences of an antibody can be prepared by making appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, insertions and / or substitutions of residues within the antibody's amino acid sequences. Any combination of deletion, insertion and substitution can be done to arrive at the final construct, as long as the final construct has the desired characteristics, for example, antigen binding. A) VARIANT SUBSTITUTIONS, INSERTIONS AND DELETIONS
[0136] [0136] In certain embodiments, variant antibodies are provided that have one or more amino acid substitutions. Sites of interest for replacement mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "conservative substitutions". More substantial changes are provided in Table 1, under the heading “exemplary substitutions”, and as further described below, in reference to the side chain classes of amino acids. Amino acid substitutions can be introduced into an antibody of interest and products selected for a desired activity, for example, retained / enhanced antigen binding, decreased immunogenicity, and enhanced ADCC or CDC.
[0137] [0137] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acid: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence the orientation of the chain: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
[0138] [0138] Non-conservative substitutions will imply the exchange of a member of one of these classes for another class.
[0139] [0139] One type of substitutional variant involves substitution in one or more residues of a hypervariable region of a parent antibody (for example, a human or humanized antibody). Generally, the resulting variant (s) selected (s) to further study if there will be changes
[0140] [0140] Changes (for example, substitutions) can be made on HVRs, for example, to improve the affinity of the antibody. These changes can be made in HVR hotspots, that is, codon-encoded residues that mutate at high frequency during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol.
[0141] [0141] In certain embodiments, substitutions, insertions or deletions can occur within one or more HVRs as long as these changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (for example, conservative substitutions as provided in the present application) that do not substantially reduce binding affinity can be made in HVRs.
[0142] [0142] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scan mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a target residue or group of residues (for example, charged residues such as arg, asp, his, lys and glu) are identified and replaced with a neutral or negatively charged amino acid (for example, alanine or polyalanine) to determine whether the antibody interaction with the antigen is affected. In addition, substitutions can be introduced at amino acid locations that demonstrate functional sensitivity to the initial substitutions. Alternatively or in addition, a crystal structure of an antigen-antibody complex to identify points of contact between the antibody and the antigen. These contact wastes and neighboring wastes can be targeted or disposed of as candidates for replacement. Variants can be selected to determine whether they contain the desired properties.
[0143] [0143] Amino acid sequence insertions include amine and / or carboxyl termination fusions, ranging in length from a residue to polypeptides containing a hundred or more residues, as well as intrassequal insertions of single or multiple amino acid residues.
[0144] [0144] In certain embodiments of the invention, an antibody provided in the present application is altered to increase or decrease the extent to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by changing the amino acid sequence, such as that in which one or more of the glycosylation sites are created or removed.
[0145] [0145] Where the antibody comprises an Fc region, the carbohydrate attached to it can be changed. Native antibodies produced by mammalian cells typically comprise a branched biantenary oligosaccharide that is generally linked by an N bond to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). The oligosaccharide can include various carbohydrates, for example, mannose, N-acetyl glucosamine (GlcNAc), galactose and sialic acid, as well as a fucose linked to a GlcNAc in the "trunk" of the biantenary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide to an antibody of the invention can be made in order to create variant antibodies with certain enhanced properties.
[0146] [0146] In one embodiment, variant antibodies are provided having a carbohydrate structure devoid of fucose linked (directly or indirectly) to an Fc region. For example, the amount of fucose in that antibody can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain in Asn297, in relation to the sum of all glycostructures linked to Asn297 (eg complex, high mannose and hybrid structures) as measured by spectrometry of MALDI-TOF grease, as described in WO 2008/077546, for example.
[0147] [0147] Variant antibodies are provided later with divided oligosaccharides, for example, in which a biantenary oligosaccharide linked to the antibody's Fc region is divided by GlcNAc. These variant antibodies may have reduced fucosylation and / or enhanced ADCC function. Examples of such variant antibodies are described, for example, in WO 2003/011878 (Jean-Mairet et al.); US patent
[0148] [0148] In certain embodiments, one or more amino acid modifications can be introduced within the Fc region of an antibody provided in the present application, thereby generating a variant Fc region. The variant Fc region can comprise a human Fc region sequence (for example, a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (for example, a substitution) at one or more amino acid positions including that of a cysteine hinge.
[0149] [0149] In certain embodiments, the invention contemplates a variant antibody that has some, but not all, effector functions, which make it a desirable candidate for applications where the half-life of the antibody in vivo is still important for certain effector functions (such as complement and ADCC) not necessary or harmful. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody is devoid of binding to Fc R (then likely to be devoid of ADCC activity), but maintains FcRn binding capacity. The main cells for ADCC mediation, NK cells, express only Fc RIII, whereas monocytes express Fc RI, Fc RII and Fc RIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991).
[0150] [0150] Antibodies with reduced effector function include those with substitutions of one or more residues from the Fc region 238, 265, 269, 270, 297, 327 and 329 (US patent 6,737,056). These Fc mutants include Fc mutants with substitutions at two or more positions of amino acids 265, 269, 270, 297 and 327, including the also known as Fc mutant "DANA" with substitutions of residues 265 and 297 for alanine (US patent 7,332,581 ).
[0151] [0151] Certain variant antibodies with improved or reduced binding to FcRs are described. (See, for example, US patent
[0152] [0152] In certain embodiments, a variant antibody comprises an Fc region with one or more amino acid substitutions that enhance ADCC, for example, substitutions at positions 298, 333 and / or 334 of the Fc region (EU residue numbering).
[0153] [0153] In some embodiments, changes are made to the Fc region that result in altered C1q binding (ie, both improved and reduced) and / or Complement-Dependent Cytotoxicity (CDC), for example, as described in US patent 6,194,551 , WO 99/51642 and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0154] [0154] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of
[0155] [0155] See also, Duncan & Winter Nature 322: 738-40 (1988); US patent 5,648,260; US patent 5,624,821; and WO 94/29351 refer to other examples of variant Fc regions. D) VARIANT ANTIBODIES GENETICALLY PREPARED WITH CYSTEINE
[0156] [0156] In certain embodiments, it may be desirable to create antibodies genetically engineered with cysteine, for example, “thioMAbs”, in which one or more residues of an antibody are replaced by cysteine residues. In specific embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, the reactive thiol groups are thereby positioned at accessible antibody sites and can be used to conjugate the antibody to other components, such as drug components or ligand-drug components, to create an immunoconjugate, as further described in the present application. In certain embodiments, any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) from the Fc region of the heavy chain. Antibodies genetically engineered with cysteine can be generated as described, for example, in the US patent
[0157] [0157] In certain embodiments, an antibody provided in the present application can be further modified to contain additional non-proteinaceous components that are known in the art and readily available. Suitable components for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly- 1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (both homopolymers and random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propopylene glycol homopolymers, polypropylene oxide / ethyl oxide copolymers , polyoxyethylated polyols (for example, glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages, due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers bound to the antibody can vary, and if more than one polymer is attached, they can have the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations that include, but are not limited to, specific properties or functions of the antibody to be improved, whether the derived antibody will be used in a therapy by defined conditions, etc.
[0158] [0158] In another embodiment, conjugates of an antibody and non-proteinaceous components are provided that can be selectively heated by exposure to radiation. In one embodiment, the non-proteinaceous component is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci USA 102: 11600-11605 (2005)). Radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not harmful to ordinary cells, but that heat the non-proteinaceous component to a temperature at which cells close to the antibody's non-proteinaceous component are destroyed. B. RECOMBINANT METHODS AND COMPOSITIONS
[0159] [0159] Antibodies can be produced using recombinant methods and compositions, for example, as described in the US patent
[0160] [0160] For recombinant production of an anti-FGFR1 antibody, the nucleic acid encoding an antibody, for example, as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. This nucleic acid can be isolated and sequenced immediately using conventional procedures (for example, using oligonucleotide probes that are able to specifically bind to the genes encoding the antibody's heavy and light chains).
[0161] [0161] Host cells suitable for cloning or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells described in the present invention. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody and polypeptide fragments in bacteria, see, for example, US patents 5,648,237,
[0162] [0162] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are cloning or suitable expression in hosts for vectors that encode antibodies, including fungi and yeasts whose glycosylation pathways have been "humanized", resulting in the production of an antibody with a pattern of glycosylation partially or totally human. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).
[0163] [0163] Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
[0164] [0164] Plant cell cultures can also be used as hosts. See, for example, US patents 5,959,177, 6,040,498,
[0165] [0165] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed by SV40 (COS-7), human embryonic kidney lineage (293 or 293 cells as described, for example, in Graham et al., J. Gen Virol 36:59 (1977)); kidney cells from hamster cubs (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals N.Y.
[0166] [0166] The anti-FGFR1 antibodies provided in the present application can be identified, selected or characterized by their physical / chemical properties and / or biological activities by various assays known in the art.
[0167] [0167] In one aspect, an antibody of the invention is tested for its antigen binding activity, for example, by methods known as ELISA, Western blot, etc.
[0168] [0168] In one aspect, assays are provided to identify anti-FGFR1 antibodies from those that have agonistic activity. For example, biological activity may include the ability to activate signal transduction of specific pathways that can be measured, for example, by determining levels of phospho-FRS2a, phospho-MEK, phospho-ERK / MAPK, phospho-STAT3 or with the use of GAL-Elk1-based luciferase assays described in the present application (see also, for example, Wu et al. J. Biol.
[0169] [0169] The invention also provides immunoconjugates that comprise an anti-FGFR1 antibody of the present application conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth-inhibiting agents, toxins (for example, protein toxins, enzymatically active toxins of origin bacterial, fungal, plant or animal, or fragments thereof), or radioactive isotopes.
[0170] [0170] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC), in which an antibody is conjugated to one or more drugs including, but not limited to, a maytansinoid (see US patents 5,208,020, 5,416,064 and European patent EP 0 425 235 B1); an auristatin as a drug component monomethyluristatin DE and DF (MMAE and MMAF) (see US patents 5,635,483 and 5,780,588 and
[0171] [0171] In another embodiment, an immunoconjugate comprises an antibody, as described in the present application, conjugated to an enzymatically active toxin or fragment thereof including, but not limited to, diphtheria toxin, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII and PAP-S), inhibitor carantia momordica, curcine,
[0172] [0172] In another embodiment, an immunoconjugate comprises an antibody as described in the present application, conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When radioconjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, for example, tc99m or I123, or a rotating marker for nuclear magnetic resonance imaging (NMR) (also known as magnetic resonance imaging, mri), as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0173] [0173] Antibody conjugates and cytotoxic agents can be produced using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithium) propionate (SPDP), succinimidil-4- (N -maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional imidoester derivatives (like dimethyl adipimidate HCl), active esters (like disuccinimidyl suberate), aldehydes (like glutaraldehyde), bis-azide compounds (like bis- (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (like bis- (p-diazoniumbenzoyl) - ethylenediamine), diisocyanates (like toluene 2,6-diisocyanate) and bis-active fluorine compounds (like 1,5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Triamino pentaacetic acid 1-isothiocyanatobenzyl-3-methyldiethylene with carbon 14 (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotide to the antibody. See document WO 94/11026. The ligand can be a "cleavable ligand" that facilitates the release of a cytotoxic drug into the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Res. 52: 127-131 (1992); US patent
[0174] [0174] The immunoconjugates or ADCs in the present application are expressly contemplated, but are not limited to those conjugates prepared with crosslinking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP , SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidil- (4-vinylsulfone) benzoate) that are commercially available (for example, from Pierce Biotechnology, Inc., Rockford, IL., USA). E. METHODS AND COMPOSITIONS FOR DIAGNOSIS AND DETECTION
[0175] [0175] In certain embodiments, any of the anti-FGFR1 antibodies provided in the present application is useful for detecting the presence of FGFR1 in a biological sample. The term "detection", as used in the present application, encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue, such as brown adipose tissue, pancreatic tissue, liver tissue, white adipose tissue, and tumor tissue.
[0176] [0176] In one embodiment, an anti-FGFR1 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method is provided to detect the presence of FGFR1 in a biological sample. In certain embodiments, the method comprises placing the biological sample in contact with an anti-FGFR1 antibody as described in the present application under permissive conditions for binding the anti-FGFR1 antibody to FGFR1, and detecting whether a complex is formed between the anti-FGFR1 antibody and FGFR1. This method can be an in vitro or in vivo method. In one embodiment, an anti-FGFR1 antibody is used to select subjects eligible for therapy with an anti-FGFR1 antibody, for example, where FGFR1 is a biomarker for patient selection.
[0177] [0177] In certain embodiments, labeled anti-FGFR1 antibodies are provided. Markers include, but are not limited to, markers or components that are directly detected (such as fluorescent, chromophoric, electrodense, chemiluminescent and radioactive markers), as well as components, such as enzymes or ligands, that are detected indirectly, for example, through an enzymatic reaction or molecular interaction. Exemplary markers 32 14 125 include, but are not limited to, the radioisotopes P, C, I, 3 131 H el, fluorophores such as the rare earth or fluorescine chelates and their derivatives, rhodamine and its derivatives, dansila, umbeliferone, luciferases, for example example, firefly luciferase and bacterial luciferase (US patent
[0178] [0178] Pharmaceutical formulations of an anti-FGFR1 antibody as described in the present application are prepared by mixing that antibody which has the desired degree of purity with one or more pharmaceutically acceptable optional vehicles (Remington’s Pharmaceutical
[0179] [0179] Exemplary lyophilized antibody formulations are described in US patent 6,267,958. Aqueous antibody formulations include those described in US patent 6,171,586 and in document
[0180] [0180] The formulation in the present application may also contain more than one active ingredient as needed for the specific indication to be treated, preferably that with complementary activities that do not adversely affect one another. For example, it may also be desirable to provide a glp-1 analogue, a synthetic amylin, a glucagon receptor antagonist (for example, an anti-GCGR antibody) or leptin. These active ingredients are suitably present in combination in amounts that are effective for the intended purpose.
[0181] [0181] The active ingredients can be retained in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively, in drug delivery systems colloidal (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.
[0182] [0182] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices of which are in the form of molded articles, for example, films or microcapsules.
[0183] [0183] The formulations to be used for in vivo administration are generally sterile. Sterility can be easily accomplished, for example, by filtration through sterile filtration membranes. G. THERAPEUTIC METHODS AND COMPOSITIONS
[0184] [0184] Any of the agonistic anti-FGFR1 antibodies provided in the present application can be used in therapeutic methods.
[0185] [0185] In one aspect, an agonistic anti-FGFR1 antibody is provided for use as a medicine. In additional aspects, an agonistic anti-FGFR1 antibody is provided for use in the treatment of a metabolic disease. In certain embodiments, an agonistic anti-FGFR1 antibody is provided for use in a treatment method. In certain embodiments, the invention provides an agonistic anti-FGFR1 antibody for use in a method of treating an individual who has a metabolic disease which comprises administering to the individual an effective amount of the anti-FGFR1 antibody. In that embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, a glp-1 analogue, a synthetic amylin, a glucagon receptor antagonist (for example an anti-GCGR antibody) or leptin. An "individual", according to any of the above achievements, is preferably a human.
[0186] [0186] In a further aspect, the invention provides the use of an agonistic anti-FGFR1 antibody in the manufacture or preparation of a medicament.
[0187] [0187] In a further aspect, the invention provides a method for treating a metabolic disease. In one embodiment, the method comprises administering to an individual who has this metabolic disease, an effective amount of an agonistic anti-FGFR1 antibody. In this embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual", according to any of the above achievements, can be a human.
[0188] [0188] In a further aspect, the invention provides pharmaceutical formulations that comprise any of the agonistic anti-FGFR1 antibodies provided in the present application, for example, for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the agonistic anti-FGFR1 antibodies provided in the present application and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the agonistic anti-FGFR1 antibodies provided in the present application and at least one additional therapeutic agent, for example, as described below.
[0189] [0189] Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is a glp-1 analogue, a synthetic amylin, a glucagon receptor antagonist (e.g., an anti-GCGR antibody) or leptin.
[0190] [0190] These therapy combinations noted above encompass combined administration (when two or more therapeutic agents are included in the same formulation or separate), and separate administration, in which case, administration of the antibody of the invention can occur before, simultaneously and / or after administration of the additional therapeutic agent and / or adjuvant.
[0191] [0191] An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired, for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. The dosage can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or continuous. Various dosage schedules that include, but are not limited to, single or multiple administrations over various periods, bolus administration and pulse infusion are contemplated in the present application.
[0192] [0192] Antibodies of the invention would be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the specific dysfunction to be treated, the specific mammal to be treated, the clinical condition of each patient, the cause of the dysfunction, the location of distribution of the agent, the method of administration, the administration schedule and other factors known to medical specialists. The antibody does not need to be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of these other agents depends on the amount of antibody present in the formulation, the type of dysfunction or treatment and the other factors discussed above. These are generally used in the same dosage and with routes of administration as described in the present application, or about 1 to 99% of the dosages described in the present application, or in any dosage and by any route that is empirically / clinically determined to be appropriate.
[0193] [0193] For disease prevention or treatment, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and disease course, whether the antibody is administered for preventive or therapeutic purposes, prior therapies, the patient's medical history and response to the antibody, and the physician's diagnosis.
[0194] [0194] In another aspect of the invention, an article of manufacture is provided containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above. The article of manufacture comprises a container and a label or package insert in or associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags for IV solution, etc. Containers can be manufactured from a variety of materials, such as glass or plastic. The container contains a composition that is by itself or combined with another composition, effective for treating, preventing and / or diagnosing the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a bottle that has a pierceable cap for a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the recommended condition. In addition, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises an additional cytotoxic agent or otherwise a therapeutic agent. The article of manufacture in that embodiment of the invention may, furthermore, comprise a package insert indicating that the compositions can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), buffered saline phosphate, Ringer's solution and dextrose solution. In addition, it may include other materials desirable from a commercial and user perspective, including other buffers, thinners, filters, needles and syringes. III. EXAMPLES
[0195] [0195] The following are examples of methods and compositions of the invention. It is understood that several other achievements can be practiced, given the general description provided above.
[0196] [0196] We generate monoclonal antibodies specific for FGFR1 using phage display technology and human FGFR1b IgD2-D3 and c as antigens. Human antibody libraries in phage with synthetic diversity in the complementarity determining regions (H1, H2, H3, L3), which mimic the natural diversity of human IgG repertoire were used for panning. Fab fragments were displayed in a bivalent manner on the surface of M13 bacteriophage particles (Lee et al., J.
[0197] [0197] We also measured the binding affinity of anti-FGFR1 antibodies to FGFR1, were measured by Biacore / SPR using a T100 BIAcore ™ instrument as described (Liang et al., Above) with the following modifications. The mouse anti-human Fc antibody was first coated on a BIAcore ™ carboxymethylated dextran CM5 chip using direct coupling to amino groups following a procedure described by the manufacturer. The antibody was then captured on CM5 biosensor chips to achieve approximately 200 response units (RU). Binding measurements were performed using a 10 mM HEPES running buffer, pH 7.4, 150 mM NaCl, 0.005% P20 surfactant (HBS-P buffer). A 2-fold serial dilution of ECD-His FGFR1 protein was injected over a range of 1,550 nM in HBS P buffer at a flow rate of µL / minute at 25ºC. Association fees (Kon, per mol / s) and dissociation rates (Koff, per s) were calculated using a Langmuir one-to-one connection model (Evaluation BIAcore ™ version 3.2 computer program). The equilibrium dissociation constant (Kd, per mol) was calculated as the Koff / Kon ratio.
[0198] [0198] Two of the anti-FGFR1 antibodies that were identified as described above in independent experiments (designated in the present application as R1MAb1 and R1MAb2) bind FGFR1b and FGFR1c to a similar affinity, but not to any other FGFR isoform (Fig.
[0199] [0199] We performed experiments to map the FGFR1 epitope linked by R1MAb1 and R1MAb2. We synthesized portions represented by 30 peptides from FGFR1 sequences with an amino-terminal biotin marker and used them for ELISA binding assays. These peptide sequences were as follows: # 1: SSSEEKETDNTKPNPVAPY (SEQ ID NO: 36); # 2: PVAPYWTSPEKMEKKLHAV (SEQ ID NO: 37); # 3: KLHAVPAAKTVKFKCPSSG (SEQ ID NO: 38); # 4: CPSSGTPNPTLRWLKNGKE (SEQ ID NO: 39); # 5: KNGKEFKPDHRIGGYKVRY (SEQ ID NO: 40); # 6: YKVRYATWSIIMDSVVPSD (SEQ ID NO: 41); # 7: VVPSDKGNYTCIVENEYGS (SEQ ID NO: 42); # 8: NEYGSINHTYQLDVVERSP (SEQ ID NO: 43); # 9: VERSPHRPILQAGLPANKT (SEQ ID NO: 44); # 10: PANKTVALGSNVEFMCKVY (SEQ ID NO: 45); # 11: MCKVYSDPQPHIQWLKHIE (SEQ ID NO: 46); # 12: LKHIEVNGSKIGPDNLPYV (SEQ ID NO: 47); # 13: NLPYVQILKTAGVNTTDKE (SEQ ID NO: 48); # 14: TTDKEMEVLHLRNVSFEDA (SEQ ID NO: 49); # 15: SFEDAGEYTCLAGNSIGLS (SEQ ID NO: 50); # 16: SIGLSHHSAWLTVLEALEE (SEQ ID NO: 51); # 17: YWTSPEKMEKKLHAVPAAK (SEQ ID NO: 52); # 18: EKMEKKLHAVPAAKTVKFK (SEQ ID NO: 53); # 19: PAAKTVKFKCPSSGTPNPT (SEQ ID NO: 54); # 20: KFKCPSSGTPNPTLRWLKN (SEQ ID NO: 55); # 21: GTPNPTLRWLKNGKEFKPD (SEQ ID NO: 56); # 22: TLRWLKNGKEFKPDHRIGG (SEQ ID NO: 57); # 23:
[0200] [0200] We tested whether the anti-FGFR1 antibodies of the invention would have anti-diabetic activity using mouse models of diabetes. All mice were purchased from the Jackson Laboratory and kept in a pathogen-free animal facility at 21ºC under a standard 12 h light / 12 h dark cycle with access to food (a standard rodent food (Labdiet 5010, 12.7 % of calories from fat) or a high-carbohydrate, high-fat diet (Harlan Teklad TD.03584, 58.4% of calories from fat) and water ad libitum.The db / db mice on the C57BLKS / J background were female and the other mice were all male. All mice were used for injection at around 9 to 11 weeks of age, except for ap2-SREBP1c mice who were 8 months old (Fig. 3E) or 4 months old ( Fig. 3F) For continuous infusion of FGF21 protein, an osmotic pump (Alzet® 2001) was subcutaneously implanted. Glucose levels were measured using the OneTouch® Ultra® glucometer. For liver lipid analysis, the lipid was extracted according to the Folch method and resuspended in PBS containing 5% Triton X-100. Total cholesterol, triglyceride, -hydroxybutylate (Thermo DMA) and non-esterified fatty acid (Roche) were determined by the use of enzymatic reactions.
[0201] [0201] We tested the agonistic activity of R1MAbs in vitro and in vivo, injecting Leptin-resistant hyperglycemic db / db mice with 3, 10 and 50 mg / kg (mpk) of R1MAb1. We observed that blood glucose levels were normalized for more than a week in all three doses, and the observed glucose-lowering effect was unexpectedly strong and long-lasting, with glucose levels remaining lower compared to control mice. for more than 30 days after a single injection (Fig. 2A). This was associated with a temporary but significant reduction in body weight (Fig. 2A). The glucose-lowering effect was probably due to an improvement in insulin sensitivity, as the level of insulin in the serum was also drastically reduced by the injection of R1MAb1 (Fig. 2B). We observed a similar R1MAb-induced reduction in blood glucose levels with R1MAb2 (Fig. 6), and in three additional mouse models with marked insulin resistance, Leptin-deficient ob / ob mice, mice fed a high-content diet. fat (HFD), and Ins2Akita mice (Hong et al., Am. J. Physiol.
[0202] [0202] To dissect the importance of IgG functionalities for R1MAb activities, we used two types of modifications. A double mutation (D265A / N297A; DANA) in the Fc region eliminates binding to Fc Rs and recruitment of immune effector cells by an IgG molecule (Gong et al., 2005). Neither the agonistic nor antidiabetic activity of R1MAb2 was affected by the introduction of DANA mutations; therefore, the effector function has no role in the antidiabetic activity of R1MAb2 (Fig. 9A-C). However, a genetically engineered (OA) version of R1Mab1 (Atwell et al., J. Mol. Biol. 270: 26-35 (1997)), devoid of one of the Fab fragments (OA-R1MAb1) (Fig .9A and D) showed decreased agonistic activity (Fig.
[0203] [0203] MAbs have emerged as a powerful therapeutic modality for the treatment of a range of human diseases. Our demonstration of potent anti-hyperglycemic and lipid-lowering activities of agonistic anti-FGFR1 MAb opens a new path towards the development of therapeutic MAbs targeting FGFR1 or the FGFR1-containing receptor complex for the treatment of type 2 diabetes and other disorders chronic diseases related to obesity. MAb based on FGFR1 targeting offers several favorable properties over recombinant FGF21 therapy. Firstly, by their nature, MAbs provide predictable, modulable and far superior pharmacokinetics compared to FGF21 or any other therapeutic protein without an antibody.
[0204] [0204] Recombinant FGF21 has been suggested to improve insulin sensitivity through adipose tissues and the liver (Berglund et al., Endocrinology 150: 4084-93 (2009); Li et al., FEBS Letters 583: 3230-34 (2009 )). The injection of FGF21 in mice induced by MEK and ERK phosphorylation in four types of tissue that express KLB, the liver, white adipose tissue (WAT), brown adipose tissue (BAT), and pancreas, as previously reported (Fig. 3A, superior ) (Kurosu et al., J. Biol. Chem. 282: 26687-95 (2007); Xu et al., Am. J. Physiol. Endocrinol. Metab. 297 (5): E1105-14 (2009)). In contrast, the injection of R1MAb1 leads to phosphorylation of the same effectors downstream in adipose and pancreas tissues, but not in the liver (Fig.
[0205] [0205] To investigate this further, we used lipoatrophic ap2-srebp1c transgenic mice, which exhibit severe insulin resistance, leptin deficiency, and hepatomegaly due to the lack of white adipose tissues and impaired brown adipose function (Fig. 3E, 11) (Shimomura et al., Genes Dev. 12: 3182-94 (1998); Shimomura et al., Nature 401: 73-76 (1999)). In line with the idea that normal adipose tissue function is necessary for the metabolic activity of R1MAb1, an i.p. single at 1 mpk (mg / kg) improved HOMA-IR and glucose tolerance only in control ob / ob mice but not in ap2-srebp1c mice (Fig. 3E). Food intake was reduced by injection of R1MAb1 in both ob / ob mice and ap2-srebp1c transgenic mice,
[0206] [0206] It has recently been suggested that FGF21 activates the transcriptional coactivating protein of the nuclear receptor in the adipose tissues and liver to induce the expression of downstream genes associated with oxidative metabolism (Chau et al., Proc. Nat'l. Acad. Sci. USA 107: 12553-58 (2010); Hondares et al., Cell Metabolism 11: 206-12 (2010); Potthoff et al. Proc.
[0207] [0207] PGC-1 transcription is regulated through cAMP response elements (CREs) in the promoter region and the CREB transcription factor that binds to CREs (Herzig et al., Nature 413: 179-83 (2001) ; Karamitri et al., J. Biol. Chem. 284: 20738-52 (2009); Muraoka et al., Am. J. Physiol.
[0208] [0208] Another important difference between our R1MAbs and FGF21 is the specificity of the target receptor. FGF21 can act on FGFR1c, 2c and 3c, but its effects are probably limited to tissues that express KLB (ie liver, adipose and pancreas) (Fon Tacer et al., Above; Kurosu et al., J.
[0209] [0209] We then tested the ability of these bispecific antibodies to provide the metabolic benefits of agonistic anti-FGFR1 antibodies. We generate transgenic mice that express human beta-Klotho (the R1MAb1 and R1MAb2 that each recognizes murine FGFR1) and confirms that the bispecific anti-KLB / FGFR1 antibodies described above improve glucose tolerance in mouse models, for example, transgenic mice hKLB fed a high fat diet. We also generate anti-beta-Klotho antibodies that react with the protein in other animal models (eg, rat, rabbit, cynomologous and rhesus monkeys) and similarly test the ability of bispecific antibodies constructed with these and anti-FGFR1 antibodies to metabolic benefits.
[0210] [0210] Although the aforementioned invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited in this application are expressly incorporated in their entirety as a reference.

权利要求:
Claims (38)
[1]
1. METHOD OF TREATING A METABOLIC DISEASE OR CONDITION in an individual, characterized by understanding to administer to the individual an effective amount of an anti-fibroblast growth factor receptor 1 (FGFR1) agonist, in which the metabolic disease is selected from of the group consisting of: polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes , type 1 diabetes, latent autoimmune diabetes (LAD) and diabetes maturity beginning in youth (MODY), in which the FGFR1 agonist does not activate FGFR2 or FGFR3.
[2]
2. METHOD according to claim 1, characterized in that the FGFR1 agonist is an anti-FGFR1 antibody.
[3]
3. METHOD according to claim 2, characterized in that the anti-FGFR1 antibody binds to peptide no 26 KLHAVPAAKTVKFKCP (SEQ ID NO: 28) or peptide no 28 FKPDHRIGGYKVRY (SEQ ID NO: 29).
[4]
4. METHOD according to claim 2, characterized in that the anti-FGFR1 antibody binds to both FGFR1b and FGFR1c.
[5]
5. METHOD according to claim 1, characterized in that the antibody is a bispecific antibody.
[6]
6. METHOD, according to claim 5, characterized in that the antibody also binds to beta-Klotho.
[7]
7. Isolated ANTIBODY that binds to FGFR1, characterized in that the antibody is an agonist of FGFR1 activity.
[8]
ANTIBODY according to claim 7, characterized in that the antibody is not an agonist of FGFR2 or FGFR3.
[9]
ANTIBODY according to one of claims 7 to 8, characterized in that the antibody is a monoclonal antibody.
[10]
ANTIBODY according to one of claims 7 to 9, characterized in that the antibody binds to peptide no 26 KLHAVPAAKTVKFKCP (SEQ ID NO: 28) or to peptide no 28 FKPDHRIGGYKVRY (SEQ ID NO: 29).
[11]
ANTIBODY according to one of claims 7 to 10, characterized in that the antibody is a human, humanized or chimeric antibody.
[12]
12. ANTI-FGFR1 ANTIBODY, characterized in that the antibody comprises (a) HVR-H3 which comprises an amino acid sequence selected from the group consisting of SSGYGGSDYAMDY (SEQ ID NO: 16), SGYGGSDYAMDY (SEQ ID NO: 17), EHFDAWVHYYVMDY (SEQ ID NO: 18), TGTDVMDY (SEQ ID NO: 19), and GTDVMDY (SEQ ID NO: 20), (b) HVR-L3 comprising the amino acid sequence QQSYTTPPT (SEQ ID NO: 23), and ( c) HVR-H2 comprising an amino acid sequence selected from the group consisting of X1X2IX3PX4DGX5TX6YADSVKG, where X1 is A or G, X2 is D or E, X3 is D or Y, X4 is N or Y, X5 is A or D, and X6 is D or Y (SEQ ID NO: 24) and X1IX2PX3DGX4TX5YADSVKG, where X1 is D or E, X2 is D or Y, X3 is N or Y, X4 is A or D, and X5 is D or Y (SEQ ID NO: 25).
[13]
13. ANTI-FGFR1 ANTIBODY, characterized in that the antibody comprises (a) HVR-H1 which comprises the amino acid sequence GFTFX1X2X3X4IX5, where X1 is S or T, X2 is N or S, X3 is N or T, X4 is W or Y , X5 is H or S (SEQ ID NO: 26), (b) HVR-H2 which comprises the amino acid sequence selected from the group consisting of X1X2IX3PX4DGX5TX6YADSVKG, where X1 is A or G, X2 is D or E, X3 is D or Y, X4 is N or Y, X5 is A or D, and X6 is D or Y (SEQ ID NO: 24) and X1IX2PX3DGX4TX5YADSVKG, where X1 is D or E, X2 is D or Y, X3 is N or Y, X4 is A or D, and X5 is D or Y (SEQ ID NO: 25), and (c) HVR-H3 which comprises an amino acid sequence selected from the group consisting of SSGYGGSDYAMDY (SEQ ID NO : 16), SGYGGSDYAMDY (SEQ ID
NO: 17), EHFDAWVHYYVMDY (SEQ ID NO: 18), TGTDVMDY (SEQ ID NO: 19), and GTDVMDY (SEQ ID NO: 20).
[14]
ANTIBODY according to claim 13, characterized in that the antibody comprises (a) HVR-H1 which comprises the amino acid sequence GFTFTSTWIS (SEQ ID NO: 7), (b) HVR-H2 which comprises an amino acid sequence selected from from the group consisting of GEIDPYDGDTYYADSVKG (SEQ ID NO: 10) and EIDPYDGDTYYADSVKG (SEQ ID NO: 11), and (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of SSGYGGSDYAMDY (SEQ ID NO : 16) and SGYGGSDYAMDY (SEQ ID NO: 17).
[15]
ANTIBODY according to claim 13, characterized in that the antibody comprises (a) HVR-H1 which comprises the amino acid sequence GFTFSNNYIH (SEQ ID NO: 8), (b) HVR-H2 which comprises an amino acid sequence selected from from the group consisting of ADIYPNDGDTDYADSVKG (SEQ ID NO: 12) and DIYPNDGDTDYADSVKG (SEQ ID NO: 13), and (c) HVR-H3 comprising the amino acid sequence EHFDAWVHYYVMDY (SEQ ID NO: 18).
[16]
16. ANTIBODY according to claim 13, characterized in that the antibody comprises (a) HVR-H1 which comprises the amino acid sequence GFTFTSNWIS (SEQ ID NO: 9), (b) HVR-H2 which comprises an amino acid sequence selected from from the group consisting of AEIDPYDGATDYADSVKG (SEQ ID NO: 14) and EIDPYDGATDYADSVKG (SEQ ID NO: 15) and (c) HVR-H3 comprising an amino acid sequence selected from the group consisting of TGTDVMDY (SEQ ID NO: 19) and GTDVMDY (SEQ ID NO: 20).
[17]
ANTIBODY according to one of claims 12 to 16, characterized in that it additionally comprises (a) HVR-L1 comprising the amino acid sequence RASQDVSTAVA (SEQ ID NO: 21);
(b) HVR-L2 comprising the SASFLYS amino acid sequence (SEQ ID NO: 22); and (c) HVR-L3 which comprises the amino acid sequence QQSYTTPPT (SEQ ID NO: 23).
[18]
18. ANTIBODY according to claim 13, characterized in that it comprises a VH sequence selected from the group consisting of SEQ ID NO: 2, 3 and 4.
[19]
ANTIBODY according to claim 18, characterized in that it additionally comprises a VL sequence of SEQ ID NO: 6.
[20]
ANTIBODY according to one of claims 7 to 19, characterized in that the antibody is a multispecific antibody.
[21]
21. ANTIBODY according to claim 20, characterized in that the antibody also binds to beta-Klotho.
[22]
22. ANTIBODY according to one of claims 7 to 21, characterized in that it is an IgG1 antibody.
[23]
23. Isolated NUCLEIC ACID, characterized by encoding the antibody, as defined in one of claims 7 to 22.
[24]
24. HOSTING CELL, characterized by comprising nucleic acid, as defined in claim 23.
[25]
25. AN ANTIBODY PRODUCTION METHOD, characterized in that it comprises the cultivation of the host cell, as defined in claim 24, so that the antibody is produced.
[26]
26. METHOD according to claim 25, characterized in that it further comprises recovering the antibody from the host cell.
[27]
27. PHARMACEUTICAL FORMULATION, characterized in that it comprises an antibody, as defined in one of claims 7 to 22, and a pharmaceutically acceptable carrier.
[28]
28. USE OF THE ANTIBODY, as defined in one of claims 7 to 22, characterized in that it is in the manufacture of a medicament.
[29]
29. USE, according to claim 28, characterized in that the drug is for the treatment of a metabolic disease or condition selected from the group consisting of: polycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity, non-steatohepatitis alcoholic (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), and youth-onset maturity diabetes (MODY).
[30]
30. USE, according to claim 28, characterized in that the drug is for sensitizing an individual to insulin.
[31]
31. METHOD OF TREATING DIABETES in an individual, characterized by comprising administering to the individual an effective amount of the antibody, as defined in one of claims 7 to 22.
[32]
32. USE OF AN AGONIST OF THE RECEPTOR 1 OF THE ANTI-FIBROBLAST GROWTH FACTOR (FGFR1), characterized by being in the manufacture of a drug to treat a disease or metabolic condition in an individual selected from the group consisting of: ovary syndrome polycystic (PCOS), metabolic syndrome (MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmune diabetes ( LAD) and childhood-onset diabetes (MODY), in which the FGFR1 agonist does not activate FGFR2 or FGFR3.
[33]
33. USE, according to claim 32, characterized in that the FGFR1 agonist is an anti-FGFR1 antibody.
[34]
34. USE according to claim 33, characterized in that the anti-FGFR1 antibody binds to peptide no 26 KLHAVPAAKTVKFKCP (SEQ ID NO: 28) or to peptide no 28 FKPDHRIGGYKVRY (SEQ ID NO: 29).
[35]
35. USE according to claim 33, characterized in that the anti-FGFR1 antibody binds to both FGFR1b and FGFR1c.
[36]
36. USE according to claim 32, characterized in that the antibody is a bispecific antibody.
[37]
37. USE according to claim 36, characterized in that the antibody also binds to beta-Klotho.
[38]
38. USE OF THE ANTIBODY, as defined in one of claims 7 to 22, characterized in that it is in the manufacture of a medicament to treat diabetes in an individual.

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法律状态:
2020-11-24| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-05-11| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-06-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161486731P| true| 2011-05-16|2011-05-16|

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US61/486,731|2011-05-16|

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US201161536936P| true| 2011-09-20|2011-09-20|

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US61/536,936|2011-09-20|

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PCT/US2012/037964|WO2012158704A1|2011-05-16|2012-05-15|Fgfr1 agonists and methods of use|

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