CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 14/421,103 filed Feb. 11, 2015, which is a national phase entry pursuant to 35 U.S.C. § 371 of International Application No. PCT/US2013/054664, which claims priority to U.S. Provisional Application No. 61/682,640, filed Aug. 13, 2012, and U.S. Provisional Application No. 61/784,332, filed Mar. 14, 2013, the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in 10 ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 20, 2018, is named 2018-09-20_01146-0026-01US_SEQLIST_ST25 and is 116,061 bytes in size.

FIELD OF THE INVENTION

The present invention relates to anti-Jagged antibodies and methods of using the same.

BACKGROUND

The Notch signaling pathway regulates a diverse array of cell functions (Kopan et al., Cell 137, 216-233 (2009)). Four Notch receptors have been identified in mammals, i.e., Notch 1-4, that share basic structural elements that include an extracellular domain, a transmembrane domain, and an intracellular domain. Similarly, the canonical ligands of Notch share certain structural similarities but a number of non-canonical ligands of Notch have also been identified (Kopan et al., Cell 137, 216-233 (2009)). The five canonical ligands in mammals are Delta-like 1, Delta-like 3, Delta-like 4, Jagged1 and Jagged2. Binding of a Notch ligand to the extracellular domain of a Notch receptor sets a signaling cascade in motion that begins with proteolytic cleavage at the extracellular S2 site by an alpha secretase of the ADAM (a disintegrin and metalloprotease) family. Cleavage at S2 is followed by proteolytic cleavage by a gamma secretase at the intracellular S3 site, which results in release of the intracellular domain and downstream events that ultimately activate Notch-dependent transcription factors such as Hes1 and Hey.

Because aberrant Notch expression and signaling has been implicated in a number of diseases, including cancer (Koch et al., Cell. Mol. Life Sci. 64, 2746-2762 (2007)), modulators of Notch signaling have been investigated as possible therapeutic agents for such diseases. For example, gamma secretase inhibitors have been tested in clinical trials for their effectiveness in treating various malignancies (Shih et al, Cancer Res. 67, 1879-1882 (2007)). Gamma secretase inhibitors prevent cleavage at S3 and thereby prevent signaling through Notch receptors. However, gamma secretase inhibitors do not distinguish individual Notch family members and therefore inhibit signaling through multiple receptors at once, as well as through unrelated pathways (Beel et al., Cell. Mol. Life Sci. 65, 1311-1334 (2008)). Consequently, administration of gamma secretase inhibitors is associated with intestinal toxicity marked by weight loss and intestinal goblet cell metaplasia, indicative of a role for Notch in determining cell fate by maintaining proliferation of intestinal crypt progenitor cells and prohibiting differentiation to a secretory cell fate (See van Es et al., Nature 435:959-963 (2005)). Similarly, inhibition of both Notch1 and Notch2 signaling via conditional Notch gene knockout (Riccio et al., EMBO Rep. 9:377-383 (2008)) or via antagonist antibody inhibition (US Patent Application Publication No. 2010/0080808) also causes intestinal goblet cell metaplasia.

Because of serious toxicity associated with inhibitors of multiple Notch receptors, there is a great need in the art for targeted inhibition of signaling through specific receptors.

SUMMARY

The invention provides anti-Jagged antibodies and methods of using the same.

In one aspect, the invention provides an isolated antibody that binds to Jagged1. In one embodiment, the antibody is an antagonist of Jagged1-mediated signaling. In one embodiment, the antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:81; (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO:84; (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO:87; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:110; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO:114. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:82; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:110; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:112. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:82; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:86; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:110; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:113. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:83; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:110; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:112.

In another aspect, the invention provides an isolated antibody that binds to Jagged2. In one embodiment, the antibody is an antagonist of Jagged2-mediated signaling. In one embodiment, the antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO:94; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:115; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO:122. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:117. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:91; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:118. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:90; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:119. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:92; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:120. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:93; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:121.

In another aspect, the invention provides an isolated antibody that binds to Jagged1 and Jagged2 (Jagged1/2). In one embodiment, the antibody is an antagonist of Jagged1/2-mediated signaling. In one embodiment, the antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:95; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:96; (c) an HVR-H3 comprising an amino acid sequence of SEQ ID NO:99; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:123; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124; and (f) an HVR-L3 comprising an amino acid sequence of SEQ ID NO:127. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:95; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:96; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:97; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:123; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:125. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:95; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:96; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:98; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:123; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:126.

In another embodiment, the antibody comprises at least one, two, three, four, five, or six HVRs selected from: (a) an HVR-H1 comprising an amino acid sequence of SEQ ID NO:105; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising an amino acid sequence of SEQ ID NO:109; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising an amino acid sequence of SEQ ID NO:134. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:100; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:107; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:130. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:100; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:108; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:131. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:101; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:107; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:132. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:102; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:107; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:133. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:103; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:107; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:132. In one embodiment, the antibody comprises: (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:104; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:107; (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:132.

In certain embodiments of the invention, any of the above embodiments is a monoclonal antibody. In certain embodiments, any of the above embodiments is a human, humanized, or chimeric antibody. In certain embodiments, any of the above embodiments is an antibody fragment.

In another aspect, the invention provides an isolated antibody as described above, further comprising a light chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO:60; FR2 comprising the amino acid sequence of SEQ ID NO:61; FR3 comprising the amino acid sequence of SEQ ID NO:62; and FR4 comprising the amino acid sequence of SEQ ID NO:135. In some embodiments, the antibody comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO:50; FR2 comprising the amino acid sequence of SEQ ID NO:136; FR3 comprising the amino acid sequence of SEQ ID NO:57; and FR4 comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the antibody comprises a heavy chain variable domain framework FR1 comprising the amino acid sequence of SEQ ID NO:50; FR2 comprising the amino acid sequence of SEQ ID NO:48; FR3 comprising the amino acid sequence of SEQ ID NO:57; and FR4 comprising the amino acid sequence of SEQ ID NO:35.

In another aspect, the invention provides an isolated antibody that binds to Jagged1, comprising (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:10; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:19; or (c) a VH sequence as in (a) and a VL sequence as in (b). In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:10. In some embodiments, the antibody comprises a VL sequence of SEQ ID NO:19. In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:10 and a VL sequence of SEQ ID NO:19. In some embodiments, the antibody comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:20; or (c) a VH sequence as in (a) and a VL sequence as in (b). In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:11. In some embodiments, the antibody comprises a VL sequence of SEQ ID NO:20. In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:11 and a VL sequence of SEQ ID NO:20.

Any of the above embodiments may be a full-length IgG1 antibody.

In another aspect, the invention provides an isolated antibody that competes with any of the above embodiments for specific binding to Jagged1. In another aspect, the invention provides an isolated antibody that competes with any of the above embodiments for specific binding to Jagged2. In another aspect, the invention provides an isolated nucleic acid encoding an isolated antibody of the above embodiments. In a further aspect, the invention provides a host cell comprising the isolated nucleic acid encoding the antibody. In a further aspect, the invention provides a method of producing an antibody comprising culturing the host cell so that the antibody is produced.

In another aspect, the invention provides an immunoconjugate comprising an antibody of any of the above embodiments and a cytotoxic agent.

In another aspect, the invention provides a pharmaceutical formulation comprising an antibody of any of the above embodiments and a pharmaceutically acceptable carrier.

In another aspect, an antibody of any of the above embodiments is provided for use as a medicament. In some embodiments, an antibody of any of the above embodiments is provided for use in treating a cancer. In some embodiments, an antibody of any of the above embodiments is provided for use in reducing cancer cell growth.

In another aspect, a method of inhibiting Jagged1-mediated signaling is provided. In one embodiment, a method of inhibiting Jagged1-mediated signaling in vitro is provided. In one embodiment, a method of inhibiting Jagged1-mediated signaling in vivo is provided.

In another aspect, a method of treating an individual having a cancer comprising administering to the individual an effective amount of an antibody of any of the above embodiments. In one embodiment, the cancer is selected from the group consisting of: breast cancer, lung cancer, brain cancer, cervical cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, skin cancer, B-cell malignancies, and T-cell malignancies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary amino acid sequences of human and murine Jagged1 protein.

FIG. 2 shows exemplary amino acid sequences of human and murine Jagged2 protein.

FIGS. 3A-D show the amino acid sequences of peptides used for phage antibody library screening and selection. All proteins were expressed as a secreted protein in BEVS cells and their sequences are listed in the N-terminal to C-terminal direction. (A) Amino acid sequence of expressed protein murine Jagged 1-DSL-EGF1-4 (Q34-D377). The bold font at the N-terminus represents a short linker sequence (ADLGS) (SEQ ID NO: 31). The bold font at the C-terminus represents a short linker sequence (EFG), a thrombin cleavage site (LVPRGS) (SEQ ID NO: 137), a G spacer and the 6-His tag (SEQ ID NO: 138). (B) Amino acid sequence of expressed protein human Jag1-DSL-EGF1-4. Only the Jag1 sequence is shown although the antigen also contained a TEV protease cleavage site and 6-His tag (SEQ ID NO: 138) at the C-terminus. (C) Amino acid sequence of expressed protein murine Jag2-DSL-EGF1-4 (M27-E388). The bold font at the N-terminus represents a short linker sequence (ADLGS) (SEQ ID NO: 31). The bold font at the C-terminus represents a short linker sequence (EFG), a thrombin cleavage site (LVPRGS) (SEQ ID NO: 137), a G spacer and the 6-His tag (SEQ ID NO: 138). (D) Amino acid sequence of expressed protein human Jag2-DSL-EGF1-4 (R2-E388). The bold font at the C-terminus represents a short linker sequence (EFG), a thrombin cleavage site (LVPRGS) (SEQ ID NO: 137), a G spacer and the 6-His tag (SEQ ID NO: 138).

FIGS. 4A-1-B-2 show an alignment of the amino acid sequences for the heavy (FIG. 4A-1 and FIG. 4A-2) and light (FIG. 4B-1 and FIG. 4B-2) chain variable domains of anti-Jagged antibodies (Example 1-2). Amino acid positions of the complementarity determining regions (CDRs) are indicated.

FIGS. 5A-B show exemplary acceptor human variable heavy (VH) consensus framework sequences for use in practicing the instant invention. Sequence identifiers are as follows:

human VH subgroup I consensus framework “A” minus Kabat CDRs (SEQ ID NOs:32, 33, 34, 35).

human VH subgroup I consensus frameworks “B,” “C,” and “D” minus extended hypervariable regions (SEQ ID NOs:36, 37, 34, 35; SEQ ID NOs:36, 37, 38, 35; and SEQ ID NOs:36, 37, 39, 35).

human VH subgroup II consensus framework “A” minus Kabat CDRs (SEQ ID NOs:40, 41, 42, 35).

human VH subgroup II consensus frameworks “B,” “C,” and “D” minus extended hypervariable regions (SEQ ID NOs:43, 44, 42, 35; SEQ ID NOs:43, 44, 45, 35; and SEQ ID NOs:43, 44, 46, and 35).

human VH subgroup III consensus framework “A” minus Kabat CDRs (SEQ ID NOs:47, 48, 49, 35).

human VH subgroup III consensus frameworks “B,” “C,” and “D” minus extended hypervariable regions (SEQ ID NOs:50, 51, 49, 35; SEQ ID NOs:50, 51, 52, 35; and SEQ ID NOs:50, 51, 53, 35).

human VH acceptor framework “A” minus Kabat CDRs (SEQ ID NOs:54, 48, 55, 35).

human VH acceptor frameworks “B” and “C” minus extended hypervariable regions (SEQ ID NOs:50, 51, 55, 35; and SEQ ID NOs:50, 51, 56, 35).

human VH acceptor 2 framework “A” minus Kabat CDRs (SEQ ID NOs:54, 48, 57, 35).

human VH acceptor 2 framework “B,” “C,” and “D” minus extended hypervariable regions (SEQ ID NOs:50, 51, 57, 35; SEQ ID NOs:50, 51, 58, 35; and SEQ ID NOs:50, 51, 59, 35).

FIG. 6 shows exemplary acceptor human variable light (VL) consensus framework sequences for use in practicing the instant invention. Sequence identifiers are as follows:

human VL kappa subgroup I consensus framework (κv1): SEQ ID NOs:60, 61, 62, 63

human VL kappa subgroup II consensus framework (κv2): SEQ ID NOs:64, 65, 66, 63

human VL kappa subgroup III consensus framework (κv3): SEQ ID NOs:67, 68, 69, 63

human VL kappa subgroup IV consensus framework (κv4): SEQ ID NOs:70, 71, 72, 63.

FIGS. 7A-F show the H1, H2, and H3 heavy chain hypervariable region (HVR) sequences of anti-Jagged antibodies, as described in the Examples. Amino acid positions are numbered according to the Kabat numbering system as described below.

FIGS. 8A-E show the L1, L2, and L3 light chain HVR sequences of anti-Jagged antibodies, as described in the Examples. Amino acid positions are numbered according to the Kabat numbering system as described below.

FIG. 9 shows light and heavy chain framework sequences of anti-Jagged antibodies described in the Examples. Numbers in superscript indicate amino acid positions according to Kabat.

FIGS. 10A-B show binding specificity of antibodies obtained from the first (FIG. 10A) and second (FIG. 10B) round of screening. (A) Results of ELISA assays measuring binding of antibody D-1 (left panel) and C-1 (right panel) to human Jagged1 (hJag-1), human Jagged2 (hJag-2), murine Jagged2 (mJag-2), human Delta-like 1 (hDLL-1), murine Delta-like 1 (mDLL-1), or human Delta-like 4 (hDLL-4). Antibody concentrations are indicated on the x-axis and OD650 on the y-axis. (B) Results of ELISA assays measuring binding specificity of Antibodies A and B, both identified during further screening using human Jag1-DSL-EGF1-4 (FIG. 3B) for antibody A and murine and human Jag2-DSL-EGF1-4 (FIGS. 3C and D) for antibody B. Black columns=binding to human Jagged1; gray columns=binding to human Jagged2. C-1 served as a control for binding to both Jagged1 and Jagged2.

FIG. 11 shows binding constants for antibodies A, A-1, A-2, B, B-1, B-2, B-3, C, C-1, D, D-1, and D-2 binding to purified human Jagged1 (human Jag1), human Jagged2 (human Jag2), and mouse Jagged2 (mouse Jag2).

FIG. 12 shows dose-dependent inhibition of Jagged1-induced signaling of Notch 1 by anti-Jagged antibodies. Results were obtained from co-culture experiments that measure Jagged1-induced signaling through the Notch1 receptor, as described in Example 4. The y-axis indicates expression levels of the Notch-dependent reporter gene firefly luciferase relative to expression of a control gene (constitutively active promoter driving expression of Renilla luciferase). The x-axis indicates concentrations for antibodies D and C (0.4-50 μg/ml). Co-culture without antibodies (J1 induced-positive control) served as positive control for Jagged1-induced signaling. An isotype control antibody served as control for specific antibody inhibition. A gamma secretase inhibitor (Compound E+) was used as control for inhibition of Notch signaling.

FIGS. 13A-B show inhibition of Notch signaling by affinity matured anti-Jagged antibodies. Co-culture assays were performed as described in FIG. 12 and Example 4. (A) Phage antibodies and their concentrations (μg/ml) are indicated on the x-axis (parental antibodies C and D served as control). The gamma-secretase inhibitors (GSIs) Compound E (CmpE) and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) at the indicated concentrations served as positive control for inhibition of Notch signaling; DMSO served as vehicle control for the GSIs; an irrelevant antibody with the same isotype as those tested in the panel served as isotype control. (B) Phage antibodies at the indicated concentration are indicated on the x-axis. DAPT at the indicated concentrations served as positive control for inhibition of Notch signaling; DMSO served as vehicle control. Signaling was induced by Jagged1 (dark gray columns) or by Jagged2 (light gray columns). Untreated=cultures that were not stimulated with ligand and not treated with antibody; No Stimulation or 3T3P=cultures not stimulated with ligand; agD or gD=isotype control antibody; Stim/no AB or No Ab=cultures stimulated with ligand but not treated with antibody; gamma-secretase inhibitor DAPT or the DAPT vehicle control of DMSO.

FIGS. 14A-B show that combined inhibition of Jagged1 and Jagged2 causes rapid weight loss. (A) Mice were dosed twice per week with the anti-Jagged1/2 antibody C-1 (anti-J1/2; 5-10 mpk), the anti-Jagged1 antibody A-2 (anti-J1; 5-20 mpk), the anti-Jagged2 antibody B-3 (anti-J2; 5-20 mpk), the antibody A-2 and B-3 together (anti-J1 & -2; 5 mpk each) or an isotype control antibody (20 mpk). Total antibody concentration of each dosing was brought up to 20 mpk with the isotype control antibody, where necessary. The average body weight changes (y-axis) are graphed as a percentage of starting body weight over time (x-axis). (B) Balb/c mice (ten per group, individually housed) were injected IP twice per week with either 30 mpk of anti-gD isotype control antibody or with a combination of 15 mpk antibody A-2 plus 15 mpk antibody B-3 for eight days. Food intake was assessed by daily weighing of the food delivered and remaining in each cage. Error bars represent standard deviations (n=10).

FIGS. 15A-B show normal intestinal histology of following anti-Jagged antibody treatment. (A) Intestinal samples of mice treated as described in Example 6 were isolated and stained with hematoxylin and eosin (FIG. 15A, H & E) or with Alcian Blue (FIG. 15A, Alcian Blue). (B) Sections of intestine samples were stained with primary antibodies to either lysozyme or Ki-67 (FIG. 15B)

FIGS. 16A-1-B-2 show inhibition of human lung cancer cell growth by an anti-Jagged1 antagonist antibody in vivo. Mice bearing human lung cancer xenografts were injected twice per week intraperitoneally (IP) with 20 mpk anti-gD isotype control antibody (Isotype control Ab) or with anti-Jagged1 antibody A-2 (Anti-Jag1), with the injections starting after average tumor volumes (measured with calipers) reached approximately 180 mm³. Tumor volumes (y-axis) were subsequently measured for 19 days. FIG. 16A-1 and FIG. 16A-2: The average tumor volumes for each group (n=10) were plotted over time (x-axis) using a linear mixed effects model (FIG. 16A-1). Tumor volumes for each mouse in each group are depicted in the two panels in FIG. 16A-2. FIG. 16B-1 and FIG. 16B-2: Total body weight of each mouse was measured and graphed as the percentage change averaged for each group (FIG. 16B-1) or for each mouse in each group (FIG. 16B-2).

FIGS. 17A-B show inhibition of human breast cancer cell growth by anti-Jagged1 and anti-Jagged2 antagonist antibodies in vivo. C.B-17 SCID.bg mice with human breast cancer xenografts were injected on days 0, 4, 7, 12, 15, 18, 22, 25, 29, 32, 36, 43, 50, and 57 with anti-gD isotype control antibody (Anti-gD), anti-ragweed isotype control antibody (anti-ragweed), anti-Jagged1 antibody A-2 in the human IgG1 backbone (anti-Jag1 A-2 (hIgG1)), anti-Jagged1 antibody A-2 in the murine IgG2a backbone (anti-Jag1 A-2 (mIgG2a)), or anti-Jagged2 antibody B-3 in the human IgG1 backbone (anti-Jag2 B-3 (hIgG1)). Tumor volumes (y-axis) of treatment groups (A) or individual animals (B) were plotted using a linear mixed effects model over time (x-axis).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. Definitions

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 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 VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

The terms “anti-Jagged antibody” and “an antibody that binds to Jagged” refer to an antibody that is capable of binding Jagged1, Jagged2, or Jagged1 and 2 (Jagged1/2) with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting Jagged. In one embodiment, the extent of binding of an anti-Jagged antibody to an unrelated, non-Jagged protein is less than about 10% of the binding of the antibody to Jagged as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to Jagged has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³M). In certain embodiments, an anti-Jagged antibody binds to an epitope of Jagged that is conserved among Jagged from different species.

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

A “blocking” antibody or an “antagonist” antibody is one which significantly inhibits (either partially or completely) a biological activity of the antigen it binds.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein 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, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

“Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising 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, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-Jagged antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying 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 disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “Jagged” or “Jag,” as used herein, refers to any native Jagged from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed Jagged as well as any form of Jagged that results from processing in the cell. The term also encompasses naturally occurring variants of Jagged, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human and murine Jagged1 and Jagged2 is shown in FIGS. 1 and 2 (SEQ ID NOS:1-4), respectively.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

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 antigen. The variable domains of the heavy chain and light chain (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, e.g., Kindt et al. Kuby Immunology, 6^thed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. 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 operatively linked. Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on the identification of anti-Jagged antibodies and fragments thereof. In certain embodiments, antibodies that bind to at least one Jagged are provided. Antibodies of the invention are useful, e.g., for the diagnosis or treatment of cancer. Accordingly, the invention provides methods, compositions, kits, and articles of manufacture related to anti-Jagged antibodies.

A. Exemplary Anti-Jagged Antibodies

In one aspect, the invention provides isolated antibodies that bind to Jagged. In certain embodiments, the anti-Jagged antibody is an anti-Jagged1 antibody.

In one aspect, the invention provides an anti-Jagged1 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:81;
- (b) HVR-H2 comprising an amino acid sequence of SEQ ID NO:84;
- (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO:87;
- (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:110;
- (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and
- (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO:114.

In a further aspect, the anti-Jagged1 antibody comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81 and at least one, two, three, four, or five HVRs selected from (b), (c), (d), (e) and (f) above. In one embodiment, the antibody comprises (a), (b), (c), (d), (e) and (f) above, wherein with respect to (b) (c), and (f) any one or more of the following embodiments are contemplated: HVR-H2 comprises an amino acid sequence selected from SEQ ID NOs: 82-83; HVR-H3 comprises an amino acid sequence selected from SEQ ID NOs: 85-86; and HVR-L3 comprises an amino acid sequence selected from SEQ ID NOs: 112-113.

In another embodiment, an antibody that specifically binds to Jagged1 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:82;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:110;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:112.

In another embodiment, an antibody that specifically binds to Jagged1 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:82;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:86;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:110;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:113.

In another embodiment, an antibody that specifically binds to Jagged1 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:81;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:83;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:85;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:110;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:111; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:112.

In certain embodiments, the anti-Jagged antibody is an anti-Jagged2 antibody.

In one aspect, the invention provides an anti-Jagged2 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:88;
- (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 89;
- (c) HVR-H3 comprising an amino acid sequence of SEQ ID NO:94;
- (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:115;
- (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and
- (f) HVR-L3 comprising an amino acid sequence of SEQ ID NO:122.

In a further aspect, the anti-Jagged2 antibody comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88 and at least one, two, three, four, or five HVRs selected from (b), (c), (d), (e) and (f) above. In one embodiment, the antibody comprises (a), (b), (c), (d), (e) and (f) above, wherein with respect to (c) and (f) any one or more of the following embodiments are contemplated: HVR-H3 comprises an amino acid sequence selected from SEQ ID NOs:90-93; and HVR-L3 comprises an amino acid sequence selected from SEQ ID NOs:117-121.

In one embodiment, an antibody that specifically binds to Jagged2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:90;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:117.

In another embodiment, an antibody that specifically binds to Jagged2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:91;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:118.

In another embodiment, an antibody that specifically binds to Jagged2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:90;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:119.

In another embodiment, an antibody that specifically binds to Jagged2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:92;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:120.

In another embodiment, an antibody that specifically binds to Jagged2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:88;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:89;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:93;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:115;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:116; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:121.

In certain embodiments, the anti-Jagged antibody is an anti-Jagged1/2 antibody.

In one aspect, the invention provides an anti-Jagged1/2 antibody comprising at least one, two, three, four, five, or six HVRs selected from an HVR-H1 comprising the amino acid sequence of SEQ ID NO:95; HVR-H2 comprising the amino acid sequence of SEQ ID NO:96; HVR-H3 comprising an amino acid sequence of SEQ ID NO:99; HVR-L1 comprising the amino acid sequence of SEQ ID NO:123; HVR-L2 comprising the amino acid sequence of SEQ ID NO:124; and HVR-L3 comprising an amino acid sequence of SEQ ID NO:127.

In one embodiment, an antibody that specifically binds to Jagged1/2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:95;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:96;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:97;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:123;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:125.

In one embodiment, an antibody that specifically binds to Jagged/1/2 is provided, wherein the antibody comprises:

- (a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:95;
- (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:96;
- (c) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:98;
- (d) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:123;
- (e) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:124; and
- (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:126.

In another aspect, the invention provides an anti-Jagged1/2 antibody comprising at least one, two, three, four, five, or six HVRs selected from an HVR-H1 comprising an amino acid sequence of SEQ ID NO:105; HVR-H2 comprising the amino acid sequence of SEQ ID NO:106; HVR-H3 comprising an amino acid sequence of SEQ ID NO:109; HVR-L1 comprising the amino acid sequence of SEQ ID NO:128; HVR-L2 comprising the amino acid sequence of SEQ ID NO:129; and HVR-L3 comprising an amino acid sequence of SEQ ID NO:134.