Anti-fchr5 antibodies

Abstract

The present application provides anti-FcRH5 antibodies and immunoconjugates and methods of using the same.

Description

[0001] This application is a divisional application of the patent application filed on June 24, 2014, with a priority date of June 24, 2013, application number 201480045475.0, entitled "Anti-FCRH5 Antibody".

[0002] Related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 838,534, filed June 24, 2013, pursuant to 35 USC §119, the disclosure of which is incorporated herein by reference in its entirety.

[0004] sequence list

[0005] This application contains a sequence list in ASCII format, which is incorporated herein by reference in its entirety. The ASCII text file was created on June 9, 2014, named GNE-0413WO_SL.txt, and is 67,909 bytes in size. Invention Field

[0006] This article provides anti-FcRH5 antibodies (e.g., bispecific antibodies) and immunoconjugates and methods for using them. background

[0007] Fc receptor-like 5 (FcRL5, also known as FcRH5 and IRTA2) belongs to a family of six recently identified genes in the immunoglobulin superfamily (IgSF). This gene family is closely associated with Fc receptors that possess conserved genomic structures, extracellular Ig domain compositions, and ITIM-like and ITAM-like signaling motifs (Davis RS et al., Eur J Immunol (2005) 35:674–80). Members of this family are also known as IFGP (from the Ig superfamily, FcR, gp42) and SPAP (phosphatase-anchored proteins containing SH2 domains). Six members of the FcRH / IRTA receptor family have been described: FcRH1 / IRTA5, FcRH2 / IRTA4, FcRH3 / IRTA3, FcRH4 / IRTA1, FcRH5 / IRTA2, and FcRH6 (Polson AG et al., Int. Immunol. (2006) 18(9):1363–1373). All FcRH / IRTAs contain combinations of typical immunoreceptor tyrosine-based inhibitory motifs and 'immunoreceptor tyrosine-based activation motif-like' signaling motifs. FcRH cDNA encodes a type I transmembrane glycoprotein with multiple Ig-like extracellular domains and cytoplasmic domains containing activation and / or inhibition signaling motifs based on shared immunoreceptor tyrosine residues. FcRH genes are structurally related, and their protein products share 28–60% extracellular identity with each other. They also share 15–31% identity with their closest FcR related genes. High homology exists between different FcRHs.

[0008] The ligand for FcRH5 is unknown, but FcRH5 has been involved in enhanced proliferation and downstream isoform expression during the development of antigen-sensitized B cells (Dement-Brown J. et al. J Leukoc Biol (2012) 91:59–67). The FcRH5 locus has three major mRNA isoforms (FcRH5a, FcRH5b, and FcRH5c). The major FcRH5 protein isoforms encoded by these transcripts share a common amino acid sequence up to residue 560, characterized by a common signal peptide and six extracellular Ig-like domains. FcRH5a represents a 759-amino acid secretory glycoprotein with eight Ig-like domains, followed by 13 distinct predominantly polar amino acids at its C-terminus. FcRH5b differs from FcRH5a at amino acid residue 560 and extends a short segment of 32 additional residues, its hydrophobicity compatible with its docking to the plasma membrane via a GPI anchor. FcRH5c is the longest isotype, and its sequence differs from FcRH5a at amino acid 746. FcRH5c encodes a 977-amino acid type I transmembrane glycoprotein with nine extracellular Ig domains (possessing eight potential N-linked glycosylation sites), a 23-amino acid transmembrane domain, and a 104-amino acid cytoplasmic domain (shared with ITIM and possessing three shared SH2 binding motifs).

[0009] FcRH genes cluster together in the middle of the classic FcR genes (FcγRI, FcγRII, FcγRIII, and FcεRI) in the 1q21–23 region of chromosome 1. This region contains one of the most common secondary chromosomal abnormalities associated with malignant phenotypes in hematopoietic neoplasms, particularly multiple myeloma (Hatzivassiliou G. et al., Immunity (2001) 14:277–89). FcRH5 is expressed only in the B-cell lineage, beginning in pre-B cells but not fully expressed until the mature B-cell stage. Unlike other most common B-cell-specific surface proteins (e.g., CD20, CD19, and CD22), FcRH5 continues to be expressed in plasma cells, while other B-cell-specific markers are downregulated (Polson AG et al., Int Immunol (2006) 18:1363–73). Furthermore, FcRH5 mRNA is overexpressed in multiple myeloma cell lines, and an 1q21 aberration was detected by oligonucleotide arrays (Inoue J., Am J Pathol (2004) 165:71–81). This expression pattern suggests that FcRH5 could be a target for antibody-based therapies for the treatment of multiple myeloma. Multiple myeloma is a malignant tumor of plasma cells characterized by bone lesions, kidney failure, anemia, and hypercalcemia. It is essentially incurable with current therapies. Current drug treatments for multiple myeloma include combinations of the proteasome inhibitor bortezomib (Velcade), the immunomodulatory agent lenalidomide (Revlimid), and the steroid dexamethasone (dexamethasone).

[0010] Therapies and detection methods based on FcRH5c-specific antibodies can be particularly effective because they specifically recognize target cell, membrane-associated FcRH5, rather than antibodies that recognize both soluble and membrane isoforms of FcRH5. However, only the last Ig-like domain of FcRH5 (Ig-like domain 9) is a unique extracellular region that differentiates the three major isoforms of FcRH5, and there is significant homology among Ig-like domains within FcRH5. Furthermore, the last Ig-like domain is highly conserved among FcRH1, FcRH2, FcRH3, and FcRH5. Any antibody-based therapy specifically targeting FcRH5 must have minimal cross-reactivity with other FcRHs to avoid undesirable off-target effects (e.g., FcRH3 expression on normal NK cells). There is a need in the art for agents that can aid in the diagnosis and treatment of cancers such as FcRH5-associated cancers.

[0011] summary

[0012] This document provides anti-FcRH5 antibodies (including bispecific antibodies), immunoconjugates, and methods of using them. This document provides isolated anti-FcRH5 antibodies that bind to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the isotype c-specific region comprises an Ig-like domain 9. In some embodiments, the isotype c-specific region comprises amino acids 743-850 of SEQ ID NO:1.

[0013] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:38, HVR-H2 containing the amino acid sequence of SEQ ID NO:62, and HVR-H3 containing the amino acid sequence of SEQ ID NO:86; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:2, HVR-L2 containing the amino acid sequence of SEQ ID NO:14, and HVR-L3 containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:50, HVR-H2 containing the amino acid sequence of SEQ ID NO:74, and HVR-H3 containing the amino acid sequence of SEQ ID NO:98.

[0014] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:39, HVR-H2 containing the amino acid sequence of SEQ ID NO:63, and HVR-H3 containing the amino acid sequence of SEQ ID NO:87; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:3, HVR-L2 containing the amino acid sequence of SEQ ID NO:15, and HVR-L3 containing the amino acid sequence of SEQ ID NO:27. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:51, HVR-H2 containing the amino acid sequence of SEQ ID NO:75, and HVR-H3 containing the amino acid sequence of SEQ ID NO:99.

[0015] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:40, HVR-H2 containing the amino acid sequence of SEQ ID NO:64, and HVR-H3 containing the amino acid sequence of SEQ ID NO:88; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:4, HVR-L2 containing the amino acid sequence of SEQ ID NO:16, and HVR-L3 containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:52, HVR-H2 containing the amino acid sequence of SEQ ID NO:76, and HVR-H3 containing the amino acid sequence of SEQ ID NO:100.

[0016] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:41, HVR-H2 containing the amino acid sequence of SEQ ID NO:65, and HVR-H3 containing the amino acid sequence of SEQ ID NO:89; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:5, HVR-L2 containing the amino acid sequence of SEQ ID NO:17, and HVR-L3 containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:53, HVR-H2 containing the amino acid sequence of SEQ ID NO:77, and HVR-H3 containing the amino acid sequence of SEQ ID NO:101.

[0017] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:42, HVR-H2 containing the amino acid sequence of SEQ ID NO:66, and HVR-H3 containing the amino acid sequence of SEQ ID NO:90; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:6, HVR-L2 containing the amino acid sequence of SEQ ID NO:18, and HVR-L3 containing the amino acid sequence of SEQ ID NO:30. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:54, HVR-H2 containing the amino acid sequence of SEQ ID NO:78, and HVR-H3 containing the amino acid sequence of SEQ ID NO:102.

[0018] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:43, HVR-H2 containing the amino acid sequence of SEQ ID NO:67, and HVR-H3 containing the amino acid sequence of SEQ ID NO:91; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:7, HVR-L2 containing the amino acid sequence of SEQ ID NO:19, and HVR-L3 containing the amino acid sequence of SEQ ID NO:31. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:55, HVR-H2 containing the amino acid sequence of SEQ ID NO:79, and HVR-H3 containing the amino acid sequence of SEQ ID NO:103.

[0019] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:44, HVR-H2 containing the amino acid sequence of SEQ ID NO:68, and HVR-H3 containing the amino acid sequence of SEQ ID NO:92; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:8, HVR-L2 containing the amino acid sequence of SEQ ID NO:20, and HVR-L3 containing the amino acid sequence of SEQ ID NO:32. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:56, HVR-H2 containing the amino acid sequence of SEQ ID NO:80, and HVR-H3 containing the amino acid sequence of SEQ ID NO:104.

[0020] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:45, HVR-H2 containing the amino acid sequence of SEQ ID NO:69, and HVR-H3 containing the amino acid sequence of SEQ ID NO:93; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:9, HVR-L2 containing the amino acid sequence of SEQ ID NO:21, and HVR-L3 containing the amino acid sequence of SEQ ID NO:33. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:57, HVR-H2 containing the amino acid sequence of SEQ ID NO:81, and HVR-H3 containing the amino acid sequence of SEQ ID NO:105.

[0021] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:46, HVR-H2 containing the amino acid sequence of SEQ ID NO:70, and HVR-H3 containing the amino acid sequence of SEQ ID NO:94; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:10, HVR-L2 containing the amino acid sequence of SEQ ID NO:22, and HVR-L3 containing the amino acid sequence of SEQ ID NO:34. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:58, HVR-H2 containing the amino acid sequence of SEQ ID NO:82, and HVR-H3 containing the amino acid sequence of SEQ ID NO:106.

[0022] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:47, HVR-H2 containing the amino acid sequence of SEQ ID NO:71, and HVR-H3 containing the amino acid sequence of SEQ ID NO:95; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:11, HVR-L2 containing the amino acid sequence of SEQ ID NO:23, and HVR-L3 containing the amino acid sequence of SEQ ID NO:35. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:59, HVR-H2 containing the amino acid sequence of SEQ ID NO:83, and HVR-H3 containing the amino acid sequence of SEQ ID NO:107.

[0023] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:48, HVR-H2 containing the amino acid sequence of SEQ ID NO:72, and HVR-H3 containing the amino acid sequence of SEQ ID NO:96; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:12, HVR-L2 containing the amino acid sequence of SEQ ID NO:24, and HVR-L3 containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:60, HVR-H2 containing the amino acid sequence of SEQ ID NO:84, and HVR-H3 containing the amino acid sequence of SEQ ID NO:108.

[0024] In some embodiments, the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:49, HVR-H2 containing the amino acid sequence of SEQ ID NO:73, and HVR-H3 containing the amino acid sequence of SEQ ID NO:97; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:13, HVR-L2 containing the amino acid sequence of SEQ ID NO:25, and HVR-L3 containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:61, HVR-H2 containing the amino acid sequence of SEQ ID NO:85, and HVR-H3 containing the amino acid sequence of SEQ ID NO:109.

[0025] In some embodiments of any antibody, the antibody comprises: a) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:111 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:110; b) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:113 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:112; c) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:115 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:114; d) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:117 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:116; e) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:119 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:110. f) A VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 121 and / or a VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 120; g) A VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 123 and / or a VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 122; h) A VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 125 and / or a VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 124; i) A VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 127 and / or a VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 126; j) A VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 128 and / or a VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 126; The amino acid sequence of NO:129 has at least 95% sequence identity with the VH sequence and / or the amino acid sequence of SEQ ID NO:128 has at least 95% sequence identity with the VL sequence; k) the amino acid sequence of NO:131 has at least 95% sequence identity with the VH sequence and / or the amino acid sequence of SEQ ID NO:130 has at least 95% sequence identity with the VL sequence; l) the amino acid sequence of NO:133 has at least 95% sequence identity with the VH sequence and / or the amino acid sequence of SEQ ID NO:132 has at least 95% sequence identity with the VL sequence;Or a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:135 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:134.

[0026] In some embodiments of any antibody, the antibody comprises: a) the VH sequence of SEQ ID NO:111 and / or the VL sequence of SEQ ID NO:110; b) the VH sequence of SEQ ID NO:113 and / or the VL sequence of SEQ ID NO:112; c) the VH sequence of SEQ ID NO:115 and / or the VL sequence of SEQ ID NO:114; d) the VH sequence of SEQ ID NO:117 and / or the VL sequence of SEQ ID NO:116; e) the VH sequence of SEQ ID NO:119 and / or the VL sequence of SEQ ID NO:118; f) the VH sequence of SEQ ID NO:121 and / or the VL sequence of SEQ ID NO:120; g) the VH sequence of SEQ ID NO:123 and / or the VL sequence of SEQ ID NO:122; h) the VH sequence of SEQ ID NO:125 and / or the VL sequence of SEQ ID NO:124; i) SEQ ID NO:111; d) the VH sequence of SEQ ID NO:113 and / or the VL sequence of SEQ ID NO:114; e) the VH sequence of SEQ ID NO:115 and / or the VL sequence of SEQ ID NO:114; i) the VH sequence of SEQ ID NO:116 and / or the VL sequence of SEQ ID NO:117; e) the VH sequence of SEQ ID NO:119 and / or the VL sequence of SEQ ID NO:118; f) the VH sequence of SEQ ID NO:121 and / or the VL sequence of SEQ ID NO:120; g) the VH sequence of SEQ ID NO:123 and / or the VL sequence of SEQ ID NO:122; h) the VH sequence of SEQ ID NO:125 and / or the VL sequence of SEQ ID NO:124; i) the VH sequence of j) VH sequence of SEQ ID NO:127 and / or VL sequence of SEQ ID NO:126; k) VH sequence of SEQ ID NO:129 and / or VL sequence of SEQ ID NO:128; k) VH sequence of SEQ ID NO:131 and / or VL sequence of SEQ ID NO:130; l) VH sequence of SEQ ID NO:133 and / or VL sequence of SEQ ID NO:132; or m) VH sequence of SEQ ID NO:135 and / or VL sequence of SEQ ID NO:134.

[0027] In some embodiments of any antibody, the antibody is a monoclonal antibody. In some embodiments of any antibody, the antibody is a human, humanized, or chimeric antibody. In some embodiments of any antibody, the antibody is an antibody fragment that binds to FcRH5. In some embodiments of any antibody, the antibody is an IgG1, IgG2a, or IgG2b antibody.

[0028] In some embodiments of any antibody, the antibody has one or more of the following properties: a) cross-reacts with full-length human and cynomolgus monkey FcRH5; b) does not cross-react with FcRH1, FcRH2, FcRH3 and / or FcRH4; c) binds to endogenous FcRH5; d) does not cross-react with FcRH5a; and e) does not cross-react with another Ig-like domain of FcRH5.

[0029] In some embodiments of any antibody, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody binds to FcRH5 and CD3.

[0030] In some embodiments, an isolated nucleic acid encoding the antibody described herein is provided. In some embodiments, a host cell containing said nucleic acid is provided. In some embodiments, a method for generating the antibody described herein is provided. In some embodiments, said method includes culturing a host cell containing a nucleic acid encoding an antibody.

[0031] In some embodiments, an immunoconjugate is provided. In some embodiments, the immunoconjugate comprises an anti-FcRH5 antibody and a cytotoxic agent. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the immunoconjugate has the formula Ab-(LD)p, wherein: (a) Ab is the antibody described herein; (b) L is a linker; (c) D is a drug selected from maytansinoid, auristatin, calicheamicin, pyrrolobenzodiazepine, and nemorubicin derivatives; and (d) p is in the range of 1-8. In some embodiments, D is auristatin. In some such embodiments, D has the formula D E

[0032]

[0033] Where R 2 and R 6 Each is a methyl group, R 3 and R 4 Each is isopropyl, R 5 For H, R 7 For sec-butyl, each R 8 Independently selected from CH3, O-CH3, OH and H; R 9 For H; and R 18 For –C(R) 8 )2–C(R 8 )2–aryl. In some embodiments, D is a MMAE having the following structure:

[0034]

[0035] In some embodiments, D is a pyrrolobenzodiazepine of formula A:

[0036]

[0037] The dashed line indicates that a double bond may be present between C1 and C2 or between C2 and C3; R 2 Independently selected from H, OH, =O, =CH2, CN, R, OR, =CH-R D =C(R) D 2. O-SO2-R, CO2R, and COR, optionally further selected from halogen or dihalogen groups, wherein R D Independently selected from R, CO2R, COR, CHO, CO2H, and halogen groups; R 6 and R 9 Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halogen groups; R 7 Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halogen groups; Q independently selected from O, S and NH; R 11 It is H or R, or SO3M when Q is O, where M is a metal cation; R and R' are each independently selected from optionally substituted C. 1-8 Alkyl, C 1-12 Alkyl, C 3-8 Heterocyclic group, C 3-20 Heterocyclic groups and C 5-20 aryl, and optionally, with respect to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6-, or 7-membered heterocycle; R 12 R 16 R 19 and R 17 For example, for R 2 R 6 R 9 and R 7 Defined; R″ is C 3-12 Alkylene, wherein the chain may be interrupted by an aromatic ring having one or more heteroatoms and / or optional substitutions; and X and X' are independently selected from O, S, and N (H). In some such embodiments, D is

[0038]

[0039] Where n is 0 or 1.

[0040] In some embodiments, D is a nemorubicin derivative. In some embodiments, D has a structure selected from the following:

[0041]

[0042] In some embodiments, the immunoconjugate contains a linker that can be cleaved by a protease. In some embodiments, the linker comprises a val-cit dipeptide or a Phe-homoLys dipeptide. In some embodiments, the immunoconjugate contains an acid-instable linker. In some such embodiments, the linker comprises a hydrazone.

[0043] In some embodiments, the immunoconjugate has a formula selected from the following:

[0044]

[0045] Where S is a sulfur atom;

[0046]

[0047]

[0048]

[0049] In some implementations, p is in the range of 2-5.

[0050] In some embodiments, a pharmaceutical formulation is provided. In some such embodiments, the pharmaceutical formulation comprises an immunoconjugate comprising, for example, an antibody that binds to FcRH5 as described herein. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the pharmaceutical formulation further comprises an additional therapeutic agent.

[0051] In some embodiments, methods are provided for treating an individual with FcRH5 (e.g., FcRH5c)-positive cancer. In some such embodiments, a method includes administering a pharmaceutical preparation comprising an immunoconjugate containing, for example, an antibody binding FcRH5 as described herein and / or an FcRH5 bispecific antibody. In some embodiments, the FcRH5 bispecific antibody comprises an FcRH5-binding arm and a CD3-binding arm. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the FcRH5-positive cancer is a B-cell proliferative disorder. In some embodiments, the FcRH5-positive cancer is a plasma cell tumor. In some embodiments, the plasma cell tumor is multiple myeloma. In some embodiments, a method includes administering an additional therapeutic agent to the individual.

[0052] In some embodiments, a method for inhibiting the proliferation of FcRH5 (e.g., FcRH5c) positive cells is provided. In some embodiments, the method includes exposing the cells to an immunoconjugate containing the antibody, allowing an FcRH5-binding antibody and / or an FcRH5 bispecific antibody to bind to FcRH5 on the cell surface. In some embodiments, the FcRH5 bispecific antibody comprises an FcRH5-binding arm and a CD3-binding arm. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the FcRH5-binding antibody is the antibody described herein. In some embodiments, FcRH5-positive cancer is a B-cell proliferative disorder. In some embodiments, FcRH5-positive cancer is a plasma cell tumor. In some embodiments, a plasma cell tumor is multiple myeloma. In some embodiments, a method includes administering an additional therapeutic agent to an individual.

[0053] In some embodiments, the antibody binding to FcRH5 is conjugated to a label. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the antibody binding to FcRH5 is the antibody described herein. In some embodiments, the label is a positron emitter. In some embodiments, the positron emitter is... 89 Zr.

[0054] In some embodiments, methods are provided for detecting human FcRH5 in a biological sample. In some embodiments, a method includes contacting a biological sample with an anti-FcRH5 antibody under conditions allowing the anti-FcRH5 antibody to bind to naturally occurring human FcRH5, and detecting whether a complex has formed between the anti-FcRH5 antibody and the naturally occurring human FcRH5 in the biological sample. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the anti-FcRH5 antibody is the antibody described herein.

[0055] In some embodiments, methods for detecting FcRH5-positive cancer are provided. In some such embodiments, a method includes (i) administering a labeled anti-FcRH5 antibody to a subject who has or is suspected of having FcRH5-positive cancer, and (ii) detecting the labeled anti-FcRH5 antibody in the subject, wherein detection of the labeled anti-FcRH5 antibody indicates FcRH5-positive cancer in the subject. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In some embodiments, the anti-FcRH5 antibody is the antibody described herein.

[0056] This invention relates to the following embodiments:

[0057] 1. An isolated anti-FcRH5 antibody that binds to the isotype c-specific region of the extracellular domain of FcRH5c.

[0058] 2. The antibody according to Embodiment 1, wherein the isotype c-specific region comprises an Ig-like domain 9.

[0059] 3. The antibody according to any one of embodiments 1-2, wherein the isotype c specific region comprises amino acids 743-850 of SEQ ID NO: 1.

[0060] 4. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:38, HVR-H2 containing the amino acid sequence of SEQ ID NO:62, and HVR-H3 containing the amino acid sequence of SEQ ID NO:86; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:2, HVR-L2 containing the amino acid sequence of SEQ ID NO:14, and HVR-L3 containing the amino acid sequence of SEQ ID NO:26.

[0061] 5. The antibody according to Embodiment 4, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:50, HVR-H2 containing the amino acid sequence of SEQ ID NO:74, and HVR-H3 containing the amino acid sequence of SEQ ID NO:98.

[0062] 6. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:39, HVR-H2 containing the amino acid sequence of SEQ ID NO:63, and HVR-H3 containing the amino acid sequence of SEQ ID NO:87; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:3, HVR-L2 containing the amino acid sequence of SEQ ID NO:15, and HVR-L3 containing the amino acid sequence of SEQ ID NO:27.

[0063] 7. The antibody according to Embodiment 6, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:51, HVR-H2 containing the amino acid sequence of SEQ ID NO:75, and HVR-H3 containing the amino acid sequence of SEQ ID NO:99.

[0064] 8. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:40, HVR-H2 containing the amino acid sequence of SEQ ID NO:64, and HVR-H3 containing the amino acid sequence of SEQ ID NO:88; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:4, HVR-L2 containing the amino acid sequence of SEQ ID NO:16, and HVR-L3 containing the amino acid sequence of SEQ ID NO:28.

[0065] 9. The antibody according to embodiment 8, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:52, HVR-H2 containing the amino acid sequence of SEQ ID NO:76, and HVR-H3 containing the amino acid sequence of SEQ ID NO:100.

[0066] 10. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:41, HVR-H2 containing the amino acid sequence of SEQ ID NO:65, and HVR-H3 containing the amino acid sequence of SEQ ID NO:89; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:5, HVR-L2 containing the amino acid sequence of SEQ ID NO:17, and HVR-L3 containing the amino acid sequence of SEQ ID NO:29.

[0067] 11. The antibody according to embodiment 10, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:53, HVR-H2 containing the amino acid sequence of SEQ ID NO:77, and HVR-H3 containing the amino acid sequence of SEQ ID NO:101.

[0068] 12. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:42, HVR-H2 containing the amino acid sequence of SEQ ID NO:66, and HVR-H3 containing the amino acid sequence of SEQ ID NO:90; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:6, HVR-L2 containing the amino acid sequence of SEQ ID NO:18, and HVR-L3 containing the amino acid sequence of SEQ ID NO:30.

[0069] 13. The antibody according to embodiment 12, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:54, HVR-H2 containing the amino acid sequence of SEQ ID NO:78, and HVR-H3 containing the amino acid sequence of SEQ ID NO:102.

[0070] 14. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:43, HVR-H2 containing the amino acid sequence of SEQ ID NO:67, and HVR-H3 containing the amino acid sequence of SEQ ID NO:91; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:7, HVR-L2 containing the amino acid sequence of SEQ ID NO:19, and HVR-L3 containing the amino acid sequence of SEQ ID NO:31.

[0071] 15. The antibody according to embodiment 14, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:55, HVR-H2 containing the amino acid sequence of SEQ ID NO:79, and HVR-H3 containing the amino acid sequence of SEQ ID NO:103.

[0072] 16. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:44, HVR-H2 containing the amino acid sequence of SEQ ID NO:68, and HVR-H3 containing the amino acid sequence of SEQ ID NO:92; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:8, HVR-L2 containing the amino acid sequence of SEQ ID NO:20, and HVR-L3 containing the amino acid sequence of SEQ ID NO:32.

[0073] 17. The antibody according to embodiment 16, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:56, HVR-H2 containing the amino acid sequence of SEQ ID NO:80, and HVR-H3 containing the amino acid sequence of SEQ ID NO:104.

[0074] 18. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:45, HVR-H2 containing the amino acid sequence of SEQ ID NO:69, and HVR-H3 containing the amino acid sequence of SEQ ID NO:93; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:9, HVR-L2 containing the amino acid sequence of SEQ ID NO:21, and HVR-L3 containing the amino acid sequence of SEQ ID NO:33.

[0075] 19. The antibody according to embodiment 18, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:57, HVR-H2 containing the amino acid sequence of SEQ ID NO:81, and HVR-H3 containing the amino acid sequence of SEQ ID NO:105.

[0076] 20. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:46, HVR-H2 containing the amino acid sequence of SEQ ID NO:70, and HVR-H3 containing the amino acid sequence of SEQ ID NO:94; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:10, HVR-L2 containing the amino acid sequence of SEQ ID NO:22, and HVR-L3 containing the amino acid sequence of SEQ ID NO:34.

[0077] 21. The antibody according to embodiment 20, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:58, HVR-H2 containing the amino acid sequence of SEQ ID NO:82, and HVR-H3 containing the amino acid sequence of SEQ ID NO:106.

[0078] 22. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:47, HVR-H2 containing the amino acid sequence of SEQ ID NO:71, and HVR-H3 containing the amino acid sequence of SEQ ID NO:95; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:11, HVR-L2 containing the amino acid sequence of SEQ ID NO:23, and HVR-L3 containing the amino acid sequence of SEQ ID NO:35.

[0079] 23. The antibody according to embodiment 22, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:59, HVR-H2 containing the amino acid sequence of SEQ ID NO:83, and HVR-H3 containing the amino acid sequence of SEQ ID NO:107.

[0080] 24. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:48, HVR-H2 containing the amino acid sequence of SEQ ID NO:72, and HVR-H3 containing the amino acid sequence of SEQ ID NO:96; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:12, HVR-L2 containing the amino acid sequence of SEQ ID NO:24, and HVR-L3 containing the amino acid sequence of SEQ ID NO:36.

[0081] 25. The antibody according to embodiment 24, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:60, HVR-H2 containing the amino acid sequence of SEQ ID NO:84, and HVR-H3 containing the amino acid sequence of SEQ ID NO:108.

[0082] 26. The antibody according to any one of embodiments 1-3, wherein the antibody comprises: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:49, HVR-H2 containing the amino acid sequence of SEQ ID NO:73, and HVR-H3 containing the amino acid sequence of SEQ ID NO:97; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:13, HVR-L2 containing the amino acid sequence of SEQ ID NO:25, and HVR-L3 containing the amino acid sequence of SEQ ID NO:37.

[0083] 27. The antibody according to embodiment 26, wherein the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:61, HVR-H2 containing the amino acid sequence of SEQ ID NO:85, and HVR-H3 containing the amino acid sequence of SEQ ID NO:109.

[0084] 28. The antibody according to any one of the foregoing embodiments, wherein the antibody comprises:

[0085] a) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:111 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:110;

[0086] b) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:113 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:112;

[0087] c) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:115 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:114;

[0088] d) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:117 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:116;

[0089] e) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:119 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:118;

[0090] f) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:121 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:120;

[0091] g) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:123 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:122;

[0092] h) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:125 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:124;

[0093] i) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:127 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:126;

[0094] j) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:129 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:128;

[0095] k) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:131 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:130;

[0096] l) A VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:133 and / or a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:132; or

[0097] m) The VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:135 and / or the VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:134.

[0098] 29. The antibody according to any one of embodiments 1-28, wherein the antibody comprises:

[0099] a) The VH sequence of SEQ ID NO:111 and / or the VL sequence of SEQ ID NO:110;

[0100] b) The VH sequence of SEQ ID NO:113 and / or the VL sequence of SEQ ID NO:112;

[0101] c) The VH sequence of SEQ ID NO:115 and / or the VL sequence of SEQ ID NO:114;

[0102] d) The VH sequence of SEQ ID NO:117 and / or the VL sequence of SEQ ID NO:116;

[0103] e) The VH sequence of SEQ ID NO:119 and / or the VL sequence of SEQ ID NO:118;

[0104] f) The VH sequence of SEQ ID NO:121 and / or the VL sequence of SEQ ID NO:120;

[0105] g) The VH sequence of SEQ ID NO:123 and / or the VL sequence of SEQ ID NO:122;

[0106] h) The VH sequence of SEQ ID NO:125 and / or the VL sequence of SEQ ID NO:124;

[0107] i) The VH sequence of SEQ ID NO:127 and / or the VL sequence of SEQ ID NO:126;

[0108] j) The VH sequence of SEQ ID NO:129 and / or the VL sequence of SEQ ID NO:128;

[0109] k) The VH sequence of SEQ ID NO:131 and / or the VL sequence of SEQ ID NO:130;

[0110] l) the VH sequence of SEQ ID NO:133 and / or the VL sequence of SEQ ID NO:132; or

[0111] m) The VH sequence of SEQ ID NO:135 and / or the VL sequence of SEQ ID NO:134.

[0112] 30. The antibody according to any one of embodiments 1-29, wherein the antibody is a monoclonal antibody.

[0113] 31. The antibody according to any one of embodiments 1-30, wherein the antibody is a human, humanized, or chimeric antibody.

[0114] 32. The antibody according to any one of embodiments 1-31, wherein the antibody is an antibody fragment that binds to FcRH5.

[0115] 33. The antibody according to any one of embodiments 1-32, wherein the antibody is an IgG1, IgG2a, or IgG2b antibody.

[0116] 34. The antibody according to any one of embodiments 1-33, wherein the antibody has one or more of the following characteristics: a) cross-reacts with full-length human and cynomolgus monkey FcRH5; b) does not cross-react with FcRH1, FcRH2, FcRH3 and / or FcRH4; c) binds to endogenous FcRH5; d) does not cross-react with FcRH5a; and e) does not cross-react with another Ig-like domain of FcRH5.

[0117] 35. The antibody according to any one of embodiments 1-34, wherein the antibody is a bispecific antibody.

[0118] 36. The antibody according to embodiment 35, wherein the bispecific antibody binds to FcRH5 and CD3.

[0119] 37. An isolated nucleic acid encoding an antibody according to any one of embodiments 1-36.

[0120] 38. A host cell comprising the nucleic acid according to embodiment 37.

[0121] 39. A method for generating an antibody, comprising culturing a host cell according to embodiment 38 to generate the antibody.

[0122] 40. An immunoconjugate comprising an antibody and a cytotoxic agent according to any one of embodiments 1-36.

[0123] 41. The immunoconjugate according to embodiment 41, having the formula Ab-(LD)p, wherein:

[0124] (a) Ab is an antibody according to any one of embodiments 1-36;

[0125] (b) L is a connector;

[0126] (c)D is a drug selected from maytansine, auristatin, calichimycin, pyrrolobenzodiazepines, and nemorubicin derivatives; and

[0127] (d)p is in the range of 1-8.

[0128] 42. The immunoconjugate according to embodiment 41, wherein D is aurestatin.

[0129] 43. The immunoconjugate according to embodiment 42, wherein D has the formula D E

[0130]

[0131] And R 2 and R 6 Each is a methyl group, R 3 and R 4 Each is isopropyl, R 5 For H, R 7 For sec-butyl, each R 8 Independently selected from CH3, O-CH3, OH and H; R 9 For H; and R 18 For C(R) 8 )2–C(R 8 )2–aryl.

[0132] 44. The immunoconjugate according to embodiment 41, wherein the drug is MMAE.

[0133] 45. The immunoconjugate according to embodiment 41, wherein D is a pyrrolobenzodiazepine of formula A:

[0134]

[0135] The dashed lines indicate double bonds that may exist between C1 and C2 or between C2 and C3;

[0136] R 2 Independently selected from H, OH, =O, =CH2, CN, R, OR, =CH-R D =C(R) D 2. O-SO2-R, CO2R, and COR, optionally further selected from halogen or dihalogen groups, wherein R D It is independently selected from R, CO2R, COR, CHO, CO2H and halogen groups;

[0137] R 6 and R 9 Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halogen groups;

[0138] R 7 Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halogen groups;

[0139] Q is independently selected from O, S, and NH;

[0140] R 11 It is H or R, or SO3M when Q is O, where M is a metal cation;

[0141] R and R' are each independently selected from the arbitrarily substituted C. 1-8 Alkyl, C 3-8 Heterocyclic groups and C 5-20 Aryl, and optionally, with respect to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocycle;

[0142] R 12 R 16 R 19 and R 17 For example, for R 2 R 6 R 9 and R 7 Defined;

[0143] R″ is C 3-12 Alkylene, wherein the chain may be interrupted by one or more heteroatoms and / or optionally substituted aromatic rings; and

[0144] X and X' are independently selected from O, S, and N (H).

[0145] 46. ​​The immunoconjugate according to embodiment 45, wherein D has the following structure:

[0146]

[0147] Where n is 0 or 1.

[0148] 47. The immunoconjugate according to embodiment 45, wherein D has a structure selected from the following:

[0149]

[0150] Where R E and R E” Each is independently selected from H or R D , where R D It is independently selected from R, CO2R, COR, CHO, CO2H and halogen groups;

[0151] Among them, Ar 1 and Ar 2 Each of the Cs can be substituted independently. 5-20 Aryl; and

[0152] Where n is 0 or 1.

[0153] 48. The immunoconjugate according to embodiment 41, wherein D is a pyrrolobenzodiazepine of formula B:

[0154]

[0155] The horizontal wavy line indicates the covalent connection site with the connector;

[0156] R V1 and R V2 Independently selected from H, methyl, ethyl, phenyl, fluorinated phenyl and C 5-6 Heterocyclic groups; and

[0157] n is 0 or 1.

[0158] 49. The immunoconjugate according to embodiment 41, wherein D is a nemorubicin derivative.

[0159] 50. The immunoconjugate according to embodiment 49, wherein D has a structure selected from the following:

[0160]

[0161]

[0162] 51. The immunoconjugate according to any one of embodiments 41 to 50, wherein the linker can be cleaved by a protease.

[0163] 52. The immunoconjugate according to embodiment 51, wherein the linker comprises val-cit dipeptide or Phe-homoLys dipeptide.

[0164] 53. The immunoconjugate according to any one of embodiments 41 to 50, wherein the linker is unstable to acid.

[0165] 54. The immunoconjugate according to embodiment 53, wherein the linker comprises hydrazone.

[0166] 55. The immunoconjugate according to embodiment 53 has the following formula:

[0167]

[0168] Where S is a sulfur atom.

[0169] 56. The immunoconjugate according to embodiment 46 has a formula selected from the following:

[0170]

[0171] 57. The immunoconjugate according to embodiment 50 has a formula selected from the following:

[0172]

[0173]

[0174]

[0175] 58. The immunoconjugate according to any one of embodiments 41 to 57, wherein p is in the range of 2-5.

[0176] 59. A pharmaceutical formulation comprising an antibody according to any one of embodiments 1-36 and / or an immunoconjugate according to any one of embodiments 41-58 and a pharmaceutically acceptable carrier.

[0177] 60. The pharmaceutical preparation according to embodiment 59 further comprises additional therapeutic agents.

[0178] 61. A method for treating an individual with FcRH5-positive cancer, the method comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1-36 and / or an immunoconjugate according to any one of embodiments 41-59.

[0179] 62. The method according to embodiment 61, wherein the FcRH5 positive cancer is a B-cell proliferative disease.

[0180] 63. The method according to any one of embodiments 61-62, further comprising administering an additional therapeutic agent to the individual.

[0181] 64. A method for inhibiting the proliferation of FcRH5 positive cells, the method comprising exposing the cells to the antibody and / or the immunoconjugate, wherein the antibody according to any one of embodiments 1-36 and / or the immunoconjugate according to any one of embodiments 41-59 are allowed to bind to FcRH5 on the surface of the cells, thereby inhibiting the proliferation of the cells.

[0182] 65. The method according to embodiment 64, wherein the cell is a B cell.

[0183] 66. The antibody according to any one of embodiments 1-36, conjugated to a label.

[0184] 67. The antibody according to embodiment 66, wherein the label is a positron emitter.

[0185] 68. The antibody according to embodiment 67, wherein the positron emitter is 89 Zr.

[0186] 69. A method for detecting human FcRH5 in a biological sample, comprising contacting the biological sample with an anti-FcRH5 antibody according to any one of embodiments 1-36 or 66-68 under conditions that allow the anti-FcRH5 antibody to bind to naturally occurring human FcRH5, and detecting whether a complex is formed between the anti-FcRH5 antibody and the naturally occurring human FcRH5 in the biological sample.

[0187] 70. The method according to embodiment 69, wherein the anti-FcRH5 antibody is an antibody as described in embodiment 9 or embodiment 15.

[0188] 71. The method according to any one of embodiments 69-70, wherein the biological sample is a blood sample.

[0189] 72. A method for detecting FcRH5-positive cancer, comprising (i) administering a labeled anti-FcRH5 antibody to a subject who has or is suspected of having FcRH5-positive cancer, wherein the labeled anti-FcRH5 antibody comprises an anti-FcRH5 antibody according to any one of embodiments 1 to 36; and (ii) detecting the labeled anti-FcRH5 antibody in the subject, wherein the detection of the labeled anti-FcRH5 antibody indicates FcRH5-positive cancer in the subject.

[0190] 73. The method according to embodiment 72, wherein the labeled anti-FcRH5 antibody comprises an anti-FcRH5 antibody conjugated to a positron emitter.

[0191] 74. The method according to any one of embodiments 72-73, wherein the positron emitter is 89 Zr. Brief description of the attached diagram

[0192] Figure 1AThe three main isotypes of FcRH5 are described: FcRH5a (IRTA2a; UniProt identifier Q96RD9-3), FcRH5b (IRTA2b; UniProt identifier Q96RD9-4), and FcRH5c (IRTA2c; UniProt identifier Q96RD9-1). The Ig-like domains are numbered and correspond to the amino acid sequences (SEQ ID NO:1) of UniProt identifier Q69RD9-1: Ig-like domain 1 (aa("amino acid") 23-100), Ig-like domain 2 (aa 105-185), Ig-like domain 3 (aa 188-271), Ig-like domain 4 (287-373), Ig-like domain 5 (aa 380-466), Ig-like domain 6 (aa 490-555), Ig-like domain 7 (aa 568-652), Ig-like domain 8 (aa 658-731) and Ig-like domain 9 (aa 754-835). Figure 1B The structure and homology of FcRH5 moiety (SEQ ID NO:136) and FcRH5c amino acids 735 to 977 (SEQ ID NO:2) are described.

[0193] Figure 2A -B shows the interaction between the FcRH5 antibody and ( ) Figure 2A ) person FcRH5 and ( Figure 2B The binding of cynomolgus monkey FcRH5 to SVT2 cells transfected at different concentrations.

[0194] Figure 3A -F indicates the interaction between the FcRH5 antibody and the human FcRH5 transfected (…). Figure 3A EJM cells or ( Figure 3B The binding of OPM2 cells, and the subclonal supernatant ( Figure 3C )5A10.1、( Figure 3D )5F1.1、( Figure 3E )3G7.1 and ( Figure 3F )6D2.2 binding to MOLP2 cells endogenously expressing FcRH5.

[0195] Figure 4A -B shows the FcRH5 subcloning supernatant and the (… Figure 4A )WT or ( Figure 4B The binding of 293 cells transfected with the mutant FcRH5, which lacks four apical extracellular domains.

[0196] Figure 5A -B shows ( Figure 5A The binding of FcRH5 antibody to FcRH5a (by ELISA), and ( Figure 5BThe binding of subclonal supernatant to human B cells.

[0197] Figure 6A -D shows the FcRH5 subcloning supernatant and the ( Figure 6A )FcRH1、( Figure 6B )FcRH2、( Figure 6C )FcRH3 or ( Figure 6D The binding of FcRH4-transfected SVT2 cells.

[0198] Figure 7 The binding of FcRH5 antibody subclonal supernatant to NK cells was shown.

[0199] Figure 8A -D indicates ( Figure 8A FcRH5 bisFab, FcRH5-TDB (clone 10A8) and ( Figure 8B HER2-TDB's killing activity against FcRH5-transfected 293 cells and ( Figure 8C )FcRH5-bisFab and ( Figure 8D The killing activity of FcRH5-TDB against FcRH5-transfected 293 cells.

[0200] Figure 9A -B shows the effects of FcRH5 bisFab and FcRH5-TDB on MOLP-2 cells. Figure 9A ) kills activity and ( Figure 9B T cell activation. Detailed Implementation

[0201] I. Definition

[0202] As used herein, the term "FcRH5" refers to any naturally occurring mature FcRH5 produced from the FcRH5 precursor protein in processing cells. Unless otherwise stated, the term includes FcRH5 from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). The term also includes naturally occurring variants of FcRH5, such as splice variants or allelic variants. In some embodiments, the amino acid sequence of the human FcRH5 protein is FcRH5a (IRTA2a; UniProt identifier Q96RD9-3; 759aa), FcRH5b (IRTA2b; UniProt identifier Q96RD9-4; 592aa), FcRH5c (IRTA2c; UniProt identifier Q96RD9-1; 977aa (SEQ ID NO:1), UniProt identifier Q96RD9-2 (124aa) and / or FcRH5d (IRTA2d; UniProt identifier Q96RD9-5; 152aa).

[0203] The term "glycosylated form of FcRH5" refers to the naturally occurring form of FcRH5 that is post-translationally modified by the addition of carbohydrate residues.

[0204] With respect to a reference polypeptide sequence, "percentage (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after alignment, with vacancies introduced where necessary to obtain the maximum percentage sequence identity, and without treating any conserved substitutions as part of the sequence identity. Alignments performed for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Those skilled in the art can determine suitable parameters for the alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this document, the sequence comparison computer program ALIGN-2 is used to generate the % amino acid sequence identity value. The ALIGN-2 sequence comparison computer program is licensed by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office, Washington DC, 20559, under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be assembled from source code. The ALIGN-2 program should be assembled for use with UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0205] When performing amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A with respect to / relative to a given amino acid sequence B (or this can be expressed in the phrase "a given amino acid sequence A has or contains a specific % amino acid sequence identity with / relative to a given amino acid sequence B") is calculated as follows:

[0206] The fraction X / Y is multiplied by 100, where X is the number of amino acid residues that the sequence alignment program ALIGN-2 rates as identical matches in the alignments of A and B, and Y is the total number of amino acid residues in B. It should be understood that when 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 be equal to the % amino acid sequence identity of B to A. Unless otherwise explicitly stated, all % amino acid sequence identity values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0207] The terms "anti-FcRH5 antibody" and "antibody bound to FcRH5" refer to antibodies that bind to FcRH5 with sufficient affinity, thereby enabling the antibody to be used as a diagnostic and / or therapeutic agent for targeting FcRH5. In one embodiment, the anti-FcRH5 antibody binds to less than about 10% of the antibody's binding to FcRH5, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody bound to FcRH5 has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or less, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (Kd) of M). In some embodiments, the anti-FcRH5 antibody binds to an epitope of FcRH5 that is conserved across FcRH5 from different species. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0208] The term “antibody” is used in the broadest sense in this document and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0209] An "antibody fragment" is a molecule other than a complete antibody that contains a portion of the complete antibody and binds to an antigen that binds to the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0210] The term "epitope" refers to a specific site on an antigen molecule that binds to an antibody.

[0211] "Antibody binding to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0212] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a largely homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or those that may appear during the production of the monoclonal antibody formulation, which are typically present in small quantities. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates the antibody characteristics obtained from a largely homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, 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 preparing monoclonal antibodies are described herein.

[0213] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to an antibody having a structure that is substantially similar to that of a natural antibody or to an antibody having a heavy chain containing an Fc region as defined herein.

[0214] "Naked antibody" refers to an antibody that is not conjugated to a heterologous portion (such as a cytotoxic portion) or radiolabeled. This naked antibody can be present in pharmaceutical formulations.

[0215] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chains of antibodies can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of their constant domains.

[0216] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0217] "Human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by humans or human cells, or an amino acid sequence derived from a non-human source using the complete set of human antibodies or other human antibody coding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0218] "Humanized" antibodies are chimeric antibodies comprising amino acid residues from nonhuman HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise substantially all at least one and typically two variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the nonhuman antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Optionally, a humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. The "humanized" form of an antibody, such as a nonhuman antibody, refers to an antibody that has undergone humanization.

[0219] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different immunoglobulin classes are respectively called α, δ, ε, γ, and μ.

[0220] The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0221] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in the binding of the antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain can be sufficient to produce antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated by screening complementary VL or VH domain libraries, respectively, using VH or VL domains from antibodies binding to that antigen. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0222] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0223] "Human common framework" refers to the framework representing the amino acid residues that most frequently occur in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of the variable domain sequence. Generally, the subgroup is one such subgroup as those in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI in Kabat et al. (ibid.). In one embodiment, for VH, the subgroup is subgroup III in Kabat et al. (ibid.).

[0224] As used herein, the term “hypervariant region” or “HVR” refers to each of the regions of an antibody variable domain that are highly variable in sequence and / or form structurally defining loops (“hypervariant loops”). Typically, a natural tetrachain antibody contains six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the “complementarity-determining region” (CDR), which has the highest sequence variability and / or is involved in antigen recognition. Exemplary hypervariant loops are located 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).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) appear 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 Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).) Except for CDR1 in VH, CDRs typically contain amino acid residues that form a hypervariable ring. CDRs also contain “specificity-determining residues” or “SDRs,” which are residues that contact the antigen. SDRs are contained within a region of the CDR called abbreviated CDRs or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are located at amino acid residues 31-34 (L1), 50-55 (L2), 89-96 (L3), 31-35B (H1), 50-58 (H2), and 95-102 (H3). (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al. (ibid.).

[0225] For the purposes of this document, a “recipient human frame” is a frame comprising an amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human common frame as defined below. A recipient human frame “derived from” the human immunoglobulin frame or the human common frame may contain the same amino acid sequence, or may contain variations in the amino acid sequence. In some embodiments, the number of amino acid variations is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer. In some embodiments, the sequence of the VL recipient human frame is identical to the VL human immunoglobulin frame sequence or the human common frame sequence.

[0226] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can typically 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 below.

[0227] "Affinity-mature" antibodies are those that have one or more alterations in one or more hypervariable regions (HVRs) that improve the antibody's affinity for the antigen compared to parent antibodies that do not have these alterations.

[0228] "Immune conjugates" are antibodies conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents).

[0229] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212Radioactive 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 inhibitors; enzymes and fragments thereof, such as lysozymes; antibiotics; toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various antitumor or anticancer agents disclosed below.

[0230] "Effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0231] "Isolated antibody" is an antibody that has been isolated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0232] "Isolated nucleic acids" refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain such molecules, but which are located outside chromosomes or at a chromosomal location different from their natural chromosomal location.

[0233] "Isolated nucleic acid encoding anti-FcRH5 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single vector or different vectors and such nucleic acid molecules present at one or more locations in the host cell.

[0234] As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of the host cell to which they are introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operably ligated. Such vectors are referred to herein as "expression vectors."

[0235] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be identical to parental cells in nucleic acid content and may contain mutations. This article includes mutant progeny with the same function or biological activity as screened or selected in the initial transformed cells.

[0236] As used herein, “treatment” (and its grammatical variations) refers to a clinical intervention that attempts to alter natural processes in the individual being treated, and may be performed for prevention or in the course of clinicopathology. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological outcomes of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some implementations, the antibodies described herein are used to delay the development of the disease or slow its progression.

[0237] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), germ cell tumor, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, small bowel cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0238] The term "B-cell malignancies" in this article includes non-Hodgkin's lymphoma (NHL), which includes low-grade / follicular NHL, small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-cleaved cell NHL, large mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia. Macroglobulinemia, non-Hodgkin's lymphoma (NHL), lymphocyte-predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), refractory NHL including relapsed refractory NHL and rituximab-refractory NHL; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloid leukemia; mantle cell lymphoma; and other hematologic malignancies. These malignancies can be treated with antibodies targeting B-cell surface markers such as FcRH5 (e.g., FcRH5c). This article anticipates the treatment of these diseases by administering antibodies targeting B-cell surface markers such as FcRH5 (e.g., FcRH5c), including the administration of unconjugated ("naked") antibodies or antibodies conjugated to the cytotoxic agents disclosed herein. This article also anticipates the use of combination therapies to treat such diseases, including combinations of anti-FcRH5 antibodies (including FcRH5 bispecific antibodies) or anti-FcRH5 antibody-drug conjugates with another antibody or antibody-drug conjugate, another cytotoxic agent, radiation or other treatments administered simultaneously or sequentially.

[0239] As used herein, the term "non-Hodgkin lymphoma" or "NHL" refers to cancers of the lymphatic system other than Hodgkin lymphoma. Hodgkin lymphoma is generally distinguished from non-Hodgkin lymphoma by the presence of Reed-Sternberg cells in Hodgkin lymphoma and the absence of said cells in non-Hodgkin lymphoma. Examples of non-Hodgkin lymphoma covered by the terminology used herein include any lymphoma that would be identified as non-Hodgkin lymphoma by a person skilled in the art (e.g., an oncologist or pathologist) according to classification schemes known in the art, such as the Revised European-American Lymphoma (REAL) scheme described in the Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). See, specifically, the lists in Figures 11.57, 11.58, and 11.59. More specific examples include, but are not limited to: relapsed or refractory NHL, first-line low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-cell lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, intranodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasmacytic myeloma, low-grade NHL. Follicular lymphoma, intermediate / follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive first-line NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary exudative lymphoma, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-cleaved cell NHL, large mass disease NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous lymphoma, anaplastic large cell lymphoma, angiogenic central lymphoma.

[0240] Plasma cell disorders are caused by the uncontrolled division or proliferation of plasma cell clones. Plasma cells originate from activated B lymphocytes (i.e., B cells). Each B cell produces unique receptors (called B cell receptors) arranged on its cell surface, which are specific to exogenous substances (i.e., antigens). When a B cell receptor binds to its homologous antigen, the cell expressing that receptor is activated and re-enters the cell cycle, generating many clonal copies of itself. These clones mature into plasma cells, which primarily remain in the bone marrow and are dedicated to producing copies of B cell receptors released into the bloodstream as antibodies. In plasma cell disorders, plasma cells or maternal B cells suffer genetic damage, resulting in inhibition or insensitivity to normal constraints on cell division and / or activity. Daughter plasma cells derived from these cells are malignant because they can divide uninhibitedly and / or produce excessive amounts of the same immunoglobulins (antibodies). The resulting immunoglobulins are often incomplete or incorrectly structured, which can cause the accumulation of proteins (also known as monoclonal proteins, M proteins, paraproteins, or amyloid proteins, depending on the specific disorder) in serum, tissues, or organs (especially the kidneys), leading to organ dysfunction and / or failure. Plasma cell disorders include monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), macroglobulinemia, heavy chain disease, and systemic light chain amyloidosis (AL), which are distinguished based on clonal proliferative capacity, the extent of bone marrow involvement, and the type of M protein expressed. Other plasma cell disorders include solitary plasmacytoma, extramedullary plasmacytoma, multiple solitary plasmacytomas, plasma cell leukemia, Waldenström macroglobulinemia, B-cell non-Hodgkin lymphoma, and B-cell chronic lymphocytic leukemia.

[0241] The term "FcRH5-positive cancer" refers to cancer contained in cells that express FcRH5 on their surface. For the purpose of determining whether cells express FcRH5 on their surface, FcRH5 mRNA expression is considered to be correlated with FcRH5 expression on the cell surface. In some embodiments, FcRH5 mRNA expression is determined by methods selected from in situ hybridization and RT-PCR (including quantitative RT-PCR). Alternatively, FcRH5 expression on the cell surface can be determined, for example, using an antibody against FcRH5 in methods such as immunohistochemistry, FACS, etc. In some embodiments, FcRH5 is one or more of FcRH5a, FcRH5b, FcRH5c (UniProt identifier Q96RD9-2), and / or FcRH5d. In some embodiments, FcRH5 is FcRH5c.

[0242] The term "FcRH5 positive cell" refers to a cell that expresses FcRH5 on its surface. In some embodiments, FcRH5 is one or more of FcRH5a, FcRH5b, FcRH5c (UniProt identifier Q96RD9-2) and / or FcRH5d. In some embodiments, FcRH5 is FcRH5c.

[0243] The “effective amount” of a drug (such as a pharmaceutical preparation) refers to the amount that is effective in achieving the desired therapeutic or preventive outcome at the necessary dose and time.

[0244] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 some embodiments, the individual or subject is a human.

[0245] The term "instructions for use" refers to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings about the use of such therapeutic products.

[0246] The term "pharmaceutical formulation" refers to a formulation which is presented in a form that enables the biological activity of the active ingredient contained therein to be effective, and which does not contain any other components that would have unacceptable toxicity to a subject who would administer the formulation.

[0247] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the subjects. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0248] "Alkyl" refers to C1-C18 hydrocarbons containing n-, tertiary, tertiary, or cyclic carbon atoms. Examples include methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (n-Pr, n-propyl, -CH2CH2CH3), 2-propyl (i-Pr, isopropyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, isobutyl, -CH2CH(CH3)2), 2-butyl (s-Bu, sec-butyl, -CH(CH3)CH2CH3), 2-methyl-2- propyl (t-Bu, tert-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1- Butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH( CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3). As used herein, the term "C1-C8 alkyl" refers to a straight-chain or branched saturated or unsaturated hydrocarbon having 1 to 8 carbon atoms.Representative "C1-C8 alkyl" groups include, but are not limited to: -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, and -n-decyl; while branched C1-C8 alkyl groups include, but are not limited to: -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl; unsaturated C1-C8 alkyl groups include, but are not limited to: -vinyl, - Allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl. C1-C8 alkyl groups may be unsubstituted or substituted with one or more groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -SO3R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0249] As used in this article, the term "C1-C" 12 "Alkyl" refers to a straight-chain or branched saturated or unsaturated hydrocarbon having 1 to 12 carbon atoms. (C1-C) 12 The alkyl group may be unsubstituted or substituted with one or more groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -SO3R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0250] As used herein, the term "C1-C6 alkyl" refers to a straight-chain or branched saturated or unsaturated hydrocarbon having 1 to 6 carbon atoms. Representative "C1-C6 alkyl" groups include, but are not limited to: -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C1-C6 alkyl groups include, but are not limited to: -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl; unsaturated C1-C6 alkyl groups include, but are not limited to: -vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, and 3-hexyl. C1-C6 alkyl groups may be unsubstituted or substituted with one or more groups as described above for C1-C8 alkyl groups.

[0251] As used herein, the term "C1-C4 alkyl" refers to a straight-chain or branched saturated or unsaturated hydrocarbon having 1 to 4 carbon atoms. Representative "C1-C4 alkyl" groups include, but are not limited to: -methyl, -ethyl, -n-propyl, -n-butyl; while branched C1-C4 alkyl groups include, but are not limited to: -isopropyl, -sec-butyl, -isobutyl, -tert-butyl; unsaturated C1-C4 alkyl groups include, but are not limited to: vinyl, -allyl, -1-butenyl, -2-butenyl, and -isobutenyl. C1-C4 alkyl groups may be unsubstituted or substituted with one or more groups as described above for C1-C8 alkyl groups.

[0252] "Alkoxy" is an alkyl group bonded to an oxygen single bond. Exemplary alkoxy groups include, but are not limited to, methoxy (-OCH3) and ethoxy (-OCH2CH3). "C1-C5 alkoxy" is an alkoxy group having 1 to 5 carbon atoms. Alkoxy groups may be unsubstituted or substituted with one or more groups as described above for alkyl groups.

[0253] "Alkenyl" refers to a C2-C group containing ortho-, secondary-, tertiary-, or cyclic carbon atoms. 18 Hydrocarbons having at least one unsaturated position, i.e., carbon-carbon sp. 2 Double bonds. Examples include, but are not limited to: ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2). "C2-C8 alkenyl" refers to hydrocarbons containing 2 to 8 normal, secondary, tertiary, or cyclic carbon atoms, having at least one unsaturated position, i.e., carbon-carbon sp. 2 Double bond.

[0254] "Alkyne" is a C2-C group containing a normal, secondary, tertiary, or cyclic carbon atom. 18Hydrocarbons having at least one unsaturated position, i.e., a carbon-carbon sp triple bond. Examples include, but are not limited to, ethynyl (-C≡CH) and propyne (-CH2C≡CH). “C2-C8 ethynyl” are hydrocarbons containing 2 to 8 normal, secondary, tertiary, or cyclic carbon atoms having at least one unsaturated position, i.e., a carbon-carbon sp triple bond.

[0255] "Alkylene" refers to a saturated branched, straight, or cyclic hydrocarbon group with 1-18 carbon atoms and two monovalent group centers, derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include, but are not limited to: methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), etc.

[0256] “C1-C 10 "alkylene" is represented by the formula -(CH2). 1-10 - a straight-chain saturated hydrocarbon group. C1-C 10 Examples of alkylene compounds include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene.

[0257] "Alkenyl" refers to an unsaturated branched, straight, or cyclic hydrocarbon group with 2-18 carbon atoms and two monovalent group centers, which are derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent olefin. Typical alkenyl groups include, but are not limited to: 1,2-ethylene (-CH=CH-).

[0258] "Imyynyl" refers to an unsaturated branched, straight, or cyclic hydrocarbon group with 2-18 carbon atoms and two monovalent group centers, derived from the removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkyne. Typical imynyl groups include, but are not limited to: acetylene (-C≡C-), propyne (-CH2C≡C-), and 4-pentynyl (-CH2CH2CH2C≡C-).

[0259] "Aryl" refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracene. The carbocyclic aromatic group or heterocyclic aromatic group may be unsubstituted or substituted with one or more groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2, and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl, and aryl.

[0260] “C5-C 20 "Aryl" refers to an aryl group having 5 to 20 carbon atoms in a carbocyclic aromatic ring. (C5-C) 20 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracene. (C5-C) 20 Aryl groups may be substituted or not substituted, as described above for aryl groups. "C5-C 14 "Aryl" refers to an aryl group having 5 to 14 carbon atoms in a carbocyclic aromatic ring. (C5-C) 14 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracene. (C5-C) 14 Aryl groups may be substituted or not substituted, as described above for aryl groups.

[0261] "Aryl" is an aryl group with two covalent bonds and can have ortho, meta, or para configurations as shown in the following structures:

[0262]

[0263] The phenyl group may be unsubstituted or substituted with up to four groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0264] "Arylalkyl" refers to an acyclic alkyl group in which the atom is bonded to a carbon atom (usually terminal or sp). 3One of the hydrogen atoms in an aryl alkyl group is replaced by an aryl group. Typical aryl alkyl groups include, but are not limited to: benzyl, 2-phenylethane-1-yl, 2-phenylvinyl-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, 2-naphthylvinyl-1-yl, naphthobenzyl, 2-naphthophenylethane-1-yl, etc. Aryl alkyl groups contain 6 to 20 carbon atoms; for example, the alkyl portion (including alkyl, alkenyl, or alkynyl) of an aryl alkyl group has 1 to 6 carbon atoms and the aryl portion has 5 to 14 carbon atoms.

[0265] "Heteroarylalkyl" refers to an acyclic alkyl group in which the atom is bonded to a carbon atom (usually terminal or sp). 3 One of the hydrogen atoms of a carbon atom is replaced by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furanylethyl, etc. Heteroarylalkyl groups contain 6 to 20 carbon atoms, for example, the alkyl part (including alkyl, alkenyl, or alkynyl) of a heteroarylalkyl group has 1 to 6 carbon atoms and the heteroaryl part has 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl part of a heteroarylalkyl group can be a monocyclic ring with 3 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: bicyclic [4,5], [5,5], [5,6], or [6,6] systems.

[0266] "Substituted alkyl", "Substituted aryl", and "Substituted arylalkyl" refer to alkyl, aryl, and arylalkyl groups in which one or more hydrogen atoms are independently replaced by substituents, respectively. Typical substituents include, but are not limited to, -X, -R, and -O. - -OR, -SR, -S - , -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, NC(=O)R, -C(=O)R, -C(=O)NR2, -SO3 - -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR, -S(=O)R, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C(=O)R, -C(=O)X, -C(=S)R, -CO2R, -CO2, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR2, -C(=S)NR2, -C(=NR)NR2, where each X is independently a halogen: F, Cl, Br or I; and each R is independently -H, C2-C 18 Alkyl, C6-C 20 Aryl, C3-C 14Heterocyclic rings, protecting groups, or prodrug moieties. Alkylene, alkenylene, and yntylide groups, as described above, can also be similarly substituted.

[0267] "Heteroaryl" and "heterocyclic" refer to ring systems in which one or more ring atoms are heteroatoms such as nitrogen, oxygen, and sulfur. Heterocyclic groups contain 3 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heterocyclic groups can be monocyclic with 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or bicyclic with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), such as bicyclic [4,5], [5,5], [5,6], or [6,6] systems.

[0268] Exemplary heterocycles are described, for example, in Paquette, Leo A., “Principles of Modern Heterocyclic Chemistry” (WA Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7 and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), specifically Volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0269] Examples of heterocycles include, but are not limited to, pyridinyl, dihydropyridinyl, tetrahydropyridine (piperidinyl), thiazolyl, tetrahydrothiophene, sulfur-oxidized tetrahydrothiophene, pyrimidinyl, furanyl, thiophene, pyrroleyl, pyrazolyl, imidazoleyl, tetrazolyl, benzofuranyl, thianaphthyl, indolyl, pseudoindolyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4- Piperidinone, pyrrolyl, 2-pyrrolidone, pyrrololinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, acryloxynyl, triazine, 6H-1,2,5-thiadiazine, 2H,6H-1,5,2-dithiazine, thienyl, thianyl, pyranyl, isobenzofuranyl 2H-pyrrole, benzopyranyl, xanthyl, phenoxathiol, 2H-pyrrole, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indoleyl, isoydinolyl, 3H-indolyl, 1H-indazolyl, purine, 4H-quinazinyl, phthalazinyl, naphthidyl, quinoxalinyl, quinazolinyl, cinnaphthyl, pteridinyl, 4aH-carbazoleyl, carbazoleyl, β-carbolinyl, phenanthridine, acridineyl, pyrimidine alkyl, phenanthroline, phenazinyl, phenothiazinyl, furazinyl, phenoxazinyl, isobenzodihydropyranyl, benzodihydropyranyl, imidazoalkyl, imidazoline, pyrazolyl, pyrazolyl, piperazinyl, dihydroindolyl, isodihydroindolyl, quininecycloyl, morpholinyl, oxazolyl, benzotriazolyl, benzoisooxazolyl, hydroxyindolyl, benzooxazolinyl, indigo (isatinoyl).

[0270] For example, and without limitation, the carbon-bonded heterocycle is bonded at the following positions: 2, 3, 4, 5, or 6 of pyridine; 3, 4, 5, or 6 of pyridazine; 2, 4, 5, or 6 of pyrimidine; 2, 3, 5, or 6 of pyrazine; 2, 3, 4, or 5 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; 2, 4, or 5 of oxazole, imidazole, or thiazole; 3, 4, or 5 of isoxazole, pyrazole, or isothiazole; 2 or 3 of aziridine; 2, 3, or 4 of acridine; 2, 3, 4, 5, 6, 7, or 8 of quinoline; or 1, 3, 4, 5, 6, 7, or 8 of isoquinoline. More typically, the heterocycles of the carbon bond include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0271] For example, and without limitation, nitrogen-bonded heterocycles are bonded at the following positions: aziridinium, acridine, pyrrole, pyrrolidine, 2-pyrrololine, 3-pyrrololine, imidazole, imidazoline, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindole, 1H-indazole at the 1 position; isoindole or isoindoline at the 2 position; morpholine at the 4 position; and carbazole or β-carbline at the 9 position. More generally, nitrogen-bonded heterocycles include 1-aziridinyl, 1-acridinyl, 1-pyrroleyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0272] "C3-C8 heterocycles" refer to aromatic or non-aromatic C3-C8 carbon rings in which one to four ring carbon atoms are independently replaced by heteroatoms selected from the group consisting of O, S, and N. Representative examples of C3-C8 heterocycles include, but are not limited to: benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrroleyl, thiopheneyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridoneyl, pyrazinyl, pyrazinyl, isothiazolyl, isoxazolyl, and tetrazolyl. The C3-C8 heterocycle may be unsubstituted or substituted with up to seven groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0273] "C3-C8 heterocyclic group" refers to the C3-C8 heterocyclic group defined above, wherein one of the hydrogen atoms of the heterocyclic group is replaced by a single bond. The C3-C8 heterocyclic group may be unsubstituted or substituted by up to six groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0274] “C3-C 20 A heterocycle is an aromatic or non-aromatic C3-C8 carbon ring in which one to four carbon atoms are independently replaced by heteroatoms selected from a group consisting of O, S, and N. (C3-C)20 The heterocycle may be unsubstituted or substituted with up to seven groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0275] “C3-C 20 "Heterocyclic group" refers to the C3-C group defined above. 20 A heterocyclic group in which one of the hydrogen atoms of the heterocyclic group is replaced by a single bond.

[0276] "Carbocyclic ring" refers to a saturated or unsaturated ring having 3 to 7 carbon atoms (monocyclic form) or 7 to 12 carbon atoms (bicyclic form). Monocyclic carbon rings have 3 to 6 ring atoms, more commonly 5 or 6 ring atoms. Bicyclic carbon rings have 7 to 12 ring atoms, for example, configured as bicyclic [4,5], [5,5], [5,6], or [6,6] systems; or 9 or 10 ring atoms configured as bicyclic [5,6] or [6,6] systems. Examples of monocyclic carbon rings include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl, and cyclooctyl.

[0277] "C3-C8 carbon rings" are 3, 4, 5, 6, 7, or 8-membered saturated or unsaturated non-aromatic carbon rings. Representative C3-C8 carbon rings include, but are not limited to: -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptanetrienyl, -cyclooctyl, and -cyclooctadienyl. The C3-C8 carbocyclic group may be unsubstituted or substituted with one or more groups, including but not limited to -C1-C8 alkyl, -O-(C1-C8 alkyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2-NHC(O)R', -S(O)2R', -S(O)R', -OH, -halogen, -N3, -NH2, -NH(R'), -N(R')2 and -CN; wherein each R' is independently selected from H, -C1-C8 alkyl and aryl.

[0278] "C3-C8 carbocyclic group" refers to the C3-C8 carbocyclic group defined above, in which one of the hydrogen atoms of the carbocyclic group is replaced by a single bond.

[0279] A "linker" is a chemical part containing a covalent bond or chain of atoms that covalently links the antibody to the drug moiety. In various embodiments, linkers include divalent groups such as alkyldiyl, aryldiyl, heteroaryldiyl, and others such as -(CR2). n O(CR2) n - Parts, repeating units of alkoxy groups (e.g., polyethylidene, PEG, polymethylidene) and repeating units of alkylamino groups (e.g., polyethylidene, Jeffamine) TM ); and esters and amides, including succinates, succinamides, diglycolates, malonates, and hexamethylenetetramine. In various embodiments, the linker may contain one or more amino acid residues, such as valine, phenylalanine, lysine, and homolysine.

[0280] The term "chirality" refers to a molecule that has a non-superimposable property of having a mirror-couple, while the term "chirality" refers to a molecule that can be superimposed on its mirror-couple.

[0281] The term "stereoisomer" refers to compounds that have the same chemical composition but whose atoms or groups are arranged differently in space.

[0282] A diastereomer is a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated using high-resolution analytical procedures such as electrophoresis and chromatography.

[0283] "Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0284] The stereochemical definitions and conventions used in this article generally follow those of SP Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds (1994), John Wiley & Sons, Inc., New York. Many organic compounds exist in an optically active form, meaning they have the ability to rotate the plane of polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule relative to its chiral center. The prefixes d and l or (+) and (-) are used to indicate that the plane-polarized light is rotated by the compound, where (-) or l indicates that the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer can also be called an enantiomer, and mixtures of such isomers are often called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can be produced when there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity.

[0285] "Leaving group" refers to a functional group that can be replaced by another functional group. Certain leaving groups are well known in the art, and examples include, but are not limited to: halides (e.g., chlorides, bromides, iodides), methanesulfonyl (methanesulfonyl), p-toluenesulfonyl (toluenesulfonyl), trifluoromethanesulfonyl (trifluoromethanesulfonate), and trifluoromethanesulfonate.

[0286] The term "protecting group" refers to a substituent commonly used to block or protect a specific function by allowing other functional groups on a compound to react. For example, an "amino protecting group" is a substituent attached to an amino group to block or protect the amino function in a compound. Suitable amino protecting groups include, but are not limited to, acetyl, trifluoroacetyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethoxycarbonyl (Fmoc). For a general description of protecting groups and their use, see T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991, or the latest version.

[0287] Those skilled in the art will understand that references to "about" a value or parameter herein include (and describe) embodiments relating to said value or parameter itself. For example, a description relating to "about X" includes a description of "X".

[0288] It should be understood that the aspects and implementations described herein include "consisting of multiple aspects and implementations" and / or "substantially consisting of multiple aspects and implementations". As used herein, the singular forms "a (a / an)" and "the" include plural indications unless otherwise stated.

[0289] II. Compositions and Methods

[0290] This article provides antibodies (including bispecific antibodies) that bind to FcRH5 and immunoconjugates containing such antibodies. The antibodies and immunoconjugates can be used, for example, for the diagnosis or treatment of FcRH5-positive cancers. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0291] Unbound by theory, the selection of the precise antigen for the antibodies of this invention is influenced by at least three important considerations. First, it is necessary to have little or no cross-reactivity with FcRH5 isotypes other than FcRH5c (e.g., isotypes a and b) to avoid the resulting therapeutic agent binding to non-target molecules and thereby reducing its potency. As shown in Figure 1, domain 9 of FcRH5 is an example of a unique sequence among the three isotypes. Second, it is necessary to have little or no cross-reactivity with FcRH family members other than FcRH5, such as FcRH1, FcRH2, FcRH3, and FcRH4. This is challenging because the final Ig-like domain is generally highly conserved across many FcRH family members. However, due to the similar requirement for FcRH5 isotype c specificity, antibodies that bind to the final Ig-like domain are sought after. Finally, for antibodies intended for use in therapeutic molecules that utilize a bispecific antibody format to bring large structures (e.g., T cells and tumor cells) close together, tumor epitopes closer to the cell membrane are known to be more effective (see, for example, Bluemel et al., Cancer Immunol Immunother. (2010) 59:1197–1209). Sometimes described as a dynamical isolation theory, the proximal cell membrane location of domain 9 of FcRH5 is a desired antigen target. To satisfy these considerations and as described in detail below, certain embodiments of the antibodies of the present invention have been developed.

[0292] This document provides an isolated anti-FcRH5 antibody that binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the isotype c-specific region comprises an Ig-like domain 9. In some embodiments, the Ig-like domain 9 is also referred to as Ig-like C2 type 8. In some embodiments, the isotype c-specific region comprises amino acids 754-835 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises amino acids 752-834 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises amino acids 743-850 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises amino acids 745-851 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids beginning at the N-terminal and / or C-terminal boundary. In some embodiments, the isotype c-specific region comprises amino acids from any one of about 750, 751, 752, 753, or 754 of SEQ ID NO:1 to about 830, 831, 832, 833, 834, 835, or 836. In some embodiments, the antibody binds to FcRH5c and / or the isotype c-specific region with an affinity of ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM, and optionally ≥0.0001 nM, ≥0.001 nM, or ≥0.01 nM.

[0293] In some embodiments of any antibody, the antibody has one or more of the following properties: a) cross-reactivity with full-length human and cynomolgus monkey FcRH5 (i.e., binding to full-length human FcRH5 and binding to full-length cynomolgus monkey FcRH5); b) non-cross-reactivity with FcRH1, FcRH2, FcRH3 and / or FcRH4 (i.e., not significantly binding to FcRH1, FcRH2, FcRH3 and / or FcRH4); c) binding to endogenous FcRH5; d) non-cross-reactivity with FcRH5a (i.e., not significantly binding to FcRH5a); and e) non-cross-reactivity with another Ig-like domain of FcRH5 (i.e., not significantly binding to another Ig-like domain of FcRH5). Methods for determining binding ability are known in the art and described below.

[0294] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:38, HVR-H2 containing the amino acid sequence of SEQ ID NO:62, and HVR-H3 containing the amino acid sequence of SEQ ID NO:86; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:2, HVR-L2 containing the amino acid sequence of SEQ ID NO:14, and HVR-L3 containing the amino acid sequence of SEQ ID NO:26. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:50, HVR-H2 containing the amino acid sequence of SEQ ID NO:74, and HVR-H3 containing the amino acid sequence of SEQ ID NO:98. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:111; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:110. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:111 and / or the VL sequence of SEQ ID NO:110. In some embodiments of any antibody, the antibody comprises six HVRs of 1C8.1. In some embodiments, the antibody comprises a VH domain and a VL domain of 1C8.1. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0295] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:39, HVR-H2 containing the amino acid sequence of SEQ ID NO:63, and HVR-H3 containing the amino acid sequence of SEQ ID NO:87; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:3, HVR-L2 containing the amino acid sequence of SEQ ID NO:15, and HVR-L3 containing the amino acid sequence of SEQ ID NO:27. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:51, HVR-H2 containing the amino acid sequence of SEQ ID NO:75, and HVR-H3 containing the amino acid sequence of SEQ ID NO:99. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:113; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:112. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:135; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:134. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:113 and / or the VL sequence of SEQ ID NO:112. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:135 and / or the VL sequence of SEQ ID NO:134. In some embodiments of any antibody, the antibody comprises six HVRs of 1G7.2. In some embodiments, the antibody comprises a VH domain and a VL domain of 1G7.2. In some embodiments of any antibody, the antibody comprises six HVRs of 1G7.2'. In some embodiments, the antibody comprises a VH domain and a VL domain of 1G7.2'. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c.In some embodiments, the antibody cross-reacts with FcRH5 in full-length humans and cynomolgus monkeys. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes. In some embodiments, the antibody does not significantly cross-react with FcRH5a.

[0296] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:40, HVR-H2 containing the amino acid sequence of SEQ ID NO:64, and HVR-H3 containing the amino acid sequence of SEQ ID NO:88; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:4, HVR-L2 containing the amino acid sequence of SEQ ID NO:16, and HVR-L3 containing the amino acid sequence of SEQ ID NO:28. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:52, HVR-H2 containing the amino acid sequence of SEQ ID NO:76, and HVR-H3 containing the amino acid sequence of SEQ ID NO:100. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:115; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:114. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:115 and / or the VL sequence of SEQ ID NO:114. In some embodiments of any antibody, the antibody comprises six HVRs of 2H7.3. In some embodiments, the antibody comprises a VH domain and a VL domain of 2H7.3. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0297] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:41, HVR-H2 containing the amino acid sequence of SEQ ID NO:65, and HVR-H3 containing the amino acid sequence of SEQ ID NO:89; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:5, HVR-L2 containing the amino acid sequence of SEQ ID NO:17, and HVR-L3 containing the amino acid sequence of SEQ ID NO:29. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:53, HVR-H2 containing the amino acid sequence of SEQ ID NO:77, and HVR-H3 containing the amino acid sequence of SEQ ID NO:101. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:117; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:116. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:117 and / or the VL sequence of SEQ ID NO:116. In some embodiments of any antibody, the antibody comprises six HVRs of 3A4.2. In some embodiments, the antibody comprises a VH domain and a VL domain of 3A4.2. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0298] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:42, HVR-H2 containing the amino acid sequence of SEQ ID NO:66, and HVR-H3 containing the amino acid sequence of SEQ ID NO:90; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:6, HVR-L2 containing the amino acid sequence of SEQ ID NO:18, and HVR-L3 containing the amino acid sequence of SEQ ID NO:30. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:54, HVR-H2 containing the amino acid sequence of SEQ ID NO:78, and HVR-H3 containing the amino acid sequence of SEQ ID NO:102. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:119; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:118. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:119 and / or the VL sequence of SEQ ID NO:118. In some embodiments of any antibody, the antibody comprises six HVRs of 3B12.1.1. In some embodiments, the antibody comprises a VH domain and a VL domain of 3B12.1.1. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes. In some embodiments, the antibody does not significantly cross-react with FcRH5a.

[0299] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:43, HVR-H2 containing the amino acid sequence of SEQ ID NO:67, and HVR-H3 containing the amino acid sequence of SEQ ID NO:91; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:7, HVR-L2 containing the amino acid sequence of SEQ ID NO:19, and HVR-L3 containing the amino acid sequence of SEQ ID NO:31. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:55, HVR-H2 containing the amino acid sequence of SEQ ID NO:79, and HVR-H3 containing the amino acid sequence of SEQ ID NO:103. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:121; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:120. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:121 and / or the VL sequence of SEQ ID NO:120. In some embodiments of any antibody, the antibody comprises six HVRs of 3C10. In some embodiments, the antibody comprises a VH domain and a VL domain of 3C10. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0300] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:44, HVR-H2 containing the amino acid sequence of SEQ ID NO:68, and HVR-H3 containing the amino acid sequence of SEQ ID NO:92; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:8, HVR-L2 containing the amino acid sequence of SEQ ID NO:20, and HVR-L3 containing the amino acid sequence of SEQ ID NO:32. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:56, HVR-H2 containing the amino acid sequence of SEQ ID NO:80, and HVR-H3 containing the amino acid sequence of SEQ ID NO:104. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:123; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:122. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:123 and / or the VL sequence of SEQ ID NO:122. In some embodiments of any antibody, the antibody comprises six HVRs of 3F10. In some embodiments, the antibody comprises a VH domain and a VL domain of 3F10. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes. In some embodiments, the antibody does not significantly cross-react with FcRH5a.

[0301] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:45, HVR-H2 containing the amino acid sequence of SEQ ID NO:69, and HVR-H3 containing the amino acid sequence of SEQ ID NO:93; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:9, HVR-L2 containing the amino acid sequence of SEQ ID NO:21, and HVR-L3 containing the amino acid sequence of SEQ ID NO:33. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:57, HVR-H2 containing the amino acid sequence of SEQ ID NO:81, and HVR-H3 containing the amino acid sequence of SEQ ID NO:105. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:125; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:124. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:125 and / or the VL sequence of SEQ ID NO:124. In some embodiments of any antibody, the antibody comprises six HVRs of 3G3. In some embodiments, the antibody comprises a VH domain and a VL domain of 3G3. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes. In some embodiments, the antibody does not significantly cross-react with FcRH5a.

[0302] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:46, HVR-H2 containing the amino acid sequence of SEQ ID NO:70, and HVR-H3 containing the amino acid sequence of SEQ ID NO:94; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:10, HVR-L2 containing the amino acid sequence of SEQ ID NO:22, and HVR-L3 containing the amino acid sequence of SEQ ID NO:34. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:58, HVR-H2 containing the amino acid sequence of SEQ ID NO:82, and HVR-H3 containing the amino acid sequence of SEQ ID NO:106. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:127; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:126. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:127 and / or the VL sequence of SEQ ID NO:126. In some embodiments of any antibody, the antibody comprises six HVRs of 3G7.1.5. In some embodiments, the antibody comprises the VH domain and VL domain of 3G7.1.5. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0303] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:47, HVR-H2 containing the amino acid sequence of SEQ ID NO:71, and HVR-H3 containing the amino acid sequence of SEQ ID NO:95; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:11, HVR-L2 containing the amino acid sequence of SEQ ID NO:23, and HVR-L3 containing the amino acid sequence of SEQ ID NO:35. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:59, HVR-H2 containing the amino acid sequence of SEQ ID NO:83, and HVR-H3 containing the amino acid sequence of SEQ ID NO:107. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:129; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:128. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:129 and / or the VL sequence of SEQ ID NO:128. In some embodiments of any antibody, the antibody comprises six HVRs of 5A10.1.3. In some embodiments, the antibody comprises a VH domain and a VL domain of 5A10.1.3. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0304] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:48, HVR-H2 containing the amino acid sequence of SEQ ID NO:72, and HVR-H3 containing the amino acid sequence of SEQ ID NO:96; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:12, HVR-L2 containing the amino acid sequence of SEQ ID NO:24, and HVR-L3 containing the amino acid sequence of SEQ ID NO:36. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:60, HVR-H2 containing the amino acid sequence of SEQ ID NO:84, and HVR-H3 containing the amino acid sequence of SEQ ID NO:108. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:131; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:130. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:131 and / or the VL sequence of SEQ ID NO:130. In some embodiments of any antibody, the antibody comprises six HVRs of 5F1.1.5. In some embodiments, the antibody comprises a VH domain and a VL domain of 5F1.1.5. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes. In some embodiments, the antibody does not significantly cross-react with FcRH5a.

[0305] In some embodiments, this document provides an antibody comprising: a) a heavy chain comprising HVR-H1 containing the amino acid sequence of SEQ ID NO:49, HVR-H2 containing the amino acid sequence of SEQ ID NO:73, and HVR-H3 containing the amino acid sequence of SEQ ID NO:97; and / or b) a light chain comprising HVR-L1 containing the amino acid sequence of SEQ ID NO:13, HVR-L2 containing the amino acid sequence of SEQ ID NO:25, and HVR-L3 containing the amino acid sequence of SEQ ID NO:37. In some embodiments, the heavy chain comprises HVR-H1 containing the amino acid sequence of SEQ ID NO:61, HVR-H2 containing the amino acid sequence of SEQ ID NO:85, and HVR-H3 containing the amino acid sequence of SEQ ID NO:109. In some embodiments, the antibody comprises a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:133; and / or a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:132. In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:133 and / or the VL sequence of SEQ ID NO:132. In some embodiments of any antibody, the antibody comprises six HVRs of 6D2. In some embodiments, the antibody comprises a VH domain and a VL domain of 6D2. In some embodiments, the antibody binds to the isotype c-specific region (e.g., Ig-like domain 9) of the extracellular domain of FcRH5c. In some embodiments, the antibody cross-reacts with full-length human and cynomolgus monkey FcRH5. In some embodiments, the antibody does not significantly cross-react with FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the antibody binds to endogenous FcRH5. In some embodiments, the antibody binds to B cells. In some embodiments, the antibody does not significantly bind to NK cells and / or monocytes.

[0306] In another aspect provided herein, the anti-FcRH5 antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one embodiment, the anti-FcRH5 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab')2 fragment. In another embodiment, the antibody is a substantially full-length antibody, such as an IgG1 antibody or other antibody class or isotype as defined herein.

[0307] In another aspect, the present invention provides antibodies that bind to the same epitopes as the anti-FcRH5 antibodies provided herein. In some embodiments, antibodies are provided that bind to the isotype c-specific region of the extracellular domain of FcRH5c, said isotype c-specific region being derived from, or overlapping with, amino acids 754-835 of SEQ ID NO:1.

[0308] In some embodiments of any anti-FcRH5 antibody, the FcRH5 antibody, especially an FcRH5 bispecific antibody (e.g., anti-CD3 / anti-FcRH5 bispecific antibody), may have single or combined features based on HEK cell line assays (HEK cells recombined with the signal transduction components required for TCR triggering, as described in James and Valle, Nature 487:64-69 (2012), which is incorporated herein by reference in its entirety). In some embodiments, single or combined features may include: tumor cell interphase / immune synapse; Lck-mediated TCR phosphorylation; ZAP70 activity, including phosphorylation status and localization; CD58 activity, including localization and binding; β2Ar activity, including localization and binding; CAAX activity, including localization and binding; CD45 activity, including localization; pMHC activity, including localization; and / or TCR activity and triggering features.

[0309] On the other hand, the anti-FcRH5 antibody according to any of the above embodiments may incorporate one or a combination of any of the features described in (a)-(e) and / or parts 1-7 below.

[0310] (a) The isotype c-specific region of the extracellular domain of FcRH5c binding

[0311] Methods for determining whether an anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c are known in the art. In some embodiments, the binding of an anti-FcRH5 antibody to the isotype c-specific region of the extracellular domain of FcRH5c can be determined by expressing an FcRH5 peptide with N-terminal and C-terminal deletions in 293 cells and / or SVT2 cells and testing the binding of the antibody to the truncated peptide by FACS as described in the examples. In some embodiments, a significant reduction (≥70% reduction) or elimination of the binding of the antibody to the truncated peptide relative to the binding to the full-length FcRH5 expressed in 293 cells indicates that the antibody does not bind to the truncated peptide.

[0312] In some embodiments, the isotype c-specific region comprises an Ig-like domain 9. In some embodiments, the Ig-like domain 9 is also referred to as Ig-like C2 type 8. In some embodiments, the isotype c-specific region comprises amino acids 754-835 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises amino acids 752-834 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises amino acids 743-850 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises amino acids 745-851 of SEQ ID NO:1. In some embodiments, the isotype c-specific region comprises any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids originating from the N-terminal and / or C-terminal boundary. In some embodiments, the isotype c-specific region comprises an amino acid from any of about 750, 751, 752, 753, or 754 of SEQ ID NO:1 to any of about 830, 831, 832, 833, 834, 835, or 836. In some embodiments, FcRH5 is human FcRH5. In some embodiments, FcRH5 is human FcRH5 or cynomolgus monkey FcRH5.

[0313] (b) Cross-reacting (binding) with human and cynomolgus monkey FcRH5 with an affinity of ≤5 nM or ≤4 nM or ≤3 nM or ≤2 nM or ≤1 nM and optionally ≥0.0001 nM or ≥0.001 nM or ≥0.01 nM.

[0314] Methods for determining binding affinity are known in the art. In some embodiments, binding affinity can be determined based on... The assay can be performed using methods such as ELISA, Facs, and IHC, for example, as described in the examples.

[0315] In some embodiments, the anti-FcRH5 antibody binds to human and / or cynomolgus monkey FcRH5 with an affinity of about ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, or ≤1 nM. In some embodiments, the anti-FcRH5 antibody binds to human and / or cynomolgus monkey FcRH5 with an affinity of about ≤5. In some embodiments, the anti-FcRH5 antibody binds to human and / or cynomolgus monkey FcRH5 with an affinity of about ≤4 nM. In some embodiments, the anti-FcRH5 antibody binds to human and / or cynomolgus monkey FcRH5 with an affinity of about ≤3 nM. In some embodiments, the anti-FcRH5 antibody binds to human and / or cynomolgus monkey FcRH5 with an affinity of about ≤2 nM. In some embodiments, FcRH5 is human FcRH5. In some embodiments, FcRH5 is cynomolgus monkey FcRH5.

[0316] (c) Does not cross-react (bind) with FcRH1, FcRH2, FcRH3 and / or FcRH4.

[0317] Methods for determining binding affinity are known in the art. In some embodiments, binding affinity can be determined based on... The assay can be performed using methods such as Facs, ELISA, and IHC, for example, as described in the examples.

[0318] In some embodiments, the binding affinity of the anti-FcRH5 antibody to FcRH5 is approximately 2, 5, 10, 20, 50, 100, 500, or 1000 times higher than that of FcRH1, FcRH2, FcRH3, and / or FcRH4. In some embodiments, the FcRH is human FcRH.

[0319] (d) Does not cross-react (bind) with FcRH5a.

[0320] Methods for determining binding affinity are known in the art. In some embodiments, binding affinity can be determined based on... The assay can be performed using methods such as Facs, ELISA, and IHC, for example, as described in the examples.

[0321] In some embodiments, the binding affinity of the anti-FcRH5 antibody to FcRH5c is approximately 2, 5, 10, 20, 50, 100, 500, or 1000 times higher than that to FcRH5a. In some embodiments, the FcRH is human FcRH.

[0322] (e) It does not cross-react (bind) with another Ig-like domain of FcRH5.

[0323] Methods for determining binding affinity are known in the art. In some embodiments, binding affinity can be determined based on... The assay can be performed using methods such as Facs, ELISA, and IHC, for example, as described in the examples.

[0324] In some embodiments, the binding affinity of the anti-FcRH5 antibody to the Ig-like domain 9 of FcRH5 is approximately 2, 5, 10, 20, 50, 100, 500, or 1000 times higher than that of the Ig-like domains 1, 2, 3, 4, 5, 6, 7, and / or 8 of FcRH5. In some embodiments, the FcRH is human FcRH. In some implementations, the Ig-like structural domains are Ig-like structural domain 1 (aa 23-100 of SEQ ID NO: 1), Ig-like structural domain 2 (aa 105-185 of SEQ ID NO: 1), Ig-like structural domain 3 (aa 188-271 of SEQ ID NO: 1), Ig-like structural domain 4 (287-373 of SEQ ID NO: 1), Ig-like structural domain 5 (aa 380-466 of SEQ ID NO: 1), Ig-like structural domain 6 (aa 490-555 of SEQ ID NO: 1), Ig-like structural domain 7 (aa 568-652 of SEQ ID NO: 1), and Ig-like structural domain 8 (aa 658-731 of SEQ ID NO: 1).

[0325] Combined measurement and other measurements

[0326] In one aspect, the antigen-binding activity of the anti-FcRH5 antibody is tested. For example, in some embodiments, the ability of the anti-FcRH5 antibody to bind to FcRH5 expressed on the cell surface is tested. This type of test can be performed using a FACS assay.

[0327] In an exemplary competitive assay, immobilized FcRH5 is incubated in a solution containing a first labeled antibody that binds to FcRH5 and a second unlabeled antibody to be tested for its ability to competitively bind FcRH5 against the first antibody. The second antibody may be present in the hybridoma supernatant. As a control, immobilized FcRH5 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to FcRH5, excess unbound antibody is removed, and the amount of label associated with the immobilized FcRH5 is measured. If the amount of label associated with the immobilized FcRH5 is substantially reduced in the test sample relative to that in the control sample, this indicates that the second antibody is competitively binding FcRH5 against the first antibody. In some embodiments, the immobilized FcRH5 is present on the surface of cells or in a membrane formulation obtained from cells expressing FcRH5 on their surface.

[0328] In one aspect, the purified anti-FcRH5 antibody can be further characterized by a series of assays, including but not limited to N-terminal sequencing, amino acid analysis, non-denaturing size-exclusion high-performance liquid chromatography (HPLC), mass spectrometry, ion-exchange chromatography, and papain digestion. In one embodiment, an antibody with some, but not all, effector functions is anticipated, making it a desirable candidate for many applications where in vivo half-life is important, and certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In some embodiments, the Fc activity of the antibody can be measured to ensure that only the desired properties are maintained. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / attenuation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The blast cells used to mediate ADCC, NK cells, express only Fc(RIII), while monocytes express Fc(RI), Fc(RII), and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). Examples of in vitro assays for assessing the ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells used in such assays include peripheral blood monocytes (PBMCs) and natural killer (NK) cells. Or alternatively... In addition, ADCC activity of the target molecule can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art.

[0329] 1. Antibody affinity

[0330] In some embodiments, the antibodies provided herein have a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM, and optionally ≥10 nM. -13 M (e.g., 10) -8 M or less, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13The dissociation constant (Kd) of M).

[0331] In some implementations, Kd can be measured by a radiolabeled antigen binding assay (RIA), which is performed using the Fab type of the target antibody and its antigen as described in the following assay. This can be done by using the minimum concentration in the presence of a titration series of unlabeled antigen. 125 I) Label the antigen to balance Fab, then use an anti-Fab antibody-coated plate to capture the bound antigen to measure the solution-binding affinity of Fab to the antigen (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, use a 5 μg / ml capture plate coated with anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6). Multiwell plates (Thermo Scientific) were incubated overnight, followed by blocking with 2% (w / v) fetal bovine serum in PBS for 2–5 hours at room temperature (approximately 23°C). In non-absorbent plates (Nunc#269620), 100 pM or 26 pM […]. 125 I] The antigen is mixed with a serially diluted solution of the target Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, incubation can be prolonged (e.g., about 65 hours) to ensure equilibration. Afterward, the mixture can be transferred to a trap plate for incubation at room temperature (e.g., for 1 hour). The solution can then be removed and treated with 0.1% polysorbate 20% in PBS. Rinse the plate 8 times. Once the plate is dry, add 150 μL / well of scintillation agent (MICROSCINT-20). TM Packard), and can be found in TOPCOUNT TM The gamma counter (Packard) counts the plates for 10 minutes. A concentration of less than or equal to 20% of the maximum binding for each Fab can be selected for competitive binding assays.

[0332] According to another embodiment, an immobilized antigen CM5 chip is used, with ~10 reaction units (RU), at 25°C. or (BLAcore, Inc., Piscataway, NJ) Kd is measured using surface plasmon resonance assays. In short, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) can be activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen can be diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to obtain approximately 10 reaction units (RU) of conjugate protein. After antigen injection, 1M ethanolamine can be injected to block unreacted groups. For kinetic measurements, a two-fold serial dilution of Fab (0.78 nM to 500 nM) can be injected at approximately 25 μL / min into a solution containing 0.05% polysorbate 20 Surfactant (PBST) in PBS. A simple one-to-one Langmuir binding model can be used. Evaluation Software version 3.2 calculates the binding rate (k) by simultaneously fitting binding and dissociation sensor maps. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) can be calculated as the ratio k. off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the bonding rate measured by the above surface plasmon resonance determination exceeds 10... 6 M -1 s -1 Therefore, fluorescence quenching techniques can be used to determine the binding rate, such as in a spectrophotometer equipped with a flow interruption device (Aviv Instruments) or the 8000 series. The fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 was measured at 25 °C in the presence of gradually increasing concentrations of antigen using a stirred cuvette in a spectrophotometer (ThermoSpectronic).

[0333] 2. Antibody fragments

[0334] In some embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as 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, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0335] Biantibodies are antibody fragments with two antigen-binding sites and can be bivalent or bispecific. See, for example, 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). Triantibodies and tetraantibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0336] A single-domain antibody is an antibody fragment containing the entire or a portion of the heavy chain variable domain or the entire or a portion of the light chain variable domain. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516B1).

[0337] Antibody fragments can be generated using various techniques, including but not limited to proteolytic digestion of intact antibodies as described herein and generation via recombinant host cells (e.g., E. coli or bacteriophages).

[0338] 3. Chimeric and humanized antibodies

[0339] In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0340] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent nonhuman antibody. Typically, a humanized antibody contains one or more variable domains, wherein the HVR, such as the CDR (or a portion thereof), is derived from the nonhuman antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may optionally also contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from the nonhuman antibody (e.g., an antibody that derives HVR residues) for example, to restore or enhance antibody specificity or affinity.

[0341] Humanized antibodies and their formation methods are reviewed in, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface remodeling”); Dall'Acqua et al., Methods 36:43-60 (2005) (describes “FR rearrangement”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer, 83:252-260 (2000) (describes “guided selection” method for FR rearrangement).

[0342] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best-fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequence of the light or heavy chain variable regions of specific subgroups of human antibodies (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol. 151:2296 (1993)); and Presta et al., J. Immunol. 151:2296 (1993)). 623 (1993)); human mature (somatic mutant) framework region or human germline framework region (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, for example, Baca et al., J. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0343] 4. Human antibodies

[0344] In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using various techniques known in the art. 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).

[0345] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, endogenous immunoglobulin loci are 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, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE. TM Technology; U.S. Patent No. 5,770,429, its description Technology; US Patent No. 7,041,870, which describes KM Technology; and U.S. Patent Application Publication No. US 2007 / 0061900, which describes (Technology). The human variable region of a complete antibody derived from such animals can be further modified, for example, by combining it with different human constant regions.

[0346] Human antibodies can also be generated using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines used to generate human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the generation of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0347] Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. The technique for selecting human antibodies from antibody libraries is described below.

[0348] 5. Library-derived antibodies

[0349] The antibodies presented herein can be isolated by screening combinatorial libraries for antibodies possessing one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., editors, Human Press, Totowa, NJ, 2001) and further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, editor, Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0350] In some phage display methods, complete sets of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library. The library can then be screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-stranded Fv (scFv) fragments or as Fab fragments. Libraries derived from immune sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, natural complete sets can be cloned (e.g., from humans) to provide single-source antibodies against a range of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be synthesized by cloning the unrearranged V gene segment from stem cells and encoding the highly variable CDR3 region using PCR primers containing random sequences, followed by in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0351] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments in this paper.

[0352] 6. Multispecific antibodies

[0353] In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity to at least two different sites. In some embodiments, one binding specificity is against FcRH5, and the other is against any other antigen. In some embodiments, one binding specificity is against FcRH5, and the other is against CD3. See, for example, U.S. Patent No. 5,821,337. In some embodiments, a bispecific antibody can bind to two different epitopes of FcRH5. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing FcRH5. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0354] In some embodiments, the FcRH5 antibody is an FcRH5 bispecific antibody. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the FcRH5 protein described herein. Other such antibodies may combine the FcRH5 binding site with a binding site targeting another protein. Alternatively, the anti-FcRH5 arm may be combined with an arm that binds to triggering molecules on leukocytes, such as T cell receptor molecules (e.g., CD3) or Fc receptors (FcγR) of IgG (e.g., FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)), in order to concentrate and localize cellular defense mechanisms to FcRH5-expressing cells. Bispecific antibodies can also be used to localize cytotoxic agents to FcRH5-expressing cells. These antibodies have an FcRH5 binding arm and an arm that binds to cytotoxic agents (e.g., saponins, interferon-α, vinca alkaloids, ricin A chain, methotrexate, or radioisotope haptens). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). In some embodiments, anti-FcRH5 antibodies bind to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, anti-FcRH5 antibodies bind to the Ig-like domain 9 of FcRH5c.

[0355] In some embodiments, the FcRH5 bispecific antibody comprises a first arm, wherein the first arm binds FcRH5, and a second arm, wherein the second arm binds Fc. The second arm of the FcRH5 bispecific antibody can be any anti-Fc antibody known in the art. For example, WO96 / 16673 describes a bispecific anti-ErbB2 / anti-FcγRIII antibody, and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-FcγRI antibody. A bispecific anti-ErbB2 / Fcα antibody is shown in WO98 / 02463. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0356] In some embodiments, the FcRH5 bispecific antibody comprises a first arm, wherein the first arm binds FcRH5, and a second arm, wherein the second arm binds CD3. The second arm of the FcRH5 bispecific antibody can be any anti-CD3 antibody known in the art. U.S. Patent Nos. 5,821,337 and 6,407,213 teach bispecific anti-ErbB2 / anti-CD3 antibodies. Other bispecific antibodies that bind to an epitope on the CD3 antigen and another epitope have been described. See, for example, U.S. Patent No. 5,078,998 (anti-CD3 / tumor cell antigen); U.S. Patent No. 5,601,819 (anti-CD3 / IL-2R, anti-CD3 / CD28, anti-CD3 / CD45); U.S. Patent No. 6,129,914 (anti-CD3 / malignant B cell antigen); U.S. Patent No. 7,112,324 (anti-CD3 / CD19); U.S. Patent No. 6,723,538 (anti-CD3 / CCR5); U.S. Patent No. 7,235,641 (anti-CD3 / EpCAM); U.S. Patent No. 7,262,276 (anti-CD3 / ovarian tumor antigen); and U.S. Patent No. 5,731,168 (anti-CD3 / CD4IgG), all of which are incorporated herein by reference in their entirety. In some embodiments, the anti-CD3 antibody of the second arm is an antibody described in any of the following: WO 2005 / 118635, WO2007 / 042261, WO2008 / 119567, US5929212, US6750325, US6491916, US7994289, US7993641, US6706265, US5585097, US5968509, US5932448, US6129914, US7381803, US5834597, and US7862813, which are incorporated herein by reference in their entirety. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0357] Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93 / 08829 and Traunecker et al., EMBO J.10:3655 (1991)), and knot-in-hole engineering (see, for example, US Patent No. 5,731,168). Engineered electrostatic manipulation effects (WO) can also be used to generate antibody Fc-heterodimeric molecules. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); generating bispecific antibodies using leucine zippers (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "dual antibody" technology for generating bispecific antibody fragments ( See, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and the use of single-chain Fv(sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies to generate multispecific antibodies as described, for example, Tutt et al., J. Immunol. 147:60 (1991).

[0358] This article also includes engineered antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[0359] The antibodies or fragments mentioned in this article also include “dual-acting FAbs” or “DAFs”, which contain antigen-binding sites that bind to FcRH5 as well as another different antigen (see, for example, US2008 / 0069820).

[0360] According to a different approach, an antibody variable domain with desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant domain sequence. Preferably, it is fused with a hinge, C... H2 and C H3 At least a portion of the Ig heavy chain constant structural domains in the region are fused. Preferably, at least one fusion contains a first heavy chain constant region (C) containing sites necessary for light chain binding. H1The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain is inserted into different expression vectors and co-transfected into suitable host cells. In embodiments that provide the desired bispecific antibody yield when the proportions of the three polypeptide chains used for construction are unequal, this offers great flexibility in adjusting the relative proportions of the three polypeptide fragments. However, when expressing at least two polypeptide chains in the same proportion results in high yield, or when this proportion has no significant effect on the yield of the desired chain combination, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0361] In some embodiments, the bispecific antibody consists of a hybrid immunoglobulin heavy chain on one arm with first binding specificity and a hybrid immunoglobulin heavy chain-light chain pair on the other arm (providing second binding specificity). This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from the unwanted immunoglobulin chain combination, as the presence of the immunoglobulin light chain in only half of the bispecific molecule provides a convenient separation pathway. This method is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0362] According to another method described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers recovered from recombinant cell cultures. A preferred interface comprises C... H3 At least a portion of the structural domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a complementary “cavity” of the same or similar size as the large side chain is generated at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other undesired end products such as homodimers. Bispecific antibodies produced according to this method are referred to herein as “cavity-protruding” antibodies.

[0363] Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in a heteroconjugation may be conjugated to avidin, while the other is conjugated to biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (US Patent No. 4,676,980) and for treating HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed, along with many cross-linking techniques, in US Patent No. 4,676,980.

[0364] The literature also describes techniques for generating bispecific antibodies from antibody fragments. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science 229:81 (1985) described a procedure in which intact antibodies are cleaved by proteolysis to produce F(ab')2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize the adjacent dithiol and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reduced back to Fab'-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of other Fab'-TNB derivatives to form a bispecific antibody. The resulting bispecific antibody can be used as a selective immobilization reagent for enzymes.

[0365] The Fab'-SH fragment from *E. coli* can be directly recovered and chemically conjugated to form bispecific antibodies. Shalaby et al., J.Exp.Med.175:217-225 (1992), described the generation of a fully humanized bispecific antibody molecule, F(ab')2. Each Fab' fragment was secreted separately by *E. coli* and directionally chemically conjugated in vitro to form a bispecific antibody. The resulting bispecific antibody was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, and trigger the lysis activity of human cytotoxic lymphocytes against human breast tumor targets.

[0366] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portion of two different antibodies via gene fusion. The antibody homodimer is reduced to a monomer in the hinge region and then oxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The "biantibody" technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. The fragments contain VH and VL linked by a linker that is too short for the two domains on the same chain to pair. Therefore, the VH and VL domains of one fragment are forced to pair with the complementary VL and V domains of another fragment, thereby forming two antigen-binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv(sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0367] Antibodies with more than two valences are expected. For example, trispecific antibodies can be prepared. (Tutt et al., J. Immunol. 147:60 (1991)).

[0368] 8. Antibody variants

[0369] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, improved binding affinity and / or other biological properties of the antibody may be desired. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0370] a) Substitution, insertion, and deletion variants

[0371] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in Table 1. More substantial variations are provided under the heading “Exemplary Substitutions” in Table 1 and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody and screen the product for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0372] Table 1

[0373]

[0374]

[0375] Based on their common side-chain characteristics, amino acids can be grouped as follows:

[0376] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0377] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0378] (3) Acidity: Asp, Glu;

[0379] (4) Alkaline: His, Lys, Arg;

[0380] (5) Residues that affect chain orientation: Gly, Pro;

[0381] (6) Aromatics: Trp, Tyr, Phe.

[0382] Non-conservative substitution would require replacing one of these categories with a member of another.

[0383] One type of alternative variant involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further research will have altered (e.g., improved) certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will retain certain biological properties substantially preserved by the parent antibody. An exemplary alternative variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques (e.g., those described herein). In short, one or more HVR residues are mutated and a variant antibody is displayed on a phage, and then screened for specific biological activities (e.g., binding affinity).

[0384] Modifications (e.g., substitutions) can be made in the HVR to, for example, improve antibody affinity. Such modifications can be made in HVR “hotspots” (i.e., residues encoded by codons that undergo frequent mutations during somatic maturation) (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or SDR (a-CDR), and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation achieved through construction and reselection from a secondary library has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for mutation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variant with the desired affinity. Another approach to introducing diversity involves HVR-guided methods, in which several HVR residues are randomized (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be explicitly identified, for example, using alanine scan mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are frequently targeted.

[0385] In some implementations, substitutions, insertions, or deletions can occur in one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes (such as the conserved substitutions described herein) can be made in HVRs that do not substantially reduce binding affinity. Such changes can occur outside of HVR "hotspots" or SDRs. In some implementations of the variants VH and VL provided above, each HVR is unchanged or contains more than one, two, or three amino acid substitutions.

[0386] A useful method for identifying residues or regions in an antibody that can be selected as mutagenic targets is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions where the initial substitution showed functional sensitivity. Alternatively, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they contain the desired properties.

[0387] Amino acid sequence insertions include fusion of amino and / or carboxyl termini, ranging in length from one residue to peptides containing one hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include fusion of the N- or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a peptide that increases the serum half-life of the antibody.

[0388] b) Glycosylation variants

[0389] In some implementations, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or eliminate one or more glycosylation sites.

[0390] In the case of an antibody containing an Fc region, the attached carbohydrate can be modified. Naturally occurring antibodies generated from mammalian cells typically contain branched biantennary oligosaccharides, generally attached to Asn297 of the CH2 domain of the Fc region via an N-linker. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies provided herein can be modified to form antibody variants with certain improved properties.

[0391] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The fucose content is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry (as described, for example, in WO 008 / 077546). Asn297 refers to the asparagine residue at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between positions 294 and 300. Such fucoidylated variants can possess improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications involving antibody variants that are “defucosylated” or “fucosylated” include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J.Mol.Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng.87:614 (2004).Examples of cell lines capable of generating defucosylated antibodies include Lec 13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines such as CHO cells with the α-1,6-fucosylation gene FUT8 knocked out (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y et al. Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0392] Further antibody variants with bifid oligosaccharides are provided, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent Nos. 6,602,684 (Umana et al.) and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0393] c) Fc region variants

[0394] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) with amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0395] In some embodiments, the present invention contemplates antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / attenuation of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primitive NK cells used to mediate ADCC express only Fc(RIII), while monocytes express Fc(RI), Fc(RII), and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, those used in flow cytometry). Non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, CA; and CytoTox) Non-radioactive cytotoxicity assays (Promega, Madison, WI). Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the target molecule can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0396] Antibodies with reduced effector function include those with substitutions for one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant where residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0397] Certain antibody variants that exhibit improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0398] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region.

[0399] In some implementations, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0400] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005 / 0014934A1 (Hinton et al.), the neonatal Fc receptor being responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies contain one or more alternative Fc regions that improve the binding of the Fc region to the FcRn. Such Fc variants include those with substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as those with a substitution at Fc region residue 434 (US Patent No. 7,371,826).

[0401] For other examples of Fc region variants, see Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0402] d) Cysteine-engineered antibody variants

[0403] In some embodiments, it is desirable to form cysteine-engineered antibodies, such as "thioMAb," in which one or more residues of the antibody are replaced by cysteine ​​residues. In a particular embodiment, the substituted residues are located at an accessible site on the antibody. By replacing those residues with cysteine, a reactive thiol group is thereby positioned at the accessible site of the antibody and can be used to conjugate the antibody to other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to create an immunoconjugate as further described herein. In some embodiments, any one or more of the following residues can be replaced with cysteine: V205 of the light chain (Kabat numbering scheme); A118 of the heavy chain (EU numbering scheme); and S400 of the Fc region of the heavy chain (EU numbering scheme). Cysteine-engineered antibodies can be generated as described, for example, U.S. Patent No. 7,521,541.

[0404] e) Antibody derivatives

[0405] In some embodiments, the antibodies provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Suitable moieties 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. Generally, the quantity and / or type of polymer used for derivatization can be determined based on a variety of considerations, including but not limited to the specific properties or functions that the antibody is to improve, and whether the antibody derivative will be used for treatment under specific conditions.

[0406] In another embodiment, an antibody is provided as a conjugate with a non-protein portion that can be selectively heated by exposure to radiation. In one embodiment, the non-protein portion is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can have any wavelength, including but not limited to wavelengths that do not harm normal cells but heat the non-protein portion to a temperature at which cells near the antibody-non-protein portion are killed.

[0407] B. Recombination methods and compositions

[0408] Antibodies can be generated using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the anti-FcRH5 antibody described herein is provided. This nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed therein): (1) a vector containing nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector containing nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector containing nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, for example, Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for preparing an anti-FcRH5 antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0409] For the recombinant generation of anti-FcRH5 antibodies, the nucleic acid encoding the antibody, such as as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. This nucleic acid can be readily isolated and sequenced using standard procedures, such as by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody.

[0410] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*). After expression, the antibody can be isolated from the bacterial cytoplasm in the soluble fraction and can be further purified.

[0411] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," thereby producing antibodies with partial or complete human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0412] Suitable host cells for expressing glycosylated antibodies also originate 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 transfecting fall armyworm (Spodoptera frugiperda) cells.

[0413] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLATNIBODIES for generating antibodies in transgenic plants). TM technology).

[0414] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 cell lines transformed with SV40 (COS-7); human embryonic kidney cell lines (such as 293 or 293 cells described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); suckling hamster kidney cells (BHK); mouse testicular supporting cells (such as TM4 cells described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); and mouse mammary tumors (MMT). 060562); such as TRI cells as described by Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255–268 (2003).

[0415] C. Measurement

[0416] The anti-FcRH5 antibodies presented herein can be identified, screened, or characterized by various assays known in the art for their physical / chemical properties and / or biological activity.

[0417] In one respect, the antigen-binding activity of the antibodies provided in this article can be tested, for example by known methods such as ELISA. FACS or Western blot.

[0418] On the other hand, competitive assays can be used to identify antibodies that compete with any of the antibodies described herein for binding to FcRH5. In some embodiments, the competing antibody binds to the same epitope (e.g., a linear or conformational epitope) that is bound by the antibody described herein. Detailed exemplary methods for mapping epitopes bound to antibodies are provided in Morris (1996), “Epitope Mapping Protocols,” Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0419] In an exemplary competitive assay, immobilized FcRH5 is incubated in a solution containing a first labeled antibody (e.g., any antibody described herein) that binds to FcRH5 and a second unlabeled antibody to be tested for its ability to competitively bind to FcRH5 against the first antibody. The second antibody may be present in the hybridoma supernatant. As a control, immobilized FcRH5 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to FcRH5, excess unbound antibody is removed, and the amount of label associated with the immobilized FcRH5 is determined. If the amount of label associated with the immobilized FcRH5 is substantially reduced in the test sample relative to that in the control sample, this indicates that the second antibody is competitively binding to FcRH5 against the first antibody. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). In some embodiments, FcRH5 is FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0420] D. Immunoconjugates

[0421] This document also provides immunoconjugates comprising the anti-FcRH5 antibody described herein, which are conjugated to one or more cytotoxic agents such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant, or animal-derived enzyme-active toxins or fragments thereof), or radioisotopes (i.e., radioconjugates). In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0422] Immunoconjugates allow for the partial targeted delivery of drugs to tumors and, in some embodiments, intracellular accumulation, where systemic administration of unconjugated drugs can lead to unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0423] Antibody-drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells (Teicher, BA (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, PJ and Senter PD (2008) The Cancer Jour. 14(3):154-169; Chari, RV (2008) Acc. Chem. Res. 41:98-107).

[0424] The ADC compounds described herein include those with anticancer activity. In some embodiments, the ADC compound includes an antibody conjugated (i.e., covalently linked) to a drug moiety. In some embodiments, the antibody is covalently linked to the drug moiety via a linker. The antibody-drug conjugates (ADCs) described herein selectively deliver an effective dose of drug to tumor tissue, thereby achieving higher selectivity (i.e., a lower effective dose) while increasing the therapeutic index (“therapeutic window”).

[0425] The pharmaceutical part (D) of an antibody-drug conjugate (ADC) may include any compound, part, or group having cytotoxic or cell-inhibitory activity. The pharmaceutical part may exert its cytotoxic and cell-inhibitory activity through a variety of mechanisms, including but not limited to: microtubule binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary pharmaceutical parts include, but are not limited to: maytansine, dolastatin, auristatin, carrichomycin, pyrrolobenzodiazepine (PBD), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycin, vinca alkaloids, taxanes, trichothecene, CC1065, camptothecin, elinafide, and their stereoisomers, isosteres, analogs, and derivatives with cytotoxic activity. Non-limiting examples of such immunoconjugates are discussed in further detail below.

[0426] 1. Exemplary antibody-drug conjugates

[0427] An exemplary embodiment of an antibody-drug conjugate (ADC) compound comprises an antibody (Ab) targeting tumor cells, a drug moiety (D), and a linker moiety (L) linking the Ab to the D. In some embodiments, the antibody is linked to the linker moiety (L) via one or more amino acid residues, such as lysine and / or cysteine.

[0428] An exemplary ADC has Equation I:

[0429] Ab-(LD) p I

[0430] Where p is from 1 to approximately 20. In some embodiments, the number of drug portions that can be conjugated to the antibody is limited by the number of free cysteine ​​residues. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADCs of Formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 engineered cysteine ​​amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine ​​residues are already present in the antibody without the use of engineering, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to conjugation to generate one or more free cysteine ​​residues. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0431] a) Exemplary connector

[0432] The "linker" (L) is a bifunctional or multifunctional portion that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an antibody-drug conjugate (ADC) of formula I. In some embodiments, the antibody-drug conjugate (ADC) can be prepared using a linker having reactive functional groups for covalently linking to the drug and the antibody. For example, in some embodiments, the cysteine ​​thiol of the antibody (Ab) can form a bond with the reactive functional group of the linker or drug-linker intermediate to form the ADC.

[0433] In one aspect, the linker has a functional group capable of reacting with free cysteine ​​present on the antibody to form a covalent bond. Non-limiting exemplary examples of such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated esters such as succinimide, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acyl chloride, sulfonyl chloride, isocyanate, and isothiocyanate. See, for example, the conjugation method in Klussman et al. (2004), Bioconjugate Chemistry 15(4):765-773, page 766, and the examples herein.

[0434] In some embodiments, the linker has a functional group capable of reacting with an electrophilic group present on the antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, acylhydrazides, oximes, amino groups, hydrazines, thioureas, hydrazide carboxylates, and aromatic hydrazides.

[0435] Linkers may comprise one or more linker components. Exemplary linker components include 6-maleimide hexanoyl (“MC”), maleimide propionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), 4-(2-pyridylthio)valerate N-succinimide ester (“SPP”), and 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid ester (“MCC”). Various linker components are known in the art, some of which are described below.

[0436] Linkers can be “cleavable linkers” that facilitate drug release. Non-limiting exemplary cleavable linkers include acid-instable linkers (e.g., containing hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), light-instable linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US5208020).

[0437] In some implementations, the connector has the following formula II:

[0438] -A a -W w -Y y - II

[0439] Where A is the "extension unit", and a is an integer from 0 to 1; W is the "amino acid unit", and w is an integer from 0 to 12; Y is the "spacer unit", and y is 0, 1, or 2. The ADC of the linker containing formula II has formula I(A): Ab-(A a -W w -Y y -D)p, where Ab, D, and p are as defined above for Formula I. Exemplary embodiments of such connectors are described in U.S. Patent No. 7,498,298, which is expressly incorporated herein by reference.

[0440] In some implementations, the linker component includes an "extension unit" (A) that links an antibody to another linker component or drug moiety. A non-limiting exemplary extension unit is shown below (where the wavy line indicates a covalently linked site with an antibody, drug, or other linker component):

[0441]

[0442] In some embodiments, the linker component comprises an “amino acid unit” (W). In some such embodiments, the amino acid unit allows the linker to be cleaved by a protease, thereby promoting the release of the drug from the immunoconjugate upon exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys), phenylalanine-homolysine (phe-homolys), and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-gly (gly-gly-gly). Amino acid units may contain naturally occurring amino acid residues and / or minor amino acids and / or non-naturally occurring amino acid analogs such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasminogen lysin.

[0443] Typically, peptide linkers can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. These peptide bonds can be prepared, for example, according to liquid-phase synthesis methods (e.g., E.). (And K. Lübke (1965), “The Peptides”, Vol. 1, pp. 76-136, Academic Press).

[0444] In some embodiments, the linker component includes a "spacer unit" (Y) that links the antibody to the drug moiety directly or via an extension unit and / or amino acid unit. The spacer unit can be "self-sacrificing" or "non-self-sacrificing." A "non-self-sacrificing" spacer unit is a unit in which part or all of the spacer unit remains bound to the drug moiety after ADC cleavage. Examples of non-self-sacrificing spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. In some embodiments, enzymatic cleavage of the ADC containing the glycine-glycine spacer unit by a tumor cell-associated protease results in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety undergoes a hydrolysis step in tumor cells, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

[0445] The “self-sacrificing” spacer unit allows for the release of a portion of the drug. In some embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is linked to the amino acid unit via an amide bond, and a carbamate and a methyl carbamate or carbonate are formed between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the spacer unit comprises a p-aminobenzyloxycarbonyl (PAB). In some embodiments, the ADC comprising the self-sacrificing linker has the following structure:

[0446]

[0447] Where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer in the range of 0 to 4; X may be one or more additional spacer units or may be absent; and p is in the range of 1 to about 20. In some embodiments, p is in the range of 1 to 10, 1 to 7, 1 to 5, or 1 to 4. A non-limiting exemplary X spacer unit includes:

[0448] R1 and R2 are independently selected from H and C1-C6 alkyl groups. In some embodiments, R1 and R2 are each –CH3.

[0449] Other examples of self-sacrificing spacer groups include, but are not limited to, aromatic compounds that are electronically similar to a PAB group, such as 2-aminoimidazolium-5-methanol derivatives (US Patent No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and o- or p-aminobenzyl acetal. In some embodiments, spacer groups that undergo cyclization after amide bond hydrolysis may be used, such as substituted or unsubstituted 4-aminobutyramide (Rodrigues et al. (1995) Chemistry Biology 2:223), suitably substituted bicyclic [2.2.1] and bicyclic [2.2.2] ring systems (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionamide (Amsberry et al. (1990) J. Org. Chem. 55:5867). The α-carbon of a drug linked to a glycine residue is another example of a self-sacrificing spacer that can be used in ADCs (Kingsbury et al. (1984) J. Med. Chem. 27: 1447).

[0450] In some implementations, the linker L can be a dendritic linker, used to covalently link more than one drug moiety to the antibody via branched, multifunctional linker portions (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody (i.e., loading), which is related to the potency of the ADC. Therefore, when the antibody contains only one reactive cysteine ​​thiol group, multiple drug moieties can be linked via dendritic linkers.

[0451] A non-limiting exemplary connector in the case of the ADC of Equation I is as follows:

[0452]

[0453]

[0454] R1 and R2 are independently selected from H and C1-C6 alkyl groups. In some embodiments, R1 and R2 are each –CH3.

[0455]

[0456] Where n is 0 to 12. In some implementations, n is 2 to 10. In some implementations, n is 4 to 8.

[0457] Other non-limiting exemplary ADCs include the following structures:

[0458]

[0459] Where X is:

[0460] -(CH2) n -、-(CH2CH2O) n -、

[0461]

[0462]

[0463] Y is:

[0464]

[0465] Each R is independently H or C1-C6 alkyl; and n is 1 to 12.

[0466] In some embodiments, the linker is replaced by a group that modifies solubility and / or reactivity. As a non-limiting example, charged substituents include, for example, sulfonate groups (-SO3). - Ammonium or other compounds can increase the water solubility of the linker reagent and promote the coupling reaction between the linker reagent and the antibody and / or drug moiety, or promote the coupling reaction between Ab-L (antibody-linker intermediate) and D or DL ​​(drug-linker intermediate) and Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, a portion of the linker is linked to the antibody and a portion of the linker is linked to the drug, then Ab-(linker moiety) is added. a Connect to drug - (connector sub-section) b ADC of Form I.

[0467] The compounds presented herein are explicitly intended, but not limited to, ADCs prepared using the following linker reagents: bismaleimide-trioxanediol (BMPEO), N-(β-maleimidepropoxy)-N-hydroxysuccinimide (BMPS), N-(ε-maleimidehexanoyloxy)succinimide (EMCS), N-[γ-maleimidebutyryloxy]succinimide (GMBS), 1,6-hexane-bisvinyl sulfone (HBVS), 4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-amidohexanoic acid succinimide) (LC-SMCC), m-maleimide... Iminobenzoyl-N-hydroxysuccinimide (MBS), 4-(4-N-maleiminophenyl)butyrylhydrazide (MPBH), 3-(bromoacetamyl)propionate succinimide (SBAP), iodoacetic acid succinimide (SIA), (4-iodoacetyl)aminobenzoic acid succinimide (SIAB), 3-(2-pyridyldithio)propionate N-succinimide (SPDP), 4-(2-pyridylthio)valerate N-succinimide (SPP), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), 4-(p-maleiminophenyl) Succinimide butyrate (SMPB), 6-[(β-maleiminopropamido)hexanoic acid]succinimide (SMPH), iminothiacyclopentane (IT), sulfonyl-EMCS, sulfonyl-GMBS, sulfonyl-KMUS, sulfonyl-MBS, sulfonyl-SIAB, sulfonyl-SMCC and sulfonyl-SMPB, and (4-vinyl sulfone)benzoic acid succinimide (SVSB), including bismaleimide reagents: dithiobismaleimide ethane (DTME), 1,4-bismaleimide butane (BMB), 1,4-bismaleimide-2,3-dihydroxybutane (BMDB) ), bismaleimide hexane (BMH), bismaleimide ethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below); bifunctional derivatives of imine esters (e.g., dimethyl hexamethyleneimine HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazide compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazo derivatives (e.g., bis(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, the bismaleimide reagent allows the thiol group of cysteine ​​in the antibody to be linked to a thiol-containing pharmaceutical moiety, linker, or linker-pharmaceutical intermediate. Other functional groups that react with thiol groups include, but are not limited to: iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0468]

[0469] Some useful linker reagents are available from various commercial sources, such as Pierce Biotechnology, Inc. (Rockford, IL) and Molecular Biosciences Inc. (Boulder, CO), or synthesized according to procedures described in the art, such as those described in the following literature: Toki et al. (2002) J. Org. Chem. 67:1866-1872; Dubowchik et al. (1997) Tetrahedron Letters, 38:5257-60; Walker, MA (1995) J. Org. Chem. 60:5352-5355; Frisch et al. (1996) Bioconjugate Chem. 7:180-186; US6214345; WO 02 / 088172; US 2003130189; US2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0470] Carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See, for example, WO94 / 11026.

[0471] b) Drug section

[0472] (1) Maytansin and Maytansin-like substances

[0473] In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more maytansine-like molecules. Maytansine-like molecules are derivatives of maytansine and are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub *Maytenus serrata* (US Patent No. 3,896,111). Later, it was discovered that certain microorganisms also produce maytansine-like molecules such as maytanol and C-3 maytanol ester (US Patent No. 4,151,042). Synthetic maytans are disclosed in, for example, U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533.

[0474] Maytansine-like drug fractions are attractive drug fractions in antibody-drug conjugates because they: (i) are relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products; (ii) can be derivatized using functional groups suitable for conjugation to antibodies via non-disulfide linkers; (iii) are stable in plasma; and (iv) are effective against a variety of tumor cell lines.

[0475] Some maytansine-like drugs suitable for use as a component are known in the art and can be isolated from natural sources or generated using genetic engineering techniques according to known methods (see, for example, Yu et al. (2002) PNAS 99:7968-7973). Maytansine-like drugs can also be prepared synthetically according to known methods.

[0476] Maytansine-like drug portions include, but are not limited to, those with modified aromatic rings, such as: C-19-dechlorination (US Patent No. 4,256,746) (e.g., prepared by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechlorination (US Patent Nos. 4,361,650 and 4,307,016) (e.g., prepared by demethylation using Streptomycetes or Actinomycetes or by dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechlorination (US Patent No. 4,294,757) (e.g., prepared by acylation using acyl chlorides), and those with modifications at other positions on the aromatic ring.

[0477] The maytansine-like drug fraction also includes those with modifications, such as: C-9-SH (US Patent No. 4,424,219) (e.g., prepared by reacting maytansine with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (US 4,331,598); C-14-hydroxymethyl or acylmethyl (CH2OH or CH2OAc) (US Patent No. 4,450,254) (e.g., prepared from Nocardia); C-15-hydroxy / acyloxy (US 4,364,866) (e.g., prepared by converting maytansine with Streptomyces); C-15-methoxy (US Patent Nos. 4,313,946 and 4,315,929) (e.g., prepared from Trewia). Nudlflora isolation); C-18-N-demethylation (US Patent Nos. 4,362,663 and 4,322,348) (e.g., prepared by demethylation of maytanol using Streptomyces); and 4,5-deoxygenation (US 4,371,533) (e.g., prepared by reduction of maytanol with titanium trichloride / LAH).

[0478] Many positions on maytansine-like compounds can be used as linking sites. For example, an ester bond can be formed by reacting with a hydroxyl group using conventional coupling techniques. In some embodiments, the reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In some embodiments, the bond is formed at the C-3 position of maytansine or maytansine analogues.

[0479] The drug moiety of maytansine includes those having the following structures:

[0480]

[0481] The wavy line indicates the covalent connection between the sulfur atom of the maytansine-like drug moiety and the linker of the ADC. Each R can be independently H or C1-C6 alkyl. The alkylene chain that connects the amide group to the sulfur atom can be methyl, ethyl, or propyl, i.e., m is 1, 2, or 3 (US 633410; US 5208020; Chari et al. (1992) Cancer Res. 52:127-131; Liu et al. (1996) Proc. Natl. Acad. Sci USA 93:8618-8623).

[0482] All stereoisomers of the maytansine-like drug moiety are anticipated for use in the ADC provided herein, i.e., any combination of the R and S configurations at the chiral carbon (US 7276497; US 6913748; US 6441163; US 633410(RE39151); US5208020; Widdison et al. (2006) J. Med. Chem. 49:4392-4408, which are incorporated herein by reference in their entirety). In some embodiments, the maytansine-like drug moiety has the following stereochemistry:

[0483]

[0484] Exemplary embodiments of the maytansine-like drug portion include, but are not limited to: DM1, DM3, and DM4, which have the following structures:

[0485]

[0486]

[0487] The wavy line indicates the covalent connection between the sulfur atom of the drug and the linker (L) of the antibody-drug conjugate.

[0488] Other exemplary maytansin-like antibody-drug conjugates have the following structures and abbreviations (where Ab is an antibody and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4):

[0489]

[0490] An exemplary antibody-drug conjugate in which DM1 is linked to the thiol group of the antibody via a BMPEO linker has the following structure and abbreviations:

[0491]

[0492] Where Ab is an antibody; n is 0, 1, or 2; and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

[0493] Immunoconjugates containing maytansine, their preparation methods, and their therapeutic uses are described, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064, US 2005 / 0276812 A1, and European Patent EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference. See also Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996); and Chari et al., Cancer Research 52:127-131 (1992).

[0494] In some embodiments, antibody-matansine conjugates can be prepared by chemically linking an antibody to a maytansine molecule without significantly attenuating the biological activity of the antibody or the maytansine molecule. See, for example, U.S. Patent No. 5,208,020 (the disclosure of which is expressly incorporated herein by reference). In some embodiments, ADCs conjugated to an average of 3-4 maytansine molecules per antibody molecule have shown enhanced cytotoxicity to target cells without adversely affecting antibody function or solubility. In some cases, even a single toxin molecule / antibody may be expected to enhance cytotoxicity compared to using a naked antibody.

[0495] Linking groups used to form antibody-maytansin conjugates include, for example, those described herein and those disclosed in the following documents: U.S. Patent No. 5,208,020; EP Patent 0,425,235 B1; Chari et al., Cancer Research 52:127-131 (1992); US 2005 / 0276812A1; and US 2005 / 016993 A1, the disclosures of which are expressly incorporated herein by reference.

[0496] (2) Aurestatin and Dolasstatin

[0497] The pharmaceutical components include dolalastatin, aurestatin, and their analogues and derivatives (US 5635483; US5780588; US 5767237; US 6124431). Aurestatin is a derivative of the marine shellfish compound dolalastatin-10. While not wishing to be bound by any particular theory, dolalastatin and aurestatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and possess anticancer activity (US 5663149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolasstatin / aurestatin drug moiety can be linked to an antibody via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172; Doronina et al. (2003) Nature Biotechnology 21(7):778-784; Francisco et al. (2003) Blood 102(4):1458-1465).

[0498] Exemplary auristatin implementations include the N-terminally linked monomethyl auristatin pharmaceutical portion D disclosed in US 7498298 and US 7659241. E and D F The publicly available content of these documents is explicitly included in its entirety through citation:

[0499]

[0500] Where D E and D F The wavy lines indicate covalently linked sites with the antibody or antibody-linker component, and are independent at each site:

[0501] R 2 Selected from H and C1-C8 alkyl groups;

[0502] R 3 Selected from H, C1-C8 alkyl, C3-C8 carbon ring, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbon ring), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

[0503] R 4 Selected from H, C1-C8 alkyl, C3-C8 carbon ring, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbon ring), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

[0504] R 5 Selected from H and methyl;

[0505] Or R 4 and R 5 They together form a carbon ring and have the formula -(CR) a R b ) n -, where R a and R b Independently selected from H, C1-C8 alkyl and C3-C8 carbon rings, and n is selected from 2, 3, 4, 5 and 6;

[0506] R 6 Selected from H and C1-C8 alkyl groups;

[0507] R 7 Selected from H, C1-C8 alkyl, C3-C8 carbon ring, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbon ring), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

[0508] Each R 8 Independently selected from H, OH, C1-C8 alkyl, C3-C8 carbon ring and O-(C1-C8 alkyl);

[0509] R 9 Selected from H and C1-C8 alkyl groups;

[0510] R 10 Selected from aryl or C3-C8 heterocycles;

[0511] Z is O, S, NH, or NR. 12 , where R 12 It is a C1-C8 alkyl group;

[0512] R 11 Selected from H, C1-C 20 Alkyl, aryl, C3-C8 heterocycles, -(R 13 O) m -R 14 or -(R) 13 O) m -CH(R 15 )2;

[0513] m is an integer in the range of 1 to 1000;

[0514] R 13 It is a C2-C8 alkyl group;

[0515] R 14 It is H or C1-C8 alkyl;

[0516] R15 Each occurrence of this substance is independently H, COOH, or -(CH2). n -N(R 16 2、-(CH2) n -SO3H or -(CH2) n -SO3-C1-C8 alkyl;

[0517] R 16 Each occurrence of this substance is independently of H, C1-C8 alkyl, or -(CH2). n -COOH;

[0518] R 18 Selected from -C(R) 8 )2-C(R 8 )2-Aryl, -C(R 8 )2-C(R 8 )2-(C3-C8 heterocycle) and -C(R 8 )2-C(R 8 )2-(C3-C8 carbon ring); and

[0519] n is an integer in the range of 0 to 6.

[0520] In one implementation, R 3 R 4 and R 7 Independently is isopropyl or sec-butyl, and R 5 It is –H or methyl. In one exemplary embodiment, R 3 and R 4 Each is isopropyl, R 5 It is -H, and R 7 It is sec-butyl.

[0521] In yet another implementation scheme, R 2 and R 6 Each is a methyl group, and R 9 Yes, it's -H.

[0522] In another implementation, R 8 Each occurrence of is -OCH3.

[0523] In some implementation schemes, R 3 and R 4 Each is isopropyl, R 2 and R 6 Each is a methyl group, R 5 -H, R 7 For sec-butyl, R 8 Each occurrence of is -OCH3, and R 9 It is -H.

[0524] In one implementation, Z is -O- or -NH-.

[0525] In one implementation, R 10 It is an aryl group.

[0526] In one exemplary implementation, R 10 It is phenyl.

[0527] In one exemplary implementation, when Z is -O-, R 11 It is –H, methyl, or tert-butyl.

[0528] In one implementation, when Z is -NH, R 11 -CH(R) 15 )2, where R 15 -(CH2) n -N(R 16 )2, and R 16 It is a -C1-C8 alkyl group or -(CH2). n -COOH.

[0529] In another implementation, when Z is -NH, R 11 -CH(R) 15 )2, where R 15 -(CH2) n -SO3H.

[0530] Formula D E An exemplary implementation of auristatin is MMAE, wherein the wavy line indicates a covalent link with the linker (L) of the antibody-drug conjugate:

[0531]

[0532] Formula D F An exemplary implementation of auristatin is MMAF, wherein the wavy line indicates a covalent link to the linker (L) of the antibody-drug conjugate:

[0533]

[0534] Other exemplary embodiments include a monomethylvaline compound having a phenylalanine carboxyl group modified at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and a monomethylvaline compound having a phenylalanine side chain modified at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0535] Non-limiting exemplary embodiments of an ADC of Formula I comprising MMAE or MMAF and different linker components have the following structures and abbreviations (where “Ab” is an antibody; p is 1 to about 8; “Val-Cit” is a valine-citrulline dipeptide; and “S” is a sulfur atom):

[0536]

[0537]

[0538] Non-limiting exemplary embodiments of the Formula I ADC comprising MMAF and different linker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates comprising MMAF linked to an antibody via a linker that is not proteolytically cleavable have been shown to have activity comparable to that of immunoconjugates comprising MMAF linked to an antibody via a linker that is proteolytically cleavable (Doronina et al. (2006) Bioconjugate Chem. 17:114-124). In some of these embodiments, drug release is thought to be achieved via antibody degradation in cells.

[0539] Typically, peptide-based drug moieties can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. These peptide bonds can be prepared, for example, by liquid-phase synthesis (see, for example, E). See K. Lübke, “The Peptides”, Vol. 1, pp. 76-136, 1965, Academic Press. The aurostatin / dolasstatin pharmaceutical fraction can be prepared according to the methods described in the following literature in some embodiments: US 7498298; US 5635483; US5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0540] In some implementation schemes, formula D E (e.g., MMAE) and formula D FThe auristatin / doralastatin drug fraction (e.g., MMAF) and drug-linker intermediates and their derivatives such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF and MC-vc-PAB-MMAE can be prepared using methods described in the following literature: US7498298; Doronina et al. (2006) Bioconjugate Chem. 17:114-124; and Doronina et al. (2003) Nat. Biotech. 21:778-784, and then conjugated to the target antibody.

[0541] (3) Calicutin

[0542] In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more calichimycin molecules. The calichimycin family of antibiotics and their analogues are capable of producing double-strand DNA breaks at subpicomolar concentrations (Hinman et al., (1993) Cancer Research 53:3336-3342; Lode et al., (1998) Cancer Research 58:2925-2928). Calichimycins have intracellular sites of action but, in some cases, do not readily cross the plasma membrane. Therefore, in some embodiments, the cytotoxic effects of these agents can be greatly enhanced by antibody-mediated internalization of cellular uptake. Non-limiting exemplary methods for preparing antibody-drug conjugates having a calichimycin drug moiety are described, for example, in US 5712374, US 5714586, US 5739116, and US 5767285.

[0543] (4) Pyrrolobenzodiazepine

[0544] In some embodiments, the ADC comprises pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. The natural product anthraquinone (a PBD) was first reported in 1965 (Leimgruber, et al., (1965) J. Am. Chem. Soc., 87: 5793-5795; Leimgruber, et al., (1965) J. Am. Chem. Soc., 87: 5791-5793). Since then, numerous PBDs (natural and analogues) have been reported (Thurston et al., (1994) Chem. Rev. 1994, 433-465), including dimers with a tricyclic PBD backbone (US 6884799; US 7049311; US ​​7067511; US ​​7265105; US 7511032; US 7528126; US 7557099). While not wishing to be bound by any particular theory, it is believed that the dimer structure provides a three-dimensional shape with isohelicality to the minor groove of B-type DNA, thereby forming a tight fit at the binding site (Kohn, In Antibiotics). III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-Van Devanter, (1986) Acc. Chem. Res., 19: 230-237. Dimeric PBD compounds containing C2 aryl substituents have been shown to be used as cytotoxic agents (Hartley et al. (2010) Cancer Res. 70 (17): 6849-6858; Antonow (2010) J. Med. Chem. 53 (7): 2927-2941; Howard et al. (2009) Bioorganic and Med. Chem. Letters 19 (22): 6463-6466).

[0545] PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to possess anticancer properties. Non-limiting exemplary linking sites on the PBD dimer include a five-membered pyrrole ring, a chain between PBD units, and an N10-C11 imine group (WO2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US ​​2011 / 0256157; WO2011 / 130598).

[0546] A non-limiting exemplary PBD dimer component of an ADC has formula A:

[0547]

[0548] and its salts and solvates, wherein:

[0549] The wavy line indicates the covalent connection site with the connector;

[0550] The dashed line indicates that a double bond may be present between C1 and C2 or between C2 and C3;

[0551] R 2 Independently selected from H, OH, =O, =CH2, CN, R, OR, =CH-R D =C(R) D 2. O-SO2-R, CO2R, and COR, optionally further selected from halogen or dihalogen groups, wherein R D It is independently selected from R, CO2R, COR, CHO, CO2H and halogen groups;

[0552] R 6 and R 9 Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halogen groups;

[0553] R 7 Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', NO2, Me3Sn and halogen groups;

[0554] Q is independently selected from O, S, and NH;

[0555] R 11 It is H or R, or SO3M when Q is O, where M is a metal cation;

[0556] R and R' are each independently selected from the arbitrarily substituted C. 1-8 Alkyl, C 1-12 Alkyl, C 3-8 Heterocyclic group, C 3-20 Heterocyclic and C 5-20 Aryl, and optionally with respect to the group NRR', R and R' together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocycle;

[0557] R 12 R 16 R 19 and R 17 For example, for R 2 R 6 R 9 and R 7 Defined;

[0558] R″ is C 3-12Alkylene, wherein the chain may be interrupted by an aromatic ring having one or more heteroatoms such as O, S, N(H), NMe and / or optionally substituted, such as benzene or pyridine; and

[0559] X and X' are independently selected from O, S, and N (H).

[0560] In some implementations, R and R' are each independently selected from the optionally substituted C. 1-12 Alkyl, C 3-20 Heterocyclic and C 5-20 The aryl group, and optionally with respect to the group NRR', R and R' together with the nitrogen atom to which they are attached, form optionally substituted 4-, 5-, 6- or 7-membered heterocycles.

[0561] In some implementation schemes, R 9 and R 19 It is H.

[0562] In some implementation schemes, R 6 and R 16 It is H.

[0563] In some implementation schemes, R 7 and R 17 All are OR 7A , where R 7A C is an optional substitute 1-4 Alkyl group. In some embodiments, R 7A It is Me. In some implementations, R 7A It is Ch2Ph, where Ph is phenyl.

[0564] In some implementations, X is O.

[0565] In some implementation schemes, R 11 It is H.

[0566] In some implementations, there is a double bond between C2 and C3 in each monomer unit.

[0567] In some implementation schemes, R 2 and R 12 It is independently selected from H and R. In some implementations, R 2 and R 12 Independently for R. In some implementations, R 2 and R 12 C can be substituted independently. 5-20 Aryl or C 5-7 Aryl or C 8-10 Aryl. In some implementations, R 2 and R 12Independently, it can be optionally substituted with phenyl, thiophene, naphthyl, pyridyl, quinolinyl, or isoquinolinyl. In some embodiments, R 2 and R 12 Independently selected from =O, =CH2, =CH-R D S = C(R) D 2. In some implementation schemes, R 2 and R 12 Each is equal to CH2. In some implementations, R 2 and R 12 Each is H. In some implementations, R 2 and R 12 Each is equal to 0. In some implementations, R 2 and R 12 Each is equal to CF2. In some implementations, R 2 and / or R 12 Independently = C(R) D 2. In some implementation schemes, R 2 and / or R 12 Independently = CH-R D .

[0568] In some implementations, when R 2 and / or R 12 =CH-R D At this time, each group can independently have any of the following configurations:

[0569]

[0570] In some implementations, =CH-R D It is in configuration (I).

[0571] In some implementations, R″ is a C3 alkylene or a C5 alkylene.

[0572] In some implementations, the exemplary PBD dimer component of the ADC has the structure of formula A(I):

[0573]

[0574] Where n is 0 or 1.

[0575] In some implementations, the exemplary PBD dimer component of the ADC has the structure of formula A(II):

[0576]

[0577] Where n is 0 or 1.

[0578] In some implementations, the exemplary PBD dimer component of the ADC has the structure of formula A(III):

[0579]

[0580] Where R E and R E” Each is independently selected from H or R D , where R D It is as defined above; and

[0581] Where n is 0 or 1.

[0582] In some implementations, n is 0. In some implementations, n is 1. In some implementations, R E and / or R E” For H. In some implementations, R E and R E” For H. In some implementations, R E and / or R E” For R D , where R D C is an optional replacement 1-12 Alkyl group. In some embodiments, R E and / or R E” For R D , where R D It is a methyl group.

[0583] In some implementations, the exemplary PBD dimer component of the ADC has the structure of formula A(IV):

[0584]

[0585] Among them, Ar 1 and Ar 2 Each of the Cs can be substituted independently. 5-20 Aryl, of which Ar 1 and Ar 2 They can be the same or different; and

[0586] Where n is 0 or 1.

[0587] In some implementations, the exemplary PBD dimer component of the ADC has the structure of formula A(V):

[0588]

[0589] Among them, Ar 1 and Ar 2 Each of the Cs can be substituted independently. 5-20 Aryl, of which Ar 1 and Ar2 They can be the same or different; and

[0590] Where n is 0 or 1.

[0591] In some implementation schemes, Ar 1 and Ar 2 Each is independently selected from optionally substituted phenyl, furanyl, thiophene, and pyridyl groups. In some embodiments, Ar... 1 and Ar 2 Each is independently an optionally substituted phenyl group. In some embodiments, Ar 1 and Ar 2 Each is independently a optionally substituted thiophene-2-yl or thiophene-3-yl. In some embodiments, Ar 1 and Ar 2 Each group is independently a optionally substituted quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group can be attached to the PBD core via any available ring position. For example, the quinolinyl group can be quinoline-2-yl, quinoline-3-yl, quinoline-4-yl, quinoline-5-yl, quinoline-6-yl, quinoline-7-yl, and quinoline-8-yl. In some embodiments, the quinolinyl group is selected from quinoline-3-yl and quinoline-6-yl. The isoquinolinyl group can be isoquinoline-1-yl, isoquinoline-3-yl, isoquinoline-4-yl, isoquinoline-5-yl, isoquinoline-6-yl, isoquinoline-7-yl, and isoquinoline-8-yl. In some embodiments, the isoquinolinyl group is selected from isoquinoline-3-yl and isoquinoline-6-yl.

[0592] Other non-limiting exemplary PBD dimer components of ADC are of formula B:

[0593]

[0594] and its salts and solvates, wherein:

[0595] The wavy line indicates the covalent connection site with the connector;

[0596] The wavy line connected to OH indicates the S or R configuration;

[0597] R V1 and R V2 Independently selected from H, methyl, ethyl, and phenyl (the phenyl group may optionally be substituted with fluorine, especially at the 4-position) and C. 5-6 Heterocyclic group; wherein R V1 and R V2 They can be the same or different; and

[0598] n is 0 or 1.

[0599] In some implementation schemes, R V1 and R V2It is independently selected from H, phenyl and 4-fluorophenyl.

[0600] In some implementations, the linker may connect to one of the different sites of the PBD dimer drug moiety, including the N10 imine of the B ring, the C-2 inner / outer position of the C ring, or a linker unit of the A ring (see structures C(I) and C(II) below).

[0601] Non-limiting exemplary PBD dimer components of ADC include formulas C(I) and C(II):

[0602]

[0603] Formulas C(I) and C(II) are shown in their N10-C11 imine form. Exemplary PBD pharmaceutical portions also include methylamine and protected methylamine forms, as shown in the table below:

[0604]

[0605] in:

[0606] X is CH2 (n = 1 to 5), N, or O;

[0607] Z and Z' are independently selected from OR and NR2, where R is a primary, secondary or tertiary alkyl chain containing 1 to 5 carbon atoms;

[0608] R1, R'1, R2, and R'2 are each independently selected from H, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C 5-20 Aryl (including substituted aryl), C 5-20 Heteroaryl, –NH2, -NHMe, -OH and -SH, wherein in some embodiments, the alkyl, alkenyl and alkynyl chains contain up to 5 carbon atoms;

[0609] R3 and R'3 are independently selected from H, OR, NHR and NR2, where R is a primary, secondary or tertiary alkyl chain containing 1 to 5 carbon atoms;

[0610] R4 and R'4 are independently selected from H, Me, and OMe;

[0611] R5 is selected from C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C 5-20 Aryl groups (including aryl groups substituted with halogens, nitro groups, cyano groups, alkoxy groups, alkyl groups, and heterocyclic groups) and C 5-20 Heteroaryl groups, wherein in some embodiments, the alkyl, alkenyl, and alkynyl chains comprise up to five carbon atoms;

[0612] R 11It is H, C1-C8 alkyl or protecting group (e.g. acetyl, trifluoroacetyl, tert-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), 9-fluorenylmethyleneoxycarbonyl (Fmoc) or a moiety containing a self-sacrificing unit such as valine-citrulline-PAB);

[0613] R 12 It is H, C1-C8 alkyl, or a protecting group;

[0614] Where R1, R'1, R2, R'2 or R 12 -OCH2CH2(X) is formed between one of the hydrogen atoms or the A ring. n One hydrogen atom of the CH2CH2O- spacer group is replaced by a single bond of the linker attached to the ADC.

[0615] Exemplary PDB dimer portions of an ADC include, but are not limited to (wavy lines indicating covalently linked sites with linkers):

[0616]

[0617] A non-limiting exemplary embodiment of an ADC comprising a PBD dimer has the following structure:

[0618]

[0619] PBD dimer-val-cit-PAB-Ab;

[0620]

[0621] PBD dimer-maleimide-acetal-Ab;

[0622]

[0623] PBD dimer - Phe-homoLys-PAB-Ab, where:

[0624] n is 0 to 12. In some implementations, n is 2 to 10. In some implementations, n is 4 to 8. In some implementations, n is selected from 4, 5, 6, 7, and 8.

[0625] The linkers for PBD dimer-val-cit-PAB-Ab and PBD dimer-Phe-homoLys-PAB-Ab are protease-cleavable, while the linker for PBD dimer-maleimide-acetal is acid-labile.

[0626] PBD dimers and ADCs containing PBD dimers can be prepared according to methods known in the art. See, for example, WO 2009 / 016516, US 2009 / 304710, US 2010 / 047257, US 2009 / 036431, US 2011 / 0256157, WO 2011 / 130598.

[0627] (5) Anthracycline compounds

[0628] In some implementations, the ADC comprises anthracycline compounds. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. While not wishing to be bound by any particular theory, studies have shown that anthracyclines can act to kill cells through a number of different mechanisms, including: 1) drug molecules embedding into the cell's DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) the drug generating free radicals, which then react with cellular macromolecules to cause cell damage; and / or 3) interactions between the drug molecule and the cell membrane (see, for example, C. Peterson et al., "Transport and Storage of Anthracycline in Experimental Systems and Human Leukemia"). Anthracycline Antibiotics in Cancer Therapy (NRBachur, “Free Radical Damage”, ibid., pp. 97-102). Due to their cytotoxic potential, anthracyclines have been used to treat a variety of cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma (see, for example, PH-Wiernik). Anthracycline:Current Status And New Developments (Page 11).

[0629] Non-limiting exemplary anthracycline compounds include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and their derivatives. Immunoreceptors and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al. (2006) Current Med. Chem. 13:477-523; Jeffrey et al. (2006) Bioorganic & Med. Chem. Letters 16:358-362; Torgov et al. (2005) Bioconj. Chem. 16:717-721; Nagy et al. (2000) Proc. Natl. Acad. Sci. USA 97:829-834; Dubowchik et al. (2002) Bioorg. & Med. Chem. Letters 12:1529-1532; King et al. (2002) J. Med. Chem. 45:4336-4343; EP 0328147; US 6630579). The antibody-drug conjugate BR96-doxorubicin specifically responds to the tumor-associated antigen Lewis-Y and has been evaluated in phase I and II studies (Saleh et al. (2000) J. Clin. Oncology 18:2282-2292; Ajani et al. (2000) Cancer Jour. 6:78-81; Tolcher et al. (1999) J. Clin. Oncology 17:478-484).

[0630] PNU-159682 is an effective metabolite (or derivative) of nemorubicin (Quintieri et al. (2005) Clinical Cancer Research 11(4):1608-1617). Nemorarubicin is a semi-synthetic analogue of doxorubicin with a 2-methoxymorpholino group on the glycosidic amino group and is under clinical evaluation (Grandi et al. (1990) Cancer Treat. Rev. 17:133; Ripamonti et al. (1992) Brit. J. Cancer 65:703, including phase II / III trials for hepatocellular carcinoma (Sun et al. (2003) Proceedings of the American Society for Clinical Oncology 22, Abs 1448; Quintieri (2003) Proceedings of the American Association of Cancer Research, 44: 1st edition, Abs 4649; Pacciarini et al. (2006) Jour. Clin. Oncology 24:14116).

[0631] A non-limiting exemplary ADC comprising Nemorubicin or a Nemorubicin derivative is shown in Formula Ia:

[0632]

[0633] R1 is a hydrogen atom, a hydroxyl group or a methoxy group, and R2 is a C1-C5 alkoxy group or a pharmaceutically acceptable salt thereof;

[0634] L1 and Z together form the connector (L) described in this paper;

[0635] T is the antibody (Ab) described in this article; and

[0636] m is from 1 to about 20. In some embodiments, m is from 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

[0637] In some implementations, both R1 and R2 are methoxy (-OMe).

[0638] Another non-limiting exemplary ADC comprising Nemorubicin or a Nemorubicin derivative is shown in Formula Ib:

[0639]

[0640] R1 is a hydrogen atom, a hydroxyl group or a methoxy group, and R2 is a C1-C5 alkoxy group or a pharmaceutically acceptable salt thereof;

[0641] L2 and Z together form the connector (L) described in this paper;

[0642] T is the antibody (Ab) described in this article; and

[0643] m is from 1 to about 20. In some embodiments, m is from 1 to 10, 1 to 7, 1 to 5, or 1 to 4.

[0644] In some implementations, both R1 and R2 are methoxy (-OMe).

[0645] In some embodiments, the nemorubicin component of the nemorubicin-containing ADC is PNU-159682. In some such embodiments, the drug portion of the ADC may have one of the following structures:

[0646]

[0647]

[0648] The wavy line indicates the connection to the connector (L).

[0649] Anthracycline compounds, including PNU-159682, can be conjugated to antibodies via several linking sites and various linkers (US 2011 / 0076287; WO2009 / 099741; US ​​2010 / 0034837; WO 2010 / 009124), including the linkers described herein.

[0650] Exemplary ADCs that include Nemorubicin stars and connectors include, but are not limited to:

[0651]

[0652] PNU-159682 Maleimide Acetal-Ab;

[0653]

[0654] PNU-159682-val-cit-PAB-Ab;

[0655]

[0656] PNU-159682-val-cit-PAB-spacer-Ab;

[0657]

[0658] PNU-159682-val-cit-PAB-spacer (R) 1 R 2)-Ab, wherein: R1 and R2 are independently selected from H and C1-C6 alkyl groups; and

[0659]

[0660] PNU-159682-Maleimide-Ab.

[0661] The linker of PNU-159682 maleimide acetal-Ab is acid-instable, while the linkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer (R) are acid-instable. 1 R 2 The linker of )-Ab is protease-cleavable.

[0662] (6) Other drug sections

[0663] The drug component also includes geldanamycin (Mandler et al. (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791); and enzymatically active toxins and their fragments, including but not limited to diphtheria toxin A chain, non-conjugated active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, absinthecin A chain, sucralose root toxin A chain, α-broomrin, tung oil protein, caryophyllin protein, and Phytolacca acinosa. Americana proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitors, jatropha toxin, croton toxin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene. See, for example, WO 93 / 21232.

[0664] The drug component also includes compounds with nucleolytic activity (such as ribonucleases or DNA endonucleases).

[0665] In some implementations, the immunoconjugate may contain highly radioactive atoms. A variety of radioactive isotopes can be used to generate radioconjugated antibodies. Examples include At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 And radioactive isotopes of Lu. In some embodiments, when using an immunoconjugate for detection, it may contain radioactive atoms such as Tc for scintillation studies. 99 Or I 123 Spin labels, such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, can be used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI). Zirconium-89 can be complexed with various metal chelating agents and conjugated to antibodies, for example, for use in PET imaging (WO2011 / 056983).

[0666] Radiolabeling or other labeling can be incorporated into immunoconjugates in known ways. For example, peptides can be biosynthesized or chemically synthesized using suitable amino acid precursors containing, for example, one or more fluorine-19 atoms in place of one or more hydrogen atoms. In some embodiments, the label is, for example, Tc. 99 I 123 Re 186 Re 188 and In 111 It can be linked via cysteine ​​residues in the antibody. In some embodiments, yttrium-90 can be linked via lysine residues in the antibody. In some embodiments, the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used for incorporation of iodine-123. Some other methods are described in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0667] In some embodiments, the immunoconjugate may include an antibody conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug-activating enzyme converts a prodrug (e.g., a peptide-based chemotherapeutic agent, see WO 81 / 01145) into an active drug, such as an anticancer drug. In some embodiments, such immunoconjugates may be used in antibody-dependent enzyme-mediated prodrug therapy (“ADEPT”). Enzymes that can be conjugated to antibodies include, but are not limited to: alkaline phosphatases, which can be used to convert phosphate-containing prodrugs into free drugs; arylsulfatases, which can be used to convert sulfate-containing prodrugs into free drugs; cytosine deaminases, which can be used to convert non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases such as Serratia protease, thermophilic protease, subtilis protease, carboxypeptidase, and cathepsins (e.g., cathepsin B and L), which can be used to convert peptide-containing prodrugs into free drugs; D-alanyl carboxypeptidase, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate lyases such as β-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs into free drugs; β-lactamases, which can be used to convert drugs derivatized using β-lactams into free drugs; and penicillin amidases such as penicillin V amidase and penicillin G amidase, which can be used to convert drugs derivatized at the ammonia nitrogen using phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. In some implementations, the enzyme can be covalently bound to the antibody using recombinant DNA techniques well known in the art. See, for example, Neuberger et al., Nature 312:604-608 (1984).

[0668] c) Drug loading

[0669] Drug loading is denoted by p, which is the average number of drug moieties per antibody in the molecule of Formula I. Drug loading can range from 1 to 20 drug moieties (D) per antibody. An ADC of Formula I comprises a collection of antibodies conjugated to a series of drug moieties from 1 to 20. The average number of drug moieties per antibody in the ADC formulation from the conjugation reaction can be characterized by conventional methods such as mass spectrometry, ELISA, and HPLC. The quantitative distribution of the ADC (denoted by p) can also be determined. In some cases, the separation, purification, and characterization of a homogeneous ADC with a certain p value from ADCs with other drug loadings can be achieved by methods such as reverse-phase HPLC or electrophoresis.

[0670] For some antibody-drug conjugates, p can be limited by the number of linker sites on the antibody. For example, if the linker is a cysteine ​​thiol (as in some exemplary embodiments above), the antibody may have only one or more cysteine ​​thiol groups, or may have only one or more sufficiently reactive thiol groups that can be attached via a linker. In some embodiments, higher drug loading (e.g., p > 5) can lead to aggregation, insolubility, toxicity, or loss of cell permeability in some antibody-drug conjugates. In some embodiments, the average drug loading of the ADC ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. Indeed, it has been shown that for some ADCs, the optimal ratio of the drug portion per antibody can be less than 8 and can be from about 2 to about 5 (US 7498298).

[0671] In some embodiments, a drug moiety less than the theoretical maximum is conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that do not react with the drug-linker intermediates or linker reagents described below. Generally, antibodies do not contain many free and reactive cysteine ​​thiols that can be linked to the drug moiety; in fact, most cysteine ​​thiols in antibodies are present as disulfide bonds. In some embodiments, the antibody may be reduced to reactive cysteine ​​thiols under partially or completely reducing conditions using a reducing agent such as dithiothreitol (DTT) or ethylphosphine tricarbonyl (TCEP). In some embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0672] The loading (drug / antibody ratio) of an ADC can be controlled in different ways, for example by: (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the time or temperature of the conjugation reaction; and (iii) using partial or limiting reduction conditions for cysteine ​​thiol modification.

[0673] It should be understood that if more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADC compounds containing one or more drug moieties linked to an antibody. The drug mean for each antibody can be calculated from the mixture using a dual ELISA antibody assay that is specific to both the antibody and the drug. Individual ADC molecules in a mixture can be identified by mass spectrometry and separated by HPLC, such as hydrophobic interaction chromatography (see, for example, McDonald et al. (2006) Prot. Engr. Design & Selection 19(7):299-307; Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Hamblett, KJ et al. “Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Vol. 45, March 2004; Alley, SC et al. “Controlling the location of drug attachment in antibody-drug conjugates,” Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting). Meeting, March 27-31, 2004 (Proceedings of the AACR, Vol. 45, March 2004). In some embodiments, a uniform ADC with a single loading value can be separated from the conjugated mixture by electrophoresis or chromatography.

[0674] d) Some methods for preparing immunoconjugates

[0675] An ADC of Formula I can be prepared via several pathways using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of an antibody with a divalent linker reagent to form Ab-L via a covalent bond, followed by a reaction with a drug moiety D; and (2) reacting a nucleophilic group of a drug moiety with a divalent linker reagent to form DL via a covalent bond, followed by a reaction with a nucleophilic group of an antibody. An exemplary method for preparing an ADC of Formula I via the latter pathway is described in US 7498298, which is expressly incorporated herein by reference.

[0676] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amino groups; (ii) side-chain amino groups such as lysine; (iii) side-chain thiols such as cysteine; and (iv) glycosyl hydroxyl or amino groups, in the case of glycosylation of the antibody. Amines, thiols, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on linker moieties and linker reagents to form covalent bonds, said linker moieties and linker reagents including: (i) reactive esters such as NHS esters, HOBt esters, halocarboxylate esters, and acyl halides; (ii) alkyl halides and benzyl halides such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleimide groups. Some antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive by treatment with reducing agents such as DTT (dithiothreitol) or ethylphosphine tricarbonyl (TCEP) to completely or partially reduce the antibody, thereby conjugating it to linker reagents. Each cysteine ​​bridge will thus theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by modifying lysine residues, for example, by reacting lysine residues with 2-iminothiacyclopentane (Traut's reagent) to convert amines into thiols. Reactive thiols can also be introduced into antibodies by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing variant antibodies containing one or more non-natural cysteine ​​amino acid residues).

[0677] The antibody-drug conjugates provided herein can also be generated by reaction between an electrophilic group, such as an aldehyde or ketone carbonyl group, on the antibody and a nucleophilic group on the linker reagent or the drug. Useful nucleophilic groups on the linker reagent include, but are not limited to, acylhydrazides, oximes, amino groups, hydrazines, thioureas, hydrazide carboxylates, and aromatic hydrazides. In one embodiment, the antibody is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on the linker reagent or the drug. In another embodiment, the sugar of the glycosylated antibody can be oxidized, for example, with a periodate oxidizing agent to form an aldehyde or ketone group capable of reacting with an amino group on the linker reagent or the drug moiety. The resulting imine Schiff base group can form a stable bond, or can be reduced, for example, by a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the carbohydrate moiety of the glycosylated antibody with galactose oxidase or sodium periodate can generate a carbonyl group (aldehyde and ketone) in the antibody, which can react with a suitable group on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can react with sodium periodate to generate an aldehyde that substitutes for the first amino acid (Geoghegan and Stroh, (1992) Bioconjugate Chem. 3:138-146; US 5362852). This aldehyde can then react with a nucleophilic reagent of the drug moiety or linker.

[0678] Exemplary nucleophilic groups on the pharmaceutical moiety include, but are not limited to: amine, thiol, hydroxyl, acylhydrazine, oxime, hydrazine, thiourea, hydrazide carboxylate, and aromatic acylhydrazine groups, which are capable of reacting with electrophilic groups on the linker moiety and linker reagent to form covalent bonds, said linker moiety and linker reagent including: (i) active esters such as NHS esters, HOBt esters, halocarbamates, and acyl halides; (ii) alkyl halides and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups, and maleimide groups.

[0679] Non-limiting exemplary cross-linking agents that can be used to prepare ADCs are described in the section entitled “Exemplary Linkers” herein. Methods for using such cross-linking agents to link two parts (including protein and chemical parts) are known in the art. In some embodiments, fusion proteins comprising antibodies and cytotoxic agents can be prepared, for example, by recombinant techniques or peptide synthesis. Recombinant DNA molecules may contain regions encoding the antibody and cytotoxic portions of the conjugate, said regions being adjacent to each other or separated by linker peptide-coding regions that do not disrupt the desired properties of the conjugate.

[0680] In yet another implementation, an antibody may be conjugated to a "receptor" (e.g., streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to a patient, followed by the removal of unbound conjugates from circulation using a scavenger, and then the administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a drug or a radioactive nucleotide).

[0681] E. Methods and compositions for diagnosis and detection

[0682] In some embodiments, any anti-FcRH5 antibody provided herein can be used to detect the presence of FcRH5 (e.g., FcRH5) in a biological sample. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some embodiments, the biological sample includes cells or tissues. In some embodiments, such tissues include normal and / or cancerous tissues, such as B cells and / or B cell-associated tissues, that express higher levels of FcRH5 relative to other tissues. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0683] In one aspect, this document provides a method for detecting the presence of FcRH5 in a biological sample. In some embodiments, the method includes contacting the biological sample with an anti-FcRH5 antibody under conditions allowing binding of the anti-FcRH5 antibody to FcRH5, and detecting whether a complex has formed between the anti-FcRH5 antibody and FcRH5. In one aspect, the present invention provides a method for diagnosing a condition associated with increased FcRH5 expression. In some embodiments, the method includes: contacting test cells with an anti-FcRH5 antibody; determining the FcRH5 expression level (quantitative or qualitative) of the test cells by detecting the binding of the anti-FcRH5 antibody to FcRH5; and comparing the FcRH5 expression level of the test cells with that of control cells (e.g., normal cells of the same tissue origin as the test cells or cells expressing FcRH5 at a level equivalent to that of the normal cells), wherein a higher FcRH5 expression level in the test cells than in the control cells indicates the presence of a condition associated with increased FcRH5 expression. In some embodiments, the test cells are obtained from an individual suspected of having a condition associated with increased FcRH5 expression. In some embodiments, the condition is a proliferative condition, such as cancer or tumor. In some embodiments, FcRH5 is FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0684] Exemplary cellular proliferative disorders that can be diagnosed using the antibodies described herein include B-cell disorders and / or B-cell proliferative disorders, including but not limited to lymphoma, multiple myeloma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed refractory NHL, refractory NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma.

[0685] In one embodiment, an anti-FcRH5 antibody is provided for use in diagnostic or detection methods. In another embodiment, a method for detecting the presence of FcRH5 in a biological sample is provided. In some embodiments, the method includes contacting the biological sample with the anti-FcRH5 antibody described herein under conditions allowing the anti-FcRH5 antibody to bind to FcRH5, and detecting whether a complex has formed between the anti-FcRH5 antibody and FcRH5 in the biological sample. This method can be in vitro or in vivo. In one embodiment, the anti-FcRH5 antibody is used to select subjects eligible for anti-FcRH5 antibody therapy, for example when FcRH5 is a biomarker used for patient selection. In another embodiment, the biological sample is cells or tissue (e.g., biopsy material). In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0686] In another embodiment, the anti-FcRH5 antibody is used in vivo to detect (e.g., by in vivo imaging) FcRH5-positive cancer in a subject, for example, for the diagnosis, prognosis, or staging of cancer, to determine appropriate treatment, or to monitor the cancer's response to treatment. One method known in the art for in vivo detection is immunopositron emission tomography (immuno-PET), as described, for example, in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel et al., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method for detecting FcRH5-positive cancer in a subject is provided, comprising administering a labeled anti-FcRH5 antibody to a subject who has or is suspected of having FcRH5-positive cancer, and detecting the labeled anti-FcRH5 antibody in the subject, wherein the detection of the labeled anti-FcRH5 antibody indicates FcRH5-positive cancer in the subject. In some such embodiments, the labeled anti-FcRH5 antibody comprises an antibody that reacts with a positron emitter (e.g., 68 Ga、 18 F,64 Cu、 86 Y、 76 Br、 89 Zr and 124 I) Conjugated anti-FcRH5 antibody. In one specific embodiment, the positron emitter is... 89 Zr. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0687] In other embodiments, a diagnostic or detection method includes contacting a first anti-FcRH5 antibody immobilized on a matrix with a biological sample in which the FcRH5 to be tested is present; exposing the matrix to a second anti-FcRH5 antibody; and detecting whether the second anti-FcRH5 binds to the complex between the first anti-FcRH5 antibody and FcRH5 in the biological sample. The matrix can be any supporting medium, such as glass, metal, ceramic, polymer beads, glass slides, chips, and other matrices. In some embodiments, the biological sample includes cells, blood, or tissue (e.g., biopsy material).

[0688] Exemplary conditions that can be diagnosed or detected according to any of the above embodiments include FcRH5-positive cancers, such as FcRH5-positive B-cell proliferative disorders, FcRH5-positive plasma cell tumors, and FcRH5-positive multiple myeloma. In some embodiments, FcRH5-positive cancers are detected by anti-FcRH5 immunohistochemistry (IHC) or in situ hybridization (ISH). In some embodiments, FcRH5-positive cancers are determined by reverse transcriptase PCR (RT-PCR) to detect FcRH5 mRNA expression in cancers. In some embodiments, the RT-PCR is quantitative RT-PCR.

[0689] In some embodiments, a labeled anti-FcRH5 antibody is provided. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. Labeling includes, but is not limited to: labels or portions that are directly detectable (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels); and portions that are indirectly detected, for example, through enzymatic reactions or molecular interactions, such as enzymes or ligands. Exemplary labels include, but are not limited to: radioisotopes. 32 P, 14 C 125 I, 3 H and 131I; fluorophores such as rare earth chelates or luciferin and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, insect luciferases such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), insect luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme; sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase, coupled to enzymes that use hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase; biotin / avidin, spin labeling, phage labeling, stable free radicals, etc. In another embodiment, the label is a positron emitter. Positron emitters include, but are not limited to, those mentioned above. 68 Ga、 18 F, 64 Cu、 86 Y、 76 Br、 89 Zr and 124 I. In one specific implementation, the positron emitter is 89 Zr.

[0690] F. Pharmaceutical preparations

[0691] The pharmaceutical formulations of the anti-FcRH5 antibodies or immunoconjugates described herein are prepared by mixing such antibodies or immunoconjugates, having the desired purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) into lyophilized or aqueous formulations. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride, hexachlorobenzyl quaternary ammonium chloride, benzalkonium chloride, benzyl chloride, phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); and low molecular weight (less than about 10 residues). Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannose, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins like rHuPH20 (…). Baxter International, Inc. Certain exemplary methods of using sHASEGP, including rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0692] Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody or immunoconjugate formulations include those described in U.S. Patent Nos. 6,171,586 and WO 2006 / 044908, the latter of which comprises histidine-acetate buffer.

[0693] The formulations described herein may also contain more than one active ingredient required to treat a specific indication, preferably those active ingredients that have complementary activities that do not adversely affect each other.

[0694] Active ingredients can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by cohesive techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980).

[0695] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing antibodies and / or immunoconjugates, said matrix being in the form of a molded article, such as a film or microcapsule.

[0696] Preparations intended for internal administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filter membrane.

[0697] G. Treatment methods and compositions

[0698] Any anti-FcRH5 antibody (e.g., an FcRH5 bispecific antibody) and / or immunoconjugate provided herein can be used in a variety of methods, such as therapeutic applications. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0699] In one aspect, the method described herein for using anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates to inhibit the proliferation of FcRH5-positive cells includes exposing the cells to the anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates under conditions allowing the anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates to bind to FcRH5 (e.g., FcRH5c) on the cell surface, thereby inhibiting the proliferation of the cells. In some embodiments, the method is an in vitro or in vivo method. In other embodiments, the cells are B-cell proliferative disorders. In some embodiments, the proliferative disorder is associated with increased expression and / or activity of FcRH5 (e.g., FcRH5c). For example, in some embodiments, B-cell proliferative disorders are associated with increased expression of FcRH5 on the surface of B cells. In some embodiments, the B-cell proliferative disorder is a tumor or cancer. In some embodiments, the B-cell proliferative disorder is a plasma cell tumor. In some embodiments, plasma cell tumors are multiple myeloma, plasmacytoma, and / or MGUS. Examples of B-cell proliferative disorders to be treated with the antibodies and / or immunoconjugates of the present invention include, but are not limited to: lymphoma, multiple myeloma, non-Hodgkin lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed refractory NHL, refractory NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and / or mantle cell lymphoma.

[0700] The presence of various biomarkers in a sample can be analyzed by a variety of methods, many of which are known in the art and understood by those skilled in the art, including but not limited to immunohistochemistry (“IHC”), Western blotting, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, DNA analysis (Southern analysis), RNA analysis (Northern analysis), whole genome sequencing, polymerase chain reaction (“PCR”) including quantitative real-time PCR (“qRT-PCR”) and other amplification-type detection methods (e.g., branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling and / or serial gene expression analysis (“SAGE”), and any of the various assays that can be performed by protein, gene and / or tissue array analysis. Typical protocols for evaluating gene status and gene products can be found, for example, in Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, Units 2 (RNA blotting), 4 (DNA blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplex immunoassays can also be used, such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”).

[0701] In vitro cell proliferation inhibition can be achieved using CellTiter-Glo, available from Promega (Madison, WI). TM The Luminescent Cell Viability Assay is used to determine the number of viable cells in a culture based on the quantification of ATP (indicating metabolically active cells). See Crouch et al. (1993) J. Immunol. Meth. 160:81-88, US Patent No. 6602677. The assay can be performed in 96-well or 384-well formats, making it suitable for automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves using a single reagent ( The reagent is added directly to the cultured cells. This causes cell lysis and the generation of a luminescent signal by a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which in turn is proportional to the number of viable cells present in the culture. Data can be recorded using a photometer or a CCD camera imaging device. The luminescence output is expressed in relative light units (RLU).

[0702] In another aspect, anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates are provided for use as pharmaceuticals. In other aspects, anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates are provided for use in treatment methods. In some embodiments, anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates are provided for treating FcRH5 (e.g., FcRH5c) positive cancers. In some embodiments, this document provides anti-FcRH5 antibodies (including FcRH5 bispecific antibodies) and / or immunoconjugates for use in methods of treating an individual with FcRH5 (e.g., FcRH5c) positive cancer, the method comprising administering an effective amount of the anti-FcRH5 antibody and / or immunoconjugate to the individual. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c. In one such implementation, the method further includes administering an effective amount of at least one other therapeutic agent, such as those described below, to the individual.

[0703] On the other hand, this document provides the use of anti-FcRH5 antibodies (e.g., FcRH5 bispecific antibodies) and / or immunoconjugates in the manufacture or preparation of medicaments. In one embodiment, the medicament is for treating FcRH5 (e.g., FcRH5c) positive cancer. In another embodiment, the medicament is a method of treating FcRH5 (e.g., FcRH5c) positive cancer, the method comprising administering an effective amount of the medicament to an individual having FcRH5 (e.g., FcRH5c) positive cancer. In one such embodiment, the method further comprises administering an effective amount of at least one other therapeutic agent, such as those described below, to the individual. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0704] On the other hand, this document provides a method for treating FcRH5 (e.g., FcRH5c) positive cancers. In one embodiment, the method includes administering an effective amount of an anti-FcRH5 antibody (e.g., an FcRH5 bispecific antibody) and / or an immunoconjugate to an individual having such FcRH5 (e.g., FcRH5c) positive cancer. In one such embodiment, the method further includes administering an effective amount of at least one other therapeutic agent as described below to the individual. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0705] FcRH5-positive cancers according to any of the above embodiments can be, for example, FcRH5-positive B-cell proliferative disorders, FcRH5-positive plasma cell tumors, and / or FcRH5-positive multiple myeloma. In some embodiments, FcRH5-positive cancers are detected by anti-FcRH5 immunohistochemistry (IHC) or in situ hybridization (ISH). In some embodiments, FcRH5-positive cancers are determined by reverse transcriptase PCR (RT-PCR) to detect FcRH5 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

[0706] In some implementation schemes of any of the above-described implementation schemes, the individual can be a person.

[0707] On the other hand, this document provides pharmaceutical formulations, for example, for any of the above-described treatment methods, comprising any anti-FcRH5 antibody and / or immunoconjugate provided herein. In one embodiment, the pharmaceutical formulation comprises any anti-FcRH5 antibody (e.g., a bispecific antibody) and / or immunoconjugate provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any anti-FcRH5 antibody (e.g., a bispecific antibody) and / or immunoconjugate provided herein and at least one other therapeutic agent, such as those described below.

[0708] The antibodies (e.g., bispecific antibodies) and / or immunoconjugates described herein can be used alone or in combination with other agents in treatment. Such combination therapies encompass both combined administration (where two or more therapeutic agents are contained in the same or different formulations) and separate administration. In the case of separate administration, the antibodies or immunoconjugates described herein may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or adjuvants. The antibodies and / or immunoconjugates described herein can also be used in combination with radiotherapy.

[0709] The antibodies (including bispecific antibodies) and / or immunoconjugates (and any other therapeutic agents) described herein may be administered by any suitable route, including parenteral, intrapulmonary, and intranasal administration, and, if intended for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or prolonged. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsatile infusions.

[0710] The antibodies (e.g., bispecific antibodies) and / or immunoconjugates described herein may be formulated, administered, and applied in accordance with good medical practice. Considerations in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The antibodies (e.g., bispecific antibodies) and / or immunoconjugates are not required, but may optionally be formulated with one or more agents currently used for the prevention or treatment of the condition in question. The effective amount of such other agents depends on the amount of antibody or immunoconjugate present in the formulation, the type of condition or treatment, and the other factors described above. These agents are typically used at the same dose and route of administration as described herein, or about 1 to 99% of the dose described herein, or at any dose and via any route determined empirically / clinically to be appropriate.

[0711] For the prevention or treatment of disease, the appropriate dosage of the antibody (e.g., bispecific antibody) and / or immunoconjugate (when used alone or in combination with one or more other additional therapeutic agents) described herein will depend on the type of disease to be treated, the type of antibody or immunoconjugate, the severity and duration of the disease, whether the antibody (e.g., bispecific antibody) and / or immunoconjugate has been administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody or immunoconjugate, and the judgment of the attending physician. The antibody (e.g., bispecific antibody) and / or immunoconjugate may be appropriately administered to the patient in a single or series of treatments. Depending on the type and severity of the disease, an antibody or immunoconjugate of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg–10 mg / kg) may be an initial candidate dose for administration to the patient, whether, for example, by single or multiple separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose may range from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, treatment generally continues until the desired suppression of disease symptoms is achieved, depending on the condition. An exemplary dose of the antibody (e.g., a bispecific antibody) and / or immunoconjugate will range from about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg can be administered to the patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., to allow the patient to receive about 2 to about 20, or for example, about 6, doses of the antibody). A higher loading dose can be administered first, followed by one or more lower doses. However, other dosing regimens may be used. Progression of this therapy is easily monitored using routine techniques and assays.

[0712] It should be understood that any of the above formulations or treatments can be implemented using the immunoconjugates and anti-FcRH5 antibodies provided herein. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0713] H. Products

[0714] In another aspect provided herein, articles comprising materials that can be used to treat, prevent, and / or diagnose the aforementioned conditions are provided. The articles comprise a container and a label or instruction manual located on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. The containers can be formed from various materials such as glass or plastic. The container contains a single composition or a composition in combination with another effective composition for treating, preventing, and / or diagnosing the condition, and may have a sterile access point (e.g., the container may be an intravenous infusion bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an antibody or immunoconjugate provided herein. The label or instruction manual indicates that the composition is used to treat a specific condition. Additionally, the articles may comprise (a) a first container containing a composition comprising an FcRH5 antibody (e.g., a bispecific antibody) and / or an FcRH5 immunoconjugate provided herein; and (b) a second container containing a composition comprising another cytotoxic agent or therapeutic agent. The articles in this embodiment provided herein may further include instruction manuals indicating that the composition can be used to treat a specific condition. Alternatively, the article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or glucose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the anti-FcRH5 antibody binds to the isotype c-specific region of the extracellular domain of FcRH5c. In some embodiments, the anti-FcRH5 antibody binds to the Ig-like domain 9 of FcRH5c.

[0715] III. Examples

[0716] The following are examples of the methods and compositions of the present invention. It should be understood that, in light of the general description provided above, many other embodiments can be practiced.

[0717] Materials and methods

[0718] Immunogen (E11-flag)

[0719] Amino acids 745-850 (SEQ ID NO:1) of human FcRH5c were cloned into the mammalian expression vector pRK5.NT.Flag using a standard protocol and transiently expressed in CHO cells. The recombinant protein with the N-terminal Flag expression tag was purified on an S200 Superdex column using anti-flag and size exclusion chromatography.

[0720] Development and characterization of mouse anti-FcRH5 E11 antibody

[0721] Balb / c mice (Charles River, Hollister, CA) were immunized via footpad injection with 2 μg of human FcRH5 E11 ECD protein (amino acid residues 743-850 of SEQ ID NO:1) (Genentech, South San Francisco, CA) mixed with MPL+TDM (Ribi) adjuvant. Mice received nine doses, followed by booster injections via footpad and intravenous routes only in PBS three days prior to fusion.

[0722] Lymphocytes from the popliteal lymph nodes of these mice (all of which had serum that showed strong binding titers to immunoproteins by ELISA and strong FACS reactivity to SVT2 cells transfected with human FcRH5 E11 ECD) were electrofused (Harvard Apparatus, Holliston, MA) with X63-Ag8.653 mouse myeloma cells (American Type Culture Collection, Rockville, MD). The fused cells were incubated overnight at 37°C / 7% CO2 in medium C (StemCell Technologies, Vancouver, BC, Canada), then suspended in semi-solid medium D (StemCell Technologies) containing 0.01 mg / ml FITC-labeled anti-mouse IgG (Jackson Immuno Research, West Grove, PA) and plated on Omniwell trays (Thermo Fisher Scientific, Rochester, NY). Nine days after plating, fluorescent colonies were screened using Clonepix FL (Genetix, New Milton, Hampshire, UK) and transferred to 96-well plates containing medium E (StemCell Technologies). Seven days after selection, the supernatant was screened for anti-mouse IgG by ELISA (MP Biomedicals, Santa Ana, CA).

[0723] Hybridomas exhibiting mouse IgG expression by ELISA were amplified, and SVT2 cells overexpressing full-length human FcRH5, cynomolgus monkey FcRH5, and human FcRH5 E11 ECD were selected using FACS. Strongly FACS-positive clones were subcloned using FACSAria (BD, Franklin Lakes, NJ). The final clone exhibiting the highest binding to the target ELISA and FACS was amplified after one or two rounds of subcloning for large-scale production in a bioreactor (Integra Biosciences, Chur, Switzerland). The supernatant was then purified by protein A affinity chromatography as previously described (Hongo et al. 2000).

[0724] The generation of bisFab

[0725] BisFab was generated by crosslinking Fab' of anti-FcRH5 Mab with Fab' of anti-CD3(UCHT1.v9) Mab at hinge cysteine ​​residues. To generate the Fab'2 fragment from hybridoma Abs, different digestion conditions were used: digestion of mIgG1 isotype Abs with 1:50 (w / w) pepsin at pH 3.5 for 1–2 hours at 37°C; digestion of mouse IgG2a Abs with cytolysin C endopeptidase at a ratio of 1:500 (w / w) at pH 8 for 2–4 hours at 37°C; and digestion of mouse IgG2b Abs with cytolysin C at a ratio of 1:100 (w / w) overnight at 37°C. In all cases, the F(ab')2 fragment was isolated from the reaction mixture by capture with an SP column and elution with a linear gradient (0–100%) of 1 M sodium chloride at 10 column volumes. Under the digestion conditions described above, mIgG1 and mIgG2b produced F(ab')2 fragments with three cysteine ​​residues in the hinge, while F(ab')2 from mIgG2a showed two cysteine ​​residues in the hinge. Two different methods were used to generate Fab' with a single reactive Cys. For fragments containing an odd number (3) hinge cysteines (mIgG1 and mIgG2b), the isolated F(ab')2 was reduced to 2-6-H at RT in 25 mM sodium acetate (pH 5), 150 mM sodium chloride, 2 mM EDTA, and 2 mM TCEP. After the reduction step, the sample was diluted to 0.2 mg / ml, the pH was raised to 7.5 by adding Tris pH 8, and 5 mM dehydroascorbic acid (DHAA) was added to promote the reoxidation of cysteine. After overnight incubation at room temperature, the presence of reduced thiols was evaluated by detection with excess NEM and by analysis of MW shifts using mass spectrometry. After confirming that each molecule contains only one reactive cysteine ​​residue, Fab' was purified by gel filtration to remove small amounts of homodimer.

[0726] For the F(ab')2 fragment derived from mIgG2a and containing two cysteine ​​residues in its hinge, a single reactive cysteine ​​residue is generated by partial blocking with N-ethylmaleimide (NEM), as described in Scheer et al. (published). In simple terms, the antibody is digested using pepsin (1% wt / wt) in sodium acetate buffer (pH 4.5). After 1 hour of digestion, F(ab')2 is separated from the digestion mixture by trapping on an SP-HP cation exchange resin and purified by a salt gradient of 10 CV 0–1 M NaCl. F(ab')2 is then reduced with 1 mM TCEP in a buffer containing 25 mM MES (pH 5.8), 2 mM EDTA, and 300 mM NaCl, and Fab is oxidized by adding 5 mM dehydroascorbic acid (DHAA) to re-form disulfide bonds between the heavy and light chains.

[0727] As described above (Scheer et al.; in publication), the effector arm of bisfab (UCHT1.v9) is generated by pepsin digestion, partial NEM blocking, and conjugation to bismaleimide. In simpler terms, the two thiols (cys residues) at the hinge are then reacted with 1 equivalent of N-ethylmaleimide (NEM) (Sigma Aldrich). Different anti-FCRH5 Fab's containing a single reactive cysteine ​​are incubated overnight at room temperature with anti-CD3 Fab' conjugated to a bismaleimide crosslinker. The ~100 kDa crosslinked Fab is separated from the unreacted species by gel filtration and then characterized by SDS-PAGE, mass spectrometry, and analytical size exclusion chromatography.

[0728] TDB expression and purification

[0729] TDBs are generated using two different methods: co-culture of bacteria expressing each of the two antibody arms, or by expressing each arm separately and then annealing them in vitro. These strategies are described in Christoph Spiess et al. 2012 and in PCT / US10 / 58958, filed May 31, 2011 (incorporated by reference). In simple terms, for the co-culture strategy, *E. coli* expressing anti-CD3 (wells) and *E. coli* expressing an anti-tumor target (knots) are grown together in a shaking flask at a predetermined ratio, resulting in similar amounts of each hemisphere. The co-cultured bacterial broth is then harvested, cells are disrupted in a microfluidic, and antibodies are purified by protein A affinity. It has been observed that during microfluidization and protein A capture, the two arms anneal and form a hinged inter-chain disulfide bridge (Christoph Spiess et al. 2012). Alternatively, antibody hemispheres are grown separately by high-cell-density fermentation and independently separated by protein A chromatography. The purified hemispheres were then combined at a 1:1 molar ratio and incubated for 4 hours in 50 mM Tris (pH 8.5) in the presence of 2 mM DTT to allow annealing and reduction of disulfides in the hin...

Claims

1. An isolated anti-Fc receptor-like 5 (FcRH5) antibody, wherein the antibody comprises: The heavy chain comprises a hypervariable region (HVR)-H1 consisting of the amino acid sequence of SEQ ID NO:39, HVR-H2 consisting of the amino acid sequence of SEQ ID NO:63, and HVR-H3 consisting of the amino acid sequence of SEQ ID NO:87; and the light chain comprises HVR-L1 consisting of the amino acid sequence of SEQ ID NO:3, HVR-L2 consisting of the amino acid sequence of SEQ ID NO:15, and HVR-L3 consisting of the amino acid sequence of SEQ ID NO:

27.

2. The antibody according to claim 1, wherein: The heavy chain comprises a heavy chain variable (VH) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:113, and the light chain comprises a light chain variable (VL) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

112.

3. The antibody according to claim 1, wherein the antibody is a monoclonal antibody.

4. The antibody according to claim 1, wherein the antibody is a human, humanized, or chimeric antibody.

5. The antibody according to claim 1, wherein the antibody is an antibody fragment that binds to FcRH5.

6. The antibody according to claim 1, wherein the antibody is an IgG1, IgG2a, or IgG2b antibody.

7. The antibody of claim 1, wherein the antibody is a bispecific antibody, and wherein the bispecific antibody binds to FcRH5 and CD3.

8. A pharmaceutical formulation comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical preparation according to claim 8, further comprising an additional therapeutic agent.

10. The antibody of claim 1, wherein the antibody is conjugated to a label.

11. The antibody of claim 10, wherein the label is a positron emitter.

12. The antibody according to claim 11, wherein the positron emitter is 89 Zr.

13. An isolated anti-FcRH5 antibody, wherein the antibody comprises: The VH sequence of SEQ ID NO:113 and the VL sequence of SEQ ID NO:

112.

14. The antibody according to claim 13, wherein the antibody is a monoclonal antibody.

15. The antibody of claim 13, wherein the antibody is a human, humanized, or chimeric antibody.

16. The antibody of claim 13, wherein the antibody is an antibody fragment that binds to FcRH5.

17. The antibody according to claim 13, wherein the antibody is an IgG1, IgG2a, or IgG2b antibody.

18. The antibody of claim 13, wherein the antibody is a bispecific antibody, and wherein the bispecific antibody binds to FcRH5 and CD3.

19. A pharmaceutical formulation comprising the antibody as described in claim 13 and a pharmaceutically acceptable carrier.

20. The pharmaceutical preparation of claim 19, further comprising an additional therapeutic agent.

21. The antibody of claim 13, wherein the antibody is conjugated to a label.

22. The antibody of claim 21, wherein the label is a positron emitter.

23. The antibody according to claim 22, wherein the positron emitter is 89 Zr.

24. An isolated nucleic acid encoding an anti-FcRH5 antibody according to any one of claims 1-7, 10-18 and 21-23.

25. A host cell comprising the nucleic acid according to claim 24.

26. A method for generating an anti-FcRH5 antibody, comprising culturing a host cell according to claim 25 to generate the anti-FcRH5 antibody.

27. Use of the anti-FcRH5 antibody according to any one of claims 1-7, 10-18 and 21-23 in the preparation of a medicament for treating an individual suffering from multiple myeloma.

28. The use according to claim 27, wherein the drug formulation is intended for administration in combination with another therapeutic agent to the individual.

29. Use of the anti-FcRH5 antibody according to any one of claims 1-7, 10-18 and 21-23 in the preparation of a kit for detecting human FcRH5 in biological samples derived from subjects who have or are suspected of having multiple myeloma.

30. The use according to claim 29, wherein the biological sample is a blood sample.

31. Use of a labeled anti-FcRH5 antibody comprising any one of claims 1-7 and 10-18 in a kit for preparing a method for detecting multiple myeloma in a subject who has or is suspected of having multiple myeloma, wherein the method comprises (i) administering the labeled anti-FcRH5 antibody to the subject, and (ii) detecting the labeled anti-FcRH5 antibody in the subject, wherein detecting the labeled anti-FcRH5 antibody indicates that the subject has multiple myeloma.

32. The use according to claim 31, wherein the labeled anti-FcRH5 antibody comprises an anti-FcRH5 antibody conjugated to a positron emitter.

33. The use according to claim 31, wherein the positron emitter is 89 Zr.

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