Polyspecific anti-polybiquitin antibodies

Abstract

Multispecific antibodies comprising a first half-antibody comprising a first antigen-binding site that binds polyubiquitin; and a second half-antibody comprising a second antigen-binding site that binds a pro-inflammatory protein, such as receptor interacting protein kinase 1 (RIP1) or receptor interacting protein kinase 2 (RIP2), and methods for using the antibodies are provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 238,749, filed August 30, 2021, and U.S. Provisional Application No. 63 / 247,776, filed September 23, 2021, each of which is incorporated by reference in its entirety into this specification for all purposes. [Background technology]

[0002] FIELD OF THEINVENTION The present invention relates to anti-polyubiquitin multispecific antibodies, and methods for making and using same. Summary of the Invention

[0003]

[0003] Ubiquitin is a small protein that plays important regulatory roles in various cellular pathways. The best known of these is its role in protein degradation, whereby ubiquitin is covalently attached to target proteins, which are then recognized and destroyed by the 26S proteasome (see Wilkinson, Semin. Cell Devel. Biol. 11(3):141-148 (2000)). The covalent attachment of ubiquitin, a 76 amino acid protein, to target proteins is a three-step enzymatic process (Pickart, Annu. Rev. Biochem. 70:503-533 (2001)). First, the ubiquitin-activating enzyme E1 forms the ubiquitin-E1 thioester in an ATP-dependent reaction. Ubiquitin is transferred from the ubiquitin-E1 thioester to a member of the ubiquitin-conjugating enzyme (E2) family in the second step. In the third step, with the help of ubiquitin protein ligase (E3), an isopeptide bond is formed between the carboxyl terminus of ubiquitin and the ε-amino group of a lysine residue on the target protein. Enzymes called deubiquitinases remove the ubiquitin moiety from the target protein (Guterman and Glickman, Curr. Prot. Pep. Sci. 5:201-210 (2004)).

[0004]

[0004] Ubiquitin contains seven lysine residues (Lys6, Lys11, Lys27, Lys33, Lys29, Lys48, and Lys63), and therefore ubiquitin itself may function as a target protein for ubiquitination (Peng et al., Nat. Biotechnol. 21:921-926 (2003); Pickart and Fushman, Curr. Opin. Chem. Biol. 8:610-616 (2004)). Molecules generated by ubiquitination of the ubiquitin protein are called polyubiquitin molecules and may contain two or more ubiquitin moieties.

[0005]

[0005] Furthermore, a linear polyubiquitin linkage (also called M1 linkage) is formed in which the C-terminal glycine of ubiquitin is conjugated to the α-amino group of the N-terminal methionine of another ubiquitin molecule. Iwai and Tokunaga, EMBO Reports 10:706-713(2009). Linear polyubiquitin is formed via the linear ubiquitin chain assembly complex (LUBAC), which consists of two RING finger proteins, HOIL-1L and HOIP. Tokunaga et al., Nat. Cell Biol. 11:123-132(2009). Genetically encoded, unanchored linear polyubiquitin is thought to be absent in cells because its C-terminus is susceptible to cleavage by isopeptidase T. Iwai and Tokunaga, EMBO Reports 10:706-713(2009). This observation suggests that linear polyubiquitin assembles post-translationally onto substrate proteins and that the conjugated linear polyubiquitin molecules are potential regulators of protein activity and function. For example, linear polyubiquitination of NF-κB essential modulator (NEMO) has been shown to be involved in NF-κB activation. Different ubiquitin chains can convey specific and distinct biochemical and biological messages that lead to activation or inhibition of cell signaling or regulation of protein stability (Ikeda, F., et al., Cell 143:677-681 (2010); Rape, M., Nat Rev Mol Cell Biol 19:59-70 (2018)).

[0006]

[0006] Receptor interacting protein-1 kinase ("RIP1" or "RIPK1") is a serine / threonine protein kinase. RIP1 is a regulator of cell signaling involved in mediating programmed cell death pathways, such as necroptosis, among others. The most well-studied form of necroptotic cell death is initiated by TNFα (tumor necrosis factor), but necroptosis can also be induced by other members of the TNFα death ligand family (Fas and TRAIL / Apo2L), interferons, Toll-like receptor (TLR) signaling, and viral infection via the DNA sensor DAI (DNA-dependent interferon regulator activator). Van den Berghe et al.(2014)Nature Reviews.Molecular cell biology 15:135-147(2014);Newton,K.Trends in Cell Biology 25:347-353(2015);de Almagro,MCand Vucic,D.Semin Cell Dev Biol.39:56-62(2015). Binding of TNFα to TNFR1 (TNF receptor 1) promotes trimerization of TNFR1 and formation of the intracellular complex Complex-I. TRADD (TNF receptor-associated death domain protein) binds to the intracellular death domain of TNFR1 and recruits the protein kinase RIP1 (receptor interacting protein 1) via the death domains present in both proteins. Chen,ZJImmunological reviews 246:95-106(2012).

[0007]

[0007] Receptor interacting serine / threonine protein kinase 2 ("RIP2" or "RIPK2") is a serine / threonine / tyrosine kinase that plays an essential role in regulating adaptive and innate immune responses. Upon recruitment by activated NOD1 and NOD2 via the CARD-CARD domain, RIPK2 autophosphorylates and undergoes K63-linked polyubiquitination by the ubiquitin ligases XIAP, BIRC2, and BIRC3. Polyubiquitinated proteins induce K63-linked polyubiquitination of IKBKG / NEMO and subsequent activation of IKBKB / IKKB. NF-kappa-B is subsequently released and translocates to the nucleus, where it activates the transcription of hundreds of genes involved in immune responses, growth control, or protection from apoptosis.

[0008] It would be beneficial to provide compositions and methods that can recognize and distinguish between modifications of RIP1 and RIP2 proteins, as well as to provide compositions and methods that are effective for targeting and regulating polyubiquitin-mediated pathways. The present disclosure is directed to meeting one or more of these needs or providing other advantages.

[0009]

[0009] The following non-limiting embodiments are provided.

[0010] Embodiment 1. A method for determining the presence of a polyubiquitinated protein in a sample suspected of containing a polyubiquitinated protein, the polyubiquitinated protein being a proinflammatory protein and comprising polyubiquitin; 1. A method comprising: exposing the sample to at least one multispecific antibody comprising: a first half-antibody comprising a first antigen-binding site that binds to polyubiquitin; and a second half-antibody comprising a second antigen-binding site that binds to a pro-inflammatory protein; and determining binding of the at least one antibody to polyubiquitinated proteins in the sample.

[0011] Embodiment 2. The method of embodiment 1, wherein the polyubiquitinated protein comprises M1-linked polyubiquitin and / or K63-linked polyubiquitin.

[0012] Embodiment 3. The method of embodiment 1 or 2, wherein the proinflammatory protein is a component of one or more signaling complexes.

[0013] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the proinflammatory protein is receptor-interacting protein kinase 1 (RIP1), receptor-interacting protein kinase 2 (RIP2), cellular inhibitor of apoptosis 1 and 2 (c-IAP1 / 2), tumor necrosis factor receptor 1 (TNFR1), linear ubiquitin chain assembly complex (LUBAC), and / or nuclear factor kappa B (NF-κB) essential modulator (NEMO).

[0014] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the pro-inflammatory protein is RIP1.

[0015] Embodiment 6. The method of any one of embodiments 1 to 4, wherein the pro-inflammatory protein is RIP2.

[0016] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the pro-inflammatory protein has an elevated ubiquitination level in an inflammatory state compared to the ubiquitination level in the absence of an inflammatory state.

[0017] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the pro-inflammatory protein has a ubiquitination level of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 1-fold to 12-fold, 2-fold to 12-fold, 3-fold to 12-fold, 4-fold to 12-fold, 5-fold to 12-fold, 6-fold to 12-fold, 7-fold to 12-fold, 8-fold to 12-fold, 9-fold to 12-fold, 10-fold to 12-fold, or 11-fold to 12-fold in an inflammatory state compared to the ubiquitination level in the absence of an inflammatory state.

[0018] Embodiment 9. The method of any one of embodiments 1 to 8, wherein increased levels of ubiquitination correlate with increased severity of the inflammatory disease state.

[0019] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the pro-inflammatory protein is associated with an inflammatory disease, such as inflammatory bowel disease, Crohn's disease, diverticulitis, and ulcerative colitis.

[0020] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the pro-inflammatory protein is associated with Crohn's disease.

[0021] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the pro-inflammatory protein is associated with ulcerative colitis.

[0022] Embodiment 13. A multispecific antibody comprising a first half antibody comprising a first antigen-binding site that binds polyubiquitin; and a second half antibody comprising a second antigen-binding site that binds receptor interacting protein kinase 1 (RIP1).

[0023] Embodiment 14. An antibody described in embodiment 13, which selectively recognizes polyubiquitinated RIP1.

[0024] Embodiment 15. An antibody described in embodiment 13 or 14, which does not recognize receptor interacting protein kinase 2 (RIP2) and / or does not recognize non-ubiquitinated RIP1.

[0025] Embodiment 16. A multispecific antibody comprising a first half antibody comprising a first antigen-binding site that binds polyubiquitin; and a second half antibody comprising a second antigen-binding site that binds receptor interacting protein kinase 2 (RIP2).

[0026] Embodiment 17. An antibody described in embodiment 16, which selectively recognizes polyubiquitinated RIP2.

[0027] Embodiment 18. An antibody described in embodiment 16 or 17, which does not recognize RIP1, XIAP and / or c-IAP1 and / or does not recognize non-ubiquitinated RIP2.

[0028] Embodiment 19. An antibody described in any one of embodiments 13 to 18, wherein the polyubiquitin has a uniform topology.

[0029] Embodiment 20. An antibody described in any one of embodiments 13 to 19, wherein the polyubiquitin comprises a K11 linkage.

[0030] Embodiment 21. An antibody described in any one of embodiments 13 to 20, wherein the polyubiquitin comprises a K48 linkage.

[0031] Embodiment 22. An antibody described in any one of embodiments 13 to 21, wherein the polyubiquitin comprises a K63 linkage.

[0032] Embodiment 23. An antibody described in any one of embodiments 13 to 22, wherein the polyubiquitin comprises an M1 linkage.

[0033] 24. a.(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14; b.(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28; c.(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42; or d. (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 24. The antibody of any one of embodiments 13 to 23, comprising a first half antibody comprising:

[0034] 25. a.(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80; or b.(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 The antibody of any one of embodiments 13 to 15 or 19 to 24, comprising a second half antibody comprising:

[0035] 26. (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 25. The antibody of any one of embodiments 16 to 24, comprising a second half antibody comprising:

[0036] 27. a. a first half antibody, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; b. a first half antibody, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; c. a first half antibody, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Contains; d. The first half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; e. the first half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; f. The first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Contains; g. The first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; h. the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; i. a first half antibody comprising: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Contains; j. a first half antibody, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; k. the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 Including, The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Contains; l. a first half antibody, (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 Including, The other second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 27. The antibody of any one of embodiments 13 to 26, comprising a first half antibody and a second half antibody comprising:

[0037] Embodiment 28. The first half antibody comprises: a. a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8; b. a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22; c. a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36; or d. A VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50. The antibody according to any one of embodiments 13 to 27, comprising:

[0038]

[0023] Embodiment 29. The second half antibody comprises: a. a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; or b. a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73. The antibody according to any one of embodiments 13 to 15, 19 to 25, or 27 to 28, comprising:

[0039]

[0026] Embodiment 30. The second half antibody comprises: (i) a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 94. The antibody according to any one of embodiments 16 to 25 or 26 to 28, comprising:

[0040] EMBODIMENT 31. a. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; b. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; c. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 94; d. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; e. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; f. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 94; g. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; h. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; i. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 94; j. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 71, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; k. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50; (i) the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 73; or l. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50; (i) The antibody of any one of embodiments 10 to 27, wherein the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 94.

[0041]

[0023] Embodiment 32. The first half antibody comprises: a. a VL sequence of SEQ ID NO: 7 and a VH sequence of SEQ ID NO: 8; b. the VL sequence of SEQ ID NO:21 and the VH sequence of SEQ ID NO:22; c. the VL sequence of SEQ ID NO: 35 and the VH sequence of SEQ ID NO: 36; or d. The VL sequence of SEQ ID NO: 49 and the VH sequence of SEQ ID NO: 50 32. The antibody according to any one of embodiments 13 to 31, comprising:

[0042]

[0023] Embodiment 33. The second half antibody comprises: a. the VL sequence of SEQ ID NO: 71 and the VH sequence of SEQ ID NO: 73; or b. The VL sequence of SEQ ID NO: 72 and the VH sequence of SEQ ID NO: 73 The antibody according to any one of embodiments 13 to 15, 19 to 25, 27 to 29, or 31 to 32, comprising:

[0043]

[0023] Embodiment 34. The second half antibody comprises: (i) a VL sequence of SEQ ID NO: 93 and a VH sequence of SEQ ID NO: 94 The antibody according to any one of embodiments 16 to 24, 26 to 28, or 30 to 32, comprising:

[0044] EMBODIMENT 35. a. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:7 and the VH sequence of SEQ ID NO:8; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 71 and the VH sequence of SEQ ID NO: 73; b. one of the first and second half antibodies comprises the VL sequence of SEQ ID NO:7 and the VH sequence of SEQ ID NO:8; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 72 and the VH sequence of SEQ ID NO: 73; c. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:7 and the VH sequence of SEQ ID NO:8; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; d. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:21 and the VH sequence of SEQ ID NO:22; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 71 and the VH sequence of SEQ ID NO: 73; e. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:21 and the VH sequence of SEQ ID NO:22; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 72 and the VH sequence of SEQ ID NO: 73; f. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:21 and the VH sequence of SEQ ID NO:22; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; g. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:35 and the VH sequence of SEQ ID NO:36; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 71 and the VH sequence of SEQ ID NO: 73; h. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:35 and the VH sequence of SEQ ID NO:36; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 72 and the VH sequence of SEQ ID NO: 73; i. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:35 and the VH sequence of SEQ ID NO:36; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; j. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:49 and the VH sequence of SEQ ID NO:50; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 71 and the VH sequence of SEQ ID NO: 73; k. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:49 and the VH sequence of SEQ ID NO:50; (i) the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 72 and the VH sequence of SEQ ID NO: 73; or l. one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO:49 and the VH sequence of SEQ ID NO:50; (i) The antibody according to any one of embodiments 13 to 34, wherein the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94.

[0045] Embodiment 36. An antibody described in any one of embodiments 13 to 35, which is a monoclonal antibody.

[0046] Embodiment 37. The antibody described in any one of embodiments 13 to 36, which is a mouse antibody, a rabbit antibody, a human antibody, a humanized antibody, or a chimeric antibody.

[0047] Embodiment 38. An antibody described in any one of embodiments 13 to 37, wherein the first antigen-binding site is human or humanized.

[0048] Embodiment 39. An antibody described in any one of embodiments 13 to 38, wherein the second antigen-binding site is human or humanized.

[0049] Embodiment 40. The antibody described in any one of embodiments 13 to 39, wherein the antibody is an IgG antibody.

[0050] Embodiment 41. The antibody described in any one of embodiments 13 to 40, wherein the antibody is an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody.

[0051] Embodiment 42. The antibody described in any one of embodiments 13 to 41, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

[0052] Embodiment 43. An antibody described in any one of embodiments 13 to 42, wherein the first half antibody comprises a first heavy chain constant region comprising a knob mutation and the second half antibody comprises a second heavy chain constant region comprising a hole mutation; or the first half antibody comprises a first heavy chain constant region comprising a hole mutation and the second half antibody comprises a second heavy chain constant region comprising a knob mutation.

[0053] Embodiment 44. The antibody described in embodiment 43, wherein the antibody is an IgG1 antibody and the knob mutation comprises a T366W mutation.

[0054] Embodiment 45. The antibody described in embodiment 43 or embodiment 44, wherein the antibody is an IgG1 antibody and the hole mutations include at least one, at least two, or three, for example 1 to 2, 1 to 3, or 2 to 3 mutations selected from T366S, L368A, and Y407V.

[0055] Embodiment 46. The antibody of embodiment 43, wherein the antibody is an IgG4 antibody and the knob mutation comprises a T366W mutation.

[0056] Embodiment 47. The antibody according to embodiment 43 or embodiment 46, wherein the antibody is an IgG4 antibody and the hole mutations include at least one, at least two, or three, for example 1 to 2, 1 to 3, or 2 to 3 mutations selected from a T366S mutation, an L368A mutation, and a Y407V mutation.

[0057] Embodiment 48. The first half antibody comprises: a. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2; b. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16; c. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30; or d. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44. 48. The antibody according to any one of embodiments 43 to 47, comprising:

[0058] Embodiment 49. The first half antibody comprises: a. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4; b. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:18; c. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; or d. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46 and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0059] Embodiment 50. The first half antibody comprises: a. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4; b. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:18; c. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; or d. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46. and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0060] Embodiment 51. The first half antibody comprises: a. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; b. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:20; c. a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; or d. A heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48. and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0061]

[0026] Embodiment 52. The first half antibody comprises: a. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; b. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20; c. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; or d. a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48. and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0062] Embodiment 53. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 35 to 52, wherein the second half antibody comprises a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60.

[0063] Embodiment 54. An antibody described in any one of embodiments 13-15, 19-15, 27-29, 31-33, or 35-53, wherein the second half antibody comprises a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 68; and optionally, the C-terminal lysine is deleted from the heavy chain.

[0064] Embodiment 55. The antibody of any one of embodiments 13-15, 19-25, 27-29, 31-33, or 35-54, wherein the second half antibody comprises a light chain sequence having at least about 95%, for example 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60, and a heavy chain sequence having at least 95%, for example 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 68; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0065] Embodiment 56. An antibody described in any one of embodiments 13-15, 19-25, 27-29, 31-33, or 33-53, wherein the second half antibody comprises a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 70; and optionally, the C-terminal lysine is deleted from the heavy chain.

[0066] Embodiment 57. The antibody of any one of embodiments 13-15, 19-25, 27-29, 31-33, 35-53 or 56, wherein the second half antibody comprises a light chain sequence having at least about 95%, for example 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60, and a heavy chain sequence having at least 95%, for example 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 70; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0067] Embodiment 58. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, or 34 to 52, wherein the second half antibody comprises a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 84.

[0068] Embodiment 59. The antibody of any one of embodiments 16-24, 26-28, 30-31, 34-52 or 5528, wherein the second half antibody comprises a heavy chain sequence having at least about 95%, e.g. 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0069] Embodiment 60. The antibody of any one of embodiments 16-24, 26-28, 30-32, 34-52, or 58-59, wherein the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0070] Embodiment 61. The antibody of any one of embodiments 16-24, 26-28, 30-32, 34-52 or 58, wherein the second half antibody comprises a heavy chain sequence having at least about 95%, e.g. 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 92; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0071] Embodiment 62. The antibody of any one of embodiments 16-24, 26-28, 30-32, 33-52, 58 or 61, wherein the second half antibody comprises a light chain sequence having at least about 95%, for example 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 84, and a heavy chain sequence having at least about 95%, for example 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 92; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0072] 63. a. a first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; b. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; c. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; d. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; e. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; f. a strand sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; g. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; h. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48; The second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68, optionally with a C-terminal lysine deleted from one or more heavy chains. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 35 to 57.

[0073] 64. a. a first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; b. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; c. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; d. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; e. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; f. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; g. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; h. the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48; The second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90, optionally with a C-terminal lysine deleted from one or more heavy chains. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, or 58 to 62.

[0074] Embodiment 65. The first half antibody comprises: a. the light chain sequence of SEQ ID NO:2; b. The light chain sequence of SEQ ID NO: 16; c. the light chain sequence of SEQ ID NO: 30; or d. Light chain sequence of SEQ ID NO:44 The antibody according to any one of embodiments 13 to 64, comprising:

[0075]

[0023] Embodiment 66. The first half antibody comprises: a. the heavy chain sequence of SEQ ID NO:4; b. The heavy chain sequence of SEQ ID NO:18; c. the heavy chain sequence of SEQ ID NO:32; or d. Heavy chain sequence of SEQ ID NO:46 and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0076] Embodiment 67. The first half antibody comprises: a. a light chain sequence of SEQ ID NO:2 and a heavy chain sequence of SEQ ID NO:4; b. a light chain sequence of SEQ ID NO:16 and a heavy chain sequence of SEQ ID NO:18; c. a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32; or d. The light chain sequence of SEQ ID NO:44 and the heavy chain sequence of SEQ ID NO:46 and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0077] Embodiment 68. The first half antibody comprises: a. The heavy chain sequence of SEQ ID NO:6; b. The heavy chain sequence of SEQ ID NO:20; c. the heavy chain sequence of SEQ ID NO:34; or d. Heavy chain sequence of SEQ ID NO:48 and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0078] Embodiment 69. The first half antibody comprises: a. a light chain sequence of SEQ ID NO:2 and a heavy chain sequence of SEQ ID NO:6; b. a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20; c. a light chain sequence of SEQ ID NO:30 and a heavy chain sequence of SEQ ID NO:34; or d. The light chain sequence of SEQ ID NO:44 and the heavy chain sequence of SEQ ID NO:48 and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0079] Embodiment 70. The antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 53, 63, or 65 to 69, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 60.

[0080] Embodiment 71. The antibody of any one of embodiments 13-15, 19-25, 27-29, 31-33, 35-54, 63, or 65-70, wherein the second half antibody comprises the heavy chain sequence of SEQ ID NO: 68; and optionally, the C-terminal lysine is deleted from the heavy chain.

[0081] Embodiment 72. The antibody described in any one of embodiments 13-15, 19-25, 27-29, 31-33, 35-55, 63, or 65-71, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0082] Embodiment 73. The antibody of any one of embodiments 13-15, 19-25, 27-29, 31-33, 35-54, 63, or 65-70, wherein the second half antibody comprises the heavy chain sequence of SEQ ID NO: 70; and optionally, the C-terminal lysine is deleted from the heavy chain.

[0083] Embodiment 74. An antibody described in any one of embodiments 13-15, 19-25, 27-29, 31-33, 35-54, 56, 63, 65-70 or 73, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0084] Embodiment 75. The antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58, or 64 to 69, wherein the second half antibody comprises the light chain sequence of SEQ ID NO: 84.

[0085] Embodiment 76. The antibody described in any one of embodiments 16-24, 26-28, 30-32, 34-52, 58-59, 64-69 or 75, wherein the second half antibody comprises a heavy chain sequence of SEQ ID NO: 90; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0086] Embodiment 77. An antibody described in any one of embodiments 16-24, 26-28, 30-32, 34-52, 58-60, 64-69 or 75-76, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0087] Embodiment 78. The antibody of any one of embodiments 16-24, 26-28, 30-32, 34-52, 58, 61, 64-69 or 75, wherein the second half antibody comprises a heavy chain of SEQ ID NO: 92; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0088] Embodiment 79. The antibody described in any one of embodiments 16-24, 26-28, 30-32, 34-52, 58, 61-62, 64-69, 75 or 78, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92; and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0089] EMBODIMENT 80. a. the first half antibody comprises the light chain sequence of SEQ ID NO:2 and the heavy chain sequence of SEQ ID NO:4; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:70; b. the first half antibody comprises the light chain sequence of SEQ ID NO:2 and the heavy chain sequence of SEQ ID NO:6; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:68; c. the first half antibody comprises the light chain sequence of SEQ ID NO: 16 and the heavy chain sequence of SEQ ID NO: 18; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:70; d. the first half antibody comprises the light chain sequence of SEQ ID NO: 16 and the heavy chain sequence of SEQ ID NO: 20; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:68; e. the first half antibody comprises the light chain sequence of SEQ ID NO:30 and the heavy chain sequence of SEQ ID NO:32; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:70; f. the first half antibody comprises the light chain sequence of SEQ ID NO:30 and the heavy chain sequence of SEQ ID NO:34; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:68; g. the first half antibody comprises the light chain sequence of SEQ ID NO:44 and the heavy chain sequence of SEQ ID NO:46; the second half antibody comprises the light chain sequence of SEQ ID NO:60 and the heavy chain sequence of SEQ ID NO:70; h. the first half antibody comprises the light chain sequence of SEQ ID NO:44 and the heavy chain sequence of SEQ ID NO:48; The antibody of any one of embodiments 16-15, 19-25, 27-29, 31-33, 35-57, 63, or 65-74, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0090] 81. a. the first half antibody comprises the light chain sequence of SEQ ID NO:2 and the heavy chain sequence of SEQ ID NO:4; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:92; b. the first half antibody comprises the light chain sequence of SEQ ID NO:2 and the heavy chain sequence of SEQ ID NO:6; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:90; c. the first half antibody comprises the light chain sequence of SEQ ID NO: 16 and the heavy chain sequence of SEQ ID NO: 18; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:92; d. the first half antibody comprises the light chain sequence of SEQ ID NO: 16 and the heavy chain sequence of SEQ ID NO: 20; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:90; e. the first half antibody comprises the light chain sequence of SEQ ID NO:30 and the heavy chain sequence of SEQ ID NO:32; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:92; f. a chain sequence of SEQ ID NO:30 and a heavy chain sequence of SEQ ID NO:34; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:90; g. the first half antibody comprises the light chain sequence of SEQ ID NO:44 and the heavy chain sequence of SEQ ID NO:46; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:92; h. the first half antibody comprises the light chain sequence of SEQ ID NO:44 and the heavy chain sequence of SEQ ID NO:48; the second half antibody comprises the light chain sequence of SEQ ID NO: 84 and the heavy chain sequence of SEQ ID NO: 90; 80. The antibody of any one of embodiments 16-24, 26-28, 30-32, 34-52, 58-62, 64-69, or 75-79, optionally having a C-terminal lysine deleted from one or more heavy chains.

[0091] Embodiment 82. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 4, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0092] Embodiment 83. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 6, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0093] Embodiment 84. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0094] Embodiment 85. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0095] Embodiment 86. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0096] Embodiment 87. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0097] Embodiment 88. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0098] Embodiment 89. An antibody described in any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48, and a second half antibody comprises a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0099] Embodiment 90. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 4, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0100] Embodiment 91. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 2 and a heavy chain sequence of SEQ ID NO: 6, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0101] Embodiment 92. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0102] Embodiment 93. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0103] Embodiment 94. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, and optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0104] Embodiment 95. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0105] Embodiment 96. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0106] Embodiment 97. An antibody described in any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein a first half antibody comprises a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48, and a second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, and optionally a C-terminal lysine is deleted from one or more heavy chains.

[0107] Embodiment 98. An antibody described in any one of embodiments 13 to 97, which is a bispecific antibody.

[0108] Embodiment 99. The antibody described in any one of embodiments 13 to 98, which is a diabody, triabody, or tetrabody.

[0109] Embodiment 100. An antibody described in any one of embodiments 13 to 99, which is conjugated to a label.

[0110] Embodiment 101. The antibody described in embodiment 100, wherein the label is a fluorescent label, an enzymatic label, or a chromogenic label.

[0111] Embodiment 102. The label is a radioisotope, optionally a positron emitter, and optionally 89 The antibody of embodiment 100, which is Zr.

[0112] Embodiment 103. A composition comprising an antibody described in any one of embodiments 13 to 102, wherein the composition is substantially free of monospecific antibodies, disassembled half antibodies, or both monospecific antibodies and disassembled half antibodies.

[0113] Embodiment 104. An immunoconjugate comprising the antibody of any one of embodiments 13 to 102 and a cytotoxic agent or an anti-inflammatory agent.

[0114] Embodiment 105. A pharmaceutical composition comprising: a) an antibody according to any one of embodiments 13 to 102; or b) An immunoconjugate according to embodiment 104 and at least one of: b. Optionally, the pharmaceutical formulation, wherein the composition is substantially free of monospecific antibodies, disassembled half antibodies, or both monospecific antibodies and disassembled half antibodies.

[0115] Embodiment 106. The pharmaceutical formulation of embodiment 105, further comprising an additional therapeutic agent.

[0116] Embodiment 107. An isolated nucleic acid encoding an antibody described in any one of embodiments 13 to 102.

[0117] Embodiment 108. A vector comprising the nucleic acid described in embodiment 107.

[0118] Embodiment 109. A host cell comprising the nucleic acid described in embodiment 107.

[0119] Embodiment 110. A method for producing an antibody, comprising culturing a host cell according to embodiment 106 under conditions in which the antibody is produced.

[0120] Embodiment 111. The method of embodiment 110, further comprising recovering the antibody from the host cell.

[0121] Embodiment 112. A method for producing an antibody described in any one of embodiments 13 to 102, comprising forming an antibody from a first half antibody and a second half antibody.

[0122] Embodiment 113. An antibody described in any one of embodiments 13 to 102 for use as a medicament.

[0123] Embodiment 114. A method for determining the presence of a polyubiquitinated protein in a sample suspected of containing polyubiquitin or a polyubiquitinated protein, comprising exposing the sample to an antibody described in any of embodiments 13 to 102 and determining binding of the antibody to the polyubiquitinated protein in the sample.

[0124] Embodiment 115. A method for separating K11-linked polyubiquitinated proteins from non-K11-linked polyubiquitinated proteins in a sample, the method comprising contacting the sample with an antibody described in any of embodiments 13 to 102.

[0125] Embodiment 116. A method for separating K48-linked polyubiquitinated proteins from non-K48-linked polyubiquitinated proteins in a sample, the method comprising contacting the sample with an antibody described in any one of embodiments 13 to 102.

[0126] Embodiment 117. A method for separating K63-linked polyubiquitinated proteins from non-K63-linked polyubiquitinated proteins in a sample, the method comprising contacting the sample with an antibody described in any one of embodiments 13 to 102.

[0127] Embodiment 118. A method for separating M1-linked polyubiquitinated proteins from non-M1-linked polyubiquitinated proteins in a sample, comprising contacting the sample with an antibody described in any of embodiments 13 to 102.

[0128] Embodiment 119. A method for determining the function and / or activity of a polyubiquitinated protein in a cell or sample, comprising contacting the cell or sample with an antibody described in any of embodiments 13 to 102 and evaluating the effect of the contacting step on the cell or sample.

[0129] Embodiment 120. A method according to any one of embodiments 114 to 119, wherein the polyubiquitinated protein comprises RIP1.

[0130] Embodiment 121. A method according to any one of embodiments 114 to 119, wherein the polyubiquitinated protein comprises RIP2.

[0131] Embodiment 122. A method for determining the presence of a polyubiquitinated protein in a sample suspected of containing a polyubiquitinated protein, wherein the polyubiquitinated protein is a pro-inflammatory protein and contains polyubiquitin, the method comprising exposing the sample to an antibody described in any of embodiments 10 to 99.

[0132] Embodiment 123. The method of embodiment 122, wherein the polyubiquitinated protein comprises M1-linked polyubiquitin and / or K63-linked polyubiquitin.

[0133] Embodiment 124. The method of embodiment 122 or 123, wherein the proinflammatory protein is a component of one or more signal transduction complexes.

[0134] Embodiment 125. A method according to any one of embodiments 122 to 124, wherein the pro-inflammatory protein is RIP1.

[0135] Embodiment 126. A method according to any one of embodiments 122 to 125, wherein the pro-inflammatory protein is RIP2.

[0136] Embodiment 127. A method according to any one of embodiments 122 to 126, wherein the pro-inflammatory protein has an elevated ubiquitination level in an inflammatory state compared to the ubiquitination level in the absence of an inflammatory state.

[0137] Embodiment 128. The method of any one of embodiments 122 to 127, wherein the pro-inflammatory protein has a ubiquitination level of at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 1-fold to 12-fold, 2-fold to 12-fold, 3-fold to 12-fold, 4-fold to 12-fold, 5-fold to 12-fold, 6-fold to 12-fold, 7-fold to 12-fold, 8-fold to 12-fold, 9-fold to 12-fold, 10-fold to 12-fold, or 11-fold to 12-fold in an inflammatory state compared to the ubiquitination level in the absence of an inflammatory state.

[0138] Embodiment 129. The method of any one of embodiments 122 to 128, wherein increased levels of ubiquitination correlate with increased severity of the inflammatory disease condition.

[0139] Embodiment 130. The method of any one of embodiments 122 to 129, wherein the pro-inflammatory protein is associated with an inflammatory disease, such as inflammatory bowel disease, Crohn's disease, diverticulitis, and ulcerative colitis.

[0140] Embodiment 131. The method of any one of embodiments 122 to 130, wherein the pro-inflammatory protein is associated with Crohn's disease.

[0141] Embodiment 132. The method of any one of embodiments 122 to 131, wherein the pro-inflammatory protein is associated with ulcerative colitis. [Brief description of the drawings]

[0142] [Figure 1A-1B]

[0010] Figures 1A-D show the interaction of RIP1-K63 bispecific antibodies with K63 ubiquitin-linked modified RIP1. Figures 1A and 1B provide Western blot analysis to determine the ability of the indicated antibodies to immunoprecipitate K63-chain ubiquitinated and linear chain ubiquitinated recombinant RIP1 proteins in vitro. Figures 1C and 1D provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP1 KO (knockout) HT29 cells. The indicated antibodies recognize RIP1 modified with K63-linked ubiquitin chains. [Fig. 1C-1D] Figures 1A-D show the interaction of RIP1-K63 bispecific antibodies with K63 ubiquitin-linked modified RIP1. Figures 1A and 1B provide Western blot analysis to determine the ability of the indicated antibodies to immunoprecipitate K63-chain ubiquitinated and linear-chain ubiquitinated recombinant RIP1 proteins in vitro. Figures 1C and 1D provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP1 KO (knockout) HT29 cells. The indicated antibodies recognize RIP1 modified with K63-linked ubiquitin chains. [Figure 2A-2B]

[0011] Figures 2A-D show the interaction of RIP1 ubiquitin chain bispecific antibody or control antibody in various cell lines including human colon carcinoma HT29 cells, fibrosarcoma HT1080 cells, and A549 cells. Specifically, Figures 2A-D show that RIP1-K63 ubiquitin chain bispecific antibody recognizes K63-chain ubiquitinated RIP1. Figures 2A and 2B provide Western blot analysis of total cell lysates (TCL) and immunoprecipitated proteins (IP) obtained with the indicated antibodies. The indicated antibodies recognize K63-chain ubiquitinated RIP1. These results were confirmed by confocal microscopy, as shown in Figure 2C (A549 cells) and Figure 2D (HT1080 cells). [Fig. 2C-2D] Figures 2A-D show the interaction of RIP1 ubiquitin chain bispecific antibody or control antibody in various cell lines including human colon carcinoma HT29 cells, fibrosarcoma HT1080 cells, and A549 cells. Specifically, Figures 2A-D show that RIP1-K63 ubiquitin chain bispecific antibody recognizes K63-chain ubiquitinated RIP1. Figures 2A and 2B provide Western blot analysis of total cell lysates (TCL) and immunoprecipitated proteins (IP) obtained with the indicated antibodies. The indicated antibodies recognize K63-chain ubiquitinated RIP1. These results were confirmed by confocal microscopy, as shown in Figure 2C (A549 cells) and Figure 2D (HT1080 cells). [Figure 3A-3B]

[0012] Figures 3A-D show the interaction of RIP1-K63 bispecific antibodies with RIP1 modified with K63-linked ubiquitin chains in various cell lines including EVSA T, Ku812F, and HT29 cells. Figures 3A and 3B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 modified with K63-linked ubiquitin chains. Figures 3C and 3D provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP1 KO (knockout) HT29 cells. The indicated antibodies recognize linear chain ubiquitinated RIP1. [Figure 3C-3D]Figures 3A-D show the interaction of RIP1-K63 bispecific antibodies with RIP1 modified with K63-linked ubiquitin chains in various cell lines including EVSA T, Ku812F, and HT29 cells. Figures 3A and 3B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 modified with K63-linked ubiquitin chains. Figures 3C and 3D provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP1 KO (knockout) HT29 cells. The indicated antibodies recognize linear chain ubiquitinated RIP1. [Figure 4A-4B]

[0013] Figures 4A-D show the interaction of RIP1 ubiquitin, K63 linear ubiquitin chain or control bispecific antibody in various cell lines including human colon cancer HT29 cells, Ku812F cells and mouse embryonic fibroblast (MEF) cells. Specifically, Figures 4A-D show that RIP1-Lin ubiquitin chain bispecific antibody recognizes linear chain ubiquitinated RIP1, whereas RIP1-gD and gD-Lin antibodies, or K63-Lin antibody without stimulation, did not immunoprecipitate ubiquitinated RIP1. Figures 4A and 4B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize linear chain ubiquitinated RIP1. These results were confirmed by confocal microscopy, as shown in Figure 4C (HT29 cells) and Figure 4D (MEF cells). [Fig. 4C-4D]Figures 4A-D show the interaction of RIP1 ubiquitin, K63 linear ubiquitin chain or control bispecific antibody in various cell lines including human colon carcinoma HT29 cells, Ku812F cells, and mouse embryonic fibroblast (MEF) cells. Specifically, Figures 4A-D show that RIP1-Lin ubiquitin chain bispecific antibody recognizes linear chain ubiquitinated RIP1, whereas RIP1-gD and gD-Lin antibodies, or K63-Lin antibody without stimulation, did not immunoprecipitate ubiquitinated RIP1. Figures 4A and 4B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize linear chain ubiquitinated RIP1. These results were confirmed by confocal microscopy, as shown in Figure 4C (HT29 cells) and Figure 4D (MEF cells). [Figure 5A-5B]

[0014] Figures 5A-C show the interaction of RIP1 ubiquitin chains, K63 linear ubiquitin chains or control bispecific antibodies in various cell lines including human colon cancer HT29 cells, D645 cells and THP1 cells. Figures 5A, 5B and 5C provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 modified with K63-linked and K63-Lin ubiquitin-linked chains. [Figure 5C] Figures 5A-C show the interaction of RIP1 ubiquitin chains, K63 linear ubiquitin chains or control bispecific antibodies in various cell lines including human colon carcinoma HT29, D645 and THP1 cells. Figures 5A, 5B and 5C provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 modified with K63-linked and K63-Lin ubiquitin-linked chains. [Figure 6A-6B]

[0015] Figures 6A-D show the interaction of RIP1 ubiquitin chains, K63 linear ubiquitin chains or a control bispecific antibody in mouse tissue samples. Figures 6A and 6B provide Western blot analysis of TCLs and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 ubiquitinated in vivo with K63-linked and linear ubiquitin chains. Figures 6C and 6D provide Western blot analysis of immunoprecipitated proteins obtained with the indicated antibodies and TCLs. The indicated antibodies recognize ubiquitinated RIP1 in wild-type (WT) or mutant RIP1 (RIP1 K376R knock-in) mouse bone marrow-derived macrophages. [Figure 6C-6D] Figures 6A-D show the interaction of RIP1 ubiquitin chains, K63 linear ubiquitin chains or a control bispecific antibody in mouse tissue samples. Figures 6A and 6B provide Western blot analysis of TCLs and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 ubiquitinated in vivo with K63-linked and linear ubiquitin chains. Figures 6C and 6D provide Western blot analysis of immunoprecipitated proteins obtained with the indicated antibodies and TCLs. The indicated antibodies recognize ubiquitinated RIP1 in wild-type (WT) or mutant RIP1 (RIP1 K376R knock-in) mouse bone marrow-derived macrophages. [Figure 7A]

[0016] Figures 7A-E show the interaction of RIP2-ubiquitin chain or control bispecific antibody in THP1 cells. Specifically, Figures 7A-D show that RIP2-ubiquitin chain bispecific antibody recognizes K63 and linear chain ubiquitinated RIP2, whereas single arm (RIP2-gD, K63-gD, Lin-gD) antibody does not recognize ubiquitinated RIP2, and even RIP2-K63, RIP2-Lin and K63-Lin bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway-related stimuli (MDP). Figures 7A and 7B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. FIG. 7C provides a Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 ubiquitinated with linear and K63-Lin ubiquitin-linked chains. The indicated antibodies recognize K63 and linear chain ubiquitinated RIP2. FIG. 7D provides a Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 modified with K63-linked ubiquitin chains. These results were confirmed by confocal microscopy, as shown in FIG. 7E. [Figure 7B]Figures 7A-E show the interaction of RIP2-ubiquitin chain or control bispecific antibodies in THP1 cells. Specifically, Figures 7A-D show that RIP2-ubiquitin chain bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2, whereas single arm (RIP2-gD, K63-gD, Lin-gD) antibodies do not recognize ubiquitinated RIP2, and even RIP2-K63, RIP2-Lin and K63-Lin bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway-related stimuli (MDP). Figures 7A and 7B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. Figure 7C provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 ubiquitinated with linear and K63-Lin ubiquitin-linked chains. The indicated antibodies recognize K63 and linear chain ubiquitinated RIP2. Figure 7D provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 modified with K63-linked ubiquitin chains. These results were confirmed by confocal microscopy, as shown in Figure 7E. [Figure 7C]Figures 7A-E show the interaction of RIP2-ubiquitin chain or control bispecific antibodies in THP1 cells. Specifically, Figures 7A-D show that RIP2-ubiquitin chain bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2, whereas single arm (RIP2-gD, K63-gD, Lin-gD) antibodies do not recognize ubiquitinated RIP2, and even RIP2-K63, RIP2-Lin and K63-Lin bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway-related stimuli (MDP). Figures 7A and 7B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. Figure 7C provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 ubiquitinated with linear and K63-Lin ubiquitin-linked chains. The indicated antibodies recognize K63 and linear chain ubiquitinated RIP2. Figure 7D provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 modified with K63-linked ubiquitin chains. These results were confirmed by confocal microscopy, as shown in Figure 7E. [Figure 7D]Figures 7A-E show the interaction of RIP2-ubiquitin chain or control bispecific antibodies in THP1 cells. Specifically, Figures 7A-D show that RIP2-ubiquitin chain bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2, whereas single arm (RIP2-gD, K63-gD, Lin-gD) antibodies do not recognize ubiquitinated RIP2, and even RIP2-K63, RIP2-Lin and K63-Lin bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway-related stimuli (MDP). Figures 7A and 7B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. Figure 7C provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 ubiquitinated with linear and K63-Lin ubiquitin-linked chains. The indicated antibodies recognize K63 and linear chain ubiquitinated RIP2. Figure 7D provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 modified with K63-linked ubiquitin chains. These results were confirmed by confocal microscopy, as shown in Figure 7E. [Figure 7E]Figures 7A-E show the interaction of RIP2-ubiquitin chain or control bispecific antibodies in THP1 cells. Specifically, Figures 7A-D show that RIP2-ubiquitin chain bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2, whereas single arm (RIP2-gD, K63-gD, Lin-gD) antibodies do not recognize ubiquitinated RIP2, and even RIP2-K63, RIP2-Lin and K63-Lin bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway-related stimuli (MDP). Figures 7A and 7B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. Figure 7C provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 ubiquitinated with linear and K63-Lin ubiquitin-linked chains. The indicated antibodies recognize K63 and linear chain ubiquitinated RIP2. Figure 7D provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 modified with K63-linked ubiquitin chains. These results were confirmed by confocal microscopy, as shown in Figure 7E. [Figure 8A]

[0017] Figures 8A-G show RIP2-K63 and RIP2-Lin bispecific antibodies tested in intestinal tissue samples from patients. Specifically, Figures 8A-F show that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to examine the ubiquitination status of RIP2 in IBD samples. Figures 8A and 8B provide Western blot analysis to determine the ability of the indicated antibodies to detect immunoprecipitated proteins from samples taken from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC). Figures 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient in Figure 8A. Figure 8E shows the expression of RIP2 in patient samples. Figure 8F provides quantification of RIP2 ubiquitination by scanning Western blots after immunoprecipitation with the antibodies shown in Figures 8C and 8D. FIG. 8G shows the data from FIG. 8F in bar graph format. [Figure 8B] Figures 8A-G show RIP2-K63 and RIP2-Lin bispecific antibodies tested in intestinal tissue samples from patients. Specifically, Figures 8A-F show that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to examine the ubiquitination status of RIP2 in IBD samples. Figures 8A and 8B provide Western blot analysis to determine the ability of the indicated antibodies to detect immunoprecipitated proteins from samples taken from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC). Figures 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient in Figure 8A. Figure 8E shows the expression of RIP2 in patient samples. Figure 8F provides quantification of RIP2 ubiquitination by scanning Western blots after immunoprecipitation with the antibodies shown in Figures 8C and 8D. Figure 8G shows the data from Figure 8F in bar graph format. [Figure 8C]Figures 8A-G show RIP2-K63 and RIP2-Lin bispecific antibodies tested in intestinal tissue samples from patients. Specifically, Figures 8A-F show that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to examine the ubiquitination status of RIP2 in IBD samples. Figures 8A and 8B provide Western blot analysis to determine the ability of the indicated antibodies to detect immunoprecipitated proteins from samples taken from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC). Figures 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient in Figure 8A. Figure 8E shows the expression of RIP2 in patient samples. Figure 8F provides quantification of RIP2 ubiquitination by scanning Western blots after immunoprecipitation with the antibodies shown in Figures 8C and 8D. Figure 8G shows the data from Figure 8F in bar graph format. [Figure 8D] Figures 8A-G show RIP2-K63 and RIP2-Lin bispecific antibodies tested in intestinal tissue samples from patients. Specifically, Figures 8A-F show that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to examine the ubiquitination status of RIP2 in IBD samples. Figures 8A and 8B provide Western blot analysis to determine the ability of the indicated antibodies to detect immunoprecipitated proteins from samples taken from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC). Figures 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient in Figure 8A. Figure 8E shows the expression of RIP2 in patient samples. Figure 8F provides quantification of RIP2 ubiquitination by scanning Western blots after immunoprecipitation with the antibodies shown in Figures 8C and 8D. Figure 8G shows the data from Figure 8F in bar graph format. [Fig. 8E-8F]Figures 8A-G show RIP2-K63 and RIP2-Lin bispecific antibodies tested in intestinal tissue samples from patients. Specifically, Figures 8A-F show that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to examine the ubiquitination status of RIP2 in IBD samples. Figures 8A and 8B provide Western blot analysis to determine the ability of the indicated antibodies to detect immunoprecipitated proteins from samples taken from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC). Figures 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient in Figure 8A. Figure 8E shows the expression of RIP2 in patient samples. Figure 8F provides quantification of RIP2 ubiquitination by scanning Western blots after immunoprecipitation with the antibodies shown in Figures 8C and 8D. Figure 8G shows the data from Figure 8F in bar graph format. [Figure 8G] Figures 8A-G show RIP2-K63 and RIP2-Lin bispecific antibodies tested in intestinal tissue samples from patients. Specifically, Figures 8A-F show that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to examine the ubiquitination status of RIP2 in IBD samples. Figures 8A and 8B provide Western blot analysis to determine the ability of the indicated antibodies to detect immunoprecipitated proteins from samples taken from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC). Figures 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient in Figure 8A. Figure 8E shows the expression of RIP2 in patient samples. Figure 8F provides quantification of RIP2 ubiquitination by scanning Western blots after immunoprecipitation with the antibodies shown in Figures 8C and 8D. Figure 8G shows the data from Figure 8F in bar graph format. [Figure 9A-9B]

[0018] Figures 9A-E show how the K63-Lin bispecific antibody can be used to detect and identify K63-linked and linear ubiquitinated proteins in different signaling pathways. Figure 9A provides a schematic of the experimental design scheme, where THP1 cells were treated with vehicle (phosphate buffered saline, PBS), tumor necrosis factor (TNF), muramyl dipeptide (MDP), or lipopolysaccharide (LPS) for the indicated time periods, then lysed, immunoprecipitated with the indicated bispecific antibodies, and analyzed by mass spectrometry (MS), identifying the proteins in Figure 9B, which were also determined by Western blot analysis in Figure 9C. Ubiquitinated RIP2 was identified from the MDP-treated condition by tandem mass spectrometry (ms / ms, Figure 9D) and extracted ion chromatography (XIC), with the matching peaks highlighted by arrows (Figure 9E). [Figure 9C] Figures 9A-E show how the K63-Lin bispecific antibody can be used to detect and identify K63-linked and linear chain ubiquitinated proteins in different signaling pathways. Figure 9A provides a schematic of the experimental design scheme, where THP1 cells were treated with vehicle (phosphate buffered saline, PBS), tumor necrosis factor (TNF), muramyl dipeptide (MDP), or lipopolysaccharide (LPS) for the indicated time periods, then lysed, immunoprecipitated with the indicated bispecific antibodies, and analyzed by mass spectrometry (MS), identifying proteins in Figure 9B that were also determined by Western blot analysis in Figure 9C. Ubiquitinated RIP2 was identified from the MDP-treated condition by tandem mass spectrometry (ms / ms, Figure 9D) and extracted ion chromatography (XIC), with matching peaks highlighted by arrows (Figure 9E). [Figure 9D]Figures 9A-E show how the K63-Lin bispecific antibody can be used to detect and identify K63-linked and linear chain ubiquitinated proteins in different signaling pathways. Figure 9A provides a schematic of the experimental design scheme, where THP1 cells were treated with vehicle (phosphate buffered saline, PBS), tumor necrosis factor (TNF), muramyl dipeptide (MDP), or lipopolysaccharide (LPS) for the indicated time periods, then lysed, immunoprecipitated with the indicated bispecific antibodies, and analyzed by mass spectrometry (MS), identifying proteins in Figure 9B that were also determined by Western blot analysis in Figure 9C. Ubiquitinated RIP2 was identified from the MDP-treated condition by tandem mass spectrometry (ms / ms, Figure 9D) and extracted ion chromatography (XIC), with matching peaks highlighted by arrows (Figure 9E). [Figure 9E] Figures 9A-E show how the K63-Lin bispecific antibody can be used to detect and identify K63-linked and linear chain ubiquitinated proteins in different signaling pathways. Figure 9A provides a schematic of the experimental design scheme, where THP1 cells were treated with vehicle (phosphate buffered saline, PBS), tumor necrosis factor (TNF), muramyl dipeptide (MDP), or lipopolysaccharide (LPS) for the indicated time periods, then lysed, immunoprecipitated with the indicated bispecific antibodies, and analyzed by mass spectrometry (MS), identifying proteins in Figure 9B that were also determined by Western blot analysis in Figure 9C. Ubiquitinated RIP2 was identified from the MDP-treated condition by tandem mass spectrometry (ms / ms, Figure 9D) and extracted ion chromatography (XIC), with matching peaks highlighted by arrows (Figure 9E). [Figure 10A-10B]

[0019] Figures 10A-E show the identification of TRADD (Figure 10A) (SEQ ID NO: 104), TNFR1 (Figure 10B) (SEQ ID NO: 105), RIP1 (Figure 10C) (SEQ ID NO: 106), NOD2 (Figure 10D) (SEQ ID NO: 107) and IRAK1 (Figure 10E) (SEQ ID NO: 108) from the conditions shown in Figures 9A and 9B. Representative ms / ms spectra for each protein are annotated. [Fig. 10C-10D] Figures 10A-E show the identification of TRADD (Figure 10A) (SEQ ID NO: 104), TNFR1 (Figure 10B) (SEQ ID NO: 105), RIP1 (Figure 10C) (SEQ ID NO: 106), NOD2 (Figure 10D) (SEQ ID NO: 107) and IRAK1 (Figure 10E) (SEQ ID NO: 108) from the conditions shown in Figures 9A and 9B. Representative ms / ms spectra for each protein are annotated. [Figure 10E] Figures 10A-E show the identification of TRADD (Figure 10A) (SEQ ID NO: 104), TNFR1 (Figure 10B) (SEQ ID NO: 105), RIP1 (Figure 10C) (SEQ ID NO: 106), NOD2 (Figure 10D) (SEQ ID NO: 107) and IRAK1 (Figure 10E) (SEQ ID NO: 108) from the conditions shown in Figures 9A and 9B. Representative ms / ms spectra for each protein are annotated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0143] Detailed Description of Specific Embodiments I. Definition As used herein, VH refers to a heavy chain variable domain and VL refers to a light chain variable domain.

[0144]

[0021] For purposes herein, an "acceptor human framework" is a framework that comprises the amino acid sequence of a VL framework or a VH framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0145]

[0022] As used herein, "about" refers to a value that is 10% more or less than the stated value, a value that gives a functionally equivalent result to the stated value, or a value that is rounded to the stated value.

[0146]

[0023] "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 inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Certain specific and exemplary embodiments for measuring binding affinity are described below.

[0147]

[0024] "Avidity" refers to the strength of the sum of non-covalent interactions between a molecule (e.g., an antibody) and its binding partner (e.g., a target molecule containing one or more antigens). The avidity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). A bispecific antibody generally has a higher avidity for a binding partner that contains an epitope recognized by both of the antigen-binding sites of the bispecific antibody than a binding partner that contains either epitope individually. Avidity can be measured by common methods known in the art, including those described herein. Certain specific and exemplary embodiments for measuring binding avidity are described below. The term "functional affinity" is sometimes used in the art to refer to avidity.

[0148]

[0025] An "affinity matured" antibody refers to an antibody that has one or more modifications in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have any modifications, and such modifications improve the affinity of the antibody for its antigen.

[0149]

[0026] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. As used herein, the term "multispecific antibody" encompasses antibodies that contain antigen-binding domains that have multi-epitope specificity (i.e., capable of binding to two or more different epitopes on one biological molecule, or capable of binding to epitopes on two or more different molecules).

[0150]

[0027] As used herein, an "agonist antibody" is an antibody that mimics at least one of the functional activities of a polypeptide of interest.

[0151]

[0028] An "antagonist antibody" or "blocking antibody" is an antibody that inhibits or reduces the biological activity of an antigen to which it specifically binds. Particular blocking or antagonist antibodies substantially or completely inhibit the biological activity of an antigen.

[0152]

[0029] As used herein, the term "antibody drug conjugate" (ADC) is equivalent to the term "immunoconjugate."

[0153]

[0030] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0154] An "antibody that binds 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.

[0155]

[0032] As used herein, the term "anti-polyubiquitin antibody" refers to an antibody that can specifically bind to a polyubiquitin molecule. In certain embodiments, an antibody that binds to polyubiquitin has a specific binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0156]

[0033] As used herein, the terms "anti-ubiquitin antibody" and "anti-monoubiquitin antibody" are used interchangeably and refer to an antibody capable of specifically binding to a ubiquitin molecule.

[0157] As used herein, the term "anti-RIP1 antibody" refers to an antibody that can specifically bind to a receptor interacting protein kinase 1 ("receptor interacting protein-1 kinase", "RIP1" or "RIPK1") molecule. In certain embodiments, an antibody that binds to RIP1 has a specific binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, for example 10 -9 M~10 -13 M, for example, 1 to 20 nM, for example, 1 to 15 nM, for example, 1 to 12 nM, for example, 1 to 10 nM).

[0158] As used herein, the term "anti-RIP2 antibody" refers to an antibody that can specifically bind to a receptor interacting protein kinase 2 ("receptor interacting serine / threonine protein kinase 2", "RIP2" or "RIPK2") molecule. In certain embodiments, an antibody that binds to RIP2 has a specific binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 ~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0159]

[0036] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, 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, cancer of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal 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, and various types of head and neck cancer.

[0160]

[0037] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular origin or species, and the remaining portions of the heavy and / or light chain are derived from a different origin or species.

[0161]

[0038] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0162]

[0039] 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 radioisotopes (e.g., 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); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents as disclosed below.

[0163] "Effector functions" refer to biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0164]

[0041] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0165]

[0042] The term "epitope" refers to a specific site on an antigen molecule to which an antibody binds.

[0166]

[0043] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within an Fc region or constant region follows EU numbering, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0167] As used herein, "first", "second", etc. are used in reference to elements of a complex structure, e.g., a protein having tertiary / quaternary structure such as an antibody, and refer to those elements (e.g., monomers, chains, domains) without any sense as to the order or positioning of the elements; thus, a "first" element may be C-terminal or N-terminal to a second element, or may be closer or further from one end or another of the structure than the second element. Thus, for example, with respect to halves of a bispecific antibody, the designation of the halves or bonds as first or second is arbitrary.

[0168] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences generally appear in VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0169]

[0046] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or an antibody having a heavy chain that includes an Fc region as defined herein.

[0170]

[0047] The term "half antibody" is used herein to refer to one arm of an antibody and contains at least one VH domain and one CH domain.

[0171]

[0048] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably herein and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0172] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody derived from a non-human source that utilizes the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0173]

[0050] A "rabbit antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a rabbit or a rabbit cell, or an antibody derived from a non-rabbit source that utilizes a rabbit antibody repertoire or other rabbit antibody-encoding sequences.

[0174]

[0051] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In some embodiments, for VL, the subgroup is subgroup kappa I as in Kabat et al. (see above). In some embodiments, for VH, the subgroup is subgroup III as in Kabat et al. (see above).

[0175]

[0052] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, e.g., one or two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to a non-human antibody and all or substantially all of the FRs correspond to a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been subjected to humanization.

[0176]

[0053] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, a natural four-chain antibody comprises six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur 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) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35B of H1, amino acid residues 50-65 of H2 and amino acid residues 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 in VH, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining regions", or "SDRs", which are residues that contact the antigen. SDRs are contained within regions of CDRs called truncated 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) occur at amino acid residues 31-34 of L1, amino acid residues 50-55 of L2, amino acid residues 89-96 of L3, amino acid residues 31-35B of H1, amino acid residues 50-58 of H2 and amino acid residues 95-102 of H3.(See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0177]

[0054] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent. Immunoconjugates are equivalent to the term "antibody drug conjugates" (ADCs).

[0178] An "individual" or "patient" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is human.

[0179]

[0056] "Inflammatory diseases" include Crohn's disease, diverticulitis, graft-versus-host disease (GVHD), inflammatory bowel disease, renal injury and delayed organ transplant function, multiple sclerosis, rheumatoid arthritis, inflammatory skin diseases, stroke and ulcerative colitis.

[0180]

[0057] An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0181]

[0058] "Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0182]

[0059] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., including naturally occurring mutations or arising during the production of a monoclonal antibody preparation, such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which usually contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced 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 as those and other exemplary methods for producing monoclonal antibodies described herein.

[0183]

[0060] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0184]

[0061] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the 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 the variable light domain or the light chain variable domain, followed by a constant light domain (CL). The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0185]

[0062] "Or" is used in its inclusive sense, ie equivalent to "and / or", unless the context requires otherwise.

[0186]

[0063] "Percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps as necessary. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to obtain maximum alignment over the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted together with user documentation to the U.S. Copyright Office, Washington DC, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from its source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0187]

[0064] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (or in other words, a given amino acid sequence A having or containing a particular % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y

[0188]

[0065] where X is the number of amino acid residues scored as identical by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then 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 specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0189]

[0066] The term "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredient contained in the preparation is effective and that does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0190]

[0067] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, additives, stabilizers, or preservatives.

[0191]

[0068] As used herein, "selectively recognizes" means that the referenced antibody does not bind to proteins other than the polyubiquitinated protein of interest, or binds to other proteins with substantially weaker affinity (e.g., 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold weaker affinity).

[0192]

[0069] As used herein, "substantially free of" means that the referenced entity is not present or, if present, (i) is present in an amount sufficiently small so as not to significantly alter the functional properties or results of the composition, method, use, or process, as the case may be; (ii) is not detectable by at least one appropriate analytical method such as mass spectrometry (e.g., MALDI-TOF or any of the MS procedures used in the Examples), blotting (e.g., Western blot of polypeptides), or electrophoresis (e.g., SDS-PAGE with Coomassie blue or silver staining); or (iii) is present in an amount of about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% or less, by mass or molar fraction, relative to the total amount of non-solvent materials in the composition.

[0193] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and remission or improving prognosis. In some embodiments, the antibodies disclosed herein are used to delay the onset of disease or to slow the progression of disease.

[0194]

[0071] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., W. H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated using the VH or VL domain from an antibody that binds the antigen and screening a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0195]

[0072] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0196] II. Compositions and Methods

[0073] In some aspects, antibodies are provided that bind to polyubiquitinated proteins, such as polyubiquitinated inflammatory proteins. Such antibodies are useful, for example, for detecting, modulating the activity, or immunoprecipitating polyubiquitinated proteins.

[0197] A. Exemplary Antibodies In some embodiments, the multispecific antibody comprises a first half antibody comprising a first antigen-binding site that binds polyubiquitin; and a second half antibody comprising a second antigen-binding site that binds receptor interacting protein kinase 1 (RIP1). In some embodiments, the antibody selectively recognizes polyubiquitinated RIP1. In some embodiments, the antibody does not recognize receptor interacting protein kinase 2 (RIP2) and / or does not recognize unubiquitinated RIP1.

[0198] In some embodiments, the multispecific antibody comprises a first half antibody comprising a first antigen-binding site that binds polyubiquitin; and a second half antibody comprising a second antigen-binding site that binds receptor interacting protein kinase 2 (RIP2). In some embodiments, the antibody selectively recognizes polyubiquitinated RIP2. In some embodiments, the antibody does not recognize receptor interacting protein kinase 1 (RIP1) and / or does not recognize unubiquitinated RIP2.

[0199] In some embodiments, the polyubiquitin comprises a K11, K48, K63, or M1 (C-terminus to N-terminus) linkage. In some embodiments, the polyubiquitin has a uniform topology.

[0200] The antibodies, antibody sequences, and sequence listing disclosed in US Patent No. 7,763,245 are incorporated herein by reference. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody disclosed in US Patent No. 7,763,245 that binds polyubiquitin. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVRs disclosed in US Patent No. 7,763,245, where the half antibody binds polyubiquitin.

[0201] The antibodies, antibody sequences, and sequence listing disclosed in US Patent No. 8,133,488 are incorporated herein by reference. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody disclosed in US Patent No. 8,133,488 that binds polyubiquitin. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVRs disclosed in US Patent No. 8,133,488, where the half antibody binds polyubiquitin.

[0202] The antibodies, antibody sequences, and sequence listing disclosed in US Patent No. 8,992,919 are incorporated herein by reference. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody disclosed in US Patent No. 8,992,919 that binds polyubiquitin. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVRs disclosed in US Patent No. 8,992,919, where the half antibody binds polyubiquitin.

[0203] The antibodies, antibody sequences, and sequence listing disclosed in US Patent No. 9,321,844 are incorporated herein by reference. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody disclosed in US Patent No. 9,321,844 that binds polyubiquitin. In some embodiments, the first half antibody comprising a first antigen binding site that binds polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVRs disclosed in US Patent No. 9,321,844, where the half antibody binds polyubiquitin.

[0204] In some embodiments, a first half antibody comprising a first antigen-binding site that binds polyubiquitin comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively. In some embodiments, a first half antibody comprising a first antigen-binding site that binds polyubiquitin comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively. In some embodiments, a first half antibody comprising a first antigen binding site that binds polyubiquitin comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively. In some embodiments, a first half antibody comprising a first antigen binding site that binds polyubiquitin comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprising the amino acid sequences of SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively.

[0205] In some embodiments, the second half antibody comprises a second antigen-binding site that binds to RIP1 and comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 that comprise the amino acid sequences of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively. In some embodiments, the second half antibody comprises a second antigen-binding site that binds to RIP1 and comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 that comprise the amino acid sequences of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively.

[0206]

[0083] In some embodiments, the second half antibody comprises a second antigen binding site that binds to RIP2 and includes HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 having the amino acid sequences of SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100, respectively.

[0207] In some embodiments, the first half antibody comprises: (i) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0208] In some embodiments, the first half antibody comprises: (i) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0209] In some embodiments, the first half antibody comprises: (i) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 9; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 12; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 13, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 14 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Includes.

[0210] In some embodiments, the first half antibody comprises: (i) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 23, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0211] In some embodiments, the first half antibody comprises: (i) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 23, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0212] In some embodiments, the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 23; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 24; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 25; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 27, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 28 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Includes.

[0213] In some embodiments, the first half antibody comprises: (i) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0214] In some embodiments, the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0215] In some embodiments, the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Includes.

[0216] In some embodiments, the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 75; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0217] In some embodiments, the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 74; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 77; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 79, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 80 Includes.

[0218] In some embodiments, the first half antibody comprises: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99, and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Includes.

[0219]

[0096] In some embodiments, the first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. In some embodiments, the first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36. In some embodiments, the first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50.

[0220] In some embodiments, the first half antibody comprises VH and VL sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 7,763,245 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. Patent No. 7,763,245, where the half antibody binds polyubiquitin.

[0221] In some embodiments, the first half antibody comprises VH and VL sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 8,133,488 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. Patent No. 8,133,488, where the half antibody binds polyubiquitin.

[0222] In some embodiments, the first half antibody comprises VH and VL sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 8,992,919 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. Patent No. 8,992,919, where the half antibody binds polyubiquitin.

[0223] In some embodiments, the first half antibody comprises VH and VL sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 9,321,844 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. Patent No. 9,321,844, where the half antibody binds polyubiquitin.

[0224] In some embodiments, the first half antibody comprises the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 7,763,245 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of the VH and VL sequences disclosed in U.S. Patent No. 7,763,245, where the half antibody binds polyubiquitin.

[0225] In some embodiments, the first half antibody comprises the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 8,133,488 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of the VH and VL sequences disclosed in U.S. Patent No. 8,133,488, where the half antibody binds polyubiquitin.

[0226] In some embodiments, the first half antibody comprises the VH and VL sequences of an antibody disclosed in U.S. Patent No. 8,992,919 that binds polyubiquitin. In some embodiments, the first half antibody comprises a combination of the VH and VL sequences disclosed in U.S. Patent No. 8,992,919, where the half antibody binds polyubiquitin.

[0227] In some embodiments, the first half antibody comprises the VH and VL sequences of the antibodies disclosed in U.S. Patent No. 9,321,844 that bind polyubiquitin. In some embodiments, the first half antibody comprises a combination of the VH and VL sequences disclosed in U.S. Patent No. 9,321,844, where the half antibody binds polyubiquitin.

[0228]

[0105] In some embodiments, the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:71, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73. In some embodiments, the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:72 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73.

[0229]

[0106] In some embodiments, the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0230] In some embodiments, a) a first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:71 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73. In some embodiments, the first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:72 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73.In some embodiments, the first half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0231] In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:71 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73. In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:72 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73.In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:21 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0232] In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:71 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73. In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:72 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73.In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:36. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0233] In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49, and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:71 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73. In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:72 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:73.In some embodiments, the first half antibody has a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50. and the second half antibody comprises a VL sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93 and a VH sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94.

[0234] In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions, but an antibody containing the sequence retains the ability to bind polyubiquitin. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the VH sequence. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVRs (i.e., FRs). Optionally, the antibody contains a VH sequence as discussed above, including post-translational modifications of the sequence.

[0235] In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions, but an antibody containing the sequence retains the ability to bind polyubiquitin. In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the VL sequence. In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the HVR (i.e., FR). Optionally, the antibody contains a VL sequence as discussed above, including post-translational modifications of the sequence.

[0236] In some embodiments, the antibody is humanized. In some embodiments, the antibody comprises an HVR as in any of the above embodiments and further comprises a human acceptor framework, such as a human immunoglobulin framework or a human consensus framework. In some embodiments, the antibody comprises an HVR as in any of the above embodiments and a rabbit framework region.

[0237] In some aspects, antibodies are provided that bind to the same epitope as the bispecific antibodies provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as an antibody comprising a first half antibody and a second half antibody, wherein: a) the first half antibody comprises the VL sequence of SEQ ID NO:7 and the VH sequence of SEQ ID NO:8; The second half antibody comprises the VL sequence of SEQ ID NO:71 and the VH sequence of SEQ ID NO:73; b) the first half antibody comprises the VL sequence of SEQ ID NO:7 and the VH sequence of SEQ ID NO:8; The second half antibody comprises the VL sequence of SEQ ID NO:72 and the VH sequence of SEQ ID NO:73; c) the first half antibody comprises the VL sequence of SEQ ID NO:7 and the VH sequence of SEQ ID NO:8; The second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; d) the first half antibody comprises the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 22; The second half antibody comprises the VL sequence of SEQ ID NO:71 and the VH sequence of SEQ ID NO:73; e) the first half antibody comprises the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 22; The second half antibody comprises the VL sequence of SEQ ID NO:72 and the VH sequence of SEQ ID NO:73; f) the first half antibody comprises the VL sequence of SEQ ID NO: 21 and the VH sequence of SEQ ID NO: 22; The second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; g) the first half antibody comprises the VL sequence of SEQ ID NO: 35 and the VH sequence of SEQ ID NO: 36; The second half antibody comprises the VL sequence of SEQ ID NO:71 and the VH sequence of SEQ ID NO:73; h) the first half antibody comprises the VL sequence of SEQ ID NO: 35 and the VH sequence of SEQ ID NO: 36; The second half antibody comprises the VL sequence of SEQ ID NO:72 and the VH sequence of SEQ ID NO:73; i) the first half antibody comprises the VL sequence of SEQ ID NO: 35 and the VH sequence of SEQ ID NO: 36; The second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; j) the first half antibody comprises the VL sequence of SEQ ID NO: 49 and the VH sequence of SEQ ID NO: 50; The second half antibody comprises the VL sequence of SEQ ID NO:71 and the VH sequence of SEQ ID NO:73; 1) the first half antibody comprises the VL sequence of SEQ ID NO: 49 and the VH sequence of SEQ ID NO: 50; the second half antibody comprises the VL sequence of SEQ ID NO: 72 and the VH sequence of SEQ ID NO: 73; or 1) the first half antibody comprises the VL sequence of SEQ ID NO: 49 and the VH sequence of SEQ ID NO: 50; The second half antibody comprises the VL sequence of SEQ ID NO:93 and the VH sequence of SEQ ID NO:94.

[0238]

[0115] In some embodiments, a bispecific antibody is provided that comprises a VH sequence and a VL sequence as in one of a) to l) in the preceding paragraphs.

[0239] In some embodiments, the antibody is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the antibody is an antibody fragment, such as a dimeric scFv, a diabody, or a F(ab')2 fragment. In another embodiment, the antibody is a substantially full-length antibody, such as an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody, or other antibody class or isotype as defined herein.

[0240]

[0117] In some embodiments, the antibody comprises at least one heavy chain with a C-terminal lysine. In some embodiments, the antibody comprises at least one heavy chain lacking a C-terminal lysine. In some embodiments, the antibody comprises only a heavy chain without a C-terminal lysine. The C-terminal lysine can be removed, for example, enzymatically, such as by carboxypeptidase treatment, or genetically, such as by deleting or substituting a lysine codon at the 3' end of the heavy chain coding sequence. Since the C-terminal lysine of the heavy chain is located away from the antigen binding site and is not required for binding activity, removing it can result in a more homogeneous antibody preparation.

[0241] In some embodiments, the first half antibody comprises: a) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4; b) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:16, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:18; c) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; or d) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46. and optionally a C-terminal lysine is deleted from one or more of the heavy chains.

[0242] In some embodiments, the first half antibody comprises: a) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; b) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20; c) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; or d) a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48. and optionally, the C-terminal lysine is deleted from one or more chains.

[0243]

[0120] In some embodiments, the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 68; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0244]

[0121] In some embodiments, the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 60, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 70; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0245]

[0122] In some embodiments, the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0246]

[0123] In some embodiments, the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84, and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; optionally, a C-terminal lysine is deleted from one or more heavy chains.

[0247] In some embodiments, the antibody comprises a first half antibody and a second half antibody, a) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; b) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; c) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; d) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; e) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; f) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; g) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:70; h) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:48; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:68; Optionally, in the above, the C-terminal lysine is deleted from one or more chains.

[0248] In some embodiments, the antibody comprises a first half antibody and a second half antibody, a) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:4; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; b) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:2 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:6; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; c) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; d) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; e) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:32; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; f) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; g) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; h) a first half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:48; the second half antibody comprises a light chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; Optionally, in the above, the C-terminal lysine is deleted from one or more chains.

[0249]

[0126] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO:2 and a heavy chain sequence of SEQ ID NO:4, and the second half antibody comprising a light chain sequence of SEQ ID NO:60 and a heavy chain sequence of SEQ ID NO:70, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0250]

[0127] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO:2 and a heavy chain sequence of SEQ ID NO:6, and the second half antibody comprising a light chain sequence of SEQ ID NO:60 and a heavy chain sequence of SEQ ID NO:68, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0251]

[0128] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18, and the second half antibody comprising a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0252]

[0129] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20, and the second half antibody comprising a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0253]

[0130] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32, and the second half antibody comprising a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0254]

[0131] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34, and the second half antibody comprising a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0255]

[0132] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46, and the second half antibody comprising a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 70, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0256]

[0133] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48, and the second half antibody comprising a light chain sequence of SEQ ID NO: 60 and a heavy chain sequence of SEQ ID NO: 68, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0257]

[0134] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO:2 and a heavy chain sequence of SEQ ID NO:4, and the second half antibody comprising a light chain sequence of SEQ ID NO:84 and a heavy chain sequence of SEQ ID NO:92, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0258]

[0135] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO:2 and a heavy chain sequence of SEQ ID NO:6, and the second half antibody comprising a light chain sequence of SEQ ID NO:84 and a heavy chain sequence of SEQ ID NO:90, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0259]

[0136] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 18, and the second half antibody comprising a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0260]

[0137] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 16 and a heavy chain sequence of SEQ ID NO: 20, and the second half antibody comprising a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0261]

[0138] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 32, and the second half antibody comprising a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0262]

[0139] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34, and the second half antibody comprising a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0263]

[0140] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 46, and the second half antibody comprising a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 92, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0264]

[0141] In some embodiments, the antibody comprises a first half antibody and a second half antibody, the first half antibody comprising a light chain sequence of SEQ ID NO: 44 and a heavy chain sequence of SEQ ID NO: 48, and the second half antibody comprising a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, optionally with a C-terminal lysine deleted from one or more heavy chains.

[0265]

[0142] In a further aspect, an antibody according to any of the above embodiments may incorporate any of the features, either alone or in combination, as described in Sections 1-7 below.

[0266] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM, and optionally, a dissociation constant of ≧10 -13 M (for example, 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M).

[0267] In some embodiments, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen, as illustrated by the following assay: The solution binding affinity of a Fab for an antigen is determined by binding the Fab to a minimal concentration of ( 125I) Equilibration with labeled antigen followed by capture of bound antigen on anti-Fab antibody-coated plates (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / mL capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23° C.). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight. However, incubation may continue for an extended period (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0268] In some embodiments, Kd is measured using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25° C. with an immobilized antigen CM5 chip at about 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (about 0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of binding protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (Tween-20™) detergent (PBST) at a flow rate of approximately 25 μl / min at 25° C. Association rates (kon) and dissociation rates (koff) are calculated by simultaneously fitting the association and dissociation sensograms using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate by the surface plasmon resonance assay described above exceeds 106 M-1 s-1, the association rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, with a band pass of 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured by a spectrometer such as a spectrophotometer equipped with a flow stop (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0269] 2. Antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, F(ab')2 fragments, dimeric single chain Fv, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (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. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments which contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0270] In some embodiments, the antibody is a diabody. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404097, 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).

[0271]

[0148] Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516).

[0272]

[0149] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0273] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. 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 some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from those of the parent antibody. The chimeric antibody includes an antigen-binding fragment thereof.

[0274] In certain embodiments, the chimeric antibody is a humanized antibody. Usually, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0275] Humanized antibodies and methods for their production are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and in, e.g., Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. 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 "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), as well as Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0276] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0277] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced 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).

[0278]

[0155] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has 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 a human immunoglobulin locus that replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is 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, XENOMOUSE. TM See also U.S. Patent Nos. 6,075,181 and 6,150,584, describing HuMab® technology, U.S. Patent No. 5,770,429, describing HuMab® technology, U.S. Patent No. 7,041,870, describing KM MOUSE® technology, and U.S. Patent Application Publication No. 2007 / 0061900, describing VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with a different human constant region.

[0279]

[0156] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing 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 produced using human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). 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).

[0280]

[0157] Human antibodies can also be made by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from an antibody library are described below.

[0281] 5. Antibodies derived from the library

[0158] Antibodies can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, various methods are known in the art for making phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, e.g., 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, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. 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).

[0282] In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immune sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences, encoding highly variable CDR3 regions, and achieving rearrangement in vitro, as described by Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0283]

[0160] Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

[0284] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0285] Techniques for making 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 "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). In some embodiments, the antibody comprises a first half antibody and a second half antibody, where the first half antibody comprises a first heavy chain constant region that includes a knob mutation and the second heavy chain comprises a second heavy chain constant region that includes a hole mutation; or the first half antibody comprises a first heavy chain constant region that includes a hole mutation and the second heavy chain comprises a second heavy chain constant region that includes a knob mutation. In some embodiments, the antibody is an IgG1 antibody and the knob mutation comprises a T366W mutation. In some embodiments, the antibody is an IgG1 antibody and the hole mutation comprises at least one, at least two or three mutations selected from T366S, L368A and Y407V. In some embodiments, the antibody is an IgG4 antibody and the knob mutation comprises a T366W mutation. In some embodiments, the antibody is an IgG4 antibody and the hole mutation comprises at least one, at least two or three mutations selected from T366S, L368A and Y407V. The numbering of the positions of the aforementioned mutations is EU numbering. The actual position of the mutation in the heavy chain sequence may vary, for example, by up to 10 positions, depending, for example, on the length of the preceding variable region.

[0286] Multispecific antibodies can be made by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 1997, 111:111-112). al., J. Immunol., 152:5368 (1994); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991). In some embodiments, the antibody is a diabody. Diabodies are antibody fragments having two antigen binding sites that may be bivalent or bispecific. See, for example, EP 404097, 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).

[0287] Triabodies and tetrabodies are described in Hudson et al., Nat. Med. 9:129-134 (2003). In some embodiments, the antibody is a triabody. In some embodiments, the triabody comprises a first antigen recognition site, a second antigen recognition site, and a third antigen recognition site, where at least one of the antigen recognition sites is different from the other antigen recognition sites. In some embodiments, the triabody comprises a first, second, and third antigen recognition site that bind to three different polyubiquitins. Each antigen recognition site can comprise a combination of HVRs or a combination of VL and VH as discussed above.

[0288] In some embodiments, the antibody is a tetrabody. In some embodiments, the tetrabody comprises a first antigen recognition site, a second antigen recognition site, a third antigen recognition site, and a fourth antigen recognition site, where at least one or at least two of the antigen recognition sites are different from the other antigen recognition sites. In some embodiments, the tetrabody comprises a first, second, and third antigen recognition site that binds to three different polyubiquitins. In some embodiments, the tetrabody comprises each antigen recognition site that can include a combination of HVRs or a combination of VL and VH as discussed above.

[0289]

[0166] Engineered antibodies having three or more functional antigen binding sites are also included herein, including "Octopus antibodies" (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576). The term Octopus antibody is used in the sense discussed in U.S. Patent Application Publication No. 2006 / 0025576A1, and is not meant to refer to antibodies produced by or obtained from octopuses.

[0290]

[0167] The antibodies or fragments herein also include "dual acting FAbs" or "DAFs" that contain two antigen binding sites that bind to two different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0291] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. 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 from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, so long as the final construct has the desired characteristics (e.g., antigen binding).

[0292] a) Substitution, insertion and deletion variants In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and as further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2024534853000002.tif161170

[0293]

[0170] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0294]

[0171] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0295] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant or variants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., improved affinity, reduced immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage are screened for a certain biological activity (e.g., binding affinity).

[0296]

[0173] Modifications (e.g., substitutions) may be made, for example, in HVRs to improve antibody affinity. Such modifications may be made to HVR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from a secondary library is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0297] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made within the HVRs. Such modifications may be outside the HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0298] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (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 interaction of the antigen with the antibody is affected. Further substitutions can also be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and the antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they have the desired properties.

[0299] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which extends the serum half-life of the antibody.

[0300] b) Glycosylation variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0301]

[0178] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may comprise a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibody may be made to generate antibody variants with specific improved properties.

[0302] In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high mannose structures) as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (Eu numbering of Fc region residues) in the Fc region, although Asn297 can be located about ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US Patent Application Publication No. 2003 / 0115614; US Patent Application Publication No. 2002 / 0164328; US Patent Application Publication No. 2004 / 0093621; US ​​Patent Application Publication No. 2004 / 0132140 No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 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 producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al, Biotechnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085107).

[0303]

[0180] Further provided are antibody variants having biantennary oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected 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-Mairett et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. 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.).

[0304] c) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid alteration (e.g., substitution) at one or more amino acid positions.

[0305] In certain embodiments, the antibody variants possess some, but not all, effector functions, making them desirable candidates for applications in which the half-life of the antibody in vivo is important, but 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 reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII, and Fc(RIII). For FcR expression on hematopoietic cells, see Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., 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), U.S. Pat. No. 5,821,337 (Bruggemann, M. et al. al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry). TMNon-radioactive cytotoxicity assays (see CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., 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 determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. Immunol. 18(12):1759-1769 (2006)).

[0306] Antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0307]

[0184] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0185] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, for example substitutions at positions 298, 333 and / or 334 of the Fc region (EU numbering of residues).

[0308]

[0186] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0309]

[0187] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include 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, e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0310]

[0188] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and International Publication No. WO 94 / 29351.

[0311] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain embodiments, the replaced residues are present in accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites of the antibody, which can be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat numbering) of the light chain; K149 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0312] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0313] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety is provided that can be selectively heated by exposure to radiation. In some embodiments, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that are not harmful to normal cells but heat the non-proteinaceous moiety to a temperature that kills cells proximal to the antibody-non-proteinaceous moiety.

[0314] B. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, an isolated nucleic acid is provided that encodes an antibody described herein. Such a nucleic acid can 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 and / or heavy chains of the antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In further embodiments, a host cell comprising such a nucleic acid is provided. In some such embodiments, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a 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 comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, methods of making the antibodies disclosed herein are provided, the methods comprising culturing a host cell comprising nucleic acid encoding the antibody as described above under conditions suitable for expression of the antibody, and, optionally, recovering the antibody from the host cell (or host cell culture medium).

[0315] In some embodiments, the components of the multispecific antibody (e.g., the first half antibody and the second half antibody) are expressed in separate cells or cell cultures and then combined in vitro. In other embodiments, all components of the multispecific antibody are expressed in the same cell or cell culture.

[0316] For recombinant production of an antibody, for example, nucleic acid encoding the above-mentioned antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0317] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BCLo ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments in E. coli.) After expression, the antibodies of the invention can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0318] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains that have been "humanized" in their glycosylation pathways to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0319]

[0197] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0320]

[0198] 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 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0321]

[0200] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals TRI cells as described in NYAcad.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, e.g., Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0322] C. Assay

[0200] The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0323]

[0201] In some embodiments, the antibody is tested for its antigen binding activity by known methods such as, for example, ELISA, FACS or Western blot.

[0324] In another aspect, a competitive assay can be used to identify antibodies that compete with any of the antibodies described herein. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) that the antibodies described herein bind. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0325] In an exemplary competitive assay, immobilized polyubiquitin is incubated with a solution containing a first labeled antibody (e.g., any of the antibodies described herein) that binds to it and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to polyubiquitin. The second antibody can be present in a hybridoma supernatant. As a control, polyubiquitin 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 polyubiquitin, excess unbound antibody is removed and the amount of label bound to immobilized polyubiquitin is measured. If the amount of label bound to immobilized polyubiquitin is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody competes with the first antibody for binding to polyubiquitin. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0326] In some embodiments, the RIP1- or RIP2-K63-linked and / or linear ubiquitin chain bispecific antibodies are used in a method for detecting selective ubiquitination of RIP1 or RIP2, respectively, polyubiquitinated with K63-linked and / or linear ubiquitin in a sample, such as a cell or tissue sample, comprising contacting the sample with the antibody. In some embodiments, the RIP1- or RIP2-linear linked ubiquitin chain bispecific antibodies allow selective ubiquitination of RIP1 or RIP2, respectively, polyubiquitinated with linear linked ubiquitin to be detected in a sample, such as a cell or tissue sample.

[0327] In some embodiments, methods are provided for detecting ubiquitination of RIP1 or RIP2, such as K63-linked and / or linear polyubiquitin, in cell or tissue samples, for example by immunofluorescence, using the antibodies disclosed herein. As shown in the examples below, detection can be performed in a time- and / or signal-dependent manner. The use of a single bispecific antibody can advantageously eliminate the need to tediously search for overlapping patterns of multiple antibodies and / or improve the specificity and accuracy of cellular localization.

[0328] The antibodies described herein can also be used in methods to assay tissue samples from subjects undergoing intestinal resection to determine the ubiquitination level of RIP2.As demonstrated in the Examples, samples from subjects with Crohn's disease and ulcerative colitis may show high levels of RIP2 ubiquitination with K63-linked and / or linear ubiquitin chains, in contrast to low levels of ubiquitination in non-IBD control samples and diverticulitis samples.

[0329] Also provided herein is a method of identifying a subject as a candidate for RIP2 targeted therapy, comprising contacting a sample from the subject with an antibody described herein and determining a level of polyubiquitinated RIP2, such as K63-linked and / or linear polyubiquitinated RIP2. In some embodiments, the subject has or is suspected of having Crohn's disease or ulcerative colitis. In some embodiments, the sample is a cell sample or a tissue sample.

[0330]

[0208] In some embodiments, methods are provided for detecting ubiquitination of RIP1 or RIP2 comprising a branched polyubiquitin chain, a hybrid polyubiquitin chain, or a mixed polyubiquitin chain, for example, the branched polyubiquitin chain, the hybrid polyubiquitin chain, or the mixed polyubiquitin chain comprises one or more K63-linked and / or linear ubiquitin chains.

[0331] D. Immunoconjugates

[0209] Immunoconjugates are provided that include the antibodies disclosed herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof) or radioactive isotopes (i.e., radioconjugates).

[0332]

[0210] Immunoconjugates allow for targeted delivery of drug moieties to tumor or other diseased cells or tissues and, in some embodiments, their intracellular accumulation when systemic administration of an unconjugated drug may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0333] Antibody drug conjugates (ADCs) are targeted chemotherapy molecules that combine the properties of both 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.

[0334] ADC compounds include those with anti-cancer activity and / or anti-inflammatory activity. In some embodiments, ADC compounds include antibodies conjugated, i.e., covalently linked, to a drug moiety. In some embodiments, the antibody is covalently linked to the drug moiety by a linker. Antibody-drug conjugates (ADCs) can selectively deliver an effective dose of drug to tumor tissue, thereby achieving higher selectivity, i.e., lower effective doses, while increasing the therapeutic index ("therapeutic window").

[0335] The drug moiety (D) of the antibody drug conjugate (ADC) may comprise any compound, moiety or group having a cytotoxic or cytostatic effect. The drug moiety may impart their cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drug moieties include, but are not limited to, maytansinoids, dolastatins, auristatins, calicheamicins, pyrrolobenzodiazepines (PBDs), nemorubicin and its derivatives, PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecins, elinafides, and their stereoisomers, isosteres, analogs, and derivatives having cytotoxic activity.

[0336]

[0214] The drug moiety (D) of the ADC may include any compound, moiety, or group that has anti-inflammatory activity. Exemplary drug moieties include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin; cox-2 inhibitors, such as celecoxib, rofecoxib, and valdecoxib; and their stereoisomers, isosteres, analogs, and derivatives that have anti-inflammatory activity.

[0337] E. Methods and Compositions for Diagnostics and Detection In some embodiments, the methods described herein are useful for determining the presence of polyubiquitinated proteins in a sample. In some embodiments, the sample is suspected of containing polyubiquitinated proteins. In some embodiments, the polyubiquitinated proteins are pro-inflammatory proteins.

[0338] The methods described herein include exposing a sample to at least one multispecific antibody comprising a first half-antibody comprising a first antigen-binding site that binds polyubiquitin; and a second half-antibody comprising a second antigen-binding site that binds a proinflammatory protein. In some embodiments, the method includes measuring binding of the at least one antibody to a polyubiquitinated protein in the sample.

[0339]

[0217] In some embodiments, the polyubiquitinated protein comprises M1-linked polyubiquitin and / or K63-linked polyubiquitin.

[0340]

[0218] In some embodiments, the methods described herein are useful for determining the presence of a polyubiquitinated protein in a sample suspected of containing the polyubiquitinated protein, wherein the polyubiquitinated protein is a pro-inflammatory protein, comprising exposing the sample to at least one multispecific antibody comprising a first half-antibody that comprises polyubiquitin and that comprises a first antigen binding site that binds to the polyubiquitin; and a second half-antibody that comprises a second antigen binding site that binds to the pro-inflammatory protein, and determining binding of the at least one antibody to the polyubiquitinated protein in the sample.

[0341]

[0219] In some embodiments, the methods described herein are useful for determining the presence of a polyubiquitinated protein in a sample suspected of containing the polyubiquitinated protein, wherein the polyubiquitinated protein is a pro-inflammatory protein, and comprises M1-linked polyubiquitin and / or K63-linked polyubiquitin, comprising exposing the sample to at least one multispecific antibody comprising: a first half-antibody comprising a first antigen-binding site that binds to polyubiquitin; and a second half-antibody comprising a second antigen-binding site that binds to the pro-inflammatory protein, and determining binding of the at least one antibody to the polyubiquitinated protein in the sample.

[0342] Proinflammatory proteins are components of one or more signaling complexes that promote inflammation by mediating inflammatory cell death and / or the release of proinflammatory cytokines, chemokines, and damage-associated molecular patterns (DAMPs). In some embodiments, the proinflammatory protein is receptor-interacting protein kinase 1 (RIP1), receptor-interacting protein kinase 2 (RIP2), cellular inhibitor of apoptosis 1 and 2 (c-IAP1 / 2), tumor necrosis factor receptor 1 (TNFR1), linear ubiquitin chain assembly complex (LUBAC), and / or nuclear factor kappa B (NF-κB) essential modulator (NEMO).

[0343]

[0221] In some embodiments, the pro-inflammatory protein is RIP1 or RIP2.

[0344]

[0222] In some embodiments, the pro-inflammatory protein has an elevated ubiquitination level in an inflammatory state compared to the ubiquitination level in the absence of an inflammatory state.

[0345]

[0223] In some embodiments, the pro-inflammatory protein has a ubiquitination level that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 1-fold-12-fold, 2-fold-12-fold, 3-fold-12-fold, 4-fold-12-fold, 5-fold-12-fold, 6-fold-12-fold, 7-fold-12-fold, 8-fold-12-fold, 9-fold-12-fold, 10-fold-12-fold, or 11-fold-12-fold higher in an inflammatory state compared to the ubiquitination level in the absence of an inflammatory state.

[0346]

[0224] In some embodiments, increased levels of ubiquitination correlate with increased severity of the inflammatory disease state.

[0347]

[0225] In some embodiments, the level of ubiquitination is increased in an inflammatory state compared to the level of ubiquitination in the absence of an inflammatory state.

[0348]

[0226] In some embodiments, the pro-inflammatory protein is associated with an inflammatory disease, such as inflammatory bowel disease, Crohn's disease, diverticulitis, and ulcerative colitis.

[0349] In some embodiments, the antibodies provided herein are useful for detecting the presence of RIP1 in a biological sample. In certain embodiments, the antibodies provided herein are useful for detecting the presence of RIP2 in a biological sample. As used herein, the term "detect" includes quantitative or qualitative detection. A "biological sample" includes, for example, a cell or tissue (e.g., a biopsy including cancerous or potentially cancerous colon, colorectal, small intestine, endometrial, pancreatic, breast, lung, prostate, or ovarian tissue).

[0350] In some embodiments, the antibodies disclosed herein are for use in methods of diagnosis or detection. In a further aspect, a method is provided for detecting the presence of RIP1 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an antibody described herein under conditions that allow binding of the antibody to RIP1, and detecting whether a complex is formed between the antibody and RIP1 in the biological sample. Such a method can be an in vitro or in vivo method. In one embodiment, the antibody is used to select subjects eligible for treatment with an anti-RIP1 antibody, for example, when RIP1 is a biomarker for selecting patients. In some embodiments, the biological sample is a cell or tissue (e.g., a biopsy containing cancerous or potentially cancerous tissue).

[0351] In some embodiments, the antibodies disclosed herein are for use in methods of diagnosis or detection. In a further aspect, a method is provided for detecting the presence of RIP2 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an antibody described herein under conditions that allow binding of the antibody to RIP2, and detecting whether a complex is formed between the antibody and RIP2 in the biological sample. Such a method can be an in vitro or in vivo method. In one embodiment, the antibody is used to select subjects eligible for treatment with an anti-RIP2 antibody, for example, when RIP2 is a biomarker for selecting patients. In some embodiments, the biological sample is a cell or tissue (e.g., a biopsy containing cancerous or potentially cancerous tissue).

[0352] In some embodiments, a method is provided for detecting polyubiquitinated RIP1 or RIP2 protein in a biological sample. In certain embodiments, the method includes contacting a biological sample with an antibody described herein under conditions that allow the antibody to bind to polyubiquitinated RIP1 or RIP2 protein, and detecting whether a complex is formed between the antibody and RIP1 or RIP2 protein in the biological sample. Such a method can be an in vitro or in vivo method. In one embodiment, the antibody is used to select subjects eligible for treatment with the antibody disclosed herein, for example, when polyubiquitinated RIP1 or RIP2 protein is a biomarker for selecting patients. In some embodiments, the biological sample is a cell or tissue (e.g., a biopsy containing cancerous or potentially cancerous tissue).

[0353] In further embodiments, the antibodies disclosed herein are used in vivo, e.g., by in vivo imaging, to detect polyubiquitinated RIP1 or RIP2 proteins, e.g., for the purpose of diagnosing, prognosing, or staging a disease, determining an appropriate course of treatment, or monitoring a response to treatment. One method known in the art for in vivo detection is immunopositron emission tomography (immunoPET), e.g., as described 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 is provided for detecting polyubiquitinated RIP1 or RIP2 proteins in a subject, the method comprising administering a labeled antibody to the subject and detecting a labeled anti-RIP1 or anti-RIP2 antibody in the subject. In certain such embodiments, the labeled antibody comprises: 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 In certain embodiments, the positron emitter comprises (e.g., is conjugated to) a positron emitter such as I. 89 Zr. 89 Non-limiting exemplary methods for making and using Zr-labeled antibodies are described, for example, in PCT Publication No. WO 2011 / 056983. In some embodiments, the labeled antibody is a cysteine ​​engineered antibody conjugated to one or more zirconium complexes. See, for example, WO 2011 / 056983.

[0354] In a further embodiment, the method of diagnosis or detection comprises contacting a first antibody as disclosed herein immobilized on a substrate with a biological sample to be tested for the presence of polyubiquitinated RIP1 or RIP2 protein, exposing the substrate to a second antibody that binds to the polyubiquitinated RIP1 or RIP2 protein, and detecting whether the second antibody binds to the complex of the first antibody and the polyubiquitinated RIP1 or RIP2 protein in the biological sample (sometimes referred to as a sandwich assay). The substrate may be any support medium, such as glass, metal, ceramic, polymer beads, slides, chips, and other substrates. In certain embodiments, the "biological sample" comprises, for example, cells or tissues (e.g., biopsies including cancerous or potentially cancerous colon, colorectal, small intestine, endometrial, pancreatic, or ovarian tissues). In certain embodiments, the first antibody or the second antibody is any of the antibodies described herein.

[0355] In certain embodiments, the antibodies disclosed herein are labeled. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and moieties, such as enzymes or ligands, that are indirectly detected, for example, via an enzymatic reaction or molecular interaction. Exemplary labels include radioisotopes, 32 P, 14 C. 125 I, 3 H, and 131These include, but are not limited to, I, rare earth chelates or fluorophores such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase conjugated to an enzyme that utilizes hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like. In another embodiment, the label is a positron emitter. Positron emitters include: 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 In certain embodiments, the positron emitter includes, but is not limited to, I. 89 Zr.

[0356] The presence of polyubiquitinated RIP1 or RIP2 protein in a sample can be analyzed by a number of methodologies using the antibodies disclosed herein, many of which are known in the art and understood by those skilled in the art, including, but not limited to, immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence-activated cell sorting ("FACS"), and quantitative blood-based assays (e.g., serum ELISA). Exemplary protocols for assessing protein status can be found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Unit 15 (Immunoblotting). Multiplexed immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD"), can also be used.

[0357] In some embodiments, compositions are provided that are substantially free of monospecific antibodies, disassembled half antibodies, or both monospecific antibodies and disassembled half antibodies. A monospecific antibody is an antibody that does not contain multiple types of antigen recognition sites, for example, an antibody that has only one set of six CDRs, or an antibody in which each set of six CDRs is identical. An antibody in which a first set of CDRs differs only slightly from another set of CDRs, for example, in terms of a small number of amino acid residues, and the difference does not result in preferential binding to different antigens, is also considered to be monospecific. A disassembled half antibody is not stably associated (covalently or non-covalently) with another half antibody and appears as a single heavy / light chain unit when analyzed by a suitable technique, for example, size exclusion chromatography, mass spectrometry, or electrophoresis.

[0358] F. Pharmaceutical Preparations

[0236] Pharmaceutical formulations of the antibodies or immunoconjugates described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or immunoconjugates having the desired purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, blood-soluble peptides, and the like. Examples of suitable pharmacopoeitic carriers include, but are not limited to, 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 dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968.In some embodiments, the sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0359] 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 No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0360]

[0238] The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.

[0361]

[0239] The active ingredient may be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., (1980).

[0362]

[0240] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0363]

[0241] Formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0364] G. Therapeutic Methods and Compositions

[0242] Any of the antibodies or immunoconjugates provided herein can be used in methods, eg, therapeutic methods.

[0365] In some aspects, there is provided an antibody or immunoconjugate disclosed herein for use as a medicament. In a further aspect, there is provided an antibody or immunoconjugate disclosed herein for use in a method of treatment. In certain embodiments, there is provided an antibody or immunoconjugate disclosed herein for use in treating a cell cycle-related disease or disorder.

[0366] In some embodiments, the antibody or immunoconjugate disclosed herein is for use in the treatment of a disease or disorder associated with an abnormal increase in cell cycle progression, provided that the disease or disorder is associated with an abnormal decrease in cell cycle progression. In some embodiments, the disease or disorder associated with an abnormal increase in cell cycle progression is cancer, such as colorectal cancer.

[0367] In some embodiments, the antibodies or immunoconjugates disclosed herein are for use in the treatment of an inflammatory disease, such as inflammatory bowel disease. In some embodiments, the inflammatory disease is selected from Crohn's disease, diverticulitis, and ulcerative colitis.

[0368]

[0246] In some aspects there is provided the use of an antibody or immunoconjugate disclosed herein in the manufacture or preparation of a medicament.

[0369]

[0247] An "individual" according to any of the above embodiments may be a human.

[0370] In some aspects, provided are pharmaceutical formulations comprising any of the antibodies or immunoconjugates provided herein, e.g., for use in any of the above-described methods of treatment. In some embodiments, the pharmaceutical formulations comprise any of the antibodies or immunoconjugates provided herein and a pharma- ceutically acceptable carrier.

[0371]

[0249] The antibodies or immunoconjugates provided herein can be used alone or in combination with other agents in therapy. For example, the antibodies or immunoconjugates provided herein can be co-administered with at least one additional therapeutic agent.

[0372]

[0250] Such combination therapy as described above includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of an antibody or immunoconjugate provided herein can occur prior to, simultaneously with, and / or after administration of an additional therapeutic agent and / or adjuvant.

[0373] The antibody or immunoconjugate (and any additional therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired for localized treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time periods, bolus administration, and pulse infusion.

[0374] The antibody or immunoconjugate will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the drug, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibody or immunoconjugate is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will be determined by the amount of antibody or immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages as those described herein, or about 1-99% of the dosages described herein, or any dosages determined empirically / clinically appropriate, and for any route of administration.

[0375] For the prevention or treatment of disease, the appropriate dosage of the antibody or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody or immunoconjugate, the severity and course of the disease, whether the antibody or immunoconjugate is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody or immunoconjugate is suitably administered to the patient at one time or over a series of treatments.

[0376]

[0254] It will be understood that any of the above formulations or treatment methods may be carried out using both immunoconjugates and antibodies.

[0377] H. Manufactured products In another aspect, an article of manufacture is provided that contains a substance useful for treating, preventing, and / or diagnosing the above-mentioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective for treating, preventing, and / or diagnosing a disorder, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody or immunoconjugate disclosed herein. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may include (a) a first container containing a composition comprising the antibody or immunoconjugate; and (b) a second container containing a composition further comprising a cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. EXAMPLES

[0378] III. Examples

[0256] The following are examples of the methods and compositions provided herein. Given the general description provided above, it is understood that various other embodiments may be implemented.

[0379] A. Analysis method Size Exclusion Chromatography - Multi-Angle Light Scattering. As an example, 50 μg of antibody is injected onto a 3.5 μm, 7.8 mm×300 mm XBridge Protein BEH analytical SEC 200 Å column (Waters) at 1 mL / min using an Agilent 1260 Infinity HPLC with 20 mM histidine acetate, 300 mM NaCl, pH 5.5 as the mobile phase. Protein eluted from the analytical SEC column is injected directly into a Wyatt DAWN HELEOS II / Optilab T-rEX multi-angle light scattering detector to measure molar mass and polydispersity.

[0380] Mass spectrometry. Prior to analysis by mass spectrometry, 30 μg of antibody is deglycosylated overnight at 37° C. using 2 units of PNGaseF (NEB). 2 μg of antibody is then injected onto a 3 μm, 4.6×50 mm reversed-phase chromatography PLRP-S column (Agilent) at 1 mL / min using an Agilent 1290 Infinity UHPLC. A 0-100% buffer B gradient over 3 min is performed using 0.05% trifluoroacetic acid (TFA) in water (buffer A) and 0.05% TFA in acetonitrile (buffer B), followed by a 1 min wash with 100% buffer B. Proteins eluted from the reversed-phase column are directly injected into an Agilent 6230 electrospray ionization time-of-flight mass spectrometer (ESI-TOF) for intact mass measurement.

[0381] B. Antibody Cloning, Expression, and Annealing

[0259] Bispecific antibodies have been generated using the knob-into-hole heterodimerization approach. For a general description of this approach, see Merchant, A. M. et al. An efficient route to human bispecific IgG. Nat Biotechnol 16, 677-681, doi:10.1038 / nbt0798-677 (1998).

[0382] Bispecific antibodies were designed using the following antibodies as building blocks: anti-M1 or linear polyubiquitin linkage-specific synthetic human antibody (clone 1F11 / 3F5 / Y102L) (Matsumoto et al., J Mol Biol. 418(3-4), 134-44. doi:10.1016 / j.jmb.2011.12.053. Epub 2011 Dec 29. PMID:22227388(2012)), anti-K63 polyubiquitin linkage-specific synthetic human antibody (clone Apu3.A8) (Newton et al., Cell. 134(4), 668-78. doi:10.1016 / j.cell.2008.07.039. PMID:18724939(2008)), mouse anti-RIP1 antibody (BD Biosciences #610459), mouse anti-RIP2 antibody (Abcam #ab75257). As a control, an anti-gD antibody recognizing an unrelated protein was used.

[0383]

[0261] T366W (knob) or T366S, L368A, and Y407V (hole) mutations were introduced into the CH3 domain. The knob and hole mutations were selected to allow preferential heterodimerization of the respective heavy chains of the antibody.

[0384] Briefly, the sequences encoding the heavy chain variable domains are SEQ ID NO:50 (for anti-M1 polyubiquitin binding specificity), SEQ ID NO:36 (for anti-K63 polyubiquitin binding specificity), SEQ ID NO:73 (for anti-RIP1), SEQ ID NO:94 (for anti-RIP2) and the sequence of a non-specific anti-gD control antibody. These variable domains were subcloned into a modified pRK vector (Genentech) containing a human IgG1 heavy chain constant domain with knob (T366W) or hole (T366S, L368A, Y407V) mutations in the CH3 domain.

[0385] Depending on the length of the variable region, the actual positions of the knob and hole mutations may vary slightly, e.g., by positions 1 to 10. For example, in SEQ ID NOs: 4 and 6, the T to W and T to S substitutions, respectively, are reflected at amino acid residue 369 rather than amino acid residue 366. It will be understood that references to knob and hole mutations at positions such as 366, 368, and 407 of the heavy chain should be interpreted with adjustments, if appropriate, taking into account the length of the variable region.

[0386] The light chain variable domain was also subcloned into a modified pRK vector (Genentech) containing the human kappa light chain constant domain. The pRK vector carries a constitutive strong signal peptide for extracellular expression in mammalian cells. The anti-M1 antibody was cloned as both a knob and hole variant (encoding heavy chains SEQ ID NOs: 46 and 48, respectively), as was the anti-K63 antibody (encoding heavy chains SEQ ID NOs: 32 and 34, respectively). The anti-RIP1 and anti-RIP2 antibodies were cloned as hole variants (encoding heavy chains SEQ ID NOs: 70 and 92, respectively), and the anti-gD antibody was cloned as a knob variant.

[0387]

[0265] The sequence listing also provides knob and hole variants of the anti-K11 heavy chain (SEQ ID NOs: 4 and 6, respectively); knob and hole variants of the anti-K48 heavy chain (SEQ ID NOs: 18 and 20, respectively); and knob variants of the anti-RIP1 heavy chain (SEQ ID NO: 68) and the anti-RIP2 heavy chain (SEQ ID NO: 90).

[0388] To maintain cognate light and heavy chain pairing, either the knob or hole heavy chain variants and their respective light chains were expressed separately in CHO cells and affinity purified individually (data not shown). Light and heavy chain plasmids for a given knob or hole half antibody were transiently co-transfected into CHO cells using PEI as previously described (see Wong, AW, Baginski, TK & Reilly, DE Enhancement of DNA uptake in FUT8-deleted CHO cells for transient production of afucosylated antibodies. Biotechnol Bioeng 106, 751-763, doi:10.1002 / bit.22749 (2010)). Half antibodies were purified on MabSelect SuRe resin (GE Healthcare) and eluted with 50 mM sodium citrate, 150 mM NaCl, pH 3.0, followed by adjustment of pH to 5.0 with 10% (v / v) 200 mM arginine, 137 mM succinate, pH 9.0.

[0389] The affinity purified knob and hole half antibodies were characterized in agreement with previously described knob and hole antibodies (not shown). See, e.g., Shatz, W. et al. MAbs 5, 872-881, doi:10.4161 / mabs.26307 (2013). Their identity was confirmed by mass spectrometry.

[0390]

[0268] Bispecific antibodies were assembled in vitro from half antibodies using annealing, reduction, and oxidation. Anti-RIP1 / anti-M1 bispecific antibodies were assembled in vitro from affinity purified anti-M1 knob and anti-RIP1 hole antibodies using a modified version of a previously described annealing, reduction, and oxidation method (Shatz, W. et al. MAbs 5, 872-881, doi:10.4161 / mabs.26307(2013)). Similarly, anti-RIP1 / anti-K63 bispecific antibodies were assembled from anti-K63 knob and anti-RIP1 hole antibodies; anti-RIP2 / anti-M1 bispecific antibodies were assembled from anti-M1 knob and anti-RIP2 hole antibodies; anti-RIP2 / anti-K63 bispecific antibodies were assembled from anti-K63 knob and anti-RIP2 hole antibodies. Briefly, the desired knob and hole half antibodies were mixed in a 1:1 mass ratio and the pH of the mixture was adjusted to approximately 8 with 15% (v / v) 800 mM arginine, pH 10.0. A 200-fold molar excess of reduced glutathione (Sigma Aldrich) in 800 mM arginine, pH 10.0 was added and the assembly reaction was incubated at room temperature with exposure to air for 72 hours to allow the knob and hole half antibodies to anneal and form hinge disulfides. Anti-M1 / anti-K63, anti-RIP1 / gD, anti-RIP2 / gD, anti-M1 / gD, and anti-K63 / gD control bispecific antibodies were similarly assembled.

[0391] C. Antibody Purification and Characterization In vitro assembled bispecific antibodies were purified by hydrophobic interaction chromatography (HIC) using a modified version of a previously described method. See Yau et al., Cell. 171(4):918-933.e20.doi:10.1016 / j.cell.2017.09.040.Epub 2017 Oct 12. PMID:29033132; PMCID:PMC5669814(2017). Briefly, 3.8 M ammonium sulfate was added to the assembly reaction to a final concentration of at least 1 M, and the reaction was filtered through a 0.22 μm filter and loaded onto a 5 μm, 7.8×75 mm ProPac HIC-10 column (Dionex). The column was then washed with Buffer A (20 mM sodium acetate, pH 5.0) and a 40 column volume (CV) linear gradient of 0-100% Buffer B (25 mM sodium phosphate, pH 6.5, 25% isopropanol) was applied to separate the bispecific antibodies from unreacted half antibodies or aggregated proteins. The identity of the eluted peak was monitored by SDS-PAGE and mass spectrometry (see below for method details) and fractions corresponding to the bispecific peak were pooled.

[0392]

[0270] The bispecific antibodies were further purified by cation exchange chromatography (CEX). Briefly, the HIC pooled material was dialyzed into 20 mM sodium acetate (pH 5.0) or 1 / 6 volume of 1 M sodium acetate (pH 5.0) was added to lower the pH to about 5, then diluted with water to a final concentration of 13 mM sodium acetate. The antibody was then loaded onto a 10 μm Mono S 5 / 50 GL column (GE Healthcare) and washed with Buffer A (20 mM sodium acetate, pH 5.0). A linear gradient of 0-100% Buffer B (20 mM sodium acetate, pH 5.0, 1 M NaCl) over 40 CV was run and the desired fractions were pooled. Purified bispecific antibodies were formulated in either 20 mM histidine acetate, 240 mM sucrose, 0.02% TWEEN®-20, or 20 mM histidine acetate, 150 mM NaCl, pH 5.5.

[0393]

[0271] The identity of the purified and annealed species was confirmed using LC-MS. To reduce heterogeneity, the antibodies were deglycosylated with PNGaseF prior to analysis.

[0394] D. RIP1-K63 ubiquitin chain bispecific antibody recognizes protein modifications Human colon cancer HT29 cells were treated for 2 hours with a combination of TNF (20 ng / ml), IAP antagonist BV6 (2 μM), and pan-caspase inhibitor zVAD (20 μM) (collectively TBZ) as generally described in Almagro et al., Cell Death and Differentiation, 24:26-37 (2017). Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain or control bispecific antibodies. Total cell lysates (TCL) and immunoprecipitated proteins (IP) were probed with the indicated antibodies as shown in Figure 2A.

[0395] Fibrosarcoma HT1080 cells were treated with PBS or TNF (100 ng / ml) for 7 min. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in Figure 2B.

[0396] A549 cells were treated with PBS or TNF (500ng / ml) for 7 min. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT), incubated in 6M urea buffer without Triton (30 min, RT), permeabilized with 0.25% Triton (10 min, RT), and then stained with the indicated RIP1-ubiquitin chain bispecific or control antibodies, followed by staining with anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. Mounted slides were analyzed with a Leica SP8 confocal microscope. All images were uniformly collected using the same settings. The results are shown in Figure 2C.

[0397] HT1080 cells were treated with PBS or TNF (500 ng / ml) for 7 min. Cells were fixed, incubated in 6 M urea buffer (30 min, RT), permeabilized, and then stained with the indicated RIP1-ubiquitin chain bispecific or control antibodies, followed by staining with anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. All images were uniformly collected using the same settings [scale bar 25 μm]. The results are shown in Figure 2D.

[0398] EVSA T cells were treated with PBS or TNF (100 ng / ml) for 7 min. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies. The results are shown in Figure 3A.

[0399] Ku812F cells were treated with PBS or Flag-TL1A (10 μg) cross-linked with anti-Flag AB for 10 min. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies. The results are shown in Figure 3B.

[0400]

[0278] This shows that the RIP1-K63 ubiquitin chain bispecific antibody recognizes K63 chain ubiquitinated RIP1 (Figures 2A-2D) and RIP1 modified with K63-linked ubiquitin chains (Figures 3A-3B).

[0401] This is also shown in Figure 1A-D. The specificity of the RIP1-K63 bispecific antibodies was verified by in vitro ubiquitinated RIP1, where RIP1 was modified with K63- or K48-tetraubiquitin molecules. After ubiquitination, RIP1 was incubated with the indicated bispecific antibodies in the presence of 6 M urea to disrupt non-covalent bonds, precipitated, and examined by Western blotting. The RIP1-K63 bispecific antibodies immunoprecipitated K63-ubiquitin chain-modified RIP1, but not K48-ubiquitin chain-modified RIP1.

[0402] E. RIP1-Lin ubiquitin chain bispecific antibody recognizes protein modifications HT29 cells were treated with PBS or TNF (20 ng / ml), BV6 (2 μM) and zVAD (20 μM) for 2 hours as described in Example D. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin, K63-linear ubiquitin chain, or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in FIG. 4A.

[0403] Ku812F cells were treated with PBS or TNF (100 ng / ml) for 10 min. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain, K63-linear ubiquitin chain or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in Figure 4B.

[0404] HT29 cells were treated with PBS or TNF (100ng / ml), BV6 (2μM) and zVAD (20μM) for 2,5 hours. Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature (RT), incubated in 6M urea buffer without Triton (30 minutes, RT), permeabilized with 0.25% Triton (10 minutes, RT) and then stained with the indicated RIP1-ubiquitin chain bispecific or control antibodies, followed by staining with anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. Mounted slides were analyzed on a Leica SPE confocal microscope. All images were uniformly collected using the same settings. The results are shown in Figure 4C.

[0405] Mouse embryonic fibroblast (MEF) cells were treated with PBS or TBZ (TNF (100 ng / ml), BV6 (1 μM) and zVAD (20 μM)) for 2.5 h. Cells were fixed, incubated in 6 M urea buffer (30 min, RT), permeabilized and then stained with the indicated RIP1-ubiquitin chain bispecific or control antibodies, followed by staining with an anti-human goat F(ab')2 fragment antibody conjugated to Cy3. Hoechst 33258 was used for nuclear staining. All images were uniformly collected using the same settings [scale bar 25 μm]. The results are shown in Figure 4D.

[0406] These results indicate that the RIP1-Lin ubiquitin chain bispecific antibody recognizes linear ubiquitinated RIP1, whereas the RIP1-gD and gD-Lin antibodies, or the unstimulated K63-Lin antibody, did not immunoprecipitate ubiquitinated RIP1.

[0407]

[0285] This is also shown in Figure 1B, where in vitro ubiquitination of RIP1 with tetra-K48 or linear tetraubiquitin molecules in 6 M urea buffer followed by immunoprecipitation demonstrated the ability of the RIP1-Lin bispecific antibody to selectively immunoprecipitate RIP1 modified with linear ubiquitin chains, but not K48-linked ubiquitin chains.

[0408] F. RIP1-Lin and K63-Lin ubiquitin chain bispecific antibodies recognize modified RIP1 HT29 cells were treated with PBS or TNF (50 ng / ml) for 7 min. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain, K63-linear ubiquitin chain or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in Figure 5A.

[0409] D645 cells were treated with PBS or TNF (100 ng / ml) for 7 min. Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain, K63-linear ubiquitin chain or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in Figure 5B.

[0410] THP1 cells were treated with PBS, TNF (100 ng / ml, 5 min), or MDP (1 μg / ml, 30 min). Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain, K63-linear ubiquitin chain bispecific antibodies, or control antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies, as shown in FIG. 5C.

[0411]

[0289] Thus, the RIP1-K63 and RIP1-Lin bispecific antibodies selectively recognize RIP1 modified with K63-linked and K63-Lin ubiquitin-linked chains, respectively, but not RIP2 or TRAF2.

[0412]

[0290] The K63-Lin bispecific antibody also recognizes modified RIP1 and modified RIP2, but there are no RIP1-specific or RIP2-specific components in this antibody.

[0413] G. RIP1-ubiquitin chain bispecific antibody recognizes RIP1 ubiquitination in vivo Mice were treated with PBS (mice M1 and M2) or TNF (500 μg / ml) for 12 min (mice M3 and M4) or 24 min (mice M5 and M6). Small intestines from the indicated mice were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP1-ubiquitin chain or control bispecific antibodies. Immunoprecipitated proteins (FIG. 6A) and whole cell lysate proteins (FIG. 6B) were probed with the indicated antibodies, showing that the RIP1-ubiquitin chain bispecific antibodies recognize RIP1 ubiquitinated in vivo with K63-linked and linear ubiquitin chains.

[0414] Bone marrow-derived macrophages (BMDMs) were isolated from mice with wild-type RIP1 or RIP1 K376R knock-in mice, treated with TNF for 5 min, lysed in 6 M urea buffer, and immunoprecipitated using the indicated RIP1-ubiquitin chain or control bispecific antibodies. Detection of total cell lysate proteins (FIG. 6C) and immunoprecipitated proteins (FIG. 6D) was performed using the indicated antibodies. This indicates that K63-linked and linear ubiquitination of RIP1 in BMDMs from RIP1 K376R knock-in mice can be detected using RIP1-ubiquitin chain bispecific antibodies.

[0415] H. RIP2-ubiquitin chain bispecific antibody recognizes modified RIP2 THP1 cells were treated with PBS or the NOD2 signaling activator MDP (1 μg / ml, 30 min). Cells were lysed in 6 M urea buffer and immunoprecipitated using the indicated RIP2-ubiquitin chains or control bispecific antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in FIG. 7A. The results showed that K63-linked ubiquitination of RIP2 was detected in a stimulation-dependent manner only when both RIP2 and K63 arms were present in the bispecific (RIP2-K63), but not in the control bispecifics containing only one of the relevant arms (RIP2-gD or K63-gD).

[0416] THP1 cells were treated with PBS or MDP (1 μg / ml, 30 min). Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP2-ubiquitin chain, K63-linear ubiquitin chain bispecific antibodies or control antibodies. Total cell lysates and immunoprecipitated proteins were probed with the indicated antibodies as shown in FIG. 7B. RIP2-Lin antibody, but not RIP2-gD or gD-Lin antibody, can successfully immunoprecipitate RIP2 modified with linear ubiquitin, but not XIAP or c-IAP1. K63-Lin bispecific antibody efficiently captures RIP2 modified with mixed and / or branched K63-linked and linear ubiquitin chains, and to some extent XIAP.

[0417] THP1 cells were treated with PBS or MDP (1 μg / ml, 30 min). Cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT), incubated in 6 M urea buffer without Triton (30 min, RT), permeabilized with 0.25% Triton (10 min, RT), and then stained with the indicated RIP2-ubiquitin chain bispecific antibodies or control antibodies, followed by staining with anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) cells and RIP2 KO (knockout) THP1 cells is shown in Figure 7C. The results confirm that RIP2-ubiquitin chain bispecific antibodies recognize RIP2 ubiquitinated by linear and K63-Lin ubiquitin-linked chains. Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) and RIP2 KO (knockout) THP1 cells is shown in Figure 7D. Mounted slides were analyzed on a Leica SPE confocal microscope. All images were uniformly collected using the same settings. These results are shown in Figure 7E. RIP2-K63, RIP2-Lin, and K63-Lin bispecific antibodies revealed stimulation-dependent localization of K63-linked and linear RIP2 ubiquitination in cells by immunofluorescence, but not RIP2-gD, gD-K63, or gD-Lin bispecific antibodies. The immunofluorescence detected with the K63-Lin antibody is likely to be primarily, but not exclusively, the result of RIP2 ubiquitination, since this antibody weakly immunoprecipitated XIAP in MDP-treated cells.

[0418] This indicates that the RIP2-ubiquitin chain bispecific antibody recognizes K63 and linear chain ubiquitinated RIP2. In contrast, single-arm (RIP2-gD, K63-gD, Lin-gD) antibodies do not recognize ubiquitinated RIP2, and even the RIP2-K63, RIP2-Lin, and K63-Lin bispecific antibodies recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway-related stimuli (MDP).

[0419] I. Ubiquitination of RIP2 in Crohn's disease and ulcerative colitis patient samples

[0297] The RIP2-K63 and RIP2-Lin bispecific antibodies were tested in intestinal tissue samples from patients (Figures 8A-G). Patients who underwent intestinal resection surgery for the treatment of colon cancer, dysplasia, diverticulitis (DVC), Crohn's disease (CD) or ulcerative colitis (UC) were enrolled in an observational study (Table 2). TIFF2024534853000003.tif64170N / A, Not Applicable. NR, not recorded.

[0420]

[0298] After surgery, intestinal tissue samples from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC) were lysed in 6M urea lysis buffer and examined by immunoprecipitation with the indicated bispecific antibodies.

[0421] FIG. 8A shows examples of immunoprecipitation from intestinal cancer, dysplasia, Crohn's disease or ulcerative colitis samples with the indicated bispecific antibodies. RIP2-K63 and RIP2-Lin bispecific antibodies immunoprecipitated ubiquitinated RIP2, with the strongest signals observed in CD and UC samples. FIG. 8C and 8D show samples 1-52 (FIG. 8C) and samples 53-92 (FIG. 8D) from individual patients. The last lane in each panel contains immunoprecipitation with a combination of antibodies RIP2-K63 and RIP2-Lin from MDP-treated (1 μg / ml, 30 min) THP1 cells and serves as a control (Ct). Tissue lysates from the above patients prepared in 6 M urea lysis buffer were examined by Western blotting with RIP2 and GAPDH antibodies. Red asterisks indicate samples that were excluded from immunoprecipitation due to poor quality and low protein levels. THP1 cell lysates serve as a control. This is shown in Figure 8E.

[0422] Samples from patients with intestinal cancer, dysplasia, diverticulitis (DIV), Crohn's disease (CD) or ulcerative colitis (UC) were lysed in 6M urea lysis buffer and the lysates were immunoprecipitated with a combination of RIP2-K63 and RIP2-Lin bispecific antibodies. Western blotting was performed with anti-RIP2 antibodies as shown in Figure 8B. To better compare RIP2 ubiquitination signals between different samples, proteins immunoprecipitated by RIP2-K63 and RIP2-Lin bispecific antibodies were compared on side-by-side gels, again demonstrating significant RIP2 ubiquitination in CD and UC samples (Figure 8B).

[0423] As shown in Figures 8C and 8D, RIP2 ubiquitination in the indicated patient samples was quantified by scanning Western blots after immunoprecipitation with the indicated antibodies. Scanned images were processed with ImageJ software, and RIP2 ubiquitination was calculated as the ratio of gel intensity from immunoprecipitation with RIP2-K63 / RIP2-Lin antibodies to gel intensity from immunoprecipitation with gD antibodies, as shown in Figure 8F. Ns indicates no significant difference, and three asterisks indicate p<0.0001. Figure 8F also showed a striking pattern of elevated K63-linked and linear ubiquitination of RIP2 in CD and UC samples compared to intestinal cancer, dysplasia, and diverticulitis samples. Risk alleles for IBD have been identified using genome-wide association studies to identify common risk alleles for disease development (Liu et al., 2015). Two CD risk alleles identified in the population as a whole, ATG16L1 and NOD2, were further evaluated in this cohort, with NOD2 being of particular interest as it is downstream of MDP.

[0424] This is shown in Figure 8G, where the RIP2 ubiquitination quantified in Figure 8F is shown in bar graph format. Below the graph is the genotype information for two common IBD risk loci: A stands for ATG16L1 and N stands for NOD2. RIP2 ubiquitination levels were found to be elevated in Crohn's disease and ulcerative colitis patient samples. Comparison of RIP2 ubiquitination levels with ATG16L1 and NOD2 risk alleles in Figure 8F showed no association, suggesting that RIP2 ubiquitination in IBD samples is not correlated with these risk loci. Taken together, these data indicate that the RIP2-K63 and RIP2-Lin bispecific antibodies can be used to investigate the ubiquitination status of RIP2 in IBD samples.

[0425] J. Proteins modified with K63-linked and linear ubiquitin chains

[0303] Figure 9A provides a schematic of the experimental design scheme. THP1 cells were treated with PBS, TNF (100 ng / ml), MDP (1 μg / ml) or LPS (1 μg / ml) for the indicated times, lysed in 6 M urea lysis buffer, immunoprecipitated with K63-Lin bispecific antibody and analyzed by mass spectrometry.

[0426] Immunoprecipitated proteins were separated by SDS-PAGE, excised, and analyzed by mass spectrometry, revealing distinct ubiquitinated substrates depending on the stimulation. Figure 9B provides proteins identified by mass spectrometry. Numbers indicate total and unique (in parentheses) number of peptides identified for each protein. TNF stimulation identified known (RIP1, TNFR1) and novel (TRADD) ubiquitinated substrates, while MDP and LPS treatment identified prominent K63-linked and linear ubiquitination of RIP2, NOD2, and IRAK1, respectively. This is also shown in Figures 10A-10E. The most robust identification signal obtained from mass spectrometry analysis was that of RIP2 after MDP treatment.

[0427] THP1 cells were treated with the indicated stimuli as in Figure 9A, lysed in 6M urea lysis buffer, and immunoprecipitated with K63-Lin bispecific antibody. Immunoprecipitated and whole cell lysate proteins were probed with the indicated antibodies as shown in Figure 9C. The immunoprecipitation and Western blot confirmed that the proteins found to be ubiquitinated by mass spectrometry were indeed modified by K63-linked and linear ubiquitination.

[0428] Selected ms / ms spectra matching the RIP2 C-terminus SPSLNLLQNKSM (SEQ ID NO: 102) were annotated with matching fragment ions (FIG. 9D) and their extracted ion chromatography (XIC) from the four treatment conditions were illustrated. Matching XIC peaks were highlighted with arrows (FIG. 9E), identifying ubiquitinated RIP2 from the MDP treatment condition. ubshows ubiquitination at position K538 of RIP2. ox shows oxidation of position M540 of the RIP2 polypeptide, indicating that K538 is the major RIP2 modification site by K63-linked and / or linear ubiquitination. Taken together, these demonstrate that the K63-Lin bispecific antibody can be used to identify specific ubiquitination sites in addition to general protein modifications.

[0429]

[0307] This demonstrates that the K63-Lin bispecific antibody can be used to detect and identify K63-linked and linear chain ubiquitinated proteins in different signaling pathways.

[0430]

[0308] Although the above-described invention has been described in some detail by way of explanation and examples for purposes of clarity of understanding, the explanations and examples should not be construed as limiting the scope of the invention. TIFF2024534853000004.tif248170TIFF2024534853000005.tif228170TIFF20245 34853000006.tif234170TIFF2024534853000007.tif229170TIFF202453485300000 8.tif234170TIFF2024534853000009.tif228170TIFF2024534853000010.tif2331 70TIFF2024534853000011.tif228170TIFF2024534853000012.tif234170TIFF2024 534853000013.tif229170TIFF2024534853000014.tif234170TIFF2024534853000015.tif252170TIFF2024534853000016.tif244170TIFF2024534853000017.tif224170TIFF2024534853000018.tif233170TIFF2024534853000019.tif252170TIFF2024534853000020.tif137170HC=heavy chain;LC=light chain;HCVR=heavy chain variable region;LCVR=light chain variable region;HVR=hypervariable region.

Claims

1. 1. A multispecific antibody comprising: a first half-antibody comprising a first antigen-binding site that binds to polyubiquitin; and a second half-antibody comprising a second antigen-binding site that binds to receptor-interacting protein kinase 2 (RIP2); (1) The first half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 40; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 41; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 or (2) the first half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 51; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 54; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 56 and The second half antibody is (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 95; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 96; (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 97; (iv) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 98; (v) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 99; and (vi) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 100 Including, antibody.

2. The first half antibody is a) a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 36; or b) a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 49, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

50. The antibody of claim 1, comprising:

3. the second half antibody having a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:93, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94; The antibody of claim 1 or 2, comprising: Claim 4: One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:49, and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:50; 3. The antibody of claim 1 or 2, wherein the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93 and a VH sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

94.

5. 3. The antibody of claim 1 or 2, wherein the first half antibody comprises the VL sequence of SEQ ID NO:49 and the VH sequence of SEQ ID NO:

50.

6. 3. The antibody of claim 1 or 2, wherein the second half antibody comprises the VL sequence of SEQ ID NO:93 and the VH sequence of SEQ ID NO:

94. Claim 7: a) one of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 35 and the VH sequence of SEQ ID NO: 36; the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; or b) one of the first and second half antibodies comprises the VL sequence of SEQ ID NO: 49 and the VH sequence of SEQ ID NO: 50; the other of the first half antibody and the second half antibody comprises the VL sequence of SEQ ID NO: 93 and the VH sequence of SEQ ID NO: 94; The antibody of claim 1 or 2.

8. The antibody of claim 1 or 2, which is a monoclonal antibody.

9. The antibody of claim 1 or 2, which is a mouse antibody, a rabbit antibody, a human antibody, a humanized antibody, or a chimeric antibody.

10. 3. The antibody of claim 1 or 2, wherein the first antigen-binding site is human or humanized and / or the second antigen-binding site is human or humanized.

11. 3. The antibody of claim 1 or 2, wherein the antibody is an IgG antibody, in particular, the antibody is an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody, or the antibody is an IgG1 or IgG4 antibody.

12. The first half antibody is a) a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34; or b) a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 48 Including; 3. The antibody of claim 1 or 2, optionally having a C-terminal lysine deleted from one or more heavy chains.

13. The first half antibody is a) a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34; or b) a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

48. Including; 3. The antibody of claim 1 or 2, optionally having a C-terminal lysine deleted from one or more heavy chains.

14. 3. The antibody of claim 1 or 2, wherein the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

84.

15. (i) the second half antibody comprises a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90; optionally, a C-terminal lysine is deleted from one or more heavy chains; (ii) the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 84 and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90; optionally, a C-terminal lysine is deleted from one or more heavy chains; (iii) the second half antibody comprises a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 92; optionally, a C-terminal lysine is deleted from one or more heavy chains; or (iv) the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 84, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 92; optionally, a C-terminal lysine is deleted from one or more heavy chains. The antibody of claim 1 or 2. Claim 16: a) a first half antibody comprising a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; b) the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 30, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90; c) the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:92; or d) the first half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:44, and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:48; the second half antibody comprises a light chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:84 and a heavy chain sequence having at least about 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90, optionally with a C-terminal lysine deleted from one or more heavy chains; The antibody of claim 1 or 2.

17. The second half antibody is (i) comprises the light chain sequence of SEQ ID NO: 84; (ii) the second half antibody comprises the heavy chain sequence of SEQ ID NO: 90; optionally, the C-terminal lysine is deleted from one or more heavy chains; (iii) the second half antibody comprises the light chain sequence of SEQ ID NO: 84 and the heavy chain sequence of SEQ ID NO: 90; optionally, a C-terminal lysine is deleted from one or more of the heavy chains; (iv) the second half antibody comprises a heavy chain of SEQ ID NO: 92; optionally, the C-terminal lysine is deleted from one or more of the heavy chains; or (v) a second half antibody comprises the light chain sequence of SEQ ID NO: 84 and the heavy chain sequence of SEQ ID NO: 92; optionally, a C-terminal lysine is deleted from one or more heavy chains. The antibody of claim 1 or 2.

18. A method for producing a human ovarian tumor graft comprising administering to a mammalian subject the invention comprising administering to said subject the invention a method for producing a human ovarian tumor graft ... the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:92; b) a chain sequence of SEQ ID NO: 30 and a heavy chain sequence of SEQ ID NO: 34; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:90; c) the first half antibody comprises the light chain sequence of SEQ ID NO: 44 and the heavy chain sequence of SEQ ID NO: 46; the second half antibody comprises the light chain sequence of SEQ ID NO:84 and the heavy chain sequence of SEQ ID NO:92; or d) the first half antibody comprises the light chain sequence of SEQ ID NO: 44 and the heavy chain sequence of SEQ ID NO: 48; 3. The antibody of claim 1 or 2, wherein the second half antibody comprises a light chain sequence of SEQ ID NO: 84 and a heavy chain sequence of SEQ ID NO: 90, and optionally a C-terminal lysine is deleted from one or more heavy chains.

19. 3. A composition comprising the antibody of claim 1 or 2, wherein the composition is substantially free of monospecific antibodies, disassembled half antibodies, or both monospecific antibodies and disassembled half antibodies.

20. An immunoconjugate comprising the antibody of claim 1 and a cytotoxic or anti-inflammatory agent.

21. a pharmaceutically acceptable carrier; and a) an antibody according to claim 1 or 2; or b) the immunoconjugate of claim 20 and at least one of: Optionally, the pharmaceutical formulation, wherein the composition is substantially free of monospecific antibodies, disassembled half antibodies, or both monospecific antibodies and disassembled half antibodies.

22. An isolated nucleic acid encoding the antibody of claim 1 or 2.

23. A vector comprising the nucleic acid of claim 22.

24. A host cell comprising the nucleic acid of claim 22.

25. 25. A method of producing an antibody, comprising culturing the host cell of claim 24 under conditions in which the antibody is produced, and optionally further comprising recovering the antibody from the host cell.

26. 3. A method of producing the antibody of claim 1 or 2, comprising forming the antibody from a first half antibody and a second half antibody.

27. 3. The antibody of claim 1 or 2 for use as a pharmaceutical.

28. A method for determining the presence of polyubiquitinated proteins in a sample suspected of containing polyubiquitin or polyubiquitinated proteins, the method comprising exposing the sample to an antibody described in claim 1 or 2 and determining binding of the antibody to polyubiquitinated proteins in the sample.

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