CN117957249A

CN117957249A - Anti-polyubiquitin multispecific antibodies

Info

Publication number: CN117957249A
Application number: CN202280058536.1A
Authority: CN
Inventors: M·L·松本; D·武契奇; T·贡恰罗夫
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2021-08-30
Filing date: 2022-08-29
Publication date: 2024-04-30

Abstract

The present disclosure provides multispecific antibodies 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, such as receptor interacting protein kinase 1 (RIP 1) or receptor interacting protein kinase 2 (RIP 2); and methods for using the antibodies.

Description

Anti-polyubiquitin multispecific antibodies

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/238,749 filed on 8-30 of 2021 and U.S. provisional application No. 63/247,776 filed on 9-23 of 2021, each of which is incorporated herein by reference in its entirety for all purposes.

Technical Field

The present invention relates to anti-polyubiquitin multispecific antibodies and methods of making and using the same.

Introduction and summary of the invention

Ubiquitin is a small protein with important regulatory roles in a variety of cellular pathways. Of these, the most notable is the role of ubiquitin in protein degradation, where covalent attachment of ubiquitin to target proteins enables targeted proteins to be recognized and destroyed by 26S proteasomes (see Wilkinson, semin. Cell development. Biol.11 (3): 141-148 (2000)). Covalent attachment of ubiquitin (a 76 amino acid protein) to a target protein is a three-step enzymatic process (Pickart, annu. Rev. Biochem.70:503-533 (2001)). First, ubiquitin activating enzyme E1 forms ubiquitin-E1 thioesters in an ATP-dependent reaction. In a second step, ubiquitin is transesterified from ubiquitin-E1 to a member of the ubiquitin conjugating enzyme (E2) family. In a third step, an isopeptide bond is formed between the carboxy terminus of ubiquitin and the epsilon-amino group of a lysine residue on the target protein with the aid of ubiquitin protein ligase (E3). An enzyme called deubiquitinase removes ubiquitin moieties from target proteins (Guterman and Glickman, curr. Prot. Pep. Sci.5:201-210 (2004)).

Ubiquitin contains seven lysine residues (Lys 6, lys11, lys27, lys29, lys48 and Lys 63) and thus ubiquitin itself can be used as a target protein for ubiquitination (Peng et al, nat. Biotechnol.21:921-926 (2003); pickurt and Fushman, curr. Opin. Chem. Biol.8:610-616 (2004)). The molecules produced after ubiquitination of ubiquitin proteins are known as polyubiquitin molecules and may comprise two or more ubiquitin moieties.

In addition, a linear polyubiquitin linkage (also called M1 linkage) is formed, wherein the C-terminal glycine of ubiquitin is conjugated to the alpha-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 a linear ubiquitin chain assembly complex (LUBAC) consisting of two ring finger proteins HOIL-1L and HOIP. Tokunaga et al, nat.cell biol.11:123-132 (2009). It is believed that genetically encoded non-anchored linear polyubiquitin is not present in the cell because its C-terminus is easily cleaved by isopeptidase T. Iwai and Tokunaga, EMBO Reports 10:706-713 (2009). This observation suggests that linear polyubiquitin is posttranslationally assembled onto the underlying protein, and that conjugated linear polyubiquitin molecules are potential modulators of protein activity and function. Linear polyubiquitination of, for example, NF- κb essential modulators (NEMO) has been shown to play a role in NF- κb activation. Different ubiquitin chains can transmit specific and different biochemical and biological messages, leading to activation or abrogation of Cell signaling or modulation of protein stability (Ikeda, f. Et al, cell 143:677-681 (2010); rape, m., nat Rev Mol Cell Biol 19:59-70 (2018)).

The receptor interacting protein-1 kinase ("RIP 1" or "RIPK 1") is a serine/threonine protein kinase. RIP1 is a modulator of cell signaling that is involved in, among other things, the pathway of programmed cell death (e.g., necrotic apoptosis). The most extensively studied necrotic cell death form is triggered by TNFa (tumor necrosis factor), but necrotic apoptosis can also be induced by other members of the TNFa death ligand family (Fas and TRAIL/Apo 2L), interferon, toll-like receptor (TLR) signaling and viral infection via the DNA sensor DAI (DNA-dependent interferon regulatory factor 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,M.C. and Vucic, D.Semin Cell Dev biol.39:56-62 (2015). Binding of TNFa to TNFRl (TNF receptor 1) promotes trimerization of TNFRl and formation of intracellular complexes, complex-I. TRADD (TNF receptor-related death domain protein) binds to the intracellular death domain of TNFRl and recruits the protein kinase RIP1 (receptor-interacting protein 1) through the death domain present in both proteins. Chen, Z.J.immunological reviews 246:95-106 (2012).

Receptor-interacting serine/threonine protein kinase 2 ("RIP 2" or "RIPK 2") is a serine/threonine/tyrosine kinase that plays an important role in the regulation of adaptive and innate immune responses. Upon recruitment by activated NOD1 and NOD2 through the CARD-CARD domain, RIPK2 autophosphorylates and undergoes K63-linked polyubiquitination by ubiquitin ligases XIAP, BIRC2 and BIRC 3. The polyubiquitin induces K63-linked polyubiquitination of IKBKG/NEMO and subsequent IKBKB/IKKB activation. NF- κb is then released and translocated into nuclei that activate transcription of hundreds of genes involved in immune responses, growth control or prevention of apoptosis.

It would be beneficial to provide compositions and methods that can recognize and differentiate modifications of RIP1 and RIP2 proteins, and to provide compositions and methods that effectively target and modulate polyubiquitin-mediated pathways. The present disclosure is directed to meeting one or more of these needs or to providing other benefits.

The following non-limiting examples are provided.

Example 1. A method of determining the presence of a polyubiquitin in a sample suspected of containing a polyubiquitin, wherein the polyubiquitin is a pro-inflammatory protein and comprises polyubiquitin,

The method comprises the following steps: 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 the binding of at least one antibody to polyubiquitin in the sample.

Example 2. The method according to example 1, wherein the polyubiquitin comprises M1-linked polyubiquitin and/or K63-linked polyubiquitin.

Embodiment 3. The method of any one of the preceding embodiments, wherein the pro-inflammatory protein is a component of one or more signaling complexes.

Embodiment 4. The method of any one of the preceding embodiments, wherein the pro-inflammatory protein is receptor interacting protein kinase 1 (RIP 1), receptor interacting protein kinase 2 (RIP 2), apoptosis inhibitors 1 and 2 (c-IAP 1/2), tumor necrosis factor receptor 1 (TNFR 1), linear ubiquitin chain assembly complex (LUBAC), and/or nuclear factor- κb (NF- κb) essential modulator (NEMO).

Embodiment 5. The method of any one of the preceding embodiments, wherein the pro-inflammatory protein is RIP1.

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

Embodiment 7. The method of any one of the preceding embodiments, wherein the pro-inflammatory protein has an elevated level of ubiquitination in the inflammatory state relative to the level of ubiquitination when not in the inflammatory state.

Embodiment 8. The method of any one of the preceding embodiments, wherein the pro-inflammatory protein has a level of ubiquitination 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 the inflammatory state relative to the level of ubiquitination when not in the inflammatory state.

Embodiment 9. The method of any one of the preceding embodiments, wherein an elevated level of ubiquitination is correlated with an increase in the severity of an inflammatory disease state.

Embodiment 10. The method of any of the preceding embodiments, wherein the proinflammatory protein is associated with an inflammatory disease such as inflammatory bowel disease, crohn's disease, diverticulitis, and ulcerative colitis.

Embodiment 11. The method of any of the preceding embodiments, wherein the proinflammatory protein is associated with crohn's disease.

Embodiment 12. The method of any of the preceding embodiments, wherein the pro-inflammatory protein is associated with ulcerative colitis.

Example 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 (RIP 1).

Example 14. The antibody of example 13, which selectively recognizes polyubiquitinated RIP1.

Example 15 the antibody of example 13 or 14, which does not recognize receptor interacting protein kinase 2 (RIP 2) and/or does not recognize non-ubiquitinated RIP1.

Example 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 (RIP 2).

Example 17 an antibody according to example 16 that selectively recognizes polyubiquitinated RIP2.

Example 18 the antibody of example 16 or 17, which does not recognize RIP1, XIAP, and/or c-IAP1, and/or does not recognize non-ubiquitinated RIP2.

Embodiment 19. The antibody of any one of embodiments 13 to 18, wherein the polyubiquitin has a homogenous topology.

Embodiment 20. The antibody of any one of embodiments 13 to 19, wherein polyubiquitin comprises a K11 bond.

Embodiment 21. The antibody of any one of embodiments 13 to 20, wherein the polyubiquitin comprises a K48 bond.

Embodiment 22. The antibody of any one of embodiments 13 to 21, wherein the polyubiquitin comprises a K63 bond.

Embodiment 23. The antibody of any one of embodiments 13 to 22, wherein the polyubiquitin comprises an M1 bond.

Embodiment 24. The antibody of any one of embodiments 13 to 23, comprising a first half antibody comprising:

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 alternatively

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.

Embodiment 25 the antibody according to any one of embodiments 13 to 15 or 19 to 24,

Comprising a second half antibody comprising:

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 alternatively

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.

Embodiment 26. The antibody of any one of embodiments 16 to 24, comprising a second half antibody comprising:

(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. .

Embodiment 27 the antibody of any one of embodiments 13 to 26 comprising a first half antibody and a second half antibody, wherein

A. The first half antibody comprises

(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,

And the second half antibody comprises

(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;

b. The first half antibody comprises

(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,

And the second half antibody comprises

(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;

c. The first half antibody comprises

(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,

And the second half antibody comprises

(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;

d. 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 comprises

(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;

e. 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 comprises

(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;

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,

And the second half antibody comprises

(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

(V) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 100;

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,

And the second half antibody comprises

(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;

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,

And the second half antibody comprises

(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;

i. 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 comprises

(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;

j. 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 comprises

(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;

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,

And the second half antibody comprises

(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;

First half antibody comprising

(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 another second half antibody comprises

(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.

Embodiment 28 the antibody of any one of embodiments 13 to 27, wherein the first half

The antibody comprises

A. A VL sequence having at least about 95%, such as 96%,97%,98%,99% or 100% sequence identity to SEQ ID No. 7, a VH sequence having at least about 95%, such as 96%,97%,98%,99% or 100% sequence identity to SEQ ID No. 8;

b. A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 21, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 22;

c. A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 35, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 36; or alternatively

D. a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 49, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 50.

Embodiment 29 the antibody of any one of embodiments 13-15, 19-25, or 27-28, wherein the second half antibody comprises

A. A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 71, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 73; or alternatively

B. A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 72, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 73.

Embodiment 30 the antibody of any one of embodiments 16 to 25 or 26 to 28,

Wherein the second half-antibody comprises

(I) A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 93, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 94.

Embodiment 31 the antibody of any one of embodiments 10 to 27, wherein:

a. One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 7, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 8,

(I) And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 71, and a VH sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 7, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 8,

(I) And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 72, and a VH sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 7, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 8,

(I) And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 93, and a VH sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22,

e. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22,

f. One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22,

g. One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36,

h. One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36,

i. One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36,

j. one of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50,

k. One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50,

(I) And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 72, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 73; or alternatively

One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50,

(I) And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 93 and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 94.

Embodiment 32 the antibody of any one of embodiments 13 to 31, wherein the first half antibody comprises

A VL sequence of SEQ ID NO. 7 and a VH sequence of SEQ ID NO. 8;

The VL sequence of SEQ ID NO. 21 and the VH sequence of SEQ ID NO. 22;

a VL sequence of SEQ ID NO. 35 and a VH sequence of SEQ ID NO. 36; or alternatively

The VL sequence of SEQ ID NO. 49 and the VH sequence of SEQ ID NO. 50.

Embodiment 33 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, or 31 to 32, wherein the second half antibody comprises

A VL sequence of SEQ ID NO. 71 and a VH sequence of SEQ ID NO. 73; or alternatively

The VL sequence of SEQ ID NO. 72 and the VH sequence of SEQ ID NO. 73.

Embodiment 34 the antibody of any one of embodiments 16 to 24, 26 to 28, or 30 to 32, wherein the second half antibody comprises

(I) The VL sequence of SEQ ID NO. 93 and the VH sequence of SEQ ID NO. 94.

Embodiment 35 the antibody of any one of embodiments 13 to 34, wherein:

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) And 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 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) And 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) And 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,

e. one of the first half antibody and the second half antibody comprises SEQ ID NO:

21 and the VH sequence of SEQ ID NO. 22,

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,

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,

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. 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,

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,

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) And 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 alternatively

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) And 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.

Embodiment 36. The antibody of any one of embodiments 13 to 35, which is a monoclonal antibody.

Embodiment 37 the antibody of any one of embodiments 13-36, which is a mouse antibody, a rabbit antibody, a human antibody, a humanized antibody, or a chimeric antibody.

Embodiment 38. The antibody of any one of embodiments 13 to 37, wherein the first antigen binding site is human or humanized.

Embodiment 39. The antibody of any one of embodiments 13 to 38, wherein the second antigen binding site is human or humanized.

Embodiment 40. The antibody of any one of embodiments 13 to 39, wherein the antibody is an IgG antibody.

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

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

Embodiment 43 the antibody of any one of embodiments 13-42, 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 knob mutation; or 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 knob mutation.

Embodiment 44. The antibody of embodiment 43, wherein the antibody is an IgG1 antibody, and wherein the knob mutation comprises a T366W mutation.

Embodiment 45. The antibody of embodiment 43 or embodiment 44, wherein the antibody is an IgG1 antibody, and wherein the mortar mutation comprises at least one mutation, at least two mutations, or three mutations, such as one to two mutations, one to three mutations, or two to three mutations, selected from T366S, L a and Y407V.

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

Embodiment 47. The antibody of embodiment 43 or embodiment 46, wherein the antibody is an IgG4 antibody, and wherein the mortar mutation comprises at least one mutation, at least two mutations, or three mutations, such as one to two mutations, one to three mutations, or two to three mutations, selected from the group consisting of T366S, L a and Y407V mutations.

Embodiment 48 the antibody of any one of embodiments 43 to 47, wherein the first half antibody comprises

A. A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2;

b. a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 16;

c. a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30; or alternatively

D. a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 44.

Embodiment 49 the antibody of any one of embodiments 13 to 48, wherein the first half antibody comprises

A. A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 4;

b. A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 18;

c. A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 32; or alternatively

D. A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 50 the antibody of any one of embodiments 13 to 49, wherein the first half antibody comprises

A. A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 4;

b. a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 16, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 18;

c. a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 32; or alternatively

D. A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 44, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 51 the antibody of any one of embodiments 13 to 48, wherein the first half antibody comprises

A. a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 6;

b. a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20;

c. A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 34; or alternatively

D. a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 52 the antibody of any one of embodiments 13 to 48 or 51, wherein the first half antibody comprises

A. a light chain sequence having at least about 95%, such as 96%,97%,98%,99% or 100% sequence identity to SEQ ID No. 2, and a heavy chain sequence having at least about 95%, such as 96%,97%,98%,99% or 100% sequence identity to SEQ ID No. 6;

b. A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 16, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20;

c. a light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 34; or alternatively

D. A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 44, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 53 the antibody of 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.

Embodiment 54 the antibody of any one of embodiments 13 to 15, 19 to 15, 27 to 29, 31 to 33, or 35 to 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; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 55 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 35 to 54, 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 and a heavy chain sequence having at least 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 68; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 56 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 33 to 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; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 57 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 53, or 56, 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 and a heavy chain sequence having at least 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 70; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 58 the antibody of 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.

Embodiment 59 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 31, 34 to 52, or 5528, 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. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 60 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, or 58 to 59, 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 61 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, or 58, 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. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 62 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 33 to 52, 58, or 61, 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 63 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 35 to 57, wherein:

a. The first 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. 2, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4,

And 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, and a heavy chain sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 2, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 6,

And 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 and a heavy chain sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 16, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 18,

And 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 and a heavy chain sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 16, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20,

e. The first 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. 30, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 32,

f. A chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 30, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 34,

And 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, and a heavy chain sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 44, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 46,

h. The first 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. 44, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 48,

And 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, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 68; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 64 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, or 58 to 62, wherein:

And 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 92;

And 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 90;

f. The first 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. 30, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 34,

And 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 65 the antibody of any one of embodiments 13 to 64, wherein the first half antibody comprises

A light chain sequence of SEQ ID NO. 2;

The light chain sequence of SEQ ID NO. 16;

c.30 light chain sequence of SEQ ID NO; or alternatively

The light chain sequence of SEQ ID NO. 44.

Embodiment 66. The antibody of any one of embodiments 13 to 65, wherein the first half antibody comprises

A heavy chain sequence of SEQ ID NO. 4;

The heavy chain sequence of SEQ ID NO. 18;

The heavy chain sequence of SEQ ID NO. 32; or alternatively

The heavy chain sequence of SEQ ID NO. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 67 the antibody of any one of embodiments 13 to 66, wherein the first half antibody comprises

A light chain sequence of SEQ ID NO. 2 and a heavy chain sequence of SEQ ID NO. 4;

the light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 18;

a light chain sequence of SEQ ID NO. 30 and a heavy chain sequence of SEQ ID NO. 32; or alternatively

The light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 68 the antibody of any one of embodiments 13-65, wherein the first half antibody comprises

A heavy chain sequence of SEQ ID NO. 6;

The heavy chain sequence of SEQ ID NO. 20;

c.the heavy chain sequence of SEQ ID NO. 34; or alternatively

The heavy chain sequence of SEQ ID NO. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 69 the antibody of any one of embodiments 13 to 65 or 68, wherein the first half antibody comprises

A light chain sequence of SEQ ID NO. 2 and a heavy chain sequence of SEQ ID NO. 6;

the light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 20;

The light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 34; or alternatively

The light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 70 the antibody of 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 the light chain sequence of SEQ ID No. 60.

Embodiment 71 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 54, 63, or 65 to 70, wherein the second half antibody comprises the heavy chain sequence of SEQ ID No. 68; optionally wherein the heavy chain lacks a C-terminal lysine.

Embodiment 72 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 55, 63, or 65 to 71, wherein the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 73 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 54, 63, or 65 to 70, wherein the second half antibody comprises the heavy chain sequence of SEQ ID No. 70; optionally wherein the heavy chain lacks a C-terminal lysine.

Embodiment 74 the antibody of any one of embodiments 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 54, 56, 63, 65 to 70, or 73, wherein the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 75 the antibody of 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.

Embodiment 76 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 59, 64 to 69, or 75, wherein the second half antibody comprises the heavy chain sequence of SEQ ID No. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 77 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 60, 64 to 69, or 75 to 76, wherein 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 wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 78 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58, 61, 64 to 69, or 75, wherein the second half antibody comprises the heavy chain sequence of SEQ ID No. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 79 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58, 61 to 62, 64 to 69, 75 or 78 wherein the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

Embodiment 80 the antibody of any one of embodiments 16 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, or 65 to 74, wherein:

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,

And 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,

And 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,

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,

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,

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,

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,

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,

And the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 68, optionally with one or more of the heavy chains missing a C-terminal lysine.

Embodiment 81 the antibody of any one of embodiments 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, or 75 to 79 wherein:

And the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 92;

And the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 90;

the chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 34,

And 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 with one or more of the heavy chains missing a C-terminal lysine.

Embodiment 82 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No.2 and the heavy chain sequence of SEQ ID No. 4, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 83 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No.2 and the heavy chain sequence of SEQ ID No. 6, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 84 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 18, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Example 85 the antibody of any one of examples 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74, or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 20, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 86 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 32, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 87 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 34, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 88 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 46, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Example 89 the antibody of any one of examples 13-15, 19-25, 27-29, 31-33, 35-57, 63, 65-74, or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 48, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 90 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 4, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 91 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 6, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 92 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 18 and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 93 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 20, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 94 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 32 and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 95 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 34, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 96 the antibody of 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 the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 46, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 97 the antibody of any 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 the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 48, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

Embodiment 98 the antibody of any one of embodiments 13-97, which is a bispecific antibody.

Embodiment 99 the antibody of any one of embodiments 13 to 98, which is a diabody, a triabody or a tetrabody.

Embodiment 100. The antibody of any one of embodiments 13 to 99, which is conjugated to a label.

Embodiment 101. The antibody of embodiment 100 wherein the label is a fluorescent label, an enzymatic label, or a chromogenic label.

Example 102. The antibody of example 100, wherein the label is a radioisotope, the radioisotope optionally being a positron emitter, the positron emitter optionally being ⁸⁹ Zr.

Embodiment 103. A composition comprising the antibody of any one of embodiments 13 to 102, wherein the composition is substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies.

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

Embodiment 105 a pharmaceutical formulation comprising a pharmaceutically acceptable carrier and at least one of:

a) The antibody of any one of embodiments 13 to 102; or alternatively

B) The immunoconjugate according to example 104;

b. optionally wherein the composition is substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies.

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

Embodiment 107 an isolated nucleic acid encoding the antibody of any one of embodiments 13 to 102.

Embodiment 108. A vector comprising the nucleic acid of embodiment 107.

Example 109 a host cell comprising a nucleic acid according to example 107.

Embodiment 110. A method of producing an antibody, the method comprising culturing the host cell of embodiment 106 under conditions wherein the antibody is produced.

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

Embodiment 112. A method of making an antibody according to any one of embodiments 13 to 102, the method comprising forming an antibody from a first half antibody and a second half antibody.

Embodiment 113 the antibody according to any one of embodiments 13 to 102 for use as a medicament.

Embodiment 114. A method of determining the presence of a polyubiquitin in a sample suspected of containing the polyubiquitin or polyubiquitin, the method comprising exposing the sample to an antibody according to any one of embodiments 13 to 102, and determining binding of the antibody to the polyubiquitin in the sample.

Embodiment 115. A method of separating a K11-linked polyubiquitin from a non-K11-linked polyubiquitin in a sample, the method comprising contacting the sample with an antibody according to any one of embodiments 13 to 102.

Embodiment 116. A method of separating a K48-linked polyubiquitin from a non-K48-linked polyubiquitin in a sample, the method comprising contacting the sample with an antibody according to any one of embodiments 13 to 102.

Embodiment 117 a method of separating K63-linked polyubiquitin from non-K63-linked polyubiquitin in a sample, the method comprising contacting the sample with an antibody according to any one of embodiments 13 to 102.

Embodiment 118. A method of separating an M1-linked polyubiquitin from a non-M1-linked polyubiquitin in a sample, the method comprising contacting the sample with an antibody according to any one of embodiments 13 to 102.

Embodiment 119. A method of determining the function and/or activity of a polyubiquitin in a cell or sample, the method comprising contacting the cell or sample with an antibody according to any one of embodiments 13 to 102, and assessing the effect of said contacting step on the cell or sample.

Embodiment 120 the method of any one of embodiments 114-119, wherein the polyubiquitin comprises RIP1.

Embodiment 121. The method of any one of embodiments 114-119, wherein the polyubiquitin comprises RIP2.

Embodiment 122. A method of determining the presence of a polyubiquitin in a sample suspected of containing a polyubiquitin, wherein the polyubiquitin is a pro-inflammatory protein and comprises polyubiquitin, the method comprising exposing the sample to an antibody according to any one of embodiments 10 to 99.

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

Embodiment 124. The method of embodiment 122 or 123, wherein the pro-inflammatory protein is a component of one or more signaling complexes.

Embodiment 125 the method of any one of embodiments 122-124, wherein the pro-inflammatory protein is RIP1.

Embodiment 126 the method of any one of embodiments 122-125, wherein the pro-inflammatory protein is RIP2.

Embodiment 127. The method of any one of embodiments 122-126, wherein the proinflammatory protein has an elevated ubiquitination level in an inflammatory state relative to the ubiquitination level when not in an inflammatory state.

Embodiment 128 the method of any one of embodiments 122-127, wherein the pro-inflammatory protein has a level of ubiquitination 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 the inflammatory state relative to the level of ubiquitination when not in the inflammatory state.

Embodiment 129 the method of any one of embodiments 122 to 128, wherein the increased level of ubiquitination is correlated with an increase in the severity of the inflammatory disease state.

Embodiment 130 the method of any one of embodiments 122-129, wherein the proinflammatory protein is associated with an inflammatory disease such as inflammatory bowel disease, crohn's disease, diverticulitis, and ulcerative colitis.

Embodiment 131 the method of any one of embodiments 122-130, wherein the proinflammatory protein is associated with crohn's disease.

Embodiment 132 the method of any one of embodiments 122-131, wherein the pro-inflammatory protein is associated with ulcerative colitis.

Drawings

FIGS. 1A to 1D show the interaction of a RIP1-K63 bispecific antibody with K63 ubiquitin bond modified RIP1. FIGS. 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. FIGS. 1C and 1D provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) and RIP1 KO (knockdown) HT29 cells. The indicated antibodies recognize RIP1 modified with K63-linked ubiquitin chains.

Figures 2A to 2D show the interaction of RIP 1-ubiquitin chain bispecific antibodies or control antibodies in various cell lines, including human colon carcinoma HT29 cells, fibrosarcoma HT1080 cells, and a549 cells. In particular, FIGS. 2A through 2D demonstrate that the RIP1-K63 ubiquitin chain bispecific antibody recognizes K63 chain ubiquitinated RIP1. FIGS. 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 fig. 2C (a 549 cells) and fig. 2D (HT 1080 cells).

FIGS. 3A-3D show the interaction of a RIP1-K63 bispecific antibody with a ubiquitin chain modified RIP1 linked with K63 in various cell lines, including EVSA T cells, ku812F cells, 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. FIGS. 3C and 3D provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) and RIP1 KO (knockdown) HT29 cells. The indicated antibodies recognize linear chain ubiquitinated RIP1.

Figures 4A to 4D show the interaction of RIP 1-ubiquitin chain bispecific antibodies, K63-linear ubiquitin chain bispecific antibodies or control bispecific antibodies in various cell lines including human colon carcinoma HT29 cells, ku812F cells and Mouse Embryo Fibroblasts (MEF) cells. In particular, FIGS. 4A through 4D demonstrate that RIP1-Lin ubiquitin chain bispecific antibodies recognize linear chain ubiquitinated RIP1, whereas RIP1-gD antibodies and gD-Lin antibodies or non-stimulated K63-Lin antibodies do not immunoprecipitate ubiquitinated RIP1. FIGS. 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 fig. 4C (HT 29 cells) and fig. 4D (MEF cells).

FIGS. 5A through 5C show the interaction of RIP 1-ubiquitin chain bispecific antibodies, K63-linear ubiquitin chain bispecific antibodies, or control bispecific antibodies in various cell lines including human colon carcinoma HT29 cells, D645 cells, and THP1 cells. FIGS. 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 a K63 connecting strand and a K63-Lin ubiquitin connecting strand.

FIGS. 6A to 6D show the interaction of a RIP 1-ubiquitin chain bispecific antibody, a K63-linear ubiquitin chain bispecific antibody or a control bispecific antibody in a mouse tissue sample. FIGS. 6A and 6B provide Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. The indicated antibodies recognize RIP1 ubiquitinated in vivo with K63 linked ubiquitin chains and linear ubiquitin chains. Figures 6C and 6D provide western blot analysis of immunoprecipitated proteins and TCL obtained as a result of the indicated antibodies. The indicated antibodies recognize ubiquitinated RIP1 in wild-type (WT) or mutant RIP1 (RIP 1K 376R knock-in) mouse bone marrow-derived macrophages.

Figures 7A to 7E show the interaction of RIP 2-ubiquitin chain bispecific antibodies or control bispecific antibodies in THP1 cells. In particular, FIGS. 7A through 7D demonstrate that the RIP 2-ubiquitin chain bispecific antibody recognizes K63 and linear chain ubiquitinated RIP2, but that the single arm (RIP 2-gD, K63-gD, lin-gD) antibodies do not recognize ubiquitinated RIP2, and even the RIP2-K63 bispecific antibody, RIP2-Lin bispecific antibody, and K63-Lin bispecific antibody recognize K63 and linear chain ubiquitinated RIP2 only after treatment with pathway dependent stimulus (MDP). Figures 7A and 7B provide western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies. FIG. 7C provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) and RIP2 KO (knockdown, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 ubiquitinated by the linear chain and the K63-Lin ubiquitin linker chain. The indicated antibodies recognize K63 and linear chain ubiquitinated RIP2. FIG. 7D provides Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) and RIP2 KO (knockdown, R2 KO) THP1 cells. The indicated antibodies recognize RIP2 modified with K63-linked ubiquitin chains. These results were confirmed by a confocal microscope shown in fig. 7E.

FIGS. 8A through 8G show the RIP2-K63 bispecific antibody and RIP2-Lin bispecific antibody tested in intestinal tissue samples from patients. In particular, FIGS. 8A through 8F demonstrate that RIP2-K63 bispecific antibodies and RIP2-Lin bispecific antibodies can be used to study 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 collected from intestinal cancer, dysplasia, diverticulitis (DIV), crohn's Disease (CD), or Ulcerative Colitis (UC) patients. Fig. 8C and 8D show samples 1-52 and samples 53-92, respectively, from the patient of fig. 8A. FIG. 8E provides expression of RIP2 in patient samples. FIG. 8F provides a quantification of RIP2 ubiquitination by scanning Western blotting after immunoprecipitation with the antibodies indicated in FIGS. 8C and 8D. Fig. 8G shows the data of fig. 8F in bar chart format.

FIGS. 9A through 9E illustrate the mechanism by which K63-Lin bispecific antibodies can be used to detect and identify proteins ubiquitinated with K63-linked chains and linear chains in different signaling pathways. Fig. 9A provides a schematic of an experimental design in which THP1 cells were treated with vehicle (phosphate buffered saline, PBS), tumor Necrosis Factor (TNF), muramyl Dipeptide (MDP) or Lipopolysaccharide (LPS) for the indicated period of time, followed by lysis and immunoprecipitation with the indicated bispecific antibody and Mass Spectrometry (MS) analysis to identify the protein in fig. 9B, as also determined in the western blot analysis of fig. 9C. Ubiquitinated RIP2 was identified from MDP-treated conditions by tandem mass spectrometry (ms/ms, fig. 9D) and extraction ion chromatography (XIC), with matching peaks highlighted by arrows (fig. 9E).

FIGS. 10A through 10E show the identification of TRADD (FIG. 10A) (SEQ ID NO: 104), TNFR1 (FIG. 10B) (SEQ ID NO: 105), RIP1 (FIG. 10C) (SEQ ID NO: 106), NOD2 (FIG. 10D) (SEQ ID NO: 107) and IRAK1 (FIG. 10E) (SEQ ID NO: 108) from the conditions indicated in FIGS. 9A and 9B. A representative ms/ms spectrum for each protein is noted.

Detailed description of certain embodiments

I. Definition of the definition

As used herein, VH refers to a heavy chain variable domain and VL refers to a light chain variable domain.

For purposes herein a "recipient human framework" is a framework comprising an amino acid sequence derived from a VL framework or a VH framework of a human immunoglobulin framework or a human consensus framework as defined below. The recipient human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence as the human immunoglobulin framework or human consensus framework, or it 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.

As used herein, "about" refers to more or less than 10% of the specified value, giving a result functionally equivalent to the specified value or rounding to the specified value.

"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). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"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 comprising one or more antigens). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Bispecific antibodies will typically have greater avidity for a binding partner comprising an epitope recognized by both antigen binding sites of the bispecific antibody than for a binding partner comprising either epitope alone. Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinities are described below. The term "functional affinity" is sometimes used in the art to refer to avidity.

An "affinity matured" antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVRs) that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody that does not have such alterations.

The term "antibody" is used herein in its broadest sense and covers 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" refers to an antibody that comprises an antigen-binding domain that has a multispecific specificity (i.e., an antibody that is capable of binding to two or more different epitopes on one molecule, or an epitope on two or more different molecules).

As used herein, an "agonist antibody" is an antibody that mimics at least one functional activity of a polypeptide of interest.

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

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

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody and binds to an 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; a diabody antibody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

By "antibody that binds to the same epitope" as the reference antibody is meant an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, whereas the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

As used herein, the term "anti-polyubiquitin antibody" refers to an antibody that is capable of specifically binding to a polyubiquitin molecule. In certain embodiments, antibodies that bind polyubiquitin have a dissociation constant (Kd) of 1. Mu.M, 100nM, 10nM, 1nM, 0.1nM, 0.01nM or 0.001nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M).

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 ubiquitin molecules.

As used herein, the term "anti-RIP 1 antibody" refers to an antibody that is capable of specifically binding to a receptor interacting protein kinase 1 ("receptor interacting protein-1 kinase", "RIP1" or "RIPK 1") molecule. In certain embodiments, the antibody that binds RIP1 has a dissociation constant (Kd) of +.1μM, +.100deg.M, +.10nM, +.1nM, +.0.1 nM, +.0.01 nM, or+.0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M, e.g., 1-20nM, such as 1-15nM, e.g., 1-12nM, e.g., 1-10 nM).

As used herein, the term "anti-RIP 2 antibody" refers to an antibody that is capable of specifically binding to a receptor-interacting protein kinase 2 ("receptor-interacting serine/threonine protein kinase 2", "RIP2" or "RIPK 2") molecule. In certain embodiments, the antibody that binds RIP2 has a dissociation constant (Kd) of +.1μM, +.100deg.M, +.10nM, +.1nM, +.0.1 nM, +.0.01 nM, or+.0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M).

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is often characterized by uncontrolled cell growth/proliferation. Examples of cancers include, but are not limited to, malignant tumors, lymphomas (e.g., hodgkin's lymphoma and non-hodgkin's lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer (LIVER CANCER), bladder cancer, liver cancer (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer (LIVER CANCER), prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatic carcinoma), leukemia and other lymphoid tissue proliferative disorders, and various types of head and neck cancer.

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

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. There are five main classes of antibodies: igA, igD, igE, igG and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG ₁、IgG₂、IgG₃、IgG₄、IgA₁ and IgA ₂. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., ,At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof such as nucleolytic enzymes; an antibiotic; 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 antitumor or anticancer agents disclosed below.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the variation of the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); b cell activation.

An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to achieve a desired therapeutic or prophylactic result at the necessary dosage and time period.

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

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al (Sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD, 1991).

As used herein, "first," "second," etc., refer to elements having complex structures, e.g., proteins having tertiary/quaternary structures such as antibodies, to refer to those elements (e.g., monomers, chains, domains) without any implication regarding ordering or positioning of the elements; thus, a "first" element may be at the C-terminus or N-terminus of a second element, or closer to or farther from one end or the other of the structure than the second element. Thus, for example, reference to half of a bispecific antibody, designating half or bond as either first or second is arbitrary.

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

The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein.

The term "half antibody" is used herein to refer to one arm of an antibody and includes at least a VH domain and a CH domain.

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

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

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 rabbit cell, or an amino acid sequence derived from a non-rabbit derived antibody that utilizes a repertoire of rabbit antibodies or other rabbit antibody coding sequences.

A "human consensus framework" is a framework that represents the amino acid residues that are most commonly present in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. In general, a subset of sequences is as described in Kabat et al Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication 91-3242, bethesda MD (1991), volumes 1-3. In some embodiments, for VL, the subgroup is subgroup κi as in Kabat et al, supra. In some embodiments, for VH, the subgroup is subgroup III as in Kabat et al, supra.

"Humanized" antibody refers to chimeric antibodies that comprise amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, such as one or two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody and all or substantially all of the FRs correspond to FRs of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a non-human antibody, in a "humanized form" refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop ("hypervariable loop"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs) that 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 50-65 of amino acid residues 31-35B, H of H1, and amino acid residues 95-102 of H3. (Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD (1991)). In addition to CDR1 in VH, CDRs typically comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues" or "SDRs," which are residues that contact an antigen. SDR is contained within CDR regions known as shortened 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, H of H1, amino acid residues 50-58 of H3, and amino acid residues 95-102. (see Almagro and Franson, front. Biosci.13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al.

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).

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

"Inflammatory diseases" include Crohn's disease, diverticulitis, graft Versus Host Disease (GVHD), inflammatory bowel disease, kidney injury and loss of graft function, multiple sclerosis, rheumatoid arthritis, skin inflammatory diseases, stroke, and ulcerative colitis.

An "isolated antibody" is an antibody that has been isolated from a component of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (isoelectric focusing, IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods of assessing antibody purity, see, e.g., flatman et al, J.chromatogrB 848:79-87 (2007).

An "isolated nucleic acid" refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

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

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabeled. Naked antibodies may be present in pharmaceutical formulations.

"Natural antibody" refers to naturally occurring immunoglobulin molecules having different structures. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminal to the C-terminal, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH 1, CH2 and CH 3). Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), also known as a variable light chain domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of its constant domain.

"Or" is used in an inclusive sense, i.e., equivalent to "and/or," unless the context requires otherwise.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the candidate sequence to the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. The alignment used to determine the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared. However, for purposes herein, the sequence comparison computer program ALIGN-2 was used to generate% amino acid sequence identity. ALIGN-2 sequence comparison computer programs were written by Genntech, inc., and the source code had been submitted with the user document to U.S. Copyright Office, washington D.C.,20559, where it was registered with U.S. copyright accession number TXU 510087. ALIGN-2 programs are publicly available from Genntech, inc. (Inc., south San Francisco, california) or may be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, which includes the digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were unchanged.

In the case of amino acid sequence comparison using ALIGN-2, the amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B (which may alternatively be expressed as having or comprising some amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

Wherein X is the number of amino acid residues scored as identical matches in the program alignment of A and B by the sequence alignment program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that in the case where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a to B will not be equal to the% amino acid sequence identity of B to a. All values of% amino acid sequence identity as used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless specifically indicated otherwise.

The term "pharmaceutical formulation" refers to a formulation that is in a form that allows for the biological activity of the active ingredient contained therein to be effective, and that is free of additional components that have unacceptable toxicity to the subject to whom the formulation is to be administered.

"Pharmaceutically acceptable carrier" refers to ingredients of the pharmaceutical formulation that are non-toxic to the subject, except for the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

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

As used herein, "substantially free" means that the recited entity is not present, or if present, is (i) in an amount sufficiently low so as not to significantly alter the functional properties or results of the composition, method, use, or step, as the case may be; (ii) Undetectable by at least one suitable analytical method, such as mass spectrometry (e.g., MALDI-TOF or any MS procedure used in the examples), blotting (e.g., western blotting for polypeptides), or electrophoresis (e.g., SDS-PAGE with coomassie blue or silver staining); or (iii) is present in an amount of less than or equal to about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% by mass or mole fraction relative to the total amount of non-solvent material in the composition.

As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treatment") refers to a clinical intervention that attempts to alter the natural course of the treated individual, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating a disease state, and alleviating or improving prognosis. In some embodiments, the antibodies disclosed herein are used to delay the progression of a disease or slow the progression of a disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVR). (see, e.g., kit et al Kuby Immunology, 6 th edition, w.h. freeman and co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated using VH or VL domains, respectively, from antibodies that bind that antigen to screen libraries of complementary VL or VH domains. See, e.g., portolano et al, J.Immunol.150:880-887 (1993); clarkson et al Nature 352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

II compositions and methods

In some aspects, antibodies that bind to polyubiquitin proteins, such as polyubiquitin pro-inflammatory proteins, are provided. Such antibodies can be used, for example, to detect polyubiquitin, to modulate the activity of polyubiquitin, or to immunoprecipitate polyubiquitin.

A. exemplary antibodies

In some embodiments, the multispecific antibody comprises 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 1 (RIP 1). In some embodiments, the antibody selectively recognizes polyubiquitinated RIP1. In some embodiments, the antibody does not recognize receptor interacting protein kinase 2 (RIP 2) and/or does not recognize non-ubiquitinated RIP1.

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 (RIP 2). In some embodiments, the antibody selectively recognizes polyubiquitinated RIP2. In some embodiments, the antibody does not recognize receptor interacting protein kinase 1 (RIP 1) and/or does not recognize non-ubiquitinated RIP2.

In some embodiments, the polyubiquitin comprises a K11, K48, K63, or M1 (C-terminal to N-terminal) bond. In some embodiments, the polyubiquitin has a homogenous topology.

The antibodies, antibody sequences, and sequence listing disclosed in U.S. patent No. 7,763,245 are incorporated herein by reference. In some embodiments, the first half of the antibody comprising a first antigen binding site that binds to polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody that binds to polyubiquitin disclosed in U.S. patent No. 7,763,245. In some embodiments, the first half antibody comprising a first antigen binding site that binds to polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVR disclosed in U.S. patent No. 7,763,245, wherein the half antibody binds to polyubiquitin.

The antibodies, antibody sequences, and sequence listing disclosed in U.S. patent No. 8,133,488 are incorporated herein by reference. In some embodiments, the first half of the antibody comprising a first antigen binding site that binds to polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody that binds to polyubiquitin disclosed in U.S. patent No. 8,133,488. In some embodiments, the first half antibody comprising a first antigen binding site that binds to polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVR disclosed in U.S. patent No. 8,133,488, wherein the half antibody binds to polyubiquitin.

The antibodies, antibody sequences, and sequence listing disclosed in U.S. patent No. 8,992,919 are incorporated herein by reference. In some embodiments, the first half of the antibody comprising a first antigen binding site that binds to polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody that binds to polyubiquitin disclosed in U.S. patent No. 8,992,919. In some embodiments, the first half antibody comprising a first antigen binding site that binds to polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVR disclosed in U.S. patent No. 8,992,919, wherein the half antibody binds to polyubiquitin.

The antibodies, antibody sequences, and sequence listing disclosed in U.S. patent No. 9,321,844 are incorporated herein by reference. In some embodiments, the first half of the antibody comprising a first antigen binding site that binds to polyubiquitin comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of the antibody that binds to polyubiquitin disclosed in U.S. patent No. 9,321,844. In some embodiments, the first half antibody comprising a first antigen binding site that binds to polyubiquitin comprises a combination of HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 selected from the HVR disclosed in U.S. patent No. 9,321,844, wherein the half antibody binds to polyubiquitin.

In some embodiments, the first half of the 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 amino acid sequence in SEQ ID NO:9, 10, 11, 12, 13 and 14, respectively. In some embodiments, the first half of the 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 amino acid sequence in SEQ ID NO:23, 24, 25, 26, 27 and 28, respectively. In some embodiments, the first half of the 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 amino acid sequence in SEQ ID NO:37, 38, 39, 40, 41 and 42, respectively. In some embodiments, the first half of the 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 amino acid sequence in SEQ ID NO:51, 52, 53, 54, 55 and 56, respectively.

In some embodiments, the second half antibody comprises a second antigen binding site that binds RIP1 and comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprising amino acid sequence 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 RIP1 and comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3 comprising amino acid sequence 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.

In some embodiments, the second half antibody comprises a second antigen binding site that binds RIP2 and comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2 and HVR-H3 comprising amino acid sequence 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.

In some embodiments, the first half antibody comprises

(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, and

The second half antibody comprises

(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.

In some embodiments, the first half antibody comprises

(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, and

The second half antibody comprises

(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.

In some embodiments, the first half antibody comprises

(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, and the second half antibody comprising

(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.

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 comprising (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.

(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,

(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,

(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 comprising (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,

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 comprising (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,

(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,

(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,

(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 comprising (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,

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 comprising (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,

(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,

(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,

(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 comprises

(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.

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.

In some embodiments, the first half antibody comprises VH and VL sequences at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL sequences of the polyubiquitin-binding antibodies disclosed in U.S. patent No. 7,763,245. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. patent No. 7,763,245, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises VH and VL sequences at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL sequences of the polyubiquitin-binding antibodies disclosed in U.S. patent No. 8,133,488. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. patent No. 8,133,488, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises VH and VL sequences at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL sequences of the polyubiquitin-binding antibodies disclosed in U.S. patent No. 8,992,919. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. patent No. 8,992,919, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises VH and VL sequences at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH and VL sequences of the polyubiquitin-binding antibodies disclosed in U.S. patent No. 9,321,844. In some embodiments, the first half antibody comprises a combination of VH and VL sequences disclosed in U.S. patent No. 9,321,844, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises the antibody VH and VL sequences 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, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises the antibody VH and VL sequences 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, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises the antibody VH and VL sequences 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, wherein the half antibody binds polyubiquitin.

In some embodiments, the first half antibody comprises the antibody VH and VL sequences 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, wherein the half antibody binds polyubiquitin.

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.

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.

In some embodiments, a) 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: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.

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, 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: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 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, 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.

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, 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: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 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, 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.

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, 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: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 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, 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.

In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an antibody comprising the sequence retains the ability to bind to polyubiquitin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted in the VH sequence. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody comprises a VH sequence discussed above, including post-translational modifications of the sequence.

In certain embodiments, VL sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but antibodies comprising the sequences retain the ability to bind to polyubiquitin. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and/or deleted in the VL sequence. In certain embodiments, the substitution, insertion, or deletion occurs in a region outside the HVR (i.e., in the FR). Optionally, the antibody comprises a VL sequence as discussed above, including post-translational modifications of the sequence.

In some embodiments, the antibody is humanized. In some embodiments, the antibody comprises an HVR in any one of the above embodiments, and further comprises a human acceptor framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the antibody comprises the HVR of any one of the above embodiments and a rabbit framework region.

In some aspects, antibodies that bind to the same epitope as the bispecific antibodies provided herein are provided. For example, in certain embodiments, antibodies that bind to the same epitope as an antibody comprising a first half antibody and a second half antibody are provided, wherein:

a) The first half antibody comprises the VL sequence of SEQ ID NO. 7 and the VH sequence of SEQ ID NO. 8, and 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,

And 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, and 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,

And 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,

f) The first half antibody comprises the VL sequence of SEQ ID NO. 21 and the VH sequence of SEQ ID NO. 22,

And 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,

h) The first half antibody comprises the VL sequence of SEQ ID NO. 35 and the VH sequence of SEQ ID NO. 36,

i) The first half antibody comprises the VL sequence of SEQ ID NO. 35 and the VH sequence of SEQ ID NO. 36,

j) The first half antibody comprises the VL sequence of SEQ ID NO. 49 and the VH sequence of SEQ ID NO. 50,

l) the first half antibody comprises the VL sequence of SEQ ID NO. 49 and the VH sequence of SEQ ID NO. 50,

And the second half antibody comprises the VL sequence of SEQ ID NO. 72 and the VH sequence of SEQ ID NO. 73; or alternatively

And the second half antibody comprises the VL sequence of SEQ ID NO. 93 and the VH sequence of SEQ ID NO. 94.

In some embodiments, bispecific antibodies comprising a VH sequence and a VL sequence in one of a) to l) in the preceding paragraph are provided.

In some embodiments, the antibody is a monoclonal antibody, including a chimeric, humanized, or human antibody. In some embodiments, the antibody is an antibody fragment, e.g., a dimeric scFv, diabody, or F (ab') ₂ fragment. In another embodiment, the antibody is a substantially full length antibody, e.g., an IgG1, igG2a, igG2b, igG3, or IgG4 antibody or other antibody class or isotype as defined herein.

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, and no C-terminal lysine. The C-terminal lysine may be removed, for example, enzymatically (such as by carboxypeptidase treatment), or genetically (such as by deletion or substitution of the lysine codon at the 3' end of the heavy chain coding sequence). The heavy chain C-terminal lysine is located remotely from the antigen binding site and is optional for binding activity, and its removal can provide a more homogeneous antibody preparation.

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 alternatively

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, 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,

Optionally wherein one or more heavy chains lacks a C-terminal lysine.

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 alternatively

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, 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,

Optionally wherein one or more of the chains lacks a C-terminal lysine.

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 wherein one or more heavy chains lacks a C-terminal lysine.

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 wherein one or more heavy chains lacks a C-terminal lysine.

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 wherein one or more heavy chains lacks a C-terminal lysine.

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 wherein one or more heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein

A) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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, and 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 wherein one or more of the chains lacks a C-terminal lysine.

A) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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 and 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) The 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, and 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 wherein one or more of the chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 4, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 6, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 18, and the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 20, and the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 32, and the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 34, and the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 46, and the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 70, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 48, and the second half antibody comprises the light chain sequence of SEQ ID NO. 60 and the heavy chain sequence of SEQ ID NO. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 2 and the heavy chain sequence of SEQ ID NO. 4, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 6, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 18, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 20, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 32, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO.30 and the heavy chain sequence of SEQ ID NO. 34, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 46, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 92, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In some embodiments, the antibody comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 48, and the second half antibody comprises the light chain sequence of SEQ ID NO. 84 and the heavy chain sequence of SEQ ID NO. 90, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

In a further aspect, an antibody according to any of the above embodiments may incorporate any of the features described in paragraphs 1-7 below, alone or in combination.

1. Affinity for antibodies

In certain embodiments, antibodies provided herein have a dissociation constant (Kd) of ∈1 μΜ, +.100 nM, +.10 nM, +.1 nM, +.0.1 nM, +.0.01 nM, or +.0.001 nM, and optionally +.10 10 ^-13 M (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M).

In some embodiments, kd is measured by radiolabeled antigen binding assay (RIA) with an antibody of interest and its antigen in Fab form as described in the assay below. The solution binding affinity of Fab to antigen is measured by equilibrating Fab with a minimum concentration of (¹²⁵ I) labeled antigen in the presence of a series of unlabeled antigen titrations, followed by capture of bound antigen with an anti-Fab antibody coated plate (see, e.g., chen et al, j. Mol. Biol.293:865-881 (1999)). To determine the conditions for the assay, 5. Mu.g/ml of capture anti-Fab antibody (Cappel Labs) in 50mM sodium carbonate (pH 9.6) was coatedThe multiwell plate (Thermo Scientific) was overnight and then blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (about 23 ℃). In a non-adsorbed plate (Nunc# 269620), 100pM or 26pM [ ¹²⁵ I ] -antigen was mixed with serial dilutions of the Fab of interest (e.g., consistent with the assessment of anti-VEGF antibody Fab-12 in Presta et al, cancer Res.57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, incubation may last longer (e.g., about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture was transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution was then removed and 0.1% polysorbate 20 in PBS The plate was washed eight times. When the plates have dried, 150 μl/well of scintillator (MICROSCINT-20 ^TM; packard) is added and the plates are counted for tens of minutes on a TOPCON ^TM gamma counter (Packard). The concentration of each Fab that gave less than or equal to 20% of maximum binding was selected for use in the competitive binding assay.

In some embodiments, the immobilized antigen CM5 chip is used at about 10 Response Units (RU) at 25 ℃-2000 Or-3000 (BIAcore, inc., piscataway, NJ), kd is measured by surface plasmon resonance assay. Briefly, carboxymethylated dextran biosensor chips (CM 5, BIACORE, inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen was diluted to 5. Mu.g/ml (about 0.2. Mu.M) with 10mM sodium acetate, pH 4.8, followed by injection at a flow rate of 5. Mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions (0.78 nM to 500 nM) of Fab in PBS containing 0.05% polysorbate 20 (TWEEN-20 ^TM) surfactant (PBST) were injected at 25℃at a flow rate of about 25. Mu.l/min. Simple one-to-one Langmuir binding model (/ >) was used by fitting the association and dissociation sensorgrams simultaneouslyThe rate of association (k _on) and rate of dissociation (k _off) were calculated by the assessment software version 3.2. The equilibrium dissociation constant (Kd) is calculated as the ratio k _off/k_on. See, for example, seyforth et al, J.mol. Biol.293:865-881 (1999). If the association rate is above 10 ⁶M^-1s^-1 as determined by the above surface plasmon resonance assay, the association rate can be determined by using a fluorescence quenching technique, i.e. an increase or decrease in fluorescence emission intensity (excitation wavelength=295 nM; emission wavelength=340 nM, bandpass=16 nM) of 20nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 ℃ in the presence of increasing concentrations of antigen as measured with a stirred cuvette in a spectrometer such as a spectrometer equipped with a flow stop device (Aviv Instruments) or 8000 series SLM-amico ^TM spectrophotometer (ThermoSpectronic).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, the F (ab') ₂ fragment, 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 reviews of scFv fragments, see, e.g., pluckth, U.S. Pat. No. The Pharmacology of Monoclonal Antibodies, volume 113, rosenburg and Moore editions, (Springer-Verlag, new York), pages 269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For a discussion of Fab and F (ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

In some embodiments, the antibody is a diabody antibody. A diabody antibody is an antibody fragment having two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat.Med.9:129-134 (2003); and Hollinger et al, proc.Natl. Acad. Sci. USA 90:6444-6448 (1993).

A single domain antibody is an antibody fragment comprising 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, the single domain antibody is a human single domain antibody (domntis, inc., waltham, MA; see, e.g., U.S. patent No. 6,248,516B1).

Antibody fragments may be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies, produced by recombinant host cells, such as e.coli (e.coli) or phage, as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Pat. 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 non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switch" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, the non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Typically, 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 optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are reviewed in, for example, almagro and Franson, front. Biosci.13:1619-1633 (2008), and further described, for example, in Riechmann et al, nature 332:323-329 (1988); queen et al, proc.Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. 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) porting); padlan, mol. Immunol.28:489-498 (1991) (describing "surface reshaping"); dall' Acqua et al, methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al, methods 36:61-68 (2005) and Klimka et al, br.J.cancer,83:252-260 (2000) (described "guide selection" Methods for FR shuffling).

Human architecture regions that may be used for humanization include, but are not limited to: the framework regions selected using the "best fit" method (see, e.g., sims et al, J. Immunol.151:2296 (1993)); framework regions of consensus sequences of human antibodies derived from specific subsets of the 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 (somatic mutation) or human germline architecture regions (see, e.g., almagro and Fransson, front. Biosci.13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., baca et al, J.biol. Chem.272:10678-10684 (1997) and Rosok et al, J.biol. Chem.271:22611-22618 (1996)).

4. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Various techniques known in the art may be used to produce human antibodies. 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).

Human antibodies can be prepared by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having a human variable region in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci, either present extrachromosomal to the animal or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For a review of methods of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. nos. 6,075,181 and 6,150,584, which describe xenomoose ^TM technology; description of the inventionU.S. patent No. 5,770,429 to the art; description of K-MTechnical U.S. Pat. No. 7,041,870 and descriptionTechnical U.S. patent application publication No. US 2007/0061900). Human variable regions from whole antibodies produced by such animals may be further modified, for example by combining with different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., kozbor J. Immunol.,133:3001 (1984); brodeur et al, monoclonal Antibody Production Techniques and Applications, pages 51-63 (MARCEL DEKKER, inc., new York, 1987); and Boerner et al, J. Immunol.,147:86 (1991)) human antibodies produced via human B cell hybridoma technology are also described in Li et al, proc. Natl. Acad. Sci. USA,103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. 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).

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human phage display library. Such variable domain sequences can then be combined with the intended human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

5. Antibodies derived from libraries

Antibodies can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed in, for example, hoogenboom et al Methods in Molecular Biology 178:1-37 (O' Brien et al, human Press, totowa, NJ, 2001) and further described in the following documents: for example, 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 248:248:161-175 (Lo, human Press, totowa, NJ, 2003); sidhu et al, J.mol.biol.338 (2): 299-310 (2004); lee et al, J.mol.biol.340 (5): 1073-1093 (2004); fellouse, proc. Natl. Acad. Sci. USA 101 (34); 12467-12472 (2004); and Lee et al, J.Immunol. Methods 284 (1-2): 119-132 (2004).

In some phage display methods, all components of the VH and VL genes are cloned individually by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library from which antigen-binding phage can then be screened as described in Winter et al, ann.rev.immunol.,12:433-455 (1994). Phage typically display antibody fragments as single chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, all natural components (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self-antigens without any immunization, as described by Griffiths et al, EMBO J,12:725-734 (1993). Finally, the initial library can also be prepared synthetically in the following manner: cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and accomplish in vitro rearrangement as described by Hoogenboom and Winter, j.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. patent No. 5,750,373, U.S. patent publication nos. 2005/007974, 2005/019455, 2005/0266000, 2007/017126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are herein considered human antibodies or human antibody fragments.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different sites. Bispecific antibodies can be made as full length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-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 "pestle and mortar" 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, wherein the first half antibody comprises a first heavy chain constant region comprising a knob mutation and the second heavy chain comprises a second heavy chain constant region comprising a knob mutation; or wherein the first half antibody comprises a first heavy chain constant region comprising a knob mutation and the second heavy chain comprises a second heavy chain constant region comprising 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 mortar mutation comprises at least one mutation, at least two mutations, or three mutations selected from T366S, L a 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 mortar mutation comprises at least one mutation, at least two mutations, or three mutations selected from the group consisting of T366S, L a and Y407V mutations. The above numbering of the positions of one or more mutations is the EU numbering. The actual position of one or more mutations in the heavy chain sequence may vary, e.g., depending on the length of the preceding variable region, such as up to 10 positions.

Multispecific antibodies can also be prepared by the following techniques: engineering electrostatic manipulation effects to produce antibody Fc-heterodimer molecules (WO 2009/089004 A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al, science 229:81 (1985)); bispecific antibodies were generated using leucine zippers (see, e.g., kostelny et al, J.Immunol.148 (5): 1547-1553 (1992)); bispecific antibody fragments were prepared using "diabody" techniques (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA,90:6444-6448 (1993)); and the use of single chain Fv (sFv) dimers (see, e.g., gruber et al, J. Immunol.152:5368 (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al J.Immunol.147:60 (1991). In some embodiments, the antibody is a diabody antibody. Diabodies are antibody fragments having two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; hudson et al, nat.Med.9:129-134 (2003); and Hollinger et al, proc.Natl. Acad. Sci. USA 90:6444-6448 (1993).

Trisomy and tetrasomy antibodies are also described in Hudson et al, nat. Med.9:129-134 (2003). In some embodiments, the antibody is a trisomy antibody. In some embodiments, the trisomy antibody comprises a first antigen recognition site, a second antigen recognition site, and a third antigen recognition site, wherein at least one antigen recognition site is different from the other antigen recognition sites. In some embodiments, the trisomy antibody comprises a first antigen recognition site, a second antigen recognition site, and a third antigen recognition site that bind to three different polyubiquitin. Each antigen recognition site may comprise an HVR or a combination of VL and VH as discussed above.

In some embodiments, the antibody is a tetrameric antibody. In some embodiments, the tetrameric antibody comprises a first antigen recognition site, a second antigen recognition site, a third antigen recognition site, and a fourth antigen recognition site, wherein at least one or at least two antigen recognition sites are different from the other antigen recognition sites. In some embodiments, the tetrameric antibody comprises a first antigen recognition site, a second antigen recognition site, and a third antigen recognition site that bind to three different polyubiquitin. In some embodiments, the inclusion of a tetrameric antibody at each antigen-recognition site may comprise a combination of HVRs or VL and VH as discussed above.

Engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies", are also included herein (see, e.g., US2006/0025576 A1). The term "octopus antibody" is used in the sense discussed in US2006/0025576A1 and is not meant to refer to antibodies produced by or obtained from octopus.

Antibodies or fragments herein also include "dual action FAb" or "DAF" comprising two antigen binding sites that bind to two different antigens (see, e.g., US 2008/0069820).

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 antibodies. Amino acid sequence variants of antibodies 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 an antibody. Any combination of deletions, insertions, and substitutions may be made to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

A) Substitution, insertion and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutations include HVRs and FR. Conservative substitutions are shown under the heading "preferred substitutions" in table 1. More substantial variations are provided under the heading "exemplary substitutions" in table 1, and are further described below with respect to the class of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

TABLE 1

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity: norleucine Met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gln;

(3) Acid: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will require exchanging members of one of these classes for the other class.

One type of substitution variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more of the resulting variants selected for further investigation will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody relative to the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

For example, HVRs can be altered (e.g., substituted) to improve antibody affinity. Such changes may be made in HVR "hot spots", i.e. residues encoded by codons that undergo high frequency mutations during the somatic maturation process (see, e.g., chowdhury, methods afol. Biol.207:179-196 (2008)), and/or SDR (a-CDR), the resulting variant VH or VL were tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, by Hoogenboom et al, in Methods in Molecular Biology 178:1-37 (O' Brien et al, human Press, totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis genes). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR targeting methods in which several HVR residues (e.g., 4 to 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. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such alterations do not substantially reduce the antigen binding capacity of the antibody. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the HVR that do not substantially reduce binding affinity. Such changes may be outside of HVR "hot spots" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or comprises no more than one, two, or three amino acid substitutions.

A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or a set 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 antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the point of contact between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution may be targeted or eliminated. Variants may be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

B) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody comprises an Fc region, the carbohydrates attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched-chain double-antenna oligosaccharides, which are typically linked by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of a double-antennary oligosaccharide structure. In some embodiments, oligosaccharides in an antibody may be modified to produce antibody variants with certain improved properties.

In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose linked (directly or indirectly) to the Fc region. For example, the fucose content of such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e. between position 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108 (Presta, l.); US 2004/0093621 (Kyowa Hakko Kogyo Co., ltd.). Examples of publications relating to "defucosylation" or "fucose deficient" antibody variants include ：US2003/0157108;WO 2000/61739;WO 2001/29246;US2003/0115614;US2002/0164328;US 2004/0093621;US2004/0132140;US2004/0110704;US2004/0110282;US2004/0109865;WO 2003/085119;WO 2003/084570;WO 2005/035586;WO 2005/035778;WO2005/053742;WO2002/031140;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 deficient in protein fucosylation (Ripka et al Arch. Biochem. Biophys.249:533-545 (1986), U.S. patent application Ser. No. 2003/0157108 A1,Presta,L, and WO 2004/056312A 1, adams et al, especially example 11), and knockout cell lines such as CHO cells knocked out of the alpha-1, 6-fucosyltransferase gene (FUT 8) (see, e.g., yamane-Ohnuki et al Biotech. Bioeng.87:614 (2004), kanda, Y. Et al, biohnol. Bioeng.,94 (4): 680-688 (2006), and WO 2003/085107).

Further provided are antibody variants having two typed oligosaccharides, e.g., wherein a dihedral oligosaccharide linked to the Fc region of an antibody is 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-Maiset et al), U.S. Pat. No. 6,602,684 (Umana et al) and U.S. 2005/0123946 (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.).

C) Variant Fc region

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) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In certain embodiments, antibody variants have some, but not all, effector functions, which makes them ideal candidates for use, where the in vivo half-life of the antibody is important, but some effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm a reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγr binding (and thus may lack ADCC activity), but retains FcRn binding capacity. Primary cells mediating ADCC express only Fc (RIII, whereas monocytes express Fc (RI, fc (RII and Fc (RIII. Hematopoietic) cells) FcR expression is summarized in Ravetch and Kinet, annu. Rev. Immunol.9:457-492 (1991) page 464. Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are 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), 5,821,337 (see Bruggemann, M. Et al, J. Exp. Med.166:1351-1361 (1987)), alternatively, non-radioactive assays (see e.g., for measuring cytotoxicity of cells of the formula 32, and of the cell type 34. View cells) may be usedNon-radioactive cytotoxicity assay (Promega, madison, wis.). 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 may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al Proc.Nat' l Acad.Sci.USA 95:652-656 (1998). A C1q binding assay 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 ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996); cragg, M.S. et al, blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determination can also be performed using methods known in the art (see, e.g., petkova, s.b. et al, int' l.immunol.18 (12): 1759-1769 (2006)).

Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

Certain antibody variants having improved or reduced binding to FcR are described. ( See, for example, U.S. Pat. nos. 6,737,056; WO 2004/056312; and Shields et al J.biol.chem.9 (2): 6591-6604 (2001). )

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region.

In some embodiments, alterations are made in the Fc region resulting in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642 and Idusogie et al J.Immunol.164:4178-4184 (2000).

Antibodies responsible for transferring maternal IgG to the fetus (Guyer et al, J.Immunol.117:587 (1976) and Kim et al, J.Immunol.24:249 (1994)) with increased half-life and improved binding to neonatal Fc receptor (FcRn) are described in US2005/0014934A1 (Hinton et al). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include Fc variants having 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, for example, substitution of the Fc region residue 434 (U.S. patent No. 7,371,826).

For other examples of variants of the Fc region, see additionally: duncan and Winter, nature 322:738-40 (1988); U.S. Pat. nos. 5,648,260; U.S. Pat. nos. 5,624,821; WO 94/29351.

D) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as "thioMAbs", in which one or more residues of the antibody are substituted with cysteine residues. In certain embodiments, the substituted residue is present at an accessible site of the antibody. As further described herein, by substituting those residues with cysteines, reactive thiol groups are thereby located at accessible sites of the antibody, and can be used to conjugate the antibody to other moieties (such as a drug moiety or linker-drug moiety) to create an immunoconjugate. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: v205 of light chain (Kabat numbering); k149 (Kabat numbering) of the heavy chain; a118 (EU numbering) of heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, for example, in U.S. patent No. 7,521,541.

E) Antibody derivatives

In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Moieties suitable 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, 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 have any molecular weight and may or may not have branching. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular characteristics or functions of the antibody to be improved, whether the antibody derivative will be used in a defined-condition therapy, and the like.

In another embodiment, conjugates of antibodies and non-protein moieties that can be selectively heated by exposure to radiation are provided. 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 may have any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells, but heat the non-proteinaceous portion to a temperature at which cells proximal to the antibody-non-proteinaceous portion are killed.

B. recombinant methods and compositions

Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. patent No. 4815567. In some embodiments, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acids may encode an amino acid sequence comprising a VL of an antibody and/or an amino acid sequence comprising a VH of an antibody (e.g., a light chain and/or a heavy chain of an antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In further embodiments, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) the following: (1) A vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and an amino acid sequence comprising a VH of an antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphocyte (e.g., Y0, NS0, sp20 cell). In some embodiments, a method of making an antibody disclosed herein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

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.

For recombinant production of antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5, 5,789,199, and 5,840,523. (see also Charlton, methods in Molecular Biology, volume 248 (B.K.C.Lo, editions, humana Press, totowa, NJ,2003, pages 245-254, describing the expression of antibody fragments in E.coli.) antibodies can be isolated from bacterial cell pastes in soluble fractions after expression and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast, including such fungi and yeast strains, are also suitable cloning or expression hosts for vectors encoding antibodies: its glycosylation pathway has been "humanized" such that antibodies with a partially or fully human glycosylation pattern are produced. See Gerngross, nat.Biotech.22:1409-1414 (2004), and Li et al, nat.Biotech.24:210-215 (2006).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ^TM techniques for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney (293 or 293 cells, as described, for example, in Graham et al, J.Gen. Virol.36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells (TM 4 cells, as described, for example, in Mather, biol. Reprod.23:243-251 (1980); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); brutro rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, for example, in Mather et al, annals N.Y. Acad. Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al, proc.Natl. Acad. Sci.usa 77:4216 (1980)); and myeloma cell lines such as Y0, 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, volume 248 (b.k.c.lo, editions, humana Press, totowa, NJ), pages 255-268 (2003).

C. Measurement

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

In some aspects, the antigen binding activity of the antibody is tested, for example, by known methods such as ELISA, FACS, or western blot.

In another aspect, a competition 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) bound by the antibodies described herein. A detailed exemplary method for mapping epitopes to which antibodies bind is provided in Morris (1996) "Epitope Mapping Protocols" in volume Methods in Molecular Biology, 66 (Humana Press, totowa, NJ).

In an exemplary competition assay, the immobilized polyubiquitin is incubated with a solution comprising a first labeled antibody (e.g., any of the antibodies described herein) to which the antigen binds and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to polyubiquitin. The second antibody may be present in the hybridoma supernatant. As a control, polyubiquitin was incubated in a solution comprising a first labeled antibody but not a 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 associated with the immobilized polyubiquitin is measured. If the amount of label associated with the immobilized polyubiquitin in the test sample is substantially reduced relative to the control sample, it is indicated that the second antibody is competing with the first antibody for binding to polyubiquitin. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, chapter 14 (Cold Spring Harbor Laboratory, cold Spring Harbor, N.Y.).

In some embodiments, a RIP 1-or RIP 2-K63-linked ubiquitin chain bispecific antibody and/or linear ubiquitin chain bispecific antibody is used in a method for detecting selective ubiquitination of K63-linked ubiquitin and/or linear ubiquitin polyubiquitination, respectively, in a sample (e.g., a cell or tissue sample), the method comprising contacting the sample with the antibody. In some embodiments, the RIP 1-or RIP 2-linearly linked ubiquitin chain bispecific antibody allows for the detection of selective ubiquitination of RIP1 or RIP2 with linearly linked ubiquitin polyubiquitination, respectively, in a sample (e.g., a cell or tissue sample).

In some embodiments, methods are provided for detecting ubiquitination of RIP1 or RIP2 (such as K63-linked polyubiquitin and/or linear polyubiquitin) using antibodies disclosed herein by immunofluorescence, e.g., in a cell or tissue sample. As presented in the examples below, detection may take the form of time and/or signal dependence. The use of a single bispecific antibody may advantageously eliminate the necessity to search for overlapping patterns of multiple antibodies in a cumbersome manner and/or may increase the specificity and accuracy of cell localization determinations.

The antibodies described herein may also be used in methods for determining the level of RIP2 ubiquitination in a tissue sample from a subject undergoing an intestinal resection procedure. As demonstrated in the examples, in samples from subjects with crohn's disease and ulcerative colitis, RIP2 can be highly ubiquitinated with K63 linked ubiquitin chains and/or linear ubiquitin chains, as opposed to lower ubiquitination levels in non-IBD control and diverticulitis samples.

Also provided herein are methods for identifying a subject as a candidate for RIP 2-targeted therapy, comprising contacting a sample from the subject with an antibody described herein and determining the level of polyubiquitinated RIP2 (such as K63-linked polyubiquitinated RIP2 and/or linear polyubiquitinated RIP 2). 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 or tissue sample.

In some embodiments, methods are provided for detecting ubiquitination of RIP1 or RIP2 comprising branched, hybrid, or mixed polyubiquitin chains, e.g., wherein the branched, hybrid, or mixed polyubiquitin chains comprise one or more K63-linked ubiquitin chains and/or linear ubiquitin chains.

D. Immunoconjugates

Immunoconjugates are provided comprising an antibody disclosed herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a bacterial, fungal, plant, or animal-derived protein toxin, an enzymatically active toxin, or fragments thereof), or a radioisotope (i.e., a radioconjugate).

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

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, b.a. (2009) Current Cancer Drug Targets 9:982-1004), thereby increasing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, p.j. And hunter p.d. (2008) THE CANCER journal.14 (3): 154-169; chari, r.v. (2008) acc.chem.res.41:98-107).

ADC compounds include those having anti-cancer activity and/or having anti-inflammatory activity. In some embodiments, the ADC compound comprises an antibody conjugated (i.e., covalently linked) to a drug moiety. In some embodiments, the antibody is covalently linked to the drug moiety through a linker. Antibody-drug conjugates (ADCs) can selectively deliver an effective dose of drug to tumor tissue, where greater selectivity, i.e., lower effective doses, can be achieved while increasing the therapeutic index ("therapeutic window").

The drug moiety (D) of the antibody-drug conjugate (ADC) may comprise any compound, moiety or group having cytotoxic or cytostatic effect. The drug moiety may confer cytotoxicity and cytostatic effects thereto 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, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine(PBD), nemorubicin and its derivatives, PNU-159582, anthracyclines, du Kamei, vinca alkaloids, taxanes, trichothecenes, CC1065, camptothecins, escitalopram Li Naifa and stereoisomers, isosteres, analogues and derivatives thereof with cytotoxic activity.

The pharmaceutical moiety (D) of the ADC may comprise any compound, moiety or group having anti-inflammatory effect. Exemplary drug moieties include, but are not limited to, non-steroidal anti-inflammatory agents (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; stereoisomers, isosteres, analogues and derivatives thereof having anti-inflammatory activity.

E. methods and compositions for diagnosis and detection

In some embodiments, the methods described herein can be used to determine the presence of polyubiquitin in a sample. In some embodiments, the sample is suspected of containing polyubiquitin. In some embodiments, the polyubiquitin protein is a pro-inflammatory protein.

In some embodiments, the methods described herein comprise exposing the 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 pro-inflammatory protein. In some embodiments, the method comprises determining the binding of at least one antibody to the polyubiquitin in the sample.

In some embodiments, the polyubiquitin comprises M1-linked polyubiquitin and/or K63-linked polyubiquitin,

In some embodiments, the methods described herein can be used to determine the presence of a polyubiquitin protein in a sample suspected of containing the polyubiquitin protein, wherein the polyubiquitin protein is a pro-inflammatory protein and comprises polyubiquitin, the method comprising: exposing the 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 pro-inflammatory protein; and determining binding of the at least one antibody to the polyubiquitin in the sample.

In some embodiments, the methods described herein can be used to determine the presence of a polyubiquitin protein in a sample suspected of containing the polyubiquitin protein, wherein the polyubiquitin protein is a pro-inflammatory protein and comprises M1-linked polyubiquitin and/or K63-linked polyubiquitin, the method comprising: exposing the 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 pro-inflammatory protein; and determining binding of the at least one antibody to the polyubiquitin in the sample.

In some embodiments, the pro-inflammatory protein is a component of one or more signaling complexes that promote inflammation by mediating inflammatory cell death and/or release of pro-inflammatory cytokines, chemokines, and risk-related molecular patterns (DAMP). In some embodiments, the pro-inflammatory protein is receptor interacting protein kinase 1 (RIP 1), receptor interacting protein kinase 2 (RIP 2), apoptosis inhibitors 1 and 2 (c-IAP 1/2), tumor necrosis factor receptor 1 (TNFR 1), linear ubiquitin chain assembly complex (LUBAC), and/or nuclear factor- κB (NF- κB) essential modulator (NEMO).

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

In some embodiments, the proinflammatory protein has an elevated level of ubiquitination in an inflammatory state relative to the level of ubiquitination when not in an inflammatory state.

In some embodiments, the pro-inflammatory protein has a level of ubiquitination 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 the inflammatory state relative to the level of ubiquitination when not in the inflammatory state.

In some embodiments, the increased level of ubiquitination is associated with an increase in the severity of the inflammatory disease state.

In some embodiments, the level of ubiquitination is increased in the inflammatory state relative to the level of ubiquitination when not in the inflammatory state.

In some embodiments, the proinflammatory protein is associated with an inflammatory disease such as inflammatory bowel disease, crohn's disease, diverticulitis, and ulcerative colitis.

In certain embodiments, the antibodies provided herein can be used to detect the presence of RIP1 in a biological sample. In certain embodiments, the antibodies provided herein can be used to detect the presence of RIP2 in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative detection. "biological sample" includes, for example, cells or tissues (e.g., biopsy material, including cancerous or potentially cancerous colon, colorectal, small intestine, endometrial, pancreatic, breast, lung, prostate, or ovarian tissue).

In some embodiments, the antibodies disclosed herein are used in diagnostic or detection methods. In a further aspect, a method of detecting the presence of RIP1 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with the antibody under conditions that allow the antibody to bind to RIP1, and detecting whether a complex is formed between the antibody and RIP1 in the biological sample. Such methods may be in vitro or in vivo. In some embodiments, the antibodies are used to select subjects eligible for treatment with an anti-RIP 1 antibody, e.g., where RIP1 is a biomarker for selecting patients. In some embodiments, the biological sample is a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous tissue).

In some embodiments, the antibodies disclosed herein are used in diagnostic or detection methods. In a further aspect, a method of detecting the presence of RIP2 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with the antibody under conditions that allow the antibody to bind to RIP2, and detecting whether a complex is formed between the antibody and RIP2 in the biological sample. Such methods may be in vitro or in vivo. In some embodiments, the antibodies are used to select subjects eligible for treatment with an anti-RIP 2 antibody, e.g., where RIP2 is a biomarker for selecting patients. In some embodiments, the biological sample is a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous tissue).

In some embodiments, methods of detecting a polyubiquitin RIP1 or polyubiquitin RIP2 protein in a biological sample are provided. In certain embodiments, the method comprises contacting a biological sample with an antibody, under conditions that allow the antibody as described herein to bind to a polyubiquitin RIP1 or polyubiquitin RIP2 protein, and detecting whether a complex is formed between the antibody and the RIP1 or RIP2 protein. Such methods may be in vitro or in vivo. In some embodiments, the antibodies are used to select subjects eligible for treatment with the antibodies disclosed herein, e.g., wherein the polyubiquitin RIP1 or polyubiquitin RIP2 protein is a biomarker for selecting patients. In some embodiments, the biological sample is a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous tissue).

In further embodiments, the antibodies disclosed herein are used in vivo to detect (e.g., by in vivo imaging) a polyubiquitin RIP1 or polyubiquitin RIP2 protein, e.g., for the purpose of diagnosing, predicting or staging a disease, determining an appropriate course of therapy, or monitoring response to therapy. One method known in the art for in vivo detection is immune positron emission tomography (immune PET), as described, for example, in van Dongen et al The Oncologies 12:1379-1389 (2007) and Verel et al, J.Nucl. Med.44:1271-1281 (2003). In such embodiments, methods for detecting polyubiquitin RIP1 or polyubiquitin RIP2 proteins in a subject are provided, the methods comprising administering a labeled antibody to the subject and detecting the labeled anti-RIP 1 or anti-RIP 2 antibody in the subject. In certain such embodiments, the labeled antibodies comprise (e.g., are conjugated to) positron emitters, such as ⁶⁸Ga、¹⁸F、⁶⁴Cu、⁸⁶Y、⁷⁶Br、⁸⁹ Zr and ¹²⁴ I. In a particular embodiment, the positron emitter is ⁸⁹ Zr. A non-limiting exemplary method of making and using ⁸⁹ Zr-labeled antibodies is 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.

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

In certain embodiments, the antibodies disclosed herein are labeled. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly (e.g., by enzymatic reactions or molecular interactions) detected moieties (such as enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes ³²P、¹⁴C、¹²⁵I、³ H and ¹³¹ I; fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone; luciferases (luceriferase), such as firefly luciferases and bacterial luciferases (U.S. Pat. No. 4,737,456); luciferin, 2, 3-dihydronaphthyridonedione, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme; sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as urate oxidase and xanthine oxidase; coupled to an enzyme (such as HRP, lactoperoxidase, or microperoxidase) that oxidizes the dye precursor with hydrogen peroxide; biotin/avidin, spin labeling, phage labeling, stable free radicals, and the like. In another embodiment, the label is a positron emitter. Positron emitters include, but are not limited to ⁶⁸Ga、¹⁸F、⁶⁴Cu、⁸⁶Y、⁷⁶Br、⁸⁹ Zr and ¹²⁴ I. In a particular embodiment, the positron emitter is ⁸⁹ Zr.

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

In some embodiments, compositions are provided that are substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies. Monospecific antibodies are antibodies that do not contain more than one type of antigen recognition site, e.g. antibodies having only one set of six CDRs or antibodies in which the six CDRs of each set are identical. Antibodies in which the first set of CDRs is only slightly different from any other set of CDRs (e.g., in terms of a small number of amino acid residues, wherein the differences do not result in preferential binding to different antigens) are also considered monospecific. The unassembled half-antibody is not stably associated (covalently or non-covalently) with another half-antibody, e.g., appears as a single heavy/light chain unit when analyzed by an appropriate technique such as size exclusion chromatography, mass spectrometry, or electrophoresis.

F. Pharmaceutical preparation

Pharmaceutical formulations of antibodies or immunoconjugates as described herein are prepared by mixing such antibodies or immunoconjugates of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington' sPharmaceutical Sciences th edition, osol, code a. 1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, including but not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates, such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; 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., zinc protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 @Baxter International, inc.). Certain exemplary shasegps and methods of use (including rHuPH 20) are described in U.S. patent publication nos. 2005/026086 and 2006/0104968. In some aspects, sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibodies or immunoconjugates include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively); in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in a coarse emulsion. Such techniques are disclosed in Remington' sPharmaceutical Sciences, 16 th edition, osol, a. Ed (1980).

A slow release preparation 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.

Formulations for in vivo administration are typically sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

G. Therapeutic methods and compositions

Any of the antibodies or immunoconjugates provided herein can be used in methods such as therapeutic methods.

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

In some embodiments, antibodies or immunoconjugates disclosed herein are provided for use in treating a disease or disorder associated with an abnormally increased cell cycle progression and a disease or disorder associated with an abnormally decreased cell cycle progression. In some embodiments, the disease or disorder associated with an abnormally increased cell cycle progression is cancer, such as colorectal cancer.

In some embodiments, antibodies or immunoconjugates disclosed herein are provided for use in treating an inflammatory disease such as inflammatory bowel disease. In some embodiments, the inflammatory disease is selected from crohn's disease, diverticulitis, and ulcerative colitis.

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

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

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 methods of treatment. In some embodiments, the pharmaceutical formulation comprises any of the antibodies or immunoconjugates provided herein and a pharmaceutically acceptable carrier.

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

Such combination therapies described above encompass the combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and the separate administration, in which case the administration of the antibodies or immunoconjugates provided herein may be performed before, simultaneously with, and/or after the administration of additional therapeutic agents and/or adjuvants.

The antibody or immunoconjugate (and any additional therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various points in time, bolus administrations, and pulse infusion.

The antibody or immunoconjugate will be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this case include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner. The antibody or immunoconjugate is not necessarily, but is optionally co-formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are typically used at the same dosages and routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosage and by any route empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of the antibody or immunoconjugate (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be 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 therapies, patient history and response to the antibody or immunoconjugate, and the discretion of the attendant physician. The antibody or immunoconjugate is suitably administered to the patient once or in a series of treatments.

It will be appreciated that any of the above formulations or methods of treatment may be performed using both immunoconjugates and antibodies.

H. Article of manufacture

In another aspect, an article of manufacture is provided that contains materials useful for treating, preventing and/or diagnosing the disorders described above. The article includes a container and a label or package insert (PACKAGE INSERT) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective in treating, preventing and/or diagnosing a condition, either by itself or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or immunoconjugate as disclosed herein. The label or package insert indicates that the composition is to be used to treat the selected condition. Further, the article of manufacture may comprise (a) a first container comprising a composition therein, wherein the composition comprises an antibody or immunoconjugate; and (b) a second container containing a composition, wherein the composition comprises a further cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment may further comprise package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, or dextrose solution. The article of manufacture may also include other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

III. Examples

The following are examples of the methods and compositions provided herein. It should be understood that various other embodiments may be practiced given the general description provided above.

A. Analysis method

Size exclusion chromatography-multi-angle light scattering. For example, 50. Mu.g of antibody was injected at 1mL/min to 3.5 μm 7.8mm x 300mm XBridge protein BEH analytical SEC using Agilent 1260 Infinicity HPLC with 20mM histidine acetate, 300mM NaCl, pH 5.5 as mobile phaseOn a column (Waters). Proteins eluted from analytical SEC columns were directly injected onto WYATT DAWN HELEOS II/Optilab T-rEX multi-angle light scattering detector to measure molar mass and polydispersity.

And (5) mass spectrometry. Prior to mass spectrometry, 30 μg of antibody was deglycosylated with 2 units of PNGaseF (NEB) overnight at 37 ℃.2 μg of antibody was then injected at 1mL/min onto a3 μm, 4.6X10 mm reverse phase chromatography PLRP-S column (Agilent) using an Agilent 1290 affinity UHPLC. A gradient of 0% to 100% buffer B was performed over 3 minutes with an aqueous solution containing 0.05% trifluoroacetic acid (TFA) (buffer A) and an acetonitrile solution containing 0.05% TFA (buffer B), followed by a 100% buffer B wash for 1 minute. Proteins eluted from the reverse phase column were directly injected onto an Agilent 6230 electrospray ionization time of flight mass spectrometer (ESI-TOF) for complete mass measurement.

B. Antibody cloning, expression and annealing

Bispecific antibodies were generated using a pestle heterodimerization method. For a general discussion of this method, see Merchant, A.M. et al AN EFFICIENT route to human bispecific IgG. Nat Biotechnol 16,677-681, doi:10.1038/nbt0798-677 (1998).

Bispecific antibodies were designed using the following antibodies as building blocks: anti-M1 or linear polyubiquitin bond specific synthetic human antibody (clone 1F11/3F 5/Y102L) (Matsumoto et al, jmol biol.418 (3-4), 134-44.Doi:10.1016/j.jmb.2011.12.053.Epub 2011Dec 29.PMID:22227388 (2012))); anti-K63 polyubiquitin bond 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)); murine anti-RIP 1 antibody (BD Biosciences # 610459); murine anti-RIP 2 antibody (abcam#ab 75257). As a control, an anti-gD antibody recognizing an unrelated protein was used.

The T366W (pestle) or T366S, L a and Y407V (mortar) mutations were introduced into the CH3 domain. The knob and socket mutations are selected to allow preferential heterodimerization of the corresponding heavy chains of the antibody.

Briefly, the sequences encoding the heavy chain variable domain are those of SEQ ID NO. 50 (specific for the anti-M1 polyubiquitin bond), SEQ ID NO. 36 (specific for the anti-K63 polyubiquitin bond), SEQ ID NO. 73 (specific for anti-RIP 1), SEQ ID NO. 94 (specific for anti-RIP 2) and the non-specific anti-gD control antibody. These variable domains were subcloned into a modified pRK vector (Genencory Co., ltd.) containing the human IgG1 heavy chain constant domain with a knob (T366W) or a knob (T366S, L A and Y407V) mutation in the CH3 domain.

The actual positions of the knob and socket mutations may vary slightly, for example 1 to 10 positions, due to the length of the variable region. For example, in SEQ ID NOS.4 and 6, the T to W and T to S substitutions are reflected in amino acid residues 369 rather than 366, respectively. It should be understood that reference to the pestle and socket mutation at positions such as 366, 368 and 407 of the heavy chain should be interpreted as adjusting according to the length of the variable region if applicable.

The light chain variable domain was similarly subcloned into a modified pRK vector (GeneTex) containing the human kappa light chain constant domain. pRK vectors carry constitutive strong signal peptides for extracellular expression in mammalian cells. The anti-M1 antibody was cloned as both a knob and a socket mutant (encoding the heavy chains of SEQ ID NOS: 46 and 48, respectively), as were the anti-K63 antibody (encoding the heavy chains of SEQ ID NOS: 32 and 34, respectively). anti-RIP 1 and anti-RIP 2 antibodies were cloned as mortar mutants (encoding SEQ ID NOs: 70 and 92, respectively) and anti-gD antibodies were cloned as pestle mutants.

The sequence listing also provides pestle and mortar mutants (SEQ ID NOs: 4 and 6, respectively) that are resistant to the K11 heavy chain; pestle and mortar mutants resistant to K48 heavy chain (SEQ ID NOs: 18 and 20, respectively); and pestle mutants directed against the RIP1 heavy chain (SEQ ID NO: 68) and against the RIP2 heavy chain (SEQ ID NO: 90).

To maintain pairing of homologous light and heavy chains, pestle or mortar heavy chain mutants and their respective light chains were expressed in CHO cells and affinity purified separately (data not shown). The light and heavy chain plasmids for a given pestle or mortar half antibody were transiently co-transfected into CHO cells using PEI as previously described (see Wong, a.w., bayinski, t.k., and Reilly,D.E.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)). the half antibody was purified on MabSelect SuRe resin (GE HEALTHCARE), eluted with 50mM sodium citrate, 150mM NaCl, pH 3.0, followed by 10% (v/v) 200mM arginine, 137mM succinate, pH 9.0 to pH 5.0.

The affinity purified knob and socket half antibodies (not shown) were characterized in agreement with the previously described knob and socket half antibodies. See, e.g., shatz, W.et al MAbs 5,872-881, doi:10.4161/MAbs.26307 (2013). The identity of which is confirmed by mass spectrometry.

Bispecific antibodies were assembled in vitro from half antibodies using annealing, reduction, and oxidation. anti-RIP 1/anti-M1 bispecific antibodies were assembled in vitro from affinity purified anti-M1 pestle and anti-RIP 1 mortar antibodies using a modified version of the annealing, reduction and oxidation methods described previously (Shatz, w. et al mabs5,872-881, doi:10.4161/mabs.26307 (2013)). Similarly, anti-RIP 1/anti-K63 bispecific antibodies were assembled from anti-K63 knob antibodies and anti-RIP 1 mortar antibodies; the anti-RIP 2/anti-M1 bispecific antibody is assembled from an anti-M1 knob antibody and an anti-RIP 2 knob antibody; and the anti-RIP 2/anti-K63 bispecific antibody was assembled from an anti-K63 knob antibody and an anti-RIP 2 mortar antibody. Briefly, the desired pestle antibody and mortar half antibody were mixed at a 1:1 mass ratio and the pH of the mixture was adjusted to about 8 with 15% (v/v) 800mM arginine, pH 10.0. 800mM arginine, pH10.0, containing 200-fold molar excess of reduced glutathione (SIGMA ALDRICH) was added and the assembly reaction was incubated at room temperature for 72 hours with exposure to air to allow annealing of the knob and socket half antibodies and formation of hinge disulfide. anti-M1/anti-K63, anti-RIP 1/gD, anti-RIP 2/gD, anti-M1/gD and anti-K63/gD control bispecific antibodies were similarly assembled.

C. antibody purification and characterization

The in vitro assembled bispecific antibody was purified by Hydrophobic Interaction Chromatography (HIC) using a modified version of the previously described method. See Yau et al ,Cell.171(4):918-933.e20.doi:10.1016/j.cell.2017.09.040.Epub 2017Oct 12.PMID:29033132;PMCID:PMC5669814(2017). briefly, 3.8M ammonium sulfate was added to the assembly reaction until the final concentration was at least 1M, and the reaction was filtered through a 0.22 μm filter and loaded onto a5 μm, 7.8x75mm ProPac HIC-10 column (Dionex). The column was then washed with buffer a (20 mM sodium acetate, pH 5.0) and a linear gradient of 0% -100% buffer B (25 mM sodium phosphate, pH 6.5, 25% isopropyl alcohol) was performed over 40 Column Volumes (CVs) to separate the bispecific antibody from any unreacted half antibodies or aggregated proteins. Identification of elution peaks was monitored by SDS-PAGE and mass spectrometry (see below for method details) and fractions corresponding to bispecific peaks were pooled.

The bispecific antibody was further purified by cation exchange Chromatography (CEX). Briefly, HIC combined material was dialyzed into 20mM sodium acetate, pH 5.0, or pH was reduced to about 5 by adding 1/6 volume of 1M sodium acetate, pH 5.0, and then diluted with water to a final concentration of 13mM sodium acetate. The antibodies were then loaded onto a 10 μm Mono S5/50 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 5.0,1M NaCl) was performed over 40 CVs and the desired fractions were pooled. At 20mM histidine acetate, 240mM sucrose, 0.02%Purified bispecific antibody was formulated in-20, pH 5.5 or 20mM histidine acetate, 150mM NaCl, pH 5.5.

LC-MS was used to confirm identification of purified, returned species. To reduce heterogeneity, antibodies were deglycosylated with PNGaseF prior to analysis.

Modification of protein recognized by RIP1-K63 ubiquitin chain bispecific antibody

Human colon cancer HT29 cells were treated with a combination of TNF (20 ng/ml), IAP antagonist BV6 (2. Mu.M) and pan-caspase inhibitor zVAD (20. Mu.M) (collectively TBZ) for 2 hours, also 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 RIP 1-ubiquitin chain bispecific antibodies or control bispecific antibodies. Total Cell Lysates (TCL) and Immunoprecipitated Proteins (IP) were probed with the indicated antibodies as shown in FIG. 2A.

Fibrosarcoma HT1080 cells were treated with PBS or TNF (100 ng/ml) for 7min. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibodies or control bispecific antibodies. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 2B.

The A549 cells were treated with PBS or TNF (500 ng/ml) for 7min. Cells were fixed in 4% paraformaldehyde at Room Temperature (RT) for 30min, incubated in 6M urea buffer without Triton (30 min, RT), permeabilized with 0.25% Triton (10 min, RT) and then stained with the indicated RIP 1-ubiquitin chain bispecific antibody or control antibody followed by anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. Mounted slides were analyzed using a Leica SP8 confocal microscope. All images are collected together using the same setup. These results are shown in fig. 2C.

HT1080 cells were treated with PBS or TNF (500 ng/ml) for 7min. Cells were fixed, incubated in 6M urea buffer (30 min, rt), permeabilized and then stained with the indicated RIP 1-ubiquitin chain bispecific antibody or control antibody followed by anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. All images were collected together using the same setup [ scale bar 25 μm ]. These results are shown in fig. 2D.

EVSA T cells were treated with PBS or TNF (100 ng/ml) for 7min. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibodies or control bispecific antibodies. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins. These results are shown in fig. 3A.

Ku812F cells were treated with PBS or Flag-TL1A (10 ug) crosslinked with anti-Flag AB for 10min. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibodies or control bispecific antibodies. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins. These results are shown in fig. 3B.

This suggests that the RIP1-K63 ubiquitin chain bispecific antibody recognizes K63 chain ubiquitinated RIP1 (fig. 2A to 2D) and RIP1 modified with K63 linked ubiquitin chains (fig. 3A to 3B).

This is also shown in fig. 1A to 1D. The specificity of the RIP1-K63 bispecific antibody was verified with in vitro ubiquitination of RIP1, wherein K63-or K48-tetraubiquitin molecules were used to modify RIP1. After ubiquitination, RIP1 was incubated with the indicated bispecific antibody in the presence of 6M urea to disrupt any non-covalent association, precipitated and checked by western blotting. The RIP1-K63 bispecific antibody immunoprecipitates K63-ubiquitin chain modified RIP1, but does not immunoprecipitate K48-ubiquitin chain modified RIP1.

Modification of protein recognized by RIP1-Lin ubiquitin chain bispecific antibody

HT29 cells were treated with PBS or TNF (20 ng/ml), BV6 (2. Mu.M) and zVAD (20. Mu.M) for 2 hours as described in example D. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibody, K63-linear ubiquitin chain bispecific antibody or control bispecific antibody. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 4A.

Ku812F cells were treated with PBS or TNF (100 ng/ml) for 10min. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibody, K63-linear ubiquitin chain bispecific antibody or control bispecific antibody. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 4B.

HT29 cells were treated with PBS or TNF (100 ng/ml), BV6 (2. Mu.M) and zVAD (20. Mu.M) for 2,5 hours. Cells were fixed in 4% paraformaldehyde at Room Temperature (RT) for 30min, incubated in 6M urea buffer without Triton (30 min, RT), permeabilized with 0.25% Triton (10 min, RT) and then stained with the indicated RIP 1-ubiquitin chain bispecific antibody or control antibody followed by anti-human IgG-Alexa 488 secondary antibody. Hoechst 33258 was used for nuclear staining. Mounted slides were analyzed with a Leica SPE confocal microscope. All images are collected together using the same setup. These results are shown in fig. 4C.

Mouse Embryonic Fibroblasts (MEF) cells were treated with PBS or TBZ (TNF (100 ng/ml), BV6 (1. Mu.M) and zVAD (20. Mu.M)) for 2.5h. Cells were fixed, incubated in 6M urea buffer (30 min, rt), permeabilized and then stained with the indicated RIP 1-ubiquitin chain bispecific antibody or control antibody followed by anti-human goat F (ab') 2 fragment antibody conjugated to Cy 3. Hoechst 33258 was used for nuclear staining. All images were collected together using the same setup [ scale bar 25 μm ]. These results are shown in fig. 4D.

These results indicate that the RIP1-Lin ubiquitin chain bispecific antibody recognizes linear chain ubiquitinated RIP1. In contrast, the non-stimulated RIP1-gD antibody and the gD-Lin antibody or the K63-Lin antibody did not immunoprecipitate ubiquitinated RIP1.

This is also shown in fig. 1B, which shows the ability of RIP1-Lin bispecific antibodies to selectively immunoprecipitate RIP1 modified with linear ubiquitin chains, but not with K48 linked ubiquitin chains, by in vitro ubiquitination of RIP1 with tetra-K48 or linear-tetraubiquitin molecules followed by immunoprecipitation in 6M urea buffer.

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 7min. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibody, K63-linear ubiquitin chain bispecific antibody or control bispecific antibody. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 5A.

D645 cells were treated with PBS or TNF (100 ng/ml) for 7min. Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibody, K63-linear ubiquitin chain bispecific antibody or control bispecific antibody. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 5B.

THP1 cells were treated with PBS, TNF (100 ng/ml,5 min) or MDP (1. Mu.g/ml, 30 min). Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibody, K63-ubiquitin chain bispecific antibody or control antibody. The indicated antibodies were used to probe the total cell lysate and immunoprecipitated proteins as shown in figure 5C.

Thus, the RIP1-K63 bispecific antibody and the RIP1-Lin bispecific antibody selectively recognize RIP1 modified with K63-linked ubiquitin chains and K63-Lin ubiquitin linked chains, respectively, but do not recognize RIP2 or TRAF2.

The K63-Lin bispecific antibody also recognizes modified RIP1 as well as modified RIP2, but no RIP 1-specific component or RIP-2-specific component of the antibody.

RIP 1-ubiquitin chain bispecific antibodies recognize in vivo ubiquitinated RIP1

Mice were treated with PBS (mice M1 and M2) or TNF (500 μg/ml) for 12min (mice M3 and M4) or 24min (mice M5 and M6). Small intestine cells from the indicated mice were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibodies or control bispecific antibodies. Immunoprecipitated proteins (FIG. 6A) and total cell lysate proteins (FIG. 6B) were probed with the indicated antibodies. This demonstrates that the RIP 1-ubiquitin chain bispecific antibody recognizes ubiquitin and linear ubiquitin chain in vivo ubiquitinated RIP1 linked with K63.

Bone marrow-derived macrophages (BMDM) were isolated from wild-type RIP1 mice or RIP 1K 376R knock-in mice, treated with TNF for 5 minutes, lysed in 6M urea buffer, and immunoprecipitated using the indicated RIP 1-ubiquitin chain bispecific antibodies or control bispecific antibodies. Total cell lysate proteins (FIG. 6C) and immunoprecipitated proteins (FIG. 6D) were detected using the indicated antibodies. This demonstrates that RIP 1-ubiquitin chain bispecific antibodies can be used to detect impaired K63 ligation ubiquitination and linear ubiquitination of RIP1 in RIP 1K 376R knock-in mouse-derived BMDM.

H.RIP2-ubiquitin chain bispecific antibodies recognize modified RIP2

THP1 cells were treated with PBS or NOD2 signaling activator MDP (1. Mu.g/ml, 30 min). Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 2-ubiquitin chain bispecific antibodies or control bispecific antibodies. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 7A. This results in that ubiquitination of the RIP2K63 link is only detected in a stimulus-dependent manner when both RIP2 and K63 arms are present in the bispecific antibody (RIP 2-K63), but not in the case of control bispecific antibodies containing only one relevant arm (RIP 2-gD or K63-gD).

THP1 cells were treated with PBS or MDP (1. Mu.g/ml, 30 min). Cells were lysed in 6M urea buffer and immunoprecipitated using the indicated RIP 2-ubiquitin chain bispecific antibody, K63-ubiquitin chain bispecific antibody or control antibody. The indicated antibodies were used to probe total cell lysates and immunoprecipitated proteins as shown in figure 7B. RIP2-Lin (but not RIP2-gD antibody or gD-Lin antibody) could successfully immunoprecipitate RIP2 modified with linear ubiquitin, but not xiAP or c-IAP 1. K63-Lin bispecific antibodies efficiently capture RIP2 and to some extent, xiAP modified by mixed and/or branched K63-linked ubiquitin chains and linear ubiquitin chains.

THP1 cells were treated with PBS or MDP (1. Mu.g/ml, 30 min). Cells were fixed in 4% paraformaldehyde at Room Temperature (RT) for 30min, incubated in 6M urea buffer without Triton (30 min, RT), permeabilized with 0.25% Triton (10 min, RT) and then stained with the indicated RIP 2-ubiquitin chain bispecific antibody or control antibody followed by 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) and RIP2KO (knock-out) THP1 cells is shown in fig. 7C. This result demonstrates that the RIP 2-ubiquitin chain bispecific antibody recognizes RIP2 ubiquitinated by the linear chain and the K63-Lin ubiquitin linker chain. Western blot analysis of TCL and immunoprecipitated proteins obtained with the indicated antibodies using WT (wild type, W) and RIP2KO (knock-out) THP1 cells is shown in fig. 7D. Mounted slides were analyzed with a Leica SPE confocal microscope. All images are collected together using the same setup. These results are shown in fig. 7E. The stimulation-dependent localization of K63-linked and linear RIP2 ubiquitination in cells was revealed by immunofluorescence by RIP2-K63, RIP2-Lin and K63-Lin, but not RIP2-gD, gD-K63 or gD-Lin bispecific antibodies. Immunofluorescence detected with the K63-Lin antibody may be primarily, but not entirely, a result of ubiquitination of RIP2, as the antibody weakly immunoprecipitates XIAP in MDP-treated cells.

This demonstrates that RIP 2-ubiquitin chain bispecific antibodies recognize K63 ubiquitinated and linear chain ubiquitinated RIP2. In contrast, single arm (RIP 2-gD, K63-gD, lin-gD) antibodies did not recognize ubiquitinated RIP2, and even RIP2-K63, RIP2-Lin, and K63-Lin bispecific antibodies recognized K63 ubiquitination and linear chain ubiquitination of RIP2 only after treatment with pathway dependent stimulus (MDP).

I. RIP2 ubiquitination in patient samples of Crohn's disease and ulcerative colitis

RIP2-K63 and RIP2-Lin bispecific antibodies were tested in intestinal tissue samples from patients (fig. 8A to 8G). Patients undergoing intestinal resection surgery for the treatment of their colon cancer, dysplasia, diverticulitis (DVC), crohn's Disease (CD) or Ulcerative Colitis (UC) were included in the observational study (table 2).

TABLE 2 clinical Properties of IBD and non-IBD groups

N/A, inapplicable.

NR, unrecorded.

After surgery, intestinal tissue samples from intestinal cancer, dysplasia, diverticulitis (DIV), crohn's Disease (CD) or Ulcerative Colitis (UC) patients were lysed in 6M urea lysis buffer and studied by immunoprecipitation with the indicated bispecific antibodies.

Figure 8A shows an example of immunoprecipitation with the indicated bispecific antibodies from intestinal cancer, dysplasia, crohn's disease or ulcerative colitis samples. The RIP2-K63 and RIP2-Lin bispecific antibodies immunoprecipitated ubiquitinated RIP2, with the strongest signal observed in CD and UC samples. FIGS. 8C and 8D show samples 1-52 (FIG. 8C) and samples 53-92 (FIG. 8D) from individual patients. The last lane in each figure contains immunoprecipitation with a combination of RIP2-K63 and RIP2-Lin antibodies from THP1 cells treated with MDP (1. Mu.g/ml, 30 min) and is used as a control (Ct). Tissue lysates from the patients listed above prepared in 6M urea lysis buffer were studied by western blotting with RIP2 and GAPDH antibodies. Red asterisks indicate samples missing from immunoprecipitation due to poor quality and low protein levels. THP1 cell lysates were used as controls. This is shown in fig. 8E.

Samples from intestinal cancer, dysplasia, diverticulitis (DIV), crohn's Disease (CD) or Ulcerative Colitis (UC) patients were lysed in 6M urea lysis buffer and lysates were immunoprecipitated with a combination of RIP2-K63 and RIP2-Lin bispecific antibodies. Western blots with anti-RIP 2 antibodies are shown in fig. 8B. To better compare RIP2 ubiquitination signals between different samples, proteins immunoprecipitated by RIP2-K63 and RIP2-Lin bispecific antibodies were run on gels adjacent to each other, again demonstrating significant RIP2 ubiquitination in CD and UC samples (fig. 8B).

Following immunoprecipitation with the indicated antibodies, RIP2 ubiquitination in the indicated patient samples was quantified by scanning western blots, as presented in fig. 8C and 8D. Scanned images were processed by ImageJ software and RIP2 ubiquitination was calculated as the gel strength ratio from immunoprecipitation with RIP2-K63/RIP2-Lin antibody versus immunoprecipitation with gD antibody, as shown in fig. 8F. ns indicates no significant difference, while three asterisks indicate p <0.0001. Figure 8F also demonstrates the striking pattern of elevated RIP 2K 63-linked ubiquitination and linear ubiquitination in CD and UC samples compared to intestinal cancer, dysplasia and diverticulitis samples. Risk alleles of IBD have been identified using whole genome association studies to identify common risk alleles for developing disease (Liu et al, 2015). Two CD risk alleles, ATG16L1 and NOD2, which have been identified across the population, were further evaluated in this group, with NOD2 being of particular interest because it is downstream of MDP.

This is shown in fig. 8G, which provides the RIP2 ubiquitination quantified in fig. 8F presented in a bar format. The figures listed below are genotype information for two common IBD risk loci: a represents ATG16L1, and N represents NOD2. Samples from patients with Crohn's disease and ulcerative colitis were found to have elevated levels of RIP2 ubiquitination. Comparison of RIP2 ubiquitination levels with ATG16L1 and NOD2 risk alleles in figure 8F did not reveal any correlation, indicating that RIP2 ubiquitination in IBD samples is not associated with these risk loci. Taken together, these data demonstrate that RIP2-K63 and RIP2-Lin bispecific antibodies can be used to study RIP2 ubiquitination status in IBD samples.

J. proteins modified with K63-linked ubiquitin chains and linear ubiquitin chains

Fig. 9A provides a schematic of the experimental design. THP1 cells were lysed in 6M urea lysis buffer for the indicated period of time by treatment with PBS, TNF (100 ng/ml), MDP (1. Mu.g/ml) or LPS (1. Mu.g/ml), immunoprecipitated with K63-Lin bispecific antibody and analyzed by mass spectrometry.

Immunoprecipitated proteins were separated on SDS-PAGE, excised and analyzed by mass spectrometry, revealing the different ubiquitinated substrates depending on the stimulus. FIG. 9B provides proteins identified by mass spectrometry. The numbers indicate the total number of identified peptides and the unique (in brackets) number for each protein. TNF stimulation resulted in the identification of known (RIP 1, TNFR 1) and novel (TRADD) ubiquitinated substrates, whereas MDP treatment and LPS treatment identified significant K63-linked ubiquitination and linear ubiquitination of RIP2, NOD2 and IRAK1, respectively. This is also demonstrated in fig. 10A to 10E. The most powerful identification signal from mass spectrometry is for RIP2 after MDP treatment.

THP1 cells were treated with the stimulus as indicated in fig. 9A, lysed in 6M urea lysis buffer, and immunoprecipitated with K63-Lin bispecific antibody. Immunoprecipitated proteins and total cell lysate proteins were probed with the indicated antibodies as shown in fig. 9C. The immunoprecipitation and western blot confirm that the ubiquitinated proteins found in mass spectrometry were indeed modified by K63 ligation ubiquitination and linear ubiquitination.

Selected ms/ms spectra matching RIP 2C-terminal SPSLNLLQNKSM (SEQ ID NO: 102) were annotated for matched fragment ions (FIG. 9D) and their ion chromatograms (XICs) extracted from the four processing conditions are shown. Matched XIC peaks are highlighted by arrows (fig. 9E) to identify ubiquitinated RIP2 from MDP treated conditions. K ^ub indicates ubiquitination at K538 of RIP2. M ^ox indicates oxidation at position M540 of the RIP2 polypeptide. This suggests that K538 is the major RIP2 modification site with K63 ligation ubiquitination and/or linear ubiquitination. Taken together, these demonstrate that in addition to general protein modifications, K63-Lin bispecific antibodies can also be used to identify specific ubiquitination sites.

This demonstrates that K63-Lin bispecific antibodies can be used to detect and identify proteins ubiquitinated with K63-linked chains and linear chains in different signaling pathways.

***

Although the invention has been described in considerable detail by way of illustration and example for the purpose of clarity of understanding, such illustration and example should not be construed to limit the scope of the invention.

Sequence listing

Hc=heavy chain; lc=light chain; HCVR = heavy chain variable region; LCVR = light chain variable region; HVR = hypervariable region.

Claims

1. A method of determining the presence of a polyubiquitin in a sample suspected of containing a polyubiquitin, wherein the polyubiquitin is a pro-inflammatory protein and comprises polyubiquitin,

Determining the binding of at least one antibody to polyubiquitin in said sample.

2. The method of claim 1, wherein the polyubiquitin protein comprises M1-linked polyubiquitin and/or K63-linked polyubiquitin.

3. The method of any one of the preceding claims, wherein the pro-inflammatory protein is a component of one or more signaling complexes.

4. The method of any one of the preceding claims, wherein the pro-inflammatory protein is receptor interacting protein kinase 1 (RIP 1), receptor interacting protein kinase 2 (RIP 2), apoptosis inhibitors 1 and 2 (c-IAP 1/2), tumor necrosis factor receptor 1 (TNFR 1), linear ubiquitin chain assembly complex (LUBAC) and/or nuclear factor- κb (NF- κb) essential modulator (NEMO).

5. The method of any one of the preceding claims, wherein the pro-inflammatory protein is RIP1.

6. The method of any one of claims 1-4, wherein the pro-inflammatory protein is RIP2.

7. The method of any one of the preceding claims, wherein the pro-inflammatory protein has an elevated level of ubiquitination in an inflammatory state relative to the level of ubiquitination when not in an inflammatory state.

8. The method of any one of the preceding claims, wherein the pro-inflammatory protein has a level of ubiquitination 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 relative to the level of ubiquitination when not in an inflammatory state.

9. The method of any one of the preceding claims, wherein an elevated level of ubiquitination is associated with an increase in severity of an inflammatory disease state.

10. The method of any one of the preceding claims, wherein the pro-inflammatory protein is associated with an inflammatory disease such as inflammatory bowel disease, crohn's disease, diverticulitis, and ulcerative colitis.

11. The method of any one of the preceding claims, wherein the pro-inflammatory protein is associated with crohn's disease.

12. The method of any one of the preceding claims, wherein the pro-inflammatory protein is associated with ulcerative colitis.

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 (RIP 1).

14. The antibody of claim 13, which selectively recognizes polyubiquitinated RIP1.

15. The antibody of claim 13 or 14, which does not recognize receptor interacting protein kinase 2 (RIP 2) and/or does not recognize non-ubiquitinated RIP1.

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 (RIP 2).

17. The antibody of claim 16, which selectively recognizes polyubiquitinated RIP2.

18. The antibody of claim 16 or 17, which does not recognize RIP1, XIAP and/or c-IAP1, and/or does not recognize non-ubiquitinated RIP2.

19. The antibody of any one of claims 13 to 18, wherein the polyubiquitin has a homogenous topology.

20. The antibody of any one of claims 13 to 19, wherein the polyubiquitin comprises a K11 bond.

21. The antibody of any one of claims 13 to 20, wherein the polyubiquitin comprises a K48 bond.

22. The antibody of any one of claims 13 to 21, wherein the polyubiquitin comprises a K63 bond.

23. The antibody of any one of claims 13 to 22, wherein the polyubiquitin comprises an M1 bond.

24. The antibody of any one of claims 13 to 23, comprising a first half antibody comprising:

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 alternatively

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.

25. The antibody of any one of claims 13 to 15 or 19 to 24, comprising a second half antibody comprising:

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 alternatively

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,

26. The antibody of any one of claims 16 to 24, comprising a second half antibody comprising:

(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 claims 13 to 26, comprising a first half antibody and a second half antibody, wherein

A) The first half antibody comprises

(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,

And the second half antibody comprises

(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;

b) The first half antibody comprises

(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,

And the second half antibody comprises

(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,

c) The first half antibody comprises

(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,

And the second half antibody comprises

(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;

d) 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 comprises

(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,

e) 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,

And the second half antibody comprises

(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,

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,

And the second half antibody comprises

(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,

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,

And the second half antibody comprises

(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,

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,

And the second half antibody comprises

(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,

i) 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,

And the second half antibody comprises

(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; j) 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 comprises

(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,

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,

And the second half antibody comprises

(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,

l) 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,

And another second half antibody comprises

(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,

28. The antibody of any one of claims 13-27, wherein the first half antibody comprises

A) Has at least about 95%, such as 96%, 97%, 98%, and,

A VL sequence having 99% or 100% sequence identity and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 8;

b) A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 21, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 22;

c) A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 35, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 36; or alternatively

D) A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 49, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 50.

29. The antibody of any one of claims 13-15, 19-25, or 27-28, wherein the second half antibody comprises

A) A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 71, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 73; or alternatively

B) A VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 72, and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 73.

30. The antibody of any one of claims 16-25 or 26-28, wherein the second half antibody comprises

A VL sequence having at least about 95%, such as 96%,97%,98%,99% or 100% sequence identity to SEQ ID No. 93, and a VH sequence having at least about 95%, such as 96%,97%,98%,99% or 100% sequence identity to SEQ ID No. 94.

31. The antibody of any one of claims 10 to 27, wherein:

a) One of the first half antibody and the second half antibody comprises a sequence identical to SEQ ID NO:

7, a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 8, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 8,

And the other of said first half antibody and said second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 71 and a VH sequence having at least about 95%, such as 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 sequence identical to SEQ ID NO:

And the other of said first half antibody and said second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 72 and a VH sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 7 and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 8, and the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 93 and a VH sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22,

e) One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22,

f) One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22,

And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 93 and a VH sequence having at least about 95%, such as 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%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36,

h) One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36,

i) One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 35, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 36,

j) One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50,

k) One of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50,

And the other of said first half antibody and said second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 72 and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 73; or alternatively

L) one of the first half antibody and the second half antibody comprises a sequence identical to SEQ ID NO:

49, such as a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity, a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50,

And the other of the first half antibody and the second half antibody comprises a VL sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 93 and a VH sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 94.

32. The antibody of any one of claims 13-31, wherein 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) A VL sequence of SEQ ID NO. 35 and a VH sequence of SEQ ID NO. 36; or alternatively

D) The VL sequence of SEQ ID NO. 49 and the VH sequence of SEQ ID NO. 50.

33. The antibody of any one of claims 13-15, 19-25, 27-29, or 31-32, wherein the second half antibody comprises

A) A VL sequence of SEQ ID NO. 71 and a VH sequence of SEQ ID NO. 73; or alternatively

B) The VL sequence of SEQ ID NO. 72 and the VH sequence of SEQ ID NO. 73.

34. The antibody of any one of claims 16-24, 26-28, or 30-32, wherein the second half antibody comprises

The VL sequence of SEQ ID NO. 93 and the VH sequence of SEQ ID NO. 94.

35. The antibody of any one of claims 13 to 34, wherein:

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,

And the other of said first half antibody and said 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 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,

And the other of said first half antibody and said 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,

And the other of said first half antibody and said 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,

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,

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,

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,

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) One of the first half antibody and the second half antibody comprises SEQ ID NO:

35 and the VH sequence of SEQ ID NO. 36,

j) One of the first half antibody and the second half antibody comprises SEQ ID NO:

49 and the VH sequence of SEQ ID NO. 50,

k) One of the first half antibody and the second half antibody comprises SEQ ID NO:

49 and the VH sequence of SEQ ID NO. 50,

And the other of said first half antibody and said second half antibody comprises the VL sequence of SEQ ID NO:72 and the VH sequence of SEQ ID NO: 73; or alternatively

L) one of the first half antibody and the second half antibody comprises SEQ ID NO:

49 and the VH sequence of SEQ ID NO. 50,

And the other of said first half antibody and said second half antibody comprises the VL sequence of SEQ ID NO. 93 and the VH sequence of SEQ ID NO. 94.

36. The antibody of any one of claims 13 to 35, which is a monoclonal antibody.

37. The antibody of any one of claims 13 to 36, which is a mouse, rabbit, human, humanized or chimeric antibody.

38. The antibody of any one of claims 13 to 37, wherein the first antigen binding site is human or humanized.

39. The antibody of any one of claims 13 to 38, wherein the second antigen binding site is human or humanized.

40. The antibody of any one of claims 13 to 39, wherein the antibody is an IgG antibody.

41. The antibody of any one of claims 13 to 40, wherein the antibody is an IgG1 antibody, an IgG2a antibody, an IgG2b antibody, an IgG3 antibody, or an IgG4 antibody.

42. The antibody of any one of claims 13 to 41, wherein the antibody is an IgG1 antibody or an IgG4 antibody.

43. The antibody of any one of claims 13-42, 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 knob mutation; or 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 knob mutation.

44. The antibody of claim 43, wherein the antibody is an IgG1 antibody, and wherein the knob mutation comprises a T366W mutation.

45. The antibody of claim 43 or claim 44, wherein the antibody is an IgG1 antibody, and wherein the mortar mutation comprises at least one mutation, at least two mutations, or three mutations, such as one to two mutations, one to three mutations, or two to three mutations, selected from T366S, L a and Y407V.

46. The antibody of claim 43, wherein the antibody is an IgG4 antibody, and wherein the knob mutation comprises a T366W mutation.

47. The antibody of claim 43 or claim 46, wherein the antibody is an IgG4 antibody, and wherein the mortar mutation comprises at least one mutation, at least two mutations, or three mutations, such as one to two mutations, one to three mutations, or two to three mutations, selected from the group consisting of T366S, L a and Y407V mutations.

48. The antibody of any one of claims 43-47, wherein the first half antibody comprises

A) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2;

b) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 16;

c) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30; or alternatively

D) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 44.

49. The antibody of any one of claims 13-48, wherein the first half antibody comprises

A) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 4;

b) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 18;

c) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 32; or alternatively

D) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

50. The antibody of any one of claims 13-49, wherein the first half antibody comprises

A) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 4;

b) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No.16, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 18;

c) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 32; or alternatively

D) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 44, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

51. The antibody of any one of claims 13-48, wherein the first half antibody comprises

A) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 6;

b) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20;

c) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 34; or alternatively

D) A heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

52. The antibody of any one of claims 13-48 or 51, wherein the first half antibody comprises

A) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 2, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 6;

b) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No.16, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 20;

c) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 30, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 34; or alternatively

D) A light chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 44, and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

53. The antibody of any one of claims 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.

54. The antibody of any one of claims 13 to 15, 19 to 15, 27 to 29, 31 to 33, or 35 to 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; optionally wherein the heavy chain lacks a C-terminal lysine.

55. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 35 to 54, 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 and a heavy chain sequence having at least 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 68; optionally wherein one or more heavy chains lacks a C-terminal lysine.

56. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 33 to 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; optionally wherein the heavy chain lacks a C-terminal lysine.

57. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 53, or 56, 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 and a heavy chain sequence having at least 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 70; optionally wherein one or more heavy chains lacks a C-terminal lysine.

58. The antibody of any one of claims 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.

59. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 31, 34 to 52 or 5528, wherein said 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. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

60. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, or 58 to 59, 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

61. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52 or 58, 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. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

62. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 33 to 52, 58, or 61, 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

63. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, or 35 to 57, wherein:

a) The first 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.2 and at least about 95%, such as 96%, 97%, 98%,

Heavy chain sequences of 99% or 100% sequence identity,

And 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 and at least about 95%, such as 96%, to SEQ ID NO. 70,

A heavy chain sequence of 97%, 98%, 99% or 100% sequence identity;

b) The first 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. 2, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 6,

c) The first 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. 16, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 18,

d) The first 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. 16, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20,

And 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 and at least about 95%, such as 96%, to SEQ ID NO. 68,

A heavy chain sequence of 97%, 98%, 99% or 100% sequence identity;

e) The first 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. 30, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 32,

f) Has at least about 95%, such as 96%, 97%, 98%, and,

A chain sequence having 99% or 100% sequence identity to SEQ ID NO. 34, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity,

g) The first 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. 44, and at least about 95%, such as 96%, 97%, 98%,

Heavy chain sequences of 99% or 100% sequence identity,

h) The first 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. 44, and at least about 95%, such as 96%, 97%, 98%,

Heavy chain sequences of 99% or 100% sequence identity,

And 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 and a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 68; optionally wherein one or more heavy chains lacks a C-terminal lysine.

64. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, or 58 to 62, wherein:

a) The first 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. 2, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4,

And 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 and at least about 95%, such as 96%, to SEQ ID NO. 92,

A heavy chain sequence of 97%, 98%, 99% or 100% sequence identity;

f) The first 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. 30, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 34,

g) The first 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. 44, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 46,

h) The first 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. 44, a heavy chain sequence having at least about 95%, such as 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 48,

65. The antibody of any one of claims 13-64, wherein 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 alternatively

D) The light chain sequence of SEQ ID NO. 44.

66. The antibody of any one of claims 13-65, wherein 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 alternatively

D) The heavy chain sequence of SEQ ID NO. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

67. The antibody of any one of claims 13-66, wherein the first half antibody comprises

A) The light chain sequence of SEQ ID NO. 2 and the heavy chain sequence of SEQ ID NO. 4;

b) The light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 18;

c) The light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 32; or alternatively

D) The light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 46; optionally wherein one or more heavy chains lacks a C-terminal lysine.

68. The antibody of any one of claims 13-65, wherein 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 alternatively

D) The heavy chain sequence of SEQ ID NO. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

69. The antibody of any one of claims 13-65 or 68, wherein the first half antibody comprises

A) The light chain sequence of SEQ ID NO. 2 and the heavy chain sequence of SEQ ID NO. 6;

b) The light chain sequence of SEQ ID NO. 16 and the heavy chain sequence of SEQ ID NO. 20;

c) The light chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 34; or alternatively

D) The light chain sequence of SEQ ID NO. 44 and the heavy chain sequence of SEQ ID NO. 48; optionally wherein one or more heavy chains lacks a C-terminal lysine.

70. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 53, 63, or 65 to 69, wherein said second half antibody comprises the light chain sequence of seq id No. 60.

71. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 54, 63, or 65 to 70, wherein the second half antibody comprises the heavy chain sequence of seq id No. 68; optionally wherein the heavy chain lacks a C-terminal lysine.

72. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 55, 63, or 65 to 71, wherein the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68; optionally wherein one or more heavy chains lacks a C-terminal lysine.

73. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 54, 63, or 65 to 70, wherein the second half antibody comprises the heavy chain sequence of seq id No. 70; optionally wherein the heavy chain lacks a C-terminal lysine.

74. The antibody of any one of claims 13-15, 19-25, 27-29, 31-33, 35-54, 56, 63, 65-70, or 73, wherein the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70; optionally wherein one or more heavy chains lacks a C-terminal lysine.

75. The antibody of any one of claims 16-24, 26-28, 30-32, 34-52, 58, or 64-69, wherein the second half antibody comprises the light chain sequence of SEQ ID No. 84.

76. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 59, 64 to 69, or 75, wherein the second half antibody comprises the heavy chain sequence of seq id No. 90; optionally wherein one or more heavy chains lacks a C-terminal lysine.

77. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 60, 64 to 69, or 75 to 76, wherein 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 wherein one or more heavy chains lacks a C-terminal lysine.

78. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58, 61, 64 to 69, or 75, wherein the second half antibody comprises the heavy chain of SEQ ID No. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

79. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58, 61 to 62, 64 to 69, 75 or 78, wherein the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92; optionally wherein one or more heavy chains lacks a C-terminal lysine.

80. The antibody of any one of claims 16 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, or 65 to 74, wherein:

a) The first half antibody comprises the light chain sequence of SEQ ID NO. 2 and SEQ ID NO:

4, the heavy chain sequence of the sequence No.,

b) The first half antibody comprises the light chain sequence of SEQ ID NO. 2 and SEQ ID NO:

6, a heavy chain sequence of a heavy chain,

c) The first half antibody comprises the light chain sequence of SEQ ID NO. 16 and SEQ ID NO:

18, a heavy chain sequence of a heavy chain,

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,

e) The first half antibody comprises the light chain sequence of SEQ ID NO. 30 and SEQ ID NO:

32, a heavy chain sequence of a heavy chain,

f) The first half antibody comprises the light chain sequence of SEQ ID NO. 30 and SEQ ID NO:

34, a heavy chain sequence of a heavy chain,

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,

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,

81. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, or 75 to 79, wherein:

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,

6, a heavy chain sequence of a heavy chain,

18, a heavy chain sequence of a heavy chain,

32, a heavy chain sequence of a heavy chain,

f) The chain sequence of SEQ ID NO. 30 and the heavy chain sequence of SEQ ID NO. 34,

82. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 4, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more heavy chains lacks a C-terminal lysine.

83. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 6, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

84. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 18, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more heavy chains lacks a C-terminal lysine.

85. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 20, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

86. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 32, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more heavy chains lacks a C-terminal lysine.

87. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 34, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

88. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 46, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 70, and optionally wherein one or more heavy chains lacks a C-terminal lysine.

89. The antibody of any one of claims 13 to 15, 19 to 25, 27 to 29, 31 to 33, 35 to 57, 63, 65 to 74 or 80, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 48, and the second half antibody comprises the light chain sequence of SEQ ID No. 60 and the heavy chain sequence of SEQ ID No. 68, and optionally wherein one or more of the heavy chains lacks a C-terminal lysine.

90. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 4, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more heavy chains are deleted of the C-terminal lysine.

91. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 2 and the heavy chain sequence of SEQ ID No. 6, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more heavy chains are deleted of the C-terminal lysine.

92. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 18, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more heavy chains are deleted of the C-terminal lysine.

93. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 16 and the heavy chain sequence of SEQ ID No. 20, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more heavy chains are deleted of the C-terminal lysine.

94. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 32, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more heavy chains are missing a C-terminal lysine.

95. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 30 and the heavy chain sequence of SEQ ID No. 34, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more heavy chains are missing a C-terminal lysine.

96. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 46, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 92, and optionally wherein one or more heavy chains are deleted of the C-terminal lysine.

97. The antibody of any one of claims 16 to 24, 26 to 28, 30 to 32, 34 to 52, 58 to 62, 64 to 69, 75 to 79 or 81, wherein the first half antibody comprises the light chain sequence of SEQ ID No. 44 and the heavy chain sequence of SEQ ID No. 48, and the second half antibody comprises the light chain sequence of SEQ ID No. 84 and the heavy chain sequence of SEQ ID No. 90, and optionally wherein one or more heavy chains are deleted of the C-terminal lysine.

98. The antibody of any one of claims 13 to 97, which is a bispecific antibody.

99. The antibody of any one of claims 13-98, which is a diabody, a triabody, or a tetrabody.

100. The antibody of any one of claims 13 to 99, which is conjugated to a label.

101. The antibody of claim 100, wherein the label is a fluorescent label, an enzymatic label, or a chromogenic label.

102. The antibody of claim 100, wherein the label is a radioisotope, optionally a positron emitter, optionally ⁸⁹ Zr.

103. A composition comprising the antibody of any one of claims 13 to 102, wherein the composition is substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies.

104. An immunoconjugate comprising the antibody of any one of claims 13 to 102 and a cytotoxic or anti-inflammatory agent.

105. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier and at least one of:

a) The antibody of any one of claims 13 to 102; or alternatively

B) The immunoconjugate according to claim 104;

Optionally wherein the composition is substantially free of monospecific antibodies, unassembled half antibodies, or both monospecific antibodies and unassembled half antibodies.

106. The pharmaceutical formulation of claim 105, further comprising an additional therapeutic agent.

107. An isolated nucleic acid encoding the antibody of any one of claims 13 to 102.

108. A vector comprising the nucleic acid of claim 107.

109. A host cell comprising the nucleic acid of claim 107.

110. A method of producing an antibody, the method comprising culturing the host cell of claim 106 under conditions wherein the antibody is produced.

111. The method of claim 110, further comprising recovering the antibody from the host cell.

112. A method of making the antibody of any one of claims 13 to 102, the method comprising forming the antibody from a first half antibody and a second half antibody.

113. An antibody according to any one of claims 13-102 for use as a medicament.

114. A method of determining the presence of polyubiquitin in a sample suspected of containing polyubiquitin or polyubiquitin, the method comprising exposing the sample to an antibody according to any one of claims 13 to 102, and determining binding of the antibody to polyubiquitin in the sample.

115. A method of separating K11-linked polyubiquitin from non-K11-linked polyubiquitin in a sample, the method comprising contacting the sample with the antibody of any one of claims 13 to 102.

116. A method of separating K48-linked polyubiquitin from non-K48-linked polyubiquitin in a sample, the method comprising contacting the sample with the antibody of any one of claims 13 to 102.

117. A method of separating K63-linked polyubiquitin from non-K63-linked polyubiquitin in a sample, the method comprising contacting the sample with the antibody of any one of claims 13 to 102.

118. A method of separating M1-linked polyubiquitin from non-M1-linked polyubiquitin in a sample, the method comprising contacting the sample with the antibody of any one of claims 13-102.

119. A method of determining the function and/or activity of a polyubiquitin in a cell or sample, the method comprising contacting the cell or sample with an antibody according to any one of claims 13 to 102, and assessing the effect of the contacting step on the cell or sample.

120. The method of any one of claims 114-119, wherein the polyubiquitin protein comprises RIP1.

121. The method of any one of claims 114-119, wherein the polyubiquitin protein comprises RIP2.

122. A method of determining the presence of a polyubiquitin protein in a sample suspected of containing the polyubiquitin protein, wherein the polyubiquitin protein is a pro-inflammatory protein and comprises polyubiquitin, the method comprising exposing the sample to the antibody according to any one of claims 10 to 99.

123. The method of claim 122, wherein the polyubiquitin protein comprises M1-linked polyubiquitin and/or K63-linked polyubiquitin.

124. The method of claim 122 or 123, wherein the pro-inflammatory protein is a component of one or more signaling complexes.

125. The method of any one of claims 122-124, wherein the pro-inflammatory protein is RIP1.

126. The method of any one of claims 122-125, wherein the pro-inflammatory protein is RIP2.

127. The method of any one of claims 122-126, wherein the pro-inflammatory protein has an elevated level of ubiquitination in an inflammatory state relative to the level of ubiquitination when not in an inflammatory state.

128. The method of any one of claims 122-127, wherein the pro-inflammatory protein has a level of ubiquitination of at least 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 1 to 12, 2 to 12, 3 to 12, 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 10 to 12, or 11 to 12 fold in an inflammatory state relative to the level of ubiquitination when not in the inflammatory state.

129. The method of any one of claims 122-128, wherein an elevated level of ubiquitination is associated with an increase in the severity of an inflammatory disease state.

130. The method of any one of claims 122-129, wherein the pro-inflammatory protein is associated with an inflammatory disease such as inflammatory bowel disease, crohn's disease, diverticulitis, and ulcerative colitis.

131. The method of any one of claims 122-130, wherein the pro-inflammatory protein is associated with crohn's disease.

132. The method of any one of claims 122-131, wherein the pro-inflammatory protein is associated with ulcerative colitis.