WO2014144616A2

WO2014144616A2 - Anti-alpha v beta 5 antibodies and uses thereof

Info

Publication number: WO2014144616A2
Application number: PCT/US2014/029100
Authority: WO
Inventors: Shelia M. Violette; Dean Sheppard; Amha Atakilit; Timothy David Jones; Francis Joseph Carr; Anja Sibylle TESSARZ
Original assignee: Biogen Idec Ma Inc.; The Regents Of The University Of California
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2014-09-18
Also published as: WO2014144616A3

Abstract

Humanized antibodies and antibody fragments that bind to ανβ5 are disclosed. Also disclosed are methods of using the disclosed antibodies and antibody fragments to treat or prevent avp5-mediated diseases.

Description

Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

ANTI-ALPHA V BETA 5 ANTIBODIES AND USES THEREOF

Cross-Reference to Related Applications

This application claims the benefit of priority to U.S. Provisional Appl. No.

61/798,216 filed March 15, 2013, and U.S. Provisional Appl. No. 61/899,001 filed November 1, 2013, the contents of both of which are incorporated by reference in their entirety herein.

Field

This invention relates generally to antibodies that bind to the alpha v beta 5 (av s) integrin and uses thereof.

Background

Integrins are cell surface glycoprotein receptors that bind extracellular matrix proteins and mediate cell-cell and cell-extracellular matrix interactions, and cell-pathogen

interactions. These receptors are composed of noncovalently associated alpha (a) and beta (β) chains that combine to give a variety of heterodimeric proteins with distinct cellular and adhesive specificities. These proteins can interact with cell surface ligands, transmembrane proteins, soluble proteases, pathogens, and growth factors. The importance of these receptors in biological processes is underscored by the pathological sequelae following integrin defects and from the often severe phenotypes of integrin subunit knockout animals.

The ανβ5 integrin is the only integrin that contains the β5 subunit. ανβ5 recognizes the RGD peptide sequence and binds vitronectin (Hynes, Cell, 69: 1 1-25 (1992). In addition, ανβ5 can activate TGF-β by a mechanism requiring an intact cytoskeleton and cell

contraction. ΤϋΡβΙ is normally secreted as a complex composed of 3 proteins, including the bioactive peptide of ΤϋΡβΙ, latency-associated peptide βΐ (LAP-βΙ), and latent ΤϋΡβ

(ΧΤϋΡβ) binding protein 1 (LTBP-1). ΤϋΡβΙ forms a noncovalent complex with LAP-βΙ, which is called small latent complex (SLC), and in this configuration, ΤϋΡβΙ is unable to bind to its receptors. ανβ5 binds the latency-associated peptide βΐ (LAP-βΙ) of the small latent complex (SLC) by recognizing an RGD motif and leads to activation of TGF-β.

Activation of TGF-β by the ανβ5 integrin occurs in scleroderma fibroblasts promoting the transition of fibroblasts into fibrogenic myofibroblasts. ανβ5 is present on mesenchymal Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

cells and is also able to activate mesenchymal TGF-β. av and β5 have both been sequenced and characterized (Hynes, 1992 supra and U.S. Patent No. 5,527,679, respectively).

Summary

This disclosure features antibodies and antigen-binding fragments thereof that specifically bind to ανβ5 and/or β5 and their use to treat, prevent, or reduce the symptoms or severity of ανβ5 -mediated diseases or conditions such as acute lung injury, acute respiratory distress syndrome (ARDS), pulmonary edema, lung fibrosis, sepsis, stroke, myocardial infarction, cancer, and ocular neovascularization disease.

In one aspect, the application discloses an isolated antibody or an antigen-binding fragment thereof that specifically binds to ανβ5, wherein the antibody or the antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: l . In certain embodiments, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain

embodiments, any of the above antibodies or antigen-binding fragments thereof further comprises or consists of a light chain variable region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 1. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: l and further comprises or consists of a light chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13. In Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: l and further comprises or consists of a light chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1 and further comprises or consists of a light chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1 , 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen- binding fragment thereof comprises light chain CDRs 1 , 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific

embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 62; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

In certain embodiments, the antibody has an isotype selected from the group

consisting of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody is modified to reduce or eliminate effector function. In a specific embodiment, the antibody is an IgGl that is modified to reduce or eliminate effector function (e.g., T299A mutation). In certain embodiments, the antigen-binding fragment is selected from the group consisting of an Fab, an Fab', an F(ab')2, an Fv, a diabody, an scFv, and an sc(Fv)2. In certain embodiments, the antibody or antigen-binding fragment thereof is conjugated to a substance selected from the group consisting of a toxin, a radionuclide, a fluorescent label, polyethylene glycol, and a cytotoxic agent. In some embodiments, the antibody or the antigen-binding fragment thereof is formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat a cancer in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments where the antibody or the antigen-binding fragment thereof is used to treat cancer, it is conjugated to a cytotoxic agent. In some embodiments, the antibody or the antigen-binding fragment thereof is administered to a human subject having a pancreatic cancer, a lung cancer, a breast cancer, a colorectal cancer, a head and neck cancer, an esophageal cancer, a skin cancer, or an

endometrial cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is administered to a human subject to inhibit angiogenesis and thereby prevent or retard the development of cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent a lung fibrosis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In a particular embodiment, the lung fibrosis is idiopathic pulmonary fibrosis (IPF). In another embodiment, the lung fibrosis is usual interstitial pneumonia (UIP). In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent stroke or myocardial infarction in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

embodiments, the antibody or the antigen-binding fragment thereof is used to treat, reduce, retard, or prevent a condition such as acute lung injury, acute respiratory disease syndrome (ARDS), pulmonary edema, or sepsis in a human subject in need thereof, comprising

administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent a viral infection wherein the viral infection proceeds, at least in part, due to an interaction between a viral RGD-containing protein and ανβ5. In certain embodiments, the antibodies or antigen binding fragments of this aspect inhibit ανβ5 binding to vitronectin, inhibit ανβ5 binding to fibronectin, osteopontin, tenascin c, or adenovirus penton base, inhibit ανβ5 binding to LAP of TGF-β, inhibit ανβ5 binding to RGD-motif containing ligands of ανβ5, inhibit TGF-β signaling, and/or inhibit TGF-β activation.

In one aspect, the application discloses an isolated antibody or an antigen-binding fragment thereof that specifically binds to ανβ5, wherein the antibody or the antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3. In certain embodiments, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:3. In certain embodiments, any of the above antibodies or antigen-binding fragments thereof further comprises or consists of a light chain variable region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: l 1. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:3 and further comprises or consists of a light chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

acid sequence set forth in SEQ ID NO: 13. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3 and further comprises or consists of a light chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3 and further comprises or consists of a light chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid

sequence set forth in SEQ ID NO: 3 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain

CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain CDRs 1 , 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

In certain embodiments, the antibody has an isotype selected from the group

endometrial cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is administered to a human subject to inhibit angiogenesis and thereby prevent or retard the development of cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent lung fibrosis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

thereof. In a particular embodiment, the lung fibrosis is idiopathic pulmonary fibrosis. In some embodiments, the lung fibrosis is usual interstitial pneumonia. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent stroke or myocardial infarction in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat, reduce, retard, or prevent a condition such as acute lung injury, acute respiratory disease syndrome (ARDS), pulmonary edema, or sepsis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some

embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent a viral infection wherein the viral infection proceeds, at least in part, due to an interaction between a viral RGD-containing protein and ανβ5. In certain embodiments, the antibodies or antigen binding fragments of this aspect inhibit ανβ5 binding to vitronectin, inhibit ανβ5 binding to LAP of TGF-β, inhibit ανβ5 binding to RGD-motif containing ligands of ανβ5, inhibit TGF-β signaling, and/or inhibit TGF-β activation.

In one aspect, the application discloses an isolated antibody or an antigen-binding fragment thereof that specifically binds to ανβ5, wherein the antibody or the antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5. In certain embodiments, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:5. In certain embodiments, any of the above antibodies or antigen-binding fragments thereof further comprises or consists of a light chain variable region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments of this aspect, the antibody or an antigen- binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:5 and further comprises or consists of a light chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5 and further comprises or consists of a light chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:5 and further comprises or consists of a light chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific

embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 or consists of comprises the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises or consists of a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding

fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments wherein the antibody or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

In certain embodiments, the antibody has an isotype selected from the group

endometrial cancer. In some embodiments, the antibody or the antigen-binding fragment Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

thereof is administered to a human subject to inhibit angiogenesis and thereby prevent or retard the development of cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent lung fibrosis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In a particular embodiment, the lung fibrosis is idiopathic pulmonary fibrosis. In some embodiments, the lung fibrosis is usual interstitial pneumonia. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent stroke or myocardial infarction in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat, reduce, retard, or prevent a condition such as acute lung injury, acute respiratory disease syndrome (ARDS), pulmonary edema, or sepsis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some

In one aspect, the application discloses an isolated antibody or an antigen-binding fragment thereof that specifically binds to ανβ5, wherein the antibody or the antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, any of the above antibodies or antigen-binding fragments thereof further comprises or consists of a light chain variable region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 7 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 and further comprises a light chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7 and further comprises or consists of a light chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7 and further comprises or consists of a light chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In a specific embodiment, the antibody or an antigen- binding fragment thereof comprises a heavy chain variable region that comprises or consists Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1 , 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1 , 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid

sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1 , 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of he amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain CDRs 1 , 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

In certain embodiments, the antibody has an isotype selected from the group

consisting of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody is modified to reduce or eliminate effector function. In a specific embodiment, the antibody is an IgGl that is modified to reduce or eliminate effector function (e.g., T299A mutation). In certain embodiments, the antigen-binding fragment is selected from the group consisting of an Fab, an Fab', an F(ab')2, an Fv, a diabody, an scFv, and an sc(Fv)2. In certain embodiments, the antibody or antigen-binding fragment thereof is conjugated to a substance selected from the group consisting of a toxin, a radionuclide, a fluorescent label, polyethylene glycol, and a cytotoxic agent. In some embodiments, the antibody or the antigen-binding fragment thereof is formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat a cancer in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments where the antibody or the antigen-binding fragment thereof is used to treat cancer, it is conjugated to a cytotoxic Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

agent. In some embodiments, the antibody or the antigen-binding fragment thereof is administered to a human subject having a pancreatic cancer, a lung cancer, a breast cancer, a colorectal cancer, a head and neck cancer, an esophageal cancer, a skin cancer, or an

endometrial cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is administered to a human subject to inhibit angiogenesis and thereby prevent or retard the development of cancer. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent lung fibrosis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In a particular embodiment, the lung fibrosis is idiopathic pulmonary fibrosis. In some embodiments, the lung fibrosis is usual interstitial pneumonia. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat or prevent stroke or myocardial infarction in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat, reduce, retard, or prevent a condition such as acute lung injury, acute respiratory disease syndrome (ARDS), pulmonary edema, or sepsis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some

In one aspect, the application discloses an isolated antibody or an antigen-binding fragment thereof that specifically binds to ανβ5, wherein the antibody or the antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, any of the above antibodies or antigen-binding fragments thereof further comprises or consists of Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a light chain variable region that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:9 and further comprises or consists of a light chain variable region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9 and further comprises or consists of a light chain variable region that is at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments of this aspect, the antibody or an antigen-binding fragment thereof comprises or consists of a heavy chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9 and further comprises or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

consists of a light chain variable region that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 17. In a specific embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain variable region that comprises or consists of the amino acid

sequence set forth in SEQ ID NO: 9 and a light chain variable region that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain

CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in

SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

antibody or the antigen-binding fragment thereof comprises light chain CDRs I, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20; and the heavy chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain CDRs 1 , 2 and 3 wherein the light chain CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56 or the amino acid sequence set forth in SEQ ID NO: 56 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 or the amino acid sequence set forth in SEQ ID NO: 57 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 56; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 ; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

sequence set forth in SEQ ID NO: 58 or the amino acid sequence set forth in SEQ ID NO: 58 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or the amino acid sequence set forth in SEQ ID NO: 59 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In certain embodiments of this aspect, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino acid sequence set forth in SEQ ID NO: 61 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions. In a specific embodiment, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments wherein the antibody or antibody-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60 or the amino acid sequence set forth in SEQ ID NO: 60 with a substitution at two or fewer amino acid positions; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or the amino Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

acid sequence set forth in SEQ ID NO: 61 with a substitution at two or fewer amino acid positions; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62 or the amino acid sequence set forth in SEQ ID NO: 62 with a substitution at two or fewer amino acid positions; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 or the amino acid sequence set forth in SEQ ID NO: 63 with a substitution at two or fewer amino acid positions; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64 or the amino acid sequence set forth in SEQ ID NO: 64 with a substitution at two or fewer amino acid positions; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 or the amino acid sequence set forth in SEQ ID NO: 65 with a substitution at two or fewer amino acid positions. In a specific embodiment wherein the antibody or antibody -binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the antibody or the antigen-binding fragment thereof comprises heavy chain alternate CDRs 1, 2 and 3 wherein the heavy chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60; the heavy chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61; and the heavy chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62; and light chain alternate CDRs 1, 2 and 3 wherein the light chain alternate CDR 1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63; the light chain alternate CDR 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 64; and the light chain alternate CDR 3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65.

In certain embodiments, the antibody has an isotype selected from the group

consisting of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody is modified to reduce or eliminate effector function. In a specific embodiment, the antibody is an IgGl that is modified to reduce or eliminate effector function (e.g., T299A mutation). In certain embodiments, the antigen-binding fragment is selected from the group consisting of an Fab, an Fab', an F(ab')2, an Fv, a diabody, an scFv, and an sc(Fv)2. In certain embodiments, the antibody or antigen-binding fragment thereof is conjugated to a substance selected from the group consisting of a toxin, a radionuclide, a fluorescent label, polyethylene glycol, and a cytotoxic agent. In some embodiments, the antibody or the antigen-binding fragment thereof Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

is formulated with a pharmaceutically acceptable carrier as a pharmaceutical composition. In some embodiments, the antibody or the antigen-binding fragment thereof is used to treat a cancer in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof. In some embodiments where the antibody or the antigen-binding fragment thereof is used to treat cancer, it is conjugated to a cytotoxic agent. In some embodiments, the antibody or the antigen-binding fragment thereof is administered to a human subject having a pancreatic cancer, a lung cancer, a breast cancer, a colorectal cancer, a head and neck cancer, an esophageal cancer, a skin cancer, or an

In another aspect, this application features an isolated nucleic acid comprising a nucleotide sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least

89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence set Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

forth in SEQ ID NOs.: 2, 4, 6, 8, or 10. The proteins encoded by these nucleic acids

specifically bind to ανβ5 or β5. This disclosure also includes proteins encoded by any of the above nucleic acids. In addition, this disclosure includes recombinant vectors comprising any of the above nucleic acids. Furthermore, this application provides host cells comprising recombinant vectors comprising any of the above nucleic acids.

In another aspect, this application features an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NOs.: 1, 3, 5, 7, or 9. The proteins encoded by these nucleic acids specifically bind to ανβ5 or β5. This disclosure also includes proteins encoded by any of the above nucleic acids. In addition, this disclosure includes recombinant vectors comprising any of the above nucleic acids.

Furthermore, this application provides host cells comprising recombinant vectors comprising any of the above nucleic acids.

In another aspect, this application features an isolated nucleic acid comprising a nucleotide sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence set forth in SEQ ID NOs.: 12, 14, 16, or 18. The proteins encoded by these nucleic acids when combined with one of the proteins of SEQ ID NOs: 1, 3, 5, 7, or 9, specifically bind to ανβ5 or β5. This disclosure also includes proteins encoded by any of the above nucleic acids. In addition, this disclosure includes recombinant vectors comprising any of the above nucleic acids. Furthermore, this application provides host cells comprising recombinant vectors comprising any of the above nucleic acids.

In another aspect, this application features an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NOs.: 1 1, 13, 15, or 17. The proteins encoded by these nucleic acids when combined with one of the proteins of SEQ ID NOs: 1, 3, 5, or 7, specifically bind to ανβ5 or β5. This disclosure also includes Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

proteins encoded by any of the above nucleic acids. In addition, this disclosure includes recombinant vectors comprising any of the above nucleic acids. Furthermore, this application provides host cells comprising recombinant vectors comprising any of the above nucleic acids.

In yet another aspect, this disclosure features a method of preparing a humanized antibody comprising culturing a host cell comprising recombinant vectors comprising the nucleic acid sequence set forth in SEQ ID NO: 2 and the nucleic acid sequence set forth in any one of SEQ ID NOs: 12, 14, 16, or 18; or the nucleic acid sequence set forth in SEQ ID NO: 4 and the nucleic acid sequence set forth in any one of SEQ ID NOs: 12, 14, 16, or 18; or the nucleic acid sequence set forth in SEQ ID NO: 6 and the nucleic acid sequence set forth in any one of SEQ ID NOs: 12, 14, 16, or 18; or the nucleic acid sequence set forth in SEQ ID NO: 8 and the nucleic acid sequence set forth in any one of SEQ ID NOs: 12, 14, 16, or 18; or the nucleic acid sequence set forth in SEQ ID NO: 10 and the nucleic acid sequence set forth in any one of SEQ ID NOs: 12, 14, 16, or 18, under conditions appropriate for expression of a humanized antibody, wherein humanized antibody chains are expressed and a humanized antibody is produced. In certain embodiments, the method further involves isolating the humanized antibody. In some embodiments, the host cell is a CHO cell.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Brief Description of the Drawings

Fig. 1 is an alignment of the VH amino acid sequences of five humanized ALULA VH regions with the VH region of murine ALULA.

Fig. 2 is an alignment of the VL amino acid sequences of four humanized ALULA VL regions with the VL region of murine ALULA.

Fig. 3 is a schematic representation of plasmid maps for light chain expression vector pANTVk and heavy chain expression vector pANTVhGl . Both VH and VK vectors contain genomic DNA fragments incorporating introns and poly A sequences. Expression of both chains is driven by a CMV promoter and selection (on the heavy chain vector) is via a DHFR mini gene.

Fig. 4 is a representation of a Coomassie Blue-stained SDS-PAGE gel of selected protein A-purified humanized ALULA antibodies. 2μg of each sample was loaded on a

NuPage 4-12% Bis-Tris gel (Invitrogen Cat. No. NP0322BOX) and run at 200 V for 35 min. Size marker is prestained protein standard Fermentas PageRuler Plus (Cat. No. SM1811).

Fig. 5 is a schematic representation of FACS competition assay with humanized

ALULA antibodies. Titrations of humanized anti-integrin ανβ5 antibodies were competed for binding to CS-1 β5 cells against a fixed concentration of PE-Fab labeled chimeric ALULA antibody.

Fig. 6 is a series of bar graphs providing the results of the functional cell adhesion assay described in Example 6. Titrations of variant antibodies were pre-incubated with

SW480 cells for 20 min at 37°C. Antibody cell complexes were dispensed into a plate coated with either vitronectin or BSA (negative control) and incubated for 1.5h at 37°C. Cell adhesion was measured using Cell Titer-Glo® Luminescent Cell Viability Assay.

Fig. 7 is a bar graph comparing the frequency of donor allotypes expressed in the study cohort (of Example 7) and the world population.

Fig. 8 is a bar graph depicting viability of PBMC from six donors cultured for seven days with chimeric or humanized ανβ5 antibodies and controls. Mean viability (n=6) was Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

assessed by Trypan Blue dye exclusion using a Beckman Coulter ViCell™ XR instrument and is expressed as a percentage (standard deviation error bars are shown).

Fig. 9 is a series of bar graphs ((a)-(d)) depicting healthy donor T cell proliferation responses to STR02 test antibodies. PBMC from bulk cultures were sampled and assessed for proliferation on days 5, 6, 7 and 8 after incubation with the three test samples.

Proliferation responses with an SI > 2.0 (p < 0.05), indicated by red dotted line that were significant (p < 0.05) using an unpaired, two sample student's t test were considered positive, (a) chimeric antibody, (b) VH1/VK1, (c) VH1/VK2, and (d) humanized A33.

Fig. 10 is a graph comparing the immunogenicity predicted using EpiScreen™

technology and immunogenicity observed in a clinical setting. Sixteen therapeutic proteins were tested for their relative risk of immunogenicity using EpiScreen™ technology. Results were plotted against the frequency of immunogenicity (anti-therapeutic antibody responses) observed for each protein when used in the clinic (data sourced from PubMed). The line of regression and the correlation coefficient is shown.

Detailed Description

This disclosure features antibodies and antigen-binding fragments that specifically bind the ανβ5 integrin. More specifically, these antibodies and antigen-binding fragments bind the β5 subunit of the ανβ5 integrin. The ανβ5 integrin recognizes the RGD peptide sequence and binds vitronectin. The antibodies and antigen-binding fragments thereof described herein block the interaction between ανβ5 and vitronectin. The antibodies and antigen-binding fragments thereof described herein can also block the interaction between ανβ5 and a ligand such as fibronectin, osteopontin, tenascin c, or adenovirus penton base.

The antibodies and antigen-binding fragments described herein are useful in treatment of o^5-associated disorders such as acute lung injury, acute respiratory distress syndrome, pulmonary edema, lung fibrosis (e.g., IPF, UIP), sepsis, stroke, myocardial infarction, cancer, and ocular neovascularization disease. In addition, the antibody or the antigen-binding fragment thereof can be used to treat or prevent a pathogenic (e.g., viral) infection, where the pathogenic infection proceeds, at least in part, by an interaction between a pathogen's RGD- containing protein and ανβ5. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

β5

The amino acid sequence of the human β5 protein (Genbank® Accession No.

NP_002204.2) is shown below:

MPRAPAPLYA CLLGLCALLP RLAGLNICTS GSATSCEECL LIHP CAWCS EDFGSPRSI TSRCDLRANL VK GCGGEIE SPASSFHVLR SLPLSS GSG SAGWDVIQMT PQEIAVNLRP GD TTFQLQV RQVEDYPVDL YYLMDLSLSM DDLDNIRSL GT LAEEMR LTSNFRLGFG SFVD DISPF SYTAPRYQTN PCIGYKLFPN CVPSFGFRHL LPLTDRVDSF NEEVR QRVS RNRDAPEGGF DAVLQAAVCK EKIGWRKDAL HLLVFTTDDV PHIALDGKLG GLVQPHDGQC HLNEANEYTA SNQMDYPSLA LLGEKLAENN INLIFAVTKN HYMLYKNFTA LIPGTTVEIL DGDSKNIIQL IINAYNSIRS KVELSVWDQP EDLNLFFTAT CQDGVSYPGQ RKCEGLKIGD TASFEVSLEA RSCPSRHTEH VFALRPVGFR DSLEVGVTYN CTCGCSVGLE PNSARCNGSG TYVCGLCECS PGYLGTRCEC QDGENQSVYQ NLCREAEGKP LCSGRGDCSC NQCSCFESEF GKIYGPFCEC DNFSCARNKG VLCSGHGECH CGECKCHAGY IGDNCNCSTD ISTCRGRDGQ ICSERGHCLC GQCQCTEPGA FGEMCEKCPT CPDACSTKRD CVECLLLHSG KPDNQTCHSL CRDEVITWVD TIVKDDQEAV LCFYKTAKDC VMMFTYVELP SGKSNLTVLR EPECGNTPNA MTILLAWGS ILLVGLALLA IWKLLVTIHD RREFAKFQSE RSRARYEMAS NPLYRKPIST

HTVDFTFNKF NKSYNGTVD (SEQ ID NO:46)

The amino acid sequence of the murine β5 protein (Genbank® Accession No.

NP_001 139356.1) is shown below:

MPRVPATLYACLLGLCALVP RLAGLNICTSGSATSCEECL LIHPKCAWCS KEYFGNPRSI TSRCDLKANLIRNGCEGEIE SPASSTHVLR NLPLS SKGS SATGSDVIQMT PQEIAVSLRP GEQTTFQLQVRQVEDYPVDLYYLMDLSLSM KDDLENIRSLGTKLAEEMRK LTSNFRLGFG SFVDKDISPFSYTAPRYQTNPCIGYKLFPN CVPSFGFRHLLPLTDRVDSFNEEVRKQRVS RNRDAPEGGF DAVLQAAVCKEKIGWRKDALHLLVFTTDDVPHIALDGKLGGLVQPHDGQC HLNEANEYTASNQMDYPSLALLGEKLAENNINLIFAVTKN HYMLYKNFTALIPGTTVEIL HGDSKNIIQLIINAYSSIRA KVELSVWDQP EDLNLFFTATCQDGISYPGQ RKCEGLKIGD TASFEVSVEARSCPGRQAAQ SFTLRPVGFR DSLQVEVAYN CTCGCSTGLE PNSARCSGNG TYTCGLCECD PGYLGTRCEC QEGENQSGYQ NLCREAEGKP LCSGRGECSC NQCSCFESEF GRIYGPFCEC DSFSCARNKG VLCSGHGECH CGECKCHAGY IGDNCNCSTD VSTCRAKDGQ ICSDRGRCVC GQCQCTEPGA FGETCEKCPT CPDACSSKRD CVECLLLHQG KPDNQTCHHQ CKDEVITWVD TIVKDDQEAV LCFYKTAKDC VMMFSYTELP NGRSNLTVLR EPECGSAPNA MTILLAWGS ILLIGMALLA IWKLLVTIHD RREFAKFQSE RSRARYEMAS NPLYRKPIST

HTVDFAFNKF NKSYNGSVD (SEQ ID NO:47) Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Αηίί-ανβ5 Antibodies

This disclosure includes antibodies and antigen-binding fragments that specifically bind to ανβ5, and more specifically to the β5 subunit. The antibodies disclosed herein are derived from the murine ALULA antibody produced by the hybridoma deposited at the ATCC on February 13, 2004, with the accession number PTA-5817. Example 2 discloses five exemplary heavy chain variable regions, VH1, VH2, VH3, VH4, and VH5, having the amino acid sequences set forth in SEQ ID NOs: l, 3, 5, 7, and 9 respectively, and four exemplary light chain variable regions, VLl, VL2, VL3, and VL4, having the amino acid sequences set forth in SEQ ID NOs: 1 1, 13, 15, and 17, respectively. Each of the VH chains can pair with any of the VL chains: i.e., VH1 can pair with VLl, VL2, VL3, or VL4; VH2 can pair with VLl, VL2, VL3, or VL4; VH3 can pair with VLl, VL2, VL3, or VL4; VH4 can pair with VLl, VL2, VL3, or VL4; and VH5 can pair with VLl, VL2, VL3, or VL4. Thus, the heavy chain variable region and light chain variable regions disclosed in Example 2 can form 20 different VH-VL pairs.

The amino acid sequences of the complementarity determining regions (CDRs) 1, 2, and 3 as well as the framework regions (FRs) 1, 2, 3, 4 of the five heavy chain variable regions and the four light chain variable regions of the exemplary antibodies described in Example 2 are provided below. The CDRs are based upon the Kabat numbering system.

Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

This application also discloses "alternate CDRs" of ALULA that can be used in the antibodies of this invention. By "alternate" CDRs are meant CDRs (CDR1, CDR2, and CDR3) defined according to any one of the Chothia from AbYsis, enhanced Chothia/AbM CDR, or the contact definitions. These alternate CDRs can be obtained, e.g., by using the AbYsis database (www.bioinf.org.uk/abysis/sequence_input/key_annotation/key_annotation.cgi). The amino acid sequences of "alternate" CDRs 1, 2, and 3 of the heavy chain variable region and the light chain variable region of ALULA are compared with the CDRs defined according to Kabat in the Table below. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

This disclosure also includes antibodies that specifically bind ανβ5 and/or β5 that have heavy chain variable regions that are: 94%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: l; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO:3; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO:5; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 7; or 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 9. In some embodiments, these antibodies inhibit the interaction between ανβ5 and vitronectin; inhibit the interaction between ανβ5 and LAP of TGF-β; and/or inhibit the activation of TGF-β. In some embodiments, these antibodies further include a light chain variable region that is: 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 1 1; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 13; 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ ID NO: 15; or 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in SEQ Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

ID NO: 17. In some embodiments wherein the antibodies comprise both a heavy and light chain that specifically bind ανβ5, these antibodies inhibit the interaction between ανβ5 and vitronectin; inhibit the interaction between ανβ5 and LAP of TGF-β; and/or inhibit the activation of TGF-β.

This disclosure also includes antibodies that specifically bind ανβ5 and/or β5 that have four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or all four of the framework regions, and/or four or fewer (e.g., four, three or fewer, two or fewer, or one) amino acid substitutions in one, two, or all three CDRs (or alternate CDRs), of the heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: l, 3, 5, 7, or 9. The application also includes antibodies that have four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or all four of the framework regions, and/or four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, or all three CDRs (or alternate CDRs), of the light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 11, 13, 15, or 17. In certain embodiments, the humanized antibodies of this disclosure include antibodies that specifically bind ανβ5 and/or β5 that have four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or four of the framework regions, and/or four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, or three CDRs (or alternate CDRs), of the heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: l, 3, 5, 7, or 9 and four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, three, or four of the framework regions, and/or four or fewer (e.g., four, three or fewer, three, two or fewer, two, or one) amino acid substitutions in one, two, or three CDRs (or alternate CDRs), of the light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs: 1 1, 13, 15, or 17. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In certain embodiments the VH and or VL region can be linked to a constant region (e.g., a wild-type human Fc region or an Fc region that includes one or more alterations). In certain embodiments, the constant region comprises a CHI domain and a hinge region. In some embodiments, the constant region comprises a CH3 domain. In some embodiments, the antibody has a constant region derived from a human kappa or lambda sequence. In a specific embodiment, the constant region comprises a human subgroup kappa 2 sequence. The constant region can be a human Fc region, e.g., a wild-type Fc region, or an Fc region that includes one or more amino acid substitutions. The constant region can have substitutions that modify the properties of the antibody Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

(e.g., increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237 (based on Kabat numbering). Antibodies may have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation. For example, antibodies may have mutations such as those described in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the antibody is modified to reduce or eliminate effector function. In a specific embodiment, the antibody is an IgGl that is modified to reduce or eliminate effector function (e.g., T299A mutation). Antibodies may also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al, Mol. Immunol, 30: 105-08 (1993)). See also, e.g., U.S. 2005-0037000.

In certain embodiments the antibodies or antigen binding fragments thereof can be linked to a cytotoxic agent. In some embodiments, the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic agent, a prodrug, an immunologically active ligand, a cytokines, an alkylating agent, an antimetabolilte, an antiproliferative agent, a tubulin binding agent, a hormone, and a hormone antagonist. Exemplary cytotoxic agents include ⁹⁰Y, ¹³¹I, Monomeihyl Auristaiin E (MMAE), mertansine (DM1), DM4, diphtheria toxin, Pseudomonas exotoxin (PE38), and A chain of ricin. In a specific embodiment, the cytotoxic agent is a maytansinoid. In certain embodiments, the antibody has an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In some embodiments, the antibody is modified to reduce or eliminate effector function. In a specific embodiment, the antibody is an IgGl that is modified to reduce or eliminate effector function (e.g., T299A mutation). Antibodies can be selected for use based on improved potency, higher affinity or avidity for β5, and/or reduced immunogenicity than previously known ανβ5 antibodies. Methods of determining potency, affinity or avidity, and immunogenicity of antibodies are within the skill of the ordinary artisan. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Methods of Obtaining Anti-ανβ 5 Antibodies

Antibodies, such as those described above, can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences. Methods of generating variants (e.g., comprising amino acid substitutions) of any of the anti-avp5 antibodies are well known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a prepared DNA molecule encoding the antibody or any portion thereof (e.g., a framework region, a CDR (an alternate CDR), a constant region). Site-directed mutagenesis is well known in the art (see, e.g., Carter et al, Nucl Acids Res., 13:4431-4443 (1985) and Kunkel et al, Proc. Natl. Acad. Sci. USA, 82:488 (1987)). PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp.177- 183 (Academic Press, 1990); and Vallette et al, Nucl. Acids Res. 17:723-733 (1989). Another method for preparing sequence variants, cassette mutagenesis, is based on the technique described by Wells et al, Gene, 34:315- 323 (1985).

Affinity Maturation

In one embodiment, an anti-av 5 antibody or antigen-binding fragment thereof described herein is modified, e.g., by mutagenesis, to provide a pool of modified antibodies. The modified antibodies are then evaluated to identify one or more antibodies having altered functional properties (e.g., improved binding, improved stability, reduced antigenicity, or increased stability in vivo). In one implementation, display library technology is used to select or screen the pool of modified antibodies. Higher affinity antibodies are then identified from the second library, e.g., by using higher stringency or more competitive binding and washing conditions. Other screening techniques can also be used.

In some implementations, the mutagenesis is targeted to regions known or likely to be at the binding interface. If, for example, the identified binding proteins are antibodies, then mutagenesis can be directed to the CDR regions (or alternate CDR regions) of the heavy or light chains as described herein. Further, mutagenesis can be directed to framework regions near or adjacent to the CDRs, e.g., framework regions, particularly within 10, 5, or 3 amino acids of a CDR (or alternate CDR) junction. In the case of antibodies, mutagenesis can also Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

be limited to one or a few of the CDRs (or alternate CDRs), e.g., to make step-wise

improvements.

In one embodiment, mutagenesis is used to make an antibody more similar to one or more germline sequences. One exemplary germlining method can include: identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the sequence of the isolated antibody. Then mutations (at the amino acid level) can be made in the isolated antibody, either incrementally, in combination, or both. For example, a nucleic acid library that includes sequences encoding some or all possible germline mutations is made. The mutated antibodies are then evaluated, e.g., to identify an antibody that has one or more additional germline residues relative to the isolated antibody and that is still useful (e.g., has a functional activity). In one embodiment, as many germline residues are introduced into an isolated antibody as possible.

In one embodiment, mutagenesis is used to substitute or insert one or more germline residues into a CDR (or alternate CDR) region. For example, the germline CDR (or alternate CDR) residue can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified. After mutagenesis, activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine if the germline residue or residues are tolerated. Similar mutagenesis can be performed in the framework regions.

Selecting a germline sequence can be performed in different ways. For example, a germline sequence can be selected if it meets a predetermined criteria for selectivity or similarity, e.g., at least a certain percentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to the donor non-human antibody. The selection can be performed using at least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2, identifying a similar germline sequence can include selecting one such sequence. In the case of CDR3, identifying a similar germline sequence can include selecting one such sequence, but may include using two germline sequences that separately contribute to the amino-terminal portion and the carboxy-terminal portion. In other implementations, more than one or two germline sequences are used, e.g., to form a consensus sequence.

Calculations of "sequence identity" between two sequences are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

In other embodiments, the antibody may be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used in this context, "altered" means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody. Addition of glycosylation sites to the presently disclosed antibodies may be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences; such techniques are well known in the art. Another means of increasing the number of carbohydrate moieties on the

antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These methods are described in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., 22:259-306. Removal of any carbohydrate moieties present on the antibodies may be accomplished chemically or enzymatically as described in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52; Edge et al. (1981) Anal.

Biochem., 1 18: 131; and Thotakura et al. (1987) Meth. Enzymol, 138:350). See, e.g., U.S.

Pat. No. 5,869,046 for a modification that increases in vivo half life by providing a salvage receptor binding epitope.

In one embodiment, an antibody has CDR sequences (e.g., a Chothia or Kabat CDR) that differ from those of SEQ ID NOs: 19, 20, 21, 22, 23, and 24. CDR sequences that differ from those of the humanized ALULA antibodies described herein include amino acid changes, such as substitutions of 1, 2, 3, or 4 amino acids if a CDR is 5-7 amino acids in length, or substitutions of 1, 2, 3, 4, 5, 6, or 7 of amino acids in the sequence of a CDR if a CDR is 10 amino acids or greater in length. The amino acid that is substituted can have similar charge, hydrophobicity, or stereochemical characteristics. In some embodiments, the amino acid substitution(s) is a conservative substitution. In other embodiments, the amino acid substitution(s) is a non-conservative substitution. Such substitutions are within the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

ordinary skill of an artisan. The antibody or antibody fragments thereof that contain the substituted CDRs can be screened to identify antibodies having one or more of the features described herein (e.g., specifically binding to β5, inhibiting the binding of ανβ5 to

vitronectin).

Unlike in CDRs, more substantial changes in structure framework regions (FRs) can be made without adversely affecting the binding properties of an antibody. Changes to FRs include, but are not limited to, humanizing a nonhuman-derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g., changing the class or subclass of the constant region, changing specific amino acid residues which might alter an effector function such as Fc receptor binding (Lund et al, J. Immun., 147:2657-62 (1991); Morgan et al, Immunology, 86:319-24 (1995)), or changing the species from which the constant region is derived.

The anti-av 5 antibodies can be in the form of full length antibodies, or in the form of low molecular weight forms (e.g., biologically active antibody fragments or minibodies) of the anti-av 5 antibodies, e.g., Fab, Fab', F(ab')2, Fv, Fd, dAb, scFv, and sc(Fv)2. Other anti- ανβ5 antibodies encompassed by this disclosure include single domain antibody (sdAb) containing a single variable chain such as, VH or VL, or a biologically active fragment thereof. See, e.g., Moller et al, J. Biol. Chem., 285(49): 38348-38361 (2010); Harmsen et al, Appl. Microbiol. Biotechnol., 77(1): 13-22 (2007); U.S. 2005/0079574 and Davies et al. (1996) Protein Eng., 9(6):531-7. Like a whole antibody, a sdAb is able to bind selectively to a specific antigen. With a molecular weight of only 12-15 kDa, sdAbs are much smaller than common antibodies and even smaller than Fab fragments and single-chain variable

fragments.

Provided herein are compositions comprising a mixture of an anti-av 5 antibody or antigen-binding fragment thereof and one or more acidic variants thereof, e.g., wherein the amount of acidic variant(s) is less than about 80%, 70%, 60%, 60%, 50%, 40%, 30%, 30%, 20%, 10%, 5% or 1%. Also provided are compositions comprising an anti-av 5 antibody or antigen-binding fragment thereof comprising at least one deamidation site, wherein the pH of the composition is from about 5.0 to about 6.5, such that, e.g., at least about 90% of the anti- ανβ5 antibodies are not deamidated (i.e., less than about 10% of the antibodies are

deamidated). In certain embodiments, less than about 5%, 3%, 2% or 1% of the antibodies Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

are deamidated. The pH may be from 5.0 to 6.0, such as 5.5 or 6.0. In certain embodiments, the pH of the composition is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5.

An "acidic variant" is a variant of a polypeptide of interest which is more acidic (e.g. as determined by cation exchange chromatography) than the polypeptide of interest. An example of an acidic variant is a deamidated variant.

A "deamidated" variant of a polypeptide molecule is a polypeptide wherein one or more asparagine residue(s) of the original polypeptide have been converted to aspartate, i.e. the neutral amide side chain has been converted to a residue with an overall acidic character.

The term "mixture" as used herein in reference to a composition comprising an anti- ανβ5 antibody or antigen-binding fragment thereof, means the presence of both the desired anti-av 5 antibody or antigen-binding fragment thereof and one or more acidic variants thereof. The acidic variants may comprise predominantly deamidated anti-av 5 antibody, with minor amounts of other acidic variant(s).

In certain embodiments, the binding affinity (¾), on-rate (¾ on) and/or off-rate (¾ off) of the antibody that was mutated to eliminate deamidation is similar to that of the wild- type antibody, e.g., having a difference of less than about 5 fold, 2 fold, 1 fold (100%), 50%, 30%, 20%, 10%, 5%, 3%, 2% or 1%.

In certain embodiments, an anti-av 5 antibody or antigen-binding fragment thereof or low molecular weight antibodies thereof specifically binds to β5, inhibits the binding of ανβ5 to vitronectin, inhibits the binding of ανβ5 to LAP of TGF-β, inhibits the activation of TGF- β, inhibits TGF-β signaling, and/or reduces the severity of symptoms when administered to human patients having one or more of, or animal models of: acute lung injury, pulmonary edema, lung fibrosis (e.g., IPF, UIP), sepsis, stroke, myocardial infarction, ocular

neovascularization disease, pancreatic cancer, lung cancer, breast cancer, colorectal cancer, head and neck cancer, esophageal cancer, skin cancer, and endometrial cancer. In some embodiments, the antibody or the antigen-binding fragment thereof inhibit or reduce

angiogenesis and thereby prevent or retard the development of cancer. In one embodiment, the anti-o^5 antibody or antigen-binding fragment thereof or low molecular weight

antibodies thereof inhibit disease development in an idiopathic pulmonary fibrosis model (Degryse et al, Am J Med Sc/„341(6):444-9 (2011)). These features of an anti-o^5 antibody or antigen-binding fragment thereof or low molecular weight antibodies thereof can be measured according to methods known in the art. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Antibody Fragments

Antibody fragments (e.g., Fab, Fab', F(ab')2, Facb, and Fv) of avp5-binding

antibodies may be prepared by proteolytic digestion of intact ανβ5 antibodies. For example, antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies yields F(ab')2 or Fab'; and plasmin digestion of whole antibodies yields Facb fragments.

Alternatively, antibody fragments can be produced recombinantly. For example, nucleic acids encoding the antibody fragments of interest can be constructed, introduced into an expression vector, and expressed in suitable host cells. See, e.g., Co, M.S. et al., J.

Immunol, 152:2968-2976 (1994); Better, M. and Horwitz, A.H., Methods in Enzymology, 178:476-496 (1989); Pluckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, 121 :652-663 (1989); Rousseaux, J. et al,

Methods in Enzymology, (1989) 121 :663-669 (1989); and Bird, R.E. et al, TIBTECH, 9: 132- 137 (1991)). Antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al, Bio/Technology, 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') 2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are

described in U.S. Pat. No. 5,869,046. Minibodies

Minibodies of anti-av 5 antibodies include diabodies, single chain (scFv), and single- chain (Fv)2 (sc(Fv)2).

A "diabody" is a bivalent minibody constructed by gene fusion (see, e.g., Holliger, P. et al, Proc. Natl. Acad. Sci. U. S. A., 90:6444-6448 (1993); EP 404,097; WO 93/1 1161).

Diabodies are dimers composed of two polypeptide chains. The VL and VH domain of each polypeptide chain of the diabody are bound by linkers. The number of amino acid residues that constitute a linker can be between 2 to 12 residues (e.g., 3-10 residues or five or about Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

five residues). The linkers of the polypeptides in a diabody are typically too short to allow the VL and VH to bind to each other. Thus, the VL and VH encoded in the same polypeptide chain cannot form a single-chain variable region fragment, but instead form a dimer with a different single-chain variable region fragment. As a result, a diabody has two antigen- binding sites.

An scFv is a single-chain polypeptide antibody obtained by linking the VH and VL with a linker (see e.g., Huston et al, Proc. Natl. Acad. Sci. U. S. A., 85:5879-5883 (1988);

and Pluckthun, "The Pharmacology of Monoclonal Antibodies" Vol.113, Ed Resenburg and Moore, Springer Verlag, New York, pp.269-315, (1994)). The order of VHs and VLs to be linked is not particularly limited, and they may be arranged in any order. Examples of arrangements include: [VH] linker [VL]; or [VL] linker [VH]. The H chain V region and L chain V region in an scFv may be derived from any anti-av 5 antibody or antigen-binding fragment thereof described herein.

An sc(Fv)2 is a minibody in which two VHs and two VLs are linked by a linker to form a single chain (Hudson, et al, J. Immunol. Methods, (1999) 231 : 177-189 (1999)). An sc(Fv)2 can be prepared, for example, by connecting scFvs with a linker. The sc(Fv)2 of the present invention include antibodies preferably in which two VHs and two VLs are arranged in the order of: VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]), beginning from the N terminus of a single-chain polypeptide; however the order of the two VHs and two VLs is not limited to the above arrangement, and they may be arranged in any order.

Examples of arrangements are listed below:

[VL] linker [VH] linker [VH] linker [VL]

[VH] linker [VL] linker [VL] linker [VH]

[VH] linker [VH] linker [VL] linker [VL]

[VL] linker [VL] linker [VH] linker [VH]

[VL] linker [VH] linker [VL] linker [VH]

Normally, three linkers are required when four antibody variable regions are linked; the linkers used may be identical or different. There is no particular limitation on the linkers that link the VH and VL regions of the minibodies. In some embodiments, the linker is a peptide linker. Any arbitrary single-chain peptide comprising about three to 25 residues (e.g., 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18) can be used as a linker. Examples of such peptide linkers include: Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

NO:48); Ser Gly Gly Gly (SEQ ID NO:49); Gly Gly Gly Gly Ser (SEQ ID NO:50); Ser Gly Gly Gly Gly (SEQ ID NO:51); Gly Gly Gly Gly Gly Ser (SEQ ID NO:52); Ser Gly Gly Gly Gly Gly (SEQ ID NO:53); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO:54); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO:55); (Gly Gly Gly Gly Ser (SEQ ID NO:50)_n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly (SEQ ID NO: 5 l)_n, wherein n is an integer of one or more.

In certain embodiments, the linker is a synthetic compound linker (chemical cross- linking agent). Examples of cross-linking agents that are available on the market include N- hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol

bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST),

disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). The amino acid sequence of the VH or VL in the minibodies may include

modifications such as substitutions, deletions, additions, and/or insertions. For example, the modification may be in one or more of the CDRs (or alternate CDRs)of the anti-av 5 antibody or antigen-binding fragment thereof. In certain embodiments, the modification involves one, two, or three amino acid substitutions in one or more CDRs (or alternate CDRs) and/or framework regions of the VH and/or VL domain of the anti-av 5 minibody. Such substitutions are made to improve the binding, functional activity, and/or reduce

immunogenicity of the anti-av 5 minibody. In certain embodiments, the substitutions are conservative amino acid substitutions. In other embodiments, one, two, or three amino acids of the CDRs (or alternate CDRs) of the anti-av 5 antibody or antigen-binding fragment thereof may be deleted or added as long as there is ανβ5 binding and/or functional activity when VH and VL are associated. The modified minibodies can inhibit ανβ5 binding to vitronectin; inhibit ανβ5 binding to LAP of TGF-β; and/or inhibit TGF-β signaling.

Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

ανβ5 protein. Other such antibodies may combine a ανβ5 binding site with a binding site for another protein (e.g., ανβό, ανβ8, tumor specific antigens (e.g., alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial tumor antigen (ETA), tyrosinase, Melanoma-associated antigen (MAGE)-l, MAGE-3, BAGE-1, GAGE-1, GnTV, KM-HN-1, KK-LC-1, LAGE-1, NA88-A, NY-ESO-1, SAGE, Spl7, SSX-2, TAG-1, TRAG- 3, TRP2, XAGE-lb, HPV 16, HPV E6, HPV E7, TAG-72, L6-antigen, CD19, CD22, CD37, CD52, EGF receptor, HER 2 receptor, Lewis Y), T-cell antigens (e.g., CD2, CD3, CD5, CD6, CD7, TCR)) . Bispecific antibodies can be prepared as full length antibodies or low molecular weight forms thereof (e.g., F(ab') 2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).

Traditional production of full length bispecific antibodies is based on the co- expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). In a different approach, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the proportions of the three polypeptide fragments. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields.

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end- products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

biotin. Heteroconjugate antibodies may be made using any convenient cross-linking

methods.

The "diabody" technology provides an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies describe herein can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. An exemplary dimerization domain comprises (or consists of) an Fc region or a hinge region. A multivalent antibody can comprise (or consist of) three to about eight (e.g., four) antigen binding sites. The multivalent antibody optionally comprises at least one polypeptide chain (e.g., at least two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VDl-(Xl)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, XI and X2 represent an amino acid or peptide spacer, and n is 0 or 1.

Conjugated Antibodies

The antibodies disclosed herein may be conjugated antibodies which are bound to various molecules including macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethylenimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers), hyaluronic acid, fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, and drugs.

In one embodiment, to improve the cytotoxic actions of anti-av 5 antibodies (and antigen-binding fragments thereof) and consequently their therapeutic effectiveness, the antibodies are conjugated with highly toxic substances, including radioisotopes and cytotoxic Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

agents. These conjugates can deliver a toxic load selectively to the target site (i.e., cells expressing the antigen recognized by the antibody) while cells that are not recognized by the antibody are spared. In order to minimize toxicity, conjugates are generally engineered based on molecules with a short serum half-life (e.g., use of antibody fragments, murine sequences,

90 125 131 123 111 and/or IgG3 or IgG4 isotypes). Exemplary radioisotopes include: Y, I, I, I, I, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, and ¹⁸⁸Re. Cytotoxic agents that can be used include cytotoxic drugs which are used for cancer therapy. As used herein, "a cytotoxic agent" means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit, or destroy a cell or malignancy. Exemplary cytotoxic agents include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents (e.g., alkylating agents, antimetabolites, anti-proliferative agents, tubulin binding agents, hormones and hormone antagonists), prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention. Exemplary cytostatics include alkylating substances, such as

mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine. In one embodiment, the cytotoxic agent that is conjugated to an antibody or antigen-binding fragment described herein is a maytansinoid. Maytansinoids are known in the art to include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogues and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441, 163).

C-3 esters of maytansinol can be naturally occurring or synthetically derived. Moreover, both naturally occurring and synthetic C-3 maytansinol esters can be classified as a C-3 ester with simple carboxylic acids, or a C-3 ester with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogues also are known in the art and described in, for example, Kupchan et al, J. Med. Chem., 21, 31-37 (1978).

Methods for generating maytansinol and analogues and derivatives thereof are described in, for example, U.S. Pat. No. 4, 151,042. In certain embodiments, the maytansinoids comprise a linking moiety that contains a reactive chemical group (e.g., C-3 esters of maytansinol and its analogs where the linking moiety contains a disulfide bond and the attachment moiety comprises a N-succinimidyl or N-sulfosuccinimidyl ester). In certain embodiments, the maytansinoid conjugated with the antibodies or antigen-binding described herein is N²'- Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

deacetyl-N²'-(-3-mercapto-l-oxopropyl)-maytansine (DM1) or N²'-deacetyl-N²'-(4-mercapto- 4-methyl-l-oxopentyl)-maytansine (DM4). In certain other embodiments, cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members of these families include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin,

methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like. In other embodiments, the cytotoxic agents include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Hormones and hormone antagonists, such as

corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g. tamoxifen, androgens, e.g. testosterone, and aromatase inhibitors, e.g. aminogluthetimide can also be conjugated with the antibodies or antigen-binding fragments thereof described herein. In certain embodiments, the cytotoxic agent comprises a member or derivative of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins, or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins, and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or

tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs. In further embodiments, the antibodies or antigen-binding fragments thereof can also be associated with a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen, or a toxic enzyme. Such biotoxins can be made using genetic Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

engineering techniques that allow for direct expression of the antibody-toxin construct. One skilled in the art could readily form such constructs using conventional techniques. Methods of conjugating cytotoxic agents are well known in the art (see, e.g., US 8,021,661).

In certain embodiments, an anti-avp5 antibody or antigen-binding fragment thereof are modified with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, the anti-av 5antibody or antigen-binding fragment thereof can be associated with (e.g.,

conjugated to) a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight.

Polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, the anti-av 5 antibody or antigen-binding fragment thereof can be conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinylalcohol or polyvinylpyrrolidone. Examples of such polymers include polyalkylene oxide

homopolymers such as polyethylene glycol (PEG) (see, e.g., Chapman et al, Nature

Biotechnology, 17: 780 - 783 (1999), or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Additional useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched

polysaccharides. The efficacy of a therapeutic antibody can be improved by increasing its serum persistence, thereby allowing higher circulating levels, less frequent administration, and reduced doses. The half-life of an IgG depends on its pH-dependent binding to the neonatal receptor FcRn. FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation. Some antibodies that selectively bind the FcRn at pH 6.0, but not pH 7.4, exhibit a higher half-life in a variety of animal models. In certain embodiments, the antibodies of the present disclosure have one or more mutations at the interface between the CH2 and CH3 domains, such as T250Q/M428L and M252Y/S254T/T256E + H433K/N434F (the numbering is according to the EU index), which increase the binding affinity to FcRn and the half-life of IgG 1 in vivo. In other embodiments, the antibodies herein have a modified Fc region comprising at least one modification relative to a wild-type human Fc region, where the modification is selected from Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

the group consisting of 434S, 252Y/428L, 252Y/434S, and 428L/434S, and the numbering is according to the EU index.

The antibodies or antigen-binding fragments thereof can also be conjugated to siRNAs, miRNAs, or anti-miRs to deliver the siRNA, miRNA, or anti-miR to cells

expressing ανβ5 (see, e.g., Song et al., Nat. BiotechnoL, 23(6):709-17 (2005); Schneider et al, Molecular Therapy Nucleic Acids, l :e46 (2012)). The siRNAs, miRNAs, or anti-miRs can target TGF-β or components of the TGF-β signaling pathway. In some embodiments, the siRNAs, miRNAs, or anti-miRs can target genes involved in the disease being treated (e.g., lung fibrosis, acute lung injury, cancer). For example, to treat lung fibrosis, one or more of the following can be targeted to av 5-expressing cells using ανβ5 antibodies or antigen- binding fragments thereof conjugated to: anti-miRs to microRNAs such as: miR-142-3p, miR-155, miR-192, miR-199a/b, miR-208, miR-21, miR-215, miR-216, miR-217, miR-23a, miR-27a, miR-27b, miR-32, miR-338, miR-34a, miR-377, miR-382; or conjugated to microRNAs such as: let-7d, miR-107, miR-132, miR-133, miR-141, miR-15b, miR-16, miR- 150, miR- 18a, miR- 19a/b, miR- 194, miR-200a/b, miR-204, miR-21 1 , miR-26a/b, miR- 29a/b/c, miR-30c, miR-335, miR-449a/b, and miR-590. To treat acute lung injury, one or more of the following can be targeted to av 5-expressing cells using ανβ5 antibodies or antigen-binding fragments thereof conjugated to: miR-127, miR-16, and/or miR-199a. In a specific embodiment, anti-miR-21 conjugated to humanized ανβ5 antibodies or antigen- binding fragments thereof can be used to treat cancer (e.g., hepatocellular carcinoma); and humanized ανβ5 antibodies or antigen-binding fragments thereof conjugated to anti-miR- 10b can be used to treat cancers such as glioblastoma.

The above-described conjugated antibodies can be prepared by performing chemical modifications on the antibodies or the lower molecular weight forms thereof described herein. Methods for modifying antibodies are well known in the art (e.g., US 5057313 and US 5156840).

Methods of Producing Antibodies

The ανβ5 antibodies (or antibody binding fragments thereof) of this disclosure may be produced in bacterial or eukaryotic cells. Some antibodies, e.g., Fab's, can be produced in bacterial cells, e.g., E. coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS). In addition, antibodies (e.g., scFv's) can be Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

expressed in a yeast cell such as Pichia (see, e.g., Powers et al, J Immunol Methods.

251 : 123-35 (2001)), Hanseula, or Saccharomyces. To produce the antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in suitable host cells. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody.

If the antibody is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have characteristics that permit amplification of the vector in the bacterial cells.

Additionally, when E. coli such as JM109, DH5a, HBlOl, or XLl-Blue is used as a host, the vector must have a promoter, for example, a lacZ promoter (Ward et al, 341 :544-546 (1989), araB promoter (Better et al, Science, 240: 1041-1043 (1988)), or T7 promoter that can allow efficient expression in E. coli. Examples of such vectors include, Ml 3 -series vectors, pUC- series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-l (Pharmacia), "QIA express system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al, J. Bacteriol, 169:4379 (1987)) may be used as the signal

sequence for antibody secretion. For bacterial expression, calcium chloride methods or electroporation methods may be used to introduce the expression vector into the bacterial cell.

If the antibody is to be expressed in animal cells such as CHO, COS, 293, 293T, and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, for example, an SV40 promoter (Mulligan et al, Nature, 277: 108 (1979)), MMLV- LTR promoter, EF 1 a promoter (Mizushima et al. , Nucleic Acids Res. , 18:5322 (1990)), or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or domain thereof, the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and

5, 179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

In one embodiment, antibodies are produced in mammalian cells. Exemplary

mammalian host cells for expressing an antibody include Chinese Hamster Ovary (CHO cells) (including dhfr^~ CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Set USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For example, the cell is a mammary epithelial cell.

In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of an anti-avp5 antibody is introduced into dhfr^~ CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate

selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and the antibody is recovered from the culture medium.

Antibodies can also be produced by a transgenic animal. For example, U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a

transgenic mammal. A transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion. The milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest. The antibody can be purified from the milk, or for some applications, used directly. Animals are also provided comprising one or more of the nucleic acids described herein.

The antibodies of the present disclosure can be isolated from inside or outside (such as medium) of the host cell and purified as substantially pure and homogenous antibodies. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Methods for isolation and purification commonly used for antibody purification may be used for the isolation and purification of antibodies, and are not limited to any particular method. Antibodies may be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel

electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography

includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption

chromatography (Strategies for Protein Purification and Characterization: A Laboratory

Course Manual. Ed Daniel R. Marshak et al, Cold Spring Harbor Laboratory Press, 1996). Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include protein A column and protein G column. Examples of columns using protein A column include Hyper D, POROS, and

Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

Characterization of the Antibodies

The av 5-binding properties of the antibodies described herein may be measured by any standard method, e.g., one or more of the following methods: OCTET^®, Surface Plasmon Resonance (SPR), BIACORE™ analysis, Enzyme Linked Immunosorbent Assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and Fluorescence Resonance Energy Transfer (FRET).

The binding interaction of a protein of interest (an anti-av 5 antibody) and a target (e.g., β5) can be analyzed using the OCTET^® systems. In this method, one of several variations of instruments (e.g., OCTET^® QK^e and QK), made by the ForteBio company are used to determine protein interactions, binding specificity, and epitope mapping. The

OCTET^® systems provide an easy way to monitor real-time binding by measuring the changes in polarized light that travels down a custom tip and then back to a sensor.

The binding interaction of a protein of interest (an anti-av 5 antibody) and a target (e.g., β5) can be analyzed using Surface Plasmon Resonance (SPR). SPR or Biomolecular Interaction Analysis (BIA) detects biospecific interactions in real time, without labeling any of the interactants. Changes in the mass at the binding surface (indicative of a binding event) Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

of the BIA chip result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The changes in the refractivity generate a detectable signal, which are measured as an indication of real-time reactions between biological molecules. Methods for using SPR are described, for example, in U.S.

Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and

Urbaniczky (1991) Anal. Chem. 63 :2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources provide by BIAcore International AB (Uppsala, Sweden).

Information from SPR can be used to provide an accurate and quantitative measure of the equilibrium dissociation constant (¾), and kinetic parameters, including K_on and K_cff, for the binding of a biomolecule to a target.

Epitopes can also be directly mapped by assessing the ability of different antibodies to compete with each other for binding to human ανβ5 or β5 using BIACORE chromatographic techniques (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160: 191-198).

When employing an enzyme immunoassay, a sample containing an antibody, for example, a culture supernatant of antibody -producing cells or a purified antibody is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, and after washing, an enzyme substrate such as p-nitrophenylphosphate is added, and the absorbance is measured to evaluate the antigen binding activity.

Additional general guidance for evaluating antibodies, e.g., Western blots and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, ed. by

Harlow and Lane, Cold Spring Harbor press (1988)).

Antibodies with Altered Effector Function The interaction of antibodies and antibody-antigen complexes with cells of the immune system triggers a variety of responses, referred to herein as effector functions.

Immune-mediated effector functions include two major mechanisms: antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Both of them are mediated by the constant region of the immunoglobulin protein. The antibody Fc domain is, therefore, the portion that defines interactions with immune effector mechanisms. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

IgG antibodies activate effector pathways of the immune system by binding to members of the family of cell surface Fey receptors and to Clq of the complement system.

Ligation of effector proteins by clustered antibodies triggers a variety of responses, including release of inflammatory cytokines, regulation of antigen production, endocytosis, and cell killing. In some clinical applications these responses are crucial for the efficacy of a

monoclonal antibody. In others they provoke unwanted side effects such as inflammation and the elimination of antigen-bearing cells. Accordingly, the present invention further relates to av 5-binding proteins, including antibodies, with altered, e.g., increased or reduced effector functions.

Effector function of an anti-avp5 antibody of the present invention may be

determined using one of many known assays. The anti-avp5 antibody's effector function may be increased or reduced relative to a second anti-avp5 antibody. In some embodiments, the second anti-av 5 antibody may be any antibody that binds ανβ5 specifically. In other embodiments, the second ανβ5 -specific antibody may be any of the antibodies of the

invention, such as the antibodies described in Example 2. In other embodiments, where the anti-av 5 antibody of interest has been modified to increase or reduce effector function, the second anti-av 5 antibody may be the unmodified or parental version of the antibody.

Effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC), whereby antibodies bind Fc receptors on cytotoxic T cells, natural killer (NK) cells, or macrophages leading to cell death, and complement-dependent cytotoxicity (CDC), which is cell death induced via activation of the complement cascade (reviewed in Daeron, Annu. Rev. Immunol, 15:203-234 (1997); Ward and Ghetie, Therapeutic Immunol, 2:77-94 (1995); and Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991)). Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using standard assays that are known in the art (see, e.g., WO 05/018572, WO 05/003175, and U.S. 6,242, 195).

Effector functions can be avoided by using antibody fragments lacking the Fc domain such as Fab, Fab'2, or single chain Fv. An alternative is to use the IgG4 subtype antibody, which binds to FcyRI but which binds poorly to Clq and FcyRII and RIII. The IgG2 subtype also has reduced binding to Fc receptors, but retains significant binding to the H131 allotype of FcyRIIa and to Clq. Thus, additional changes in the Fc sequence are required to eliminate binding to all the Fc receptors and to Clq. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Several antibody effector functions, including ADCC, are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. The affinity of an antibody for a particular FcR, and hence the effector activity mediated by the antibody, may be modulated by altering the amino acid sequence and/or post-translational modifications of the Fc and/or constant region of the antibody.

FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FceR, for IgA as FcaR and so on. Three subclasses of FcyR have been identified: FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16). Both FcyRII and FcyRIII have two types: FcyRIIA (CD32) and FcyRIIB (CD32); and

FcyRIIIA (CD 16a) and FcyRIIIB (CD 16b). Because each FcyR subclass is encoded by two or three genes, and alternative RNA splicing leads to multiple transcripts, a broad diversity in FcyR isoforms exists. For example, FcyRII (CD32) includes the isoforms Ila, Ilbl, IIb2 IIb3, and lie.

The binding site on human and murine antibodies for FcyR has been previously mapped to the so-called "lower hinge region" consisting of residues 233-239 (EU index numbering as in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), Woof et al, Molec.

Immunol. 23:319-330 (1986); Duncan et al, Nature 332:563 (1988); Canfield and Morrison, J. Exp. Med. 173: 1483-1491 (1991); Chappel et al, Proc. Natl. Acad. Sci USA 88:9036-9040 (1991)). Of residues 233-239, P238 and S239 are among those cited as possibly being involved in binding. Other previously cited areas possibly involved in binding to FcyR are: G316-K338 (human IgG) for human FcyRI (Woof et al, Mol. Immunol, 23:319-330 (1986)); K274-R301 (human IgGl) for human FcyRIII (Sarmay et al, Molec. Immunol. 21 :43-51 (1984)); and Y407-R416 (human IgG) for human FcyRIII (Gergely et al, Biochem. Soc.

Trans. 12:739-743 (1984) and Shields et al, J Biol Chem 276: 6591-6604 (2001), Lazar GA et al, Proc Natl Acad Sci 103: 4005-4010 (2006). These and other stretches or regions of amino acid residues involved in FcR binding may be evident to the skilled artisan from an examination of the crystal structures of Ig-FcR complexes (see, e.g., Sondermann et al. 2000 Nature 406(6793):267-73 and Sondermann et al. 2002 Biochem Soc Trans. 30(4):481-6).

Accordingly, the anti-av 5 antibodies of the present invention include modifications of one or more of the aforementioned residues (to increase or decrease effector function as needed). Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Another approach for altering monoclonal antibody effector function include mutating amino acids on the surface of the monoclonal antibody that are involved in effector binding interactions (Lund, J., et al. (1991) J. Immunol. 147(8): 2657-62; Shields, R. L. et al. (2001) J. Biol. Chem. 276(9): 6591-604).

Methods of increasing effector function of antibodies are well known in the art (see, e.g., Kelley et al, Methods Mol. Biol, 901 :277-93 (2012); Natsume et al, Drug Des Devel Ther., 3:7-16 (2009); US 8, 188,231, US 7,960,512). In one embodiment, the ανβ5 antibodies have one, two, three, four, five, six, seven, or more amino acid substitutions at a position selected from the group consisting of 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, and 337, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. In certain embodiments, the ανβ5 antibodies have one, two, three, four, five, six, seven, or more of the amino acid substitutions selected from the group

consisting of: D221K, D221Y, K222E, K222Y, T223E, T223K, H224E, H224Y, T225E, T225K, T225W, P227E, P227G, P227K, P227Y, P228E, P228G, P228K, P228Y, P230A, P230E, P230G, P230Y, A231E, A231G, A231K, A231P, A231Y, P232E, P232G, P232K, P232Y, E233A, E233D, E233F, E233G, E233H, E233I, E233K, E233L, E233M, E233N, E233Q, E233R, E233S, E233T, E233V, E233W, E233Y, L234A, L234D, L234E, L234F, L234G, L234H, L234I, L234K, L234M, L234N, L234P, L234Q, L234R, L234S, L234T, L234V, L234W, L234Y, L235A, L235D, L235E, L235F, L235G, L235H, L235I, L235K, L235M, L235N, L235P, L235Q, L235R, L235S, L235T, L235V, L235W, L235Y, G236A, G236D, G236E, G236F, G236H, G236I, G236K, G236L, G236M, G236N, G236P, G236Q, G236R, G236S, G236T, G236V, G236W, G236Y, G237D, G237E, G237F, G237H, G237I, G237K, G237L, G237M, G237N, G237P, G237Q, G237R, G237S, G237T, G237V, G237W, G237Y, P238D, P238E, P238F, P238G, P238H, P238I, P238K, P238L, P238M, P238N, P238Q, P238R, P238S, P238T, P238V, P238W, P238Y, S239D, S239E, S239F, S239G, S239H, S239I, S239K, S239L, S239M, S239N, S239P, S239Q, S239R, S239T, S239V, S239W, S239Y, V240A, V240I, V240M, V240T, F241D, F241E, F241L, F241R, F241 S, F241W, F241Y, F243E, F243H, F243L, F243Q, F243R, F243W, F243Y, P244H, P245A, Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

K246D, K246E, K246H, K246Y, P247G, P247V, D249H, D249Q, D249Y, R255E, R255Y, E258H, E258S, E258Y, T260D, T260E, T260H, T260Y, V262A, V262E, V262F, V262I, V262T, V263A, V263I, V263M, V263T, V264A, V264D, V264E, V264F, V264G, V264H, V264I, V264K, V264L, V264M, V264N, V264P, V264Q, V264R, V264S, V264T, V264W, V264Y, D265F, D265G, D265H, D265I, D265K, D265L, D265M, D265N, D265P, D265Q, D265R, D265S, D265T, D265V, D265W, D265Y, V266A, V266I, V266M, V266T, S267D, S267E, S267F, S267H, S267I, S267K, S267L, S267M, S267N, S267P, S267Q, S267R,

S267T, S267V, S267W, S267Y, H268D, H268E, H268F, H268G, H268I, H268K, H268L, H268M, H268P, H268Q, H268R, H268T, H268V, H268W, E269F, E269G, E269H, E269I, E269K, E269L, E269M, E269N, E269P, E269R, E269S, E269T, E269V, E269W, E269Y, D270F, D270G, D270H, D270I, D270L, D270M, D270P, D270Q, D270R, D270S, D270T, D270W, D270Y, P271A, P271D, P271E, P271F, P271G, P271H, P271I, P271K, P271L, P271M, P271N, P271Q, P271R, P271S, P271T, P271V, P271W, P271Y, E272D, E272F, E272G, E272H, E272I, E272K, E272L, E272M, E272P, E272R, E272S, E272T, E272V, E272W, E272Y, V273I, K274D, K274E, K274F, K274G, K274H, K274I, K274L, K274M, K274N, K274P, K274R, K274T, K274V, K274W, K274Y, F275L, F275W, N276D, N276E, N276F, N276G, N276H, N276I, N276L, N276M, N276P, N276R, N276S, N276T, N276V, N276W, N276Y, Y278D, Y278E, Y278G, Y278H, Y278I, Y278K, Y278L, Y278M, Y278N, Y278P, Y278Q, Y278R, Y278S, Y278T, Y278V, Y278W, D280G, D280K, D280L, D280P, D280W, G281D, G281E, G281K, G281N, G281P, G281Q, G281Y, V282E, V282G, V282K, V282P, V282Y, E283G, E283H, E283K, E283L, E283P, E283R, E283Y, V284D, V284E, V284L, V284N, V284Q, V284T, V284Y, H285D, H285E, H285K, H285Q, H285W, H285Y, N286E, N286G, N286P, N286Y, K288D, K288E, K288Y, K290D, K290H, K290L, K290N, K290W, P291D, P291E, P291G, P291H, P291I, P291Q, P291T, R292D, R292E, R292T, R292Y, E293F, E293G, E293H, E293I, E293L, E293M, E293N, E293P, E293R, E293S, E293T, E293V, E293W, E293Y, E294F, E294G, E294H, E294I, E294K, E294L, E294M, E294P, E294R, E294S, E294T, E294V, E294W, E294Y, Q295D, Q295E, Q295F, Q295G, Q295H, Q295I, Q295M, Q295N, Q295P, Q295R, Q295S, Q295T, Q295V, Q295W, Q295Y, Y296A, Y296D, Y296E, Y296G, Y296H, Y296I, Y296K, Y296L, Y296M, Y296N, Y296Q, Y296R, Y296S, Y296T, Y296V, N297D, N297E, N297F, N297G, N297H, N297I, N297K, N297L, N297M, N297P, N297Q, N297R, N297S, N297T, N297V, N297W, N297Y, S298D, S298E, S298F, S298H, S298I, S298K, S298M, S298N, S298Q, S298R, S298T, S298W, Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

S298Y, T299A, T299D, T299E, T299F, T299G, T299H, T299I, T299K, T299L, T299M, T299N, T299P, T299Q, T299R, T299S, T299V, T299W, T299Y, Y300A, Y300D, Y300E, Y300G, Y300H, Y300K, Y300M, Y300N, Y300P, Y300Q, Y300R, Y300S, Y300T, Y300V, Y300W, R301D, R301E, R301H, R301Y, V302I, V303D, V303E, V303Y, S304D, S304H, S304L, S304N, S304T, V305E, V305T, V305Y, W313F, K317E, K317Q, E318H, E318L, E318Q, E318R, E318Y, K320D, K320F, K320G, K320H, K320I, K320L, K320N, K320P, K320S, K320T, K320V, K320W, K320Y, K322D, K322F, K322G, K322H, K322I, K322P, K322S, K322T, K322V, K322W, K322Y, V323I, S324D, S324F, S324G, S324H, S324I, S324L, S324M, S324P, S324R, S324T, S324V, S324W, S324Y, N325A, N325D, N325E, N325F, N325G, N325H, N325I, N325K, N325L, N325M, N325P, N325Q, N325R, N325S, N325T, N325V, N325W, N325Y, K326I, K326L, K326P, K326T, A327D, A327E, A327F, A327H, A327I, A327K, A327L, A327M, A327N, A327P, A327R, A327S, A327T, A327V, A327W, A327Y, L328A, L328D, L328E, L328F, L328G, L328H, L328I, L328K, L328M, L328N, L328P, L328Q, L328R, L328S, L328T, L328V, L328W, L328Y, P329D, P329E, P329F, P329G, P329H, P329I, P329K, P329L, P329M, P329N, P329Q, P329R, P329S,

P329T, P329V, P329W, P329Y, A330E, A330F, A330G, A330H, A330I, A330L, A330M, A330N, A330P, A330R, A330S, A330T, A330V, A330W, A330Y, P331D, P331F, P331H, P331I, P331L, P331M, P331Q, P331R, P331T, P331V, P331W, P331Y, I332A, I332D, I332E, I332F, I332H, I332K, I332L, I332M, I332N, I332P, I332Q, I332R, I332S, I332T, I332V, I332W, I332Y, E333F, E333H, E333I, E333L, E333M, E333P, E333T, E333Y,

K334F, K334I, K334L, K334P, K334T, T335D, T335F, T335G, T335H, T335I, T335L, T335M, T335N, T335P, T335R, T335S, T335V, T335W, T335Y, I336E, I336K, I336Y, S337E, S337H, and S337N, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. In a particular embodiment, the ανβ5 antibodies comprise one, two, or three of the following mutations: S239D, S239D/I332E, S239D/I332E/A330L,

S239D/I332E/G236A, S298A, A330L I332E, E333A, and K334A.

The presence of oligosaccharides— specifically, the N-linked oligosaccharide at asparigine-297 in the CH2 domain of IgGl— is important for binding to FcyR as well as Clq. Reducing the fucose content of antibodies improves effector function (see, e.g., US

8,163,551). In certain embodiments the ανβ5 antibodies have reduced fucosylation and amino acid substitutions that increase effector function (e.g., one, two, or three of the following mutations: S298A; E333A, and K334A). Effector function can also be achieved by Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

preparing and expressing the anti-avp5 antibodies described herein in the presence of alpha- mannosidase I inhibitors (e.g., kifunensine) at a concentration of the inhibitor of about 60-200 ng/mL (e.g., 60 ng/mL, 75 ng/mL, 100 ng/mL, 150 ng/ml). Antibodies expressed in the presence of alpha-mannosidase I inhibitors contain mainly oligomannose-type glycans and generally demonstrate increased ADCC activity and affinity for FcyRIIIA, but reduced Clq binding.

Αηίί-ανβ5 antibodies of the present disclosure with increased effector function include antibodies with increased binding affinity for one or more Fc receptors (FcRs) relative to a parent or non-variant anti-av 5 antibody. Accordingly, anti-av 5 antibodies with increased FcR binding affinity includes anti-av 5 antibodies that exhibit a 1.5-fold, 2- fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or higher increase in binding affinity to one or more Fc receptors compared to a parent or non-variant anti-av 5 antibody. In some embodiments, an anti-av 5 antibody with increased effector function binds to an FcR with about 10-fold greater affinity relative to a parent or non-variant antibody. In other embodiments, an anti- ανβ5 antibody with increased effector function binds to an FcR with about 15-fold greater affinity or with about 20-fold greater affinity relative to a parent or non-variant antibody.

The FcR receptor may be one or more of FcyRI (CD64), FcyRII (CD32), and FcyRIII, and isoforms thereof, and FceR, FcμR, Fc5R, and/or an FcaR. In particular embodiments, an anti-av 5 antibody with increased effector function exhibits a 1.5-fold, 2-fold, 2.5-fold, 3- fold, 4-fold, or 5 -fold or higher increase in binding affinity to FcyRIIa.

To reduce effector function, one can use combinations of different subtype sequence segments (e.g., IgG2 and IgG4 combinations) to give a greater reduction in binding to Fey receptors than either subtype alone (Armour et al, Eur. J. Immunol, 29:2613-1624 (1999); Mol Immunol, 40:585-593 (2003)). In addition, sites of N-linked glycosylation can be removed as a means of reducing effector function. A large number of Fc variants having altered and/or reduced affinities for some or all Fc receptor subtypes (and thus for effector functions) are known in the art. See, e.g., US 2007/0224188; US 2007/0148171 ; US

2007/0048300; US 2007/0041966; US 2007/0009523; US 2007/0036799; US 2006/0275283; US 2006/0235208; US 2006/0193856; US 2006/0160996; US 2006/0134105; US

2006/0024298; US 2005/0244403; US 2005/0233382; US 2005/0215768; US 2005/0118174; US 2005/0054832;US 2004/0228856; US 2004/132101;US 2003/158389; see also US

7, 183,387; 6,737,056; 6,538,124; 6,528,624; 6, 194,551; 5,624,821 ; 5,648,260. In certain Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

embodiments amino acids at positions 232, 234, 235, 236, 237, 239, 264, 265, 267, 269, 270, 299, 325, 328, 329, and 330 (numbered according to Kabat) are substituted to reduce effector function. Non-limiting examples of substitutions that reduce effector function include one or more of: K322A; L234A/L235A; G236T; G236R; G236Q; H268A; H268Q; V309L;

A330S;P331 S; V234A/G237A/P238S/ H268A/V309L/A330S/P331S;

E233P/L234V/L235A/G236Q + A327G/A330S/P33 IS; and L235E + E318A/K320A/K322A.

Αηίί-ανβ5 antibodies of the present invention with reduced effector function include antibodies with reduced binding affinity for one or more Fc receptors (FcRs) relative to a parent or non-variant anti-av 5 antibody. Accordingly, anti-av 5 antibodies with reduced FcR binding affinity includes anti-av 5 antibodies that exhibit a 1.5-fold, 2-fold, 2.5-fold, 3- fold, 4-fold, or 5-fold or higher decrease in binding affinity to one or more Fc receptors compared to a parent or non-variant anti-av 5 antibody. In some embodiments, an anti-av 5 antibody with reduced effector function binds to an FcR with about 10-fold less affinity relative to a parent or non-variant antibody. In other embodiments, an anti-av 5 antibody with reduced effector function binds to an FcR with about 15 -fold less affinity or with about 20-fold less affinity relative to a parent or non- variant antibody. The FcR receptor may be one or more of FcyRI (CD64), FcyRII (CD32), and FcyRIII, and isoforms thereof, and FceR, FcμR, Fc5R, and/or an FcaR. In particular embodiments, an anti-av 5 antibody with reduced effector function exhibits a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, or 5-fold or higher decrease in binding affinity to FcyRIIa.

In CDC, the antibody-antigen complex binds complement, resulting in the activation of the complement cascade and generation of the membrane attack complex. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen; thus the activation of the complement cascade is regulated in part by the binding affinity of the immunoglobulin to Clq protein. To activate the complement cascade, it is necessary for Clq to bind to at least two molecules of IgGl, IgG2, or IgG3, but only one molecule of IgM, attached to the antigenic target (Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995) p. 80). To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al, J. Immunol. Methods, 202: 163 (1996), may be performed.

Various residues of the IgG molecule are involved in binding to Clq including the Glu318, Lys320 and Lys322 residues on the CH2 domain, amino acid residue 331 located on Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a turn in close proximity to the same beta strand, the Lys235 and Gly237 residues located in the lower hinge region, and residues 231 to 238 located in the N-terminal region of the CH2 domain (see e.g., Xu et al, J. Immunol. 150: 152A (Abstract) (1993),W094/29351; Tao et al, J. Exp. Med., 178:661-667 (1993); Brekke et al, Eur. J. Immunol, 24:2542-47 (1994);

Burton et al; Nature, 288:338-344 (1980); Duncan and Winter, Nature 332:738-40 (1988);

Idusogie et al J Immunol 164: 4178-4184 (2000; U.S. 5,648,260, and U.S. 5,624,821).

Αηίί-ανβ5 antibodies with improved Clq binding can comprise an amino acid substitution at one, two, three, or four of amino acid positions 326, 327, 333 and 334 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat. In one embodiment, the anti-av 5 antibodies include the following amino acid substitutions: K326W/E333S, which are known to increase binding of an IgGl antibody to Clq (Steurer W. et al, J Immunol, 155(3): 1 165- 74 (1995)).

Αηίί-ανβ5 antibodies with reduced Clq binding can comprise an amino acid

substitution at one, two, three, or four of amino acid positions 270, 322, 329 and 331 of the human IgG Fc region, where the numbering of the residues in the IgG Fc region is that of the EU index as in Kabat. As an example in IgGl, two mutations in the COOH terminal region of the CH2 domain of human IgGl— K322A and P329A— do not activate the CDC pathway and were shown to result in more than a 100 fold decrease in Clq binding (US 6,242, 195).

Accordingly, in certain embodiments, an anti-av 5 antibody of the present invention exhibits increased or reduced binding to a complement protein relative to a second anti-av 5 antibody. In certain embodiments, an anti-av 5 antibody of the invention exhibits increased or reduced binding to Clq by a factor of about 1.5-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7- fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, or about 15- fold or more, relative to a second anti-av 5 antibody.

Thus, in certain embodiments of the invention, one or more of these residues may be modified, substituted, or removed or one or more amino acid residues may be inserted so as to increase or decrease CDC activity of the anti-av 5 antibodies provided herein.

In certain other embodiments, the present invention provides an anti-av 5 antibody that exhibits reduced binding to one or more FcR receptors but that maintains its ability to bind complement (e.g., to a similar or, in some embodiments, to a lesser extent than a native, non- variant, or parent anti-av 5 antibody). Accordingly, an anti-av 5 antibody of the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

present invention may bind and activate complement while exhibiting reduced binding to an FcR, such as, for example, FcyRIIa (e.g., FcyRIIa expressed on platelets). Such an antibody with reduced or no binding to FcyRIIa (such as FcyRIIa expressed on platelets, for example) but that can bind Clq and activate the complement cascade to at least some degree will reduce the risk of thromboembolic events while maintaining perhaps desirable effector functions. In alternative embodiments, an anti-avp5 antibody of the present invention exhibits reduced binding to one or more FcRs but maintains its ability to bind one or more other FcRs. See, for example, US 2007-0009523, 2006-0194290, 2005-0233382, 2004- 0228856, and 2004-0191244, which describe various amino acid modifications that generate antibodies with reduced binding to FcRI, FcRII, and/or FcRIII, as well as amino acid

substitutions that result in increased binding to one FcR but decreased binding to another FcR.

Accordingly, effector functions involving the constant region of an anti-av 5

antibody may be modulated by altering properties of the constant region, and the Fc region in particular. In certain embodiments, the anti-av 5 antibody having increased or decreased effector function is compared with a second antibody with effector function and which may be a non-variant, native, or parent antibody comprising a native constant or Fc region that mediates effector function.

A native sequence Fc or constant region comprises an amino acid sequence identical to the amino acid sequence of a Fc or constant chain region found in nature. Preferably, a control molecule used to assess relative effector function comprises the same type/subtype Fc region as does the test or variant antibody. A variant or altered Fc or constant region

comprises an amino acid sequence which differs from that of a native sequence heavy chain region by virtue of at least one amino acid modification (such as, for example, post- translational modification, amino acid substitution, insertion, or deletion). Accordingly, the variant constant region may contain one or more amino acid substitutions, deletions, or insertions that results in altered post-translational modifications, including, for example, an altered glycosylation pattern. A parent antibody or Fc region is, for example, a variant having normal effector function used to construct a constant region (i.e., Fc) having altered, e.g., increased effector function.

Antibodies with altered (e.g., increased) effector function(s) may be generated by engineering or producing antibodies with variant constant, Fc, or heavy chain regions. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Recombinant DNA technology and/or cell culture and expression conditions may be used to produce antibodies with altered function and/or activity. For example, recombinant DNA technology may be used to engineer one or more amino acid substitutions, deletions, or insertions in regions (such as, for example, Fc or constant regions) that affect antibody function including effector functions. Alternatively, changes in post-translational

modifications, such as, e.g. glycosylation patterns, may be achieved by manipulating the host cell and cell culture and expression conditions by which the antibody is produced.

Certain embodiments of the present invention relate to an anti-av 5 antibody

comprising one or more heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO: 19, VH CDR2 of SEQ ID NO:20, and VH CDR3 of SEQ ID NO:21 ; or one or more heavy chain alternate CDR sequences selected from: VH CDR1 of SEQ ID NO:56, VH

CDR2 of SEQ ID NO:57, and VH CDR3 of SEQ ID NO:21 ; or one or more heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO:58, VH CDR2 of SEQ ID NO:59, and VH CDR3 of SEQ ID NO:21 ; or one or more heavy chain CDR sequences selected from VH CDR1 of SEQ ID NO:60, VH CDR2 of SEQ ID NO:61, and VH CDR3 of SEQ ID

NO:62, wherein the antibody further comprises a variant Fc region that confers increased or reduced effector function compared to a native or parental Fc region. In further

embodiments, the anti-av 5 antibody comprises at least two of the CDRs (or alternate

CDRs), and in other embodiments the antibody comprises all three of the heavy chain CDR (or alternate CDR) sequences. These anti-av 5 antibodies inhibit the interaction between ανβ5 and vitronectin, inhibit the interaction between ανβ5 and LAP of TGF-β, inhibit TGF-β signaling, inhibit TGF-β activation, and/or inhibit the interaction between ανβ5 and its RGD- motif containing ligands.

Other embodiments of the present invention relate to an anti-o^5 antibody

comprising one or more light chain CDR sequences selected from VL CDR1 of SEQ ID

NO:22, VL CDR2 of SEQ ID NO:23, and VL CDR3 of SEQ ID NO:24; or one or more light chain alternate CDR sequences selected from VL CDR1 of SEQ ID NO:63, VL CDR2 of SEQ ID NO:64, and VL CDR3 of SEQ ID NO:65, the antibody further comprising a variant Fc region that confers increased or reduced effector function compared to a native or parental Fc region. In further embodiments, the anti-o^5 antibody comprises at least two of the light chain CDRs (or alternate CDRs), and in other embodiments the antibody comprises all three of the light chain CDR (or alternate CDR) sequences. These anti-o^5 antibodies inhibit the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

interaction between ανβ5 and vitronectin, inhibit the interaction between ανβ5 and LAP of TGF-β, inhibit TGF-β signaling, inhibit TGF-β activation, and/or inhibit the interaction between ανβ5 and its RGD-motif containing ligands.

In further embodiments of the present invention, the anti-o^5 antibody with

increased or reduced effector function comprises all three light chain CDR sequences (CDRs 1, 2, and 3) or alternate CDRs of SEQ ID NO: 11 and comprises all three heavy chain CDR sequences (CDRsl, 2, and 3) or alternate CDRs of SEQ ID NO: 1. In certain embodiments, the anti-o^5 antibody with increased or reduced effector function comprises: three or fewer, two or fewer, or one amino acid substitution in one, two, or three CDRs (or alternate CDRs) of SEQ ID NO: 11 and three or fewer, two or fewer, or one amino acid substitution in one, two, or three CDRs (or alternate CDR) of SEQ ID NO: 1. These anti-o^5 antibodies inhibit the interaction between ανβ5 and vitronectin, inhibit the interaction between ανβ5 and LAP of TGF-β, inhibit TGF-β signaling, inhibit TGF-β activation, and/or inhibit the interaction between ανβ5 and its RGD-motif containing ligands.

In other embodiments, the invention relates to an anti-o^5 antibody comprising a VL sequence selected from the group consisting of: SEQ ID NOs: 1 1, 13, 15, and 17, the antibody further comprising a variant Fc region that confers reduced effector function compared to a native or parental Fc region. In yet other embodiments, the invention relates to an anti-o^5 antibody comprising a VH sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9, the antibody further comprising a variant Fc region that confers reduced effector function compared to a native or parental Fc region. These anti-o^5 antibodies inhibit the interaction between ανβ5 and vitronectin, inhibit the interaction between ανβ5 and LAP of TGF-β, inhibit TGF-β signaling, inhibit TGF-β activation, and/or inhibit the interaction between ανβ5 and its RGD-motif containing ligands.

Αηύ-ανβ5 Antibodies with Altered Glycosylation

Glycan removal produces a structural change that should greatly reduce binding to all members of the Fc receptor family across species. In glycosylated antibodies, including anti- ανβ5 antibodies, the glycans (oligosaccharides) attached to the conserved N-linked site in the CH2 domains of the Fc dimer are enclosed between the CH2 domains, with the sugar residues making contact with specific amino acid residues on the opposing CH2 domain.

Different glycosylation patterns are associated with different biological properties of Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

antibodies (Jefferis and Lund, 1997, Chem. Immunol, 65: 1 11-128; Wright and Morrison, 1997, Trends Biotechnol. , 15: 26-32). Certain specific glycoforms confer potentially

advantageous biological properties. Loss of the glycans changes spacing between the domains and increases their mobility relative to each other and is expected to have an inhibitory effect on the binding of all members of the Fc receptor family. For example, in vitro studies with various glycosylated antibodies have demonstrated that removal of the CH2 glycans alters the Fc structure such that antibody binding to Fc receptors and the complement protein C1Q are greatly reduced. Another known approach to reducing effector functions is to inhibit production of or remove the N-linked glycans at position 297 (EU numbering) in the CH2 domain of the Fc (Nose et al., 1983 PNAS 80: 6632; Leatherbarrow et al, 1985 Mol. Immunol. 22: 407; Tao et al, 1989 J. Immunol. 143: 2595; Lund et al, 1990 Mol. Immunol. 27: 1 145; Dorai et al, 1991 Hybridoma 10:21 1; Hand et al, 1992 Cancer Immunol.

Immunother. 35: 165; Leader et al, 1991 Immunology 72: 481 ; Pound et al, 1993 Mol.

Immunol. 30:233; Boyd et al, 1995 Mol. Immunol. 32: 1311). It is also known that different glycoforms can profoundly affect the properties of a therapeutic, including pharmacokinetics, pharmacodynamics, receptor-interaction and tissue-specific targeting (Graddis et al, 2002, Curr Pharm Biotechnol. 3: 285-297). In particular, for antibodies, the oligosaccharide structure can affect properties relevant to protease resistance, the serum half-life of the antibody mediated by the FcRn receptor, phagocytosis and antibody feedback, in addition to effector functions of the antibody (e.g., binding to the complement complex CI, which induces CDC, and binding to FcyR receptors, which are responsible for modulating the

ADCC pathway) (Nose and Wigzell, 1983; Leatherbarrow and Dwek, 1983; Leatherbarrow et al., 1985; Walker et al, 1989; Carter et al, 1992, PNAS, 89: 4285-4289).

Accordingly, another means of modulating effector function of antibodies includes altering glycosylation of the antibody constant region. Altered glycosylation includes, for example, a decrease or increase in the number of glycosylated residues, a change in the pattern or location of glycosylated residues, as well as a change in sugar structure(s). The oligosaccharides found on human IgGs affects their degree of effector function (Raju, T.S.

BioProcess International April 2003. 44-53); the microheterogeneity of human IgG

oligosaccharides can affect biological functions such as CDC and ADCC, binding to various Fc receptors, and binding to Clq protein (Wright A. & Morrison SL. TIBTECH 1997, 15 26- 32; Shields et al. J Biol Chem. 2001 276(9):6591-604; Shields et al. J Biol Chem. 2002; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

277(30):26733-40; Shinkawa et al. J Biol Chem. 2003 278(5):3466-73; Umana et al. Nat Biotechnol. 1999 Feb; 17(2): 176-80). For example, the ability of IgG to bind Clq and activate the complement cascade may depend on the presence, absence or modification of the carbohydrate moiety positioned between the two CH2 domains (which is normally anchored at Asn297) (Ward and Ghetie, Therapeutic Immunology 2:77-94 (1995).

Glycosylation sites in an Fc-containing polypeptide, for example an antibody such as an IgG antibody, may be identified by standard techniques. The identification of the

glycosylation site can be experimental or based on sequence analysis or modeling data.

Consensus motifs, that is, the amino acid sequence recognized by various glycosyl

transferases, have been described. For example, the consensus motif for an N-linked

glycosylation motif is frequently NXT or NXS, where X can be any amino acid except proline. Several algorithms for locating a potential glycosylation motif have also been described. Accordingly, to identify potential glycosylation sites within an antibody or Fc- containing fragment, the sequence of the antibody is examined, for example, by using publicly available databases such as the website provided by the Center for Biological

Sequence Analysis (see NefNGlyc services for predicting N-linked glycosylation sites and NetOGlyc services for predicting O-linked glycosylation sites).

In vivo studies have confirmed the reduction in the effector function of aglycosyl antibodies. For example, an aglycosyl anti-CD8 antibody is incapable of depleting CD8- bearing cells in mice (Isaacs, 1992 J. Immunol. 148: 3062) and an aglycosyl anti-CD3 antibody does not induce cytokine release syndrome in mice or humans (Boyd, 1995 supra; Friend, 1999 Transplantation 68: 1632).

Importantly, while removal of the glycans in the CH2 domain appears to have a significant effect on effector function, other functional and physical properties of the

antibody remains unaltered. Specifically, it has been shown that removal of the glycans had little to no effect on serum half-life and binding to antigen (Nose, 1983 supra; Tao, 1989 supra; Dorai, 1991 supra; Hand, 1992 supra; Hobbs, 1992 Mol. Immunol. 29:949).

Although there is in vivo validation of the aglycosyl approach, there are reports of residual effector function with aglycosyl mAbs (see, e.g., Pound, J. D. et al. (1993) Mol.

Immunol. 30(3): 233-41; Dorai, H. et al. (1991) Hybridoma 10(2): 21 1-7). Armour et al.

show residual binding to FcyRIIa and FcyRIIb proteins (Eur. J. Immunol. (1999) 29: 2613- 1624; Mol. Immunol. 40 (2003) 585-593). Thus a further decrease in effector function, Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

particularly complement activation, may be important to guarantee complete ablation of activity in some instances. For that reason, aglycosyl forms of IgG2 and IgG4 and a G1/G4 hybrid are envisioned as being useful in methods and antibody compositions of the invention having reduced effector functions.

The anti-av 5 antibodies of the present invention may be modified or altered to elicit reduced effector function(s) (compared to a second ανβ5 -specific antibody) while optionally retaining the other valuable attributes of the Fc portion.

Accordingly, in certain embodiments, the present invention relates to aglycosyl anti- ανβ5 antibodies with decreased effector function, which are characterized by a modification at the conserved N-linked site in the CH2 domains of the Fc portion of the antibody. A modification of the conserved N-linked site in the CH2 domains of the Fc dimer can lead to aglycosyl anti-avp5 antibodies. Examples of such modifications include mutation of the conserved N-linked site in the CH2 domains of the Fc dimer, removal of glycans attached to the N-linked site in the CH2 domains, and prevention of glycosylation. For example, an aglycosyl anti-avp5 antibody may be created by changing the canonical N-linked Asn site in the heavy chain CH2 domain to a Gin residue (see, for example, WO 05/03175 and US 2006- 0193856).

In one embodiment of present invention, the modification comprises a mutation at the heavy chain glycosylation site to prevent glycosylation at the site. Thus, in one embodiment of this invention, the aglycosyl anti-av 5 antibodies are prepared by mutation of the heavy chain glycosylation site, i.e., mutation of N298Q (N297 using Kabat EU numbering) and expressed in an appropriate host cell. For example, this mutation may be accomplished by following the manufacturer's recommended protocol for unique site mutagenesis kit from Amersham-Pharmacia Biotech® (Piscataway, NJ, USA).

The mutated antibody can be stably expressed in a host cell (e. g. NSO or CHO cell) and then purified. As one example, purification can be carried out using Protein A and gel filtration chromatography. It will be apparent to those of skill in the art that additional methods of expression and purification may also be used.

In another embodiment of the present invention, the aglycosyl anti-av 5 antibodies have decreased effector function, wherein the modification at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody or antibody derivative comprises the removal of the CH2 domain glycans, i.e. , deglycosylation. These aglycosyl anti-av 5 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

antibodies may be generated by conventional methods and then deglycosylated

enzymatically. Methods for enzymatic deglycosylation of antibodies are well known to those of skill in the art (Williams, 1973; Winkelhake & Nicolson, 1976 J. Biol Chem. 251 : 1074- 80.)·

In another embodiment of this invention, deglycosylation may be achieved by growing host cells which produce the antibodies in culture medium comprising a

glycosylation inhibitor such as tunicamycin (Nose & Wigzell, 1983). That is, the

modification is the reduction or prevention of glycosylation at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody.

In other embodiments of this invention, recombinant X polypeptides (or cells or cell membranes containing such polypeptides) may be used as an antigen to generate an anti-avp5 antibody or antibody derivatives, which may then be deglycosylated.

In alternative embodiments, agyclosyl anti-avp5 antibodies or anti-avp5 antibodies with reduced glycosylation may be produced by the method described in Taylor et al. (WO 05/18572 and US 2007-0048300). For example, in one embodiment, an anti-av 5 aglycosyl antibody may be produced by altering a first amino acid residue (e.g., by substitution, insertion, deletion, or by chemical modification), wherein the altered first amino acid residue inhibits the glycosylation of a second residue by either steric hindrance or charge or both. In certain embodiments, the first amino acid residue is modified by amino acid substitution. In further embodiments, the amino acid substitution is selected from the group consisting of

Gly, Ala, Val, Leu, He, Phe, Asn, Gin, Trp, Pro, Ser, Thr, Tyr, Cys, Met, Asp, Glu, Lys, Arg, and His. In other embodiments, the amino acid substitution is a non-traditional amino acid residue. The second amino acid residue may be near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS. In one exemplary embodiment, the first amino acid residue is amino acid 299 and the second amino acid residue is amino acid 297, according to the Kabat numbering. For example, the first amino acid substitution may be T299A, T299N, T299G, T299Y, T299C, T299H, T299E, T299D, T299K, T299R, T299G, T299I, T299L, T299M, T299F, T299P, T299W, and T299V, according to the Kabat numbering. In particular embodiments, the amino acid substitution is T299C.

Effector function may also be reduced by modifying an antibody of the present invention such that the antibody contains a blocking moiety. Exemplary blocking moieties Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

include moieties of sufficient steric bulk and/or charge such that reduced glycosylation occurs, for example, by blocking the ability of a glycosidase to glycosylate the polypeptide. The blocking moiety may additionally or alternatively reduce effector function, for example, by inhibiting the ability of the Fc region to bind a receptor or complement protein. In some embodiments, the present invention relates to an avp5-binding protein, e.g., an anti-avp5 antibody, comprising a variant Fc region, the variant Fc region comprising a first amino acid residue and an N-glycosylation site, the first amino acid residue modified with side chain chemistry to achieve increased steric bulk or increased electrostatic charge compared to the unmodified first amino acid residue, thereby reducing the level of or otherwise altering glycosylation at the N-glycosylation site. In certain of these embodiments, the variant Fc region confers reduced effector function compared to a control, non-variant Fc region. In further embodiments, the side chain with increased steric bulk is a side chain of an amino acid residue selected from the group consisting of Phe, Trp, His, Glu, Gin, Arg, Lys, Met and Tyr. In yet further embodiments, the side chain chemistry with increased electrostatic charge is a side chain of an amino acid residue selected from the group consisting of Asp, Glu, Lys, Arg, and His.

Accordingly, in one embodiment, glycosylation and Fc binding can be modulated by substituting T299 with a charged side chain chemistry such as D, E, K, or R. The resulting antibody will have reduced glycosylation as well as reduced Fc binding affinity to an Fc receptor due to unfavorable electrostatic interactions.

In another embodiment, a T299C variant antibody, which is both aglycosylated and capable of forming a cysteine adduct, may exhibit less effector function (e.g., FcyRI binding) compared to its aglycosylated antibody counterpart (see, e.g., WO 05/18572). Accordingly, alteration of a first amino acid proximal to a glycosylation motif can inhibit the glycosylation of the antibody at a second amino acid residue; when the first amino acid is a cysteine residue, the antibody may exhibit even further reduced effector function. In addition, inhibition of glycosylation of an antibody of the IgG4 subtype may have a more profound effect on FcyRI binding compared to the effects of agycosylation in the other subtypes.

In additional embodiments, the present invention relates to anti-av 5 antibodies with altered glycosylation that exhibit reduced binding to one or more FcR receptors and that optionally also exhibit increased or normal binding to one or more Fc receptors and/or complement— e.g., antibodies with altered glycosylation that at least maintain the same or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

similar binding affinity to one or more Fc receptors and/or complement as a native, control anti-av 5 antibody). For example, anti-avp5 antibodies with predominantly

Man₅GlcNAc₂N-glycan as the glycan structure present (e.g., wherein Man₅GlcNAc₂N-glycan structure is present at a level that is at least about 5 mole percent more than the next

predominant glycan structure of the Ig composition) may exhibit altered effector function compared to an anti-avp5 antibody population wherein Man₅Glc Ac₂ -glycan structure is not predominant. Antibodies with predominantly this glycan structure exhibit decreased binding to FcyRIIa and FcyRIIb, increased binding to FcyRIIIa and FcyRfflb, and increased binding to Clq subunit of the CI complex (see US 2006-0257399). This glycan structure, when it is the predominant glycan structure, confers increased ADCC, increased CDC, increased serum half-life, increased antibody production of B cells, and decreased

phagocytosis by macrophages.

In general, the glycosylation structures on a glycoprotein will vary depending upon the expression host and culturing conditions (Raju, TS. BioProcess International April 2003. 44-53). Such differences can lead to changes in both effector function and pharmacokinetics (Israel et al. Immunology, 1996; 89(4):573-578; Newkirk et al. P. Clin. Exp., 1996;

106(2):259-64). For example, galactosylation can vary with cell culture conditions, which may render some immunoglobulin compositions immunogenic depending on their specific galactose pattern (Patel et al, 1992. Biochem J. 285: 839-845). The oligosaccharide

structures of glycoproteins produced by non-human mammalian cells tend to be more closely related to those of human glycoproteins. Further, protein expression host systems may be engineered or selected to express a predominant Ig glycoform or alternatively may naturally produce glycoproteins having predominant glycan structures. Examples of engineered protein expression host systems producing a glycoprotein having a predominant glycoform include gene knockouts/mutations (Shields et al, 2002, JBC, 277: 26733-26740); genetic engineering in (Umana et al, 1999, Nature Biotech., 17: 176-180) or a combination of both. Alternatively, certain cells naturally express a predominant glycoform— for example,

chickens, humans and cows (Raju et al., 2000, Glycobiology , 10: 477-486). Thus, the expression of an anti-av 5 antibody or antibody composition having altered glycosylation (e.g., predominantly one specific glycan structure) can be obtained by one skilled in the art by selecting at least one of many expression host systems. Protein expression host systems that may be used to produce anti-av 5 antibodies of the present invention include animal, plant, Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

insect, bacterial cells and the like. For example, US 2007-0065909, 2007-0020725, and

2005-0170464 describe producing aglycosylated immunoglobulin molecules in bacterial cells. As a further example, Wright and Morrison produced antibodies in a CHO cell line deficient in glycosylation (1994 J Exp Med 180: 1087-1096) and showed that antibodies produced in this cell line were incapable of complement-mediated cytolysis. Other examples of expression host systems found in the art for production of glycoproteins include: CHO cells: Raju WO 99/22764 and Presta WO 03/35835; hybridoma cells: Trebak et al, 1999, J. Immunol. Methods, 230: 59-70; insect cells: Hsu et al, 1997, JBC, 272:9062-970, and plant cells: Gerngross et al, WO 04/74499. To the extent that a given cell or extract has resulted in the glycosylation of a given motif, art recognized techniques for determining if the motif has been glycosylated are available, for example, using gel electrophoresis and/or mass

spectroscopy.

Additional methods for altering glycosylation sites of antibodies are described, e.g., in US 6,350,861 and US 5,714,350, WO 05/18572 and WO 05/03175; these methods can be used to produce anti-av 5 antibodies of the present invention with altered, reduced, or no glycosylation.

The aglycosyl anti-av 5 antibodies with reduced effector function may be antibodies that comprise modifications or that may be conjugated to comprise a functional moiety. Such moieties include a blocking moiety (e.g., a PEG moiety, cysteine adducts, etc.), a detectable moiety (e.g., fluorescent moieties, radioisotopic moieties, radiopaque moieties, etc., including diagnostic moieties), a therapeutic moiety (e.g., cytotoxic agents, anti-inflammatory agents, immunomodulatory agents, anti-infective agents, anti-cancer agents, anti-neurodegenerative agents, radionuclides, etc.), and/or a binding moiety or bait (e.g., that allows the antibody to be pre-targeted to a tumor and then to bind a second molecule, composed of the

complementary binding moiety or prey and a detectable moiety or therapeutic moiety, as described above).

Indications

The anti-av 5 antibodies or antigen-binding fragments thereof described herein can be used to treat, prevent, or reduce the symptoms or severity of av 5-mediated diseases or conditions. av 5-mediated diseases or conditions include acute lung injury, acute respiratory Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

distress syndrome, pulmonary edema, lung fibrosis, sepsis, stroke, myocardial infarction, cancer, and ocular neovascularization disease.

In certain embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat or reduce the symptoms or severity of acute lung injury. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat or reduce the symptoms or severity of pulmonary edema (e.g., edema associated with lung injury). In certain embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat or reduce the symptoms or severity of sepsis. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat lung fibrosis (e.g., IPF, UIP). In other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to protect against epithelial and/or endothelial cell injury. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to reduce or prevent alveolar epithelial injury. In yet other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to treat epithelial cancers (e.g., head and neck (including oral, laryngeal, pharyngeal, esophageal), breast, lung, prostate, cervical, colon, pancreatic, skin (basal cell carcinomas) and ovarian cancers). In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be used as anti-angiogenic agents. In further

embodiments, the antibodies or antigen-binding fragments thereof described herein can be used to block interaction of the ανβ5 receptor with RGD-containing ligands, e.g., proteins on the surface of viruses or other pathogens, thereby reducing or preventing infection.

The efficacy of the antibodies of the invention can be assessed in various animal models. Animal models for acute lung injury include: the pulmonary ischemia/reperfusion model (Sakuma T. et al., Am J Physiol Lung Cell Mol Physiol, 276: L137-L145, (1999); WO 2005/094391); the non-pulmonary ischemia/reperfusion model (Koike K. et al, J Surg Res., 52:656-662 (1992)); the oleic acid model (Schuster, Am JRespir Crit Care Med, 149: 245- 260 (1994)); the LPS model (Wiener-Kronish et al, J Clin Invest, 88: 864-875 (1991); the acid aspiration model (Modelska K. et al, Am JRespir Crit Care Med, 160: 1450-1456, (1999)); the hyperoxia model (Frank L. et al, J Appl Physiol, 45: 699-704 (1978)); the bleomycin model (Moore et al, Am J Physiol Lung Cell Mol Physiol, 294: L152-L160

(2008)); the saline lavage model (Lachmann B. et al., Acta Anaesthesiol Scand., 24: 231-236, (1980)); the cecal ligation and puncture model (Villar J. et al, Crit Care Med., 22: 914-921 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

(1994)); and the intrapulmonary bacteria model (Fox-Dewhurst R. et al., Am JRespir Crit Care Med., 155: 2030-2040 (1997). Mouse models for lung fibrosis include bleomycin- (Pittet et al, J. Clin. Invest., 107(12): 1537-1544 (2001); and Munger et al, Cell, 96:319-328 (1999)) and irradiation-inducible lung fibrosis (Franko et al, Rad. Res., 140:347-355 (1994)). Animal models for sepsis are known in the art and include toxaemia models (e.g., LPS injection), bacterial infection models, host-barrier disruption models (Doi et al, J. Clin.

Invest., 1 19(10):2868-2878 (2009); WO 201 1/011775). Finally, the ανβ5 antibodies

described herein can be assessed for their ability to inhibit tumor growth, progression, and metastasis in standard in vivo tumor growth and metastasis models. See, e.g., Rockwell et al, J. Natl. Cancer Inst., 49:735 (1972); Guy et al, Mol. Cell Biol, 12:954 (1992); Wyckoff et al, Cancer Res., 60:2504 (2000); and Oft et al, Curr. Biol, 8: 1243 (1998).

The efficacy of treatments may be measured by a number of available diagnostic tools, including physical examination, blood tests, pulmonary function tests, observation and scoring of scarring or fibrotic lesions, deposition of extracellular matrix such as collagen, smooth muscle actin and fibronectin, ultrasound, magnetic resonance imaging (MRI), and CT scan.

Pharmaceutical Compositions

An anti-av 5 antibody or antigen-binding fragment thereof described herein can be formulated as a pharmaceutical composition for administration to a subject, e.g., to treat a disorder described herein. Typically, a pharmaceutical composition includes a

pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19).

Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20^th ed., Lippincott,

Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al, Pharmaceutical Dosage

Forms and Drug Delivery Systems, 7^th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3^rd ed. (2000) (ISBN: 091733096X). Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.

In one embodiment, an anti-avp5 antibody described herein is formulated with excipient materials, such as sodium citrate, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate, Tween-80, and a stabilizer. It can be provided, for example, in a buffered solution at a suitable concentration and can be stored at 2-8°C. In some other embodiments, the pH of the composition is between about 5.5 and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5).

The pharmaceutical compositions can also include agents that reduce aggregation of the ανβ5 antibody or antigen-binding fragment thereof when formulated. Examples of aggregation reducing agents include one or more amino acids selected from the group consisting of methionine, arginine, lysine, aspartic acid, glycine, and glutamic acid. These amino acids may be added to the formulation to a concentration of about 0.5 mM to about 145 mM (e.g., 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM). The pharmaceutical compositions can also include a sugar (e.g., sucrose, trehalose, mannitol, sorbitol, or xylitol) and/or a tonicity modifier (e.g., sodium chloride, mannitol, or sorbitol) and/or a surfactant (e.g., polysorbate-20 or polysorbate-80).

Such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). In one embodiment, the anti-av 5 antibody or antigen-binding fragment thereof compositions are administered subcutaneously. In one embodiment, the anti-av 5 antibody or antigen-binding fragment thereof compositions are administered intravenously. The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous,

intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients

enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the anti-avp5 antibody or antigen-binding fragment thereof may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate,

polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel

Dekker, Inc., New York (1978).

In one embodiment, the pharmaceutical formulation comprises an anti-avp5 antibody or antigen-binding fragment thereof at a concentration of about 0.5 mg/mL to 500 mg/mL (e.g., 0.5 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/ mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 350 mg/mL, 400 mg/mL, 450 mg/mL, 500 mg/mL), formulated with a pharmaceutically acceptable carrier. In some embodiments, the anti-av 5 antibody or antigen-binding fragment thereof is formulated in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation may be between 5.5 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 6.3, 6.4 6.5, 6.6 6.7, 6.8, 6.9 7.0, 7.1, 7.3, 7.4, 7.5).

Administration

The anti-av 5 antibody or antigen-binding fragment thereof can be administered to a subject, e.g., a subject in need thereof, for example, a human subject, by a variety of methods. For many applications, the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection. It is also possible to use intra-articular delivery. Other modes of parenteral administration can also be used. Examples of such modes include: intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular,

subarachnoid, intraspinal, and epidural and intrasternal injection. In some cases,

administration can be oral.

The route and/or mode of administration of the antibody or antigen-binding fragment thereof can also be tailored for the individual case, e.g., by monitoring the subject, e.g., using tomographic imaging, e.g., to visualize a tumor.

The antibody or antigen-binding fragment thereof can be administered as a fixed dose, or in a mg kg dose. The dose can also be chosen to reduce or avoid production of antibodies against the anti-av 5 antibody. Dosage regimens are adjusted to provide the desired

response, e.g., a therapeutic response or a combinatorial therapeutic effect. Generally, doses of the anti-av 5 antibody or antigen binding fragment thereof (and optionally a second agent) can be used in order to provide a subject with the agent in bioavailable quantities. For example, doses in the range of 0.1-100 mg/kg, 0.5-100 mg/kg, 1 mg/kg -100 mg/kg, 0.5-20 mg/kg, 0.1-10 mg/kg, or 1-10 mg/kg can be administered. Other doses can also be used. In certain embodiments, a subject in need of treatment with an anti-av 5 antibody or antigen binding fragment thereof is administered the antibody at a dose of 1 mg/kg to 30 mg/kg. In some embodiments, a subject in need of treatment with an anti-av 5 antibody or antigen- binding fragment thereof is administered the antibody at a dose of 1 mg/kg, 2 mg/kg, 4 mg/kg, 5 mg/kg, 7 mg/kg 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 28 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, or 50 mg/kg. In certain embodiments, a subject in need of treatment with a toxin-conjugated anti-av 5 antibody or antigen binding fragment thereof is administered the toxin-conjugated antibody or antigen binding fragment thereof at a dose of Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

0.1 mg/kg to 30 mg/kg. In some embodiments, a subject in need of treatment with a toxin- conjugated anti-av 5 antibody or antigen-binding fragment thereof is administered the toxin- conjugated antibody or antigen binding fragment thereof at a dose of 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, 5 mg/kg, 7 mg/kg 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 28 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, or 50 mg/kg. In a specific embodiment, the antibodies or antigen-binding fragments thereof are administered subcutaneously at a dose of 1 mg/kg to 3 mg/kg. In another embodiment, the antibodies or antigen-binding fragments thereof are administered intravenously at a dose of 4 mg/kg to 30 mg/kg. In certain embodiments, the toxin-conjugated versions of the antibodies or antigen-binding fragments thereof are administered intravenously at a dose of 0.1 mg/kg to 30 mg/kg.

A composition may comprise about 1 mg/mL to 100 mg/ml or about 10 mg/mL to 100 mg/ml or about 50 to 250 mg/mL or about 100 to 150 mg/ml or about 100 to 250 mg/ml of anti-av 5 antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-av 5 antibody or antigen-binding fragment thereof in a composition is predominantly in monomeric form, e.g., at least about 90%, 92%, 94%, 96%, 98%, 98.5% or 99% in

monomeric form. Certain anti-av 5 antibody or antigen-binding fragment thereof

compositions may comprise less than about 5, 4, 3, 2, 1, 0.5, 0.3 or 0.1% aggregates, as detected, e.g., by UV at A280 nm. Certain anti-av 5 antibody or antigen-binding fragment thereof compositions comprise less than about 5, 4, 3, 2, 1, 0.5, 0.3, 0.2 or 0.1% fragments, as detected, e.g., by UV at A280 nm.

Dosage unit form or "fixed dose" as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of anti-av 5 antibody calculated to produce the desired therapeutic effect in

association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. Alternatively, or in addition, the antibody may be administered via continuous infusion.

An anti-av 5 antibody or antigen-binding fragment thereof dose can be administered, e.g., at a periodic interval over a period of time (a course of treatment) sufficient to

encompass at least 2 doses, 3 doses, 5 doses, 10 doses, or more, e.g., once or twice daily, or about one to four times per week, or preferably weekly, biweekly (every two weeks), every three weeks, monthly, e.g., for between about 1 to 12 weeks, preferably between 2 to 8 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. Factors that may influence the dosage and timing required to effectively treat a subject, include, e.g., the severity of the disease or disorder, formulation, route of delivery, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments.

If a subject is at risk for developing a disorder described herein, the antibody can be administered before the full onset of the disorder, e.g., as a preventative measure. The duration of such preventative treatment can be a single dosage of the antibody or the

treatment may continue (e.g., multiple dosages). For example, a subject at risk for the disorder or who has a predisposition for the disorder may be treated with the antibody for days, weeks, months, or even years so as to prevent the disorder from occurring or

fulminating.

A pharmaceutical composition may include a "therapeutically effective amount" of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disorder parameter or amelioration of at least one symptom of the disorder. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

In certain embodiments, the anti-avp5 antibody or antigen-binding fragment thereof is administered subcutaneously at a concentration of about 1 mg/mL to about 500 mg/mL (e.g., 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL _t5 mg/mL , 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL, 75 mg/mL, 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 175 mg/mL, 200 mg/mL, 225 mg/mL, 250 mg/mL, 275 mg/mL, 300 mg/mL, 325 mg/mL, 350 mg/mL, 400 mg/mL, 450 mg/mL). In one embodiment, the anti-av 5 antibody or antigen-binding fragment thereof is administered subcutaneously at a concentration of 50 mg/mL. In another embodiment, the anti-av 5 antibody or antigen- binding fragment thereof is administered intravenously at a concentration of about 1 mg/mL Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

to about 500 mg/niL. In a particular embodiment, the anti-av 5 antibody or antigen-binding fragment thereof is administered intravenously at a concentration of 50 mg/mL.

The anti-av 5 antibody or antigen-binding fragment thereof can be administered to a patient in need thereof (e.g., a patient with lung fibrosis) in combination with a second therapeutic agent. The second therapeutic agent depends on the type of disease or disorder being treated. For example, the second therapeutic agent can be an antagonist (e.g.,

antibodies, polypeptide antagonists, and/or small molecule antagonists) of one or more: other integrin receptors (e.g., αΐ βΐ, α4β1, ανβ8, ανβό, ανβΐ, etc.); cytokines (e.g., TGF-β, IL-4, IL-13, IL-17); chemokines (e.g., CCL2, CXCL8, CXCL12); growth factors (e.g., Connective tissue growth factor (CTGF), Platelet-derived growth factor (PDGF), Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF), Insulin-like growth factor- 1 (IGF- 1)), and/or small secreted signaling proteins (e.g., Wnt proteins, endothelin-1). The anti- ανβ5 antibody or antigen-binding fragment thereof and the second therapeutic agent may be administered simultaneously or sequentially. In certain embodiments, the anti-o^5 antibody or antigen-binding fragment thereof and the second therapeutic agent can each be

administered at either a subtherapeutic dose or a therapeutic dose.

In certain embodiments, where a patient has, or is at risk of developing acute lung injury, pulmonary edema, or ARDS, the anti-o^5 antibody or antigen-binding fragment thereof can be administered in combination with diuretic agents, bronchodilating agents, narcotics, oxygen, and selective tourniquet application. In addition, the anti-o^5 integrin antibodies disclosed herein may be administered in conjunction with a second therapeutic agent that targets metabolic pathways that are implicated in acute lung injury, ARDS, or PE. For example, an anti-o^5 integrin antibody or antigen-binding fragment thereof may be administered in conjunction with ΤϋΡβ pathway inhibitors, activated Protein C, steroids, GM-CSF, platelet inhibitors, β-2 agonists, surfactants, other antibodies that specifically bind to ανβ5 integrin or β5, a second antagonist of ανβ5 integrin, antibodies that specifically bind to a ανβό integrin, antagonists of ανβό integrin, thrombin receptor antagonists, anti -thrombin agents, rho kinase inhibitors, and nucleic acids that inhibit expression of ανβ5 integrin including e.g., the antisense oligonucleotides, ribozymes, miRNA, and siRNA. Suitable

TGFfi pathway inhibitors include, e.g., TGF-β antibodies (including those that specifically block TGF-β 1, TGF^2, TGF^3 or any combination thereof) as described in e.g., Ling et al., J. Amer. Soc. Nephrol., 14: 377-388 (2003), McCormick et al, J. Immunol, 163:5693-5699 Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

(1999), and Cordeiro, Curr. Opin. Mol. Ther., 5(2): 199-203 (2003); TGF-β receptor type II inhibitors or TGF-β receptor type I kinase inhibitors as described in, e.g., DaCosta Bayfield, Mol. Pharmacol, 65(3):744-52 (2004), Laping, Curr. Opin. Pharmacol, 3(2):204-8 (2003), Laping, Mol. Pharmacol, 62(l):58-64 (2002); soluble TGF-β receptor type II as described in, e.g., Pittet, J. Clin. Invest., 107: 1537-1544 (2001); Wang et al, Exp Lung Res., 28(6):405-17 (2002) and Wang, Thorax, 54(9):805-12 (1999); soluble latency associated peptides as described in, e.g., Zhang, J Invest. Dermatol, 121(4):713-9 (2003); thrombospondin I inhibitors as described in, e.g., Crawford et al, Cell, 93 : 1 159-1170 (1998), Riberiro et al, J.

Biol. Chem., 274: 13586-13593 (1999), and Schultz-Cherry et al, J. Biol. Chem., 269: 26775- 26782 (1994). Suitable β-2 agonists include, e.g., albuterol, bitolterol, formoterol,

isoproterenol, levalbuterol, metaproterenol, pirbuterol, salmeterol, and terbutaline. Suitable surfactants include, e.g., exosurf, infasurf, KL-4, pumactant, survanta, venticute, and

surfactant TA, as described in Taeusch et al, Acta Pharmacol Sin 23 Supplement: 11-15

(2002). Suitable anti-thrombin agents include, e.g., hirudin, Hirulog (Biogen), argatroban, efegatran, and compounds described in U.S. Patent No. 6,518,244. Suitable thrombin receptor antagonists are described in, e.g., U.S. Patent Nos. 6,544,982; 6,515,023; 6,403,612;

6,399,581 ; and 5,446, 131. Suitable rho kinase inhibitors include, e.g., Y-27632 as described in e.g., Tasaka et al, Am JRespir Cell Mol Biol, 32(6):504-10 (2005); fasudil as described in, e.g., Nishikimi et al, J Hypertens., 22(9): 1787-96 (2004); l-(5-isoquinolinesulfonyl)- homopiperazine (HA- 1077), (S)-(+)-2-methyl- 1 -[(4-methyl-5-isoquinoline)sulfonyl]- homopiperazine (H-l 152P) as described in Sasaki et al, Pharmacol Ther., 93(2-3):225-32 (2002), and additional rho kinase inhibitors as described in, e.g., U.S Patent Nos. 6,451,825 and 6,218,410 and U.S. Patent Publication Nos. 20050014783 and 20030134775. In

addition, the antagonist of ανβ5 integrin may be administered combination with an

adenovirus expressing ATPase as described in U.S. Patent Application No. 20020192186;

with a β2 adrenergic receptor as described in U.S. Patent Application No. 20020004042; with νΕϋΡβ antagonists as described in U.S. Patent No. 6,284,751; with lipid peroxidation inhibitors as described in U.S. Patent No. 5,231, 114; and with small molecule inhibitors for ανβ6, ανβ5, and ανβ3 integrins as described in, e.g., US Patent Application Nos.

2000/40019206, 2004/0019037, 2004/0019035, 2004/0018192, 2004/0010023,

2003/0181440, 2003/0171271, 2003/0139398, 2002/0037889, 2002/0077321, 2002/0072500, U.S. Patent No. 6, 683,051 and Goodman et al, J. Med Chem. 45(5): 1045-51 (2002). Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

In certain embodiments, where a patient has, or is at risk of developing sepsis, the anti-av 5 antibody or antigen-binding fragment thereof can be administered in combination with any of the standard treatments for sepsis including, e.g., antibiotics, statins, steroids, activated Protein C, diuretic agents, vasoconstrictors, or inotropic drugs. Antibiotic therapies are common, and can best be selected by the medical professional to specifically target a particular infection. Exemplary antibiotics include, e.g., penicillin, erythromycin, cyclic lipopeptides (daptomycin), glycylcyclines (tigecycline), and oxazolidinones (linezolid).

Statins (HMG-CoA reductase inhibitors) include, e.g., simvastatin or atorvastatin. In addition, an anti-av 5 integrin antibody or antigen binding fragment thereof may be

administered in conjunction with agents that target metabolic pathways that are implicated in sepsis. For example, an antagonist of ανβ5 integrin may be administered in conjunction with TGF pathway inhibitors, activated Protein C, GM-CSF, antibodies that specifically bind to ανβ5 integrin or β5, a second antagonist of ανβ5 integrin, antibodies that specifically bind to a ανβό integrin, antagonists of ανβό integrin, thrombin receptor antagonists, anti-thrombin agents, rho kinase inhibitors, and nucleic acids that inhibit expression of ανβ5 integrin including e.g., antisense oligonucleotides, ribozymes, siR A, microRNA.

Devices and Kits for Therapy

Pharmaceutical compositions that include the anti-o^5 antibody or antigen-binding fragment thereof can be administered with a medical device. The device can be designed with features such as portability, room temperature storage, and ease of use so that it can be used in emergency situations, e.g., by an untrained subject or by emergency personnel in the field, removed from medical facilities and other medical equipment. The device can include, e.g., one or more housings for storing pharmaceutical preparations that include anti-o^5 antibody or antigen-binding fragment thereof, and can be configured to deliver one or more unit doses of the antibody. The device can be further configured to administer a second agent, e.g., a chemo therapeutic agent, either as a single pharmaceutical composition that also includes the anti-o^5 antibody or antigen-binding fragment thereof or as two separate pharmaceutical compositions.

The pharmaceutical composition may be administered with a syringe. The

pharmaceutical composition can also be administered with a needleless hypodermic injection device, such as the devices disclosed in US 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules include: US 4,487,603, which discloses an implantable micro-infusion pump for dispensing

medication at a controlled rate; US 4,486, 194, which discloses a therapeutic device for administering medicaments through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; US 4,439, 196, which discloses an osmotic drug delivery system having multi-chamber

compartments; and US 4,475, 196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems, and modules are also known.

An anti-av 5 antibody or antigen-binding fragment thereof can be provided in a kit.

In one embodiment, the kit includes (a) a container that contains a composition that includes anti-av 5 antibody, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of the agents for therapeutic benefit.

In an embodiment, the kit also includes a second therapeutic agent for treating a disorder described herein (e.g., an antagonist (e.g., antibodies, polypeptide antagonists, and/or small molecule antagonists) of one or more: other integrin receptors (e.g., αΐ βΐ, α4β1, ανβ8, ανβ5, ανβΐ, etc.); cytokines (e.g., TGF-β, IL-4, IL-13, IL-17); chemokines (e.g.,

CCL2, CXCL8, CXCL12); growth factors (e.g., Connective tissue growth factor (CTGF), Platelet-derived growth factor (PDGF), Vascular endothelial growth factor (VEGF),

Fibroblast growth factor (FGF), Insulin-like growth factor-1 (IGF-1)), small secreted

signaling proteins (e.g., Wnt proteins, endothelin-1) a steroid, a cytotoxic compound, a radioisotope, a prodrug-activating enzyme, colchicine, oxygen, an antioxidant (e.g., N- acetylcysteine), a metal chelator (e.g., terathiomolybdate), IFN-β, IFN-γ, alpha-antitrypsin). For example, the kit includes a first container that contains a composition that includes the anti-o^5 antibody, and a second container that includes the second therapeutic agent.

The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the anti-o^5 antibody or antigen-binding fragment thereof, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

administration described herein), to treat a subject who has had or who is at risk for an immunological disorder described herein. The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio

recording, or information that provides a link or address to substantive material, e.g., on the internet.

In addition to the antibody, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The antibody can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile. When the agents are provided in a liquid solution, the liquid solution preferably is an aqueous solution. In certain embodiments, the antibody or antigen binding fragment thereof in the liquid solution is at a concentration of about 25 mg/mL to about 250 mg/mL (e.g., 40 mg/mL, 50 mg/mL, 60 mg/mL, 75 mg/mL, 85 mg/mL, 100 mg/mL, 125 mg/mL, 150 mg/mL, 200 mg/mL). When the antibody or antigen binding fragment is provided as a lyophilized product, the antibody or antigen binding fragment is at about 75 mg/vial to about 200 mg/vial (e.g., 100 mg/vial, 108.5 mg/vial, 125 mg/ vial, 150 mg/vial). The lyophilized powder is generally reconstituted by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer (e.g., PBS), can optionally be provided in the kit. In certain embodiments, the

lyophilized product is at 108.5 mg/vial and reconstituted to a liquid solution at a

concentration of 75 mg/mL.

The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual

containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes both the anti-av 5 antibody or antigen-binding fragment thereof and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

dose. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.

Diagnostic Uses

Αηίί-ανβ5 antibodies or antigen-binding fragments thereof can be used in a diagnostic method for detecting the presence of ανβ5 in vitro or in vivo (e.g., in vivo imaging in a subject). For example, anti-avp5 antibodies can be administered to a subject to detect ανβ5 within the subject. For example, the antibody can be labeled, e.g., with an MRI detectable label or a radiolabel. The subject can be evaluated using a means for detecting the detectable label. For example, the subject can be scanned to evaluate localization of the antibody within the subject. For example, the subject is imaged, e.g., by NMR or other tomographic means.

Examples of labels useful for diagnostic imaging include radiolabels such as ¹³¹I, ^mIn, ¹²³1, ^99mTc, ³²P, ³³P, ¹²⁵1, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes, can also be employed. The protein ligand can be labeled with such reagents using known techniques. For example, see Wensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York for techniques relating to the radiolabeling of antibodies and Colcher et al. (1986) Meth.

Enzymol. 121 : 802-816.

The subject can be "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. See e.g., A.R. Bradwell et al, "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al, (eds.), pp 65-85 (Academic Press 1985). Alternatively, a positron emission transaxial tomography scanner, such as designated Pet VI located at

Brookhaven National Laboratory, can be used where the radiolabel emits positrons (e.g., ^UC, ¹⁸F, ¹⁵0, and ¹³N). Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Magnetic Resonance Imaging (MRI) uses NMR to visualize internal features of living subject, and is useful for prognosis, diagnosis, treatment, and surgery. MRI can be used without radioactive tracer compounds for obvious benefit. Some MRI techniques are summarized in EP0 502 814 A. Generally, the differences related to relaxation time

constants Tl and T2 of water protons in different environments are used to generate an image. However, these differences can be insufficient to provide sharp high resolution images.

The differences in these relaxation time constants can be enhanced by contrast agents. Examples of such contrast agents include a number of magnetic agents, paramagnetic agents (which primarily alter Tl) and ferromagnetic or superparamagnetic agents (which primarily alter T2 response). Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (and reduce toxicity) of some paramagnetic substances (e.g., Fe³⁺, Mn²⁺, Gd³⁺). Other agents can be in the form of particles, e.g., less than 10 μιη to about 10 nm in diameter). Particles can have ferromagnetic, anti-ferromagnetic or superparamagnetic properties. Particles can include, e.g., magnetite (Fe₃0₄), y-Fe₂0₃, ferrites, and other magnetic mineral compounds of transition elements. Magnetic particles may include one or more magnetic crystals with and without nonmagnetic material. The nonmagnetic material can include synthetic or natural polymers (such as sepharose, dextran, dextrin, starch and the like).

The anti-av 5 antibodies or antigen-binding fragments thereof can also be labeled with an indicating group containing the NMR-active ¹⁹F atom, or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the ¹⁹F isotope and, thus, substantially all fluorine-containing compounds are NMR-active; (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost, and (iii) many fluorinated compounds have been found medically acceptable for use in humans such as the perfluorinated polyethers utilized to carry oxygen as hemoglobin replacements. After permitting such time for incubation, a whole body MRI is carried out using an apparatus such as one of those described by Pykett (1982) Scientific American, 246:78-88 to locate and image ανβ5 distribution.

In another aspect, the disclosure provides a method for detecting the presence of ανβ5 in a sample in vitro (e.g., a biological sample, such as serum, plasma, tissue, biopsy). This method can be used to diagnose a disorder, e.g., acute lung injury, lung fibrosis, or cancer (e.g., pancreatic, lung, breast, colorectal, head and neck, esophageal, skin, or endometrial). Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

The method includes: (i) contacting the sample or a control sample with the anti-av 5 antibody; and (ii) evaluating the sample for the presence of ανβ5, e.g., by detecting formation of a complex between the anti-av 5 antibody and ανβ5, or by detecting the presence of the antibody or ανβ5. For example, the antibody can be immobilized, e.g., on a support, and retention of the antigen on the support is detected, and/or vice versa. The antibody used may be labeled e.g., with a fluorophore. A control sample can be included. The positive control can be a sample known to have the disease or disorder being assessed, and a negative control can be a sample from a subject who does not have the disease or disorder being assessed. A statistically significant change in the formation of the complex in the sample relative to the control sample can be indicative of the presence of ανβ5 in the sample. Generally, an anti- ανβ5 antibody can be used in applications that include fluorescence polarization, microscopy, ELISA, centrifugation, chromatography, and cell sorting (e.g., fluorescence activated cell sorting). In certain embodiments, the anti-o^5 antibody is a humanized ALULA antibody or an antigen-binding fragment thereof. The tissue sample can be, e.g., skin biopsies from human patients with cancer, e.g., pancreatic, lung, breast, colorectal, head and neck,

esophageal, skin, or endometrial.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1; Construction and Analysis of Chimeric Anti-Integrin ανβ5 Antibodies

The variable region sequences of ALULA were used to construct two chimeric antibodies comprising murine anti-integrin ανβ5 V regions with human IgG4 or human IgGl heavy chain and human kappa light chain constant regions. The IgG4 constant regions additionally contain a mutation in the hinge region (S241P, Kabat numbering) to reduce the formation of half antibodies. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

The amino acid sequence of the murine anti-integrin ανβ5 VH sequence is provided below:

EVQVQQSGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGN SDTSYNQKFKGKAKLTAVTSPNTAYMELSSLTNEDSAVYYCTTTTYGYDWFAYWG QGTLVTVSA (SEQ ID NO:42)

The nucleic acid sequence encoding the murine anti-integrin ανβ5 VH sequence is provided below:

GAGGTTCAGGTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAA ATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAG TCACATCCCCCAACACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACT CTGCGGTCTATTACTGTACAACCACTACATATGGTTACGACTGGTTTGCTTACTGG GGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:43)

The amino acid sequence of the murine anti-integrin ανβ5 VL sequence is provided below:

NIMMTQSPSSLTVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYW ASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSLTFGAGTKLELK

(SEQ ID NO:44)

The nucleic acid sequence encoding the murine anti-integrin ανβ5 VL sequence is provided below:

AACATTATGATGACACAGTCGCCATCATCTCTGACTGTGTCTGCAGGAGAAAAGG TCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAA CTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC TGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTG GGACAGATTTTACTCTTACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTA TTACTGTCATCAATACCTCTCCTCGCTCACGTTCGGTGCTGGGACCAAGCTGGAG CTGAAA (SEQ ID NO:45)

The murine VH nucleotide sequences were transferred into mammalian expression vectors for expressing IgGl or IgG4 (S241P) VH chains, respectively. Similarly, the murine VK region was transferred into an expression vector for expressing VK chains. All of the constructs were confirmed by sequencing. The VK expression vector was paired with either Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

the IgGl or the IgG4(S241P) expression vector and stably co-transfected into NS0 cells via electroporation.

The transfected cells were selected using 200nM methotrexate (Sigma, Cat. No.

M8407) and methotrexate-resistant colonies for each construct were tested for IgG expression levels using an IgGl or IgG4 ELISA, as appropriate. The best expressing cell lines were selected, expanded into NS0 growth medium containing 500nM methotrexate and frozen under liquid nitrogen. Cell lines expressing both IgGl and IgG4 (S241P) chimeric antibodies were successfully generated. NS0 cells expressing either IgGl or IgG4 (S241P) chimeric antibody were observed to grow almost entirely in suspension, as compared to non- transfected NS0 cells that loosely attach to tissue culture flasks. It was theorized that the anti- ανβ5 integrin antibody was cross reacting with murine integrins on the surface of NS0 cells thereby preventing them from adhering to the plastic. This theory was corroborated by FACS.

Chimeric anti-av 5 antibodies were purified from cell culture supernatants on a

Protein A sepharose column (GE Healthcare, Cat. No. 1 10034-93). Antibodies were eluted in a low pH buffer (0.1M Sodium Citrate pH 3.0) and immediately buffer exchanged into PBS pH 7.4 and quantified by OD280nm using an extinction coefficient based on the predicted amino acid sequences (Ec₍o_.i_%) = 1.66). IgGl and IgG4 (S241P) chimeric antibodies were analyzed by SDS-PAGE under both reducing and non-reducing conditions. For both samples, under reducing conditions, two bands could be visualized by Coomassie Blue staining corresponding to the antibody heavy and light chains. Whereas the light chain migrated according to its predicted molecular weight of 25 kDa, the heavy chain migrated somewhat slower than predicted (50 kDa) as a result of glycosylation.

The binding of chimeric anti-integrin ανβ5 antibodies to human integrin ανβ5 was assessed by competition FACS using CS-1 β5 cells. The binding of chimeric anti-integrin ανβ5 antibodies to human integrin ανβ5 was found to be similar to the murine reference antibody (ALULA).

The biological activity of murine and chimeric anti-integrin ανβ5 antibodies was assessed in an assay measuring cell adhesion to vitronectin. The objective was to demonstrate that anti-integrin ανβ5 antibodies block the adhesion of cells to vitronectin-coated plates.

Briefly, murine or chimeric antibody at a range of concentrations (3 - 0.1 1 μg/ml) was pre- incubated with SW480 cells (HPACC, Cat. No. 87092801) for 20 min at 37°C. Cells were dispensed into wells of a plate coated with 100 μΐ of vitronectin at 1 μg/ml, at 3.5xl0⁴ cells Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

per well (four replicates per condition) and incubated for 1.5 h at 37°C. Non-adherent cells were removed by discarding the supematants and centrifuging the plate top-side down at 45 g for five minutes. Cell adhesion was measured using Cell Titer-Glo® Luminescent Cell

Viability Assay (Promega, Cat. No. 7572). Luminescence was read on a BMG LABTECH FLUOstar OPTIMA plate reader (Figure 6). Both human IgGl and IgG4 (S241P) chimeric anti-integrin ανβ5 antibodies demonstrated a similar efficiency for blocking cell adhesion compared to the murine antibody.

Analytical size exclusion chromatography (SEC) was performed using a Superdex 200 5/150 GL column (GE Healthcare, Cat. No. 28-9065-61) on an Akta Purifier (GE

Healthcare) run in lx PBS buffer pH 7.4. The results (Figure 7) indicated that there was no propensity of IgGl and IgG4 (S241P) chimeric antibodies to aggregate or degrade and both chimeric antibodies ran as a single peak consistent with whole antibody.

Example 2; Humanized ALULA Heavy and Light Chains

Structural models of the mouse anti-av 5 antibody ALULA V regions were produced using Swiss PDB and analyzed in order to identify important "constraining" amino acids in the V regions that were likely to be essential for the binding properties of the antibody.

Residues contained within the CDRs (using Kabat definition) together with a number of framework residues were considered to be important. Both the VH and VK sequences of ALULA contain typical framework residues and the CDR 1, 2 and 3 motifs are comparable to many murine antibodies.

A set of sequence segments that could be used to create ALULA Composite Human Antibody™ variants was selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class III alleles (Perry et al, Drugs R D 9(6):385- 396 (2008)), and using the TCED™ of known antibody sequence-related T cell epitopes (Bryson et al, Biodrugs, 24(1): 1-8 (2010)). Sequence segments that were identified as significant non-human germline binders to human MHC class II or that scored significant hits against the TCED™ were discarded. This resulted in a reduced set of segments, and

combinations of these were again analyzed, as above, to ensure that the junctions between segments did not contain potential T cell epitopes. Selected segments that were predicted to be non-immunogenic were then combined to produce the five heavy and four light chain V region sequences shown below. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

hALULA VH1 Heavy Chain Amino Acid Sequence

EVQVVQSGTELKKPGASVKMSCKASGYTFTSYWMHWVKQAPGQGLEWIGAIYPGN SDTSYNQKFKGKAKLTAVTSPNTAYMELSSLRSEDSAVYYCTTTTYGYDWFAYWG QGTLVTVSS (SEQ ID NO:l)

hALULA VH1 Heavy Chain Nucleotide Sequence

GAGGTTCAGGTCGTGCAGTCTGGGACTGAGCTGAAGAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGGCCCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAA ATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAG TCACATCCCCCAACACTGCCTACATGGAGCTCAGCAGCCTGAGAAGCGAGGACT CTGCGGTCTATTACTGTACAACCACTACATATGGTTACGACTGGTTTGCTTACTGG GGCCAAGGGACTCTGGTCACTGTCTCTAGC (SEQ ID NO:2) hALULA VH2 Heavy Chain Amino Acid Sequence

EVQVVQSGAEVKKPGASVKMSCKASGYTFTSYWMHWVKQAPGQGLEWIGAIYPG NSDTSYNQKFKGKAKLTAVTSTNTAYMELSSLRSEDTAVYYCTTTTYGYDWFAYW GQGTLVTVSS (SEQ ID NO:3)

hALULA VH2 Heavy Chain Nucleotide Sequence

GAGGTTCAGGTCGTGCAGTCTGGGGCCGAGGTGAAGAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGGCCCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAA ATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAG TCACATCCACCAACACTGCCTACATGGAGCTCAGCAGCCTGAGAAGCGAGGACA CCGCGGTCTATTACTGTACAACCACTACATATGGTTACGACTGGTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTAGC (SEQ ID NO:4) hALULA VH3 Heavy Chain Amino Acid Sequence

EVQVVQSGAEVKKPGASVKMSCKASGYTFTSYWMHWVKQAPGQGLEWIGAIYPG NSDTSYNQKFKGKAKLTAVTSTSTAYMELSSLRSEDTAVYYCTTTTYGYDWFAYW GQGTLVTVSS (SEQ ID NO:5)

hALULA VH3 Heavy Chain Nucleotide Sequence

GAGGTTCAGGTCGTGCAGTCTGGGGCCGAGGTGAAGAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGGCCCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAA ATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAG TCACATCCACCAGCACTGCCTACATGGAGCTCAGCAGCCTGAGAAGCGAGGACA CCGCGGTCTATTACTGTACAACCACTACATATGGTTACGACTGGTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTAGC (SEQ ID NO:6) Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

hALULA VH4 Heavy Chain Amino Acid Sequence

EVQVVQSGAEVKKPGASVKMSCKASGYTFTSYWMHWVKQAPGQGLEWIGAIYPG NSDTSYNQKFKGKVKLTAVTSTSTAYMELSSLRSEDTAVYYCTTTTYGYDWFAYW GQGTLVTVSS (SEQ ID NO:7)

hALULA VH4 Heavy Chain Nucleotide Sequence

GAGGTTCAGGTCGTGCAGTCTGGGGCCGAGGTGAAGAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGGCCCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAA ATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGTGAAACTGACTGCAG TCACATCCACCAGCACTGCCTACATGGAGCTCAGCAGCCTGAGAAGCGAGGACA CCGCGGTCTATTACTGTACAACCACTACATATGGTTACGACTGGTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTAGC (SEQ ID NO:8) hALULA VH5 Heavy Chain Amino Acid Sequence

EVQVVQSGAEVKKPGASVKMSCKASGYTFTSYWMHWVKQAPGQGLEWIGAIYPG NSDTSYNQKFKGKVKLTADTSTSTAYMELSSLRSEDTAVYYCTTTTYGYDWFAYW GQGTLVTVSS (SEQ ID NO:9)

hALULA VH5 Heavy Chain Nucleotide Sequence

GAGGTTCAGGTCGTGCAGTCTGGGGCCGAGGTGAAGAAGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGGCCCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAA ATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGTGAAACTGACTGCAG ACACATCCACCAGCACTGCCTACATGGAGCTCAGCAGCCTGAGAAGCGAGGACA CCGCGGTCTATTACTGTACAACCACTACATATGGTTACGACTGGTTTGCTTACTG GGGCCAAGGGACTCTGGTCACTGTCTCTAGC (SEQ ID NO: 10) hALULA V I Light Chain Amino Acid Sequence

NIMMTQSPATLTVSAGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYW ASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSLTFGQGTKLEIK

(SEQ ID NO: 11)

hALULA VKI Light Chain Nucleotide Sequence

AACATTATGATGACACAGTCGCCAGCCACCCTGACTGTGTCTGCAGGAGAAAGA GCCACTCTGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGA ACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTA CTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTTACTCTTACCATCAGCAGTCTGCAAGCTGAAGACGTGGCAGTTT ATTACTGTCATCAATACCTCTCCTCGCTCACGTTCGGTCAGGGGACCAAGCTGGA GATCAAA (SEQ ID NO: 12) Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

1IALULAV 2 Light Chain Amino Acid Sequence

NIMMTQSPATLSVSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQPPKLLIYW ASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSLTFGQGTKLEIK

(SEQ ID NO:13) hALULA V 2 Light Chain Nucleotide Sequence

AACATTATGATGACACAGTCGCCAGCCACCCTGAGCGTGTCTCCCGGAGAAAGA GCCACTCTGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGA ACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCCCCTAAACTGCTGATCT ACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATC TGGGACAGATTTTACTCTTACCATCAGCAGTCTGCAAGCTGAAGACGTGGCAGTT TATTACTGTCATCAATACCTCTCCTCGCTCACGTTCGGTCAGGGGACCAAGCTGG AGATCAAA (SEQ ID NO: 14) hALULA V 3 Light Chain Amino Acid Sequence

NIMMTQSPATLSVSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQPPKLLIYW ASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSLTFGQGTKLEIK

(SEQ ID NO:15) hALULA V 3 Light Chain Nucleotide Sequence

AACATTATGATGACACAGTCGCCAGCCACCCTGAGCGTGTCTCCCGGAGAAAGA GCCACTCTGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGA ACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCCCCTAAACTGCTGATCT ACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCAGCGGCAGTGGATC TGGGACAGATTTTACTCTTACCATCAGCAGTCTGCAAGCTGAAGACGTGGCAGTT TATTACTGTCATCAATACCTCTCCTCGCTCACGTTCGGTCAGGGGACCAAGCTGG AGATCAAA (SEQ ID NO: 16) hALULA V 4 Light Chain Amino Acid Sequence

NIVMTQSPATLSVSPGERATLSCKSSQSVLYSSNQKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSLTFGQGTKLEIK

(SEQ ID NO:17)

hALULA V 4 Light Chain Nucleotide Sequence

AACATTGTGATGACACAGTCGCCAGCCACCCTGAGCGTGTCTCCCGGAGAAAGA GCCACTCTGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGA ACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCCCCTAAACTGCTGATCT ACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCAGCGGCAGTGGATC TGGGACAGATTTTACTCTTACCATCAGCAGTCTGCAAGCTGAAGACGTGGCAGTT TATTACTGTCATCAATACCTCTCCTCGCTCACGTTCGGTCAGGGGACCAAGCTGG AGATCAAA (SEQ ID NO:18) Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Alignments of the five VH amino acid sequences and the four VL amino acid

sequences with the counterpart murine ALULA antibody VH and VL sequences are shown in Figures 1 and 2, respectively.

Example 3; Construction of ALULA Humanized Antibodies

All variant Composite Human Antibody VH and VK region genes for anti-integrin ανβ5 were synthesized using a series of overlapping oligonucleotides that were annealed, ligated and PCR amplified to give full length synthetic V regions. The assembled variants were then cloned directly into the pANT expression vector system for IgGl heavy chain and kappa light chain (Figure 3). The VH region was cloned using M and Hindlll sites, and the VK region was cloned using BssHII and BamHI restriction sites. All constructs were confirmed by sequencing.

Example 4; Expression and Purification of Antibodies

All combinations of composite IgGl VH and VK chains (i.e. a total of 20 pairings) were stably transfected into NS0 cells via electroporation. The stable transfections were selected using 200nM methotrexate (Sigma Cat. No. M8407), methotrexate-resistant colonies for each construct were tested for IgG expression levels using an IgGl ELISA, and the best expressing lines were selected, expanded and frozen under liquid nitrogen. Successful transfection and stable clone selection was achieved for all 20 Composite Human Antibodies.

Composite variants of anti-integrin ανβ5 were purified from cell culture supernatants on a Protein A sepharose column (GE Healthcare Cat. No. 1 10034-93), buffer exchanged into PBS pH 7.4 and quantified by OD₂80nm using an extinction coefficient (Ec₍o.i%) = 1.66) based on the predicted amino acid sequences. Five selected candidate composite variants were analyzed by reducing SDS-PAGE. Bands corresponding to the predicted sizes of the VH and VK chains were observed (Figure 4).

Example 5; Binding of Humanized ALULA Antibodies to CS-1 β5 cells

The binding of humanized ALULA Antibodies to human integrin ανβ5 was assessed by competition FACS using CS-1 β5 cells. In order to compare the binding properties of IgGl chimeric and humanized ALULA variant antibodies in a FACS competition assay, chimeric ALULA antibody was labeled with PE-labeled Fab fragments that specifically bind Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

to human antibody using a Zenon® R-Phycoerythrin Human IgG Labeling Kit (Invitrogen, Cat. No. Z-25455).

A dilution series of unlabeled humanized ALULA antibody (concentration range 6 - 0.001 μg/ml) was mixed with a constant concentration (0.04 μg/ml) of PE-Fab labeled IgGl chimeric anti-integrin ανβ5 antibody and incubated with 3x105 CS-1 β5 cells per dilution in FACS buffer. After incubating on ice for 1 hour, cells were washed and resuspended in 300 μΐ FACS buffer and analyzed on a Beckton Dickinson FACScalibur. The geometric mean fluorescence intensity was plotted against antibody concentration (Figure 5). The IC50 of each of the 20 humanized ALULA Antibodies was calculated relative to the IC5₀ of the chimeric ALULA antibody and is provided in Table 1 below.

TABLE 1

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The data showed that the four humanized antibodies containing the VH1 chain retained similar binding properties to the reference chimeric antibody with relative IC50's of 0.8 to 1.1.

Example 6; Biological Activity of Humanized ALULA Antibodies

Based on the FACS competition data described in Example 5, eight humanized

ALULA antibodies were selected (i.e., VHWKI, VH1/VK2, VH 1/VK3, VH1/VK4,

VH2/VK1 , VH2/VK2, VH2/VK3, and VH2/VK4) for testing in the cell adhesion blocking assay. The objective was to test whether humanized ALULA antibodies block the adhesion of cells to vitronectin-coated plates comparably to the chimeric ALULA antibody. Humanized ALULA antibodies at a range of concentrations (1 - 0.11 μg/ml) were preincubated with SW480 cells (HPACC, Cat. No. 87092801) for 20 min at 37°C. Cells were dispensed into wells of a plate coated with 100 μΐ of vitronectin at 1 μg/ml, at 3.5x10⁴ cells per well (four replicates per condition) and incubated for 1.5 h at 37°C. Non-adherent cells were removed by discarding the supernatants and centrifuging the plate top-side down at 45 g for five minutes. Cell adhesion was measured using Cell Titer-Glo® Luminescent Cell Viability Assay (Promega, Cat. No. 7572). Luminescence was read on a BMG LABTECH FLUOstar OPTIMA plate reader (Figure 6). All four antibodies comprising VH1 as well as VH2/VK1 demonstrated a similar blocking effect to the chimeric antibody whereas VH2/VK2, VH2/VK3 and VH2/VK4 demonstrated slightly less potent blocking especially at the highest antibody concentration tested (^g/ml). Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Example 7; EpiScreen™ Time Course Assay of Humanized ALULA Antibodies

The objective of the current project was to assess the immunogenic potential of humanized anti-av 5 antibodies (VH1/VK1 and VH1/VK2) compared to the reference chimeric ALULA antibody by measuring ex vivo T cell responses against the whole

antibodies (compared to the chimeric antibody) using the EpiScreen™ time course T cell assay.

Methods

Preparation and selection of donor PBMC

PBMC were isolated from healthy community donor buffy coats (from blood drawn within 24 hours) obtained from the UK National Blood Transfusion Service (Addenbrooke's

Hospital, Cambridge, UK) and according to approval granted by Addenbrooke's Hospital Local Research Ethics Committee. PBMC were isolated from buffy coats by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation and CD8⁺ T cells were depleted using

CD8⁺

RosetteSep™ (StemCell Technologies Inc, London, UK). Donors were characterized by identifying HLA-DR haplotypes using an HLA SSP-PCR based tissue-typing kit (Biotest, Solihull, UK). T cell responses to a 'reproducibility' control antigen (Keyhole Limpet

Haemocyanin (KLH), Pierce (Perbio), Cramlington, UK), and a clinical standard antibody (humanized A33) were also determined (Welt et a\ 2003). PBMC were then frozen and stored in liquid nitrogen until required.

A cohort of 20 donors was selected (study cohort STR02) to best represent the number and frequency of HLA-DR allotypes expressed in the world population. Analysis of the allotypes expressed in the cohort against those expressed in the world population revealed that all major HLA-DR alleles (individual allotypes with a frequency >5% expressed in the world

population) were well represented. Details of individual donor haplotypes and a comparison of the frequency of MHC class II haplotypes expressed in the world population and the sample population are shown in Table 2 and Figure 7, respectively. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

TABLE 2

Figure 7 also shows responses of donor PBMC to KLH before storage ("Test 1") and prior to use in the present study ("STR02"). This showed a concordance in KLH responses in 18 out of 20 donors (90%) which is within the normal range.

Purification of Antibodies

Chimeric and humanized antibodies were prepared from up to 2L cultures of antibody expressing NSO cell-lines grown to saturation. Supernatants were separated from cells and debris by centrifugation, adjusted to pH 7.4 and 150 mM NaCl with 1/9 volumes lOx PBS Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

(Invitrogen, Paisley, UK), filter sterilized and run through 1 ml Hi-Trap Mab Select Sure protein A affinity columns (GE Healthcare, Amersham, UK), which had previously been sanitized with 0.5 M NaOH and equilibrated into PBS, at a flow rate of 1 ml/min. The columns were washed with 20 ml PBS pH 7.4 and antibody was eluted in 1 ml fractions with 0.1 M sodium citrate pH 3.0 followed by immediate neutralization with 0.1 ml 1 M Tris pH 9.0. The protein content of each fraction was monitored by UV absorption at 280 nm and protein containing fractions were pooled and immediately buffer exchanged into lx PBS pH 7.4 (PAA Laboratories, Yeovil, UK) using PD10 desalting columns (GE Healthcare). The antibodies were further purified by size exclusion chromatography using a 16/60 Superdex S200 column (GE Healthcare) in lx PBS. The major peak fractions were collected, pooled, filter sterilized and stored at +4°C. Final concentrations were determined by UV absorption using calculated molar extinction coefficients where A280 1.0 = 1.50 mg/ml.

Endotoxin levels were analyzed for all four preparations using an Endosafe®-PTS™ (Charles River, Margate, UK) and found to be within the tolerances of the EpiScreen assay

(<5.0 EU/mg).

EpiScreen™ time course T cell proliferation assays

PBMCs from each donor were thawed, counted and viability assessed. Cells were revived in room temperature AIM-V® culture medium (Invitrogen, Paisley, UK), washed and resuspended in AIM-V® to 4-6x106 PBMC/ml. For each donor, bulk cultures were

established in which 1 ml proliferation cell stock was added to the appropriate wells of a 24 well plate. Culture medium, 0.5 ml, and 0.5 ml of each diluted test sample were added to the PBMC to give a final concentration of 50 μg/ml per sample. For each donor, a

reproducibility control (cells incubated with 100 μg/ml KLH), and a culture medium only well

were also included. Cultures were incubated for a total of 8 days at 37°C with 5% C02. On days 5, 6, 7 and 8, the cells in each well were gently resuspended and 3 x 100 μΐ aliquots transferred to each well of a round bottomed 96 well plate. The cultures were pulsed with 0.75 μα [3H]-Thymidine (Perkin Elmer®, Beaconsfield, UK) in 100 μΐ AIM-V® culture medium and incubated for a further 18 hours before harvesting onto filter mats (Perkin

Elmer®) Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

using a TomTec Mach III cell harvester. Counts per minute (cpm) for each well were determined by Meltilex™ (Perkin Elmer®) scintillation counting on a 1450 Microbeta

Wallac Trilux Liquid Scintillation Counter (Perkin Elmer®) in paralux, low background counting.

EpiScreen™ data analysis

For proliferation assays, an empirical threshold of an SI equal to or greater than 2 (SI > 2.0) has been previously established whereby samples inducing proliferative responses above

this threshold are deemed positive (where included, borderline Sis > 1.90 are highlighted).

Extensive assay development and previous studies have shown that this is the minimum signal to noise threshold allowing maximum sensitivity without detecting large numbers of false positive responses. For proliferation data sets (n=3), positive responses were defined by statistical and empirical thresholds:

1. Significance (p<0.05) of the response by comparing cpm of test wells against medium

control wells using unpaired two sample student's t-test.

2. SI equal to or greater than 2 (SI > 2.0).

In addition, intra-assay variation was assessed by calculating the CVs and SDs of the raw data from replicate cultures.

Results

The humanized and chimeric anti-av 5 antibodies were tested against a cohort of 20 healthy donors using EpiScreen™ time course T cell assay in order to determine the relative risk of immunogenicity. The samples were tested at a final concentration of 50 μg/ml based on previous studies showing that this saturating concentration is sufficient to stimulate detectable antibody-specific T cell responses. In order to assess the immunogenic potential of each sample, the EpiScreen™ time course T cell assay was used with analysis of proliferation to measure T cell activation.

Since the samples had not been previously assessed in a PBMC -based assay, an initial Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

assessment of any gross toxic effect of the samples on PBMC viability was determined. Cell viabilities were calculated using Trypan Blue dye exclusion of PBMC 7 days after culture with

the test samples. Figure 8 shows the mean viabilities of cells from 6 donors. It was clear that the test samples did not significantly affect the viability of the cells since PBMC from medium alone cultures had a mean viability similar to that of the test samples.

Table 3 provides a summary of the results obtained in the EpiScreen™ time course T cell

proliferation assay of CD4+ T cell responses induced by the test antibodies.

TABLE 3

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Figure 9 illustrates the donor SI responses to each of the test antibodies throughout the time course. The humanized anti-avp5 antibodies VHWK1 and VH1/VK2 induced no positive responses using SI > 2.0, p < 0.05 threshold in any of the donors in the proliferation assay, whereas the chimeric anti-avp5 antibody induced positive T cell proliferation responses in 30% of donors. This is comparable to an immunogenic clinical standard antibody humanized A33 which induced 35% of donors to respond positively (Welt et al, Clin. Cancer Res., 9(4): 1338-1346 (2003)). Results with the reproducibility control antigen KLH show that there was a good correlation (<10% interassay variability) between positive and negative results in repeat studies Test 1 and STR02 (Table 2), which indicates a high level of

reproducibility in the assay. In addition, the frequency of T cell proliferation responses to KLH was in the expected range (80-90%).

Conclusions

The EpiScreen™ time course T cell proliferation assay was used to determine the potential for clinical immunogenicity of two humanized anti-av 5 antibodies. The

humanized antibodies were tested for their ability to induce CD4⁺ T cell responses as measured by proliferation against a panel of 20 HLA-typed donors compared to a chimeric antibody control. Frequent and potent positive CD4⁺ T cell proliferation responses were observed in the EpiScreen™ time course T cell assay against the reproducibility control antigen, KLH, which indicated that the assay functioned as expected. T cell proliferation responses were also observed against the chimeric antibody in 30% of the study cohort, thus showing that the chimeric antibody has a significant immunogenic potential. No responses were seen to the humanized anti-av 5 antibodies indicating that they have a very low

potential for immunogenicity.

Previous EpiScreen™ time course T cell assays with a range of biologies (Figure 10) have shown a clear correlation between the percentage of donor T cell responses in the

EpiScreen™ assay and the level of immunogenicity (anti-protein therapeutic antibody responses) observed in the clinic. High frequency donor responses were observed in

EpiScreen™ assays for immunogenic antibodies such as Campath, whereas relatively low frequency donor responses were observed for non-immunogenic antibodies such as Xolair and Herceptin. In general, protein therapeutics that induce <10% positive responses in the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

EpiScreen™ assay are associated with a low risk of immunogenicity in the clinic. The current study shows that, in comparison to other protein therapeutics tested in EpiScreen™ assays (Figure 10), the humanized anti-avp5 antibodies fall into the same range as Xolair, Herceptin and Avastin, and would be considered as having a low risk of immunogenicity. In comparison, the chimeric antibody stimulated 30% of donors to respond in the EpiScreen™ assay and would therefore fall into the same range as the moderately immunogenic antibodies such as Humira and Synagis. Humanized A33 induced 35% of the study cohort to respond positively in the T cell proliferation assay, this compares with the typical range of 25-35% anti-drug antibody responses seen in clinical trials. In sum, the humanized anti- av 5antibodies exhibit a clinically acceptable immunogenicity profile from the EpiScreen™ assay.

Example 8; Efficacy of Different Fc versions of Humanized ALULA in the Prevention of Renal Ischemia in the Rat Unilateral Ischemic Model with Pre-clamp Treatment Objective:

This study tested the efficacy of humanized ALULA expressed as a chimeric mAb with two different murine Fc tails (IgG2a or agly IgGl) compared to murine ALULA (murine IgG2b) or an isotype control mAb in the rat kidney unilateral ischemic clamp model after subcutaneous (SQ) injection administration 6 hr pre clamp release. This study compared the activity of the agly IgGl mAb to one containing a murine IgG2a Fc tail because murine

IgG2a Fc is expected to have full effector function in rat. The original murine ALULA was included as a control.

Antibodies:

1. Murine ALULA mAb {murine IgG2b) Below is an exemplary heavy chain of a murine ALULA IgG2b construct (with constant regions from a C57B1/6 mouse). The three CDRs of the heavy chain are boldened.

Heavy chain:

EVQVQQSGTVLARPGASV MSCi ASGYTFTSYWMHWV QRPGQGLEWIGAIYPGNSDTSYNQKFK GKA.B LTAVTSPNTAYMELSSLTNEDSAVYYCTTTTYGYDWFAYWGQGTLVTVSAA TTPPSVYPLA PGCGDTTGSSVTSGCLV GYFPEPVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVT CSVAHPASSTTVD i LEPSGPISTINPCPPCi ECH CPAPNLEGGPSVFIFPPNIKDVLMISLTP VTCVW Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRWSTLPIQHQDWMSGi EF C VNNKDLPSPI ERTIS I GLVRAPQVYTLPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSD GSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 66)

Light chain: Below is an exemplary light chain of a murine ALULA IgG2b construct (with constant regions from a C57B1/6 mouse). The three CDRs of the light chain are boldened.

NIMMTQSPSSLTVSAGE VTMSCKSSQSVLYSSNQKNYLAWYQQKTGQSPi LLIYWASTRESGVPDR FTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSLTFGAGTi LELi RADAAPTVSIFPPSSEQLTSGGASV VCFLNNFYPKDINV WKJDGSERQNGVLNSWTDQDS DSTYSMSSTLTLTKDEYERHNSYTCEATH T STSPIV SFNRNEC (SEQ ID NO :67)

2. Humanized ALULA HI /LI as a murine IgGl agly

This antibody has the following heavy and light chain sequences. These are mature

sequences without signal peptides. The variable domains are underlined. The three CDRs of the heavy chain and light chain are boldened. Heavy chain:

EVOWOSGTELKKPGASVKMSCKA.SGYTFTS MH KQAPGOGLEWIGAIYPGNSDTSYNOKFK GKAKLTAVTSPNTAYMELS SLRSED S AVYYCTTTTYGYDWFAYWGOGTLVTVS SA TTPP S VYPLAP GSAAQTNSMVTLGCLV GYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTC NVAHPASST VDKKIVPRDCGCKTCICTVPEVSSVFIFPP PKDVLTITLTP VTCVWDISKDDPEVQFS WFVDDVEVHTAQTQPREEQFQSTFRSVSELPIMHQDWLNGi EF CRVNSAAFPAPIE TIS T GRPKA. PQVYTIPPP EQMAKD VSLTCMITDFFPEDITVEWQWNGQPAENYi TQPIMDTDGSYFVYSi LNVQ SNWEAGNTFTCSVLHEGLHNHHTE SLSHSPG (SEQ ID NO:68)

Light chain:

NIMMTOSPATLTVSAGERATLSCKSSOSVLYSSNOKNYLAWYOOKTGOSPI LLIYWASTRESGVPDR FTGSGSGTDFTLTISSLQAEDVAVYYCHQYLSSLTFGOGT LEII RADAAPTVSIFPPSSEOLTSGGASV VCFLNNFYPKDINV WKJDGSERQNGVLNSWTDQDS DSTYSMSSTLTLTKDEYERHNSYTCEATH T STSPIVKSFNRNEC (SEQ ID NO:69)

3. Humanized ALULA HI /LI as a murine IgG2a (not agly) This antibody has the following heavy and light chain sequences. These are mature

sequences without signal peptides. The variable domains are underlined. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Heavy chain:

EVOWOSGTELKKPGASVKMSCKA.SGYTFTS MHWVKQAPGOGLEWIGAIYPGNSDTSYNOKFK GKAKLTAVTSPNTAYMELS SLRSED S AVYYCTTTTYGYDWFAYWGOGTLVTVS S AKTTAP S VYPL AP VCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN VAHPASSTKVDKXIEPRGPTIKTCPPCKCPAPNLLGGPSVFIFPPKTKDVLMISLSPIVTCVWDVSEDDPD VQISWFVNNVEVHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKXiFKCKVNNKDLPAPIERTISKTK GSVRAPQVWLPPPEEEMTKXQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMY SKLRVEKKN ERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO:70)

Light chain:

NIMMTQSPATLTVSAGERATLSCKSSOSVLYSSNOKNYLAWYOQKPGOSPKLLIYWASTRESGVPDR FTGSGSGTDFTLTISSLOAEDVAVYYCHOYLSSLTFGOGTKLEIKRADAAPTVSIFPP S SEQLT SGGASV VCFLNNFYPKDINVKWKTDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC (SEQ ID NO: 71)

Methods: Unilateral Clamp Ischemia Model Protocol

The animal was anesthetized with an Isofluorane / O2 mixture, 5% for induction and 1-2% for maintenance of anesthesia. An induction chamber was used for induction and an anesthesia circuit was used during surgery. The abdomen was shaved with clippers and washed with germicidal soap and water, towel dried and swabbed with Betadine. The animal was placed on a sterile disposable absorbent towel over a warming pad thermostatically controlled by a rectal thermometer. The animal was monitored continuously for pulse, oximetry, respiratory rate, and blood pressure. The abdomen was opened using a 3 cm midline incision. Each kidney was isolated and the fat and connective tissue surrounding the renal artery and vein were dissected away using sterile cotton swabs. The right kidney was removed and the renal artery and vein sutured off.

Ischemia of the left kidney was initiated by clamping the renal artery and vein for 30 minutes using non-traumatic clamps on each renal pedicle. For sham surgery, the kidneys were isolated as above but not clamped. The incision was covered with sterile saline

saturated gauze sponge during the ischemic period. At the conclusion of the ischemic period, the clamps were removed and the kidneys were observed to insure rapid re-establishment of blood flow. The animal was rehydrated Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

with 2 cc of sterile saline introduced into the abdominal cavity. The muscle layer was closed with 3-0 silk; the skin was closed with surgical 3-0 silk.

The test agents (ALULA antibodies described above or the control antibody) was administered by subcutaneous injection, in an injection volume of 300 μΐ, 6 hours prior to clamping.

Serum creatinine was measured at 0, 24, 48, and 72 hours post-surgery and the results were reported as mg per deciliter.

Animals:

Species/ Strain: Sprague Dawley Rats

Source: Harlan Laboratories

P.O. Box 29176

Indianapolis, Indiana 46229-0176

USA

Age: 50-60 days

Body Weight Range: 250-320 g

Sex: Males

Group Size: 6 rats

Total number: 24 rats

Diet: Animals were provided an ad libitum commercial rodent diet and free access to

drinking water.

Environment: (i) Acclimatization of at least 5 days.

(ii) All the animals were confined in a limited access facility with environmentally-controlled housing conditions throughout the entire study period, and maintained in accordance with approved standard operating procedures (SOPs).

Blood Sampling

A 0.15 mL venous blood sample was drawn at study initiation for baseline creatinine measurement and at 0, 24, 48, and 72 hours post-surgery for pathological serum creatinine levels evaluation. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Study Termination

72 hours post-surgery, the study was terminated and the rats were euthanized by pentobarbital overdose followed by cervical dislocation.

Creatinine Measurement

Serum creatinine of all rats was measured at baseline and at days 1, 2, and 3 post- clamp. A 0.15 ml venous blood sample was drawn and centrifuged. The serum was removed and stored at +4°C and saved for analysis. Creatinine concentration was measured on a

Creatinine Analyzer 2 from Beckman Inc. The machine was standardized with a known control and the samples were run using a picric acid reaction.

Study Design

mg/kg

Group Sample BW kg mg/rat N Doses

Isotype negative mAb-

1 IgG2b 1 0.3 0.3 6 1

anti-avb5mAb, ALULA

2 IgG2b (original mu mAb) 1 0.3 0.3 6 1

anti-avb5mAb, ALULA

agly IgGl (humanized/mu

3 Fc) 1 0.3 0.3 6 1

anti-avb5mAb, ALULA

4 IgG2a (humanized/mu Fc) 1 0.3 0.3 6 1

Dosed: 24

Animals: 24

Results:

The creatinine measurements (in mg/dL) at baseline, 24 hours, 48 hours, and 72 hours post-surgery from this study are summarized in the Tables below. A value of 2.5-3.0 at 24 hours in untreated ischemic rats is common. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

Groupl: Antibody: Isotype negative mAb-IgG2b; Dose: lmg/kg/BW (6hr pre-clamp).

Group2: Antibody: ALULA IgG2b; Dose: lmg/kg/BW (6hr pre-clamp).

Group3: Antibody: chimeric humanized ALULA agly IgGl; Dose: lmg/kg/BW (6hr clamp).

Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

The serum creatinine data above indicates that there is no difference in potency when comparing all three versions of the mAb ALULA. The fact that there is no loss of activity after eliminating effector function suggests that the effector function is not required as part of the mechanism of action producing efficacy.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001 WHAT IS CLAIMED IS:

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to ανβ5, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region that is: at least 94% identical to the amino acid sequence set forth in SEQ ID NO: l ; at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3; at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5; at least 89% identical to the amino acid sequence set forth in SEQ ID NO:7; or at least 88% identical to the amino acid sequence set forth in SEQ ID NO:9.

2. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises a heavy chain variable region that is: at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; or at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9.

3. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises a heavy chain variable region that is: at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 1; at least 94% identical to the amino acid sequence set forth in SEQ ID NO:3; at least 94% identical to the amino acid sequence set forth in SEQ ID NO:5; at least 94% identical to the amino acid sequence set forth in SEQ ID NO:7; or at least 94% identical to the amino acid sequence set forth in SEQ ID NO:9. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

4. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises a heavy chain variable region that is: at least 95% identical to the amino acid sequence set forth in SEQ ID NO: l ; at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3; at least 95% identical to the amino acid sequence set forth in SEQ ID NO:5; at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7; or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:9.

5. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises a heavy chain variable region that is: at least 98% identical to the amino acid sequence set forth in SEQ ID NO: l ; at least 98% identical to the amino acid sequence set forth in SEQ ID NO:3; at least 98% identical to the amino acid sequence set forth in SEQ ID NO:5; at least 98% identical to the amino acid sequence set forth in SEQ ID NO:7; or at least 98% identical to the amino acid sequence set forth in SEQ ID NO:9.

6. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; or a heavy chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11.

7. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11.

8. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; or a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11.

9. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; or a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11.

10. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11 ; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11.

11. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; or a heavy chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13.

12. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; or a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13.

13. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13.

14. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13; or a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13.

15. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 13; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 13; or a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 13.

16. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15.

17. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15; or a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 15.

18. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15; or a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15.

19. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 15.

20. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 15; or a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 15.

21. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; or a heavy chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17.

22. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 89% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 17.

23. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 3 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 5 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17; or a heavy chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 17.

24. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 17; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 17; or a heavy chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 17.

25. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 17; or Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 17.

26. The antibody or the antigen-binding fragment thereof of any one of claims 1 to 25, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions, the heavy chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions, and the heavy chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid positions.

27. The antibody or the antigen-binding fragment thereof of any one of claims 1 to 25, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDRs 1, 2 and 3 wherein the heavy chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the heavy chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and the heavy chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 21.

28. The antibody or the antigen-binding fragment thereof of any one of claims 6 to 25, wherein the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions, the light chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions, and the light chain CDR 3 comprises the Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions.

29. The antibody or the antigen-binding fragment thereof of any one of claims 6 to 25, wherein the antibody or the antigen-binding fragment thereof comprises light chain CDRs 1,

2 and 3 wherein the light chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 22, the light chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and the light chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 24.

30. The antibody or the antigen-binding fragment thereof of any one of claims 6 to 25, wherein the antibody or the antigen-binding fragment thereof comprises: heavy chain CDRs 1 , 2 and 3 wherein the heavy chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 19 or the amino acid sequence set forth in SEQ ID NO: 19 with a substitution at two or fewer amino acid positions, the heavy chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 20 or the amino acid sequence set forth in SEQ ID NO: 20 with a substitution at two or fewer amino acid positions, and the heavy chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 21 or the amino acid sequence set forth in SEQ ID NO: 21 with a substitution at two or fewer amino acid

positions; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 22 or the amino acid sequence set forth in SEQ ID NO: 22 with a substitution at two or fewer amino acid positions, the light chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 23 or the amino acid sequence set forth in SEQ ID NO: 23 with a substitution at two or fewer amino acid positions, and the light chain CDR

3 comprises the amino acid sequence set forth in SEQ ID NO: 24 or the amino acid sequence set forth in SEQ ID NO: 24 with a substitution at two or fewer amino acid positions. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

31. The antibody or the antigen-binding fragment thereof of any one of claims 6 to 25, wherein the antibody or the antigen-binding fragment thereof comprises: heavy chain CDRs 1 , 2 and 3 wherein the heavy chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 19, the heavy chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and the heavy chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 21 ; and light chain CDRs 1, 2 and 3 wherein the light chain CDR 1 comprises the amino acid sequence set forth in SEQ ID NO: 22, the light chain CDR 2 comprises the amino acid sequence set forth in SEQ ID NO: 23, and the light chain CDR 3 comprises the amino acid sequence set forth in SEQ ID NO: 24.

32. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the

antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: l; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:3; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:5; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:7; or a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:9.

33. The antibody or the antigen-binding fragment thereof of claim 32, wherein the antibody or the antigen-binding fragment thereof comprises: Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 1; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 1; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 1; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 1; or a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 1.

34. The antibody or the antigen-binding fragment thereof of claim 32, wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 13; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 13; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 13; or a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:9 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 13.

35. The antibody or the antigen-binding fragment thereof of claim 32, wherein the antibody or the antigen-binding fragment thereof comprises: a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 15; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 15; or a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 15.

36. The antibody or the antigen-binding fragment thereof of claim 32, wherein the antibody or the antigen-binding fragment thereof comprises: Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:3 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:5 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 17; a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 17; or a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 17.

37. The antibody or the antigen-binding fragment thereof of any one of claims 1 to 36, wherein the antibody has an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

38. The antibody or the antigen-binding fragment thereof of any one of claims 1 to 36, wherein the antigen-binding fragment is selected from the group consisting of an Fab, an Fab', an F(ab')2, an Fv, a diabody, an scFv, and an sc(Fv)2.

39. The antibody or the antigen-binding fragment thereof of any one of claims 1 to 38, wherein the antibody is conjugated to a substance selected from the group consisting of a toxin, a radionuclide, a fluorescent label, polyethylene glycol, and a cytotoxic agent. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

40. A pharmaceutical composition comprising the antibody or the antigen-binding fragment thereof of any one of claims 1 to 39 and a pharmaceutically acceptable carrier.

41. A method of treating a cancer in a human subject in need thereof, comprising

administering to the human subject the antibody or the antigen-binding fragment thereof of any one of claims 1 to 39.

42. The method of claim 41, wherein the cancer is a pancreatic cancer, a lung cancer, a breast cancer, a colorectal cancer, a head and neck cancer, an esophageal cancer, a skin cancer, and an endometrial cancer.

43. A method of treating pulmonary edema in a human subject in need thereof,

comprising administering to the human subject the antibody or the antigen-binding fragment thereof of any one of claims 1 to 39.

44. A method of treating lung fibrosis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof of any one of claims 1 to 39.

45. The method of claim 44, wherein the lung fibrosis is usual interstitial pneumonia.

46. The method of claim 45, wherein the lung fibrosis is idiopathic pulmonary fibrosis. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

47. A method of treating acute lung injury in a human subject in need thereof,

48. A method of inhibiting angiogenesis in a human subject in need thereof, comprising administering to the human subject the antibody or the antigen-binding fragment thereof of any one of claims 1 to 39.

49. An isolated nucleic acid comprising a nucleotide sequence that is at least 85%

identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

50. The isolated nucleic acid of claim 49, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs.: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

51. An isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 85% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 1, 3, 5, 7, 9, 1 1, 13, 15, and 17.

52. The isolated nucleic acid of claim 51, comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs.: 1, 3, 5, 7, 9, 11, 13, 15, and 17.

53. An isolated protein encoded by the nucleic acid of any one of claims 49 to 52.

54. A recombinant vector comprising the nucleic acid of any one of claims 49 to 52. Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

A host cell comprising the recombinant vector of claim 54.

56. A method of preparing a humanized antibody comprising culturing a host cell comprising recombinant vectors comprising: the nuc eic acid sequences set forth n SEQ ID NOs: 2 and 12; the nuc eic acid sequences set forth n SEQ ID NOs: 2 and 14 the nuc eic acid sequences set forth n SEQ ID NOs: 2 and 16 the nuc eic acid sequences set forth n SEQ ID NOs: 2 and 18 the nuc eic acid sequences set forth n SEQ ID NOs: 4 and 12 the nuc eic acid sequences set forth n SEQ ID NOs: 4 and 14 the nuc eic acid sequences set forth n SEQ ID NOs: 4 and 16 the nuc eic acid sequences set forth n SEQ ID NOs: 4 and 18 the nuc eic acid sequences set forth n SEQ ID NOs: 6 and 12 the nuc eic acid sequences set forth n SEQ ID NOs: 6 and 14 the nuc eic acid sequences set forth n SEQ ID NOs: 6 and 16 the nuc eic acid sequences set forth n SEQ ID NOs: 6 and 18 the nuc eic acid sequences set forth n SEQ ID NOs: 8 and 12 the nuc eic acid sequences set forth n SEQ ID NOs: 8 and 14 the nuc eic acid sequences set forth n SEQ ID NOs: 8 and 16 the nuc eic acid sequences set forth n SEQ ID NOs: 8 and 18 the nuc eic acid sequences set forth n SEQ ID NOs: 10 and 12; Attorney Docket No .: 13751 -0144 WO 1 /P 1056 WO 001

the nucleic acid sequences set forth in SEQ ID NOs: 10 and 14; the nucleic acid sequences set forth in SEQ ID NOs: 10 and 16; or the nucleic acid sequences set forth in SEQ ID NOs: 10 and 18; under conditions appropriate for expression of a humanized antibody, wherein humanized antibody chains are expressed and a humanized antibody is produced.

57. The method of claim 56, further comprising isolating the humanized antibody.

58. The method of claim 56 or 57, wherein the host cell is a CHO cell.